YY1/Asprosin/PFKP axis regulates glycolytic metabolic and exacerbates pathological cardiac hypertrophy

Domain: biology
Published: 2026-04-01
Editorial outcome: major_revision

Abstract
In hypertrophic and failing hearts, fuel metabolism is reprogrammed toward enhanced glycolysis. Here, we identify asprosin, an adipokine, as a critical regulator of this process in pathological cardiac hypertrophy. In patients, circulating asprosin levels correlate with NT-proBNP and EF. Cardiomyocyte-specific asprosin overexpression aggravates hypertrophy, abnormal glycolysis, and impairs mitochondrial ATP production in male mice, whereas its deficiency confers protection against TAC- or Ang II-induced remodeling in male/female mice.

Mechanistically, asprosin binds PFKP and inhibits the K48-linked ubiquitination by DTX3L, thereby stabilizing PFKP and driving aberrant glycolysis, PDK4 induction, PDH inhibition, and impaired respiration. Genetic knockdown of PFKP mitigates hypertrophy and fibrosis in male mice, whereas PFKP overexpression abolishes the protective effects conferred by FBN1 knockdown. Moreover, YY1 is identified as a transcriptional activator of asprosin in the hypertrophic hearts of male mice. Here we show that the YY1–asprosin–PFKP–PDK4–PDH axis underlies metabolic remodeling, and we highlight plasma asprosin as a potential early biomarker and therapeutic target for cardiac dysfunction.

Introduction
During the progression of decompensated pathological myocardial hypertrophy, the myocardium undergoes extensive metabolic and structural remodeling. A key feature of this process is the reprogramming of ATP production pathways in cardiomyocytes, characterized by a metabolic shift from fatty acid oxidation (FAO) to increased glycolysis and a reversion to a fetal-like energy metabolism pattern1. While the reactivation of fetal-like metabolic programs may initially serve as an adaptive response, it can ultimately contribute to cardiac dysfunction and accelerate the progression to heart failure (HF)2.

Studies have shown that myocardial glycolytic flux increases in hypertrophic and failing hearts, providing a temporary benefit in maintaining energy supply3-7. However, the contribution of enhanced glycolysis to total ATP production remains limited and is insufficient to compensate the overall decline in ATP levels8-10, as glucose oxidation either remains unchanged or decreases11-14. Furthermore, pathological hypertrophy induced by mechanical overload—such as hypertension, valvular disease, or post-myocardial infarction—leads to sustained upregulation of glycolysis and suppression of FAO, ultimately reducing ATP synthesis capacity. Persistent metabolic disturbances result in a cascade of pathological changes, including impaired oxidative metabolism, increased oxidative stress, insulin resistance, lipid accumulation, and energy deficiency, all of which contribute to the progression of HF3,5,15,16.

Recent studies have identified key molecular regulators of metabolic remodeling in pathological hypertrophy. Ma et al. demonstrated that cardiac-specific deletion of lin28a reduced glycolytic flux by downregulating PCK2, thereby alleviating pathological hypertrophy17. Similarly, Zhou et al. found that cardiomyocyte-specific deletion of ATPIF1 in mice prevented the metabolic shift and inhibited pathological remodeling under chronic stress18. These findings underscore the importance of investigating the mechanisms underlying energy metabolism remodeling in pathological myocardial hypertrophy, which may offer therapeutic targets for preventing disease progression.

Asprosin, a secreted glucogenic adipokine, encoded by exon 65 and exon 66 of the Fibrillin-1 (FBN1) gene, was originally identified in a study by Romer et al. in patients with neonatal progeroid syndrome19,20. It is a C-terminal cleavage product of precursor-fibrillin protein cleaved by furin-mediated and is mainly secreted by white adipose tissue21. There is increasing evidence that asprosin plays a key role in the development of metabolic diseases, such as diabetes, obesity, non-alcoholic fatty liver disease, cancer, myocardial infarction, atherosclerosis, and polycystic ovary syndrome22,23. Previous studies have proved that asprosin activates the cAMP signalling pathway and promotes hepatic glucose release in the liver by interacting with the OLR734 receptor21. Moreover, recent studies have shown that asprosin promotes glycolysis in the testicular cells, providing an energy supply for steroid hormone synthesis and sperm synthesis24.

In summary, asprosin plays an important and complex role in the regulation of glucose metabolism and homeostasis. However, the exact function of asprosin in pathological cardiac hypertrophy and specific mechanisms by which it regulates glucose metabolism are unclear.

In this work, we revealed that asprosin is highly expressed in the pathological hypertrophic model. Meanwhile, uncover a previously unrecognized role of asprosin as a key glycolytic driver of cardiac hypertrophy under pressure overload, as demonstrated through using gain-and-loss of-function studies conducted in vivo and in vitro. Furthermore, asprosin directly disrupted the DTX3L-PFKP interaction and inhibited the K48-linked ubiquitination and proteasomal degradation of PFKP. The stabilization and accumulation of PFKP drove aberrant glycolysis and upregulated PDK4 expression, which in turn inhibited PDH activity via phosphorylation, exacerbated the insufficiency of ATP production, ultimately leading to cardiac metabolic remodeling.

Results
Asprosin participates in the development of cardiac hypertrophy
To examine asprosin's role in pathological cardiac hypertrophy, we measured plasma asprosin levels and found that plasma asprosin levels were significantly higher in hypertensive heart failure (HF) patients than non-heart failure (non-HF) patients (Fig. 1A). Baseline characteristics of the patients are summarized in Table S1. In patients with hypertensive HF, asprosin plasma levels were positively correlated with patient’s NT-proBNP plasma concentrations and negatively correlated with patient’s cardiac ejection fraction (EF) (Fig. 1B, C). Besides, we also observed markedly elevated plasma levels of asprosin in thoracic aortic ligation (TAC) mice (Fig. 1F). Since asprosin is primarily synthesized and released by white adipocytes20,21,25, we also assessed its expression in white adipocytes under the TAC mouse model. qRT-PCR and immunohistochemistry results showed no significant change in asprosin expression in subcutaneous white adipose tissue (Fig. S1A, B). Additionally, western blot and qRT-PCR results showed markedly elevated asprosin expression in TAC mouse hearts (Fig. 1D, E), a finding further confirmed by immunohistochemistry (Fig. 1H). Meanwhile, we observed that asprosin protein was also upregulated in hypertrophic cardiac tissue induced by isoproterenol (ISO) (Fig. 1G). This finding suggests that asprosin may play a role in modulating pathological cardiac hypertrophy.

Primary neonatal mouse-derived cardiomyocytes (NMCMs), Primary neonatal mouse-derived fibroblasts (NMCFs), mouse aortic smooth muscle cells (SMC), and mouse aortic endothelial cells (EC) are the main cell types in cardiac tissue. In the basal state, we observed higher asprosin mRNA expression in NMCMs (Fig. 1I). Interestingly, Ang II treatment significantly increased asprosin expression in NMCMs, but not in other cell types (Fig. 1J-L). Therefore, we hypothesize that in the pathological model of cardiac hypertrophy, asprosin expression is upregulated, predominantly synthesized and secreted by cardiomyocytes.

To evaluate the role of asprosin in pathological cardiac hypertrophy, we overexpressed asprosin in cardiomyocytes by tail vein injection of cardiophilic adeno-associated virus serotype 9 (AAV9-cTnT-Asprosin and AAV9-cTnT- NC) three weeks before TAC surgery in male mice, designating this group as AAV9-Asprosin or AAV9-NC (Fig. S2A). We isolated adult mouse cardiomyocytes using the langendorff perfusion method and confirmed efficient asprosin overexpression in these cells via qRT-PCR and western blot analysis (Fig. S2B-D). The aortic blood flow velocity was significantly increased in the AAV9-TAC group, confirming the success of the TAC model (Fig. S2E). Four weeks after TAC, AAV9-Asprosin TAC mice exhibited marked increases in heart weight/body weight (HW/BW), lung weight/body weight (LW/BW), and heart weight/tibia length (HW/TL) ratios compared to AAV9-NC TAC mice (Fig. 1N). Two independent batches of echocardiographic analysis revealed that cardiomyocyte-specific overexpression of asprosin exacerbated cardiac dysfunction, evidenced by decreased EF and FS, along with increased LVIDd and LVIDs, diastolic posterior wall thickness (LVPWd), and systolic posterior wall thickness (LVPWs) (Fig. 1M&O and Fig. S2F-G). Cardiac morphology and hematoxylin and eosin (H&E) staining revealed that AAV9-Asprosin TAC mice had larger heart sizes and more severe inflammation (Fig. 1P, Q). Masson’s trichrome staining revealed left ventricular interstitial more fibrosis in the heart of AAV9-Asprosin TAC mice (Fig. 1R, S). Wheat germ agglutinin (WGA) staining showed that these mice also had significantly larger cardiomyocyte cross-sectional areas compared to AAV9-NC TAC mice (Fig. 1T, U). Moreover, the expression levels of cardiac hypertrophy markers atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC), as well as fibrosis markers collagen type I alpha 1 (Col1α1), collagen type I alpha 3 (Col3α1), and alpha-smooth muscle actin (α-SMA), were further elevated in the AAV9-Asprosin TAC mice (Fig. 1V, W).

Consistently, we further investigated whether asprosin alone could induce cardiomyocytes hypertrophy. NMCMs were transfected with asprosin or vector overexpression plasmids, and immunofluorescence results demonstrated that asprosin overexpression significantly induced NMCMs hypertrophy (Fig. S3A-C). Besides, asprosin overexpression upregulated the expression level of ANP, BNP, and β-MHC genes (Fig. S3D, E). Given that asprosin is generated by the cleavage of precursor-fibrillin-1 (FBN1) by activated furin, we aimed to exclude the potential influence of fibrillin-1 on cardiomyocytes hypertrophy. By transfecting NMCMs with the FBN1 overexpression plasmid, we observed that overexpression of wild-type full-length FBN1 significantly induced NMCMs hypertrophy (Fig. S4A, B). However, treatment with the furin inhibitor BOS-318 reversed this effect (Fig. S4B). In contrast, transfection with an FBN1 mutant plasmid lacking the asprosin-encoding region (Flag-FBN1-del-asprosin) did not induce hypertrophy in NMCMs (Fig. S4C, D). These findings indicate that asprosin, rather than FBN1 itself, is the critical factor responsible for inducing cardiomyocytes hypertrophy. In conclusion, these data indicate that cardiomyocyte-specific overexpression of asprosin exacerbates pathological cardiac hypertrophy in vivo and in vitro.

Asprosin overexpression specifically in cardiomyocytes does not induce pathological cardiac hypertrophy in sham-operated mice.
In our study, we overexpressed asprosin in cardiomyocytes by tail vein injection of cardiophilic adeno-associated virus serotype 9 (AAV9-cTnT-Asprosin and AAV9-cTnT-NC) three weeks before Sham surgery in mice, designating this group as AAV9-Asprosin or AAV9-NC (Fig. S5A). The efficiency of asprosin overexpression in isolated cardiomyocytes from adult mice was verified by western blot (Fig. S5B). Unexpectedly, we observed that, compared with the Sham+AAV9-NC group, mice in the Sham+AAV9-Asprosin group exhibited no significant impairments in cardiac function or in the heart weight-to-body weight ratio (Fig. S5C-E). Furthermore, H&E/Masson’s staining results demonstrated that cardiomyocyte-specific overexpression of asprosin did not significantly promote cardiac hypertrophy, inflammation, or fibrosis in sham-operated mice (Fig. S5F-I). Above all, we further observed that cardiomyocyte-specific overexpression of asprosin had no significant hypertrophic effect on cardiomyocytes of sham-operated mice (Fig. S5J, K). Collectively, our results demonstrate that cardiomyocyte-specific overexpression of asprosin alone does not suffice to induce pathological cardiac hypertrophy in sham-operated mice.

Cardiomyocyte-specific asprosin deficiency prevents TAC-induced cardiac hypertrophy and fibrosis
To further evaluate the protective role of asprosin deficiency in pathological cardiac hypertrophy, we specifically delivered adeno-associated virus serotype 9 (AAV9-cTnT-shFBN1 and AAV9-cTnT-shNC) via tail vein injection to knock down FBN1 in cardiomyocytes, thereby achieving asprosin deficiency (Fig. 2A-C). As anticipated, cardiomyocyte-specific asprosin deficiency significantly alleviated TAC-induced pathological cardiac hypertrophy, cardiac insufficiency, and myocardial fibrosis compared to AAV9-shNC TAC mice (Fig. 2D-N). Furthermore, the expression of endogenous asprosin in NMCMs was silenced using small interfering RNAs (siFBN1) (Fig. S6A, B) . After 48 hours of Ang II treatment, we observed marked hypertrophy of NMCMs accompanied by the expression level upregulation of ANP, BNP and β-MHC, whereas asprosin knockdown partially normalised this effect (Fig. S6C-E). Both in vivo and in vitro results highlight the crucial role of asprosin deficiency in mitigating pathological cardiac hypertrophy

Asprosin enhances glycolytic flux of NMCMs
Given asprosin's essential role in pathological cardiac hypertrophy, we aimed to elucidate its molecular mechanisms in regulating this condition. RNA-seq analysis was performed on the heart tissues of AAV9-Asprosin TAC and AAV9-NC TAC mice to profile gene expression differences. The transcriptomic profiles were categorized into two clusters through unsupervised hierarchical clustering analysis (Fig. S7A). A total of 1,329 up-regulated and 1,234 down-regulated differentially expressed genes were identified in the TAC+AAV9-Asprosin group compared to the TAC+AAV9-NC group (defined by P value < 0.05; Fig. 3A and Fig. S7B). Pathway enrichment analysis of the RNA-seq differential genes was performed using Kyoto Encyclopedia of Genes and Genomes (KEGG) database, which showed that the significantly up-regulated pathways were metabolic pathways, ubiquitin-mediated proteolysis and hypertrophic cardiomyopathy (HCM) (Fig. 3B). Further pathway and gene set enrichment analysis of the differentially expressed metabolic genes revealed extensive alterations across several metabolic processes, including carbon metabolism, central carbon metabolism in cancer and glycolysis/gluconeogenesis pathways (Fig. 3C). Mechanical overload-induced pathological hypertrophy results in an increase in myocardial glycolytic flux, which helps maintain cardiac energy supply in the short-term3-7. However, prolonged continuous declines in FAO and increase glycolysis lead- to metabolic disturbances that progressively reduce mitochondrial oxidative metabolism, exacerbate oxidative stress, and ultimately diminish ATP synthesis capacity, contributing to heart failure3,5,15,16. Next, to investigate whether asprosin might be involved in the regulation cardiac hypertrophy by influencing glycolytic in cardiomyocytes. We extracted genes related to HCM and glycolysis/gluconeogenesis from the KEGG enrichment analysis and compared their expression levels between the two groups. Genes associated with HCM and glycolysis/gluconeogenesis were significantly upregulated in the heart tissues of AAV9-Asprosin TAC mice (Fig. 3D, E). In addition, we further verified these differential expression transcripts by qRT-PCR assays (Fig. 3F).

To directly examine the role of asprosin in regulating glycolysis, we measured metabolic flux in adult mouse cardiomyocytes by assessing the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). The ECAR and OCR results demonstrated that asprosin overexpression in cardiomyocytes significantly increased glycolytic activity in TAC mice while concurrently impaired mitochondrial respiratory and ATP production capacity (Fig. S7C, D). In particular, these findings were further validated in an in vitro Ang II-induced NMCMs pathological hypertrophy model (Fig. 3G, H). Simultaneously, we further found that asprosin overexpression alone increased glycolytic capacity while decreasing respiratory capacity and ATP production in NMCMs (Fig. S3F&H). And, forced expression of asprosin significantly enhanced extracellular glucose uptake, glut1 expression, lactate secretion and reduced cardiomyocytes ATP contents (Fig. S3I-K &G). Excessive glycolysis induces mitochondrial dysfunction by promoting superoxide overproduction, oxidative stress, and metabolic imbalance26-30. Asprosin overexpression significantly decreased the mitochondrial membrane potential, as indicated by tetramethylrhodamine ethyl ester (TMRE) staining in NMCMs (Fig. S3L). On the contrary, knockdown of asprosin produced opposite effects in Ang II-treated NMCMs (Fig. S6F-I). To futher verify that asprosin directly regulated glycolytic and promoted cardiomyocyte hypertrophy, we used the glycolysis inhibitor 2-DG to inhibit cardiomyocyte glycolysis. Blocking glycolysis notably eliminated the hypertrophic effects induced by asprosin (Fig. S3M). Taken together, these findings suggest that asprosin up-regulates glycolytic flux in cardiomyocytes, thereby disrupting normal mitochondrial ATP synthesis and contributing to the development of pathological cardiac hypertrophy.

Asprosin directly interacts with PFKP
To investigate the downstream target proteins that interact with asprosin to regulate glycolytic in NMCMs, we performed co-immunoprecipitation (CO-IP) combined with mass spectrometry (MS) to identify asprosin-associated proteins in NMCMs (Fig. 4A). A total of 134 potential target proteins interacting with asprosin were identified after excluding non-specific binding from negative control IgG (Fig. 4A and Table S4). KEGG database analysis of the interacting proteins showed significant enrichment of asprosin's interactome in cell growth and death, energy metabolism, and carbohydrate metabolism pathways (Fig. S8A). Among these, two glycolytic rate-limiting enzymes, PFKP and LDHA, were found to potentially interact with asprosin (Fig. S9A, B). Notably, PFKP’s protein abundances and unique peptides showed significantly higher than LDHA (Table S4). Therefore, we hypothesized that PFKP may be a potential target through which asprosin regulated glycolytic in cardiomyocytes. As expected, the specific interaction between asprosin and PFKP through immunofluorescence colocalization assays and both endogenous and exogenous immunoprecipitation were verified (Fig. 4B-D&F). In addition, His-tag pull-down assay confirmed the direct interaction between asprosin and PFKP (Fig. 4E). Furthermore, CO-IP assays confirmed the specific interaction between asprosin and PFKP, effectively excluding the possibility of binding to other PFK1 subtypes, specifically PFKM/PFKL (Fig. 4G, H).

PFKP consists of two domains: the N-terminal catalytic PFK domain 1 and the C-terminal regulatory PFK domain 2 (Fig. 4I). To determine which domain bound to asprosin, we constructed two PFKP truncations. Through co-transfecting HEK-293T cells with Flag-Asprosin and HA-PFKP truncated overexpression plasmid, the PFKP D1 structural domain was found to bind specifically to asprosin (Fig. 4J). Molecular docking results revealed that amino acids 2756, 2758, 2766, 2768, 2779, 2780, and 2871 of the asprosin protein form interactions with amino acids 196, 199, 303, 304, 307 of the PFKP D1 structural domain (Fig. S8B). Therefore, we mutated the amino acids of the predicted binding site of asprosin. Co-IP assays suggested that the binding ability of asprosin to PFKP was significantly abolished in Flag-Asprosin-MUT group (Fig. 4L). These results demonstrate that asprosin directly interacts with PFKP.

Asprosin inhibits PFKP ubiquitination degradation by competitively inhibiting DTX3L-PFKP interaction
To explore how asprosin induces cardiomyocyte hypertrophy through the regulation of PFKP expression, we assessed PFKP expression levels by overexpressing asprosin in NMCMs. Western blot and qRT-PCR results indicated that asprosin overexpression enhanced PFKP protein levels without altering mRNA levels (Fig. 5A and Fig. S11A, B). Even more important, we observed that asprosin overexpression did not significantly alter the expression levels of PFKM or PFKL (Fig. S10A, B), thereby supporting the hypothesis that asprosin specifically targets and regulates PFKP expression to participate in the development of pathological cardiac hypertrophy. It was reported that USP5 promoted triple-negative breast cancer progression by activating aerobic glycolysis through PFKP deubiquitination31. In addition, KEGG analysis of RNA-seq data revealed that asprosin affected the ubiquitin-mediated proteolysis pathway (Fig. 3B). Hence, we supposed that asprosin may upregulate PFKP protein expression by inhibiting PFKP ubiquitin proteasomal degradation in NMCMs. To test this hypothesis, we inhibited protein translation by treating NMCMs with actinomycin ketone (CHX) and determined PFKP protein level and stability by western blot. The results showed that asprosin overexpression significantly prolonged the half-life of PFKP protein degradation (Fig. 5B and Fig. S11C). Moreover, MG132 treatment abolished the effect of asprosin overexpression on PFKP protein levels (Fig. 5C and Fig. S11D). CO-IP assays demonstrated that asprosin overexpression reduced PFKP ubiquitination in both NMCMs and HEK-293T cells (Fig. 5D, E). However, the decrease in PFKP ubiquitination resulting from asprosin overexpression was not observed in the Asprosin-mut group (Fig. 5F). The above results suggest that asprosin maintains PFKP protein homeostasis by regulating its ubiquitin-proteasomal degradation.

