bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Tu127: Very low-density lipoprotein receptor mediates triglyceride-rich lipoprotein induced oxidative stress and insulin resistance.
Authors: Hajri Tahar , Gharib Mohamed , Fungwe Thomas
Published: October 2024

Background:
Very-low-density lipoprotein receptor (VLDLR), a member of the LDL receptor family, binds and increases the catabolism of triglyceride–rich lipoproteins. Although VLDLR is highly expressed in the heart, its role in VLDLR in oxidative stress and insulin resistance is unclear. Here, we used lean (WT), genetically obese leptin-deficient (ob/ob), and leptin–VLDLR double-null (ob/ob-VLDLR-/-) mice to determine the role of VLDLR in VLDL-induced oxidative stress and insulin resistance in the heart.

Methods:
Insulin resistance was investigated using glucose tolerance test and uptake of labeled deoxyglucose. Uptake VLDL was assessed in vivo and in isolated cardiomyocytes using double labeled ([3H]-TG, cholesteryl oleoyl ether ([14C])-VLDL. Oxidative stress was evaluated by the measurement of lipid peroxidation products (LPO) and hydrogen peroxides (HPO). NADPH-dependent superoxide production was measured in tissue homogenates and cardiomyocytes with the lucigenin chemiluminescent assay. Superoxide production in cardiomyocytes was investigated using fluorescent probe.

Results:
While insulin sensitivity and glucose uptake were reduced in the hearts of ob/ob mice, VLDLR expression was upregulated and was associated with increased VLDL uptake and excess lipid deposition in the heart. This was accompanied by an upregulation of cardiac NADPH oxidase (Nox) expression (+100%), increased oxidative stress markers LPO and HPO (+300%) and Nox-dependent superoxide production (+300%). Silencing VLDLR in ob/ob mice had reduced VLDL uptake and prevented excess lipid deposition in the heart, in addition to a reduction of superoxide production and the normalization of insulin sensitivity and glucose uptake. In isolated cardiomyocytes, VLDLR deficiency had prevented VLDL-mediated induction of Nox activity and superoxide overproduction while improving insulin sensitivity and glucose uptake.

Conclusions:
Our findings indicate that VLDLR deficiency prevents excess lipid accumulation and moderates oxidative stress and insulin resistance in the heart of obese mice. This effect is linked to the active role of VLDLR in VLDL uptake, which triggers a cascade of events leading to increased NOX activity, overproduction superoxide and insulin resistance.
Abstract Tu130: Intracellular Galectin-3 Interacts with Cytosolically Exposed Glycans to Promote the Injury of Cardiomyocytes Under Oxidative Stress
Authors: Yang Chiu-fen , Ho Li-Chen , Chiou Jyun-Yu , Meng Tzu-Ching
Published: October 2024

Background:
Oxidative stress is a hallmark of cardiovascular (CV) diseases. The potential involvement of Galectin-3 (Gal-3) has been proposed in CV pathological conditions, but its role in reactive oxygen species (ROS)-induced cardiomyocyte damage remains elusive.

Hypothesis:
Oxidative stress leads to ruptures of membrane-bound organelles including lysosome. Intracellular Gal-3 of cardiomyocytes may interact with glycans exposed by ROS-damaged organelles to modulate downstream cellular signaling.

Aims:
To investigate whether intracellular Gal-3-glycan interactions may occur in ROS-treated cardiomyocytes and how such interactions may regulate cell survival or death.

Methods and Results:
Using cardiomyoblast H9c2 cells exposed to hydrogen peroxide (H
2
O
2
) to be the cell model, we found that oxidative stress induced significant lysosomal damage followed by cell death. The immunofluorescence imaging showed that cytosolic Gal-3 aggregated into small puncta, which were colocalized with LAMP2. To examine whether the aggregation of Gal-3 was contributed by ROS-induced damage, catalase was ectopically expressed in H
2
O
2
-treated cells and the formation of Gal-3 puncta was attenuated. These results suggest that intracellular Gal-3 interacts with glycans presented by the ruptured lysosomes in H
2
O
2
-treated H9c2 cells, leading to puncta formation. To further delineate whether these Gal-3 puncta show a functional role in cell survival under oxidative stress, we knocked endogenous Gal-3 down in H9c2 cells by RNAi and found that Gal-3 ablation protected the cells from ROS-induced injuries. Moreover, the ectopic expression of N-terminally truncated Gal-3, which loses its protein-interaction function but preserved the carbohydrate-recognition ability, also prevented H9c2 cells from oxidative stress-caused damage.

