Restoration of RBM22 overcomes the transcriptional and epigenetic barriers of cardiomyocyte proliferation for heart regeneration

Domain: chemistry
Published: 2026-03-09
Editorial outcome: major_revision

Abstract
Stimulating endogenous cardiomyocyte proliferation holds great therapeutic promise for
cardiac repair, but how chromatin remodeling governs this process remains poorly
understood. RNA-binding motif 22 (RBM22) participates in the regulation of various
biological contexts, whereas its role in cardiac regeneration and repair is largely unknown.
Here, we identify RBM22 as a pivotal regulator of cardiomyocyte proliferation.
Cardiomyocyte-specific deletion of Rbm22 impairs neonatal heart regeneration and
exacerbates post-infarction ventricular remodeling in adult mice. Mechanistically, RBM22
selectively binds to the proximal promoters of key cell cycle genes (Cdk4, Ccna2, and Ccne1),
where it cooperates with chromatin remodeler SMARCA4 to enhance transcriptional
accessibility. Furthermore, RBM22 is essential for the gene-specific recruitment of RNA
Polymerase II to these gene loci to drive transcription. AAV9-mediated delivery of Rbm22
promotes cardiomyocyte proliferation in vivo following cardiac damage and increases the
proliferation of human induced pluripotent stem cell-derived cardiomyocytes. Our findings
establish RBM22 as a transcriptional and epigenetic regulator that overcomes cell-cycle
barriers in cardiomyocytes, highlighting its therapeutic potential for cardiac injury.

Introduction
Heart failure is the leading cause of mortality globally, with ischemic cardiomyopathy being
the predominant condition contributing to this adverse outcome1. Following myocardial
infarction (MI) in adults, a significant number of cardiomyocytes are irreversibly lost. For
decades, the prevailing dogma holds that the mammalian heart exists as a post-mitotic
organ, completely devoid of regenerative capacity beyond the neonatal period2,3. Emerging
evidence now demonstrates the relatively low-level cardiomyocyte turnover in adult
mammalian hearts, though this endogenous regenerative potential proves insufficient to
compensate for catastrophic cell loss after ischemic injury4,5. The absence of regenerative
potential in adult cardiomyocytes prompts fibroblasts to rapidly proliferate and stabilize
damaged myocardium through extracellular matrix deposition. However, persistent fibrotic
remodeling drives pathological tissue stiffening, ventricular impairment, and heart failure
progression6. Current therapies only mitigate symptoms but fail to regenerate functional
cardiomyocytes, highlighting the need for regenerative strategies targeting cardiomyocyte
replenishment.

Previous studies have shown that the hearts of some adult vertebrates and neonatal mice
possess robust regenerative capabilities, and pre-existing cardiomyocytes re-enter the cell
cycle to proliferate and replenish lost cardiomyocytes7,8. However, the regenerative capacity
of neonatal mouse hearts markedly diminishes at postnatal day 7 (P7). Hence, an in-depth
investigation into the mechanisms underlying facilitating endogenous cardiomyocyte
proliferation and myocardial regeneration is crucial for promoting cardiac repair after
myocardial injury. Within the adult mouse heart, the complete exit of cardiomyocytes from
the cell cycle and remaining non-dividing markedly limit their proliferative capacity. This
phenomenon suggests that the diminished capacity for cardiomyocyte proliferation is
predominantly attributed to the exit from the cell cycle9. Cell cycle regulatory proteins play
a pivotal role in the critical phases, facilitating cell growth and division, which serve as the
potential therapeutic targets for enhancing the proliferation of adult mammalian
cardiomyocytes3,10. Identifying the key factors that promote cardiomyocytes to re-enter the
cell cycle is therefore crucial for preventing adverse cardiac remodeling after MI.

In eukaryotic cells, transcriptional regulation relies on dynamic control of chromatin
accessibility, which determines the extent to which DNA is exposed and available for
interaction with transcriptional machinery11. This accessibility is continuously modulated by
nucleosome positioning, histone modifications, and higher-order chromatin organization.
As the ATPase subunit of the SWI-SNF complex, SMARCA4 (SWI/SNF related BAF chromatin
remodeling complex subunit ATPase 4) drives local chromatin remodeling in a locus- and
cofactor-dependent manner. Through nucleosome repositioning or eviction, SMARCA4
modulates the accessibility of promoters and enhancers, thereby regulating gene expression
in a context-dependent and functionally selective manner12,13. Modulating the accessibility
of chromatin is essential for the precise regulation of specific gene expression, a conserved
epigenetic mechanism that has been shown to play a crucial role in various biological
processes, including cardiac regeneration and development14. The profiling of
cardiomyocyte transcriptional program uncovers that adult mouse cardiomyocytes fail to
re-activate the neonatal proliferative network post-infarction, which is closely associated
with loss of chromatin accessibility around cell cycle genes during postnatal maturation15.
For example, dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) loss-
of-function in cardiomyocytes leads to open and accessible chromatin, which facilitates
expression of several transcription factors involved in promoting cardiomyocyte cell cycle
activation16. In addition, YAP5SA, a version of YAP in which all LATS1/2 phosphorylation sites
are mutated from serine (S) to alanine (A), also enhances chromatin accessibility and
upregulates the expression of genes characteristic of fetal development, thereby partially
reprogramming cardiomyocytes in vivo towards a more primitive, fetal-like state with
enhanced proliferative capacity17. Despite these findings, a thorough comprehension of the
underlying epigenetic regulatory networks is still required to develop strategies aimed at
promoting desirable cardiomyocyte proliferation.

RNA-binding proteins (RBPs) have been established as critical effectors of controlling gene
expression, and as such, their malfunction underlies the origin of many diseases18. RBPs
orchestrate post-transcriptional gene expression by regulating alternative splicing (AS),
transport, localization, stability, polyadenylation, and translation of pre-mRNA19. RNA-
binding motif 22 (RBM22), a highly conserved RBP, regulates gene expression at the post-
transcriptional level19. Furthermore, RBM22 participates in gene regulation by binding DNA
and functioning as a non-canonical transcription factor on certain target genes20. RBM22
has been found to play a critical role in regulating embryonic heart development in both
zebrafish and drosophila21,22. Moreover, RBM22 has both tumor-promoting and tumor-
suppressing effects in various cancer types, including glioblastoma and lung cancer20.
However, the role of RBM22 in the regenerative capacity of the postnatal heart remains
unclear.

Here, we demonstrate that RBM22 expression is significantly increased during postnatal
heart regeneration upon injury, which is correlated with the enhanced cardiomyocyte
proliferation. Rbm22 deficiency in neonatal cardiomyocytes impairs heart regeneration after
apical resection (AR) and MI, whereas the loss of Rbm22 in adult cardiomyocytes
compromises cardiac repair post-MI. RBM22 can selectively bind to the proximal promoters
of genes encoding cell cycle regulators, increasing chromatin accessibility through the
interaction with SMARCA4, which in turn enhances their transcription and promotes
cardiomyocyte cell cycle activity. This study reveals a significant role of RBM22 in mediating
cardiomyocyte proliferation and establishes it as a potential therapeutic target for cardiac
regeneration after myocardial injury.

Results
RBM22 expression profile in postnatal and injured hearts
It is universally acknowledged that neonatal mice exhibit a transient regenerative capacity in
response to cardiac injury within the first postnatal week8,17. Given that transcriptional
regulation plays a critical role in controlling cardiomyocyte proliferation during this
regenerative window, RNA-binding motif (RBM) proteins are likely to be key modulators
involved in these processes18,23,24. This prompts us to investigate the expression patterns of
RBM proteins in the postnatal and injured mouse heart. We conducted cross-species
transcriptomic analyses using published datasets. Comparative profiling identified 34 and 28
developmental-stage-regulated RBM genes in murine (GSE51483) and porcine (GSE145346)
hearts, respectively (Supplementary Fig. 1a, b). Analysis of neonatal mouse hearts post-MI
(GSE221539) revealed 39 injury-responsive RBM genes (Supplementary Fig. 1c, d).
Integrated analysis of murine and porcine transcriptomes identified seven conserved RBM
genes showing biphasic expression patterns: progressive downregulation during postnatal
maturation (from P1 to adulthood) with re-activation following myocardial injury in neonatal
mice (Fig. 1a). Notably, RBM22 exhibited the most prominent induction among these
candidates in neonatal mouse hearts post-MI (Fig. 1b). Analysis of human myocardial tissue
from ischemic cardiomyopathy patients (GSE5406) also showed that RBM22 expression was
significantly increased compared with non-failing controls (Fig. 1c). This finding was
consistent across both human and mouse models, supporting the potential role of RBM22
in cardiac disease.

To validate the dynamic expression of RBM22 during heart injury, we resected the ventricular
apex of the mouse hearts at P3 and examined the expression change of RBM22 at day 7
post-resection (dpr). The above regulatory pattern was functionally validated in AR models,
where RBM22 was displayed the most significant upregulation among candidate genes at 7
dpr (Fig. 1d and Supplementary Fig. 1e). Moreover, developmental profiling revealed the
progressive decrease of RBM22 protein level in murine hearts from P1 to P7 (Fig. 1e), which
coincides with the regenerative windows of hearts in mice8.However, RBM22 protein was
markedly induced at 7 dpr (Fig. 1f).

Consistent with these temporal patterns, analysis of the GSE130699 dataset showed that
Rbm22 is predominantly expressed in cardiomyocytes (Supplementary Fig. 1f, g).
Furthermore, immunofluorescence analysis further demonstrated that this injury-induced
RBM22 expression was mainly localized to cardiomyocytes rather than non-myocytes (Fig.
1g). Additionally, RBM22 was found to be predominantly enriched in the border zone post-
MI in neonatal mice, with relatively lower expression in the remote areas (Supplementary Fig.
1h, i). In addition, as RBM22 is primarily expressed in cardiomyocytes, we isolated
cardiomyocytes across multiple postnatal ages and confirmed that RBM22 expression
progressively declined with maturation at both the mRNA and protein levels (Fig.1h, i).
Furthermore, immunofluorescence staining also visualized this pattern and revealed a
marked decline in the proportion of RBM22⁺ cardiomyocytes during postnatal maturation,
whereas cardiac injury induced a significant increase in the proportion of RBM22⁺
cardiomyocytes (Fig. 1j, k). A previous study has shown that hypoxia promotes
cardiomyocyte proliferation25. Consistently, we found that hypoxia exposure could induce a
significant upregulation of Rbm22 expression in primary neonatal mouse cardiomyocytes
(Fig. 1l). Therefore, the dynamic expression pattern of RBM22 during heart development and
damage suggests that it might play a role in cardiomyocyte proliferation.

RBM22 is required for neonatal heart regeneration
These above observations prompted us to examine whether there is a correlation between
RBM22 and cardiomyocyte proliferation. By conducting gene expression correlation analysis,
we identified 4 genes that were correlated with Rbm22, including proliferating cell nuclear
antigen (Pcna), marker of proliferation Ki-67 (Mki67), cyclin-dependent kinase inhibitor 1A
(Cdkn1a), and transformation related protein 53 (Trp53), according to the published RNA-
seq data (GSE51483). Rbm22 mRNA expression demonstrated strong positive correlations
with proliferative markers (Supplementary Fig. 1j, k), but inverse correlations with cell cycle
inhibitory factors (Supplementary Fig. 1l, m). To elucidate the potential role of RBM22 in
cardiomyocyte proliferation in postnatal hearts, we first generated cardiomyocyte-specific
Rbm22-deficient mice by crossing Rbm22fl/fl mouse with Myh6-iCre mouse (Supplementary
Fig. 2a). During the generation of the conditional knockout mice, it revealed complete
embryonic lethality in homozygous mutants (Rbm22fl/fl Myh6-iCre+), as evidenced by ratio
analysis (Supplementary Fig. 2b, c), suggesting the essentiality of RBM22 in murine
cardiogenesis. Supporting this developmental effects, bioinformatic analysis of neonatal
cardiomyocyte transcriptomes found the significant co-expression correlation between
Rbm22 and the cardiac determinant NK2 homeobox 5 (Nkx2-5), according to the published
RNA-seq data from GEO dataset GSE193137 (Supplementary Fig. 2d). To further interrogate
this relationship, analysis of RNA-seq data from dataset GSE77547 showed that Rbm22
expression is reduced following Nkx2-5 knockout (Supplementary Fig. 2e), suggesting that
RBM22 may be transcriptionally regulated by NKX2-5. Consistently, Chromatin
Immunoprecipitation-sequencing (ChIP-seq) profiling from dataset GSE77548 showed an
enrichment of NKX2-5 chromatin signals around the transcription start site of the RBM22
gene locus, further supporting the hypothesis that NKX2-5 may regulate RBM22 expression
in cardiomyocytes (Supplementary Fig. 2f). This suggests that RBM22 is critical for cardiac
development.

To circumvent embryonic lethality, we generated tamoxifen-inducible cardiomyocyte
conditional knockout (iCKOT) mice through cross-breeding Rbm22fl/fl mice with αMHC-
MerCreMer (Cre+/-) mice (Supplementary Fig. 2g, h). To exclude potential tamoxifen toxicity,
tamoxifen administration was performed from P0 to P3 in Cre+/- mice, and cardiac function
was assessed at P21. The results showed normal cardiac function with no significant change
in cardiac morphology (Supplementary Fig. 2i, j). Next, tamoxifen administration from P0 to
P3 was performed in two kinds of control (Rbm22fl/fl mice and Cre+/- mice) and Rbm22-iCKO
mice (Supplementary Fig. 2k). RBM22 protein expression was markedly reduced after
tamoxifen administration for four consecutive days after birth (Supplementary Fig. 2l).
Echocardiography also revealed no alterations in cardiac function and ventricular wall
thickness in control mice and Rbm22-iCKO mice (Supplementary Fig. 2m, n). The cardiac
morphology showed no obvious change in hearts from Rbm22-iCKO mice, compared to the
control mice at P21 (Supplementary Fig. 2o), suggesting that cardiac-specific knockout of
Rbm22 in mice after birth has no effect on heart morphology under physiological conditions.
To further explore the role of RBM22 in cardiomyocyte proliferation and cardiac
regeneration in mice, we administrated control and Rbm22-iCKO mice with tamoxifen and
then performed AR at P3 for evaluation of regenerative capacity (Fig. 2a). Echocardiography
analysis displayed the worse heart function in Rbm22-iCKO mice at 21 dpr, compared to
Rbm22fl/fl and Cre+/-controls (Fig. 2b-f), as measured by left ventricular ejection fraction (LVEF),
left ventricular fraction shortening (LVFS), left ventricular internal diameter at end-systolic
(LVIDs), and left ventricular volume at systole (LV Vols). Masson trichrome staining analysis
revealed the markedly increased fibrosis and impaired regenerative capacity in Rbm22-iCKO
mice, compared to Rbm22fl/fl and Cre+/- controls at 21 dpr (Fig. 2g-i). Furthermore, neonatal
mice underwent permanent ligation of the left anterior descending (LAD) coronary artery to
induce MI at P3 (Supplementary Fig. 3a). Similar to the AR model, Rbm22 deficiency
compromised heart regeneration, leading to impaired cardiac function and increased scar
formation in neonatal mice post-MI (Supplementary Fig. 3b-h). These data imply that
Rbm22-specific deletion in cardiomyocytes impedes the recovery of heart function in
neonatal mice following cardiac injury. Furthermore, wheat germ agglutinin (WGA) staining
showed that Rbm22 knockout increased the cardiomyocyte cross-sectional area (CSA) at 21
dpr (Fig. 2j, k). Despite the heart weight remaining unchanged, this increase in CSA
suggested a reduction in the number of cardiomyocytes (Fig. 2l).

Previous studies have shown that the central feature of heart regenerative response is the
activation of cardiomyocyte proliferation following injury26. We further evaluated the cell
cycle entry of cardiomyocytes by measuring the nuclear incorporation of 5-ethynyl-2’-
deoxyuridine (EdU). EdU incorporation analysis revealed that the percentage of EdU-positive
cardiomyocytes significantly decreased in Rbm22-iCKO mice compared with their controls
at 7 dpr or day post-infarction (dpi) (Fig. 2m, n, and Supplementary Fig. 3i). To evaluate
mitotic progression, we examined phosphorylated histone H3 (pH3), a marker of transition
through G2/M27. The percentage of pH3-positive cardiomyocytes was significantly
decreased by Rbm22 knockout (Fig. 2o, p, and Supplementary Fig. 3j), suggesting that
Rbm22 knockout inhibits the proliferation of cardiomyocytes. Additionally, Aurora B staining
was performed to evaluate cytokinesis, as Aurora B is a core component of the chromosomal
passenger complex and localizes to the cleavage furrow and midbody during late mitosis,
thereby serving as a definitive marker of cardiomyocytes that have completed the terminal
stage of cell division28,29. Our data showed that Rbm22 knockout significantly reduced the
percentage of Aurora B-positive cardiomyocytes, indicating impaired cytokinesis in Rbm22-
iCKO hearts (Fig. 2q, r, and Supplementary Fig. 3k). Taken together, these data demonstrate
that RBM22 is required for neonatal heart regeneration and endogenous cardiomyocyte
proliferation after AR and MI injury.

Rbm22 knockout compromises cardiac repair in adult mice
The above observation in early postnatal mice prompts us to explore whether Rbm22
knockout influences cardiac repair in adult mice post-MI. Consistent with the neonatal injury
model, RBM22 expression was elevated in adult hearts post-MI and predominantly localized
to the border zone (Supplementary Fig. 4a, b). Previous studies have shown that tamoxifen
administration at an inappropriate dosage in αMHC-MerCreMer+ adult mice can induce a
DNA damage response in cardiomyocytes, leading to apoptosis and cardiac dysfunction30,31.
In light of these findings, we optimized the tamoxifen dosing regimen and determined that
a 30 mg/kg dosage, administered intermittently over three days, was the optimal condition
to induce recombination in the αMHC-MerCreMer system without causing adverse effects
such as cardiac hypertrophy or fibrosis30. Tamoxifen was used to induce Cre expression in
eight-week-old adult Rbm22-iCKO mice, leading to the deletion of Rbm22 from
cardiomyocytes (Supplementary Fig. 4c). Immunoblotting revealed a significant reduction in
RBM22 protein abundance in the cardiomyocytes of Rbm22-iCKO mice following tamoxifen
administration, relative to control mice (Rbm22fl/fl and Cre+/-) (Supplementary Fig. 4d). And
there was no remarkable difference in cardiac morphology and size between Rbm22-iCKO
and control mice (Supplementary Fig. 4e).

Rbm22-iCKO and control mice were administered tamoxifen at eight-week-old and
subsequently used to generate MI model (Fig. 3a). The echocardiographic assessment
revealed that Rbm22 deletion impaired cardiac function post-MI, as LVEF and LVFS values
were significantly lower than those in controls measured at 4 weeks post-MI (Fig. 3b, c).
Rbm22 knockout increased LVIDs, LV Vols, left ventricular internal diameters at end-diastole
(LVIDd), and left ventricular volume at diastole (LV Vold), indicating significant left ventricular
dilation and deteriorated cardiac function (Fig. 3b, c, and Supplementary Fig. 4f, g). Masson
trichrome staining showed the increased fibrotic area in Rbm22-iCKO mice (Fig. 3d, e). To
investigate whether RBM22 affects cardiomyocyte number, we isolated cardiomyocytes from
adult Rbm22-iCKO and control mice using collagenase digestion. Compared to control mice,
Rbm22-iCKO mice exhibited a significant decrease in the total number of cardiomyocytes
(Fig. 3f, g). Diploid cardiomyocytes are identified as the primary subpopulation capable of
proliferation and contributing to regeneration32. We then investigated whether Rbm22
knockout affected the ploidy of cardiomyocytes. There was a significantly decreased fraction
of mononucleated and diploid cardiomyocytes, as indicated by cTnT and DAPI staining of
isolated cardiomyocytes in Rbm22-iCKO mice (Fig. 3h-j). WGA staining revealed an
increased cross-sectional area of cardiomyocytes in Rbm22-iCKO mice (Fig. 3k, l). We further
evaluated cardiomyocyte proliferation in adult Rbm22-iCKO and control mouse hearts at
day 14 post-MI. Analysis of EdU incorporation, pH3, and Aurora B immunostaining showed
significant decreases in the percentages of cardiomyocytes positive for EdU, pH3, or Aurora
B at the MI border zone in adult Rbm22-iCKO mice compared with control mice (Fig. 3m-
r).

Moreover, Rbm22 reduction also compromised adult myocardial repair post-MI, as
Rbm22fl/+Myh6-iCre mice exhibited worsened cardiac function and increased fibrosis
(Supplementary Fig. 5a-d). Consistent with this phenotype, the total number of
cardiomyocytes was reduced, accompanied by a marked decrease in the mononuclear
diploid population (Supplementary Fig. 5e-h). Cardiomyocyte proliferation was also
significantly reduced in Rbm22fl/+Myh6-iCre mice, as indicated by reduced EdU- and Ki67-
positive cardiomyocytes, compared with that in controls (Supplementary Fig. 5i, j). Together,
these findings demonstrate that the loss of Rbm22, whether through heterozygous
reduction or complete genetic ablation, leads to cardiomyocyte cell-cycle arrest, thereby
impairing myocardial repair and exacerbating adult cardiac dysfunction post-MI.