Ubiquitination is a crucial and dynamic multifaceted protein post-translational modification involved in the development of common cardiac diseases and typically requires the involvement of multiple E3 ubiquitin ligases32. Since asprosin does not function as an E3 ubiquitin ligase, we hypothesized that ubiquitinating or deubiquitinating enzymes mediated the post-translational modification of PFKP. NMCMs were transfected PFKP overexpression plasmid and conducted IP-MS experiments to identify E3 ubiquitin ligases interacting with PFKP. Among the candidate targets, DTX3L, a member of the DELTEX family with a RING domain, was the only E3 ubiquitin ligase that interacted with PFKP in NMCMs (Table S5 and Fig. S9C). We co-transfected HEK-293T cells with PFKP and DTX3L overexpression plasmids. CO-IP assays demonstrated that DTX3L was co-immunoprecipitated with PFKP and vice versa (Fig. 5G). Overexpression of DTX3L significantly decreased PFKP protein levels in NMCMs and HEK-293T cells (Fig. 5H, I and Fig. S11E, F) and accelerated PFKP protein degradation (Fig. 5J and Fig. S11H). However, qRT-PCR results indicated that DTX3L overexpression did not affect PFKP mRNA expression levels (Fig. S11G). Additionally, MG132 treatment reversed DTX3L overexpression-induced PFKP protein degradation and maintained protein homeostasis in NMCMs (Fig. 5K and Fig. S11I). Moreover, the results showed that DTX3L significantly increased the PFKP ubiquitination level (Fig. 5L). Apart from this, CO-IP result also showed that PFKP ubiquitination was upregulated to a level comparable to that of Myc-Ub in cells expressing Myc-K48, but DTX3L had no significant effect on PFKP ubiquitination in cells expressing Myc-K63 (Fig. 5M). In addition, western blot assay revealed a significant upregulation of PFKP levels under pathological cardiac hypertrophy stimulation (Fig. S12A, B). Consistently, PFKP protein levels were significantly increased in the hearts of TAC+AAV9-shNC model mice, whereas this effect was reversed in the hearts of TAC+AAV9-shFBN1 mice (Fig. S12C). E3 ubiquitin-linking enzymes catalyze the covalent binding of substrates and ubiquitin molecules primarily through their active sites33. The active site residues C569 and C572 of DTX3L, located in the Ring structural domain, were evolutionarily conserved across species (Fig. 5N). Therefore, we constructed the C569S and C572S mutants of DTX3L (replacing cysteines at positions 569 and 572 with serines), which lacked ubiquitinating enzyme activity34. The DTX3L-MUT variants failed to promote PFKP ubiquitination compared to the wild-type DTX3L (Fig. 5O) and lost their regulatory effect on PFKP protein levels (Fig. 5P and Fig. S11J). Given that asprosin does not directly affect PFKP ubiquitination, we hypothesized that asprosin competed with DTX3L for binding to PFKP. As shown in Fig. 5Q, R and Fig. S11K, overexpression of asprosin significantly inhibited the interaction between DTX3L and PFKP, and reduced DTX3L-induced ubiquitination of PFKP. Collectively, these results suggest that asprosin directly interacts with PFKP by disrupting DTX3L binding, thereby inhibiting PFKP ubiquitination and stabilizing its proteostasis.

Cardiomyocyte-specific knockdown of PFKP alleviates TAC-induced aberrant cardiac glycolysis and pathological hypertrophy.
Next, to further clarify whether targeted intervention with PFKP can protect mice from TAC-induced aberrant cardiac glycolysis and pathological hypertrophy. AAV9-cTnT-shPFKP and AAV9-cTnT-shNC virus were administered via the tail vein to specifically knockdown PFKP in cardiomyocytes of 8-week-old C57BL/6 (wild type (WT) male mice, followed by a 4-week TAC challenge (Fig. 6A-C). As anticipated, cardiomyocyte-specific PFKP deficiency significantly alleviated TAC-induced pathological cardiac hypertrophy, cardiac dysfunction, and fibrosis compared to AAV9-shNC TAC mice (Fig. 6D-M). Correspondingly, the result revealed that glucose and lactate levels were lower in AAV9-shPFKP TAC mice than in AAV9-shNC TAC mice. (Fig. 6N, O).

We further investigated the specific role of PFKP in regulating NMCMs hypertrophy. Notably, PFKP overexpression significantly promoted NMCMs hypertrophy, increased glycolytic capacity while decreasing respiratory capacity, enhanced NMCMs extracellular glucose uptake, glut1 expression levels, lactate secretion, and decreased ATP content and mitochondrial membrane potential (Fig. S13A-N). However, knockdown of PFKP completely reversed Ang II-induced cardiomyocyte hypertrophy (Fig. S14A-D). The expression of genes related to cardiac hypertrophy mRNA and protein levels were upregulated by Ang II-treated in cardiomyocytes, which was abrogated by silencing PFKP (Fig. S14C&E). As shown in Fig. S14F and S14G, knocking down of PFKP restrained extracellular glucose uptake and lactate secretion in NMCMs induced by Ang II. TMRE experiments demonstrated that knockdown of PFKP protected against mitochondrial membrane potential decrease under Ang II treatment (Fig. S14H). Conversely, PFKP knockdown rescued the Ang II-induced ATP deficit in NMCMs (Fig. S14I). In summary, the in vitro and in vivo studies demonstrate that silencing PFKP expression not only directly suppresses the upregulation of glycolysis in NMCMs but also restores mitochondrial ATP production, consequently mitigating cardiac hypertrophy.

PFKP promotes pathological cardiac hypertrophy through activation of the PDK4-PDH signaling axis.
In pressure overload-induced cardiac hypertrophy, a hallmark metabolic alteration is the diminished capacity for mitochondrial pyruvate oxidation3,35-38. This process is predominantly regulated by the pyruvate dehydrogenase kinase (PDK) – pyruvate dehydrogenase (PDH) axis39. Activated PDK, especially the PDK4 isoform, inhibits the PDH activity, blocking the decarboxylation of pyruvate to acetyl-coenzyme A entry into the TCA cycle40. This leads to mitochondrial dysfunction and energy metabolism disturbances, contributing to pathological cardiac hypertrophy41-43. To investigate the mechanism by which PFKP overexpression promotes pathological cardiac hypertrophy, we assessed the phosphorylation status of PDK4 and its downstream target, PDH. As shown in Fig. S13O and P, PDK4 protein levels were significantly increased in NMCMs transfected PFKP-overexpressing plasmid, accompanied by a marked rise in PDH phosphorylation, indicating suppressed PDH enzymatic activity (Fig. S13O, P). This finding was further validated by PDH activity assay using a commercial enzyme kit (Fig. S13Q). Furthermore, we measured the levels of pyruvate, acetyl-CoA, and ATP in NMCMs. The results demonstrated that PFKP overexpression significantly increased pyruvate accumulation while reducing acetyl-CoA synthesis, thereby impairing TCA cycle-dependent ATP production (Fig. S13R, S&I). Conversely, we observed that PFKP knockdown significantly suppressed Ang II-induced upregulation of PDK4 expression and PDH phosphorylation, reduced pyruvate accumulation, and enhanced acetyl-CoA synthesis, thereby facilitating its entry into the TCA cycle for ATP production (Fig. S14I-O). These findings align with the established mechanism of metabolic remodeling in cardiac hypertrophy, whereby pathological stimuli activate upstream signaling pathways (e.g., HIF-1α or FOXO) to induce PDK expression, leading to PDH inactivation and impaired pyruvate utilization in the tricarboxylic acid cycle44-50.

DCA inhibits PDK4 activity, thereby allowing PDH to be in its unphosphorylated active form and promotes pyruvate metabolism via the TCA cycle39. Our results showed that DCA treatment markedly alleviated pathological hypertrophy, mitochondrial membrane potential disruption, and impaired cellular respiration induced by PFKP overexpression (Fig. S13T–W). These findings show that PFKP drives pathological cardiac hypertrophy primarily via PDK4-PDH axis-mediated mitochondrial dysfunction, ultimately inducing cardiac metabolic remodeling.

Asprosin induces cardiac hypertrophy by regulating glycolytic through PFKP
To further clarify the regulating glycolytic processes in cardiomyocytes effect of asprosin in vivo and whether such effect is mediated by PFKP, cardiotropic recombinant adenovirus-associated viruses 9 containing PFKP overexpression plasmid (AAV9-cTnT-PFKP) were established and designated as AAV9-PFKP. AAV9-cTnT-shFBN1 and AAV9-cTnT-shNC viruses were via the tail vein injected with or without AAV9-cTnT-PFKP, AAV9-cTnT-NC virus three weeks before administration of TAC (Fig. S15A-D). As foreseen, AAV9-shFBN1 TAC mice significantly inhibited TAC-induced cardiac hypertrophy and recovered impaired cardiac function compared to AAV9-shNC TAC mice. Conversely, these protective effects were completely lost with the addition of PFKP (Fig. 7A-D and Fig. S15E). Masson’s staining showed that cardiomyocyte-specific knockdown FBN1 significantly attenuated left ventricle interstitial collagen deposition of TAC mice, however, it did not have an ameliorative effect on AAV9-shFBN1+AAV9-PFKP TAC mice (Fig. 7E, F). Consistent with these results, the knockdown of FBN1 attenuated the TAC-induced cardiomyocyte cross-sectional areas increase, and this inhibition was effectively restored by the addition of AAV9-PFKP (Fig. 7G, H). qRT-PCR results showed that knockdown of FBN1 restrained the TAC-induced mRNA expression levels upregulation of ANP, BNP, β-MHC, Col1α1, Col1α3, and α-SMA. Nevertheless, these regulatory changes were attenuated by the addition of AAV9-PFKP (Fig. 7I). The results showed that glucose and lactate levels in the AAV9-shFBN1 TAC group were significantly lower than AAV9-shNC TAC group, however, this effect was reversed by the addition of PFKP (Fig. 7J, K). As shown in Fig. S17A-B, cardiomyocyte-specific deficiency of asprosin significantly ameliorated TAC-induced glycolytic dysfunction and impaired respiratory capacity in adult male mice cardiomyocytes. This protective effect is markedly reversed by additional PFKP overexpression (Fig. S17A-B).

Asprosin modulates pathological cardiac hypertrophy via the PFKP-PDK4-PDH signaling axis.
Subsequently, in vitro experiments showed that the knockdown of FBN1 inhibited Ang II-induced cellular hypertrophy in NMCMs, but overexpression of PFKP reversed this protective effect (Fig. S16A, B&D). ECAR and OCR results showed that knockdown of FBN1 abolished Ang II-induced decreasing respiratory and ATP production capacity and upregulated glycolytic capacity of NMCMs. However, these effective changes were abolished by the addition of PFKP (Fig. S17C, D). As shown in Fig. S16C&E-G, FBN1 knockdown inhibited Ang II-induced glucose uptake, glut1 levels and lactate secretion of NMCMs, and this effect was restored by the addition of PFKP. Moreover, TMRE and ATP content results supported the same experimental trends (Fig. S16H, I). To further elucidate the specific molecular mechanism through which asprosin modulates mitochondrial function and contributes to pathological cardiac hypertrophy, we assessed the expression levels of PDK4 and pPDH in NMCMs transfected with siFBN1 and PFKP overexpression plasmids. As shown in Fig. S18A–D, FBN1 silencing significantly suppressed Ang II-induced upregulation of PDK4 and phosphorylation of PDH, accompanied by an enhancement of PDH activity (Fig. S18A–D). This led to a reduction in pyruvate accumulation, mitigation of acetyl-CoA deficiency, and an increase in ATP content. Importantly, these beneficial effects were largely abolished by concurrent PFKP overexpression (Fig. S18B–D and S16I). Furthermore, qRT-PCR and western blot analyses revealed that DCA treatment ameliorated asprosin-induced pathological hypertrophy in NMCMs, concomitant with increased ATP production, enhanced PDH activity, and restoration of pyruvate metabolism and acetyl-CoA synthesis (Fig. S18E–K). Taken together, these data collectively indicate that the detrimental effects of asprosin on cardiac hypertrophy are primarily underpinned by the dysregulation of the PDK4-PDH axis in a PFKP-dependent manner.

YY1 regulates asprosin expression and mediates cardiac hypertrophy
To explore the mechanism behind the asprosin expression upregulation induced by hypertrophic stimulation, we used the CHIP-Atlas (https://chip-atlas.org/), JASPAR (https://jaspar.elixir.no), and TFDB (http://bioinfo.life.hust.edu.cn/AnimalTFDB4/) databases to predict transcription factors that potentially bind to FBN1 promoter. We identified two intersecting transcription factors (Fig. 8A) that were implicated in pathological cardiac hypertrophy.51,52 Based on predicted FBN1 promoter binding sites from the JASPAR, we performed chromatin immunoprecipitation (CHIP) analysis and designed primers to amplify these regions. As shown in Fig. 8B and 8C, YY1 bound to the FBN1 promoter at the -1022 to -1011 bp, while KLF4 does not bind to any site in the FBN1 promoter. Likewise, YY1 overexpression significantly enhanced transcriptional activity of the FBN1 promoter, however, mutations of the predicted binding site (-1022 to -1011 bp) significantly abolished the effect of YY1 on the FBN1 promoter transcriptional activity (Fig. 8D, E). More importantly, YY1 protein expression level was upregulated in the hearts of TAC model mice and Ang II-treated NMCMs (Fig. 8F, G), and YY1 overexpression significantly promoted asprosin expression (Fig. 8H, I). To further investigate the role of YY1 in mediating the regulatory effects of asprosin on pathological cardiac hypertrophy, we constructed a smart silencer (siYY1) to inhibit its endogenous expression in NMCMs. Western blot and qRT-PCR results showed that asprosin overexpression promoted cardiac hypertrophy marker expression, however, these regulatory changes were attenuated by the addition of siYY1 (Fig. 8JN, K). At the same time, as shown in Fig. 8L, knockdown of YY1 significantly inhibited cell hypertrophy induced by asprosin. Overall, YY1 acts as an upstream regulator of asprosin, promoting its role in pathological cardiac hypertrophy.

Cardiomyocyte-specific asprosin deficiency ameliorates TAC-induced pathological cardiac hypertrophy in female mice.
To assess the generality of our findings, we extended our investigation to female mice. As shown in Fig. S19A–C, asprosin expression in both cardiac tissue and serum samples increased in a time-dependent manner in female mice subjected to TAC (Fig. S19A–C). To determine whether the cardioprotective effects of asprosin knockdown are sex-dependent, we subsequently subjected female mice with successful cardiac asprosin silencing – achieved via cardiac-specific AAV9-mediated FBN1 knockdown three weeks prior – to TAC for a period of four weeks (Fig. S19D). Notably, asprosin knockdown similarly conferred robust protection against TAC-induced cardiac dysfunction, pathological hypertrophy and remodeling in female mice, demonstrating that its beneficial effects are not sex-specific (Fig. S19E–O).

Methods
Human clinical samples
The use of human plasma samples of 47 subject for this study (collected from June to August 2024) was approved by the Ethics Committee Institutional Review Board (IRB) of Harbin Medical University's College of Pharmacy (IRB5063S724) and provided by the Harbin Medical university's Second Affiliated Hospital. All participants complied with the rules guiding human experimentation in the Declaration of Helsinki and gave informed consent in writing. All participants participated in the study process without compensation.
According to the 2018 Chinese Heart Failure (HF) Diagnosis and Treatment Guidelines, plasma samples were collected from hypertensive HF patients with cardiac hypertrophy and normal healthy adults for experimental gene expression analysis. Inclusion criteria for patients with HF were as follows: (1) aged 18 – 85 years old are restricted to individuals with high blood pressure, regardless of gender; (2) NT-proBNP greater than 125 ng/L was accompanied by left ventricular hypertrophy or a ventricular septal thickness greater than 11 mm and diastolic dysfunction. Exclusion criteria were as follows: (1) patients with severe liver and kidney dysfunction combined with malignant tumors. (2) female patients with polycystic ovary syndrome or ovarian cancer, and male patients with infertility. (3) patients with anorexia nervosa.

Animals
To account for potential sex-based differences in the study of pathological cardiac hypertrophy and given that female mice typically exhibit more pronounced disease progression compared to males, this study included male and female C57BL/6 mice aged 8 to 10 weeks, purchased from Liaoning Changsheng Biotechnology Co., Ltd (Benxi, China). The neonatal mice (1- to 3-day-old) were purchased from the Experimental Animal Center of The Second Affiliated Hospital of Harbin Medical University (Harbin, China). Mice were housed in ventilated cages under a temperature (20 – 24°C) and humidity (40 – 60%) with a 12h light/dark cycle and were fed with commercial formula feed satisfying the standard mice nutritional requirements and water ad libitum. All the mice used in present study were randomly assigned to experimental groups via a computerized, body weight-based allocation procedure, and all investigators involved in the surgical procedures, cardiac function assessments, and data analysis were blinded to both the genotype and treatment conditions of the animals. No mice were excluded from the study unless died. All animal experiment protocols complied with the guiding principles for the care and use of laboratory animals at Harbin Medical University and were approved by the Ethics Committee for Animal Experimentation (IRB5045724). Experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (U.S. NIH) and the ARRIVE guidelines.

Isolation of cardiomyocytes from adult mice
Adult mouse cardiomyocytes were isolated using the lengendorff perfusion method as previously described76. In brief, heart was exposed first, and EDTA (MA0232, Meilunbio, Dalian, China) buffer is injected steadily into the right ventricle after cutting of descending artery. The heart then was removed from the mice, perfused with EDTA buffer and collagenase buffer. After digestion completed, cardiac myocytes may be collected by gravity settling. The identification of isolated adult mouse cardiomyocytes was performed using α-actinin (GTX11007, Genetex, Texas, USA, 1: 300) immunofluorescence staining.

Mouse TAC model and echocardiography
Male/femal C57BL/6 (8 – 10 weeks old) mice were anesthetized by a single intraperitoneal injection of sodium pentobarbital (60 mg/kg) (P3761, Sigma-Aldrich, St. Louis, MO, USA) followed by TAC surgery. In brief, the mice’s chest cavities were opened, and the transverse aorta was ligated using a 7-0 silk suture wire and a 27-gauge needle in a transverse direction. The needle was then carefully removed, the muscle and skin were sutured, and the chest cavity was closed. The Sham-operated group underwent the same procedure but without ligating the aortic arch. After four weeks of the TAC model, M-mode echocardiography was performed using a Doppler ultrasound imaging system (VINNO 6 VET, Suzhou, China), and measurements of left ventricle (LV) long-axis left ventricular diastolic internal dimensions (LVIDd), and systolic internal dimensions (LVIDs), ejection fraction (EF ) and fractional shortening (FS), and LV wall thickness were obtained. Four weeks after TAC surgery, all mice were euthanized, and cardiac tissues were collected and fixed in 4% paraformaldehyde (BL539A, Biosharp, Guangzhou, China) for histological analysis.

Animal adeno-associated virus (AAV)-treatment
All experimental mice were randomly inoculated with adeno-associated virus serotype 9 carrying the cardiomyocyte promoter cTnT-specific overexpression of asprosin (AAV9-cTnT-Asprosin) or negative control overexpression plasmid (AAV9-cTnT-NC) at a dose of 2 × 1011 v.g. per animal via tail vein injection. These constructs were prepared by Guangzhou Cyagen Co (Cyagen, Guangzhou, China). AAV9 viruses carrying cardiomyocyte-specific knockdown of a short fragment of FBN1 (AAV9-cTnT-shFBN1) or a negative control transcribed RNA fragment (AAV9-cTnT-shNC), and cardiomyocyte-specific overexpression of PFKP (AAV9-cTnT-PFKP) or a negative control overexpression plasmid (AAV9-cTnT-NC) were produced by Shanghai GENECHEM Co (GENECHEM, Shanghai, China). AAV9-cTnT-shFBN1, AAV9-cTnT-PFKP and the corresponding control viruses were administered by tail vein injection at a dose of 2 x 1011 v.g. per animal.