Conclusions:
ROS-induced lysosomal rupture leads to intracellular Gal-3-glycan interactions, further recruiting cytosolic proteins and activating downstream cell death signaling cascades. Such intracellular Gal-3-glycan interactions may contribute to the cardiomyocyte injuries under oxidative stress.
Abstract Tu131: Counteracting Cardiac Ischemia and Reperfusion Injury: the role of Sialidase Neu3
Authors: Piccoli Marco , Coviello Simona , Borella Rossana , Lavota Ivana , Cirillo Federica , Ghiroldi Andrea , Pappone Carlo , Anastasia Luigi
Published: October 2024

Coronary reperfusion procedures are crucial for restoring blood flow to the heart tissue after an acute myocardial infarction. However, they also lead to ischemia and reperfusion injury (IRI), which exacerbate the damage and promote heart failure. Despite the urgent need for cardioprotective strategies against IRI, none have been clinically implemented, emphasizing the need for a comprehensive understanding of the pathophysiology of IRI. Our research team found that sialidase Neu3, an enzyme responsible for the removal of sialic acid from glycosphingolipids, is modulated during IRI in vivo. Constitutive overexpression of Neu3 in rat cardiomyoblasts was shown to attenuate IRI through activation of the reperfusion injury salvage kinase (RISK) and HIF-1α signaling pathways.

To further investigate the role of Neu3 in promoting cardioprotection, in this study we developed an inducible Neu3 overexpression model in human cardiac cells and mice.

Inducible upregulation of Neu3 significantly improved cell resistance and reduced oxidative stress in AC16 cells exposed to IRI in vitro. These effects were attributed to Neu3-mediated maintenance of mitochondrial membrane potential, which was impaired in control cells. When looking at the upregulated genes exclusively in cells overexpressing the sialidase Neu3, RNAseq analysis revealed the predominant activation of MAP kinases typical of the RISK pathway.

We also generated, for the first time, αMHC-Cre/LSL-Neu3 mice overexpressing Neu3 in an inducible and cardiomyocyte-specific manner. These mice showed no significant differences in cardiac morphology and functionality compared to wild-type animals. However, the Neu3-overexpressing mice exhibited a significant reduction in infarct size and improved cardiac functionality after surgical induction of IRI.

These results suggest that Neu3 is a promising target for improving cardioprotection and attenuating the incidence and severity of myocardial infarction.
Abstract Tu134: Oncostatin M favors cardiomyocyte survival by promoting glycolysis
Authors: Li Ruopu , Bai Xue , Wu Yunxiaoxiao , Feng Jie , Hu Shengshou , Nie Yu
Published: October 2024

Introduction:
Cardiomyocytes is in high demand of energy and is susceptible to low oxygen environment. Timely switch of energy metabolism after cardiac ischaemia is helpful for cardiomyocyte preservation. We previously identified a critical role of Oncostatin M (OSM) in cardiomyocyte proliferation. Since the level of OSM quickly rises in myocardium after myocardial injury, OSM may play a protective role in early phase after myocardial injury through metabolic regulation.

Hypothesis:
We hypothesized that Oncostatin M regulates cardiomyocyte energy metabolism to promote its resistance to hypoxia.

Methods and Results:
We cultivated cardiomyocytes under hypoxic condition observed that in OSM-treated group, the percentage of TUNEL positive cardiomyocytes significantly decreased compared with that in vehicle-treated group. Published RNA sequencing data from OSM-treated neonatal cardiomyocytes revealed alterations in genes related to energy metabolism. Our Seahorse metabolic stress assays demonstrated that OSM-treated cardiomyocytes exhibited higher glycolytic metabolism and lower mitochondrial respiration than vehicle-treated cells. Pyruvate kinase M2 (PKM2), among the regulatory enzymes, was significantly upregulated in OSM-treated cardiomyocytes as was detected via western-blotting. Interestingly, PKM2 has lower catalytic activity compared to its isotype PKM1, suggesting that the augmented glycolysis might be mediated by PKM2. The mutually exclusive splicing of the gene
Pkm
between exons 9 and 10 led to the switch between PKM1 and PKM2. qPCR results indicated a 1.5-fold increase in
Pkm
transcripts and a 2-fold increase in the ratio of
Pkm2
to
Pkm1
, suggesting that OSM concomitantly promoted
Pkm
transcription as well as alternative splicing of
Pkm2
. Furthermore, we performed RNA interference to knock down
Stat3
, the canonical downstream signal of OSM, in cardiomyocytesand observed that OSM failed to upregulate
Pkm
transcription. ChIP-qPCR analysis on cardiomyocyte cell line HL-1 confirmed an increased STAT3 binding signal upstream of
Pkm
after OSM treatment. Additionally, RIP-qPCR assays revealed a 3-fold increase in PTBP1-binding signal to
Pkm
transcripts in cardiomyocytes following OSM treatment.

Conclusions:
Our results suggest that OSM promotes glycolysis and suppresses mitochondrial respiration in cardiomyocytes by upregulates PKM2 through STAT3 mediated transcription of
Pkm
and PTBP1-mediated alternative splicing of
Pkm
transcripts.
Abstract Tu137: Reductive Stress Impedes Neonatal Cardiomyocyte Regeneration
Authors: Trouten Allison , Li Shuang , Sucov Henry , Tao Ge
Published: October 2024