RBM22 induces cardiomyocyte transcriptional reprogramming
We further isolated primary cardiomyocytes from neonatal mice to validate the role of
RBM22 in cardiomyocyte proliferation in vitro. Primary cardiomyocytes were isolated from
neonatal Rbm22-iCKO and control mice to assess their proliferative capacity. Consistent with
the in vivo findings, a significant reduction in cardiomyocyte proliferation was observed in
the Rbm22-iCKO group, compared to the controls (Fig. 4a-c). Subsequently, the siRNA
targeting Rbm22 (si-ARbm22 #1) and negative control (si-Ctrl #1) were transfected into the
primary cardiomyocytes isolated from neonatal mice at P1. The silencing efficiency of si-
Rbm22 #1 was confirmed at protein levels (Supplementary Fig. 6a, b). Immunofluorescence
analysis confirmed that Rbm22 silencing resulted in a significant decrease in cardiomyocyte
numbers (Supplementary Fig. 6c). The percentages of EdU-, Ki67-, and pH3-positive
cardiomyocytes were markedly decreased by Rbm22 silencing (Supplementary Fig. 6d-f),
suggesting that Rbm22 silencing inhibits the proliferation of cardiomyocytes in vitro. To
address potential off-target effects and further validate the observed phenotypes, we used
a second independent siRNA targeting Rbm22 (si-Rbm22 #2). Consistent with the initial
results, knockdown of Rbm22 using this second siRNA also resulted in a significant reduction
in the proliferative capacity of primary cardiomyocytes, as evidenced by decreased numbers
of EdU-, pH3-, and Aurora B-positive cardiomyocytes (Supplementary Fig. 6g-i). Cell cycle
analysis by flow cytometry following propidium iodide (PI) staining revealed that Rbm22
knockdown decreased the percentage of cardiomyocytes in S and G2/M phases (Fig. 4d).
To gain insights into the mechanisms by which RBM22-induced cardiomyocyte proliferation,
we performed RNA-seq on primary neonatal mouse cardiomyocytes treated with si-Rbm22
#1 and si-Ctrl #1 for 48 h (Supplementary Fig. 7a). Scatter plot analysis further revealed
significant differential gene expression between the two groups, highlighting genes with
substantial changes in expression (Supplementary Fig. 7b). Both Kyoto Encyclopedia of
genes and genomes (KEGG) and Gene set enrichment analysis (GSEA) analyses identified the
DNA replication pathway as significantly enriched (Fig. 4e, f, and Supplementary Fig. 7c).
Notably, si-Rbm22 #1 treatment resulted in downregulation of gene sets associated with
cardiomyocyte proliferation, including mitotic cell cycle and cell division (Supplementary Fig.
7d), suggesting a potential dysregulation of cell cycle progression. Heatmap analysis of cell
cycle genes revealed a consistent downregulation of genes involved in DNA replication
pathway following Rbm22 silencing (Fig. 4g). Furthermore, a circular heatmap analysis
demonstrated that the expression levels of most cell cycle and cyclin-dependent kinases
(CDKs), cell division cycle genes (Cdcs), and Cyclin genes were decreased by Rbm22 silencing
(Fig. 4h). The gene regulatory network analysis revealed that differentially expressed genes
in the si-Rbm22 #1 group were predominantly enriched in pathways related to cardiac
development, cardiomyocyte dedifferentiation, cell proliferation, and other biological
processes relevant to heart regeneration (Fig. 4i). Selected genes and proteins were
comprehensively validated through qRT-PCR and immunoblotting analyses. Specifically,
Rbm22-silencing cardiomyocytes showed significant suppression of key cell cycle-
promoting genes, with cyclin-dependent kinase 4 (CDK4), cyclin A2 (CCNA2), cyclin E1
(CCNE1), and PCNA mRNA and protein levels exhibiting the most pronounced reductions
compared to scramble controls (Fig. 4j, k). Similarly, treatment with si-Rbm22 #2 also
resulted in decreased mRNA expression and protein levels of these proliferative genes
(Supplementary Fig. 7e, f). Furthermore, we isolated primary cardiomyocytes from Rbm22-
iCKO and control mice, confirming that Rbm22 knockout led to the reduced expression of
mRNA and protein levels of these cell cycle genes (Supplementary Fig. 7g, h). These results
indicate that RBM22 regulates cardiomyocyte proliferation by triggering transcription
reprogramming of many genes that drive cardiomyocyte cell cycle activation.

Since RBM22 is a canonical RNA-binding protein, we wondered whether it regulates gene
expression through RNA post-transcriptional processing and turnover. We treated
cardiomyocytes with Actinomycin D (ActD) to observe the effects on mRNA levels of cell
cycle genes in si-Rbm22 #1 and si-Ctrl #1 cardiomyocytes. The results showed that RBM22
did not influence the mRNA degradation of Cdk4, Ccna2, and Ccne1 (Supplementary Fig.
8a). Moreover, RBM22‐mediated RNA alternative splicing events were investigated. The
relative fractions of transcription with AS events induced by RBM22 showed no difference
between si-Rbm22 #1 and si-Ctrl #1 groups, including alternative 3’ splice sites (A3SS),
alternative 5’ splice sites (A5SS), mutually exclusive exons (MXE), retained introns (RI), and
skipped exons (SE) (Supplementary Fig. 8b). AS analysis revealed SE as the predominant
splicing variant among the detected AS events (Supplementary Fig. 8b, c). Subsequent
volcano plot analysis of SE-associated transcripts displayed no significant difference in
expression levels of cell cycle-related genes (including Cdk4, Ccna2, and Ccne1) between
si-Rbm22 #1 and si-Ctrl #1 groups (Supplementary Fig. 8d).

RBM22 contains three major functional domains: Zinc Finger domain (ZnF), RNA-
Recognition Motif (RRM), and Proline-Rich domain. Apart from the established role of
RBM22 as the classical RNA-binding protein, it has also been reported to function as a
transcriptional co-factor, directly binding chromatin to regulate gene expression33. To
investigate the specific domain responsible for its transcriptional regulation of cell cycle
genes, cardiomyocytes were transfected with either RBM22 mutants with RRM domain
(residues 232-305) deletion or ZnF domain (residues 1-232) deletion (Supplementary Fig.
8e). Transfection of these mutants into cardiomyocytes revealed that deletion of the ZnF
domain, but not the RRM domain, significantly altered the transcription level of cell cycle-
related genes compared to wild-type (WT) RBM22 (Supplementary Fig. 8f), suggesting that
other mechanisms, rather than the post‐transcriptional splicing, were involved in the cell
cycle activation mediated by RBM22.

RBM22 modulates cell cycle genes by binding their promoters
To further confirm the above conjecture, subcellular fractionation combined with
immunoblotting revealed that RBM22 was enriched in the chromatin fraction of
cardiomyocytes (Fig. 5a). Based on these findings, we propose that RBM22 regulates
cardiomyocyte cell cycle activation through epigenetic mechanisms, primarily modulating
chromatin states at its target genes. To test this, we performed Cleavage Under Targets &
Tagmentation (CUT & Tag) sequencing with an anti-RBM22 antibody in neonatal
cardiomyocytes at P1 (which retain high regenerative capacity) and P8 (which lose
regenerative capacity) to generate genome-wide RBM22 binding profiles (Supplementary
Fig. 9a). CUT & Tag analysis showed that RBM22 binding sites included a subset located in
the gene promoters in both P1 and P8 cardiomyocytes (Fig. 5b, Supplementary Fig. 9b).
Gene Ontology (GO) analysis of the differentially binding genes in CUT & Tag showed
significant enrichment in positive regulation of the cell cycle (Fig. 5c), suggesting that RBM22
might directly enhance cardiomyocyte proliferation by promoting cell cycle-related gene
expression. Integration of CUT & Tag and RNA-seq data revealed that 18% of the RBM22-
bound genes were significantly downregulated upon Rbm22 silencing, suggesting direct
transcriptional regulation (Fig. 5d). Integrative Genomics Viewer (IGV) visualization of CUT &
Tag data revealed that RBM22 bound to the promoter of positive cell cycle regulatory genes,
including Cdk4, Ccna2, Ccne1, Mki67, and Pcna (Fig. 5e and Supplementary Fig. 9c). The
published Assay for Transposase-Accessible Chromatin with high-throughput sequencing
(ATAC-seq) data (GSE178673) were further used to identify chromatin accessible regions at
RBM22 binding sites within the promoters of positive cell cycle regulatory genes in P3
neonatal mouse cardiomyocytes, and to compare the accessibility of these specific sites with
that in P7 cardiomyocytes (Fig. 5e and Supplementary Fig. 9c). Furthermore, the binding
regions identified by ATAC-seq were overlapped with H3K4me3 ChIP-seq signals in
neonatal mouse cardiomyocytes (GSE155658), supporting that RBM22 selectively associated
with the active promoters of cell cycle-related genes (Fig. 5e, Supplementary Fig. 9c). The
binding of RBM22 to the promoter of cell cycle gene was notably elevated in P1
cardiomyocytes, aligning with the enhanced gene expression observed in the RNA-seq
results presented in Figure 4h. Interestingly, we found that RBM22 did not bind to the
promoters of house-keeping genes (Hprt and Actb) or cell cycle inhibitory genes (Cdkn1a
and Cdkn2a) in cardiomyocytes, according to the CUT & Tag analysis (Supplementary Fig.
9d, e). ChIP-qPCR results confirmed that RBM22 could bind to the promoter regions of Cdk4,
Ccna2, and Ccne1 (Fig. 5f). Motif analysis of CUT & Tag peaks identified evolutionarily
conserved RBM22-binding motifs within cardiomyocyte cell cycle gene promoters (Fig. 5g).
To establish direct DNA-protein interaction, we performed electrophoretic mobility shift
assays (EMSA) using nuclear extracts from Flag-tagged RBM22-transfected cardiomyocytes.
Biotin-labeled probes containing the conserved motif exhibited specific binding to RBM22
in Flag-tagged RBM22-expressing cardiomyocytes compared with control cardiomyocytes
(Fig. 5h). Functional validation through luciferase reporter assays demonstrated that RBM22
enhanced WT Cdk4, Ccna2, and Ccne1 promoter-induced reporter gene activation (Fig. 5i).
Strikingly, deletion of the conserved motifs in either mutant construct abrogated this
transcriptional activation effect (Fig. 5i). These integrated data demonstrate that RBM22
activates the transcription of cell cycle genes via sequence-specific binding to their active
promoter regions.

To investigate the roles of the Cdk4, Ccna2, and Ccne1 in mediating the cardiomyocyte-
proliferative effects of RBM22, we knocked down these genes in Rbm22-overexpressed
cardiomyocytes. Either knocking down Cdk4, Ccna2, or Ccne1 significantly reduced the
proliferative effect of RBM22, as determined by the percentage of EdU-positive
cardiomyocytes (Fig. 5j). Moreover, it blocked Rbm22 overexpression-induced increased
expression of cell cycle genes, including Pcna, Pole2, Aurkb, Chek1, and Ccnb1 (Fig. 5k).
These functional rescue experiment data further confirm that RBM22 promotes
cardiomyocyte proliferation primarily via transcriptional activation of key regulators of the
cell cycle.

RBM22 increases promoter accessibility of cell cycle genes
Given that CUT & Tag analysis identified RBM22 occupancy at the promoters of cell cycle-
related genes, we next employed ATAC-seq in si-Rbm22 #1 and si-Ctrl #1 cardiomyocytes
to assess whether RBM22 influences chromatin accessibility at these regions, thereby
contributing to transcriptional regulation (Supplementary Fig. 10a).

Analysis of ATAC-seq peak enrichment across genome-wide regions surrounding
transcription start sites (± 1.5 kb; center) showed a reduction in chromatin accessibility at a
large fraction of promoters following Rbm22 knockdown (Supplementary Fig. 10b, c).
Additionally, 2.26% of gene promoters were closed in si-Rbm22 cardiomyocytes
(Supplementary Fig. 10d). We annotated the differential peaks based on their genomic
distribution and found that the majority of differentially regulated chromatin accessible
peaks were located in promoter regions, rather than in intergenic or intronic regions
(Supplementary Fig. 10e, f). GO analysis of RBM22-associated differentially regulated
chromatin accessible peaks identified by ATAC-seq revealed significant enrichment in cell
cycle-associated pathways, suggesting that RBM22 may directly facilitate the expression of
cell cycle genes by enhancing chromatin accessibility at their transcription regulatory regions
(Fig. 6a). Furthermore, we classified cell proliferation-related genes into the targeted clusters.
Our analysis showed the significantly reduced chromatin accessibility in the promoter
regions of corresponding genes following Rbm22 knockdown (Fig. 6b). Numerous
downregulated genes from the RNA-seq data that were strongly associated with the cell
cycle and cell division presented less accessible promoter regions (Fig. 6c). Moreover, pie
chart analysis revealed that 28% of the RBM22-bound genes exhibited decreased chromatin
accessibility upon Rbm22 knockdown. Notably, a subset of these genes is involved in cell
cycle progression and mitosis, including Cdk4, Ccna2, Ccne1, Cdk5, Cdk9, Cdk14, Cdk19,
Cdk20, Cdc25b, Ccng2, Ccnl1, Myc, Mki67, Aurkb, Pcna, and Chek2, all of which showed
pronounced accessibility changes (Fig. 6d). Moreover, consistent with our RNA-seq results,
a snapshot of the ATAC-seq profiles revealed less accessible peaks at promoters of known
cell cycle (Cdk4, Ccna2, and Ccne1) genes in Rbm22-silenced cardiomyocytes than that in
control cardiomyocytes (Fig. 6e). Re-analysis of published ChIP-Seq data revealed that
specific peaks corresponding to H3K4me3 and RNA Polymerase II (RNA Pol II) (from
cardiomyocytes, GSE155658), and H3K27ac (from cardiac tissues, GSE123867), were
observed within the promoter regions of cell cycle genes at genomic sites that, critically,
exhibited significantly reduced chromatin accessibility upon Rbm22 knockdown (si-Rbm22
#1), as determined by our ATAC-seq analysis (Fig. 6e). RBM22 did not affect the chromatin
accessibility on the promoters of cell cycle inhibitory genes (Cdkn1a and Cdkn2a) or house-
keeping genes (Hprt and Actb) (Supplementary Fig. 10g). ATAC-qPCR demonstrated that
Rbm22 knockdown reduced chromatin accessibility at the promoter regions of key cell cycle
genes, including Cdk4, Ccna2, Ccne1, Mki67, and Pcna (Supplementary Fig. 10h). These
findings indicate that RBM22 promotes chromatin accessibility at the promoters of cell
cycle-related genes, thereby facilitating their transcriptional activation.

RBM22 interacts with SMARCA4 to remodel cell cycle gene promoters
Next, we explored the molecular mechanism through which RBM22 regulated chromatin
accessibility at cell cycle gene promoters. Given the established role of SWI/SNF chromatin
remodeling complexes in modulating nucleosome positioning and chromatin accessibility
to regulate gene expression34, we first investigated potential interactions between RBM22
and SWI/SNF components. Immunoprecipitation (IP) analysis in cardiomyocytes revealed
that RBM22 interacted with multiple SWI/SNF complex components, including SMARCA4,
SMARCC1, and SMARCC2 (Fig. 6f). Intriguingly, Co-IP assays demonstrated that SMARCA4
specifically interacted with RBM22 (Fig. 6g, h). Using the HDOCKlite v1.1 server for protein-
protein docking, our analysis suggested a direct interaction between the Proline-rich
domain of RBM22 and SMARCA4 (Fig. 6i). Further investigation of cardiomyocytes isolated
from Rbm22-iCKO mice revealed a significant reduction in the interaction between RBM22
and SMARCA4 compared to controls, indicating the functional relevance of this interaction
in the context of chromatin remodeling (Supplementary Fig. 11a). Additionally, Rbm22
knockdown by siRNA treatment similarly resulted in a marked decrease in the RBM22-
SMARCA4 interaction, reinforcing the critical role of RBM22 in facilitating chromatin
remodeling through SMARCA4 (Supplementary Fig. 11b). As SMARCA4 serves as the
catalytic ATPase subunit of SWI/SNF complex and has been implicated in transcriptional
activation of secondary response genes, including cell cycle regulators35, we hypothesized
that RBM22 might recruit SMARCA4 to specific genomic regions. To test this hypothesis, we
performed sequential chromatin immunoprecipitation (Re-ChIP) assays, which revealed co-
occupancy of RBM22 and SMARCA4 at promoter regions of key cell cycle regulators (Cdk4,
Ccna2, and Ccne1) in cardiomyocytes (Fig. 6j-l). Furthermore, we isolated cardiomyocytes
from Rbm22-iCKO mice and found that Rbm22 knockout significantly reduced the
recruitment of SMARCA4 to the promoters of Cdk4, Ccna2, and Ccne1, further supporting
the functional role of RBM22 in mediating SMARCA4 recruitment and chromatin remodeling
for gene transcription (Supplementary Fig. 11c-e).

To validate the interaction of RBM22 with SMARCA4 and determine the domain of RBM22
responsible for the physical binding, we constructed plasmids encoding Flag-tagged full-
length RBM22 and several mutated RBM22 with deletion of the ZnF (232-420),
RRM and Proline-Rich (1-186), Proline-Rich (1-305), or N termination (159-420), and
V5-tagged full-length SMARCA4 (Fig. 6m). Co-IP assay confirmed that deletion of the Proline-Rich domain
in RBM22 disrupted the interaction between RBM22 and SMARCA4, whereas mutations in
the ZnF or RRM domains did not impact the interactions (Fig. 6m). Furthermore, WT RBM22
but not RBM22 mutant with a Proline-Rich domain deletion enhanced Cdk4, Ccna2, and
Ccne1 promoter reporter activation (Supplementary Fig. 11f, g). WT RBM22 rescued Cdk4,
Ccna2, and Ccne1 mRNA expression in si-Rbm22 #1 treated cardiomyocytes, while the
RBM22-Proline-Rich domain mutant could not (Supplementary Fig. 11h), suggesting that
the Proline-Rich domain was required for the regulation of cell cycle genes expression
mediated by RBM22. Moreover, ChIP assays confirmed that Rbm22 knockdown reduced the
recruitment of SMARCA4 on the Cdk4, Ccna2, and Ccne1 promoters (Fig. 6n). These results
demonstrate that RBM22 recruits SMARCA4 to the cell cycle gene promoters and enhances
transcription activation of these genes through SMARCA4-mediated chromatin remodeling.

The above results imply that chromatin with a more open conformation of the cell cycle
gene promoter, mediated by the RBM22 and SMARCA4 complex, might provide more
access for recruitment of the basal transcription machinery. Interestingly, we observed the
reduced enrichments of both total RNA polymerase II (Pol II) and serine 2 phosphorylated-
RNA Pol II (RNA Pol II S2P) near TSS of Cdk4, Ccna2, and Ccne1 in Rbm22-silenced
cardiomyocytes compared to control cardiomyocytes, which indicated a significant decrease
of transcriptional initiation and elongation (Fig. 6o, p). These data indicate that RBM22
recruits SMARCA4 to form an epigenetic complex at the Cdk4, Ccna2, and Ccne1 promoters
with an open chromatin conformation, which contributes to the enhanced binding of RNA
Pol II, thereby promoting cell cycle gene expression (Supplementary Fig. 11i).

RBM22 gene therapy improves adult cardiac repair
To explore the potential therapeutic application of RBM22 in heart diseases, we used an
AAV9-mediated delivery system harboring the Rbm22 gene with a cardiomyocyte-specific
cTnT promoter (termed AAV9-Rbm22) to specifically overexpress RBM22 in mouse
cardiomyocytes. Immunoblotting showed the protein expression level of RBM22 increased
significantly in AAV9-Rbm22 group (Supplementary Fig. 12a). Immunostaining showed the
cell number of cardiomyocytes increased following AAV9-Rbm22 treatment for 48 h
(Supplementary Fig. 12b). Assessment of proliferation using EdU incorporation assays or
immunostaining for Ki67, pH3, or Aurora B showed a significant increase in the proliferation
of cardiomyocytes after AAV9-Rbm22 treatment (Supplementary Fig. 12c-f). Flow cytometry
analysis using PI staining showed that Rbm22 overexpression led to an increased proportion
of cardiomyocytes in the S and G2/M phases (Supplementary Fig. 12g). We next performed
Cell Trace Violet (CTV) dilution assays to directly assess cardiomyocyte proliferation. In this
assay, the fluorescent intensity of cytoplasmic labeling with CTV is progressively halved
following each cell division and quantified in the cytoplasm of cardiomyocytes, creating a
bona fide index of the duplication rate36,37. Consistently, CTV analysis further indicated that
Rbm22 overexpression enhanced cardiomyocyte division (Supplementary Fig. 12g).

In MI model, the AAV9-Rbm22 or control virus was injected directly into the peri-infarct
region of the myocardium to observe the in vivo effect of Rbm22 overexpression (Fig. 7a).
Immunoblotting analysis showed the protein levels of RBM22 were significantly increased in
cardiac tissues below the ligation suture at 14 dpi (Supplementary Fig. 12h). Compared with
AAV9-Ctrl, administration of AAV9-Rbm22 led to a gradual and significant improvement in
cardiac function, as evidenced by increased LVEF and LVFS values, along with reduced LVIDs,
LV Vols, LVIDd, and LV Vold (Fig. 7b-d and Supplementary Fig. 12i). In mouse hearts treated
with AAV9-Rbm22, Masson trichrome staining displayed the reduced collagen deposition
(Fig. 7e), consistent with the pro-regenerative effects of RBM22 post-MI.

Furthermore, WGA staining revealed decreased cardiomyocyte size at 28 dpi after AAV9-
Rbm22 administration (Supplementary Fig. 12j). The cardiomyocyte number per heart was
increased in AAV9-Rbm22-administrated mice (Supplementary Fig. 12k).