Histological staining
Fresh heart tissues were fixed with 4% paraformaldehyde (P1110, Solarbio, Beijing, China) for 72 h, dehydrated with graded ethanol, and paraffin-embedded cut into 5 μm thick sections for H&E (G1120, Solarbio, Beijing, China) and Masson’s staining (G1340, Solarbio, Beijing, China). Histological analysis images were captured using a light microscope (OLYMPUS DP80, Olympus, Tokyo, Japan). Quantification of myocardial collagen was performed on cardiac tissue sections using Image-Pro Plus software.

Wheat germ agglutinin (WGA) staining
WGA (W11261, Invitrogen, CA, USA) staining method: heart tissues were treated with dewaxed with endogenous peroxidase blocking buffer (E-IR-R115, Elabscience, Wuhan China) for 10 min, incubated with sodium citrate antigen restoration solution (C1032, Solarbio, Beijing, China) for 20 min, and rinsed with PBS. WGA staining working solution (1:200) was configured, incubated at room temperature for 10 min, and photographed with fluorescence microscope (OLYMPUS DP80, Olympus, Tokyo, Japan). Image-Pro Plus software was used to measure the mean optical density of WGA-stained cardiac tissue sections for quantitative assessment of cardiomyocyte size.

Western blot
Total protein was extracted from tissues or cells using RIPA (P0013B, Beyotime, Beijing, China) lysate. Protein concentration was quantified by BCA Protein Concentration Measurement Kit (P0011, Beyotime, Beijing, China) to standardize the mass uploaded mass, and protein SDS-PAGE Protein Loading Buffer (5X) (P0015, Beyotime, Beijing, China) was added and heated at 100 °C for 7 min. SDS-polyacrylamide gels were performed, and after electrophoresis, the proteins were transferred to nitrocellulose membranes (TM-NC-R-22, LABSELECT, Beijing, China) closed in 5% skimmed milk. The protein membranes were incubated with primary antibodies overnight at 4 °C. Primary antibodies as follows: anti-Asprosin (FNab09797, Finetest, Wuhan, China, 1:500), anti-β-MHC (22280-1-AP, Proteintech, Wuhan, China, 1:500), anti-ANP (DF6497, Affinity, Changzhou, 1:500), anti-PFKP (13389-1-AP, Proteintech, Wuhan, China,1:500), anti-PFKP (68129-1-Ig, Proteintech, Wuhan, China,1:500), anti-DTX3L (A18551, Abclonal, Wuhan, China, 1:500), anti-HA (81290-1-RR, Proteintech, Wuhan, China, 1:500), anti-Flag (66008-4-Ig, Proteintech, Wuhan, China, 1:500), anti-Myc (16286-1-AP, Proteintech, Wuhan, China, 1:500), PDK4 (12949-1-AP, Proteintech, China, 1:500), p-PDH (ab177461, Abcam, USA, 1:500), PDH (18068-1-AP, Proteintech, China, 1:500), PFKL (GTX105697, Genetex, USA, 1:500), PFKM (55028-1-AP, Proteintech, China, 1:500), Anti-β-actin (66009-1-Ig, Proteintech, China, 1:500), Anti-α-Tubulin (FNab0033, Finetest, China, 1:500), Anti-His (66005-1-Ig, Proteintech, China, 1:500), Anti-FBN1(YT1684, Immunoway, China, 1:500), and anti-Ubiquitin (Ab134953, Abcam, Cambridge, UK, 1:500). Next, the protein membranes were incubated with secondary fluorescence-conjugated anti-rabbit IgG or anti-mouse IgG secondary antibodies (926-32211/926-32210, LI-COR Bio-Science, Lincoln, USA, 1:8000) at room temperature away from light for 1 h. Protein bands were imaged using an Odyssey infrared imaging system (LI-COR Odyssey CLx 9140, Biosciences, Lincoln, USA), and the resultant band intensities were quantified via Image Studio 6.0 software based on grayscale values. Pre-stained protein markers used in this western blot assay were commercially obtained from the following suppliers: Thermo Fisher Scientific (Cat#26619 and 26661, USA), Yeasen Biotechnology (Cat#WJ103L, China), and Titan Scientific (Cat#E8010, China).

qRT-PCR and Chromatin Immunoprecipitation (ChIP)
Total RNA was extracted from C57BL/6 WT mice heart tissues and NMCMs, using TRIzol reagent (15596018CN, Invitrogen, CA, USA). The concentration and purity of the extracted RNA were determined by NanoDrop 8000 (ThermoFisher, Waltham, MA, USA) and reverse-transcribed to cDNA using a reverse transcription kit (FSQ-101, Toyobo, Osaka, Japan). Real-time polymerase chain reaction was performed using Toyobo Universal SYBR qPCR Master Mix (QPX-201, Toyobo, Osaka, Japan). All cDNA samples were prepared into a 20 μL reaction system. Reaction conditions: first, 95 ℃ for 10 min; 40 cycles of 95 ℃ for 30 s, 65 ℃ for 30 s, and 72 ℃ for 30 s. Real-time quantitative polymerase chain reaction (qPCR) was performed with a real-time PCR system (Quant Studio™ 6 Flex, ABI, MA, USA). The comparative cycle threshold (Ct) method was utilized to analyze the target gene expression using the 2-ΔΔCT method normalized to the amount of endogenous Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin.
ChIP assay was performed using a ChIP assay kit (P2078, Beyotime, Beijing, China). According to the manufacturer’s instructions, NMCMs were treated with 37% formaldehyde (F8775, Sigma-Aldrich, St. Louis, MO, USA) under serum-free conditions to a final concentration of 1%, crosslinked for 10 minutes in a 37 °C incubator, and quenched with a final concentration of 125 mmol/L glycine (CAS: 56-40-6, Solarbio, Beijing, China). After the cells were lysed with PBS supplemented with PMSFN (ST505, Beyotime, Beijing, China), the sheared genomic DNA was sonicated to break most of the DNA into 200-1000 bp size. Magnetic bead DNA complexes were mixed with anti-YY1 (Cat#46395, CST, MA, USA, 1:50), and anti-KLF4 (11880-1-Ap, Proteintech, Wuhan, China, 1:50) antibodies incubated overnight at 4 °C. The DNA complexes of the magnetic beads were washed with low salt, high salt, Licl, TE buffer, and elution buffer to elute the DNA-protein complexes. Cross-links between proteins and genomic DNA were removed by heating at 65°C for 4 h. The samples were purified with a DNA purification kit (D0033, Beyotime, Beijing, China), and the DNA was eluted with a small amount of DEPC (R1600, Solarbio, Beijing, China) water and used for PCR detection of the target gene. The qRT-PCR and CHIP-PCR primer sequences used are listed in supplemental Table S2 and Table S3.

ELISA
The asprosin enzyme-linked immunosorbent assay kit (EM1707, Finetest, Wuhan, China) was used to determine asprosin levels in human plasma or mouse plasma and supernatants of NMCMs, according to the manufacturer’s protocol.

Measurement of acetyl-CoA, pyruvate, and ATP contents
Acetyl-CoA measurement was performed according to the manufacturer’s protocol using the Acetyl-CoA assay kit (E-EL-0125, Elabscience, Wuhan, China). Pyruvate measurement was performed according to the manufacturer’s protocol using the pyruvate assay kit (KTB1121, Abbkine, Wuhan, China). ATP measurement was performed according to the manufacturer’s protocol using the ATP assay kit (KTB1016, Abbkine, Wuhan, China).

Measurement of PDH activity
The PDH activity microplate assay kit (KTB1270, Abbkine, Wuhan, China) was used to measure the PDH activity of cells, according to the manufacturer’s protocol.

Isolation and culture of primary neonatal mouse cardiomyocytes (NMCMs)
Cardiomyocytes were extracted from neonatal mice. NMCMs were isolated from 1- to 3-day-old neonatal mice were purchased from the Experimental Animal Center of The Second Affiliated Hospital of Harbin Medical University (Harbin, China). Briefly, heart tissues were sequentially digested with 0.25% trypsin-EDTA (C0201, Beyotime, Beijing, China) and type II collagenase (17101015, Gibco, Grand Island, USA) at 37°C with agitation. The resulting cell precipitates were resuspended in DMEM (C11885500BT, Gibco, Grand Island, USA) supplemented with 10% FBS (A5256701, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (SC118, Seven, Beijing, China), plated at a density of 1.2×10⁵ cells/cm², and incubated for 1–2 h to facilitate purification by selective adhesion. All animal experiment protocols complied with the guiding principles for the care and use of laboratory animals at Harbin Medical University and were approved by the Ethics Committee for Animal Experimentation (IRB5045724). Experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (U.S. NIH) and the ARRIVE guidelines.

Cell culture and treatment
Mouse aortic vascular smooth muscle cells (SMCs; Cat# BNCC338213, Beijing, China) and mouse aortic vascular endothelial cells (ECs; Cat# BNCC338463, Beijing, China) were obtained from Beina Biotechnology Co., Ltd. Human embryonic kidney 293T (HEK-293T) cells (Cat# CL-0005, Wuhan, China) were purchased from Procell Life Science & Technology Co., Ltd. All cells were cultured in DMEM supplemented with 10% FBS, maintained at 37 ± 0.5°C in a humidified incubator containing 5% CO₂. The identity of the aforementioned cell lines was confirmed by short tandem repeat (STR) profiling, and mycoplasma contamination was not detected.
SiFBN1, siPFKP and negative control RNA (siNC) were synthesized by Guangzhou RiboBio Co (Guangzhou, China). Flag-Asprosin (NM_007993(1-27/2732-2873aa)) overexpression plasmid of GV657 vector and its mutant plasmid were constructed by Shanghai GENECHEM Co. In addition, HA-PFKEP (NM_019703) overexpression plasmid and HA-PFKP structural domain deletion overexpression plasmid of GV657 vector were constructed by GENECHEM (Shanghai, China). Similarly, Flag-DTX3L (NM_001013371) overexpression plasmid of GV657 vector and its mutant overexpression plasmid were constructed by GENECHEM. HA-YY1(NM_009537), Flag-FBN1(NM_007993.2) and Flag-FBN1-del-asprosin (MUT) overexpression plasmid of pcDNA3.1 vector and negative control overexpression plasmid were constructed by HonorGene (Wuhan, China). Plasmids or siRNAs were transfected using Lipofectamine™ 2000 Transfection Reagent (11668030, Invitrogen, CA, USA) according to the manufacturer’s instructions. Transfection conditions were used to transfect NMCMs with 2000 ng overexpression plasmid or siRNA at a final concentration of 50 μM. 24 h after transfection, NMCMs were incubated for 48 h with Ang II at a final concentration of 2 μM. The target sequence of siFBN1 was GTACATCGGCACTCACTGT, the target sequence of si-PFKP was CCAATATTGTGGAAGCCAA, and the target senseSeq sequence of si-YY1 was CACAUCUUAACACACGCUAAA and antiSeq is UUUAGCGUGUGUUAAGAUGUG.

Immunofluorescence staining
NMCMs were inoculated and cultured in confocal dishes for 48 h. Cell morphology was fixed with 4% paraformaldehyde, treated with 0.2% Triton-X100 cell penetrant (T8787, Sigma-Aldrich, St. Louis, MO, USA) for 10 min, and blocked with 5% goat serum (AR0009, Boster, Wuhan, China). The cells were washed in PBS and then incubated with a primary antibody anti-α-actinin (GTX11007, Genetex, Texas, USA, 1: 300) incubated for cardiomyocyte marker staining. Alexa-Fluor 594/488-linked anti-Rabbit/Mouse IgG antibody (A23410, A23420, A23220, A23210, Abbkine, Wuhan, China) was used to incubate for 1 h at room temperature, and stained with DAPI (C1005, Beyotime, Beijing, China) for 5 min. Finally, the images were collected by fluorescence microscope (FV300, Olympus, Tokyo, Japan).

RNA-sequencing analysis
Mouse heart tissue was collected for RNA sequencing. Briefly, the total RNA from each sample was extracted using either CTrizol Reagen. A total of 1 µg of RNA per sample was used as input material for RNA sample preparation. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) and index codes were added to attribute sequences to each sample. Index codes were added to the sequence attributes for each sample. Finally, PCR products were purified (AMPure XP system), and library quality was assessed with an Agilent Bioanalyzer 2100 system and quantified with a 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).

Co-immunoprecipitation (Co-IP)
For Co-IP assays, the Pierce™ Immunoprecipitation Kit (26149, Thermo Scientific, California, USA) was used in present study. NMCMs and HEK-293T cells were extracted by IP lysis/wash buffer. According to the reagent’s instructions, control coupling resin was used to remove non-specific binding, and immobilized agarose resin was added for antibody coupling. Protein complexes were eluted by the eluent, and 1× Protein Sampling Buffer was added, and the samples were boiled at 95 ℃ for 7min, and then were subjected to SDS-PAGE for separation.

Ubiquitylation assays
NMCMs or HEK-293T cells were transfected with HA-PFKP, Flag-Asprosin, Flag-DTX3L, Myc-Ub, or vector overexpression plasmids. Following 36 h post-transfection, 50 μM MG132 (HY-13259, MCE, Shanghai, China) was introduced into the culture medium for 6 h duration. Subsequently, the cells were lysed using cell lysis buffer to facilitate co-immunoprecipitation assays. The protein complexes were captured using Protein A/G Magnetic Beads (HY-K0202, MCE, Shanghai, China) in conjunction with an anti-PFKP antibody (13389-1-AP, Proteintech, Wuhan, China, 1:500). The protein samples were then subjected to 10% SDS-PAGE electrophoresis and subsequently blotted with an anti-Ubiquitin antibody (Ab134953, Abcam, Cambridge, UK, 1: 500). IgG was employed as a negative control.

LC-MS/MS analysis
Briefly, NMCMs were transfected with Flag-Asprosin overexpression plasmid, and the cells were completely lysed by RIPA (89901, ThermoFisher, Waltham, MA, USA) after 48 h. Lysates were subjected to COA-IP assay with anti-Asprosin, anti-PFKP and anti-IgG antibodies, and the resulting samples were subjected to SDS-PAGE electrophoresis. The electrophoresis of the samples was stopped at 1 cm through the separation gel, and the gel strips were stained with Coomassie Blue Fast Staining (P0017, Beyotime, Beijing, China) and analyzed by LC-MS/MS. LC-MS/MS was done by Beijing Novogene Technology Co (Beijing, China).

Lactate production and glucose content determination
Lactate products in NMCMs and cardiac tissues were determined using the Lactate Detection Kit (KTB1100, Abbkine, Wuhan, China). For cardiac glucose determination, the glucose content in cardiac tissues was measured using the Glucose Assay Kit (S0201S, Beyotime, Beijing, China).

Glucose uptake
Glucose uptake in NMCMs was assessed using a commercial assay kit (UP02, Dojindo, Kyushu, Japan). Briefly, 48 h post-transfection with plasmids or siRNA, the cells were washed twice with pre-warmed glucose-free medium and incubated in the same medium for 1 h. Subsequently, they were incubated with the pre-warmed probe working solution for 15 min, washed twice with pre-chilled WI buffer, and left at room temperature for 5 min. Fluorescence imaging was finally performed using a Live cell Imaging System (FV300, Olympus, Tokyo, Japan) to quantify glucose uptake. The fluorescence intensity was analyzed by Image-pro plus.

Mitochondrial membrane potential
Mitochondrial membrane potential was assessed using tetramethylrhodamine ethyl ester (TMRE) staining (C2001S, Beyotime, Beijing, China). According to the manufacturer's instructions, the TMRE working solution was prepared immediately before use by diluting TMRE (1000X) with assay buffer at a ratio of 1 µL per mL, protected from light. For adherent NMCMs, cells were washed with PBS and incubated with the TMRE working solution at 37 °C for 30 min, followed by two washes with pre-warmed DMEM and resuspension in serum-containing medium for observation under a Live cell Imaging System (FV300, Olympus, Tokyo, Japan). The fluorescence intensity was analyzed by Image-pro plus.

Seahorse
The energy metabolism of cardiomyocytes was assessed using the Agilent Seahorse XFe 96 Cell Energy Metabolism Analysis System (Agilent, California, USA). Mitochondrial respiration of cardiomyocytes was analyzed by measuring oxygen consumption rate (OCR) using the Seahorse XF Cell Mito Stress Test Kit (103015-100, Agilent Technologies, CA, USA). Glycolysis in cardiomyocytes were measured by measuring the extracellular acidification rate (ECAR) using the Seahorse XF Glycolytic Stress Test Kit (103020-100, Agilent Technologies, CA, USA). The ECAR) and OCR data were analyzed using the Wave v2.6.3 package (Agilent Technologies).

Molecular docking
Hex 8.0.0 software was used to study the binding mode of PFKP and asprosin. The 3D structure of murine PFKP protein was obtained by AlphaFold construction, while the 3D structure of murine asprosin protein was obtained by SWISS-MODEL server construction. Default parameters were used for molecular docking unless otherwise stated. Docking results were analyzed for binding patterns using PyMoL 1.7.6 software (www.pymol.org)

Dual-luciferase reporter assay
FBN1 or FBN1-MUT promoter luciferase reporter plasmids were constructed using the pGL3-Basic vector from Abiowell BioTech Inc (Changsha, China). HEK-293T cells were co-transfected with luciferase reporter plasmid, YY1, and vector overexpression plasmids. Cells were collected 48 h post-transfection according to the manufacturer’s protocol of the Dual-Luciferase System Kit (E1910, Promega, WI, USA), and relative luciferase activity was calculated by measuring optical density.

His-Pulldown
His-pull down assays were performed using the Pierce™ Pull-Down PolyHis Protein Kit (#21277, Thermo Scientific, California, USA). Briefly, polyhistidine-tagged asprosin was incubated with pre-equilibrated HisPur™ Cobalt Resin in spin columns for 2 hours at 4 °C under gentle rotation. After centrifugation at 1300 g for 1 minute, the resin was washed five times with wash buffer. Then, 1000 μg of lysate from HEK-293T cells transfected with HA-tagged PFKP plasmid was diluted to 600 μL and added to the columns, followed by incubation overnight at 4 °C with gentle shaking. The resin was centrifuged again and washed five times. Bound proteins were eluted with 250 μL of 290 mM imidazole elution buffer via centrifugation at 1300 g for 1 minute. Eluates were analyzed by SDS-PAGE and immunoblotting.

Statistical analysis
All data were statistically analyzed using GraphPad Prism 8.0 software. Results are presented as mean ± SEM of at least three independent experiments and normal distribution was tested. For normally distributed variables, two groups of data were tested between two groups using an unpaired two-tailed/sided t-test, and a nonparametric Mann-Whitney test was used for non-normally distributed variables. Data were analyzed by Bonferroni's test after one-way analysis of variance (ANOVA) for multiple groups conforming to normal distribution, and by Kruskal-Wallis test with Dunn's multiple comparisons test after nonparametric tests for nonnormally distributed data. To assess the associations between cardiac function parameters (e.g., EF, NT-proBNP) and plasma asprosin levels, we selected correlation methods based on the distribution of each continuous variable. Data following a normal distribution were analyzed using Pearson correlation, while non-normally distributed data were analyzed using Spearman correlation. For both methods, the correlation coefficient (r), corresponding P-value, and 95% confidence interval (CI) were reported. P < 0.05 was considered indicative of a statistically significant difference.