Background:
The elevated level of reactive oxygen species (ROS) has been reported to drive the cell-cycle-exit of neonatal mouse cardiomyocytes and impede their regeneration. On the contrary, the roles of reductive stress in perinatal development and regeneration of cardiomyocytes are still poorly understood. To counterbalance increased oxidative stress, antioxidant enzymes are expressed to remove excessive ROS. Our previous work uncovered a role of the transcription factor Pitx2 as a redox regulator in the ventricular myocardium. Pitx2 protects myocardium by positively regulating genes that encode antioxidant scavengers. In regenerating neonatal hearts, Pitx2 expression is activated by Nrf2, a known antioxidant regulator. Under oxidative stress, Nrf2 dissociates from the Keap1-Cul3-Rbx1 degradation complex and translocates to the nucleus where it promotes Pitx2 expression. Interestingly, our RNA-Seq and ChIP-Seq studies showed that Pitx2 promotes Rbx1 expression which degrades Nrf2. We hypothesize that this negative feedback loop (
Image 1
) is required to prevent reductive stress in regenerating cardiomyocytes.

Methods:
Cre-Lox
models were used to specifically knock out
Nrf2
in cardiomyocytes. Constitutively active Nrf2 (caNrf2) was expressed specifically in cardiomyocyte using AAV9. Guide RNAs were delivered into
Myh6Cas9
mouse heart using AAV9 to knock out
Rbx1
in cardiomyocytes. Neonatal ROS was transiently suppressed using high dose of N-acetyl-cysteine (NAC). Following left anterior descending artery occlusion (LAD-O), cardiac regeneration was assessed by Masson’s trichrome staining and echocardiography. Markers of cell cycle activity and cell death were assessed by immunofluorescence. RNA-Seq was performed on isolated cardiomyocytes from NAC and saline treated hearts after sham or LAD-O procedure.

Results:
Pitx2 promotes the degradation of Nrf2 by inducing the expression of Rbx1. In cardiomyocytes,
Rbx1
, but not
Nrf2
, is required for neonatal heart regeneration. Meanwhile, expression of
caNrf2
in cardiomyocytes increases mortality after LAD-O. Removing ROS in neonatal heart prevents cardiomyocyte maturation and disrupts injury response after LAD-O.

Conclusions:
Together, these results suggest that a fine balance between reductive and oxidative activity is required for neonatal heart regeneration and postnatal heart development. Excessive antioxidant activity will cause reductive stress and impede neonatal heart regeneration after MI.
Abstract Tu142: Pannexin-2 Deficiency Exacerbates Stress-induced Cell Injury in Cardiomyocytes
Authors: Sarkar Anusua , Kim Seulhee , Zhou Lufang
Published: October 2024

Pannexins are a family of channel proteins involved in a variety of biological processes, including cell death signaling and immune functions. While Pannexin isoforms 1 and 3 have been widely studied, mainly in the brain, much less is known about the role of isoform 2 (Pannexin2, or Panx2). A recent study showed that deficiency in Panx2 sensitizes pancreatic beta cells to cytokine-induced apoptosis, while another study revealed that Panx2 promotes ultraviolet-induced apoptosis in keratinocytes. The role of Panx2 in the regulation of cardiomyocyte function has not been investigated. In the current study, we explored the subcellular distribution profile of Panx2 expression in cardiomyocytes (AC16 cells and adult cardiomyocytes) and examined the role of Panx2 in regulating cardiomyocyte response to stress. Western blotting analysis revealed palmitoylation of Panx2 in the heart, suggesting that this channel is located intracellularly, instead of in the plasma membrane. Confocal imaging revealed that Panx2 expression is in the proximity of the sarcoplasmic reticulum and mitochondria, confirming its intracellular localization. The stress test results indicated that the knockdown of Panx2 by siRNA exacerbated FCCP-reduced oxidative stress and cell injury in AC16 cells. Similarly, adult cardiomyocytes isolated from Panx2-KO mice were more vulnerable to ischemia-reperfusion injury compared with wild-type cells. Finally, we found that the level of Panx2 expression altered in the cardiac tissue of a mouse model with pressure overload-induced heart failure, implying that Panx2 may be involved in the pathological remodeling of a failing heart.
Abstract Tu144: DIAPH1 regulates stress induced senescence in Human cardiomyocytes
Authors: Yepuri Gautham , Mangar Kaamashri , Schmidt Ann Marie , Ramasamy Ravichandran
Published: October 2024

Introduction:
Stress induced senescence (SIS), defined as the loss of cell physiology/function associated with increased senescence associated secretory phenotypes (SASPs), is a potential risk factor for cardiac dysfunction and failure. Human Diaphanous 1(DIAPH1), a formin molecule is known to regulate actin-cytoskeleton dynamics, trafficking of organelles and signal transduction. Our recent work demonstrated the importance of DIAPH1-MFN2 interactions in regulating actin-cytoskeleton dynamics and the mitochondria-sarcoplasmic reticulum(Mito-SR) distance thereby regulating cardiomyocyte function and pathological responses to ischemia reperfusion injury. We tested the hypothesis that DIAPH1 regulated stress induced senescence in cardiomyocytes under aging and hypoxia/reoxygenation (H/R) conditions.