Immunofluorescence staining further showed that AAV9-Rbm22 treatment increased the
fraction of mononucleated and diploid cardiomyocytes in injured hearts (Fig. 7f, g). EdU
incorporation assays or immunostaining of Ki67 in different groups at 14 dpi showed that
EdU- and Ki67-positive cardiomyocytes dramatically increased in the border zones of
AAV9-Rbm22-treated hearts (Fig. 7h, i). These results further suggest that RBM22 effectively
enhances cardiac recovery by promoting cardiomyocyte proliferation and myocardial
regeneration in adult mice post-MI.

Given the pronounced enhancement of cardiomyocyte proliferation and cardiac repair
observed following AAV9-mediated Rbm22 overexpression, we next investigated whether
corresponding changes in chromatin accessibility using ATAC-seq accompanied these
functional improvements (Supplementary Fig. 13a). Volcano-plot analysis showed that a
higher proportion of chromatin regions exhibited increased openness following Rbm22
overexpression (Supplementary Fig. 13b). GO enrichment analysis of accessible regions
revealed a significant enrichment of pathways related to cell cycle regulation and
cardiomyocyte proliferation (Fig. 7j). KEGG pathway analysis further supported this analysis,
demonstrating marked enrichment of cell cycle-associated pathways in Rbm22-
overexpressing cardiomyocytes (Fig. 7k). Heatmap visualization confirmed that promoter
accessibility of major cell cycle regulators was prominently elevated following Rbm22
overexpression (Fig. 7l). Consistent with these chromatin-level alternations, inspection of IGV
tracks revealed increased ATAC-seq signal intensity at the promoter regions of Cdk4, Ccna2,
Ccne1, Mki67, and Pcna in Rbm22-overexpressing adult cardiomyocytes compared with
controls (Fig. 7m and Supplementary Fig. 13c). Integration of these results with ChIP-seq
datasets, including H3K4me3 and RNA polymerase II profiles from isolated cardiomyocytes
(GSE155658) and H3K27ac profiles from adult cardiac tissues (GSE231014), revealed that the
regions displaying increased accessibility in Rbm22-overexpressing adult cardiomyocytes
coincide with genomic loci marked by active promoter signatures (Fig. 7m and
Supplementary Fig. 13c). These observations further indicate that Rbm22 overexpression
enhances chromatin accessibility at transcriptionally positive regulatory regions. Importantly,
promoter accessibility of house-keeping genes (Hprt and Actb) remained unchanged,
indicating that RBM22 selectively modulated chromatin accessibility at cell cycle-promoting
loci (Supplementary Fig. 13d). Supporting this mechanistic link, adult cardiomyocytes
isolated from AAV9-Rbm22-treated mice showed increased interaction between RBM22
and SMARCA4, and enhanced recruitment of SMARCA4 to the promoters of Cdk4, Ccna2,
and Ccne1, reinforcing a role for RBM22 in facilitating SMARCA4-dependent chromatin
remodeling and transcriptional activation during cardiac repair (Fig. 7n, o).

RBM22 promotes the proliferation of hiPSC-CMs
So far, strategies aimed at stimulating cardiomyocyte proliferation for cardiac repair have
primarily been explored in rodents. However, it remains uncertain whether these approaches
can be translated to restore heart function in humans. To assess the translational potential
of RBM22, we investigated the effects of RBM22 on hiPSC-CMs. Analysis of human and
mouse RBM22 protein sequences from NCBI databases revealed exceptionally high amino
acid sequence conservation between these two species. Rbm22 overexpression in hiPSC-
CMs was verified at both the protein and mRNA levels by immunoblotting and qRT-PCR,
respectively (Fig. 8a, b). Rbm22 overexpression significantly increased the number of EdU-
and Aurora B-positive cardiomyocytes (Fig. 8c, d). Furthermore, hiPSC-CMs treated with
AAV9-Rbm22 displayed significant decreases in the gene expression levels of a panel of
cardiac function and maturation markers, including CACNA1C, GJA1, KCNJ2, S100A1,
SCN5A, MYOM2, MYOM3, COX6A2, and CASQ2 (Fig. 8e). AAV9-Rbm22 treatment also
increased the expression levels of cell cycle genes in hiPSC-CMs, including CCNA2, CDK4,
CCNE1, and CDK6 (Fig. 8e). In addition, CTV dilution assays in hiPSC-CMs demonstrated a
marked increase in dye dilution associated with cell division upon Rbm22 overexpression,
suggesting enhanced proliferative activity (Fig. 8f). These data indicate that RBM22 promotes
a more regenerative phenotype in human cardiomyocytes. To sum up, our study identifies
RBM22 as an essential factor in maintaining cardiac regenerative capacity. RBM22
overcomes the transcriptional and epigenetic barriers of cardiomyocyte proliferation to
facilitate cell cycle re-entry, highlighting its therapeutic potential for cardiac injury (Fig. 8g).

Methods
Animals
All animal experiments and procedures were carried out in strict accordance with the Guide
for the Care and Use of Laboratory Animals, as established by the National Institutes of
Health, and were approved by the Institutional Animal Care and Use Committee of Tongji
University School of Medicine. Mice were housed in a specific pathogen-free (SPF) animal
facility at Tongji University under a 12 h light/dark cycle, a temperature range of 25 ± 1 °
C, and a humidity of 50 ± 5%. Neonatal 1-day-old mice of both sexes were used in the
study. For experiments using adult animals, male mice were used in this study. The C57BL/6J
mice used in WT studies were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd.
(Shanghai, China). The Rbm22fl/fl mouse line (C57BL/6 background) was generated by
Cyagen Co., Ltd (Suzhou, China). Exon 4 of the Rbm22 gene (NCBI Reference Sequence:
NM_025776) was selected as a conditional knockout region. Tamoxifen-inducible αMHC-
MerCreMer (Cre+/-) mice were obtained from Jackson Laboratory, and Myh6-iCre (Myh6-
improved Cre) mice were purchased from Jiangsu Gempharmatech Co., Ltd (Jiangsu, China).
Rbm22fl/fl mice were crossed with αMHC-MerCreMer (Cre+/-) mice to generate Rbm22fl/fl
αMHCMerCreMer+/- mice, which exhibit an inducible deletion of RBM22 in cardiomyocytes
(termed Rbm22 inducible Knockout, Rbm22-iCKO mice). Tamoxifen (Sigma-Aldrich, T5648)
was dissolved in corn oil. Recombination of floxed alleles in postnatal 7-day (P7d) and P21d
mice was achieved by daily tamoxifen administration (30 mg/kg body weight) from P0 to
P3 via an insulin needle47. Rbm22-iCKO mice were subjected to AR and MI at P3. To achieve
recombination of floxed alleles in adult mice, tamoxifen was administered via intraperitoneal
injection (30 mg/kg, once a day for 3 consecutive days). Following tamoxifen treatment, a
14-day period was allowed for tamoxifen to induce recombination before MI was induced.
Adult Rbm22-iCKO mice at 8 weeks of age were subjected to MI. The Rbm22fl/fl mice and
Cre+/- mice adult mice were used as the control in this study. Rbm22fl/fl mice crossed with
Myh6-iCre mice did not generate pure bred cardiomyocyte conditional knockout RBM22
mice, but rather heterozygous cardiomyocyte conditional knockout RBM22 mice
(Rbm22fl/+Myh6-iCre). Adult Rbm22fl/fl and Rbm22fl/+Myh6-iCre mice were constructed with
MI at 8 weeks of age. Animals were randomly assigned to different experimental groups
based on their genotypes. The genotype of the animals from which individual samples were
taken was not known to the investigators at the time of the experiments, and all experiments
were performed in a blinded manner. All mice were euthanized by cervical dislocation after
anaesthetization with an intraperitoneal injection of sodium pentobarbital (70 mg/kg body
weight).

Apical resection (AR)
The AR procedure in mice was performed as previously described48. In this study, AR was
conducted at P3 to allow for prior pharmacological induction with tamoxifen. Briefly, P3 mice
were anaesthetized by hypothermia, achieved through immersion in an ice-water mixture
for 4 min. Under the fluoroscope, the mice were positioned at the left fourth intercostal
space, and an incision was made to sequentially separate the tissues and muscles, thereby
opening the chest cavity to expose the heart. A suction device was used to securely aspirate
15% of the myocardial tissue at the apex of the heart in each neonatal mouse. Following AR,
neonates were removed from the ice bed, and the thoracic wall was sutured using 8-0
surgical thread, and the skin incision was closed with 6-0 suture. The mice were rewarmed
on a heating pad (37 °C) for several min until they recovered. Sham procedures excluded
apex amputation. And the heart tissues were harvested at designated time points for
subsequent experiments.

Myocardial infarction (MI)
For the neonatal Rbm22fl/fl control, Cre+/- control and Rbm22-iCKO mice, MI was performed
at P3. The mice were anaesthetized using hypothermia by immersion in an ice-water mixture
for 4 min, which induced reduced heart and respiratory rates. As previously described, eight-
week-old Rbm22fl/fl, Cre+/-, Rbm22-iCKO, Rbm22fl/+, and Rbm22fl/+Myh6-iCre were
anesthetized with 1% isoflurane49. The mice were then placed in a left supine position on the
stereotaxic frame, and the heart was exposed through a thoracotomy at the fourth left
intercostal space. Following pericardial opening, the LAD coronary artery was permanently
ligated using a 7-0 suture. Successful ligation was confirmed when the left ventricle became
pale. In virus-injected mice, either the cardiomyocyte-targeted RBM22-expressing adeno-
associated virus (AAV9-cTnT-Rbm22-GFP, 5 × 1011 viral genomes per mouse), which was
purchased from Genechem Company (Shanghai, China), or a control vector (AAV9-cTnT-
GFP) was administered into the myocardium adjacent to the infarct zone immediately after
ligation. AAV9-cTnT-Rbm22-GFP is hereafter referred to as AAV9-Rbm22. The chest cavity
was then closed with a 6-0 suture, and the mice were put in feeding cages after recovery.
The ligation was at a similar location in sham-operated mice, but was not tied.

EdU incorporation assay
For neonatal mouse cardiomyocytes, 10 μM EdU (Invitrogen, c10339) was added to the
culture medium 48 h post-transfection, and the cells were subsequently incubated for 6-8
h. For the EdU assay of neonatal heart sections, a single injection of 50 mg/kg EdU per
animal was induced intraperitoneally for 8 h prior to execution. For EdU labelling of
proliferating adult cardiomyocytes in vivo, adult mice were intraperitoneally injected with
EdU at 50 mg/kg body weight every 2 days for a total of seven times before the heart was
harvested according to previously described methods50. According to the manufacturer’s
instructions, cardiac sections and cardiomyocytes, after incubating with secondary
antibodies, were incubated with the Click-iT EdU 594 Imaging Kit (Invitrogen, c10339) to
show EdU incorporation.

Echocardiography
Echocardiographic imaging was performed using a digital ultrasound system (Vevo2100
Imaging System, Visual Sonics Vevo 770). Isoflurane (1%) was used to anesthetize the mice
before echocardiography. Cardiac ultrasound protocols integrated B-mode anatomical
imaging for longitudinal plane acquisition with synchronized M-mode recordings, ensuring
precise spatiotemporal correlation of ventricular wall dynamics. A series of cardiac functional
parameters, such as LVEF, LVFS, LVIDs, and LVIDd, as well as LV Vols and LV Vold, were
evaluated and derived from M-mode echocardiographic measurements of end-diastolic
and end-systolic dimensions.

Histology
Hearts were perfused with phosphate-buffered saline (PBS) to remove blood from residual
hearts, fixed in 4% paraformaldehyde (PFA) at 4°C overnight, dehydrated through graded
ethanol and xylene, and subsequently embedded in paraffin. For the AR and MI models in
neonatal mice, hearts were serially sectioned longitudinally at a thickness of 4 μm until the
site of injury was observed. For the adult mouse cardiac regeneration following the MI
procedure, the slices were sectioned at a thickness of 4 µm, and slides were collected at
100-300 µm intervals along the direction of the heart from the ligation site to the apex, with
five cross-sectional frozen slides collected from each heart. Slides were stained with Masson
trichrome staining to identify fibrotic areas51. The myocardial scar dimensions were assessed
through systematic analysis of sequential tissue sections spanning from the cardiac apex to
the ligation point, with quantitative evaluation performed by computing the mean ratio of
collagenous tissue to total myocardial area via Image J software (v 1.51m9).

Immunofluorescence staining
For cultured cardiomyocytes, cells were fixed with 4% PFA for 15 min at room temperature.
And then permeabilized and blocked with PBS containing 0.2% Triton X-100 and 1% bovine
serum albumin (BSA) for 1 h at room temperature. Incubate overnight at 4°C with primary
antibodies: anti-RBM22 (Novus Biologicals, NBP1-85379, 1:100), anti-cTnT (Abcam,
ab209813, 1:400), anti-α-actinin (Abcam, ab9465, 1:400), anti-pH3 (Abcam, ab80612, 1:100),
anti-Ki67(Abcam, ab15580, 1:100), and anti-Aurora B (Abcam, ab2254, 1:100). Wash three
times with PBS, then apply fluorescent-labeled secondary antibody for 1 h at room
temperature. The secondary antibodies used were as follows: donkey anti-mouse Alexa
Fluor 488 (Invitrogen, A-11001, USA, 1:500), donkey anti-rabbit Alexa Fluor 594 (Invitrogen,
A-11012, 1:500). Finally, counterstain with 4’,6-diamidino-2-phenylindole dihydrochloride
(DAPI) (Southern Biotech, 0100-20) for nuclear visualization. For paraffin-embedded mouse
heart sections, deparaffinize with xylene, then rehydrate and rinse with water. Heat in a
100°C water bath for 30 min, then cool to room temperature. Incubate frozen slides with
sodium citrate antigenic solution at 37°C for 1 h. Block with 1% BSA for 1 h, then incubate
overnight at 4°C with primary antibodies. Apply fluorescent-labeled secondary antibody
(Invitrogen) for 1 h at room temperature, wash three times with PBS, and counterstain with
DAPI. Stained tissue sections were viewed using a fluorescence microscope (Leica v 3.8).

Wheat germ agglutinin staining (WGA)
For WGA staining, the slides were first deparaffinized and rehydrated. Heart tissue sections
were incubated for 1 h at room temperature in PBS containing WGA conjugated to Alexa
Fluor 488 (Thermo Fisher Scientific, W11261). Following incubation, the slides were rinsed
three times with PBS to ensure thorough washing. Image J was used to quantify the size of
cardiomyocytes that were round and contained a nucleus.

Neonatal cardiomyocyte isolation, culture, viral transduction, and siRNA transfection
Primary cardiac cells were isolated from P1 C57BL/6J neonatal mouse hearts using the
Neonatal Heart Dissociation Kit in combination with gentle MACS and Octo Dissociator
(Miltenyi Biotec, Germany) according to the manufacturer’s instructions. The same isolation
procedure was performed for control mice and Rbm22-iCKO neonatal mice to obtain
genotype-matched primary cardiomyocytes for comparative analyses. The cells were
resuspended in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Fetal Bovine
Serum (FBS) at 37 ℃ with 5% CO2. For cardiomyocytes isolated from Rbm22-iCKO neonatal
mice, 4-hydroxytamoxifen (4-OHT) was administered for 48 h to induce Cre-mediated
recombination prior to subsequent analyses. After 24 h of culture, WT cardiomyocytes were
treated with RBM22 adeno-associated virus (AAV9-Rbm22) at a multiplicity of infection
(MOI) of 50 for 48 h, and harvested for proliferation analysis. Silencing of Rbm22 was
performed using two distinct siRNAs obtained from Dharmacon (siGENOME SMART pool
siRNA M-055902-01-0005, referred to as siRNA #1) and Santa Cruz Biotechnology (sc-
15273, referred to as siRNA #2). siRNAs targeting Cdk4, Ccna2, and Ccne1 (GenePharma)
were incorporated as indicated to interrogate downstream regulatory pathways. All
transfections were performed using Lipofectamine RNAiMAX for 48 h, and cells were
harvested for further experiments. The target siRNA sequences were as follows: siRNA-Cdk4
sense 5’-CCCUAGUGUUUGAGCAUAUTT- 3’, siRNA-Cdk4 antisense 3’-
AUAUGCUCAAACACUAGGGTT-5’, siRNA-Ccna2 sense 5’-GGCUGCACCAACAGUAAAUTT-
3’, siRNA- Ccna2 antisense 3’-AUUUACUGUUGGUGCAGCCTT-5’, siRNA-Ccne1 sense 5’-
GCCCUGGGAUGAUAAUUCATT- 3’, siRNA-Ccne1 antisense 3’-
UGAAUUAUCAUCCCAGGGCTT-5’.

Isolation of adult mouse cardiomyocytes
The adult mouse cardiomyocytes were isolated with Langendorff perfusion equipment, with
a minor modification50,52-54. Eight-week-old mice were injected with 200 μL of heparin before
being anesthetized with pentobarbital. The hearts were excised and perfused with a calcium-
free buffer at 37°C until the effluent was clear. The Ca²⁺-free Tyrode’s solution containing
(in mM): NaCl 135, KCl 5.4, MgCl₂·6H₂O 1, NaH₂PO₄ 0.33, glucose 10, and HEPES 10 (pH 7.3).
Enzymatic digestion was performed using collagenase type II (300 U/mL, Worthington) for
20 min. After the hearts appeared pale and flaccid, the ventricles were excised, minced in a
stop buffer (KB solution with 10% BSA), and filtered through a 100 µm cell strainer. The KB
solution was composed of (in mM): L-glutamic acid 120, KOH 120, KCl 10, KH₂PO₄ 10,
MgSO₄·7H₂O 1.8, K-EGTA 0.5, glucose 20, taurine 10, HEPES 10, and 0.2% BSA (pH 7.2), and
it helps prevent hypercontraction while preserving both structural and nuclear integrity. The
myocytes were then centrifuged to obtain purified cardiomyocytes for subsequent
experiments. Cardiomyocytes yields and the percentage of viable rod-shaped cells were
quantified using a hemocytometer.

Quantification of cardiomyocyte nucleation and ploidy
Following DAPI staining, single-cell suspensions were washed with PBS and deposited onto
slides for manual quantification of cardiomyocyte nuclei under fluorescence microscopy. For
ploidy analysis, single-cell suspensions of cardiomyocytes were stained with DAPI as
described above. To define nuclear boundaries, images were processed using Image J
software, with a standardized threshold applied to accurately delineate individual nuclei. The
DAPI fluorescence intensity of nuclei from both cardiomyocytes and non-cardiomyocytes
was measured within the same imaging field. The intensity values of non-cardiomyocyte
nuclei, assumed to be diploid, were used to establish a reference standard. Cardiomyocyte
nuclei were classified based on their relative fluorescence intensity compared to the non-
cardiomyocyte reference. Nuclei were considered diploid if their normalized intensity values
were within 0.5 to 1.5 times the reference value, tetraploid if within 1.5 to 2.5 times the
reference, and polyploid if the intensity exceeded 2.5 times the reference55.

Extraction of chromatin proteins from cardiomyocytes
The subcellular fractionation of cardiomyocytes into nuclear and cytoplasmic fractions was
conducted using well-established methods with minor experimental modifications56. Briefly,
cultured cardiomyocytes at a density of 1×107 cells/mL were diluted with RSB buffer (10 mM
Tris-HCl, pH 7.2; 10 mM NaCl; 1.5 mM MgCl₂), and the cytoplasmic membranes were
disrupted by adding 0.5% NP-40 supplemented with a protease inhibitor cocktail (Millipore,
539134) and PMSF (Sigma-Aldrich, P7626). After centrifugation, the supernatant was
collected as the cytoplasmic fraction. Pelleted nuclei were successively lysed in IPH buffer
(50mM Tris-HCl, pH 8.0; 150mM NaCl; 5mM EDTA; 0.5% NP-40) for 30 minutes on ice.
Nucleoplasmic and chromatin fractions were separated by 15 minutes of centrifugation at
4 °C. The precipitate was resuspended in cell lysis buffer (Cell Signaling Technology) and
disrupted by sonication to obtain cardiomyocyte chromatin protein.

Immunoprecipitation and western blotting
The cultured cardiomyocytes and HEK293T cells (ATCC, USA) were lysed in cell lysis buffer
supplemented with protease inhibitor cocktail and PMSF. The lysates were cleared by
centrifugation, and proteins were sequentially incubated with the anti-RBM22,
anti-SMARCA4, anti-V5-tag, and anti-Flag-tag antibodies at 4°C overnight and protein A/G
agarose (Santa Cruz Biotechnology) at room temperature for 2 h. The beads were washed
thrice with the lysis buffer and centrifuged to obtain the indicated protein complexes, which
were then subjected to immunoblotting.

Tissue lysates were prepared using ice-cold TPER buffer (Thermo Fisher Scientific),
supplemented with a protease inhibitor cocktail and PMSF. Protein concentrations were
determined using the BCA protein assay kit (Thermo Fisher Scientific, 23227). Proteins were
separated by SDS-PAGE and transferred to nitrocellulose membranes (Cytiva, 10600002),
which were blocked with 5% non-fat dry milk for 1 h. Membranes were incubated overnight
at 4°C with primary antibodies: anti-RBM22 (Proteintech, 22103-1-AP, 1: 1000), anti-CDK4
(Proteintech,11026-1-AP, 1: 1000), anti-CCNA2 (Proteintech, 18202-1-AP, 1: 1000), anti-
CCNE1 (Proteintech, 11554-1-AP, 1: 1000), anti-PCNA (Proteintech, 10205-2-AP, 1: 1000),
anti-SMARCA4 (Abcam, ab110641, 1: 1000), anti-SMARCC1 (Abcam, ab305037, 1: 1000),
anti-SMARCC2 (Abways, AY4215, 1: 1000), anti-Lamin A/C (Thermo Fisher Scientific, PA5-
17113, 1: 1000), anti-Histone H3 (Cell Signaling Technology, #9715S, 1: 1000), anti-β-Actin
(Cell Signaling Technology, #3700, 1: 2000), anti-GAPDH (Cell Signaling Technology, #5174,
1: 2000), anti-Flag-tag (Cell Signaling Technology, #2044, 1: 1000), and anti-V5-tag (Cell
Signaling Technology, #13202, 1: 1000). On the next day, the membrane was washed with
Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with the secondary
antibody for 1 h at room temperature. Protein bands were detected using Pierce ECL
western blot Substrate (Thermo Fisher Scientific, 34096) and analyzed with Image J. The
dilution doses of antibodies were according to the instructions.