Statistics and reproducibility
Each experiment was repeated 3 times or more independently with similar results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The study reveals the key role of YY1-Asprosin-PFKP axis in pathological cardiac hypertrophy, enhancing glycolysis and\ninhibiting mitochondrial function by stabilizing PFKP, providing a new target for the treatment of cardiac hypertrophy.\nHowever, the study still has some limitations that need to be further validated and expanded.\n(1) Does AAV9-Asprosin affect the cardiac function and other parameters in sham mice? Fig1M-W lacks Sham+AAV9-\nAsprosin group.\n(2) The representative graphs of Fig 1M and Fig 2D did not show that the heart LVPWd/s in TAC-AAV9-NC and TAC-AAV9-\nAsprosin groups was further thickened compared with the corresponding control group.\n(3) It is recommended to verify glycolysis rates at the in vivo level, including fig3 and fig6, and not only at the in vitro level.\n(4) The manuscript involves the assay of OCR, and it is suggested that statistics of ATP production are more convincing.\n(5) Table S3 lists Cpt1b, which is much more abundant than PFKP. Why not choose Cpt1b? Does Asprosin have an effect\non fatty acid oxidation?\n(6) Methods for isolating CMs, CFs, SMC and EC from adult mouse hearts and identification of cell types are lacking.\n(7) The discussion section should also focus on the current research status of the role of Asprosin in the heart, including\nischemic cardiomyopathy, diabetic cardiomyopathy, etc., and whether its mechanism of action is relevant to this study?\n(8) While DTX3L is identified as the E3 ligase for PFKP, other ubiquitin-modifying enzymes (e.g., deubiquitinases) may\ncontribute.", "reviewer_2": "Ming Tong et al. found that plasma asprosin levels were significantly higher in patients with hypertensive heart failure than in\nnon-heart failure patients. These findings align with previous studies showing that serum asprosin levels are elevated in\nheart failure patients, including those with hypertensive heart failure (PMID: 38097977), as well as in patients with coronary\nartery disease (PMID: 34419063) and dilated cardiomyopathy (DCM) (PMID: 32894050). However, I am surprised that the\nauthors neither discussed nor referenced any existing literature associating this adipokine with cardiovascular disease in\ntheir manuscript.\nThe novelty of this study lies in the authors' demonstration that overexpressing asprosin induces pathological hypertrophy,\nwhile transgenic mice with reduced asprosin expression are protected against TAC-induced hypertrophy. They also\nobserved that an inhibitor, BOS-318, reversed hypertrophy in a cell model. However, despite conducting multiple in vivo\nexperiments, they have not yet tested the therapeutic potential of this inhibitor in a TAC model, which would be a critical step\nin evaluating its translational relevance for heart failure treatment.\nThe authors propose that asprosin binds to and inhibits the ubiquitination-mediated degradation of phosphofructokinase\nplatelet (PFKP). Consistent with a study published earlier this year (Biochimica et Biophysica Acta - Molecular Basis of\nDisease, DOI: 10.1016/j.bbadis.2024.167542), they demonstrate that cardiac-specific knockout of PFKP alleviates cardiac\nhypertrophy.\nI also have concerns regarding the reproducibility of the TAC control group between experiments. In Figure 1.O, the ejection\nfraction of the TAC control group (TAC + AAV9-NC) is around 60% when compared to the TAC-Asprosin group, which has a\nlower ejection fraction. However, in Figure 2.F, the ejection fraction of the TAC control group (TAC + AAV9-NC) is\napproximately 40% when compared to the TAC + AAV9-shFBN1 group, which has a higher ejection fraction. This\ndiscrepancy raises concerns about the consistency of the TAC control group across different experimental conditions.\nI also have questions regarding the methodology. The asprosin immunoprecipitation shown in Figure 4.A did not\ndemonstrate enrichment of asprosin, which is understandable, as immunoprecipitation experiments can be technically\nchallenging. However, the Western blot signal for the co-immunoprecipitated (Co-IP) PFKP was more intense than the\nasprosin signal. This raises important questions about how the immunoprecipitation was conducted. Were the input and IP\nsamples run on the same gel? Was the same amount of protein loaded for all samples? Additionally, were appropriate\nloading controls included to ensure equal protein input and confirm the specificity of the pull-down? The authors should\nclarify these aspects to ensure the validity of their findings and rule out potential technical artifacts.\nIn Figure 3K, the authors report increased glucose uptake in neonatal mouse cardiomyocytes (NMCM) overexpressing\nasprosin. However, it is unclear whether this increased glucose uptake is accompanied by upregulation of glucose\ntransporters such as GLUT1 or GLUT4. The authors should clarify whether the expression levels of these transporters were\nassessed.\nIn Figure 3M, which marker did the authors use to measure cell surface area?\nI understand that Nature Communications is not a cardiovascular specialist journal, and providing a broad context of\nasprosin’s role is appropriate. However, given that this study focuses on cardiovascular research, the authors should\ndiscuss relevant literature on asprosin in a cardiovascular context. For example, previous studies have reported increased\nasprosin levels in plasma from patients with hypertensive cardiomyopathy and other types of cardiomyopathies, this\nliterature should be included. Additionally, the authors state, \"we discovered that its (asprosin) expression was markedly\nincreased in neonatal cardiomyocytes, suggesting that cardiac major cells also contribute to asprosin production.\" However,\nthere is existing literature that has investigated the localization and distribution of asprosin protein in human atrial and\nventricular heart tissue (https://doi.org/10.1093/cvr/cvae088.217), which should also be discussed to provide a more\ncomprehensive perspective on asprosin’s role in the heart.", "reviewer_4": "This manuscript by Ming Tong et al. introduces a novel regulatory axis—YY1–Asprosin–PFKP—that links transcriptional\nand metabolic remodeling in cardiomyocytes during pathological hypertrophy. Using both in vivo and in vitro models, the\nauthors propose that Asprosin, derived from FBN1, binds to PFKP, enhancing glycolysis and suppressing oxidative\nmetabolism. This interaction contributes to mitochondrial dysfunction and the development of cardiac hypertrophy. Overall,\nthe manuscript is well-structured and addresses a topic of significant relevance to cardiovascular biology. However, several\nkey mechanistic gaps and missing experimental controls must be addressed before the manuscript can be considered for\npublication in Nature Communications.\nMajor Comments\n1. In Figure S6, the authors use IP-Mass to explore mechanisms underlying Asprosin-induced glycolysis. However, as\nAsprosin is a secreted hormone-like molecule, it is more appropriate to first investigate whether its effects are mediated via\nspecific paracrine or autocrine pathways through defined receptors. The rationale for conducting a binding partner screen\nshould be explicitly described and supported by appropriate background information.\n2. In Figure 3H, the authors claim a \"direct\" interaction between Asprosin and PFKP. However, the current evidence—based\non intracellular overexpression, structural prediction, and mutational analysis—is insufficient to conclusively demonstrate a\ndirect physical interaction. To substantiate this claim, in vitro reconstitution assays using purified proteins (e.g., GST pull-\ndown) are required. Until such data are provided, the term “direct binding” should be used cautiously.\n3. In Figure 4, the authors demonstrate that Asprosin competes with DTX3L for PFKP binding, affecting its protein levels.\nThey also suggest this regulation is important for glycolytic shifts under cardiac stress. However, in unstressed\ncardiomyocytes, DTX3L may suppress PFKP levels and promote a FAO-dominant metabolic state. The basal metabolic role\nof DTX3L should be explicitly addressed.\n4. In Figures 3 and 4, the authors show Asprosin binds to PFKP, but it remains unclear how this interaction occurs. Asprosin\nis typically secreted following cleavage of FBN1 by Furin and acts in a paracrine/autocrine manner. The authors must clarify\nwhether the observed interaction occurs intracellularly before secretion or after reuptake of secreted Asprosin.\n5. In Figures 4–6, the authors focus exclusively on the PFKP (platelet-type) isoform among the PFK family. No comparisons\nare made with other isoforms, such as PFKL (liver-type) or PFKM (muscle-type), despite PFKM being highly expressed in\nthe heart. To establish specificity of Asprosin–PFKP binding, the authors should assess interactions and expression\npatterns of other isoforms, especially PFKM.\n6. In Figure 6, the authors show that shFBN1 leads to decreased Asprosin and PFKP expression and mitigates hypertrophy\nand metabolic disturbances. However, without Asprosin reconstitution experiments in FBN1 knockdown models, it is unclear\nwhether the phenotype results from reduced Asprosin levels or loss of FBN1 function itself. Such experiments are necessary\nto establish causality.\n7. The authors report that FBN1 knockdown modulates glucose uptake, lactate production, and mitochondrial membrane\npotential (Δψm) via PFKP (Figure 6G–I). However, the impact of PFKP overexpression on mitochondrial respiration and the\nmechanism by which knockdown restores Δψm require further discussion. Given that TAC-induced hypertrophy involves\nHIF-1α/FOXO-driven PDK expression and PDH inhibition (Zhou et al., Circulation, 2018), the authors should evaluate\nPDK–PDH axis involvement using qPCR or Western blot (e.g., PDK1/4 levels, PDH phosphorylation).\n8. In Figures S10A–J, PFKP overexpression in neonatal mouse cardiomyocytes (NMCMs) promotes glucose uptake, lactate\nsecretion, and reduces Δψm. Figure S11H shows PFKP knockdown attenuates Ang II-induced Δψm reduction. However,\nthe potential impact of PFKP on mitochondrial respiration beyond glycolysis and the mechanism of Δψm protection upon\nknockdown remain unclear. Given known metabolic shifts following TAC (Zhou et al., 2018), the authors should assess\nwhether PFKP alters pyruvate entry into mitochondria and explore PDK–PDH pathway changes.\n9. In addition to JC-1 staining for Δψm, correlating ATP production and oxygen consumption rate (OCR) would strengthen\nthe metabolic interpretation. If glycolysis is upregulated in the absence of PDH inhibition, the role of LDHA in determining\npyruvate fate should also be considered. At present, it is unclear whether PFKP knockdown improves Δψm via reduced\nlactate production or enhanced TCA flux through PDH. Further pathway analyses are required.\n10. The authors propose that the Asprosin–PFKP axis enhances glycolysis, suppresses oxidative metabolism, and thereby\nreduces Δψm and promotes hypertrophy. If so, inhibiting PDK or LDHA would promote TCA flux and oxidative metabolism,\npotentially alleviating hypertrophy. Conversely, PDH inhibition might worsen pathology. Pharmacological modulation of the\nPDK–PDH axis would help clarify the role of this metabolic switch. Supplementary experiments using agents like\ndichloroacetate (DCA) are warranted and could have translational implications.\n11. In Figures S13A–C and 6O,P, FBN1 knockdown suppresses Ang II-induced hypertrophy, which is reversed by PFKP\noverexpression. While intriguing, the link between FBN1–PFKP and classical hypertrophy pathways (NFAT, ERK1/2, Akt,\nmTOR) remains unexplored. Evaluating these pathways would enhance understanding of the interplay between metabolism\nand signal transduction.\nMinor Comments\n1. Figures 3, 4, and 6 include more than 10 panels each, making them difficult to interpret. Splitting them into separate\nfigures would improve clarity.\n2. On page 20, line 2, \"Glycogen\" appears to be a typographical error and should be corrected to \"Glucose\"."}, "author_response": "Responses to Reviewers’ Comments\nCorresponding Author: Professor Yan Zhang\nDear Reviewers:\nThank you for your thoughtful letter and constructive advice. We sincerely appreciate\nthe comments provided by you and the reviewers regarding our manuscript\n(NCOMMS-25-14208) entitled “YY1/Asprosin/PFKP Axis Regulates Glycolytic\nMetabolism and Exacerbates Pathological Cardiac Hypertrophy.”\nWe have carefully considered all the feedback and have made revisions accordingly. In\naddition, we performed several new experiments as suggested, which further strengthen\nour conclusions.\nBelow, we provide detailed, point-by-point responses to all reviewer comments. We\nhope that the changes and additional data presented in the revised manuscript address\nthe concerns raised and demonstrate that this work represents a strong candidate for\npublication in Nature Communications.\nPoint-by-point Responses to The Reviewer’s Criticisms\nReviewer #1: (Remarks to the Author):\nThe study reveals the key role of YY1-Asprosin-PFKP axis in pathological cardiac\nhypertrophy, enhancing glycolysis and inhibiting mitochondrial function by stabilizing\nPFKP, providing a new target for the treatment of cardiac hypertrophy. However, the\nstudy still has some limitations that need to be further validated and expanded.\nMajor concerns:\n(1) Does AAV9-Asprosin affect the cardiac function and other parameters in sham mice?\nFig1M-W lacks Sham+AAV9-Asprosin group.\nResponse: Thank you very much for your insightful comments. We sincerely\nappreciate your emphasis on assessing the effects of AAV9-Asprosin in sham-operated\nmice, which is indeed a crucial aspect for evaluating the treatment's specificity. In the\n1 / 37\nprevious experimental design, in addition to the Fig.1M experimental group, we\nspecifically established the \"sham + AAV9-Asprosin\" and \"sham + AAV9-NC\" control\ngroups to systematically investigate the regulatory mechanism of asprosin on cardiac\nfunctional homeostasis under physiological conditions. The data from these groups\nwere originally omitted from the manuscript because AAV9-Asprosin overexpression\nunder sham conditions did not elicit significant changes in cardiac function or\nmorphology—that is, the results were negative. Nonetheless, we fully agree with your\nassessment that inclusion of these data is essential to ensure the rigor and\ncomprehensiveness of our study.\nAs summarized in the figure below, eight-week-old male C57BL/6 mice received\nequal doses of either AAV9-Asprosin (driven by the cardiomyocyte-specific cTNT\npromoter) or control AAV9-NC. Successful overexpression was confirmed three weeks\npost-injection, followed by sham surgery (Figure R1A–B) (page11，line 207-211).\nCardiac function and structure were evaluated four weeks after surgery. Compared to\nSham+AAV9-NC controls, Sham+AAV9-Asprosin mice showed no significant\ndifferences in cardiac function (LVEF, LVFS, LVPW; Figure R1C&E) (page11, line\n212-214) or organ weight ratios (HW/BW, LW/BW, HW/TL; Figure R1D) (page11,\nline 212-214). Histological examination revealed no notable changes in heart\nmorphology, ventricular thickness, inflammation (H&E; Figure R1F, G), or fibrosis\n(Masson’s trichrome; Figure. R1H, I) (page11, line 214-217).\nMoreover, WGA staining indicated no signs of pathological cardiomyocyte\nhypertrophy (Figure R1J, K) (page11, line 217-220). Collectively, these data confirm\nthat cardiomyocyte-specific overexpression of asprosin does not induce detectable\ncardiac dysfunction, structural remodeling, or fibrosis in sham-operated mice. The\naforementioned data have been integrated into the revised manuscript (Figure\nS5A-K). Finally, it is important to note that in Figure 1 of our manuscript, the group\nlabeled as “Sham” should in fact be designated as “Sham+AAV9-NC”. We apologize\nfor this oversight in the figure labeling during the preparation of the manuscript.\n2 / 37\nFigure R1. Cardiomyocyte-specific, AAV9-mediated overexpression of asprosin does not induce pathological\ncardiac hypertrophy in sham-operated mice under baseline conditions.\n(A) Experimental design for male mice treated with AAV9-cTnT-Asprosin or AAV9-cTnT-NC virus via tail vein\ninjection. (B) Asprosin expression levels in isolated cardiomyocytes from adult mice infected with AAV9-Asprosin\nor AAV9-NC virus were detected by western blot (n=6). (D) HW/BW, LW/BW and HW/TL ratios in AAV9-Asprosin\nor AAV9-NC mice 4 weeks after Sham surgery (n=8). (C&E) Representative M-mode echocardiogram of mice and\nquantitative analysis of EF, FS, LVIDd, LVIDs LVPWd, and LVPWs in different groups of mice described in Fig. C\n(EF, FS, LVIDd, LVIDs: n=8; LVPWd, LVPWs: n=6). (F) Representative morphological images of mouse hearts\ninfected with AAV9-Asprosin or AAV9-NC virus after four weeks of Sham surgery. (G) Representative H&E\nstaining images of hearts from the indicated groups (n=6). (H, I) Representative images of Masson’s staining and\nquantitative analysis of left ventricular interstitial collagen volume from the indicated groups (n=6). (J, K)\nRepresentative cardiomyocyte cross-sectional area images of WGA staining and quantitative analysis of hearts from\nthe indicated groups (n=6). Statistical tests were performed using the unpaired two-tailed t-test in (B, D, E, I, K). ns,\nnot significant.\nAdditionally, we would like to highlight the in vitro experimental results presented\nin our study, which may also be of relevance. In cultures of neonatal mouse primary\ncardiomyocytes, asprosin overexpression significantly induced a hypertrophic response,\nas demonstrated by increased cell surface area and upregulation of the hypertrophy\nmarkers ANP and BNP. These in vitro findings provide clear evidence supporting a\ndirect pro-hypertrophic effect of asprosin on cardiomyocytes. We note the apparent\ndiscrepancy between these results and the in vivo data, where no significant induction\nof pathological hypertrophy was observed in sham-operated mice following\ncardiomyocyte-specific asprosin overexpression.\n3 / 37\nSuch discrepancies in biological research may arise from a combination of the\nfollowing factors. Firstly, systemic homeostasis and compensatory mechanisms: The\nintact in vivo environment possesses complex neurohormonal regulatory networks,\nhemodynamic adaptations, and multi-organ crosstalk. In sham-operated healthy mice,\nthe heart may counterbalance the direct hypertrophic stimuli of asprosin by activating\nintrinsic compensatory pathways—such as antioxidant and anti-apoptotic mechanisms,\nor metabolic reprogramming involving other organs—thereby maintaining myocardial\nstructural and functional homeostasis. Secondly, cellular heterogeneity and\nmicroenvironment: In vitro models typically use purified cardiomyocyte cultures,\nwhereas the in vivo heart consists of multiple interacting cell types, including\ncardiomyocytes, fibroblasts, endothelial cells, and immune cells. This integrated\nmicroenvironment may secrete factors that antagonize asprosin’s actions or modulate\nits bioactivity.\n(2) The representative graphs of Fig 1M and Fig 2D did not show that the heart\nLVPWd/s in TAC-AAV9-NC and TAC-AAV9-Asprosin groups was further thickened\ncompared with the corresponding control group.\nResponse: We sincerely appreciate the reviewer’s insightful comment concerning the\nrepresentative images in Figures 1M and 2D, which highlight the observation that the\napparent increase in left ventricular posterior wall thickness during diastole (LVPWd)\nin the TAC+AAV9-NC and TAC+AAV9-Asprosin groups, as compared to the control\ngroups, is not clearly discernible. We agree that this is an appropriate observation, and\nwe acknowledge that clearly discernible intergroup differences are essential for the\naccurate interpretation of these experimental results. In response to the reviewer’s\nsuggestion, we have replaced the more representative echocardiographic images\nshowing left ventricular posterior wall thickness at diastole (LVPWd) in Figures 1M\nand 2D in the revised manuscript. The updated figures clearly demonstrated that under\nTAC-induced hypertrophic conditions, LVPWd is further increased in the TAC+AAV9-\nAsprosin group compared to the TAC+AAV9-NC group (Figure R2A) (page9-10, line\n173-177). Conversely, the TAC+AAV9-shFBN1 group showed a reduction in\n4 / 37\nventricular wall thickness and improved ventricular function relative to the\nTAC+AAV9-shNC group (Figure R2B) (page12, line 229-231). These updated images\nprovide direct visual evidence supporting our conclusion that AAV9-mediated\ncardiomyocyte-specific asprosin overexpression exacerbates pressure overload-\ninduced cardiac hypertrophy, while cardiomyocyte-specific asprosin knockdown\nattenuates this pathological remodeling. The aforementioned data have been\nintegrated into the revised manuscript (Figure 1M and Figure 2D).\nFigure R2. Representative left ventricular ultrasound images (echocardiograms) of male mice hearts are\nshown.\n(3) It is recommended to verify glycolysis rates at the in vivo level, including fig3 and\nfig6, and not only at the in vitro level.\nResponse: We thank the reviewer for this valuable comments and thorough review of\nour manuscript. The suggestion to validate glycolytic rate in vivo (in relation to Figures\n3 and 6), is both critical and highly valuable. We fully agree that direct measurement of\nglycolytic flux in a robust in vivo model is essential for translating our cellular findings\ninto broader physiological and pathological contexts.