Methods:
Human cardiomyocytes (HiPSC-CMs) from two different sources of HiPSCs which are double positive for TNT2 and TNNI3 were used. To understand the role of DIAPH1 in SIS, we have used two different approaches. HiPSC-CMs were either kept for 120days in culture or at day25 were treated with mitomycin(200nM/72hrs). DIAPH1 was silenced using shRNA approach (
shDIAPH1
).

Results:
We observed increased
DIAPH1
gene expression at 120 days of culture vs day 30. Interestingly,
shDIAPH1
HiPSC-CMs, attenuated the number of SA-β-galactosidase positive cells, reduced expression of senescence markers
SERPINE, CDKN1A, CDKN2A, TP53
,
COL1A2
along with improved mitochondrial function when compared to shScr controls. In experiments where HiPSC-CMs at day 25 were treated with mitomycin to induced senescence, we observed that silencing DIAPH1 attenuated the number of SA-β-galactosidase positive cells, reduced F/G actin ratio and robustly decreased DIAPH1-MFN2 interactions as measured by DUOLINK proximity ligation assay. We probed the RNA sequencing datasets from our recent publication (Yepuri et.al.,
Nat. Comm.
2023) and observed that senescence markers, SASPs and cytokines including
CDKN1A, H2AX, LMNA,
IL1A, IL6, SERPINE
were significantly down regulated in
shDIAPH1
HiPSC-CMs exposed to H/R conditions.

Conclusion:
Taken together our data demonstrates that DIAPH1 mediates stress induced senescence in cardiomyocytes and is linked to actin-cytoskeleton dynamics and DIAPH1-MFN2 interactions. Importantly, our data indicates a causative role of DIAPH1 in SIS and sets the stage for probing DIAPH1 as a therapeutic target for protection of hearts from stress induced senescence.
Abstract Tu146: GJA1-20k Promotes Formation of Mitochondrial Actin Cages
Authors: Nguyen Vu , Baum Rachel , Maalouf Mario , HUNTER JENNIFER , Shimura Daisuke , Shaw Robin
Published: October 2024

Introduction:
GJA1-20k, an N-terminus truncated isoform of the gap junction protein Connexin 43, is stress responsive and mimics ischemia preconditioning, reducing ischemia-reperfusion (IR) injury. GJA1-20k localizes to the outer membrane of mitochondria, stabilizes actin, and mediates an actin mediated mitochondrial protection. The mechanism is not known how GJA1-20k and actin work towards mitochondrial protection.

Methods:
Cell-free TIRF imaging was performed to study direct interactions between actin and purified GJA1-20k. High-resolution confocal microscopy was applied to visualize GJA1-20k and actin organization around mitochondria in intact HEK cells and cell-free suspensions wherein rhodamine-labeled actin, purified GJA1-20k, and isolated mitochondria were incubated together.

Results:
Cell-free TIRF imaging establishes that
GJA1-20k directly binds to actin
, forming both actin clusters and stabilized actin filaments. As expected, live-cell imaging reveals that GJA1-20k is enriched at the mitochondrial outer membrane. The direct binding of GJA1-20k and actin results in a rich collection of actin around mitochondria, as evidenced in both live-cell imaging and cell-free suspensions containing only purified actin, GJA1-20k, and isolated mitochondria. 3D reconstruction reveals that mitochondrial localization of GJA1-20k causes formation of dense actin sheets enveloping mitochondria, which we call “mitochondrial actin cages” which appear to limit the ability of mitochondria to swell under stress conditions.

Conclusions:
GJA1-20k induced actin cages could be critical to GJA1-20k mediated protection against IR injury. The observed actin around mitochondria could prevent pathological mitochondrial swelling, preserving mitochondrial integrity and function in oxidative stress conditions.
Abstract Tu147: Serial measurement of circulating cardiovascular enriched miR-1 and -34a reflects the changes in cardiac function in patients with ischemic heart disease – a five year follow-up study
Authors: Bellae Papannarao Jayanthi , Coffey Sean , Gray Andrew , Williams Michael , Katare Rajesh
Published: October 2024

Background:
Patients with ischemic heart disease (IHD) require frequent monitoring, as the transition from stable disease to acute life-threatening events remains largely unpredictable. Currently available tests are either not sensitive enough to detect early changes or not easily incorporated into routine clinical practice. MicroRNAs (miRNAs), small non-coding RNAs involved in both physiological and pathological processes, are released into circulation and remain stable.

Research Question:
In this study, we aimed to determine if the serial measurement of cardiovascular-enriched circulating miRNAs could reflect changes in cardiac function in patients with IHD.

Methods:
Fifty-four new IHD patients joined a 5-year study, providing informed consent for regular echocardiography, blood tests and follow-ups at 12-month intervals. Plasma RNA was extracted and RT-PCR analysis used to assess expression of cardiovascular-enriched miR-1, miR-126, miR-132, and miR-34a.