Quantitative reverse transcriptase PCR (qRT-PCR)
Total RNA was extracted from heart samples or cardiomyocytes using TRIzol reagent
(Invitrogen, 15596018CN). cDNA synthesis was then performed using the PrimeScript RT
Master Mix ( Takara, RR036A). Gene expression was quantified by qRT-PCR, using SYBR
Green qPCR Master Mix (Toyobo, QPK-201) and amplified on a QuantStudio 6 Real-Time
PCR System. The list of all specific primers used in the reactions is provided in Supplementary
Table 1.

RNA stability study
The cultured cardiomyocytes were transfected with si-Ctrl #1 or si-Rbm22 #1. After 48 h,
the medium was replaced with medium containing 1 µM actinomycin D (Selleck, S8964) to
block transcription. Total RNA was then extracted at every 40 min intervals using TRIzol
reagent. qRT-PCR was performed using specific primer sets and SYBR Green Master Mix.
The specific gene expression levels were analyzed by qRT-PCR and were normalized using
Actb levels.

Electrophoretic Mobility Shift Assay (EMSA)
Cardiomyocytes were transfected with RBM22-Flag and control vector plasmids for 48 h,
and then nuclear extracts from cells were prepared using the RSB buffer, supplemented with
protease inhibitor cocktail and PMSF. The nuclear extracts were incubated with a biotin-
labeled RBM22-binding motif probe (5’-Biotin-
GCCCCGCCCCAGGGCGGGGCGGGGCGGGAC-3’). The experiments were performed using
the LightShift Chemiluminescent EMSA Kit according to the manufacturer’s instructions
(Thermo Fisher Scientific, 20148). Briefly, binding reactions were prepared with the
appropriate binding buffer, nuclear protein extract, and biotin-labeled probe. For the super-
shift assay, 2 µg of rabbit anti-RBM22 antibody (Bethyl, A303-923A) or control rabbit IgG
was added. After incubating for 20 min at room temperature, the reactions were separated
on a 5% polyacrylamide gel, transferred to a nylon membrane, and incubated with
streptavidin-conjugated HRP. The blot was developed as described above.

Culture of hiPSC-CMs
Human iPSC-derived cardiomyocytes (CSM-H230110), which are terminally differentiated
cells with very limited regenerative capacity, were supplied by Nanjing Cosmos
Biotechnology Co., Ltd (Nanjing, China). The cells were thawed and plated on 1% gelatin-
coated 24-well optical bottom plates at an appropriate density. Cells were incubated to allow
adhesion for 48 to 72 h before medium replacement with cardiac maintenance medium
(Nanjing Cosmos Biotechnology). The medium was refreshed every 2 days and treated with
AAV9-Rbm22. After 10 days, the cells were stained for immunofluorescent microscopy, and
flow cytometry analysis was performed to assess proliferation.

PI analysis and CTV dilution by flow cytometry
Neonatal mouse cardiomyocytes were enzymatically dissociated and resuspended in
complete culture medium. After centrifugation, the cells were stained with PI for DNA
content analysis and subjected to permeabilization using the Cell Cycle Detection Kit
(Vazyme, # AC101) according to the manufacturer’s instructions. Following incubation at
room temperature (protected from light), cells were analyzed by flow cytometry. Red
fluorescence was measured to assess cell proliferation, and light scatter properties were used
to determine cell cycle distribution.

Neonatal mouse cardiomyocytes and hiPSC-CMs were labeled with 5 μM CTV (Thermo
Fisher Scientific, C34557) according to the manufacturer’s instructions. CTV is a cell-
permeable intracellular stain characterized by high fluorescence intensity. Briefly, cells were
washed twice with cold PBS, resuspended in CTV solution, and incubated at 37 °C for 20
min. After staining, excess dye was removed by adding fresh culture medium, and cells were
incubated on ice for 5 min. Cells were then washed, resuspended in PBS, and counted.
Therefore, loss of fluorescence was attributed to a cell-specific mechanism other than
halving at each division. Fluorescence intensities were measured using cells resuspended in
PBS on a BD FACSCelesta Cell Analyzer (BD Biosciences), and raw data were analyzed with
FlowJo (v 10.8.1).

Dual luciferase reporter gene assay
For the construction of luciferase reporter plasmids for the Cdk4, Ccna2, and Ccne1
promoters, 664 bp, 651 bp, and 897 bp DNA fragments, containing the RBM22 binding sites
in the corresponding promoter regions of Cdk4, Ccna2, and Ccne1, were amplified from
mouse genomic DNA by PCR. The fragment was then inserted into the pGL3-Basic luciferase
reporter plasmid between the KpnI and XhoI loci. To further validate the potential RBM22
binding sites, mutant luciferase reporter plasmids were generated by mutating the
corresponding RBM22 binding motifs. Neonatal mouse cardiomyocytes were cotransfected
with a mixture of the indicated luciferase reporter plasmids following treatment with AAV9-
Rbm22 or AAV9-Ctrl virus for 48 h. Cells were harvested 24 h after plasmid transfection, and
luciferase activities were measured via the Dual Luciferase Reporter Assay System (Promega,
E1910) according to the manufacturer’s instructions. The data were normalized for
transfection efficiency by dividing firefly luciferase activity by Renilla luciferase activity.

RNA-sequencing (RNA-seq) and data Analysis
Neonatal cardiomyocytes were isolated from the pooled hearts of multiple P1 C57BL/6J mice
and transfected with si-Rbm22 #1 or si-Ctrl #1 using Lipofectamine RNAiMAX, followed by
culture for 48 h. The RNA-seq was performed on pooled cell samples with three technical
replicates. Total RNA was extracted from 1×106 cardiomyocytes using TRIzol reagent. The
quantity and quality of total RNA were measured by the Nanodrop 2000 spectrophotometer
and the Agilent 2100 Bioanalyzer, respectively. Then the libraries were constructed and
subsequently sequenced using a paired-end approach on the Illumina Novaseq 6000
platform. The transcriptome sequencing and analysis were conducted by OE Biotech Co.,
Ltd. (Shanghai, China). RNA-seq reads were aligned to the mouse genome by HISAT2, and
the gene expression level was quantified as fragments per kilobase of exon per million
fragments (FPKM) using HTSeq-count. DESeq2 was used to determine genes differentially
expressed between the si-Ctrl and si-Rbm22 groups. q value＜0.05 and |log fold-change| >
1 were set as the threshold for significantly differentially expressed genes (DEGs). Heatmaps
of gene expression, GO, and KEGG pathway analyses were conducted using R (v 3.2.0). RNA-
seq data are available in the Gene Expression Omnibus (GEO) database under the accession
number GSE295695.

Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and data
analysis
The ATAC-seq experiments were conducted utilizing the Hyperactive ATAC-Seq Library
Prep Kit for Illumina (Vazyme, TD711) following the manufacturer’s instructions. Briefly,
cardiomyocytes were transfected with si-Ctrl #1 or si-Rbm22 #1 using Lipofectamine
RNAiMAX transfection reagent and cultured for 48 h. Adult cardiomyocytes from virus-
injected mice, in which AAV9-Rbm22 or AAV9-Ctrl was directly injected into the peri-infarct
myocardial area at the time of MI, were isolated 14 dpi using the Langendorff perfusion
system for ATAC-seq analysis. The cells (5×104) were washed with TW Buffer and lysed with
Lysis Buffer. The cells were fragmented with 50 µL fragmentation reaction mixture (TW Buffer,
10% Tween 20, 1% Digitonin, 5 × TTBL, TTE Mix V50) at 37 ℃ for 30 min. The fragmentation
reaction was terminated with the stop buffer. The samples were purified with ATAC DNA
Extract Beads and washed with 80% ethanol. The elution sample reaction was amplified by
PCR. After the PCR reaction, the libraries were purified with ATAC DNA Clean Beads, and the
quality of the libraries was assessed by Qubit. After that, the obtained samples were
sequenced and analyzed by Shanghai Biotechnology Corporation (Shanghai, China), and
ATAC-qPCR was performed for validation57. Primers used for this analysis are shown in
Supplementary Table 1. For ATAC-seq, the raw data were filtered via Fastx (version: 0.0.13),
and low-quality reads contaminated by adaptor sequences were trimmed. Clean reads were
then mapped to the reference mouse genome mm10 via Bowtie (version: 0.12.8) using
default parameters. MACS (version 2.2.7.1) was applied to call peaks. Motif analysis was
performed using the “HOMER” tool. The bam file and R package DiffBind were used to
analyze the differences between the peaks of the specified two samples. The default
parameters were set as fold change ≥ 2 or ≤ 1/2, and P value < 0.05. The differences
between the obtained peaks were annotated. The sequencing data are available in the GEO
database under accession number GSE295696 and GSE311241.

CUT & Tag and data analysis
The CUT & Tag was conducted utilizing the Hyperactive Universal CUT & Tag Assay Kit for
Illumina (Vazyme, TD903-0T1) following the manufacturer’s instructions. For cell preparation,
the P1 and P8 mouse cardiomyocytes were isolated as described above. Afterward, the cells
were incubated at 37°C in complete medium for 12 h before proceeding with the
subsequent detection. Cardiomyocytes (1×105) were coupled to magnetic beads coated with
concanavalin A and incubated separately with anti-RBM22 antibody (Bethyl, A303-923A,
1:200) at 4°C overnight. Subsequently, samples were incubated with secondary antibodies
and Hyperactive pA/G-Transposon for 1 h. DNA was treated with TTBL for 1 h at 37°C, then
extracted and purified for library amplification. After that, the obtained samples were
sequenced and analyzed by Shanghai Biotechnology Corporation (Shanghai, China).
Genome mapping was performed using Bowtie software (version: 0.12.8), and clean reads
were matched to the reference genome mm10. The default parameter was set as fold
change ≥ 2 or ≤ 1/2 and P value < 0.05, and the differences between the obtained peaks
were annotated. The annotation program was the same as the above peak annotation
program, including different peak location information, annotation information, etc. Motif
analysis was performed using the “HOMER” tool, and the exact position of the Motif on the
peak was analyzed using the findmotifsGenome.pl tool. The sequencing data are accessed
in the GEO database under accession number GSE295697.

ChIP and Re-ChIP assays
Chromatin immunoprecipitation (ChIP) and Re-ChIP assays were performed as previously
described58. Briefly, to prepare the samples for ChIP, 5×106 cardiomyocytes /sample were
cross-linked with 1% methanol-free formaldehyde for 10 min using the ChIP kit (Millipore).
The antibodies against RBM22 (Bethyl, A303-923A, 1:500), SMARCA4 (Abcam, ab110641,
1:500), RNA Poly II (Active Motif, 39497, 1:500), RNA Poly II S2P (Abcam, ab5095, 1:500), and
IgG (Millipore, PP64) were incubated overnight at 4°C with gentle rotation. On the next day,
ChIP Grade Protein G Magnetic Beads (Cell Signaling Technology, #9006) were added and
incubated for 2 h with rotation. The beads were washed with buffers containing varying salt
concentrations and then eluted with a buffer containing 1% SDS. The antibody-chromatin
complex underwent reverse cross-linking and DNA purification.

The Re-ChIP assay was carried out using the Active Motif Re-ChIP-IT kit (Active Motif, 53016)
with antibodies against RBM22 or SMARCA4 at a dilution ratio of 1:500, as directed by the
manufacturer. The obtained DNA samples were subjected to qRT-PCR using the primers
listed in the Supplementary Table 1.

Statistical analysis
All data are presented as the mean ± SD. The numbers of repeated samples were given in
the individual figure legends. The experiments in Fig. 1g, 5h, 6f-h, and 6j-m were
independently repeated three times with similar results. For comparing 2 groups, a two-
tailed unpaired t-test was applied; for comparing among more than two groups, one-way
ANOVA with Tukey’s multiple comparisons test or two-way ANOVA with Sidak’s multiple
comparisons test was employed. GraphPad Prism 9 software was used to generate all graphs.
The results with a P value < 0.05 were considered statistically significant. No statistical
method was used to predetermine sample size. Sample sizes were determined based on
established practices and applicable standards. No data were excluded from the analyses.