\nAs suggested, we isolated cardiomyocytes from in vivo models and conducted\nSeahorse XF metabolic assays to further examine the role of the asprosin–PFKP axis in\nregulating cardiomyocyte glycolytic metabolism. Extracellular acidification rate\n(ECAR) measurements revealed a significant increase in glycolytic capacity in the\nTAC+AAV9-Asprosin group compared to the TAC+AAV9-NC group (Figure R3A)\n(page14, line 267–272). We also assessed the effects of cardiomyocyte-specific\nasprosin overexpression on mitochondrial respiration in TAC-induced hypertrophic\n5 / 37\ncardiomyocytes. Oxygen consumption rate (OCR) analysis indicated that asprosin\noverexpression markedly suppressed mitochondrial respiratory function, as shown by\nreduced basal respiration, maximal respiration, spare respiratory capacity, and ATP\nproduction-linked oxygen consumption (Figure R3B) (page14, line 267–272). The\naforementioned data have been integrated into the revised manuscript (Figure\nS7C-D). These additional data provide in vivo evidence that asprosin enhances\nglycolytic metabolism while impairing mitochondrial respiration, thereby supporting\nour central conclusion.\nFigure R3. ECAR and OCR assays are conducted to investigate the glycolytic and respiratory ability of adult\nmice cardiomyocytes.\n(A-B) ECAR (n=3) and OCR (n=6) of the cardiomyocytes isolated from adult mice were detected by seahorse in\nthe indicated groups. Statistical tests were performed using the unpaired two-tailed t-test in (A, B). **, P <0.01. ***, P\n<0.001.\nIn parallel, we further assessed glycolytic flux and mitochondrial respiratory\nfunction in vivo in the experimental groups associated with Figure 6 using Seahorse\nXF technology. Results demonstrated that cardiomyocyte-specific knockdown of\nasprosin significantly attenuated the TAC-induced elevation in both glycolytic capacity\nand mitochondrial respiratory dysfunction (Figure R4A–B; page23, line 475–479).\nImportantly, concomitant overexpression of PFKP largely reversed these protective\neffects (Figure R4A–B; page23, line 475–479). These results further reinforce our\nconclusion that asprosin promotes pathological cardiac hypertrophy through PFKP-\n6 / 37\nmediated enhancement of glycolytic metabolism. The aforementioned data have\nbeen integrated into the revised manuscript (Figure S17A-B).\nFigure R4. ECAR and OCR assays are conducted to investigate the glycolytic and respiratory ability of adult\nmice cardiomyocytes.\n(A-B) ECAR (n=3-4) and OCR (n=5) of the cardiomyocytes isolated from adult mice were detected by seahorse in\nthe indicated groups. Statistical tests were performed using the one-way ANOVA with Bonferroni's multiple\ncomparisons test in (A, B). *, P <0.05. **, P <0.01. ***, P <0.001.\n(4) The manuscript involves the assay of OCR, and it is suggested that statistics of ATP\nproduction are more convincing.\nResponse: We thank the reviewer for their insightful suggestions. As recommended,\nwe have reanalyzed the existing data and conducted additional experiments to more\nrigorously evaluate the ATP production derived from our OCR measurements. The\nresults indicated that overexpression of either asprosin or PFKP led to a significant\nreduction in ATP production capacity in NMCMs (Figures R5A, B) (page 14, line 274–\n276 & page20, line 402–403). Furthermore, under angiotensin II (Ang II)-induced\nhypertrophic conditions, asprosin overexpression markedly exacerbated the impairment\nof ATP production (Figure R5C) (page14, line 272–274). In the rescue experiments,\nknockdown of asprosin significantly ameliorated the Ang II-induced mitochondrial\ndysfunction and concomitant decline in ATP generation. This protective effect was,\nhowever, effectively abolished by concomitant PFKP overexpression (Figure R5D)\n(page24, line 484–487). Moreover, treatment with dichloroacetate (DCA) significantly\nreversed the PFKP overexpression-induced suppression of both mitochondrial\nrespiratory capacity and ATP production in NMCMs (Figure R5E) (page22, line 447–\n450). Together, these data provide functional evidence that the asprosin–PFKP axis\n7 / 37\nimpairs mitochondrial ATP generation, thereby contributing to pathological remodeling.\nThe aforementioned data have been integrated into the revised manuscript (Figure\nS3H, Figure S13H, Figure 3H, Figure S17D, and Figure S13W).\nFigure R5. OCR assays are conducted to investigate the NMCMs respiratory ability.\n(A) OCR assay was performed to assess the impact of asprosin overexpression on the respiratory capacity of\nNMCMs (n=3). (B) OCR assay was performed to assess the impact of PFKP overexpression on the respiratory\ncapacity of NMCMs (n=3-4). (C) Analysis and quantification of OCR (n=5) of the NMCMs in the indicated groups.\n(D) Analysis and quantification of OCR (n=3) of the NMCMs in the indicated groups. (E) Analysis and quantification\nof OCR (n=5) of the NMCMs in the indicated groups. Statistical tests were performed using the unpaired two-tailed\nt-test in (A, B, C), and the one-way ANOVA with Bonferroni's multiple comparisons test in (D, E). *, P <0.05. **,\nP <0.01. ***, P <0.001.\nImportantly, we also quantitatively assessed intracellular ATP levels across all\nexperimental groups using a commercial ATP detection kit. Results showed that\n8 / 37\nphysiological overexpression of either asprosin or PFKP was sufficient to reduce ATP\ncontent in NMCMs (Figures R6A, B) (page14, line 276–278 & page20, 402–406).\nConversely, targeted knockdown of asprosin or PFKP significantly attenuated the Ang\nII–induced reduction in ATP content of NMCMs (Figures R6C, D) (page14, line 282–\n283 & page20, line 414–415). Furthermore, in vitro rescue experiments confirmed that\nasprosin knockdown alleviated the Ang II–induced deficit in ATP supply, an effect that\nwas reversed by concomitant PFKP overexpression (Figure R6E) (page24, line 490–\n491). The aforementioned data have been integrated into the revised manuscript\n(Figure S3G, Figure S13I, Figure S6F, Figure S14I, and Figure S16I).\nFigure R6. Analysis of the regulatory role of the asprosin-PFKP axis in modulating ATP content in NMCMs.\n(A-E) The ATP contents in NMCMs was quantified using an ATP content assay kit (A, B, D: n=5; C: n=7; E: n=6).\nStatistical tests were performed using the unpaired two-tailed t-test in (A, B), and the one-way ANOVA with\nBonferroni's multiple comparisons test in (C, D, E). *, P <0.05; ***, P <0.001.\n(5) Table S3 lists Cpt1b, which is much more abundant than PFKP. Why not choose\nCpt1b? Does Asprosin have an effect on fatty acid oxidation?\nResponse: Thank you for your valuable comment. We fully agree that selecting a\nprotein with high expression is important; however, target selection must also consider\nits biological relevance. First, accumulating evidence has demonstrated that asprosin\nplays a critical role in regulating glucose metabolism homeostasis (PMID: 27087445,\n31230984, 37769508, 36056803). Second, other studies have also confirmed that\nasprosin significantly contributes to the regulation of fatty acid oxidation (FAO),\nincluding modulating inflammatory responses via the AMPK signaling pathway, which\npromotes the progression of non-alcoholic fatty liver disease (NAFLD) (PMID:\n37647445). However, KEGG pathway enrichment analysis of differentially expressed\n9 / 37\ngenes from myocardial tissue RNA-seq in this study showed that the major pathways\ninfluenced by asprosin were predominantly enriched in metabolic processes,\nparticularly those involved in glucose metabolism, central carbon metabolism in cancer,\npyruvate metabolism, and various amino acid metabolic pathways. In contrast, no\nsignificant enrichment was observed in FAO-related pathways. This finding suggests\nthat asprosin may primarily regulate glucose metabolism in the context of pathological\ncardiac hypertrophy. More importantly, ECAR experiments demonstrated that asprosin\noverexpression enhanced glycolytic metabolism of NMCMs in this study.\nPFKP, a key platelet isoform of phosphofructokinase (PFK1), is closely associated\nwith critical biological processes such as glycolytic metabolic shifts, energy\nmetabolism reprogramming, and downstream molecular mechanisms linked to\ncardiovascular disease progression (PMID: 39419453). Importantly, existing literature\nhas established the regulatory roles of the liver-type (PFKL) and muscle-type (PFKM)\nisoforms of phosphofructokinase in cardiovascular diseases (PMID: 36810848,\n40222696, 40569576, 35450439), further supporting the involvement of PFKP in\npathological cardiac hypertrophy. More importantly, the latest study has further\nhighlighted the critical role of PFKP in the regulation of pathological cardiac\nhypertrophy (PMID: 39419453). In contrast, CPT1B primarily participates in FAO and\nexhibits a relatively weak association with the core metabolic phenotypes observed in\nour study. Therefore, the selection of PFKP—over CPT1B—as the downstream target\nof asprosin is supported by the aforementioned evidence, as it not only aligns with its\nwell-established function in glucose homeostasis but also offers greater mechanistic\nrelevance and scientific merit.\nIn response to the reviewer’s suggestion, we performed additional experiments\ninvolving asprosin overexpression in cardiomyocytes to evaluate its effects on key\nmarkers of FAO. qRT-PCR analysis revealed that asprosin overexpression led to a\nmodest decrease in the mRNA expression of several critical FAO-related genes,\nincluding CPT1B, LCAD, PGC-1α, ACOX1, and PPARα (Figure R7A). However,\nthese changes did not reach statistical significance. Importantly, western blot analysis\n10 / 37\nfurther confirmed that asprosin overexpression did not significantly alter CPT1B\nprotein expression (Figure R7B).\nFigure R7. Asprosin does not exert a significant regulatory effect on FAO-related genes in NMCMs.\n(A) qRT-PCR was performed to detect the FAO-related genes mRNA expression levels (CPT1B: n=5; LCAD,\nPPARα, PGC1α, ACOX1: n=6). (B) Western blot was performed to detect CPT1B protein expression levels (n=6).\nStatistical tests were performed using the unpaired two-tailed t-test in (A, B). ns, not significant.\nWe sincerely appreciate your insightful question. Dysregulation of FAO represents\na hallmark of metabolic remodeling in heart failure (PMID：28192373). Although our\ndata do not support a significant role for asprosin in the transcriptional regulation of\nFAO genes, further investigations at the functional level may yield valuable insights\ninto its potential involvement in FAO. We are deeply grateful for your valuable\ncomments on our manuscript. Your thoughtful suggestions will contribute to a more\ncomprehensive understanding of the specific mechanisms by which asprosin modulates\nFAO in cardiac diseases and provide meaningful references for the development of\npotential cardiovascular therapeutic targets.\n(6) Methods for isolating CMs, CFs, SMC and EC from adult mouse hearts and\nidentification of cell types are lacking.\nResponse: We are grateful for your valuable comments. Your meticulous attention to\nmethodological details has encouraged us to provide a clearer and more thorough\nexplanation regarding the experimental approach. We would like to clarify that the cells\nutilized in Figure 1J consisted of neonatal mouse-derived primary cardiomyocytes\n11 / 37\n(NMCMs), neonatal mouse-derived primary cardiac fibroblasts (NMCFs), as well as\nmouse aortic smooth muscle cell lines (SMCs) and mouse aortic endothelial cell lines\n(ECs). The aim of this experiment was to efficiently evaluate the effect of Ang II on\nasprosin expression across various cardiac cell types. The use of neonatal mouse\nprimary cells is a well-established approach in cardiovascular research, owing to their\nhigher viability and enhanced proliferative capacity in vitro, which makes them\nparticularly suitable for phenotypic assays. We recognize that the original description\nin the Methods and Results sections may not have sufficiently emphasized the\ndistinction between cell sources, which could have led to misunderstanding. We\nsincerely apologize for this lack of clarity. In the revised version of the manuscript,\nwe will ensure that the origin of the cells used in Figure 1J is explicitly stated to prevent\nany potential ambiguity and to improve the overall precision and readability of the study.\nWe fully concur with your perspective on the importance of using adult mouse\ncardiac cells to validate the physiological relevance of our findings. Indeed, this\napproach constitutes a central element of our experimental design. As described in later\nsections of the manuscript, adult mouse cardiomyocytes were isolated using the\nestablished langendorff perfusion technique (PMID: 23861485, 27502479), and\ncarefully characterized through immunofluorescence staining—including confirmation\nwith anti-α-actinin antibody (Figure R8A). These rigorously validated cells provided\nthe essential foundation for subsequent functional analyses, such as Seahorse metabolic\nassays. Data obtained from experiments utilizing adult cardiomyocytes are consistent\nwith and strongly support the NMCMs results, together reinforcing the main\nconclusions of this study. The aforementioned data have been integrated into the\nrevised manuscript (Figure S2B).\n12 / 37\nFigure R8. (A) Representative immunofluorescence images showing α-actinin staining for the identification\nof isolated adult mouse cardiomyocytes. Red: α-actinin; Blue: DAPI; Light：Light cells\n(7) The discussion section should also focus on the current research status of the role\nof Asprosin in the heart, including ischemic cardiomyopathy, diabetic cardiomyopathy,\netc., and whether its mechanism of action is relevant to this study?\nResponse: Thanks for reviewer`s invaluable advice. We sincerely appreciate your\nmeticulous review and insightful comments on our manuscript. Your suggestion that\n\"the discussion section should further focus on the mechanism of asprosin in heart\ndiseases (ischemic cardiomyopathy and diabetic cardiomyopathy) and its relevance to\nthe findings of this study\" is highly constructive. In response to your recommendation,\nwe have incorporated the following additions and in-depth discussions in the revised\nmanuscript (page 27-28, line 552-578).\n(8) While DTX3L is identified as the E3 ligase for PFKP, other ubiquitin-modifying\nenzymes (e.g., deubiquitinases) may contribute.\nResponse: Thanks for reviewer`s invaluable advice. Indeed, multiple studies have\nreported PFKP ubiquitination by different regulators, such as USP5, Trim21, HRD1,\nand RNF123, in various pathological contexts (PMIDs: 38217030, 39308018,\n33588886, 39725718). In this study, we employed Co-IP coupled with mass\nspectrometry (MS) to identify E3 ligases interacting with PFKP in NMCMs. As shown\nin Table S3, enzymes including USP5, Trim21, HRD1, and RNF123 were not\nsignificantly enriched in the PFKP immunoprecipitation complex. This may be\nattributed to weak or transient interactions between these enzymes and PFKP, or their\n13 / 37\nlow basal expression levels in NMCMs. Significantly, our functional experiments\nprovided robust evidence for the DTX3L–PFKP interaction; DTX3L overexpression\nenhanced PFKP ubiquitination and proteasomal degradation, whereas the catalytically\ninactive DTX3L mutant failed to regulate PFKP ubiquitination. Collectively, these\nfindings establish DTX3L as a key E3 ubiquitin ligase involved in the regulation of\nPFKP stability.\nWe sincerely appreciate your insightful comments on the regulatory mechanisms\nof ubiquitination. The perspective you highlighted — that \"other ubiquitination-\nmodifying enzymes, such as deubiquitinating enzymes or yet-to-be-characterized E3\nligases, may also participate in the regulation of PFKP\" — is highly valuable. We\ncompletely concur that the ubiquitination process is inherently complex and typically\ninvolves the coordinated interplay of multiple enzymatic components. This study is\nprimarily aimed at identifying the core E3 ligase responsible for mediating PFKP\ndegradation in NMCMs. However, we also recognize that the ubiquitination status of\nPFKP may be dynamically regulated by additional factors, such as both well-\ncharacterized and yet-to-be-identified E3 ubiquitin ligases. Importantly, we have\nincorporated in-depth discussions in the revised manuscript (page30, line 627-629).\nReviewer #2: (Remarks to the Author):\nMing Tong et al. found that plasma asprosin levels were significantly higher in patients\nwith hypertensive heart failure than in non-heart failure patients. These findings align\nwith previous studies showing that serum asprosin levels are elevated in heart failure\npatients, including those with hypertensive heart failure (PMID: 38097977IF: 2.3 Q2),\nas well as in patients with coronary artery disease (PMID: 34419063IF: 4.2 Q1) and\ndilated cardiomyopathy (DCM) (PMID: 32894050IF: 2.3 Q2). However, I am surprised\nthat the authors neither discussed nor referenced any existing literature associating this\nadipokine with cardiovascular disease in their manuscript. The novelty of this study lies\nin the authors' demonstration that overexpressing asprosin induces pathological\nhypertrophy, while transgenic mice with reduced asprosin expression are protected\nagainst TAC-induced hypertrophy. They also observed that an inhibitor, BOS-318,\nreversed hypertrophy in a cell model. However, despite conducting multiple in vivo\nexperiments, they have not yet tested the therapeutic potential of this inhibitor in a TAC\nmodel, which would be a critical step in evaluating its translational relevance for heart\nfailure treatment.\nThe authors propose that asprosin binds to and inhibits the ubiquitination-mediated\ndegradation of phosphofructokinase platelet (PFKP). Consistent with a study published\n14 / 37\nearlier this year (Biochimica et Biophysica Acta - Molecular Basis of Disease, DOI:\n10.1016/j.bbadis.2024.167542), they demonstrate that cardiac-specific knockout of\nPFKP alleviates cardiac hypertrophy.\nMajor concerns:\n(1) I also have concerns regarding the reproducibility of the TAC control group between\nexperiments. In Figure 1.O, the ejection fraction of the TAC control group (TAC +\nAAV9-NC) is around 60% when compared to the TAC-Asprosin group, which has a\nlower ejection fraction. However, in Figure 2.F, the ejection fraction of the TAC control\ngroup (TAC + AAV9-NC) is approximately 40% when compared to the TAC + AAV9-\nshFBN1 group, which has a higher ejection fraction. This discrepancy raises concerns\nabout the consistency of the TAC control group across different experimental conditions.\nResponse: We sincerely thank the reviewer for the thoughtful comments and\nmeticulous evaluation of our manuscript. The concern regarding variability in ejection\nfraction (EF) values of the TAC control group across experimental batches is highly\npertinent. Upon careful review, we acknowledge this observation and provide the\nfollowing clarification.\nIn Figures 1O and 2F, EF values of the TAC control group (TAC+AAV9-NC)\nindeed show inter-batch variation. This variability may primarily be attributed to the\ninherent biological heterogeneity among individual mice subjected to TAC surgery,\nwhich manifests as differences in cardiac compensatory capacity, the extent of fibrosis,\nand the progression of pathological changes. It is worth noting that batch-to-batch\nvariations in functional readouts are not uncommon in cardiovascular research—\nparticularly in pressure overload models such as TAC (PMID: 32862709, 39557830,\n35652349). The cardiac remodeling process is influenced by multiple factors, and its\ndynamic and complex nature can contribute to fluctuations in experimental outcomes\nacross batches. Most importantly, our experimental data consistently exhibited a low\ndegree of intra-group variability across all independent replicates, further confirming\nthe high reproducibility of the results. We are confident that the current data, given the\ninternal controls and statistical rigor applied, are fully supportive of our conclusions.\nWe hope this clarification alleviates the reviewer's concern regarding the\nreproducibility of our model.\n15 / 37\n(2) I also have questions regarding the methodology. The asprosin immunoprecipitation\nshown in Figure 4.A did not demonstrate enrichment of asprosin, which is\nunderstandable, as immunoprecipitation experiments can be technically challenging.\nHowever, the Western blot signal for the co-immunoprecipitated (Co-IP) PFKP was\nmore intense than the asprosin signal. This raises important questions about how the\nimmunoprecipitation was conducted. Were the input and IP samples run on the same\ngel? Was the same amount of protein loaded for all samples? Additionally, were\nappropriate loading controls included to ensure equal protein input and confirm the\nspecificity of the pull-down? The authors should clarify these aspects to ensure the\nvalidity of their findings and rule out potential technical artifacts.\nResponse: We are grateful for these critical methodological questions, which have\ngreatly improved the rigor and reliability of our study. We especially appreciate your\ncareful scrutiny of the co-immunoprecipitation (Co-IP) experiments. Below we address\neach of your concerns point by point.\n1) Question: Regarding whether the Input and IP samples were run on the\nsame gel?\nAnswer: Yes. In each independent Co-IP experiment, the Input, specific antibody\nimmunoprecipitate (using anti-Asprosin), and isotype IgG control samples were\nelectrophoresed and transferred simultaneously on the same gel. This approach ensured\nidentical electrophoretic and transfer conditions across all samples, thereby maintaining\ncomparability of the results. The experiments were conducted using the Pierce™ Co-\nIP Kit (Thermo Fisher, catalog #26149), which significantly reduces interference from\nantibody heavy and light chains. As presented in Figure R9A (IP: Asprosin; blot:\nPFKP), we have included the full, uncropped western blot membrane to further validate\nthe accuracy and reliability of the Co-IP results.\n2) Question: Regarding whether the protein contents of all samples are the\nsame?