Results:
Serial echocardiography analysis showed the development of diastolic dysfunction in the participants starting from 48 months (E/e’-10.7±4.3 vs 9.7±3.6 at baseline, P<0.04). RT-PCR analysis revealed significant downregulation of miR-1 and miR-126 starting from 24 months of follow-up (P<0.03 vs. baseline). Associations between each miRNA and each cardiac function measure were examined using linear mixed models without and with adjustment for changes in BMI and HbA1c during the same period. None of the lagged models found statistically significant associations (all p ≥ 0.126). From the concurrent models, two miRNA were associated with cardiac function: miR-1 and MV E Vel in the unadjusted (p = 0.045) and adjusted models (slope = -0.004, 95% CI -0.007 to -0.000, p = 0.026) models and miR-34a and FS(%) in the adjusted model only (slope = -0.151, 95% CI -0.301 to -0.001, p = 0.048).

Conclusion:
Results from this first-ever five-year follow-up study have identified a link between cardiovascular enriched miRNAs, miR-1 and -34a and cardiac function in patients with IHD, providing a knowledge platform for prognostic tests for IHD and laying the foundation for further studies in larger population size.
Abstract We006: Metabolic Reprogramming to Increase Mitochodnrial Mass, Fusion and Energetics Represents an Important Rate Limiting Step in Direct Cardiac Reprogramming
Authors: Spurlock Brian , Xie Yifang , Song Yiran , Hua James , Nishtala Meenakshi , Salmenov Rustem , Liu Jiandong , Qian Li
Published: October 2024

Direct reprogramming of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) shows promise for treating myocardial infarction (MI). However, low conversion efficiency and relative immaturity of iCMs have so far precluded its clinical translation. Native CM maturation and cell cycle exit is associated with a switch from glycolytic metabolism to mitochondrial fatty acid oxidation (FAO), accompanied by a shift in the dynamic balance of mitochondrial fission (dividing) and fusion (joining) toward fusion. Because CFs lack the energy requirements of CMs, we hypothesized that transdifferentiation of CFs to iCMs requires a similar switch. Combining confocal microscopy with metabolic flux assays, we directly compared native CF and CM mitochondrial morphology and energetics. CMs showed significantly higher mitochondrial mass, fusion and energetics, all metrics relevant for increased reliance on mitochondrial FAO. Applying these techniques to study reprogramming, we found that at later reprogramming time points key metrics move significantly toward the phenotypes of native CMs. We then hypothesized that facilitating metabolic reprogramming by inhibiting fission and/or promoting fusion could improve iCM conversion. A loss of function screen identified certain fission genes, particularly the little studied mitochondrial fission regulator 1-like protein (Mtfr1l), as potent barriers to iCM conversion and maturation. In contrast, the core fusion genes were necessary for successful generation of iCMs, particularly the key fusion effector optical atrophy 1 (Opa1). Mtfr1l has been shown to promote cleavage of Opa1, and indeed concurrent knockdown of Opa1 ablated improvements to reprogramming efficiency. In conclusion, we showed both that iCMs are associated with increased mitochondrial mass, fusion and energetics approaching those of native CMs and that inducing CM-like mitochondria by knocking down Mtfr1l dramatically improved reprogramming, potentially through increased fusion activity of Opa1. By incorporating metabolic reprogramming into traditional direct cardiac reprogramming strategies, we may be able to move the field closer to clinical application to improve outcomes after MI.
Abstract We009: Cardiac Fibroblast Autophagy Is Required for Neonatal Heart Regeneration
Authors: Feng Jie , Li Yandong , Nie Yu
Published: October 2024

Introduction:
During heart regeneration, a switch between the synthesis, secretion and degradation of extracellular matrix (ECM) is essential, which relies on highly coordinated biosynthetic and catabolic processes. Although many biosynthetic signaling have been identified, catabolic processes in heart regeneration is still largely unexplored.

Hypothesis:
Autophagy is a key catabolic process necessary for depredating unwanted cellular components, dysfunctional organelles or aggregated proteins, and providing the needed substances for newly synthesizing ECM. We assessed the hypothesis that autophagy was involved in heart regeneration through regulating ECM remodeling.

Methods:
Apical resection (AR) and myocardial infarction (MI) models in neonatal mice were used. Three mouse stains,

GFP-LC3
tg/+

,

Atg7
fl/fl
and Col1a2-2A-CreER

mice were induced
.
Transmission electron microscopy was applied to observe autophagic vesicles. Immunostaining was used to detect autophagy activation and cardiomyocyte proliferation. Cardiac fibrosis was evaluated by Masson staining. Liquid chromatography-mass spectrometry was utilized for ECM components analysis.

Results:
We performed AR injury on postnatal day 1 (P1) mice, and observed an obvious increase in the number and size of autophagic vesicles in the injured heart tissue at 1 day post-resection using transmission electron microscopy (TEM). Co-localization analysis of GFP-LC3 with cardiomyocyte marker α-actinin and fibroblast (FB) marker PDGFRα revealed that autophagy was predominantly activated in cardiac FBs following AR. Cardiac FB-specific Atg7 conditional knockout (
Atg7-
cKO) resulted in impaired heart regeneration after AR or MI injury in neonatal mice, accompanied by reduced cardiomyocyte proliferation. Cardiac ECM (cECM) derived from
Atg7
-cKO mice hindered cardiomyocyte proliferation compared to

Atg7
fl/fl

mice, indicating that cardiac FB autophagy regulated cardiomyocyte proliferation by modulating the ECM profile. Through liquid chromatography-mass spectrometry analysis, we revealed that cardiac FB-specific Atg7 deficiency induced the downregulation of ECM components associated with regeneration, cell cycle, and muscle cell proliferation.