Data availability
The data presented in the study have been provided in the main text, supplementary
materials, and source data files. The RNA-seq, ATAC-seq, and CUT & Tag data have been
deposited in Gene Expression Omnibus with accession numbers GSE295695
[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE295695], GSE295696
[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE295696], GSE311241
[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE311241], and GSE295697
[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE295697], respectively. Publicly
available datasets used in this study are listed in Supplementary Table 2. Source data are
provided with this paper. All other data that support the findings of this study are available
from the corresponding authors upon reasonable request.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "REQUIRES SIGNIFICANT REVISIONS\nThis manuscript by Duan et al. presents a comprehensive study on the role of RBM22 in promoting cardiomyocyte (CM) proliferation and heart regeneration. The authors utilize a wide range of experimental approaches—including conditional knockout and overexpression models, bulk and single-cell transcriptomics, CUT&Tag, ATAC-seq, ChIP-qPCR, and luciferase assays—to support their central claim that RBM22 regulates the transcription of key cell cycle genes through modulation of chromatin accessibility. They propose that RBM22 directly binds to promoter regions of Cdk4, Ccna2, and Ccne1, and promotes transcriptional activation in cooperation with SMARCA4. The manuscript is extensive and includes consistent phenotypic findings across neonatal, adult, and hiPSC-derived cardiomyocytes.\nWhile the dataset is large and the conclusions are generally well-supported at the phenotypic level, I have several concerns about the mechanistic interpretation and experimental framing:\nMajor Concerns\n1. Mechanistic studies in neonatal cardiomyocytes cannot reflect the in vivo regenerative context.\n2. While the binding of RBM22 to chromatin is convincingly shown, the possibility that RBM22 acts indirectly via another DNA-binding factor (e.g., via protein complexes) should be discussed.\n3. The authors use Rbm2 fl/+; Myh6-iCre+ mice to study RBM22 function in adult heart regeneration, citing concerns about tamoxifen-induced DNA damage in αMHC-MerCreMer mice. However, this inducible Cre model is commonly and safely used in the field, with many studies showing that proper low-dose tamoxifen regimens avoid toxicity. Therefore, this reasoning is not sufficient to avoid using the inducible model in adults. Using a heterozygous model introduces ambiguity, as partial loss of RBM22 may not reflect its true function in adult regeneration and could be influenced by developmental compensation.\n4. The authors do not show any baseline data for tamoxifen-treated neonatal Rbm22-iCKO or control mice. This makes it difficult to separate the effects of RBM22 deletion from potential Cre or tamoxifen effects in early postnatal stages.\n5. Where does RBM22 express after MI (border zone or remote region)?\n6. The embryonic lethality in full KO mice suggests developmental roles of RBM22 beyond postnatal proliferation. These roles are mentioned but not explored.\n7. Isolation of primary cardiomyocytes through collagenase digestion is not a reliable method to identify the total number of cardiomyocytes. It is not stated clearly if only the rod-shaped (healthy) cardiomyocytes were counted or both rod-shaped and round-shaped (dead) cardiomyocytes were counted.\n8. The authors state that RBM22 binding sites are enriched at promoter regions of cell cycle genes in figure 5E, but they only show the chromatin accessible region by ATAC-seq without any ChIP-seq data supporting the presence of the promoters.\n9. Line 617-622: The authors discuss the potential Nkx2.5 regulation on RBM22 in adult hearts. However, this is not supported by the analyses made previously.\n10. The working model in figure 8 is not accurate. There is no data in the paper indicating CM dedifferentiation in RBM22 overexpression model. The vectors of RBM22 and SMARCA4 should stick together as the authors show there is direct interaction by Co-IP.\n11. The authors may consider presenting their data in a more effective way, for example, moving key supporting data to the main figures and removing the unnecessary data from the main figures.\n12. Please make the accurate citation and please cite the papers that support the authors’ statements and the ones that provide datasets used in the manuscript.\nMinor concerns:\n1. Figure 3I: the Edu+ CMs is higher in WT compared to the published paper.\n2. Figure 6I: SMARC2 is mis-spelled.\n3. Line 305-7: Do KEGG and GSEA analyses identified upregulated, or downregulated, or both genes in DNA replication pathway?\n4. Line 854-7: Please clarify what the calcium-free buffer and the high K+ buffer are.\n5. Figure S7B: Rbm22 binding sites more in the promoter region in P1 than P8 (8.73% vs 8.19%). Is there any significance?\n6. Figure S8E: There is no difference in chromatin accessibility in the promoter of Mik67 and Pcna upon Rbm22 knockdown.\n7. Figure S9B: Data presentation is confusing. Consider using bar plots and putting the four groups together.", "reviewer_2": "The study by Duan and colleagues offers a comprehensive exploration of RBM22 in cardiomyocyte proliferation, spanning a broad range of molecular, in vitro, and in vivo approaches. The authors propose that RBM22 acts not only as a splicing factor but also as a transcriptional regulator of cell cycle genes via interaction with SMARCA4. The dataset is rich and the experimental scope is ambitious. However, the manuscript falls short of the rigor required to substantiate its mechanistic conclusions in the context of cardiac regeneration. The omission of essential control groups—particularly the αMHC-MerCreMer-only and Myh6-iCre-only mice—combined with inconsistent genetic modeling and insufficient specificity in the proliferation assays, represents a fundamental limitation. These issues are well recognized in the field and must be addressed before the authors’ conclusions can be considered robust. As the literature has already seen multiple claims of cardiomyocyte cell cycle reactivation that failed under independent scrutiny, and as experimental artifacts in this area are now better understood, it is essential that this work adhere to the highest standards.\nMajor Concerns\n1. Inadequate Cre Controls in Neonatal and Adult Models. The central mechanistic claim—that RBM22 deletion impairs cardiac regeneration and repair—is undermined by limitations in genetic modeling and control group design. Most notably, the authors do not include a tamoxifen-treated αMHC-MerCreMer-only control group in their neonatal apical resection experiments. While they cite known MerCreMer toxicity in adult hearts, they assert that no such effect occurs in neonates. However, this contradicts existing evidence: Bersell et al. (PMID: 23929941) demonstrated that neonatal αMHC-MerCreMer mice treated with tamoxifen exhibit dose-dependent fibrosis and impaired regeneration even in the absence of a floxed allele. The inclusion of Rbm22^fl/fl^ mice lacking Cre addresses tamoxifen and background controls but fails to account for MerCreMer-driven toxicity. A tamoxifen-treated αMHC-MerCreMer; Rbm22^+/+^ group is essential to isolate the effects of the gene deletion from those of the Cre system. Without it, the interpretation of regenerative defects remains ambiguous.\n2. Inconsistency Across Injury Models. The manuscript uses different genetic models for neonatal and adult injury paradigms. In neonates, the authors employ homozygous Rbm22^fl/fl^ deletion with tamoxifen-inducible αMHC-MerCreMer. In adults, they use heterozygous Rbm22^fl/+^ mice with constitutive Myh6-iCre, citing embryonic lethality and MerCreMer toxicity. This introduces discrepancies in timing of gene deletion, dosage, and Cre driver specificity. While practical constraints are understandable, the use of divergent models limits mechanistic interpretation across developmental stages. The authors should acknowledge this limitation clearly and temper their cross-model conclusions accordingly.\n3. Missing Myh6-iCre-Only Control in Adult MI Studies. In the adult myocardial infarction model, the comparison is made between Rbm22^fl/+^ and Rbm22^fl/+^; Myh6-iCre+ mice, without inclusion of a Myh6-iCre+; Rbm22^+/+^ group. Given that Myh6-iCre is a constitutively active Cre driver, and previous studies have shown Cre expression alone can cause cardiac remodeling or dysfunction, this is a critical omission. Without this control, it is not possible to determine whether the observed phenotype results from RBM22 haploinsufficiency or Cre-mediated toxicity. This confound must be addressed experimentally or, at a minimum, clearly acknowledged.\n4. Insufficient Rigor in Proliferation Assays. The authors assess cardiomyocyte proliferation using EdU incorporation, Ki67 immunostaining, and phospho-Histone H3 (pH3). While pH3 offers modest specificity for mitotic entry, EdU and Ki67 are widely recognized as inadequate for detecting productive cardiomyocyte proliferation, as they may reflect DNA synthesis associated with cell cycle re-entry, polyploidization, or DNA repair. Even pH3 cannot distinguish cytokinesis from abortive mitosis or binucleation. To support the claim that RBM22 deletion impairs cardiomyocyte proliferation, additional assays are needed. Specifically, Aurora B kinase or anillin staining should be used to assess cleavage furrow formation. Ideally, this would be validated by genetic lineage tracing (e.g., Confetti, MADM) under αMHC control to directly visualize cardiomyocyte division.\n5. Lack of Rigor in In Vitro Proliferation Assays. The above concern is particularly pertinent to the in vitro experiments, where rigorous quantification of proliferation is more tractable. In neonatal mouse cardiomyocytes (NMCMs) and hiPSC-derived cardiomyocytes (hiPSC-CMs), proliferation should be assessed using validated readouts such as dye dilution or cell cycle reporter systems. This is especially important in the hiPSC-CM experiments, which currently rely solely on EdU incorporation and therefore cannot distinguish proliferation from DNA synthesis related to stress or partial re-entry.\nSecondary Concerns:\n1. Only one siRNA sequence targeting RBM22 appears to have been used. Given the known risk of off-target effects, key phenotypes (e.g., Fig. 4K, L) should be validated using a second independent siRNA or with orthogonal strategies such as CRISPRi, which can be effectively delivered to NMCMs using AAV vectors.\n2. The authors claim that RBM22 physically interacts with SMARCA4, but do not assess the specificity of this interaction under conditions of RBM22 silencing. A co-IP of endogenous proteins in knockdown conditions would help validate the interaction’s dependence on RBM22.\n3. The discussion of RBM22 function in hiPSC-CMs should be more cautious. These cells are highly immature, and evidence of increased proliferation or delayed maturation does not imply similar effects in adult cardiomyocytes. While possibly beyond the current scope, testing in more mature systems—such as engineered heart tissues or in vivo grafts—would help substantiate translational relevance.\n4. The discussion on clinical applications is confusing and speculative. The authors point out the impracticality of viral vectors in clinical settings, yet this is the approach they demonstrate here. They later refer vaguely to “small molecules and synthetic oligonucleotides designed to target RBM22,” but no candidates or rationale are offered. This section should be revised for clarity and realism.\nMinor Concerns:\n1. The abstract should mention that RBM22 has a previously reported noncanonical transcriptional role, to avoid confusing the readter and/oroverstating the novelty of this finding.\n2. Lines 78–81: The text implies that SMARCA4 regulates chromatin’s only two states. This is a massive oversimplification and should be nuanced.\n3. Line 93: Please define YAP5SA for non-experts.\n4. Lines 147–148: The clinical relevance of the analysis is not clear. The conclusion drawn appears counterintuitive. Please rephrase or clarify.\n5. Line 153: Define AR for non-specialist readers.\n6. Lines 159–160: Single-cell RNA-seq is now a widely accepted standard to provide evidence of cells type-specific expression, and multiple published human and mouse heart atlases exist that could be referenced or reanalyzed without the need for new data generation.\n7. Fig. 1G and other immunofluorescence images: please provide grayscale individual channels and use colorblind-friendly merged images (e.g., cyan/magenta/yellow) to ensure accessibility. See: https://www.ascb.org/science-news/how-to-make-scientific-figures-accessible-to-readers-with-color-blindness/\n8. Line 172 and Line 371: Terms like “implies” and “indicates” suggest causation. These should be softened to “suggests” or “is consistent with.”\n9. Figures S2D and S2E appear contradictory regarding the relationship between RBM22 and NKX2-5. This discrepancy should be addressed in the relevant results and discussion sections.\n10. Figure S2J: Please specify the time point of the analysis.\n11. Figure 4D shows an unusually large number of differentially expressed genes (~1/3 to 1/5 of the transcriptome). Could this reflect differences in cardiomyocyte vs. non-cardiomyocyte proportions?\n12. Line 347: The “ZnF deletion” construct removes residues 1–232, which includes far more than the zinc finger motif (residues 159–182). This should be clearly acknowledged and discussed in terms of interpretation.\n13. Data plotting for CUT&Tag and ATAC-seq dataa in Fig. 5E and Fig. S7 lacks conventional visualization. Please include representative read pileups or genome browser snapshots to assess signal and background.\n14. Line 416: The phrase “global decrease” is misleading, as only a small fraction of promoters are affected. Please rephrase.\n15. Line 462 and Line 597: Terms like “direct interaction” and “directly” are too strong based solely on co-IP or EMSA. Please moderate or support with additional biophysical assays.\n16. The Discussion section could be more concise. Redundant summaries of background information should be minimized to make room for a deeper comparison of RBM22 with other known inducers of cardiomyocyte proliferation (e.g., YAP5SA, miR-206, etc.).\n17. Line 598: Clarify whether the authors are referring to the ZnF domain or another motif. The RRM appears dispensable, so this interpretation should be specified.\n18. Overall, while the manuscript is generally well-written, there are numerous grammatical issues and awkward phrasings throughout. Examples include\n- Line 48: “attach of myocardial infarction”\n- Line 61: “The previous sstudies show”\n- Line 66: “imperative” (used oddly multiple times)\n- Line 84: “conversed”\n- Line 593: “carcinogenic” (possibly meant to say “oncogenic” or “tumorigenic”)"}, "author_response": "Point-by-Point Response\nReviewer #1:\nWe are very grateful for your positive and constructive comments. According to your\ninsightful comments and recommendations, we have performed the additional\nexperiments and made extensive corrections to strengthen our manuscript.\nMajor Concerns\nQuestion 1. Mechanistic studies in neonatal cardiomyocytes cannot reflect the in vivo\nregenerative context.\nResponse: Thank you for your valuable feedback on our manuscript. Based on a\nthorough review of the literature (EBioMedicine. 2022. PMID: 35810562; Basic Res\nCardiol. 2024. PMID: 38147128; Sci Adv. 2025. PMID: 40408476; Nat Commun. 2025.\nPMID: 40128196; Cell Res. 2021. PMID: 32973339; Cell Stem Cell. 2022. PMID:\n35395187), we found that many studies on cardiomyocyte proliferation have employed\nexperimental approaches in which cells are treated with siRNA, AAV9, or a chemical\ncocktail of five small molecules followed by transcriptomic or mechanistic analyses.\nThese approaches are widely recognized as effective models for dissecting molecular\npathways in an experimentally controlled environment.\nWe fully acknowledge that our model system, which utilizes the simplified\nneonatal mouse cardiomyocytes (NMCMs) system, does not fully recapitulate the\ncomplexity of the in vivo heart, such as multi-cellular interactions, paracrine signaling,\nmechanical loading, and immune modulation. These limitations, however, allow us to\nfocus specifically on cardiomyocyte-intrinsic responses. That is the primary objective\nof our study on the proliferation and regeneration of neonatal mouse cardiomyocytes.\nIn this way, we manipulated RBM22 expression and systematically examined its\ndownstream transcriptional and chromatin-related effects under defined conditions,\n1\nsuch as small-interfering RNA-mediated RBM22 silencing or AAV9-cTnT-mediated\nRBM22 overexpression in primary mouse cardiomyocytes/human iPSC-derived\ncardiomyocytes in vitro. To address your concerns, we further validated the key\nmechanisms by isolating cardiomyocytes from neonatal RBM22 knockout gene-edited\nmice (Figure 4a, Supplementary Fig. 7g, h, and Supplementary Fig. 11a, c-e) and\nadult wild-type mice treated with AAV9-RBM22 (Figure 7j-o and Supplementary\nFig. 13a-d). , These complementary approaches thereby strengthen the physiological\nrelevance of our findings and ensure that our in vitro observations are not artifacts of\ncell culture, but reflect biologically relevant mechanisms underlying cardiac\nregeneration.\nOn one hand, using an in vitro model with tamoxifen-inducible knockout of\nRBM22 in primary cardiomyocytes, we isolated cardiomyocytes from Rbm22-iCKO\nneonatal mice and induced RBM22 knockout using 4-hydroxytamoxifen. Subsequent\nimmunofluorescence assays revealed a significant reduction in the proportion of\nproliferating cardiomyocytes (Figure R1-1a), accompanied by marked decreases in the\nmRNA and protein levels of key proliferation-related genes (Figure R1-1b, 1c).\nMechanistically, co-immunoprecipitation (Co-IP) assays demonstrated a diminished\ninteraction between RBM22 and SMARCA4 in these RBM22-deficient\ncardiomyocytes (Figure R1-1d). Furthermore, Chromatin Immunoprecipitation\nquantitative PCR (ChIP-qPCR) results suggested that RBM22 knockout reduced the\nrecruitment of SMARCA4 to the promoter regions of crucial cell cycle regulators,\nincluding Cdk4, Ccna2, and Ccne1 (Figure R1-1e). Therefore, we concluded that\nRBM22 is essential for regulating cell cycle progression and proliferation in neonatal\ncardiomyocytes, acting through its interaction with SMARCA4 and recruitment to the\npromoters of key cell cycle genes.\nWe have now included this data and added descriptions in the revised manuscript\n(Pages 15, lines 292-296, revised Fig. 4a-c). In addition, the corresponding\nsupplementary figures have been updated accordingly (revised Supplementary Fig. 7g,\nh; Supplementary Fig. 11a, c–e), and the textual descriptions have been added in the\nmanuscript (Pages 17, lines 338-341; Pages 24, lines 487-490, 500-504).\nOn the other hand, to directly validate these findings in a pathological context,\nwild-type mice were subjected to myocardial infarction (MI) model and administered\nwith intramyocardial injection of AAV9-cTnT-Rbm22 to achieve cardiomyocyte-\nspecific RBM22 overexpression. Assay for Transposase-Accessible Chromatin with\n3\nsequencing (ATAC-seq) of adult primary cardiomyocytes isolated post-MI revealed\nthat RBM22 overexpression resulted in altered chromatin accessibility, including 1319\npeaks with open chromatin accessibility and 159 peaks with closed chromatin\naccessibility (Figure R1-2a-b). Gene Ontology (GO) enrichment analysis suggested\nthat the genes with differentially accessible regions were predominantly enriched in\npathways related to cardiomyocyte proliferation and tissue regeneration (Figure R1-\n2c). Consistently, KEGG pathway analysis demonstrated these regions were involved\nin cell cycle regulation (Figure R1-2d). Heatmap and IGV trajectory analyses further\nconfirmed the enhanced chromatin openness at the transcription regulatory regions,\nespecially gene promoters, with remarkable H3K4me3 (GSE155658) /H3K27ac signals\n(GSE231014) and RNA Pol II binding (GSE155658), of several cell cycle-related genes\nin RBM22-overexpressed adult cardiomyocytes, especially Cdk4, Ccna2, and Ccne1\n(Figure R1-2e-h). Critically, co-IP and ChIP-qPCR validation experiments\ncorroborated our in vitro data, showing that RBM22 overexpression facilitated the\nSMARCA4 recruitment and chromatin accessibility of the promoter regions of Cdk4,\nCcna2, and Ccne1 for heart regeneration (Figure R1-2i, j).\ngenes involved in adult cardiomyocyte proliferation\nWe have now included this data in the revised manuscript (Pages 28-29, lines\n580-603, revised Fig. 7j-o) and supplementary materials (Pages 28-29, lines 575-599,\nrevised Supplementary Fig. 13a-d) and added descriptions in the section. Our data\nsuggest that RBM22 binds to the promoter regions of cell cycle genes in\ncardiomyocytes, where it likely plays a pivotal role in enhancing their transcriptional\n5\nexpression. Through recruiting SMARCA4, a core subunit of chromatin remodeling\ncomplexes, RBM22 improves the chromatin accessibility at these critical gene loci.\nAnd subsequently, the increased chromatin accessibility facilitates the transcriptional\nactivation of key cell cycle genes, ultimately promoting cardiomyocyte proliferation.\nThis mechanistic pathway, supported by both in vitro and in vivo findings, provides\nconvincing evidence that RBM22/SMARCA4 interaction is central to the transcription\nregulatory network governing heart regeneration.\nWe hope that our response and the additional data provided in the revised\nmanuscript might address your concerns. We appreciate your valuable feedback and\nbelieve that these additional experiments enhance the robustness of our study and\nprovide a clearer understanding of the molecular mechanisms involved in RBM22-\nmediated myocardial proliferation and regeneration.\nQuestion 2. While the binding of RBM22 to chromatin is convincingly shown, the\npossibility that RBM22 acts indirectly via another DNA-binding factor (e.g., via\nprotein complexes) should be discussed.\nResponse: We appreciate your thoughtful comments regarding the possibility that\nRBM22 may act indirectly via other DNA-binding factors. We isolated chromatin\nfractions and performed immunoblotting, which revealed that RBM22 predominantly\nlocalizes to the chromatin and nucleoplasm fractions in cardiomyocytes (Figure 5a).\nAdditionally, CUT & Tag sequencing showed that RBM22 binds to the promoter\nregions of key cell cycle genes in proliferative P1 cardiomyocytes, and this was further\nvalidated by ChIP-qPCR experiments (Figure 5b-f). While our above data provide\nstrong evidence for the binding of RBM22 to chromatin, we fully agree that your\ninsightful hypothesis for indirect regulation via protein complexes or other transcription\nfactors should be considered. In the revised manuscript, we have expanded the\nDiscussion section to supplement this possibility. We now mention that RBM22 could\npotentially interact with other DNA-binding proteins, forming epigenetic-associated\nprotein complexes that mediate chromatin remodeling or transcriptional regulation.\ncardiomyocytes, we acknowledge that future experiments, such as mass spectrometry\nfollowed by co-immunoprecipitation, will be required to definitively identify the\npotential interacting partners and elucidate their role in RBM22-mediated\ntranscriptional regulation of genes involved in cell cycle progression in cardiomyocytes.\nThis addition enhances the depth of our discussion and provides an improved\ninterpretation of our experimental data. We thank you for this valuable suggestion,\nwhich has improved the comprehensiveness of our analysis. The corresponding\ndescriptions were included in the discussion section of the revised manuscript (Pages\n32-33, lines 675-680).\nQuestion 3. The authors use Rbm2 fl/+; Myh6-iCre+ mice to study RBM22 function\nin adult heart regeneration, citing concerns about tamoxifen-induced DNA damage\nin αMHC-MerCreMer mice. However, this inducible Cre model is commonly and\nsafely used in the field, with many studies showing that proper low-dose tamoxifen\nregimens avoid toxicity. Therefore, this reasoning is not sufficient to avoid using the\ninducible model in adults. Using a heterozygous model introduces ambiguity, as\npartial loss of RBM22 may not reflect its true function in adult regeneration and\ncould be influenced by developmental compensation.\nResponse: Thank you for your insightful comments. We fully acknowledge that the\nαMHC-MerCreMer+ system is a well-established and widely used tool for inducible\ncardiomyocyte-specific gene targeting, and that several studies have demonstrated its\nsafety under optimized low-dose tamoxifen regimens (Dis Model Mech. 2013. PMID:\n23929941).