\nAnswer: We greatly appreciate your attention to this critical technical detail. The\nsample loading volumes were carefully standardized as follows: For the input lane, 2–\n5% of the total cell lysate was loaded, with protein concentration precisely determined\nby BCA assay prior to immunoprecipitation (IP). For the IP and IgG control lanes, the\nentire elution fraction from the IP reaction was loaded.\n16 / 37\n3) Question: Regarding whether appropriate loading controls were included\nto ensure input consistency and pull-down specificity?\nAnswer: This is indeed a critical aspect, and we have given it thorough\nconsideration. Our experimental design included the following controls: An IgG isotype\ncontrol was included in every experiment. As shown in Figure R9A, the IgG control\ngroup exhibited almost no detectable PFKP signal, demonstrating that the co-\nprecipitation of PFKP and asprosin represents specific protein–protein interaction\nrather than non-specific binding. Additionally, all Input samples were normalized based\non BCA-determined protein concentrations to ensure consistent loading across samples.\n4) Question: Regarding the phenomenon that \"PFKP signal is stronger than\nAsprosin\"\nAnswer: We sincerely appreciate your observation of this phenomenon. This\nobservation is not uncommon in Co-IP experiments and can be attributed to several\ntechnical factors. One plausible explanation is that during immunoprecipitation with\nthe asprosin antibody, variables such as antibody concentration or binding affinity may\nlead to a relatively weak signal for asprosin itself. In contrast, PFKP, which interacts\nwith asprosin with high affinity, exhibits a stronger detection signal. Similar\nexperimental phenomena have been documented in the literature (e.g., PMID:\n40192103, 37153745, 37098980). Importantly, we conducted an additional validation\nexperiment using the asprosin antibody for IP (Figure R9B) (Page15, line 305), which\nclearly demonstrated a robust asprosin band, confirming the specificity of the\ninteraction between asprosin and PFKP. We believe this explanation, together with the\nsupplementary data, adequately addresses your concern. Once again, thank you for your\nrigorous and constructive feedback, which has significantly strengthened the quality\nand reliability of our study. The aforementioned data have been integrated into the\nrevised manuscript (Figure 4B).\n17 / 37\nFigure R9. Representative western blot image demonstrating the interaction between asprosin and PFKP.\n(A) Original Western blot image. (B) Co-IP experiment confirming the interaction between asprosin and PFKP.\n(3) In Figure 3K, the authors report increased glucose uptake in neonatal mouse\ncardiomyocytes (NMCM) overexpressing asprosin. However, it is unclear whether this\nincreased glucose uptake is accompanied by upregulation of glucose transporters such\nas GLUT1 or GLUT4. The authors should clarify whether the expression levels of these\ntransporters were assessed.\nResponse: We thank the reviewer for raising this important point. Cellular glucose\nuptake is facilitated by a family of glucose transporter proteins that mediate energy-\nindependent diffusion across the plasma membrane. Among more than 10 known\nglucose transporters, GLUT1 serves as a fundamental glucose transporter ubiquitously\nexpressed across various human tissues and is critically involved in basal glucose\nuptake (PMID: 11882521). To examine whether the asprosin/PFKP axis influenced\nglucose uptake in NMCMs through the regulation of glucose transporters, we assessed\nGLUT1 mRNA expression via qRT-PCR. Our results indicated that overexpression of\neither asprosin or PFKP significantly upregulates GLUT1 mRNA expression in\nNMCMs (Figure R10A, B) (page14, line 276–278 & page20, line 402–406).\nFurthermore, knockdown of FBN1 (resulting in asprosin deficiency) markedly\nattenuated Ang II-induced GLUT1 upregulation—an effect that was significantly\nrescued by concomitant overexpression of PFKP (Figure R10C) (page24, line 488–\n490). The aforementioned data have been integrated into the revised manuscript\n18 / 37\n(Figure S3K, Figure S13M, and Figure S16C).\nFigure R10. Asprosin/PFKP regulatory axis modulates GLUT1 expression levels in NMCMs.\n(A-B) qRT-PCR was performed to detect the GLUT1 gene mRNA expression levels (n=5-6). (C) GLUT1 mRNA\nexpression levels of NMCMs in the indicated groups (n=5). Statistical tests were performed using the unpaired two-\ntailed t-test in (A, B), and the one-way ANOVA with Bonferroni's multiple comparisons test in (C). *, P <0.05. **,\nP <0.01. ***, P <0.001.\nNotably, our previous findings and existing literature have indicated that asprosin\nand its downstream effector PFKP can modulate the ERK1/2 signaling pathway (PMID:\n38677690, 38982480). More importantly, our current research has identified the\nasprosin-PFKP axis as a critical regulator of the ERK1/2 signaling pathway (Figure\nR20A). As a central kinase governing cell proliferation and metabolic processes,\nERK1/2 directly phosphorylates and stabilizes HIF-1α—even under normoxic\nconditions—thereby enhancing its transcriptional activity (PMID: 3438829, 33821668,\n31695767, 19060905, 30854566, 29891648). Given that HIF-1α serves as a master\ntranscriptional regulator of glucose metabolism and directly targets GLUT1, we\nspeculate that asprosin-PFKP axis may enhance GLUT1 expression through ERK1/2-\nmediated activation and stabilization of HIF-1α.\n(4) In Figure 3M, which marker did the authors use to measure cell surface area?\nResponse: We sincerely appreciate your meticulous review of the manuscript and your\nthoughtful inquiry regarding the markers used to measure cardiomyocyte surface area\nin Figure 3M. We would like to clarify that the quantitative analysis of cardiomyocyte\nsurface area presented in Figure 3M was conducted using immunofluorescence\nstaining with α-actinin, a well-established and specific marker for cardiomyocytes.\nFurthermore, in all relevant figures of the revised manuscript that involve\n19 / 37\nimmunofluorescence staining for cardiomyocyte membrane area, we have clearly\nannotated both the figures and their corresponding captions to indicate the use of α-\nactinin as the identification and quantification marker. This marker has been widely\nutilized in numerous published studies (PMID: 34098726, 39846173, 35437018). We\nbelieve that the additional information provided can address your concerns. Once again,\nwe sincerely thank you for your valuable feedback, which has made a significant\ncontribution to enhancing the rigor and clarity of our research.\n(5) I understand that Nature Communications is not a cardiovascular specialist journal,\nand providing a broad context of asprosin’s role is appropriate. However, given that this\nstudy focuses on cardiovascular research, the authors should discuss relevant literature\non asprosin in a cardiovascular context. For example, previous studies have reported\nincreased asprosin levels in plasma from patients with hypertensive cardiomyopathy\nand other types of cardiomyopathies, this literature should be included. Additionally,\nthe authors state, \"we discovered that its (asprosin) expression was markedly increased\nin neonatal cardiomyocytes, suggesting that cardiac major cells also contribute to\nasprosin production.\" However, there is existing literature that has investigated the\nlocalization and distribution of asprosin protein in human atrial and ventricular heart\ntissue (https://doi.org/10.1093/cvr/cvae088.217), which should also be discussed to\nprovide a more comprehensive perspective on asprosin’s role in the heart.\nResponse: Thanks for reviewer`s invaluable advice. We fully agree that the existing\nevidence regarding asprosin should be more comprehensively discussed within the\ncontext of cardiovascular diseases. In response to your recommendation, we have\nincorporated the following additions and in-depth discussions in the revised manuscript\n(page27-28, line 558-584).\nReviewer #3: (Remarks to the Author):\nThis manuscript by Ming Tong et al. introduces a novel regulatory axis—YY1–\nAsprosin–PFKP—that links transcriptional and metabolic remodeling in\ncardiomyocytes during pathological hypertrophy. Using both in vivo and in vitro\nmodels, the authors propose that asprosin, derived from FBN1, binds to PFKP,\nenhancing glycolysis and suppressing oxidative metabolism. This interaction\ncontributes to mitochondrial dysfunction and the development of cardiac hypertrophy.\nOverall, the manuscript is well-structured and addresses a topic of significant relevance\nto cardiovascular biology. However, several key mechanistic gaps and missing\nexperimental controls must be addressed before the manuscript can be considered for\npublication in Nature Communications.\n20 / 37\nMajor Comments:\n(1) In Figure S6, the authors use IP-Mass to explore mechanisms underlying Asprosin-\ninduced glycolysis. However, as Asprosin is a secreted hormone-like molecule, it is\nmore appropriate to first investigate whether its effects are mediated via specific\nparacrine or autocrine pathways through defined receptors. The rationale for conducting\na binding partner screen should be explicitly described and supported by appropriate\nbackground information.\nResponse: We sincerely appreciate your insightful and constructive comments. The\nresearch approach you highlighted—that is, to prioritize investigating whether asprosin\nacts through specific receptors—is indeed a classical and essential paradigm in the\nstudy of secreted proteins, and we fully agree with its importance.\nIn this study, we opted to begin with co-immunoprecipitation followed by mass\nspectrometry (IP-MS) to screen for binding partners, based on the following\nconsiderations. IP-MS represents an unbiased screening strategy that enables a\ncomprehensive exploration of asprosin’s binding candidates, including both membrane\nreceptors and intracellular effector proteins. The use of IP-MS to identify intracellular\nbinding partners of secreted proteins is not without precedent; for example, Wang et al.\nsuccessfully applied this approach in their study of secretory SIRT1 (PMID: 40343797),\nwhich served as a methodological reference for our work.\nWe have conducted preliminary experiments on several membrane proteins, such\nas TFRC, ITGB1, ITM2B, and etc, based on our IP-MS result. However, these did not\nyield the expected outcome—that is, evidence supporting asprosin’s regulation of\ncardiomyocyte function through paracrine or autocrine mechanisms mediated by\nspecific membrane receptors. Moreover, our preliminary RNA-Seq data suggested that\nasprosin is likely involved in regulating glycolysis in pathological cardiac hypertrophy.\nConsistent with this, our Co-IP results revealed an interaction between asprosin and\nPFKP, a key glycolytic enzyme, suggesting a cooperative role in modulating glycolysis\nin cardiomyocytes. Therefore, the present study focuses primarily on the direct\ninteraction between asprosin and intracellular targets. In addition, previous studies have\nindicated that asprosin may exhibit non-classical modes of action within cells (PMID:\n21 / 37\n40231957, 35902881). These findings led us to hypothesize that, beyond potential\nreceptor-mediated mechanisms, asprosin might also engage in direct interactions with\nintracellular proteins. We once again thank you for your valuable feedback, which will\nbe highly beneficial for further refinement of our study.\n(2) In Figure 3H, the authors claim a \"direct\" interaction between Asprosin and PFKP.\nHowever, the current evidence—based on intracellular overexpression, structural\nprediction, and mutational analysis—is insufficient to conclusively demonstrate a\ndirect physical interaction. To substantiate this claim, in vitro reconstitution assays using\npurified proteins (e.g., GST pull-down) are required. Until such data are provided, the\nterm “direct binding” should be used cautiously.\nResponse: We sincerely appreciate the reviewers' insightful suggestions concerning the\nevidence for the \"direct\" interaction between asprosin and PFKP presented in Figure\n3H. As accurately noted, data derived from intracellular overexpression, structural\nprediction, and mutational analysis alone are insufficient to definitively exclude the\npossibility of indirect effects or the influence of other intracellular components. To\nrigorously establish a direct physical interaction between these two proteins, in vitro\nbinding assays employing purified proteins are essential. In response to this important\nsuggestion, we have conducted the required in vitro protein pulldown assay. In this\nexperiment, purified His-Asprosin recombinant protein was incubated with HA-PFKP\noverexpressed in HEK-293T cells lysates under physiological in vitro binding\nconditions. Our results demonstrate that His-Asprosin, used as the bait protein,\neffectively pulled down HA-PFKP, thereby providing direct evidence supporting a\nphysical interaction between asprosin and PFKP (Figure R11A) (Page15, line 306-\n307). The aforementioned data have been integrated into the revised manuscript\n(Figure 4E).\nFigure R11. Asprosin directly interacts with PFKP.\n(A) His-Tag pulldown assays for the direct binding between asprosin and PFKP (n=3).\n22 / 37\n(3) In Figure 4, the authors demonstrate that Asprosin competes with DTX3L for PFKP\nbinding, affecting its protein levels. They also suggest this regulation is important for\nglycolytic shifts under cardiac stress. However, in unstressed cardiomyocytes, DTX3L\nmay suppress PFKP levels and promote a FAO-dominant metabolic state. The basal\nmetabolic role of DTX3L should be explicitly addressed.\nResponse: We sincerely appreciate the insightful feedback provided by the reviewers.\nYour suggestion to clarify the basal metabolic role of DTX3L in unstressed\ncardiomyocytes is of great importance, as it contributes to a more comprehensive\nunderstanding of the physiological relevance of the asprosin-DTX3L-PFKP axis. In\nresponse to this valuable input, we conducted additional analyses of metabolic\nphenotypes to better define the physiological implications of DTX3L regulation under\nbasal conditions. Our results showed that DTX3L overexpression led to a modest\nreduction in basal ATP levels; however, this change did not reach statistical significance\n(Figure R12A). Furthermore, assessment of FAO capacity in NMCMs revealed that\nphysiological overexpression of DTX3L, while capable of down-regulating PFKP\nexpression, does not significantly alter overall FAO capacity (Figure R12B). These\nfindings suggest that, although DTX3L may exert a minor influence on PFKP levels\nand ATP production under basal conditions, the DTX3L-PFKP regulatory axis does not\ndrive a substantial metabolic shift in the absence of stress. Your suggestions have\nensured that our conclusions remain firmly grounded in the experimental data, while\nemphasizing the central finding of this work—the critical role of the asprosin-DTX3L-\nPFKP axis in regulating metabolic remodeling during cardiac stress.\nFigure R12. The regulatory role of DTX3L in basal metabolic processes of NMCMs.\n(A, B) ATP content (n=5) and FAO activity (n=6) in NMCMs overexpressing DTX3L were measured using ATP\nassay kit and FAO activity assay kit, respectively. Statistical tests were performed using the unpaired two-tailed t-\ntest in (A, B). ns; not significant.\n23 / 37\n(4). In Figures 3 and 4, the authors show Asprosin binds to PFKP, but it remains unclear\nhow this interaction occurs. Asprosin is typically secreted following cleavage of FBN1\nby Furin and acts in a paracrine/autocrine manner. The authors must clarify whether the\nobserved interaction occurs intracellularly before secretion or after reuptake of secreted\nAsprosin.\nResponse: We are grateful to the reviewer for raising important and thought-provoking\nquestions. As correctly noted, \"asprosin is typically secreted following cleavage of\nFBN1 by Furin and functions in a paracrine/autocrine manner,\" which prompted us to\ninvestigate whether the observed asprosin-PFKP interaction occurs via a post-secretion\nreuptake mechanism. To determine the subcellular localization of this interaction, we\nperformed a key experiment. Specifically, we treated NMCMs with high-purity\nrecombinant His-Asprosin protein (purity > 95%, endotoxin content <1 EU/μg) and\nassessed its intracellular distribution using cell cytoplasmic membrane separation\nfollowed by Western blot analysis. Our results showed no detectable enrichment of His-\nAsprosin in either the membrane or cytoplasmic fractions (Figure R13A). Furthermore,\nour previous immunofluorescence data (Figure 4F) showed that endogenous asprosin\nis primarily localized in the cytoplasm, with minimal detection on the cell membrane.\nTogether, these results indicate that exogenous asprosin is not internalized by\ncardiomyocytes to mediate its regulatory effects. Thus, our datas provide evidence that\nasprosin does not interact with PFKP via a post-secretion reuptake mechanism, but\nrather binds to it directly within the cell before secretion occurs.\nFigure R13. Exogenous asprosin exhibits limited capacity for cellular internalization into NMCMs (n=3).\n(A) Cytoplasmic membrane separation experiment was performed on NMCMs to detect the expression of His-\nasprosin recombinant protein within these cells.\n24 / 37\n(5) In Figures 4–6, the authors focus exclusively on the PFKP (platelet-type) isoform\namong the PFK family. No comparisons are made with other isoforms, such as PFKL\n(liver-type) or PFKM (muscle-type), despite PFKM being highly expressed in the heart.\nTo establish specificity of Asprosin–PFKP binding, the authors should assess\ninteractions and expression patterns of other isoforms, especially PFKM.\nResponse: We sincerely thank the reviewer for raising this important point. As correctly\nnoted, PFKM is highly expressed in the heart, and it is essential to evaluate the\ninteraction between asprosin and other PFK isoforms—such as PFKL and PFKM—to\nconfirm the specificity of its binding to PFKP. In response to this comment, we\nperformed additional Co-IP assays to assess potential interactions between asprosin and\nPFKL or PFKM. Our results clearly demonstrated that neither PFKL nor PFKM co-\nprecipitated with asprosin (Figure R14A-B) (page15-16, line 307-309). To further rule\nout the possibility that asprosin influences the expression of these isoforms, we\noverexpressed asprosin in NMCMs and examined PFKL and PFKM protein levels via\nWestern blot. As shown in Figure R14C-D (page16-17, line 328-332), asprosin\noverexpression did not significantly alter the expression of either isoform. Collectively,\nthese findings indicate that asprosin specifically interacts with PFKP and does not bind\nto other cardiac-expressed PFK isoforms, namely PFKL or PFKM. Moreover, asprosin\ndoes not modulate the expression levels of these alternative isoforms. We are deeply\ngrateful for the reviewer’s insightful and constructive feedback, which has significantly\nstrengthened the rigor and completeness of our study. Their emphasis on isozyme\nspecificity has been invaluable in clarifying the molecular mechanism underlying the\nasprosin–PFKP interaction and enhancing the overall scientific quality of the\nmanuscript. The aforementioned data have been integrated into the revised\nmanuscript (Figure 4G-H, and Figure S10A-B).\n25 / 37\nFigure R14. Overexpression of asprosin has no effect on the expression levels of PFKM and PFKL proteins.\n(A, B) CO-IP analysis of PFKM/PFKL and asprosin interaction in NMCMs (n=3). (C, D) Western blotting was\nperformed to assess the expression levels of PFKM (n=6) and PFKL (n=7) in NMCMs. Statistical tests were\nperformed using the unpaired two-tailed t-test in (C, D). ns, not significant.\n(6) In Figure 6, the authors show that shFBN1 leads to decreased Asprosin and PFKP\nexpression and mitigates hypertrophy and metabolic disturbances. However, without\nAsprosin reconstitution experiments in FBN1 knockdown models, it is unclear whether\nthe phenotype results from reduced Asprosin levels or loss of FBN1 function itself.\nSuch experiments are necessary to establish causality.\nResponse: We sincerely thank the reviewers for their insightful comments. The\nsuggestion to “reconstitute asprosin in the FBN1 knockdown model to clarify the\nattribution of the phenotype” is critically important for establishing a clear causal\nrelationship. In response, we would like to clarify the following: asprosin is a secreted\nprotein derived from the C-terminal fragment of FBN1 via Furin-mediated proteolytic\ncleavage. To determine whether the observed pathological cardiac hypertrophy is\nspecifically driven by asprosin rather than by FBN1 itself, a series of gain- and loss-of-\nfunction experiments were conducted in our earlier studies to verify its specific role.\nThe results showed that overexpression of full-length FBN1 in NMCMs significantly\npromoted pathological hypertrophy; however, this effect was completely abolished\nupon treatment with the Furin inhibitor BOS-318, which blocks FBN1 cleavage and\nprevents asprosin generation (Figure R15A, B) (page10, line 193-198). Moreover,\n26 / 37\ntransfection with a mutant FBN1 construct lacking the Asprosin-coding region (FBN1-\ndel-asprosin) failed to induce hypertrophic responses (Figure R15C, D) (Figure S4C,\nD) (page10-11, line 199-201). These results collectively demonstrate that the\nhypertrophic effects attributable to FBN1 are strictly dependent on its cleavage product\nasprosin, and are independent of other biological functions of FBN1. We deeply\nappreciate your insightful comments, which have significantly strengthened the causal\nrigor and mechanistic clarity of our study. Your expertise has been invaluable in guiding\nus toward more precise conclusions regarding the role of asprosin in cardiac\nhypertrophy. The aforementioned data have been integrated into the revised\nmanuscript (Figure S4A-D).