Conclusion:
In conclusion, our research highlights that cardiac FB autophagy plays a crucial role in neonatal heart regeneration by controlling the ECM remodeling. These findings suggest that targeting autophagy could be a promising therapeutic approach for individuals with ischemic heart disease.
Abstract We015: Modeling Cardiac Arrhythmogenicity of hiPSC-CMs and Cardiac Fibroblasts Nanopatterned Coculture and Machine Learning
Authors: Yang Huaxiao , Rogozinski Nicolas , Wo Hung-Ta , Mohanraj Gautham , Wu Joseph , Abilez Oscar
Published: October 2024

Background:
Sudden cardiac death is one of the most threatening heart conditions in the U.S. Most individuals have an underlying structural cardiac disease associated with cardiac fibrosis and ventricular arrhythmia. The relationship between fibrosis and arrhythmias is found to be related to cardiovascular cell couplings, especially cardiomyocytes (CMs) and cardiac fibroblasts (CFs). While animal models have been applied to model cardiac arrhythmias, the disparities between animal models and humans limit the accuracy of pro-arrhythmic predictions. The use of human induced pluripotent stem cells (hiPSCs)-derived CMs has permitted highly modular testing conditions, specifically the ratio of CFs to CMs in an engineered coculture system. Moreover, machine learning (ML) has become an emerging tool in cardiology, such as cardiac toxicity and function evaluation and classification, with its unique advantages of high accuracy and efficiency with less human bias and intuition.

Hypothesis:
We hypothesize that cardiac fibrosis-associated arrhythmia will be modeled by nanopatterned coculture of hiPSC-CMs and CFs at varied ratios, and the arrhythmic samples will be accurately and efficiently classified by ML algorithms.

Approach:
The hiPSC-CMs were cocultured and nanopatterned with CFs at ratios of 0 to 30% for 7 days. Upon the electrical pacing at 1Hz, the action potential was recorded by live-cell optical mapping with FluoVolt. The recorded membrane potential and electrical propagation were analyzed by Matlab-based Electomap software.

Results:
The cocultured hiPSC-CMs and CFs were aligned with aligned sarcomeres and uni-directional contraction (
Fig. 1a
). The cardiac arrhythmia was observed in 15% CFs with spiral wave (
Fig. 1b
) in comparison to the hiPSC-CMs with 0% CF. Beat-to-beat and depolarization durations were found significant with top ranks in Feature Importance to facilitate the distinction between arrhythmic and non-arrhythmic samples by the unsupervised K-means Clustering (
Figs. 1c and d
).

Conclusions:
We have successfully established a nanopatterned coculturing system of hiPSC-CMs and CFs for modeling cardiac fibrosis-induced arrhythmia, which was further analyzed and classified by ML algorithms. This system reveals great potential for better understanding the relationship between cardiac fibrosis-associated arrhythmia and sudden cardiac death and also drug evaluation for anti-cardiac arrhythmia.
Abstract We017: Tnni3k mediates postnatal reactive oxygen species through Prdx2 signaling to induce polyploidization in cardiomyocytes
Authors: Westbury Baylee , Sucov Henry
Published: October 2024

Fetal CMs are characteristically diploid and regenerative, while adult CMs are polyploid and nonregenerative. Increased polyploidy in postmitotic CMs remains a significant barrier to regeneration in the adult heart. Previously, through the analysis of multiple mouse strains, our lab has shown that Tnni3k, a cardiac-specific kinase, increases polyploidy in the adult heart. The mechanisms that induce CM polyploidization are unknown but could be due to a multitude of changes including the postnatal increase in reactive oxygen species (ROS). ROS is essential in cardiac development and works to promote maturation and facilitate cell signaling. Using HEK293 cells, I have found that Tnni3k physically interacts with Prdx2, a prominent cytoplasmic ROS sensor. This interaction results in hyperactivated Tnni3k kinase activity. Comparing Tnni3k kinase activity with interaction of WT or catalytically dead Prdx2 showed hyperactivity dependent on Prdx2 catalysis. Polyploidization increases along with the increase in ROS after birth, and Tnni3k contributes to increased ploidy in mice. I hypothesize that Tnni3k mediates postnatal ROS through Prdx2 signaling to induce polyploidization in postnatal CMs. Discovering the consequences of this interaction could lead to advanced knowledge of CM cell cycle and provide new therapeutic targets.
Abstract We019: YAP Overcomes Mechanical Barriers to Induce Adult Cardiomyocyte Division
Authors: Morikawa Yuka , Kim Jong , Li Rich Gang , Martin James
Published: October 2024

Adult mammalian cardiomyocytes (CMs) are among the least able to renew of all cell types. CMs exit the cell cycle soon after birth and remain cell cycle arrested by unknown mechanisms. We previously found that in adult CMs, the active form of the Hippo signaling pathway effector YAP, YAP5SA, induces cell division
in vivo
. Here, we analyze the mechanism by which YAP5SA regulates the cell cycle in adult CMs. YAP5SA-expressing adult CMs reenter the cell cycle at the G1/S transition and have a prolonged S-phase lasting approximately 48 hours. Molecular and functional analysis revealed that progression from S to G2 phase, and the induction of mitotic rounding, requires sarcomere disassembly. Transition from G2 to M phase requires overcoming DNA damage checkpoint facilitated by the activation of the P21 degradation pathway. Clonal analysis showed that approximately 20% of CMs progress through cytokinesis.