\nIn our initial attempts to generate the inducible Rbm22fl/flαMHC-MerCreMer+\n(Rbm22-iCKO) mice, we used the standard tamoxifen dosage (75 mg/kg) commonly\napplied in the fields outside cardiovascular diseases. However, tamoxifen\nadministration (75 mg/kg) consistently resulted in premature mortality before adult MI\nmodel establishment, preventing us from assessing the capacity of cardiac regeneration\n7\nin Rbm22-iCKO mice. To address this issue, we first conducted our analyses using adult\nRbm22fl/+; Myh6-iCre+ mice, which allowed us to evaluate the functional consequences\nof RBM22 knockdown in the absence of tamoxifen exposure. According to your kind\nsuggestion, we next performed a detailed dose response optimization of tamoxifen in\nadult Rbm22-iCKO mice to determine a safe and effective dosage. Our new\nexperimental results revealed that we injected tamoxifen intraperitoneally on three\nconsecutive days at doses of 0 mg/kg, 30 mg/kg, 40 mg/kg and 60 mg/kg. The results\nrevealed that doses of 60 mg/kg × 3 caused marked systolic dysfunction, inflammation,\nand apoptosis, and even 40 mg/kg × 3 led to detectable myocardial fibrosis. In contrast,\nthe administration of tamoxifen at 30 mg/kg × 3 achieved efficient RBM22 knockout\nin adult cardiomyocytes without overt baseline toxicity (Figure R1-3a-j).\n8\nMerCreMer+ mice\nWe further isolated adult cardiomyocytes and confirmed that this low-dose\ntamoxifen treatment effectively induced RBM22 deletion in cardiomyocytes. In\naddition, hematoxylin and eosin (H&E) staining showed no apparent morphological\nabnormalities in the hearts after tamoxifen administration in either Rbm22fl/fl mice,\nαMHC-MerCreMer+ mice, or Rbm22-iCKO mice (Figure R1-4a-c).\nFigure R1-4. Low-dose tamoxifen (30 mg/kg) effectively induces RBM22 deletion in\ncardiomyocytes\nUsing this optimized low-dose strategy, we conducted new experiments in adult\nRbm22fl/fl αMHC-MerCreMer+ (RBM22-iCKO) mice and controls (Rbm22fl/fl and\nαMHC-MerCreMer+) subjected to MI. At day 28 post-MI, RBM22 deletion in\ncardiomyocytes led to significantly worsened cardiac function and increased fibrosis.\nIsolated cardiomyocyte analysis revealed a reduction of the absolute number of\ncardiomyocytes and a lower proportion of mononuclear diploid cardiomyocytes\n(Figure R1-5a-i). Immunofluorescence staining further confirmed that RBM22\ndeletion decreased cardiomyocyte proliferation in border zones at day 14 post-MI\n(Figure R1-6a-h).\npost-MI\nWe agree with you that partial loss of RBM22 could raise the possibility of\ndevelopmental compensation or haploinsufficiency. Nevertheless, the robust\nregenerative defects observed even after partial, acutely inducible loss of RBM22\nunderscore its essential role in adult cardiac regeneration. We therefore believe that our\nexperimental strategy represents a valid and physiologically relevant approach that\nappropriately balances genetic precision and experimental safety.\nWe have now included this data in the revised manuscript (Pages 13-14, lines 255-\n277, revised Figure 3a-r; Page 13-14, lines 248-262, Supplementary Fig. 4c-g) and\nadded descriptions in the section.\nQuestion 4. The authors do not show any baseline data for tamoxifen-treated\n12\nneonatal Rbm22-iCKO or control mice. This makes it difficult to separate the effects\nof RBM22 deletion from potential Cre or tamoxifen effects in early postnatal stages.\nResponse: Thank you for your valuable comments and suggestions. In our preliminary\nexperiments, we showed that tamoxifen-treated neonatal Rbm22-iCKO and control\nmice exhibited no obvious morphological abnormalities in the heart following\ntamoxifen administration. Following your suggestion, we have conducted additional\nexperiments to further clarify the baseline effects of tamoxifen or Cre expression in the\nneonatal stage. Specifically, neonatal Rbm22-iCKO and control mice were\nadministered tamoxifen via intraperitoneal injection, and cardiac function was assessed\n21 days later by echocardiography (Figure R1-7a, b). The results demonstrated that\neither αMHC-MerCreMer+ Cre or tamoxifen effects did not affect cardiac systolic\nfunction, compared with controls (Figure R1-7c, d). Consistently, histological analysis\nusing H&E staining revealed no detectable changes in cardiac morphology (Figure R1-\n7e). These findings confirm that neither neonatal tamoxifen administration nor Cre\nactivation alone impact normal cardiac function and physiology.\nFigure R1-7. The effect of knockout of RBM22 on cardiac function and morphology\n13\nin neonatal mice at postnatal day 21\nTo further exclude potential tamoxifen toxicity, we treated αMHC-MerCreMer+\nmice with tamoxifen from postnatal day 0 to day 3 and monitored cardiac morphology\nand function at P21. The results demonstrated that tamoxifen administration did not\ninduce any toxicity, as there was no observable damage to the cardiac morphology or\nfunction in early postnatal stages (Figure R1-8a-b).\nFigure R1-8. Validation of tamoxifen safety in neonatal αMHC-MerCreMer+ mice at\npostnatal day 21\nWe have included these new data in the revised supplementary material to provide\na more comprehensive understanding of the role of RBM22 in cardiac function and\nrepair (Pages 10-11, lines 184-196, Supplementary Fig. 2i-o).\nQuestion 5. Where does RBM22 express after MI (border zone or remote region)?\nResponse: Thank you for your insightful comments and suggestions.\nImmunofluorescence staining of heart sections from neonatal mice at day 3 post-MI\nand adult mice at day 7 post-MI revealed that RBM22 is predominantly expressed in\ncardiomyocytes located within the border zone, rather than in those of the remote\nmyocardium (Figure R1-9a-d). However, RBM22 expression was also observed in a\nsmall number of cardiomyocytes within the remote region. The proportion of RBM22-\npositive cardiomyocytes was significantly higher in the border zone, indicating its\nenrichment in regions actively involved in cardiac repair. The higher proportion of\nRBM22-positive cardiomyocytes in the border zone aligns with our observation of a\n14\nhigher proportion of proliferating cardiomyocytes in the border zone of neonatal hearts.\nThese data confirmed that the differences in cardiomyocyte proliferation capacity\nbetween regions are primarily due to the higher expression of RBM22 in the border\nzone. We have incorporated these new results into the revised manuscript and\nsupplementary materials (Page 8, lines 137-141, Supplementary Fig. 1g-h; Page 13,\nlines 239-241, Supplementary Fig. 4b).\nFigure R1-9. RBM22 is primarily localized to the infarct border zone post-MI\nQuestion 6. The embryonic lethality in full KO mice suggests developmental roles of\nRBM22 beyond postnatal proliferation. These roles are mentioned but not explored.\nResponse: We appreciate this thoughtful comment. During the breeding of\ncardiomyocyte-specific RBM22 conditional knockout mice in the absence of tamoxifen\ninduction, we found that crossing Rbm22fl/fl mice with Myh6-iCre mice did not yield\nhomozygous cardiomyocyte-specific RBM22 knockout mice (Rbm22fl/flMyh6-iCre)\n(Supplementary Fig. 2c).\nseveral key cardiac developmental regulators, Nkx2-5 may be involved in the control\nof RBM22 expression (Supplementary Fig. 2d-f). Consistent with this, previous work\nhas shown that RBM22 is broadly expressed during zebrafish development without\nclear spatial or temporal restriction (Genet Mol Res. 2009. PMID: 20013661). Similar\nembryonic lethality and defects in cardiomyocyte proliferation have also been reported\nin knockout models of genes such as Cbx7 (Circulation. 2023. PMID: 37158107) and\nMrps5 (Circulation. 2023. PMID: 37905452), supporting the notion that RBM22 may\nplay an essential role during early cardiac development.\nIn the present study, we do not further investigate the developmental function of\nRBM22, as our primary focus is to elucidate its regulatory role in cardiomyocyte\nproliferation and cardiac regeneration in the postnatal and adult heart, especially in\npathological conditions such as myocardial infarction. We have now clarified this point\nand included a discussion of the potential developmental significance of RBM22 in the\nrevised manuscript (Page 33, lines 686-693, 696-698).\nQuestion 7. Isolation of primary cardiomyocytes through collagenase digestion is not\na reliable method to identify the total number of cardiomyocytes. It is not stated\nclearly if only the rod-shaped (healthy) cardiomyocytes were counted or both rod-\nshaped and round-shaped (dead) cardiomyocytes were counted.\nResponse: Thank you for your insightful comments. Isolation of adult cardiomyocytes\nusing collagenase digestion has been widely used and validated in previous high-level\nstudies (Nature. 2017. PMID: 27798600; Nature. 2013. PMID: 23594737; Nat\nCommun. 2025. PMID: 40087279; and Cell Res. 2021. PMID: 32973339). In our\nexperiments, we employed collagenase II to digest the heart tissue, which efficiently\nyields highly viable cardiomyocytes (Figure R1-10). Importantly, for quantification,\nwe counted only rod-shaped cardiomyocytes, which are indicative of healthy,\nfunctional cells, as indicated by the arrows in Figure 3f, Supplementary Fig. 5e, and\nSupplementary Fig. 12k. Round-shaped or non-rod-shaped cardiomyocytes, which\n16\ntypically represent damaged or dead cells, were excluded from the analysis to ensure\naccurate assessment of viable cardiomyocyte numbers.\nFigure R1-10. Isolation and characterization of adult cardiomyocytes\nWe have improved the Methods section and the corresponding figure legend in\nthe revised manuscript to better reflect these points, as suggested by your valuable\nfeedback (Page 44, lines 933-934).\nQuestion 8. The authors state that RBM22 binding sites are enriched at promoter\nregions of cell cycle genes in figure 5E, but they only show the chromatin accessible\nregion by ATAC-seq without any ChIP-seq data supporting the presence of the\npromoters.\nResponse: We thank you for this important comment. In our previous data, ATAC-seq\nanalysis demonstrated that knockdown of RBM22 reduced chromatin accessibility at\nthe promoters of cell cycle genes (Figure 6e). We apologize for any confusion caused\nby the presentation of these data. In our manuscript, we analyzed RBM22 binding\nsignals from CUT & Tag experiments and observed strong, specific RBM22 signals\nnear the transcription start sites (TSS) in both P1 and P8 cardiomyocytes (Figure 5b).\nThis finding suggests that RBM22 exhibits a relatively specific binding pattern at TSS\nloci, which suggests that the binding sites of RBM22 are primarily located in the\npromoter regions of cell cycle-regulated genes. To further support our definition of the\ngene promoter, we integrated CUT & Tag data with publicly available ChIP-seq\ndatasets (GSE155658), which provide the information of histone H3K4me3\nmodification, a well-established marker of active promoters in cardiomyocytes. The\n17\nchromatin-binding sites of RBM22 were highly overlapped with H3K4me3 signals,\nindicating that RBM22 selectively binds to the promoters of cell cycle-related genes,\nand this binding is reduced at P8 (Figure R1-11). These results collectively support the\nconclusion that RBM22 directly supports the transcriptional activation of cell cycle-\nrelated genes through binding to their active promoters, respectively. This promoter-\nspecific chromatin binding pattern enables SMARCA4-dependent chromatin\naccessibility of these target genes in cardiomyocytes.\nFigure R1-11. Integrative Genomics Viewer (IGV) visualization shows RBM22\nbinding at the promoter regions of cell cycle genes\nWe have now included this data in the revised manuscript (Page 20, lines 397-400,\nFigure 5e). We greatly appreciate your guidance in ensuring that our presentation and\ninterpretation of the data are as clear and accurate as possible.\nQuestion 9. Line 617-622: The authors discuss the potential Nkx2.5 regulation on\nRBM22 in adult hearts. However, this is not supported by the analyses made\npreviously.\nResponse: We apologize for any confusion regarding our statement. To date, there have\nbeen no reports demonstrating regulation of RBM22 by NKX2-5, which underscores\nthe novelty of our findings. By analyzing publicly available transcriptomic datasets\n18\n(GSE77547), we observed that RBM22 expression is reduced in NKX2-5 knockout\n(NKO) cardiomyocyte (Figure R1-12b). Furthermore, integration with ChIP-seq data\n(GSE77548) revealed the abundance of NKX2-5 chromatin signals around the\ntranscription start sites of the RBM22 gene locus, suggesting that NKX2-5 may\ncontribute to the transcriptional regulation of RBM22 in the adult heart (Figure R1-\n12b-c). We have incorporated these new results into the revised supplementary\nmaterials (Page 10, lines 174-180, Supplementary Fig 2e, f).\nFigure R1-12. NKX2-5 binds to the promoter region of RBM22 and modulates its\ntranscription\nQuestion 10. The working model in figure 8 is not accurate. There is no data in the\npaper indicating CM dedifferentiation in RBM22 overexpression model. The vectors\nof RBM22 and SMARCA4 should stick together as the authors show there is direct\ninteraction by Co-IP.\nResponse: In accordance with your suggestion, we have revised the working model in\nFigure 8g. Specifically, we have removed the CM dedifferentiation step, as our current\ndata do not directly demonstrate this process. In addition, we have repositioned the\nRBM22 and SMARCA4 components to display them together, highlighting their direct\ninteraction as evidenced by our experimental data, mainly in Figure 6. The revised\nschematic now illustrates that the RBM22-SMARCA4 complex facilitates\ncardiomyocyte cell cycle re-entry, proliferation, and subsequent heart regeneration.\nand depth of our manuscript.\nQuestion 11. The authors may consider presenting their data in a more effective way,\nfor example, moving key supporting data to the main figures and removing the\nunnecessary data from the main figures.\nResponse: You suggested that we present our data in a more effective way by moving\nkey supporting data to the main figures and removing unnecessary data from them.\nFollowing your suggestion, we have reorganized the figures to improve clarity and\nlogical flow. Specifically, the previous Figure 4a–c have been moved to revised\nFig. 10b-d, and Figure 7a–c, 7h-i, and 7k-l have been moved to revised\nSupplementary Fig. 12c-e, 12i, and 12k. We believe that these changes make the data\npresentation clearer and more coherent. We appreciate your suggestion, as it has helped\nus enhance the clarity and reliability of our manuscript. Thank you for your constructive\nfeedback.\nQuestion 12. Please make the accurate citation and please cite the papers that support\nthe authors’ statements and the ones that provide datasets used in the manuscript.\nResponse: We appreciate your suggestion to provide accurate citations and include\nreferences that support our claims as well as the datasets used in the manuscript.\nSpecifically, we have cited the appropriate references in the manuscript to support our\nstatements, including Signal Transduct Target Ther. 2024. PMID: 39627201, Circ Res.\n2018. PMID: 30355161, Nat Cardiovasc Res. 2022. PMID: 37692772, Nat Commun.\n2018. PMID: 29453456, and Sci Transl Med. 2022. PMID: 35675436, and additional\nreferences. These references have been incorporated into the corresponding sections of\nthe manuscript and are highlighted in the revised Reference list for clarity.\nIn addition, we have added the GEO accession numbers for the public datasets\nused in this study in the revised supplementary materials (Supplementary Table 2).\nDataset Data Type Sample Description Figure\nhuman left ventricular\nGSE5406 RNA-seq Figure 1c\nmyocardium\nFigure 5e and\nneonatal mouse\nGSE178673 ATAC-seq Supplementary Fig.\ncardiomyocytes\n9c-e\nFigure 5e, 6e, and\nH3K4me3 neonatal mouse\nGSE155658 Supplementary Fig.\nChIP-seq cardiomyocytes\n9c-e and 10g\nGSE155658 Figure 6e and\nChIP-seq cardiomyocytes\nSupplementary Fig.\nH3K27ac\nGSE123867 neonatal mouse hearts 10g\nChIP-seq\nH3K4me3 P56 mouse\nGSE155658\nChIP-seq cardiomyocytes\nFigure 7m and\nRNA Pol II P56 mouse\nGSE155658 Supplementary Fig.\nChIP-seq cardiomyocytes\n13c, d\nH3K27ac\nGSE231014 P56 mouse hearts\nChIP-seq\nmouse left ventricle\nSupplementary Fig.\nGSE51483 RNA-seq\n1a and i-m\ntissue\npig neonatal left\nSupplementary Fig.\nGSE145346 RNA-seq\n1b\nventricular tissues\nSupplementary Fig.\nGSE221539 RNA-seq neonatal mice hearts\n1c, d\nSingle cell Supplementary Fig.\nGSE130699 neonatal mice hearts\nmRNA-seq 1f\nneonatal murine\nSupplementary Fig.\nGSE193137 RNA-seq\n2d\ncardiomyocyte\nmouse embryonic stem\nSupplementary Fig.\nGSE77547 RNA-seq (ES) cells-derived\n2e\ncardiomyocytes\nNKX2-5 mouse ES-derived\nSupplementary Fig.\nGSE77548\n2f\nChIP-seq cardiomyocytes\nSupplementary Table 2. Publicly available GEO datasets used in this paper\nQuestion 1. Figure 3I: the Edu+ CMs is higher in WT compared to the published\npaper.\nResponse: We appreciate your valuable comment. In adult MI models, the proportion\nof EdU⁺ cardiomyocytes can vary considerably depending on the experimental protocol,\nincluding the EdU dosage, injection frequency, and labeling duration. In our study, EdU\n(50 mg/kg) was administered every other day for a total of seven injections over 14\ndays post-MI, creating a cumulative labeling window that increases the likelihood of\ndetecting cardiomyocytes that entered the DNA synthesis phase during the repair\nprocess. Moreover, our quantification was specifically performed in the border zone, a\nregion known to exhibit higher cardiomyocyte renewal and DNA synthesis activity than\nremote or sham areas. These factors likely explain the relatively higher percentage of\nEdU⁺ cardiomyocytes observed in the WT group. Therefore, the observed difference\nreflects methodological and analytical variations rather than biological inconsistency.\nFurthermore, our EdU staining results are consistent with those reported by multiple\nindependent studies using EdU or BrdU, which are based on the same principle of\nlabeling cells in the S-phase of the cell cycle. This consistency supports the reliability\nand validity of our experimental findings (Nature. 2017. PMID: 28581497;\nTheranostics. 2025. PMID: 39816679; Adv Sci (Weinh). 2025. PMID: 40145861; Cell\nRes. 2021. PMID: 32973339; Circulation. 2020. PMID: 32078387; Nat Commun. 2018.\nPMID: 29453456). We thank you for this insightful comment, which prompted us to\nclarify these methodological details and enhance the transparency and rigor of our study.\nQuestion 2. Figure 6I: SMARC2 is mis-spelled.\nResponse: We sincerely apologize for this oversight and have corrected the spelling to\n“SMARCC2” in the revised figure (Figure 6f). Thank you for your careful review and\nfor helping us improve the accuracy and rigor of our manuscript.\nQuestion 3. Line 305-7: Do KEGG and GSEA analyses identified upregulated, or\n23\ndownregulated, or both genes in DNA replication pathway?\nResponse: Our results revealed that RBM22 silencing by siRNAs markedly inhibited\nthe DNA replication pathway, with almost all genes involved being significantly\ndownregulated. To better illustrate this finding, we have added a heatmap depicting the\nmRNA expression of all genes involved in the DNA replication pathway in the revised\nmanuscript (Figure R1-13c), which clearly shows a general decrease in the expression\nof DNA replication-related genes.\nFigure R1-13. RBM22 knockdown markedly inhibited genes associated with the\nDNA replication pathway\nWe have included the corresponding data in Figure 4 of the revised manuscript\n24\n(revised Figure 4g). We hope this additional analysis clarifies your concern and\nprovides a more comprehensive view of the gene expression changes in this pathway.\nQuestion 4. Line 854-7: Please clarify what the calcium-free buffer and the high K+\nbuffer are.\nResponse: We apologize for not providing sufficient details in the original submission.\nSpecifically, the calcium-free buffer (Ca²⁺-free Tyrode’s solution) used for perfusion\nand digestion contained (in mM): NaCl 135, KCl 5.4, MgCl₂·6H₂O 1, NaH₂PO₄ 0.33,\nglucose 10, and HEPES 10 (pH 7.3). Following isolation, cardiomyocytes were\nstabilized in a high-K⁺ buffer (KB solution) containing (in mM): L-glutamic acid 120,\nKOH 120, KCl 10, KH₂PO₄ 10, MgSO₄·7H₂O 1.8, K-EGTA 0.5, glucose 20, taurine 10,\nHEPES 10, and 0.2% BSA (pH 7.2). While many studies typically use calcium-free\nTyrode’s solution for cardiomyocyte isolation, we employed the KB solution buffer to\nstabilize the cardiomyocytes during subsequent ploidy analysis by immunofluorescence\n(IF). This buffer helps prevent hypercontraction and preserves both structural and\nnuclear integrity after isolation, which is crucial for accurate ploidy assessment\n(Circulation. 2014. PMID: 24895455; Sci Transl Med. 2022, PMID: 35675436; Cell\nRes. 2021. PMID: 32973339; Channels (Austin). 2020. PMID: 32491968).\nIn the revised supplementary material, we have specified the exact composition\nand purpose of these buffers in the Methods section (Page 44, lines 923-925, 928-931).\nWe have included these details to make the experimental procedures more transparent.\nQuestion 5. Figure S7B: Rbm22 binding sites more in the promoter region in P1 than\nP8 (8.73% vs 8.19%). Is there any significance?\nResponse: We apologize for the inappropriate wording in the original manuscript. Our\nintention was merely to provide a descriptive summary of the data rather than to imply\na statistical comparison.\nIn the revised manuscript, we have rephrased this sentence to clarify that no\nstatistical comparison was performed and that the percentages were presented only for\n25\ndescriptive purposes (Page 19, lines 381-383).\nQuestion 6. Figure S8E: There is no difference in chromatin accessibility in the\npromoter of Mik67 and Pcna upon Rbm22 knockdown.\nResponse: We appreciate this insightful comment. Mechanistically, RBM22 primarily\nregulates cell-cycle progression by directly targeting Cdk4, Ccna2, and Ccne1, while\nMki67 and Pcna function as effector genes of cell cycle, rather than the direct targets\nof RBM22-dependent transcriptional regulation. Therefore, the lack of a significant\ndifference in chromatin accessibility at the promoters of Mki67 and Pcna in the IGV\nvisualization is expected. Following your suggestion, we performed additional\nexperiments to further investigate this issue. Specifically, we re-isolated neonatal\ncardiomyocytes and conducted multi-sample ATAC-qPCR in Rbm22-knockdown\ncardiomyocytes, as described previously (Nat Methods. 2018. PMID: 29702633). The\nresults showed that chromatin accessibility in the promoter regions of Cdk4, Ccna2,\nCcne1, Mki67, and Pcna was significantly decreased compared with the control group,\nwhich further supports the notion that RBM22 affects chromatin accessibility at\ndifferent levels of the cell cycle gene regulatory network (Figure R1-14).\nFigure R1-14. ATAC-qPCR analysis reveals decreased chromatin accessibility of\ncardiomyocyte proliferation-associated genes upon Rbm22 knockdown\naccessibility of key cell-cycle regulatory and effector genes, thereby suppressing\ncardiomyocyte proliferation. The corresponding data descriptions were included in the\nResults section of the revised manuscript (Pages 23, lines 469-471, Supplementary\nFig. 10h).\nQuestion 7. Figure S9B: Data presentation is confusing. Consider using bar plots\nand putting the four groups together.\nResponse: We apologize for the confusion caused by our previous figure design.\nFollowing your suggestion, we have replaced the line plots with bar plots and presented\nthe four groups together in the Figure R1-15a-c (revised Supplementary Fig. 11f-h).\nWe sincerely appreciate your valuable suggestion. This modification makes the data\nvisualization clearer and facilitates direct comparison among the groups.\nFigure R1-15. RBM22 regulates the transcriptional activity of downstream cell-cycle\ngenes through its Proline-Rich domain\nMany thanks for the insightful and valuable comments. According to your suggestions,\nwe have performed additional experiments and revised the manuscript with new data.\nMajor Concerns\nQuestion 1. Inadequate Cre Controls in Neonatal and Adult Models. The central\nmechanistic claim—that RBM22 deletion impairs cardiac regeneration and repair—\nis undermined by limitations in genetic modeling and control group design. Most\nnotably, the authors do not include a tamoxifen-treated αMHC-MerCreMer-only\ncontrol group in their neonatal apical resection experiments. While they cite known\nMerCreMer toxicity in adult hearts, they assert that no such effect occurs in neonates.\nHowever, this contradicts existing evidence: Bersell et al. (PMID: 23929941)\ndemonstrated that neonatal αMHC-MerCreMer mice treated with tamoxifen exhibit\ndose-dependent fibrosis and impaired regeneration even in the absence of a floxed\nallele. The inclusion of Rbm22^fl/fl^ mice lacking Cre addresses tamoxifen and\nbackground controls but fails to account for MerCreMer-driven toxicity. A\ntamoxifen-treated αMHC-MerCreMer; Rbm22^+/+^ group is essential to isolate the\neffects of the gene deletion from those of the Cre system. Without it, the interpretation\nof regenerative defects remains ambiguous.