\nFigure R15. Asprosin promotes pathological hypertrophy of NMCMs, with no involvement of fibrillin-1.\n(A) Western blot analysis was performed to evaluate the expression efficiency of the wild-type full-length FBN1\nplasmid in NMCMs (n=5). (B) Representative α-actinin staining immunofluorescence images and relative\nquantification of the cell surface area in NMCMs transfected with wild-type full-length FBN1 or vector plasmid,\nfollowed by treatment with BOS-318 (pcDNA3.1, n=119 cells; pcDNA3.1-FBN1, n=94 cells; pcDNA3.1-\nFBN1+BOS-318, n=104 cells;). (C) Western blot analysis was performed to evaluate the expression efficiency of\nthe FBN1 mutant plasmid (pcDNA3.1-Flag-FBN1-del-asprosin) in NMCMs (n=4). (D) Representative α-actinin\nstaining immunofluorescence images and relative quantitation results of cell surface area of NMCMs transfected\nwith pcDNA3.1-FBN1-del-asprosin plasmid (n=115 cells) or pcDNA3.1 (n=141 cells) plasmid. Statistical tests were\nperformed using the unpaired two-tailed t-test in (A, C, D), the one-way ANOVA with Bonferroni's multiple\ncomparisons test in (B). ***, P <0.001; ns, not significant.\n27 / 37\n(7) The authors report that FBN1 knockdown modulates glucose uptake, lactate\nproduction, and mitochondrial membrane potential (Δψm) via PFKP (Figure 6G–I).\nHowever, the impact of PFKP overexpression on mitochondrial respiration and the\nmechanism by which knockdown restores Δψm require further discussion. Given that\nTAC-induced hypertrophy involves HIF-1α/FOXO-driven PDK expression and PDH\ninhibition (Zhou et al., Circulation, 2018), the authors should evaluate PDK–PDH axis\ninvolvement using qPCR or Western blot (e.g., PDK1/4 levels, PDH phosphorylation).\nResponse: We sincerely appreciate your valuable suggestion. Your suggestion\nregarding the critical role of the PDK–PDH axis in TAC-induced cardiac hypertrophy—\nparticularly its connection to the HIF-1α/FOXO pathway (Zhou et al., Circulation,\n2018)—provided key mechanistic insight into how PFKP regulates mitochondrial\nfunction. In response to your suggestion, we performed additional Western blot\nanalyses to evaluate the PDK–PDH axis. Our results clearly demonstrate that PFKP\noverexpression significantly increased PDK4 protein expression, PDH phosphorylation\nand reduced PDH activity levels in NMCMs (Figure R16A, B) (page21, line 427-434).\nConversely, PFKP knockdown markedly attenuated Ang II-induced upregulation of\nPDK4, phosphorylation of PDH and reduced PDH activity levels (Figure R16C, D)\n(page21-22, line 438-442). Furthermore, in rescue experiments performed in NMCMs,\nFBN1 knockdown (which silences asprosin expression) significantly suppressed Ang\nII-induced PDK4 upregulation, PDH phosphorylation and reduced PDH activity levels.\nImportantly, these effects were reversed by concurrent PFKP overexpression, which\nrestored PDK4 expression, PDH phosphorylation and reduced PDH activity levels\n(Figure R16E, F) (page24, line 491-497). These findings align closely with the\nmechanism highlighted in the cited reference. Our results demonstrate that activation\nof the asprosin/PFKP signaling pathway induces a metabolic shift from oxidative\nphosphorylation to glycolysis, which is mediated through the upregulation of PDK4\nand subsequent inhibition of PDH activity. This mechanism provides a direct\nexplanation for our observations of suppressed mitochondrial respiration, enhanced\nlactate production, and alterations in mitochondrial membrane potential (ΔΨm). Your\ncomments have substantially strengthened the mechanistic depth and completeness of\nour study, enabling us to delineate a coherent signaling pathway from asprosin–PFKP\n28 / 37\nto downstream metabolic reprogramming. We thank you once again for your highly\ninstructive feedback. The aforementioned data have been integrated into the\nrevised manuscript (Figure S13O-Q, Figure S14J-M, and Figure S18A-B).\nFigure R16. The Asprpsoin-PFKP axis regulates the expression of PDK4/PDH.\n(A) The protein levels of PFK4 and pPDH/PDH in NMCMs transfected with PFKP or vector overexpression plasmid\nwere detected by western blot (n=6). (B) Quantitative examination of PDH activity levels of NMCMs in the indicated\ngroups (n=6). (C) Western blot was subjected to dectect PDK4 (n=5) and pPDH/PDH (n=8) protein levels in NMCMs\ntransfected with siNC or siFBN1 and treated with Ang II for 24h. (D) Quantitative examination of PDH activity\nlevels of NMCMs in the indicated groups (n=6). (E) Western blot was subjected to dectect PDK4 (n=6) and\npPDH/PDH (n=5) protein levels of NMCMs in the indicated groups. (F) Quantitative examination of PDH activity\nlevels of NMCMs in the indicated groups (n=6). Statistical tests were performed using the unpaired two-tailed t-test\nin (A, B), and the one-way ANOVA with Bonferroni's multiple comparisons test in (C, D, E, F) *, P <0.05; ** P\n<0.05; ***, P <0.001.\nFurthermore, to directly assess whether PFKP regulates mitochondrial function via\nPDK4, we employed the PDK4 inhibitor dichloroacetate (DCA). By inhibiting PDK4\nactivity, DCA promoted the dephosphorylated active form of PDH, thereby enhancing\npyruvate flux through the tricarboxylic acid cycle (PMID: 12621116). Results\ndemonstrated that DCA treatment significantly attenuated PFKP overexpression-\ninduced pathological cardiac hypertrophy, loss of mitochondrial membrane potential,\n29 / 37\nand impairment of cellular respiration (Figure. R17A-D) (page22, line 446-450).\nThese findings provide compelling evidence that PFKP predominantly mediates\nmitochondrial dysfunction and the subsequent development of pathological cardiac\nhypertrophy through the PDK4/PDH signaling axis. The aforementioned data have\nbeen integrated into the revised manuscript (Figure S13T-W).\nFigure R17. PFKP directly interacts with and regulates PDK4, thereby playing a role in the pathological\ndevelopment of cardiac hypertrophy.\n(A) qRT-PCR was performed to detected the expression level of hypertrophy marker mRNA in NMCMs transfected\nwith the PFKP overexpression plasmid subsequently treated with DCA (5mM) (n=5). (B) Representative α-actinin\nstaining immunofluorescence images (left) and quantitative results of the cell surface area (right) of NMCMs\ninfected with the PFKP overexpression plasmid and treated with DCA (Vector: n=117 cells; PFKP: n=88 cells;\nPFKP+DCA: n=116 cells). (C) Analysis and quantification of OCR (n=5) of the NMCMs in the indicated groups.\n(D) Representative fluorograms and quantification of mean fluorescence intensity of TMRE in NMCMs transfected\nwith PFKP or vector overexpression plasmid and and subsequently treated with DCA (n=8, independent\nexperiments). Statistical tests were performed using the one-way ANOVA with Bonferroni's multiple comparisons\ntest in (A, C, D). and the Kruskal-Wallis test with Dunn's multiple comparisons test in (B). *, P <0.05. **, P <0.01.\n***, P <0.001.\n(8) In Figures S10A–J, PFKP overexpression in neonatal mouse cardiomyocytes\n(NMCMs) promotes glucose uptake, lactate secretion, and reduces Δψm. Figure S11H\nshows PFKP knockdown attenuates Ang II-induced Δψm reduction. However, the\npotential impact of PFKP on mitochondrial respiration beyond glycolysis and the\nmechanism of Δψm protection upon knockdown remain unclear. Given known\nmetabolic shifts following TAC (Zhou et al., 2018), the authors should assess whether\nPFKP alters pyruvate entry into mitochondria and explore PDK–PDH pathway changes.\nResponse: We sincerely appreciate your suggestions. In accordance with your advice,\nwe have performed additional experiments to evaluate changes in pyruvate metabolites\nand acetyl-CoA levels. The new results clearly demonstrated that PFKP overexpression\n30 / 37\nsignificantly upregulated pyruvate accumulation along with a reduction in acetyl-CoA\nlevels (Figure R18A, B) (page21, line 434-438). Conversely, PFKP knockdown\nmarkedly attenuated Ang II-induced upregulation of PDK4 expression and PDH\nphosphorylation, and normalized the altered pyruvate and acetyl-CoA contents (Figure\nR18C, D) (page21-22, line 438-442). These findings demonstrate that PFKP directly\nmodulates pyruvate metabolism by regulating its conversion to acetyl-CoA via the\nPDK4-PDH pathway, thereby influencing its entry into the TCA cycle. Furthermore, in\nrescue experiments conducted in neonatal mouse cardiac myocytes (NMCMs), we\nobserved that FBN1 knockdown, which silences asprosin expression, significantly\nsuppressed Ang II-induced upregulation of PDK4, phosphorylation of PDH, pyruvate\naccumulation, and reduction in acetyl-CoA levels. Importantly, these effects were\nreversed by concomitant overexpression of PFKP. (Figure R18E, F) (page24, line 495-\n500) Collectively, our data demonstrate that activation of the asprosin-PFKP axis\nupregulates PDK4 and inhibits PDH activity, thereby impairing the conversion of\npyruvate to acetyl-CoA. This results in pyruvate accumulation and reduced acetyl-CoA\nlevels, providing a direct mechanistic explanation for the observed suppression of\nmitochondrial respiration, elevated lactate production, and alterations in ΔΨm. Your\nvaluable comments have greatly enhanced the mechanistic depth and completeness of\nour study, and we are grateful for your guidance in strengthening this work. The\naforementioned data have been integrated into the revised manuscript (Figure\nS13R-S, Figure S14N-O, and Figure S18C-D).\n31 / 37\nFigure R18. The Asprosin-PFKP axis dynamically regulates pyruvate accumulation and acetyl-CoA\nproduction, thereby influencing mitochondrial metabolic flux.\n(A-F) Quantitative examination of pyruvate and Acetyl CoA levels of NMCMs in the indicated groups (n=6).\nStatistical tests were performed using the unpaired two-tailed t-test in (A, B), and the one-way ANOVA with\nBonferroni's multiple comparisons test in (C, D, E, F). *, P <0.05; ***, P <0.001.\n(9) In addition to JC-1 staining for Δψm, correlating ATP production and oxygen\nconsumption rate (OCR) would strengthen the metabolic interpretation. If glycolysis is\nupregulated in the absence of PDH inhibition, the role of LDHA in determining\npyruvate fate should also be considered. At present, it is unclear whether PFKP\nknockdown improves Δψm via reduced lactate production or enhanced TCA flux\nthrough PDH. Further pathway analyses are required.\nResponse: Thank you very much for your valuable comments. The question you\nraised—regarding the need to clarify the metabolic fate of pyruvate following PFKP\nknockout (i.e., whether the improvement in Δψm is mediated through reduced lactate\nproduction or enhanced TCA cycle flux)—is crucial for further refining the metabolic\nmechanisms underlying our findings. In this study, our previous experimental results\nhave demonstrated that PFKP plays a pivotal role in pathological cardiac hypertrophy\nby modulating the PDK4/PDH axis (Figure R16A-F and Figure R17A-D). Besides,\nwe demonstrated that the mechanism underlying the benefits of PFKP knockdown lies\nin its capacity to reduce Ang II-induced PDH phosphorylation, leading to enhanced\nPDH activity, increased TCA cycle flux, and improved mitochondrial function, as\n32 / 37\nopposed to a mechanism involving LDHA-directed pyruvate-to-lactate conversion. We\nhighly value your suggestions. Building upon these insights, we plan to incorporate\nmore comprehensive detection and analysis of LDHA in future studies to further\nelucidate the role of the asprosin-PFKP axis in regulating cardiovascular diseases\nthrough lactate production. This line of investigation will not only expand the current\nunderstanding of this research field but also provide novel perspectives into the\nmetabolic regulatory network in cardiovascular pathophysiology.\n(10) The authors propose that the Asprosin–PFKP axis enhances glycolysis, suppresses\noxidative metabolism, and thereby reduces Δψm and promotes hypertrophy. If so,\ninhibiting PDK or LDHA would promote TCA flux and oxidative metabolism,\npotentially alleviating hypertrophy. Conversely, PDH inhibition might worsen\npathology. Pharmacological modulation of the PDK–PDH axis would help clarify the\nrole of this metabolic switch. Supplementary experiments using agents like\ndichloroacetate (DCA) are warranted and could have translational implications.\nResponse: We sincerely thank the reviewers for their insightful and constructive\ncomments. The suggestion to pharmacologically modulate the PDK–PDH axis was\ninstrumental in enhancing the mechanistic and translational depth of our study. In\nresponse, we performed additional experiments using the PDK inhibitor DCA. NMCMs\nwere divided into the following groups: 1) empty vector control (Vector), 2) asprosin\noverexpression (Asprosin), and 3) asprosin overexpression with DCA treatment\n(Asprosin+DCA). Western blot and qPCR analyses revealed that DCA treatment\nsignificantly attenuated asprosin-induced cardiomyocyte hypertrophy, as indicated by\nreduced expression of hypertrophy markers (ANP, BNP, and β-MHC) and decreased\ncell surface area (Figure R19A–C) (page24, line 500-504). Moreover, asprosin\noverexpression resulted in pyruvate accumulation, decreased acetyl-CoA levels,\nsuppressed PDH activity, and reduced ATP content. Importantly, DCA treatment\neffectively reversed these metabolic alterations: it diminished pyruvate accumulation,\nelevated acetyl-CoA levels, restored PDH activity, and increased ATP production\n(Figure R19D–G) (page24, line 500-504). These findings demonstrate that\npharmacological inhibition of PDK by DCA corrects the metabolic reprogramming\ninduced by the asprosin–PFKP axis and mitigates subsequent pathological hypertrophy.\n33 / 37\nOur results not only confirm the PDK–PDH axis as a key downstream effector of this\npathway but also underscore its potential as a therapeutic target. The aforementioned\ndata have been integrated into the revised manuscript (Figure S18E-K).\nFigure. S19 Asprosin is involved in the regulation of pathological cardiac hypertrophy through direct\nmodulation of PDK4.\n(A, B) qRT-PCR and western blot were subjected to detect cardiac hypertrophy markers mRNA (ANP: n=5; BNP,\nβ-MHC: n=6) and protein (n=5-6) levels in NMCMs transfected with asprosin or vector overexpression plasmid and\ntreated with DAC. (C) Representative α-actinin staining immunofluorescence images and quantitative cell surface\narea of NMCMs in the indicated groups (Vector: n=100 cells; Asprosin: n=128 cells; Asprosin+DCA: n=108 cells).\n(D-G) Quantitative examination of ATP, PDH activity, pyruvate and Acetyl CoA levels of NMCMs in the indicated\ngroups (PDH activity: n=6; Pryruvate: n=6; Acetyl CoA: n=8; ATP: n=6). Statistical tests were performed using the\none-way ANOVA with Bonferroni's multiple comparisons test in (A, B, D, E, F, G), and the Kruskal-Wallis test with\nDunn's multiple comparisons test in (C). *, P <0.05; ** P <0.05; ***, P <0.001.\n(11) In Figures S13A–C and 6O,P, FBN1 knockdown suppresses Ang II-induced\nhypertrophy, which is reversed by PFKP overexpression. While intriguing, the link\nbetween FBN1–PFKP and classical hypertrophy pathways (NFAT, ERK1/2, Akt,\nmTOR) remains unexplored. Evaluating these pathways would enhance understanding\nof the interplay between metabolism and signal transduction.\nResponse: Thank you sincerely for your insightful and constructive comments. Your\nsuggestion to investigate the connection between the FBN1 (asprosin)-PFKP axis and\nclassical cardiac hypertrophy signaling pathways is highly valuable, as it will\nsignificantly deepen our understanding of the crosstalk between metabolic\nreprogramming and signal transduction. We concur with your perspective and wish to\nhighlight that accumulating evidence indicates both asprosin and PFKP independently\ncontribute to the activation of the ERK1/2 signaling pathway (PMID: 38677690,\n34 / 37\n31194992, 38982480). Based on this knowledge, we have further explored whether the\nasprosin-PFKP axis contributes to the pathogenesis of pathological cardiac hypertrophy\nthrough modulation of the ERK1/2 signaling pathway. Our experimental results clearly\ndemonstrated (Figure R20A): in the Ang II-induced NMCMs pathological cardiac\nhypertrophy model, the ERK1/2 pathway was significantly activated. However,\nknockdown of FBN1 (which leads to asprosin deficiency) markedly suppressed the\nactivation of this pathway. Crucially, overexpression of PFKP in FBN1-knockdown\ncells effectively reversed this inhibition and restored ERK1/2 signaling activity (Figure\nR20A). These findings indicated that ERK1/2 serves as a key downstream signaling\nnode of the asprosin-PFKP axis.\nTo further validate the functional involvement of the ERK1/2 pathway in this\ncontext, the specific ERK1/2 inhibitor ulixertinib was administered concurrently in the\nangiotensin II-induced model of pathological cardiac hypertrophy in NMCMs. Our\nresults showed that blocking ERK1/2 activity significantly attenuated the upregulation\nof cardiac hypertrophy-related genes induced by the asprosin-PFKP axis (Figure\nR20B), thereby supporting the conclusion that the asprosin-PFKP axis at least partially\nmediated pathological cardiac hypertrophy via ERK1/2 activation. In summary, this\nstudy not only confirms the presence of the asprosin-PFKP axis in the context of cardiac\nhypertrophy but also establishes a direct link between this metabolic axis and the\nclassical ERK1/2 signaling pathway, thereby providing initial mechanistic insights into\nhow metabolic regulators interface with core signaling cascades.\nHowever, we also recognize that other classical signaling pathways—such as those\nmediated by NFAT, Akt, and mTOR—may interplay with asprosin in regulating\npathological cardiac hypertrophy, which represents an important direction for future\nresearch. We greatly appreciate your valuable insights, which have significantly\nenhanced the mechanistic depth and overall rigor of our study.\n35 / 37\nFigure R20. The Asprosin-PFKP axis activates the ERK1/2 signaling pathway.\n(A) Western blot was performed to detect pERK/tERK protein expression (n=7). (B) The mRNA expression of\ncardiac hypertrophy markers were detected by qRT-PCR indicated groups (n=5-7). Statistical tests were performed\nusing the the one-way ANOVA with Bonferroni's multiple comparisons test in (A, B). **, P <0.01. ***, P <0.001.\nMinor Comments:\n(1). Figures 3, 4, and 6 include more than 10 panels each, making them difficult to\ninterpret. Splitting them into separate figures would improve clarity.\nResponse: Thanks for reviewer`s invaluable advice. As you pointed out, Figures 3, 4,\nand 6 were difficult to interpret due to the excessive number of panels, and we fully\nagree with this assessment. In response, we have reorganized these figures by splitting\nthem into separate, more focused figures in the revised manuscript to improve clarity\nand readability.\n(2) On page 20, line 2, \"Glycogen\" appears to be a typographical error and should be\ncorrected to \"Glucose\".\nResponse: Thank you sincerely for your careful and thoughtful review of our\nmanuscript. We truly appreciate your attention to detail, which helped us identify the\nincorrect use of the term \"Glycogen\" on line 2 of page 20. Upon review, we confirmed\nthat this was a typographical error, and the correct term should be \"Glucose\"(Page20,\nline 399). We have carefully proofread and made the necessary correction in the revised\n36 / 37\nmanuscript.\nBelieving that all issues raised by you and the reviewers have been fixed, we now\nresubmit the manuscript for further consideration. Thank you and the reviewers for your\ninterest in our work.\nSincerely,\nYan Zhang, Ph.D.\nProfessor in Pharmacology, College of Pharmacy\nHarbin Medical University\nHarbin, Heilongjiang 150081 P. R. China\nE-mail: zhangyan@ems.hrbmu.edu.cn\n37 / 37"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The author have addressed most of the concerns of the reviewers', and the manuscript is now acceptable.", "reviewer_2": "I appreciate the authors’ extensive dataset and the scope of experimentation, and I thank them for addressing many of my\nprior comments. Their clarifications were helpful and several suggestions appear to have been incorporated. That said, two\nsubstantive concern remains unresolved.\nThe reported ejection fraction for the same control condition (TAC + AAV9-NC) differs markedly between Figures 1.O and\n2.F (≈40% vs ≈55%). If these panels are meant to represent the same control condition, such a discrepancy would likely be\nstatistically significant under an appropriate test. While variability is expected in vivo, the pattern here is troubling: the control\nEF appears to move opposite to the experimental group (higher when the experimental EF is lower, and vice versa) and\ncould raise concerns about analysis bias.\nIt remains unclear whether the immunoprecipitation (IP) in Figure 4B worked as intended and I remain concerned about the\nvalidity of the co-IP. In a well-performing IP, especially a FLAG-IP designed for purification, the Flag-protein should be\nenriched in the IP fraction relative to the input. However, the uncropped blot (R9A) shows asprosin markedly reduced versus\ninput, while the PFKP co-IP band appears stronger than Flag-asprosin, a pattern more consistent with inefficient pull-down or\nnon-specific carry-over (e.g., inadequate wash stringency). To resolve this, please provide full uncropped blots for Figure\n4B with molecular-weight markers and matched exposures and include essential controls: anti-FLAG beads incubated with\nlysate lacking FLAG-asprosin (negative control) ± His-tagged PFKP and beads-only control. A total-protein stain (Ponceau\nor Coomassie) to document loading would further strengthen the evidence. Without such controls and clear enrichment of\nasprosin, it is difficult to conclude that the IP was successful or that the PFKP co-IP reflects a specific interaction rather than\nartefact.", "reviewer_4": "The authors have provided a detailed and constructive rebuttal, together with substantial new experimental data. I greatly\nappreciate their sincere efforts to address my previous concerns in a thorough and transparent manner.