Our results show that multiple barriers are involved in suppressing the adult CM cell cycle. These include the structural block caused by the highly structured CM cytoskeleton and the P21 regulated DNA damage checkpoint.
Abstract We024: Spatial Transcriptomics Identifies a Regeneration-Permissive Microenvironment in the Neonatal Mammalian Heart
Authors: Miriyala Saradha , Cui Miao
Published: October 2024

Ischemic heart disease is the leading cause of death globally, largely driven by inability of the adult heart to restore tissue structure and function after injury. However, the neonatal mouse heart is able to regenerate up to one week after birth, circumventing scarring and functional impairment. Neonatal heart regeneration is a spatially and temporally regulated process, involving coordinated communication of different cardiac cell types. Our previous work identified a regenerative cardiomyocyte population (CM4) that proliferates after myocardial infarction (MI), but the intercellular communication that induces their regenerative response is not fully understood. To study the cellular environment of CM4 cells during neonatal heart regeneration, we profiled spatial transcriptomes of regenerative P1 hearts at 3 days and 7 days after MI and compared them to nonregenerative P8 hearts. We identified unique spatial regions with distinct gene expression signatures, that we refer to as “molecular niches”, in the ischemic and border zones (IZ, BZ) of the P1 heart post-MI compared to P8 hearts. Cell type deconvolution of molecular niches showed a distinct temporal change of fibroblast and macrophage abundance in the IZ of P1 hearts compared to P8 hearts and also showed the localization of CM4 cells to the BZ of P1 hearts at 7 days post-MI. To identify intercellular communication that contributes to the differential injury response in P1 and P8 hearts, we analyzed ligand-receptor pairs that were expressed at higher levels in the IZ and BZ of P1 compared to P8 hearts. Among the nearly 50 signaling pairs identified, OSM and SLIT signaling have been previously implicated in heart regeneration. Notably, many of these signaling factors, including cytokines, chemokines and growth factors, have receptors expressed in multiple cardiac cell types. Thus, ongoing studies are systematically distilling the effect of each secreted factor on individual cell types in the heart using a cardiac cell co-culture and multiplexed single-cell RNA sequencing approach. In summary, our study provides insight into the spatial organization and tissue microenvironment of neonatal heart regeneration and identifies a number of candidate signaling pathways that potentially mediate critical intercellular communication during neonatal heart regeneration.
Abstract We028: Decoding m6a RNA methylomes identifies Igf2bp1 as a common barrier to direct reprogramming
Authors: Xie Yifang , Wang Qiaozi , Spurlock Brian , Liu Jiandong , Qian Li
Published: October 2024

Direct reprogramming has revolutionized the fields of stem cell biology and regenerative medicine. Targeted transdifferentiation of cells
in situ
holds significant promise in treating a large swathe of illness and injury including cardiovascular disease. However, much remains unknown about the molecular mechanisms of direct reprogramming, particularly its post-transcriptional regulation, including the role of m6a RNA modification. Here, by characterizing early changes in the m6a RNA methylome during reprogramming of fibroblasts toward three distinct lineages—cardiac, hepatic and neuronal—we identify lineage-specific features as well as common regulatory patterns. Our data reveal extensive changes in m6a methylome of direct reprogramming with clear impacts on cell fate decisions. Through loss-of-function screening of m6a readers, we uncovered Igf2bp1(insulin-like growth factor 2 mRNA binding protein 1) as a shared barrier to direct reprogramming. Further mechanistic studies will show how Igf2bp1 influences the reprogramming process mediated via m6a modifications. Collectively, our results present a fascinating portrait of m6a RNA methylome during direct reprogramming and reveal a key role of Igf2bp1 in maintaining cell fate, enhancing our mechanistic understanding of the reprogramming process.
Abstract We031: Optimizing Immunosuppressive Strategies for Enhanced Survival of Allogeneic Pluripotent Stem Cell-Derived Cardiomyocytes in Non-Human Primate Transplantation Model
Authors: Chino Shuji , Ichimura Hajime , Tohyama Shugo , Fukuda Keiichi , Shiina Takashi , Chong James , NAITO TAKAFUMI , Ohya Maki , Kobayashi MIkiko , Tomita Takuro , Yamada Mitsuhiko , Kobayashi Hideki , Shiba Naoko , Seto Tatsuichiro , Kadota Shin , Shiba Yuji
Published: October 2024

Introduction:
Accumulating evidence suggests that the transplantation of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) regenerates injured hearts in animal studies. Currently, several clinical studies are underway, transplanting allogeneic human PSC-CMs. In these clinical studies, patients have been treated with the same combination of immunosuppressants used after heart transplantation, including steroids, calcineurin inhibitors (CNIs), and mycophenolate mofetil (MMF). However, the optimal treatment following PSC-CM transplantation has yet to be established. The purpose of this study is to determine the optimal treatment for grafted PSC-CM survival by regulating the immune response using an allogeneic non-human primate transplantation model.