\nResponse: We respectfully note that the study cited by you (Dis Model Mech. 2013.\nPMID: 23929941) investigated 6-week-old αMHC-MerCreMer+ mice, and therefore\ndoes not directly address tamoxifen effects in neonates. As reported in previous studies,\nno detectable phenotype or cardiac dysfunction was observed in αMHC-Cre mice up to\nsix months of age, with normal appearance and physical behavior, indicating no early\ntamoxifen-induced cardiac injury (Int J Mol Sci. 2023. PMID: 36834504). With this\ncontext in mind, we truly appreciate your important concern regarding the lack of\ntamoxifen-treated αMHC-MerCreMer+ only controls in our neonatal AR and MI\nmodels. We fully agree with your opinion that the absence of a tamoxifen-treated\nactivation from those of RBM22 deletion.\nIn response, we conducted additional experiments by incorporating αMHC-\nMerCreMer+ control mice alongside Rbm22-iCKO mice in both neonatal AR and MI\nmodels. The new data demonstrated that RBM22 deletion in cardiomyocytes markedly\nimpaired cardiac regeneration and repair, as evidenced by sustained cardiac dysfunction\nand fibrotic scar replacement, rather than functional recovery (Figure R2-1a-g, Figure\nR2-2a-f). In contrast, tamoxifen-treated αMHC-MerCreMer+ control mice exhibited\nnormal cardiac regeneration and preserved cardiac function, thereby confirming that\nthe observed regenerative defect is specifically attributable to RBM22 deficiency rather\nthan αMHC-MerCreMer+ mediated toxicity. Furthermore, immunofluorescence\nanalysis revealed a significantly reduced fraction of proliferating cardiomyocytes in\nRbm22-iCKO hearts compared with αMHC-MerCreMer+ controls (Figure R2-1h-p,\nFigure R2-2g-i), supporting the conclusion that RBM22 deficiency suppresses\ncardiomyocyte proliferation, leading to fibrotic scar formation instead of regeneration.\nregeneration\ncardiomyocyte proliferation post-MI\nAfter optimizing the tamoxifen dosage for safety, we included Cre controls in the\nadult mouse model. Using this optimized low-dose strategy, we conducted new\nexperiments in adult Rbm22-iCKO mice and controls (Rbm22fl/fl and αMHC-\nMerCreMer+/-) subjected to MI. RBM22 deletion in cardiomyocytes significantly\ninhibited cardiomyocyte proliferation, worsened cardiac function, and increased\nfibrosis post-MI (Figure R2-3a-r).\nnew experiments, which now include data from mice with the Rbm22fl/fl mice, αMHC-\nMerCreMer+ (Cre+/-) mice, and Rbm22-iCKO mice. These new results are included in\nthe revised version of our manuscript (Pages 11-12, lines 197-232, Figure 2; Pages 13-\n14, lines 255-277, Figure 3). Please see more details in the supplementary material\n(Pages 11-12, lines 206-232, Supplementary Fig. 3). We are grateful for your\nsuggestion and believe that these additional experiments not only address your concerns\nbut also enhance the depth and breadth of our study.\nQuestion 2. Inconsistency Across Injury Models. The manuscript uses different\ngenetic models for neonatal and adult injury paradigms. In neonates, the authors\nemploy homozygous Rbm22^fl/fl^ deletion with tamoxifen-inducible αMHC-\nMerCreMer. In adults, they use heterozygous Rbm22^fl/+^ mice with constitutive\nMyh6-iCre, citing embryonic lethality and MerCreMer toxicity. This introduces\ndiscrepancies in timing of gene deletion, dosage, and Cre driver specificity. While\npractical constraints are understandable, the use of divergent models limits\nmechanistic interpretation across developmental stages. The authors should\nacknowledge this limitation clearly and temper their cross-model conclusions\naccordingly.\nResponse: You raised a valid concern regarding the use of different genetic models in\nour neonatal and adult injury paradigms, which could potentially introduce\ndiscrepancies in gene dosage, timing of deletion, and Cre driver specificity. We\nsincerely appreciate this thoughtful comment and fully acknowledge this as an\nimportant consideration.\nIn our initial attempts to generate the inducible Rbm22fl/flαMHC-MerCreMer+\n(Rbm22-iCKO) mice, we used the standard tamoxifen dosage (75 mg/kg) commonly\napplied in the fields outside cardiovascular diseases. However, tamoxifen\nadministration (75 mg/kg) consistently resulted in premature mortality before adult MI\nmodel establishment, preventing us from assessing the capacity of cardiac regeneration\n34\nin Rbm22-iCKO mice. To address this issue, we first conducted our analyses using adult\nRbm22fl/+; Myh6-iCre+ mice, which allowed us to evaluate the functional consequences\nof RBM22 knockdown in the absence of tamoxifen exposure. According to your kind\nsuggestion, we next performed a detailed dose response optimization of tamoxifen in\nadult Rbm22-iCKO mice to determine a safe and effective dosage. Our new\nexperimental results revealed that we injected tamoxifen intraperitoneally on three\nconsecutive days at doses of 0 mg/kg, 30 mg/kg, 40 mg/kg and 60 mg/kg. The results\nrevealed that doses of 60 mg/kg × 3 caused marked systolic dysfunction, inflammation,\nand apoptosis, and even 40 mg/kg × 3 led to detectable myocardial fibrosis. In contrast,\nthe administration of tamoxifen at 30 mg/kg × 3 achieved efficient RBM22 knockout\nin adult cardiomyocytes without overt baseline toxicity (Figure R2-4a-j).\nMerCreMer+ mice\ntamoxifen treatment effectively induced RBM22 deletion in cardiomyocytes. In\naddition, hematoxylin and eosin (H&E) staining showed no apparent morphological\nabnormalities in the hearts after tamoxifen administration in either Rbm22fl/fl mice,\nαMHC-MerCreMer+ mice, or Rbm22-iCKO mice (Figure R2-5a-c).\nFigure R2-5. Low-dose tamoxifen (30 mg/kg) effectively induces RBM22 deletion in\ncardiomyocytes\nUsing this optimized low-dose strategy, we conducted new experiments in adult\nRbm22fl/fl αMHC-MerCreMer+ (RBM22-iCKO) mice and controls (Rbm22fl/fl and\nαMHC-MerCreMer+) subjected to MI. At day 28 post-MI, RBM22 deletion in\ncardiomyocytes led to significantly worsened cardiac function and increased fibrosis.\nIsolated cardiomyocyte analysis revealed a reduction of the absolute number of\ncardiomyocytes and a lower proportion of mononuclear diploid cardiomyocytes\n(Figure R2-6a-i). Immunofluorescence staining further confirmed that RBM22\ndeletion decreased cardiomyocyte proliferation in border zones at day 14 post-MI\n(Figure R2-7a-h).\npost-MI\nTogether, these new data demonstrate that RBM22 deletion aggravates post-MI\nremodeling by suppressing cardiomyocyte proliferation, leading to fibrotic rather than\nregenerative repair. Importantly, by re-establishing comparable genetic backgrounds\nand induction conditions across neonatal and adult models, the revised experiments\nprovide a more consistent and reliable assessment of the molecular and cellular\nmechanisms underlying cardiac repair and regeneration. We have updated the revised\nmanuscript (Pages 13-14, lines 255-277, Figure 3a-r) and supplementary material with\nthese new representative images (Page 13-14, lines 248-262, Supplementary Fig. 4c-\ng).\nWe appreciate your careful attention in identifying this important methodological\nissue and in helping us improve the consistency of our experimental design. The\nadditional experiments and clarification now ensure that the genetic and technical\nconditions are comparable across models, allowing for a clearer interpretation of the\nresults and a more meaningful comparison between developmental stages.\nQuestion 3. Missing Myh6-iCre-Only Control in Adult MI Studies. In the adult\nmyocardial infarction model, the comparison is made between Rbm22^fl/+^ and\nRbm22^fl/+^; Myh6-iCre+ mice, without inclusion of a Myh6-iCre+; Rbm22^+/+^\ngroup. Given that Myh6-iCre is a constitutively active Cre driver, and previous studies\nhave shown Cre expression alone can cause cardiac remodeling or dysfunction, this\nis a critical omission. Without this control, it is not possible to determine whether the\nobserved phenotype results from RBM22 haploinsufficiency or Cre-mediated toxicity.\nThis confound must be addressed experimentally or, at a minimum, clearly\nacknowledged.\nResponse: We appreciate this important comment. As addressed in our response to\nincluded tamoxifen-treated αMHC-MerCreMer+ control mice in both neonatal and\nadult experiments. These additional controls effectively exclude Cre-mediated effects\nand confirm that the observed cardiac phenotypes result from RBM22 deletion rather\nthan Cre-related toxicity. To avoid redundancy, the corresponding results and revised\nfigures are presented in the updated section and figures described in our response to\nyour Question 2. Thank you again for your insightful comments, which have helped us\nto refine our experimental controls and improve the overall rigor and clarity of our study.\nQuestion 4. Insufficient Rigor in Proliferation Assays. The authors assess\ncardiomyocyte proliferation using EdU incorporation, Ki67 immunostaining, and\nphospho-Histone H3 (pH3). While pH3 offers modest specificity for mitotic entry,\nEdU and Ki67 are widely recognized as inadequate for detecting productive\ncardiomyocyte proliferation, as they may reflect DNA synthesis associated with cell\ncycle re-entry, polyploidization, or DNA repair. Even pH3 cannot distinguish\ncytokinesis from abortive mitosis or binucleation. To support the claim that RBM22\ndeletion impairs cardiomyocyte proliferation, additional assays are needed.\nSpecifically, Aurora B kinase or anillin staining should be used to assess cleavage\nfurrow formation. Ideally, this would be validated by genetic lineage tracing (e.g.,\nConfetti, MADM) under αMHC control to directly visualize cardiomyocyte division.\nResponse: We appreciate your insightful comments regarding the rigor of our\nproliferation assays. We fully agree that EdU and Ki67 labeling alone cannot\ndistinguish true cardiomyocyte division from DNA synthesis associated with\npolyploidization or DNA repair, and that phospho-Histone H3 (pH3) provides only\nlimited specificity for mitosis.\nIn response, we performed Aurora B staining, which allows the identification of\ncardiomyocytes undergoing cytokinesis through visualization of the cleavage furrow in\nthe previously acknowledged studies (Circ Res. 2018. PMID: 30355161; Nature. 2019.\nPMID: 31068698). The new results demonstrated that RBM22 deletion markedly\n40\nsuppressed cardiomyocyte proliferation in neonatal AR, neonatal MI, and adult MI\nmodels (Figure R2-8a-b).\nFigure R2-8. RBM22 deletion suppresses cardiomyocyte proliferation in neonatal and\nadult mice after myocardial injury\nConsistently, the proportion of proliferating cardiomyocytes was significantly\nreduced in Rbm22-deficient neonatal hearts. And we observed similar phenomenon in\nisolated neonatal cardiomyocytes treated with Rbm22-specific siRNA (Figure R2-9a,\nb). Conversely, RBM22 overexpression by adeno-associated virus significantly\nenhanced cardiomyocyte proliferation in both neonatal cardiomyocytes (Figure R2-9c)\nand human iPSC-derived cardiomyocytes (hiPSC-CMs) (Figure R2-9d). These new\nfindings further substantiate that RBM22 plays a critical role in promoting\ncardiomyocyte proliferation during cardiac repair and regeneration.\nWe have updated the revised manuscript (Page 12, lines 226-232, Figure 2q,\nPage 14, lines 274-277, Figure 3q, Page 15, lines 292-296, Figure 4c, and Page 30,\nlines 615-617, Figure 8d) and supplementary material with these new representative\nimages (Page 12, lines 230-232, Supplementary Fig. 3k, Page 15-16, lines 303-309,\nSupplementary Fig. 6i, and Pages 26-27, lines 544-547, Supplementary Fig. 12f).\nThank you again for your valuable feedback, which has helped us to strengthen the\nrigor of our proliferation analyses and to improve the overall clarity and impact of our\nstudy.\nQuestion 5. Lack of Rigor in In Vitro Proliferation Assays. The above concern is\nparticularly pertinent to the in vitro experiments, where rigorous quantification of\nproliferation is more tractable. In neonatal mouse cardiomyocytes (NMCMs) and\nhiPSC-derived cardiomyocytes (hiPSC-CMs), proliferation should be assessed using\nvalidated readouts such as dye dilution or cell cycle reporter systems. This is\nespecially important in the hiPSC-CM experiments, which currently rely solely on\nsynthesis related to stress or partial re-entry.\nResponse: We appreciate your insightful comments regarding the rigor of our in vitro\nproliferation assays. To address your concerns, we have strengthened our analysis by\nincorporating additional validated assays to assess cardiomyocyte proliferation.\nIn response to your suggestion, we have incorporated Aurora B staining, which\nprovides strong evidence that cardiomyocytes have entered the cell cycle. Since\ncardiomyocyte proliferation is a key phenotype in our study, we fully acknowledge your\nconcern. Therefore, we conducted additional parallel experiments to further validate\nthis finding. Specifically, we introduced propidium iodide (PI) staining to evaluate cell\ncycle distribution in neonatal mouse cardiomyocytes (NMCMs) following RBM22\nknockdown or overexpression. PI staining represents a classical and widely adopted\nassay for evaluating cardiomyocyte cell cycle status and is routinely employed in many\nhigh-impact studies (Adv Sci (Weinh). 2025. PMID: 39559922; Nat Commun. 2025.\nPMID: 40128196). PI-based cell cycle analysis enabled the discrimination of G0/G1,\nS, and G2/M phases, providing quantitative evidence of cell cycle progression. The\nresults showed that, in control cardiomyocytes, the proportion of cells in the S and\nG2/M phases was higher, indicating active cell cycle progression and proliferative\nactivity. In contrast, RBM22 overexpression significantly increased the proportion of\ncells in these phases, reflecting enhanced cell cycle progression and proliferation\n(Figure R2-10a, b).\nSecond, to further validate the proliferative effect of RBM22, we performed the\nCell Trace Violet (CTV) dilution assay, a widely recognized method for tracking cell\ndivision (Cell. 2024. PMID: 38211590; Nat Immunol. 2020. PMID: 32807943; Mol\nTher. 2024. PMID: 38720457). This assay is based on the same dye-dilution principle\nas carboxyfluorescein succinimidyl ester (CFSE) (Cell Rep. 2018. PMID: 29768213)\nand CellTrace Yellow (CTY) (Circ Res. 2019. PMID: 31648614), commonly used in\nhiPSC-CM proliferation studies. These methods use fluorescent dyes to track cell\ndivision through the dilution of the fluorescent signal as cells divide. CTV analysis\n43\ndemonstrated that RBM22 overexpression significantly promoted cell division in both\nNMCMs and hiPSC-derived cardiomyocytes (hiPSC-CMs) (Figure R2-10c, d).\nTogether, these additional assays provide rigorous and quantitative evidence that\nRBM22 promotes cardiomyocyte proliferation in vitro.\nFigure R2-10. Assessment of RBM22-mediated regulation of cardiomyocyte\nproliferation by PI cell-cycle and CTV dye-dilution assays\nWe have now included this data in the revised manuscript (Page 16, lines 309-311,\nFigure 4d, Page 30, lines 622-625, Figure 8f) and supplementary materials (Page 27,\nlines 547-554, Supplementary Fig. 12g) and added descriptions. Thank you again for\nyour constructive feedback, which has helped us to enhance the methodological rigor\nand scientific robustness of our proliferation analyses.\nSecondary Concerns:\nQuestion 1. Only one siRNA sequence targeting RBM22 appears to have been used.\nGiven the known risk of off-target effects, key phenotypes (e.g., Fig. 4K, L) should be\nvalidated using a second independent siRNA or with orthogonal strategies such as\nResponse: We appreciate your valuable suggestion regarding the potential off-target\neffects associated with using a single siRNA sequence. We would like to clarify that\nthe siRNA used in our experiments was purchased from Dharmacon SMARTpool 4-in-\n1, a widely recognized strategy designed to minimize off-target effects in gene silencing.\nTo enhance the robustness of our findings, we utilized an additional independent siRNA\ntargeting Rbm22 (Santa Cruz, sc-152734; referred to as siRNA-Rbm22 #2) to validate\nour findings. After transfecting cardiomyocytes with siRNA-Rbm22 #2,\nimmunofluorescence staining demonstrated that Rbm22 knockdown significantly\nreduced the proportion of proliferating cardiomyocytes (Figure R2-11a). Furthermore,\nthe expression of downstream cell cycle–related genes was markedly decreased at both\nthe mRNA and protein levels, confirming the inhibitory effect of RBM22 silencing on\ncardiomyocyte proliferation (Figure R2-11b, c). These results verify that the observed\nphenotypes are consistent across independent siRNA sequences, thereby excluding off-\ntarget artifacts and reinforcing the reliability of our conclusions.\nFigure R2-11. RBM22 knockdown with a second siRNA reduces cardiomyocyte\nproliferation and cell cycle-related genes expression\nWe have included these new results in the revised version of our manuscript.\nPlease see more details in the supplementary material (Pages 15-16, lines 303-309,\nSupplementary Fig. 6g-i, Pages 17, lines 336-338, Supplementary Fig. 7e-f). We are\n45\ngrateful for your constructive suggestion and believe that these additional experiments\nnot only address your concern regarding off-target effects but also strengthen the rigor\nand reproducibility of our findings.\nQuestion 2. The authors claim that RBM22 physically interacts with SMARCA4,\nbut do not assess the specificity of this interaction under conditions of RBM22 silencing.\nA co-IP of endogenous proteins in knockdown conditions would help validate the\ninteraction’s dependence on RBM22.\nResponse: We appreciate your insightful suggestion regarding the need to assess the\nspecificity of the RBM22–SMARCA4 interaction under RBM22-silencing conditions.\nIn the previously submitted version of our manuscript, we primarily characterized the\nchromatin-remodeling complexes associated with RBM22, and both endogenous and\nexogenous co-immunoprecipitation (co-IP) experiments demonstrated that RBM22\ninteracts with SMARCA4. Following your suggestion, we performed co-IP assays after\nsiRNA-mediated RBM22 knockdown in cultured cardiomyocytes. The results showed\nthat the interaction between RBM22 and SMARCA4 was markedly reduced upon\nRBM22 silencing, indicating that the association depends on RBM22 expression\n(Figure R2-12a). Furthermore, we isolated cardiomyocytes from Rbm22-iCKO\nneonatal mice, and co-IP analysis similarly revealed a significant reduction in\nSMARCA4 binding after RBM22 deletion (Figure R2-12b). Conversely, when we\noverexpressed RBM22 in adult cardiomyocytes using AAV9-cTnT-Rbm22, co-IP\nassays showed a notable increase in SMARCA4 binding (Figure R2-12c). These\nresults collectively confirm the specificity and RBM22-dependence of the RBM22–\nSMARCA4 interaction.\nFigure R2-12. RBM22–SMARCA4 interaction depends on RBM22 expression\nlevel in cardiomyocytes\n29, lines 599-603, Figure 7n), and additional details are provided in the supplementary\nmaterial (Page 24, lines 487-493, Supplementary Fig. 11a, b). We sincerely appreciate\nyour insightful comment, which prompted us to further validate the RBM22–\nSMARCA4 interaction under different expression conditions.\nQuestion 3. The discussion of RBM22 function in hiPSC-CMs should be more\ncautious. These cells are highly immature, and evidence of increased proliferation or\ndelayed maturation does not imply similar effects in adult cardiomyocytes. While\npossibly beyond the current scope, testing in more mature systems—such as\nengineered heart tissues or in vivo grafts—would help substantiate translational\nrelevance.\nResponse: We appreciate your thoughtful comment regarding the interpretation of\nRBM22 function in hiPSC-CMs. We fully agree that hiPSC-CMs are relatively\nimmature compared with adult cardiomyocytes, and that evidence of enhanced\nproliferation or delayed maturation in these cells should not be directly extrapolated to\nadult cardiac physiology. In the revised manuscript, we have carefully revised the\nDiscussion section to adopt a more cautious tone when describing the findings from\nhiPSC-CMs (Pages 35, lines 729-736). We now clearly state that the results obtained\nin hiPSC-CMs mainly reflect the role of RBM22 in regulating cardiomyocyte cell-cycle\nactivity at an immature stage. Further validation in more mature systems, such as\nengineered heart tissues or in vivo transplantation models, will be required to establish\nthe translational relevance of these findings in our future studies.\nQuestion 4. The discussion on clinical applications is confusing and speculative. The\nauthors point out the impracticality of viral vectors in clinical settings, yet this is the\napproach they demonstrate here. They later refer vaguely to “small molecules and\nsynthetic oligonucleotides designed to target RBM22,” but no candidates or rationale\n47\nare offered. This section should be revised for clarity and realism.\nResponse: We appreciate your constructive comment regarding the discussion of\npotential clinical applications. We agree that our original discussion may have been\noverly speculative and lacked sufficient clarity. In the revised manuscript, we have\nsubstantially rewritten this section to present a more balanced and realistic perspective\n(Page 35, lines 722-736). While our study highlights the clinical potential of RBM22\nas a therapeutic target, which demonstrates that AAV9-mediated delivery of RBM22\nsignificantly delays myocardial fibrosis progression, induces cardiomyocyte cell cycle\nre-entry, alleviates ischemic injury, and improves adverse ventricular remodeling in\nadult mice. However, we did not conduct long-term observations following AAV9-\nRBM22 treatment post-myocardial infarction in adult mice. Consequently, the long-\nterm safety and potential side effects of this treatment remain to be assessed. Further\ninvestigation into more effective and safer strategies for targeting RBM22 is necessary.\nMinor Concerns:\nQuestion 1. The abstract should mention that RBM22 has a previously reported\nnoncanonical transcriptional role, to avoid confusing the readter and/oroverstating\nthe novelty of this finding.\nResponse: We appreciate your insightful comment regarding the need to clarify the\npreviously reported noncanonical transcriptional role of RBM22. We agree that\nacknowledging this background will help avoid confusion and prevent overstating the\nnovelty of our findings. In the revised abstract (Page 1, lines 5-8), we have added a\nsentence noting that RBM22 has been reported to play a noncanonical role in\ntranscriptional regulation in other cellular contexts (Genome Biol. 2024.\nPMID:38641822); however, its involvement in cardiac regeneration has not been\npreviously defined. We then clarify that our study extends this knowledge by\nidentifying RBM22 as a key transcriptional regulator of cardiomyocyte proliferation\n48\nand heart regeneration. This revision provides appropriate context for previous research\non RBM22 and clearly highlights the new contribution of our study to the understanding\nof heart regeneration.\nQuestion 2. Lines 78–81: The text implies that SMARCA4 regulates chromatin’s only\ntwo states. This is a massive oversimplification and should be nuanced.\nResponse: We appreciate your insightful comment highlighting the oversimplified\ndescription of chromatin regulation. We fully agree that depicting chromatin as having\nonly two states (“open” and “closed”) is an inaccurate representation of its highly\ndynamic and context-dependent nature. In the revised manuscript, we have refined this\nsection to emphasize that chromatin accessibility exists along a continuous spectrum\nshaped by nucleosome positioning, histone modifications, and higher-order chromatin\narchitecture. We now clarify that SMARCA4 (also named as BRG1), the catalytic\nATPase subunit of the BAF/SWI-SNF chromatin-remodeling complex, regulates\nchromatin structure in a locus- and cofactor-dependent manner. Specifically,\nSMARCA4 can either promote or restrict transcription by repositioning or evicting\nnucleosomes, thereby modulating accessibility at promoters and enhancers in a context-\nspecific fashion. This revision provides a more accurate and mechanistically grounded\nexplanation of the role of SMARCA4 in chromatin remodeling and transcriptional\nregulation (Page 4, lines 55-64). We appreciate this valuable comment, which helped\nus to refine the mechanistic description and improve the scientific accuracy of the\nmanuscript.\nQuestion 3. Line 93: Please define YAP5SA for non-experts.\nResponse: We appreciate this helpful suggestion. In the revised manuscript, we have\nadded the full name of YAP5SA for clarity. It is now defined as Yes-associated protein\n(YAP) 5SA, a constitutively active form of YAP that has all LATS1/2 phosphorylation\nsites mutated from S to A. This clarification appears in Line 74-75 (Page 5) of the\nrevised manuscript.