\nKey findings:\nThis study convincingly demonstrates that asprosin stabilizes PFKP and induces metabolic remodeling in pathological\ncardiac hypertrophy via the PDK4–PDH pathway. Of particular importance, the authors provide compelling in vivo and in\nvitro evidence that FBN1-derived asprosin acts specifically in cardiomyocytes and contributes to the mechanisms underlying\ncardiac hypertrophy. These findings add both novelty and persuasiveness to the field.\nSignificance and originality:\nThe study goes beyond the established concept of asprosin as an adipose-derived adipokine and instead introduces the\nnovel idea that cardiomyocyte-derived asprosin directly contributes to metabolic remodeling and pathological hypertrophy.\nThe significance and originality of this concept are clearly demonstrated.\nSupport for conclusions:\nThe additional experiments—including PFKP knockdown and overexpression, FBN1 mutant analysis, and pharmacological\ninhibition with a Furin inhibitor—strongly support the asprosin dependency of the observed effects. While rescue\nexperiments with exogenous asprosin in the shFBN1 knockdown model were not performed, the current evidence is\nsufficient to support the conclusions.\nData analysis and interpretation:\nNo major issues are identified. The asprosin–PFKP interaction is supported by new pulldown data; however, the evidence is\nnot sufficient to definitively conclude “direct binding.” More cautious terminology such as “interaction” or “binding” would\ntherefore be more appropriate.\nMethodological rigor:\nThe overall methodology is sound and consistent with standards in the field. The rationale for IP-MS screening is well\njustified by the biological background, the absence of interactions with candidate membrane proteins, and references to\nprevious studies reporting secreted proteins interacting with intracellular molecules. In addition, the roles of DTX3L under\nbasal conditions, isoform specificity among PFK family members, the PDK–PDH axis, and classical hypertrophic signaling\npathways have all been carefully examined, providing strong support for the conclusions.\nReproducibility and clarity:\nThe methods are described in sufficient detail in the Supplemental Information to ensure reproducibility. The reorganization\nof Figures 3, 4, and 6 has improved clarity, and typographical errors have been appropriately corrected.\nOverall assessment:\nIn conclusion, the authors’ responses are both sufficient and appropriate, convincingly addressing the major concerns raised in the previous round.\nAlthough some questions remain for future investigation, the current revision provides robust evidence and offers clear value\nfor publication. I recommend acceptance after minor editorial revisions."}, "author_response": "Responses to Reviewers’ Comments\nCorresponding Author: Professor Yan Zhang\nDear Reviewers:\nThank you for your thoughtful letter and constructive advice. We sincerely appreciate\nthe comments provided by you and the reviewers regarding our manuscript\n(NCOMMS-25-14208A) entitled “YY1/Asprosin/PFKP Axis Regulates Glycolytic\nMetabolism and Exacerbates Pathological Cardiac Hypertrophy.”\nWe have carefully considered all the feedback and have made revisions accordingly. In\naddition, we performed several new experiments as suggested, which further strengthen\nour conclusions.\nBelow, we provide detailed, point-by-point responses to all reviewer comments.\nPoint-by-point Responses to The Reviewer’s Criticisms\nReviewer #1: (Remarks to the Author):\nThe author have addressed most of the concerns of the reviewers', and the manuscript\nis now acceptable.\nResponse: We are thrilled to receive the reviewer's positive evaluation. Their guidance\nthroughout the review process has been instrumental in strengthening our manuscript,\nand we are grateful for their time and expertise.\nReviewer #2: (Remarks to the Author):\nI appreciate the authors’ extensive dataset and the scope of experimentation, and I thank\nthem for addressing many of my prior comments. Their clarifications were helpful and\nseveral suggestions appear to have been incorporated. That said, two substantive\nconcern remains unresolved.\nMajor concerns:\n1 / 9\n(1) The reported ejection fraction for the same control condition (TAC + AAV9-NC)\ndiffers markedly between Figures 1.O and 2.F (≈40% vs ≈55%). If these panels are\nmeant to represent the same control condition, such a discrepancy would likely be\nstatistically significant under an appropriate test. While variability is expected in vivo,\nthe pattern here is troubling: the control EF appears to move opposite to the\nexperimental group (higher when the experimental EF is lower, and vice versa) and\ncould raise concerns about analysis bias.\nResponse:\nWe sincerely appreciate your meticulous review of our data and your insightful\ncomments regarding the variation in ejection fraction values for the control group\nbetween Figures 1.O and 2.F. We fully understand the concerns this discrepancy may\nraise, particularly regarding analytical consistency and data interpretation. To ensure\nthe absolute reliability of our data, upon receiving the first round of reviewers'\ncomments, we promptly conducted a new and independent repeat experiment for the\nentire set of experimental groups (Sham+AAV9-NC; TAC+AAV9-NC; TAC+AAV9-\nAsprosin). This replication was designed to rigorously re-evaluate the regulatory role\nand functional significance of cardiomyocyte-specific asprosin overexpression in TAC-\ninduced pathological hypertrophy.\nThe new experimental results showed that, compared with the Sham + AAV9-NC\ngroup (mean EF: 78%), the model control group (TAC + AAV9-NC) exhibited a mean\nEF of 42.6%, while the experimental group (TAC + AAV9-Asprosin) showed a further\nreduced mean EF of 27.9% (Figure R1A-B). These findings consistently reaffirm the\nregulatory role of cardiomyocyte-specific asprosin overexpression in exacerbating\nTAC-induced pathological cardiac hypertrophy. Importantly, the mean EF of the newly\ngenerated model control group (TAC + AAV9-NC) closely aligns with the value\nobserved for the TAC + AAV-shNC group in Figure 2F (mean EF: 41.5%) and falls\nwithin the same baseline range as other established model groups. This concordance\nestablishes a coherent and reliable internal control framework, thereby strengthening\nthe consistency and reproducibility of our experimental data.\nConsistent with your earlier concerns, our in-depth investigation revealed that the\n2 / 9\ninitial discrepancies in cardiac function measurements across control groups were\nattributable to biological variability within a specific experimental batch, rather than\nsystematic analytical bias. To ensure data integrity and reliability, we have\ncomprehensively updated the statistical analyses and experimental results from the new\nanimal cohort in this group within the revised manuscript (Figure 1M&O). These new\nfindings provide a more reliable and solid foundation for our central conclusions.\nFigure R1. Assessment of cardiac function in mice using echocardiography.\n(A-B) Representative M-mode echocardiogram of mice and quantitative analysis of EF, FS, LVPWd, LVPWs,\nLVIDd, and LVIDs in different groups of mice described in indicated groups (Sham+AAV9-NC: n=10; TAC+AAV9-\nNC: n=12; TAC+AAV9-Asprosin: n=13). Statistical tests were performed using the one-way ANOVA with\nBonferroni's multiple comparisons test in (B). ***, P <0.001.\n3 / 9\n(2) It remains unclear whether the immunoprecipitation (IP) in Figure 4B worked as\nintended and I remain concerned about the validity of the co-IP. In a well-performing\nIP, especially a FLAG-IP designed for purification, the Flag-protein should be enriched\nin the IP fraction relative to the input. However, the uncropped blot (R9A) shows\nasprosin markedly reduced versus input, while the PFKP co-IP band appears stronger\nthan Flag-asprosin, a pattern more consistent with inefficient pull-down or non-specific\ncarry-over (e.g., inadequate wash stringency). To resolve this, please provide full\nuncropped blots for Figure 4B with molecular-weight markers and matched exposures\nand include essential controls: anti-FLAG beads incubated with lysate lacking FLAG-\nasprosin (negative control) ± His-tagged PFKP and beads-only control. A total-protein\nstain (Ponceau or Coomassie) to document loading would further strengthen the\nevidence. Without such controls and clear enrichment of asprosin, it is difficult to\nconclude that the IP was successful or that the PFKP co-IP reflects a specific interaction\nrather than artefact.\nResponse: We sincerely appreciate the reviewer's insightful comment regarding the\nimmunoprecipitation data. This has prompted us to clarify that Figure 4B was intended\nto show an endogenous interaction in cardiomyocytes and did not employ a Flag-tag\nantibody Co-IP. We recognized that the specific issues raised regarding the Flag-IP\nprocedure rightly pertain to Figure 4D, where we overexpressed Flag-asprosin in HEK-\n293T cells. Below, we provided a comprehensive response to these pertinent concerns\nabout the experimental design and interpretation of the results in Figure 4D. We would\nlike to clarify that the western blot analysis was performed using the ODYSSEY dual-\nchannel infrared fluorescence detection system, and the images were acquired and\nprocessed with Image Studio software. During the preparation of Figure 4D, the\ndisplay intensity of the FLAG-asprosin channel was appropriately reduced to clearly\nvisualize band morphology and prevent signal saturation. We acknowledge that the data\npresentation may have obscured a clear interpretation of the immunoprecipitation\nresults, and we apologize for any valid questions it has raised regarding the reliability\nof the apparently stronger PFKP signal compared to Flag-asprosin.\nIn this study, all co-immunoprecipitation (Co-IP) experiments investigating the\ninteraction between Flag-asprosin and HA-PFKP were performed using the Pierce™\nCo-Immunoprecipitation Kit (Thermo Fisher, #26149), which effectively circumvented\nthe non-specific interference from co-eluting antibody heavy and light chains inherent\n4 / 9\nto traditional Protein A/G magnetic bead methods by covalently immobilizing\nantibodies onto amine-reactive resins. As recommended, the following co-\nimmunoprecipitation experimental groups were included to further validate the binding\ninteraction between Flag-asprosin and HA-PFKP:\n1) Experiment group lane: Immunoprecipitation (IP) was performed using anti-Flag\ncoupling resins on lysates co-expressing Flag-asprosin and HA-PFKP.\n2) Antibody negative control lane: The same co-expression lysate was subjected to IP\nusing mouse IgG isotype control antibody coupled to resins.\n3) Flag-tag specificity control lane: Lysates expressing HA-PFKP alone (without\nFlag-asprosin) were immunoprecipitated with anti-Flag coupling resins to assess\nbinding specificity dependent on the Flag tag.\n4) Coupling resins background control lane: HA-PFKP lysates were incubated with\ncoupling resins alone (no antibody) to evaluate non-specific adsorption to the resins.\n5) Cellular background control lane: Lysates from cells transfected with empty\nvector were used for IP with anti-Flag coupling resins to rule out non-specific\ninteractions mediated by overexpression or cellular components.\n6) Absolute negative control lane: Pure RIPA lysis buffer was incubated with\nantibody-free conjugated resins to confirm absence of contamination or signal from\nresins.\nFor western blot analysis, the entire Co-IP eluate was loaded, while the input\ncontrol represented 1% of the total cell lysate volume. The results are shown in Figure\nR2A. Based on the complete uncropped blots (Figure R2A), the following conclusions\ncan be drawn: 1) Specific Enrichment and Interaction: In the experiment group lane\n(Lane 1), anti-Flag coupling resins efficiently immunoprecipitated Flag-asprosin from\nlysates co-expressing Flag-asprosin and HA-PFKP, with clear co-precipitation of HA-\nPFKP. The markedly stronger signal of Flag-asprosin relative to HA-PFKP further\nsupports effective pull-down and a specific interaction. 2) Absence of Antibody-\n5 / 9\nMediated Non-Specific Binding: No Flag-asprosin or HA-PFKP bands were detected\nwhen anti-IgG coupling resins were used (Lane 2), indicating that the observed\ninteraction is not attributable to non-specific binding. 3) Dependency on Flag-asprosin\nExpression: In all samples lacking Flag-asprosin expression (Lanes 3–6), HA-PFKP\nwas not pulled down, irrespective of whether anti-Flag (Lanes 3&5) or bare coupling\nresins (Lanes 4&6) were used, demonstrating that the interaction strictly requires the\npresence of Flag-asprosin.\nWe performed Coomassie Brilliant Blue staining on the eluted Co-IP samples as\nsuggested by the reviewer. As shown in Figure R2B (Lane 1), two specific protein\nbands (with molecular weights below 35 kDa and approximately 100 kDa, respectively)\nwere clearly observed in the lysates co-expressing Flag-asprosin and HA-PFKP\nfollowing immunoprecipitation with anti-Flag conjugated resin. Crucially, the\nmolecular sizes of these bands correspond to the expected sizes of Flag-asprosin and\nHA-PFKP, respectively. In contrast, no characteristic bands for HA-PFKP or Flag-\nasprosin were detected at the corresponding positions in any of the control lanes\n(Figure R2B, Lane 2-6). Collectively, these results strongly support that the interaction\nbetween Flag-asprosin and HA-PFKP is highly specific and reproducible.\n6 / 9\nFigure R2. Representative CO-IP original Coomassie Brilliant Blue staining and western image.\nTo further substantiate the rigor of our study, we performed additional co-\nimmunoprecipitation (Co-IP) assays. As shown in the Figure R3A (now included as\nFigure 4D in the revised manuscript), we observed efficient and specific co-\nprecipitation between HA-PFKP and Flag-asprosin. This result effectively excludes\nnon-specific binding as a major contributing factor. We believe these additional data\nsatisfactorily address the reviewer's legitimate concerns and provide compelling\nevidence for a specific interaction between asprosin and PFKP.\nFigure R3. Representative image demonstrating the interaction between Flag-asprosin and HA-PFKP.\nReviewer #4: (Remarks to the Author):\nThe authors have provided a detailed and constructive rebuttal, together with substantial\nnew experimental data. I greatly appreciate their sincere efforts to address my previous\nconcerns in a thorough and transparent manner.\nKey findings:\nThis study convincingly demonstrates that asprosin stabilizes PFKP and induces\nmetabolic remodeling in pathological cardiac hypertrophy via the PDK4–PDH pathway.\nOf particular importance, the authors provide compelling in vivo and in vitro evidence\nthat FBN1-derived asprosin acts specifically in cardiomyocytes and contributes to the\nmechanisms underlying cardiac hypertrophy. These findings add both novelty and\npersuasiveness to the field.\nSignificance and originality:\nThe study goes beyond the established concept of asprosin as an adipose-derived\nadipokine and instead introduces the novel idea that cardiomyocyte-derived asprosin\ndirectly contributes to metabolic remodeling and pathological hypertrophy. The\nsignificance and originality of this concept are clearly demonstrated.\nSupport for conclusions:\nThe additional experiments—including PFKP knockdown and overexpression, FBN1\nmutant analysis, and pharmacological inhibition with a Furin inhibitor—strongly\nsupport the asprosin dependency of the observed effects. While rescue experiments with\nexogenous asprosin in the shFBN1 knockdown model were not performed, the current\nevidence is sufficient to support the conclusions.\nData analysis and interpretation:\nNo major issues are identified. The asprosin–PFKP interaction is supported by new\npulldown data; however, the evidence is not sufficient to definitively conclude “direct\nbinding.” More cautious terminology such as “interaction” or “binding” would\ntherefore be more appropriate.\nMethodological rigor:\nThe overall methodology is sound and consistent with standards in the field. The\nrationale for IP-MS screening is well justified by the biological background, the\nabsence of interactions with candidate membrane proteins, and references to previous\nstudies reporting secreted proteins interacting with intracellular molecules. In addition,\nthe roles of DTX3L under basal conditions, isoform specificity among PFK family\nmembers, the PDK–PDH axis, and classical hypertrophic signaling pathways have all\nbeen carefully examined, providing strong support for the conclusions.\nReproducibility and clarity:\nThe methods are described in sufficient detail in the Supplemental Information to\nensure reproducibility. The reorganization of Figures 3, 4, and 6 has improved clarity,\nand typographical errors have been appropriately corrected.\nOverall assessment:\nIn conclusion, the authors’ responses are both sufficient and appropriate, convincingly\naddressing the major concerns raised in the previous round. Although some questions\nremain for future investigation, the current revision provides robust evidence and offers\nclear value for publication. I recommend acceptance after minor editorial revisions.\nResponse: We sincerely thank the reviewer for their highly positive feedback.\nBelieving that all issues raised by you and the reviewers have been fixed, we now\nresubmit the manuscript for further consideration. Thank you and the reviewers for your\ninterest in our work.\nSincerely,\nYan Zhang, Ph.D.\nProfessor in Pharmacology, College of Pharmacy\n8 / 9\nHarbin Medical University\nHarbin, Heilongjiang 150081 P. R. China\nE-mail: zhangyan@ems.hrbmu.edu.cn\n9 / 9"}, {"round": 3, "reviewer_comments": {"reviewer_2": "Thank you for addressing my previous comments and for repeating the experiment. In the revised dataset, the model control group (TAC + AAV9-NC) now shows a mean EF of 42.6%, which is noticeably lower than the mean of approximately 55% reported in your earlier dataset and is now consistent with the same control condition shown in Figure 2F. I noticed that the previous dataset was not included or shown alongside the new results in the revised figure. Could you please clarify why the earlier data were not incorporated in the new figure? Including both datasets, or explaining the rationale for focusing solely on the repeated experiment, would help readers understand the variability and ensure transparency in the interpretation of the findings.\nI also want to thank you for replacing the IP shown in Figure 4D in the revised manuscript; the updated result is more convincing to demonstrate that the IP experiment worked."}, "author_response": "Responses to Reviewers’ Comments\nCorresponding Author: Professor Yan Zhang\nDear Editor and Reviewers:\nThank you for your thoughtful letter and constructive advice. We sincerely appreciate\nthe comments provided by you and the reviewers regarding our manuscript\n(NCOMMS-25-14208) entitled “YY1/Asprosin/PFKP Axis Regulates Glycolytic\nMetabolism and Exacerbates Pathological Cardiac Hypertrophy.”\nWe have carefully considered all the feedback and have made revisions accordingly.\nBelow, we provide detailed, point-by-point responses to all reviewer comments.\nPoint-by-point Responses to The Reviewer’s Criticisms\nReviewer #2: (Remarks to the Author):\nMajor Comments:\n(1) Thank you for addressing my previous comments and for repeating the experiment.\nIn the revised dataset, the model control group (TAC + AAV9-NC) now shows a mean\nEF of 42.6%, which is noticeably lower than the mean of approximately 55% reported\nin your earlier dataset and is now consistent with the same control condition shown in\nFigure 2F. I noticed that the previous dataset was not included or shown alongside the\nnew results in the revised figure. Could you please clarify why the earlier data were not\nincorporated in the new figure? Including both datasets, or explaining the rationale for\nfocusing solely on the repeated experiment, would help readers understand the\nvariability and ensure transparency in the interpretation of the findings.\nResponse: We sincerely thank the reviewer for highlighting the important issue\nregarding data transparency. To address this concern and ensure the comprehensiveness\nand reproducibility of our findings, we have made the following revisions: The full set\nof echocardiographic data from the first independent experiment has been incorporated\ninto the supplementary materials (Supplementary Figure S2F, G), while the data from\nthe second independent experiment are preserved in the main text (Figure 1M, O).\nWe deeply appreciate the reviewer’s constructive suggestions, which have significantly\n1 / 2\nenhanced the rigor and transparency of our study. The relevant descriptions have been\nupdated accordingly in the Results section of the manuscript (page 7–8, lines 138–142).\n(2) I also want to thank you for replacing the IP shown in Figure 4D in the revised\nmanuscript; the updated result is more convincing to demonstrate that the IP experiment\nworked.\nResponse: We sincerely thank the reviewer for this positive feedback. We are pleased\nthat the revised Figure 4D now more convincingly demonstrates the success of the co-\nimmunoprecipitation experiment, thereby providing clearer support for our conclusions.\nBelieving that all issues raised by you and the reviewers have been fixed, we now\nresubmit the manuscript for further consideration. Thank you and the reviewers for your\ninterest in our work.\nSincerely,\nYan Zhang, Ph.D.\nProfessor in Pharmacology, College of Pharmacy\nHarbin Medical University\nHarbin, Heilongjiang 150081 P. R. China\nE-mail: zhangyan@ems.hrbmu.edu.cn\n2 / 2"}]

Decision letter