\nconclusion drawn appears counterintuitive. Please rephrase or clarify.\nResponse: We appreciate your thoughtful comment. We agree that the previous\nwording was imprecise and could have led to confusion. In response, we have revised\nthis section to provide a more accurate and detailed explanation. Specifically, we now\nstate that myocardial tissue samples from patients with cardiomyopathy were analyzed\nto assess RBM22 expression levels, and we found a significant association between\naltered RBM22 expression and pathological cardiac remodeling. Furthermore, we\nhighlight that the increased expression of RBM22 observed in both human and mouse\nmodels is conserved, underscoring the consistency and translational relevance of this\nfinding across species. This revised explanation enhances the logical flow and clinical\nrelevance of the discussion, offering a clearer and more balanced interpretation of the\ndata. The updated text can be found in the revised manuscript (Page 7, lines 123-126).\nQuestion 5. Line 153: Define AR for non-specialist readers.\nResponse: We appreciate your helpful suggestion. In the revised manuscript, we have\nadded the full term for “AR” to improve clarity for non-specialist readers. It is now\ndefined as apical resection (AR), a neonatal cardiac injury model commonly used to\nstudy heart regeneration. This clarification has been included in the revised manuscript\n(Page 6, line 97).\nQuestion 6. Lines 159–160: Single-cell RNA-seq is now a widely accepted standard\nto provide evidence of cells type-specific expression, and multiple published human\nand mouse heart atlases exist that could be referenced or reanalyzed without the need\nfor new data generation.\nResponse: We appreciate your constructive suggestion regarding the use of publicly\navailable single-cell RNA-sequencing (scRNA-seq) datasets to support cell–type–\nspecific expression analysis. We fully agree that scRNA-seq is a powerful and widely\naccepted approach for defining transcriptomic profiles at cellular resolution. In the\n50\nrevised manuscript, we have incorporated and referenced published single-cell heart\natlases (GSE130699) to validate the expression pattern of RBM22 across different\ncardiac cell types. Reanalysis of these datasets confirmed that RBM22 expression is\nenriched in cardiomyocytes, consistent with our own results (Figure R2-13a, b). These\nadditions provide stronger evidence for cell–type–specific expression of RBM22\nwithout the need for additional data generation.\nFigure R2-13. Single-cell analysis of RBM22 cellular localization in cardiac tissue\nThe relevant information and citations have been added in the revised manuscript\n(Page 8, lines 135-137, Supplementary Fig. 1f).\nQuestion 7. Fig. 1G and other immunofluorescence images: please provide grayscale\nindividual channels and use colorblind-friendly merged images (e.g.,\ncyan/magenta/yellow) to ensure accessibility. See: https://www.ascb.org/science-\nnews/how-to-make-scientific-figures-accessible-to-readers-with-color-blindness/\nResponse: We appreciate your thoughtful suggestion regarding the presentation of\nimmunofluorescence images and accessibility for colorblind readers. We fully agree\nthat including grayscale individual channels can improve figure transparency in many\ncontexts, and we offer our sincerest apologies to potential readers with color blindness.\nHowever, the fluorescence images in our study were directly captured using a Leica\nDM3000 microscope, which produces standard RGB (red, green, blue) fluorescence\nchannels without pseudo-color adjustment capability. Therefore, post hoc modification\nof color channels is technically not feasible.\nextremely low, converting the images to grayscale would markedly decrease the\nvisibility of the few positively stained cells. To ensure both clarity and accessibility,\nwe have retained the original RGB color scheme but added yellow arrows to clearly suggest\nthe positively stained proliferating cells. Moreover, we have optimized image contrast\nand labeling to enhance visualization without altering the original data. In addition, we\nhave included statistical data on the right side of the immunofluorescence images to\nreflect the expression of RBM22 and its regulatory role in cardiomyocyte proliferation.\nIt is also worth noting that many studies published in Nature Communications\nand similar journals have adopted RGB fluorescence images for comparable\nexperimental settings (Nat Commun. 2025. PMID: 41068075, Nat Commun. 2024.\nPMID: 39516197, Nat Commun. 2025. PMID: 39837836, Nat Commun. 2024. PMID:\n38459019, Nat Commun. 2025. PMID: 40128196, and Nat Commun. 2025. PMID:\n40087279). While we acknowledge that the RGB color scheme we currently use may\nnot be as accessible for readers with color blindness, we have made efforts to mitigate\nany potential visual barriers by using yellow arrows and providing statistical data. We\nbelieve that our current presentation maintains scientific rigor, visual clarity, and\naccessibility for all readers. We would like to express our sincerest apologies to you\nand the readers once again.\nQuestion 8. Line 172 and Line 371: Terms like “implies” and “suggests” suggest\ncausation. These should be softened to “suggests” or “is consistent with.”\nResponse: We appreciate your careful reading and constructive suggestion regarding\nwording precision. We agree that terms such as “implies” and “indicates” may\ninadvertently suggest causation. In the revised manuscript, we have carefully reviewed\nthe relevant sentences and replaced these expressions with more appropriate, non-\ncausal terms such as “suggests” or “is consistent with” (Page 9, line 151; Page 9, line\n169; Page 12, line 225; Page 15, line 302; Page 16, line 322; Page 18, line 368; Page\n19, line 388; Page 25, line 517; Page 27, line 564). These revisions ensure that our\n52\ninterpretations remain descriptive and evidence-based without overstating causal\ninference. Thank you for helping us improve the linguistic rigor and clarity of the\nmanuscript.\nQuestion 9. Figures S2D and S2E appear contradictory regarding the relationship\nbetween RBM22 and NKX2-5. This discrepancy should be addressed in the relevant\nresults and discussion sections.\nResponse: We appreciate your careful observation and constructive comment\nregarding the apparent inconsistency between Figures S2D and S2E in depicting the\nrelationship between RBM22 and NKX2-5. After re-evaluating the data, we found that\nthe previous Supplementary Fig. 2E could be misleading due to differences in\nexperimental context, as we did not perform embryonic-stage experiments. Therefore,\nwe have removed this panel from the revised manuscript. To further investigate the\npotential regulation between RBM22 and NKX2-5, we integrated publicly available\ntranscriptomic datasets (GSE77547) and found that Nkx2-5 knockout significantly\nreduced RBM22 transcription, suggesting a regulatory relationship between these\nfactors (Figure R2-14b). Additionally, analysis of ChIP-seq data (GSE77548) revealed\nan enrichment of NKX2-5 chromatin signals around the transcription start site of the\nRBM22 gene locus, suggesting its involvement in transcriptional regulation (Figure\nR2-14c).\nFigure R2-14. NKX2-5 binds to the promoter region of RBM22 and modulates its\n53\ntranscription\nThese findings, together with Supplementary Fig. 2d, consistently support a\nregulatory association between NKX2-5 and RBM22. We have incorporated these new\nresults into the revised supplementary materials (Page 10, lines 174-180,\nSupplementary Fig. 2e, f). We believe that the revised presentation improves clarity\nand strengthens the mechanistic rationale of our study. We sincerely thank you for this\nvaluable comment, which helped us to refine our data presentation and reinforce the\nlogical consistency of the manuscript.\nQuestion 10. Figure S2J: Please specify the time point of the analysis.\nResponse: We appreciate your helpful comment. In the revised manuscript, we have\nspecified the time point for the analysis shown in Supplementary Fig. 2k-o\n(corresponding to the original Figure S2J-related experiments) as postnatal day 21\n(P21). This information has been added to both the figure legend and the corresponding\nsection of the Results to ensure clarity.\nQuestion 11. Figure 4D shows an unusually large number of differentially expressed\ngenes (~1/3 to 1/5 of the transcriptome). Could this reflect differences in\ncardiomyocyte vs. non-cardiomyocyte proportions?\nResponse: We appreciate your insightful comment regarding the large number of\ndifferentially expressed genes (DEGs) in Figure 4D. Comprehensive quality control\nconfirmed the robustness of our RNA-seq data, with Q30 scores >94% and sequencing\ndepth of 40–65 million reads per sample. Cardiomyocytes were isolated using a\nstandardized protocol reported previously, yielding comparable purity and viability\nacross groups (Figure R2-15a).\nPCA and sample correlation analyses revealed tight intra-group clustering and\ndistinct separation between si-Ctrl and si-Rbm22 samples, confirming that\ntranscriptional changes reflect genuine RBM22-dependent regulation (Figure R2-15b).\nDNA replication, cell-cycle progression, and chromatin remodeling-consistent with our\nexperimental phenotypes. The relatively high DEG number likely results from the\npooled-sample design, which reduces intra-group variance and enhances statistical\npower. To improve clarity, the scatter plot was replaced with a correlation-based\nvisualization format, which more effectively illustrates the overall transcriptional\ndifferences between groups (Figure R2-15c). Collectively, these results demonstrate\nthat the DEG profile reflects authentic biological regulation rather than differences in\ncell composition or technical artifacts.\nFigure R2-15. RNA-seq analysis of neonatal mouse cardiomyocytes following\nRBM22 knockdown\nWe have included these new results in the revised supplementary material (Page\n16, lines 314-317, Supplementary Fig. 7b). We sincerely appreciate this valuable\ncomment, which prompted us to perform additional validation and clarify the\nrobustness of our transcriptomic analysis.\nQuestion 12. Line 347: The “ZnF deletion” construct removes residues 1–232, which\nincludes far more than the zinc finger motif (residues 159–182). This should be\nclearly acknowledged and discussed in terms of interpretation.\nResponse: We appreciate your insightful comment and fully agree that the ΔZnF\nconstruct (residues 1–232) removes a region larger than the canonical zinc finger motif\n(residues 159–182). We have clarified this point in the revised manuscript and added\nan explanatory note in both the Results and Discussion sections. Specifically, we now\nstate that the deletion extends beyond the zinc finger domain to include part of the N-\nterminal region; however, based on our structural analysis and functional evidence, the\nmajor regulatory activity within this segment is primarily attributed to the ZnF domain\nitself. This clarification does not alter our main conclusion that the integrity of the ZnF-\ncontaining region is essential for the regulatory function of RBM22, but it ensures a\nmore accurate and cautious interpretation of the data (Page 18, lines 362–365). In\naddition, we have revised the labeling in the corresponding figure to ensure clearer and\nmore standardized annotation (Supplementary Fig. 8e, f).\nQuestion 13. Data plotting for CUT&Tag and ATAC-seq data in Fig. 5E and Fig. S7\nlacks conventional visualization. Please include representative read pileups or\ngenome browser snapshots to assess signal and background.\nResponse: We appreciate your constructive suggestion regarding the visualization of\nCUT & Tag and ATAC-seq data. In the revised manuscript, we have included\nrepresentative genome browser snapshots (IGV tracks) for the CUT & Tag and ATAC-\nseq datasets presented in Figure 5e and Supplementary Fig. 9. These new panels\ndisplay normalized read pileups for representative loci, including cell-cycle-related\ngenes such as Cdk4, Ccna2, and Ccne1, and clearly illustrate the differences in\nchromatin accessibility and histone-mark enrichment between P7 and P1\ncardiomyocytes. The signal patterns in these tracks are consistent with the peak-calling\nand differential-enrichment analyses shown in the main figures, thereby validating the\nreproducibility and specificity of the data. The updated visualization enhances\ntransparency and allows readers to directly assess both signal intensity and background\nlevels (Figure R2-16a-d). The new images have been incorporated into the revised\nmanuscript (Figure 5e) and supplementary figures (Supplementary Fig. 9c-e).\nthe promoter regions of cell cycle–related genes\nQuestion 14. Line 416: The phrase “global decrease” is misleading, as only a small\nfraction of promoters are affected. Please rephrase.\nResponse: We appreciate your careful reading and agree that the phrase “global\ndecrease” overstates the extent of promoter changes observed in the CUT & Tag and\nATAC-seq analyses. In the revised manuscript, we have rephrased this statement to\nmore accurately describe the data, now stating that only a subset of promoters exhibited\ndecreased chromatin accessibility and histone-mark enrichment upon RBM22 depletion\n(Page 22, lines 437-438). This modification provides a more precise representation of\nthe results and avoids any implication of genome-wide effects. We thank you for this\nhelpful comment, which has improved the accuracy and balance of our interpretation.\nQuestion 15. Line 462 and Line 597: Terms like “direct interaction” and “directly”\nare too strong based solely on co-IP or EMSA. Please moderate or support with\nadditional biophysical assays.\nResponse: We appreciate your insightful comment regarding the use of the terms\n57\n“direct interaction” and “directly.” We agree that these expressions may overstate the\nevidence provided by co-immunoprecipitation (co-IP) and EMSA assays, which\nsuggest an association but do not conclusively demonstrate direct physical binding.\nIn the revised manuscript, we have moderated the wording throughout the relevant\nsections, replacing “direct interaction” with “interaction” or “association”, and\nclarifying that our data suggest a functional relationship between RBM22 and\nSMARCA4, the precise molecular mechanism of which remains to be further\ninvestigated (Page 24, lines 484-485).\nQuestion 16. The Discussion section could be more concise. Redundant summaries\nof background information should be minimized to make room for a deeper\ncomparison of RBM22 with other known inducers of cardiomyocyte proliferation\n(e.g., YAP5SA, miR-206, etc.).\nResponse: We appreciate your valuable suggestion regarding the structure and focus of\nthe Discussion section. We agree that some portions of the background information\nwere redundant and could be streamlined to allow for a deeper comparative analysis.\nIn the revised manuscript, we have condensed the background content to make the\nDiscussion more concise and focused on the main findings. We have also expanded the\ncomparative discussion by analyzing how RBM22-mediated regulation of\ncardiomyocyte proliferation relates to other known inducers such as YAP5SA, FOXK1\nand FOXK2. Similar to the previously mentioned molecules, RBM22 promotes\ncardiomyocyte proliferation by altering chromatin accessibility. However, it\npredominantly achieves this through a non-canonical mechanism that is independent of\nits traditional role in RNA splicing (Page 31-32, lines 636-660). These revisions provide\na clearer contextual framework and strengthen the mechanistic interpretation of the role\nof RBM22 in cardiac regeneration.\nQuestion 17. Line 598: Clarify whether the authors are referring to the ZnF domain\nor another motif. The RRM appears dispensable, so this interpretation should be\n58\nspecified.\nResponse: We appreciate your careful reading and thank you for pointing out the\nambiguity in our description. We have corrected the statement in Line 598 to specify\nthat the relevant structural region is the Proline-Rich domain, rather than the ZnF\ndomain. The revised text now accurately reflects that the Pro-rich domain is required\nfor the transcriptional regulatory activity of RBM22, whereas the RRM domain appears\ndispensable under the experimental conditions tested (Page 32, lines 668-672). This\nclarification ensures precision in the structural interpretation and avoids confusion with\nthe ZnF motif.\nQuestion 18. Overall, while the manuscript is generally well-written, there are\nnumerous grammatical issues and awkward phrasings throughout. Examples\ninclude\n- Line 48: “attach of myocardial infarction”\n- Line 61: “The previous sstudies show”\n- Line 66: “imperative” (used oddly multiple times)\n- Line 84: “conversed”\n- Line 593: “carcinogenic” (possibly meant to say “oncogenic” or “tumorigenic”)\nResponse: We sincerely appreciate your careful reading and constructive feedback\nregarding the grammatical and phrasing issues throughout the manuscript. We have\nthoroughly re-examined the entire text and corrected all typographical, grammatical,\nand stylistic errors, including those specifically noted. The phrase “attach of myocardial\ninfarction” has been revised to “Following myocardial infarction” (Page 3, lines 26-27).\nThe phrase “The previous studies show” has been changed to “Previous studies have\nshown” (Page 3, line 40). The word “imperative” has been replaced with “crucial”\n(Page 3, line 45). And the word “conversed” has been modified to “conserved” (Page\n4, line 65). Regarding the term “carcinogenic”, in response to Question 16 (Minor\nConcerns), we have removed redundant summaries of background information in the\nrevised manuscript. The “carcinogenic” has been removed from the Discussion section\n59\n(Page 32, lines 663-672). The authors have carefully polished the revised version to\nensure fluency, clarity, and stylistic consistency throughout the manuscript. We believe\nthese revisions have substantially improved the overall readability and precision of the\nmanuscript.\nWe extend our sincere thanks to the reviewers for the insightful comments and\nrecommendations, which have been pivotal in refining our manuscript. We have\ncarefully considered and responded to each query, incorporating the changes into the\nrevised version. We are confident that these amendments have significantly improved\nthe quality of our work.\nReviewer #2:\nQuestion 1. Supplementary Fig. 1f does not clearly support the statement that\n“Rbm22 is predominantly expressed in cardiomyocytes” (line 155). Please include a\nviolin plot or another quantitative visualization to compare Rbm22 expression across\nthe different lineages.\nResponse: Thank you for your thoughtful feedback. We appreciate your suggestion to\ninclude a more quantitative visualization of Rbm22 expression. In response, we have\nadded a bubble plot (Supplementary Fig. 1f, g) comparing Rbm22 expression across\ndifferent lineages. This visualization clearly demonstrates the higher expression of\nRbm22 in cardiomyocytes, supporting our statement that “RBM22 is predominantly\nexpressed in cardiomyocytes”.\nWe have now included this data in the supplementary figures (Page 8, lines 131-\n133, Supplementary Fig. 1g). We believe this additional data enhances the clarity of\nour findings.\nQuestion 2. Lines 568–572: The sentence beginning with “In this assay …” is\nconfusing, as it appears to refer back to the PI-staining experiment while it actually\ndescribes the CTV results introduced later. I recommend inverting the order of these\nsentences to eliminate this ambiguity.\nResponse: Thank you for pointing out this ambiguity in writing. We have revised the\nrelevant description by adding the sentence “We next performed Cell Trace Violet\n(CTV) dilution assays to directly assess cardiomyocyte proliferation” before the phrase\n“In this assay” (Pages 26-27, lines 538-543). This adjustment eliminates the confusion\nand clarifies that the CTV results are being described, rather than referring back to the\nPI-staining experiment.\nQuestion 3. The discussion of other regenerative models is much improved; however,\nseveral of the studies referenced in the text are not accompanied by citations. Please\nadd the key references to support this section.\nResponse: Thank you for your positive comment on the improvement of the discussion\nregarding other regenerative models. We apologize for the omission of citations in the\nDiscussion section. In response, we have added the key references to support those\nstudies discussed (Dev Cell. 2019. PMID: 30773489; Nat Rev Genet. 2009. PMID:\n19274050; Development. 2016. PMID: 27965437; Nat Commun. 2025. PMID:\n40128196). These additions enhance the credibility of the Discussion section and\nprovide proper context for the cited work.\nQuestion 4. Line 742: The phrase “, which demonstrates” reads awkwardly.\n“Demonstrating” would improve readability.\nResponse: Thank you for your suggestion. We have revised it to “demonstrating” as\nrecommended, to enhance the flow of the sentence (Page 34, line 707).\nQuestion 5. In the rebuttal, when describing Supplementary Fig. 7b, the authors\nstate: “The relatively high DEG number likely results from the pooled-sample design,\nwhich reduces intra-group variance and enhances statistical power.” I would\nappreciate clarification on this point. If biological samples were pooled prior to\nsequencing and only technical replicates were generated, this would preclude a\nstatistical assessment of inter-biological variability.\nResponse: Thank you for your valuable comment. To clarify, the number of neonatal\ncardiomyocytes isolated from heart tissues from a single P1 mouse was insufficient for\nRNA sequencing, due to the required cell quantity. To overcome this limitation, we\npooled cardiomyocytes from multiple P1 C57BL/6J mice, cultured and transfected with\nsi-Rbm22 #1 or si-Ctrl #1. After 48 hours, the cardiomyocytes were collected, and RNA\nsequencing was performed. We have revised the Methods section to provide a clearer\ndescription of the sample preparation process for better reader understanding (Pages\n49-50, lines 1044-1047).\nThe use of pooled samples is common in similar studies to overcome limitations\nin cell number. For instance, P3 SD rat cardiomyocytes were pooled, and RNA was\nisolated from these pooled samples for sequencing (Cell Stem Cell. 2022, PMID:\n35395187). In another study, neonatal cardiomyocytes in primary culture were\ntransfected with siRNA against DYRK1A, followed by RNA-seq (EBioMedicine. 2022.\nPMID: 35810562). Similarly, neonatal mouse cardiomyocytes were pooled for BMP2\ntreatment and sequencing (Cell Stem Cell. 2025. PMID: 41205597), and primary\nneonatal mouse cardiomyocytes were treated with versican and subsequently\nsequenced (Circulation. 2024. PMID: 37886839). Furthermore, the researchers used\npooled neonatal cardiomyocytes treated with OSM for RNA sequencing (Circulation.\n2020. PMID: 32600062). To validate the RNA-seq findings, we also performed mRNA\nand protein expression analyses, which confirmed that Rbm22 knockdown (Fig. 4j, k)\nand knockout (Supplementary Fig. 7g, h) led to the decreased expression of key\ncardiomyocyte proliferation and cell cycle genes, consistent with the RNA sequencing\nresults. We hope this clarification addresses your concern and provides a better\nunderstanding of our experimental design.\nQuestion 6. The authors did not implement my recommendation to adjust the\nimmunofluorescence color scheme to make the figures more accessible for color-\nblind readers. This is unfortunate, as this reviewer—and approximately 1 in 12 men\nand 1 in 200 women—cannot fully interpret the images in their current form. The\nuse of yellow arrows helps but does not resolve the issue. The authors state that color\nadjustment is not technically feasible because the Leica DM3000 produces standard\nRGB fluorescence images. I disagree: it is straightforward to convert images to\ngrayscale or to remap color channels (e.g., red to magenta) using freely available\nsoftware such as Fiji/ImageJ. Citing precedent from other papers in the journal does\nnot, in my view, justify maintaining a non-inclusive visualization practice. While I\ndefer to the editor regarding whether the main figures must be revised, I strongly\nrequest that the authors at least provide grayscale versions of all relevant images in\nthe source data.\nResponse: Thank you for your important suggestion. We sincerely apologize for not\nimplementing your previous recommendation to adjust the immunofluorescence color\nscheme for color-blind accessibility. To address this, we have revised all\nimmunofluorescence images by adjusting the color scheme to a red-green color-blind\nfriendly palette (cyan, magenta, and blue). These adjusted images have been provided\nin the Source Data to ensure accessibility for all readers. We hope this revision resolves\nthe concern and improves the accessibility of our figures."}]

Decision letter