Methods and Results:
We prepared 9 male cynomolgus monkeys as recipient animals. Donor cardiomyocytes were generated from induced pluripotent stem (iPS) cells derived from another monkey, with MHC class I and class II types differing from those in the recipients. The recipient animals underwent ischemia/reperfusion injury of the left anterior descending artery two weeks before iPSC-CMs transplantation. Initially, animals were treated with the same combination of immunosuppressants (steroid/CNIs/MMF: N=3), and grafted PSC-CMs survived without apparent immune rejection. Subsequently, we conducted experiments aiming to reduce immunosuppressants. The animals were treated with the same combination, but steroids were terminated at 4 weeks post-transplantation (N=4). Histological analysis at 8 or 12 weeks post-transplantation revealed that grafted cardiomyocytes were rejected, with a significant infiltration of CD3-positive lymphocytes. When recipients were treated with another combination of immunosuppressants (steroid/abatacept/MMF), and steroids were terminated at 4 weeks post-transplantation, grafted cardiomyocytes survived without apparent infiltration of CD3-positive lymphocytes at 8 weeks post-transplantation (N=2).

Conclusion:
A different combination of immunosuppressants from heart transplantation allows transplanted allogeneic PSC-CMs to survive without the long-term use of steroids.
Abstract We038: Metal-organic frameworks as next-generation cardiac therapeutics
Authors: Baseeruddin Alvi Syed , Mergaye Muhamad , Dholaniya Pankaj Singh , Sameer Ghazala , Xu Xianyao , Islam Helena , Khan Mahmood
Published: October 2024

Background:
Zinc (Zn) is an essential trace element for the normal functioning of the heart. Long-term dietary supplementation of Zn has been shown to improve cardiac function and prevent myocardial damage caused by ischemia-reperfusion injury. However, the major limitation in delivering Zn to the cardiac tissue is the off-targeted absorption and the duration of treatment to achieve a therapeutic effect.

Hypothesis:
Cardiac-targeted delivery of trace elements like Zn may attenuate myocardial damage caused by ischemia-reperfusion injury, improve cardiac function, and exert a cardioprotective effect.

Methods:
Zn-based metal-organic frameworks (ZIF-NPs) were fabricated and tested on Microvascular Endothelial Cells (MVECs), and human-induced pluripotent derived cardiomyocytes (hiCMs) for cytoprotective effect against hypoxic stress using Live/dead assay and multi-electrode array (MEA) analysis respectively. The in vivo efficacy of ZIF-NPs was tested in the rodent myocardial infarction (MI) model and cardiac function was assessed via M-mode echocardiography.

Results:
The fabricated ZIF-NPs were biocompatible and showed significant intracellular uptake causing the induction of cytoprotective metallothioneins. ZIF-NP treatment showed enhanced cell survival in serum-starved MVECs and exhibited pro-angiogenic effects in vitro. Similarly, hiCMs treated with ZIF-NPs displayed enhanced endurance and electrophysiological function, when subjected to hypoxic stress and protected the cells against ROS-induced DNA damage. Intramyocardial delivery of ZIF-NPs in the cardiac tissue improved cardiac function and attenuated fibrosis post-MI.

Conclusions:
ZIF-NPs exerted a pro-survival, cytoprotective response in both in vitro and in vivo conditions. Our study highlights the significance of cardiac targeted Zn delivery by ZIF-NPs for cardioprotection as next-generation cardiac therapeutics.
Abstract We041: In vitro characterization of different human-induced pluripotent stem cell-derived cardiac progenitors
Authors: Ahmed Uzair , Sridharan Divya , Khan Mahmood
Published: October 2024

Introduction:
Myocardial Infarction (MI) is the number one cause of death in the United States. The heart undergoes severe trauma during MI, which results in the death of cardiac cells. Although the cardiac tissue cannot regenerate on its own, regenerative medicine approaches involving cell transplantation have been shown to improve the cardiac function. Among different cell types, human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSCs) have been shown to have the ability to proliferate and differentiate into cardiac lineage cells and therefore, are a promising source for myocardial repair. Here, we characterized two distinct populations of hiPSC-CPCs and assessed their differentiation potential.

Methods:
We compared hiPSC-CPCs differentiated using the small molecule, isoxazole-9 (ISX-9), ISX9-hiPSC-CPCs with the commercially acquired iCell hiPSC-CPCs (Fujifilm Cellular Dynamics Int.). The expression of different cardiac lineage markers were assessed via immunostaining, qPCR and flow cytometry. Furthermore, we assessed the proliferation of the cells using immunostaining and XTT assays. We also evaluated their in vitro differentiation to cardiomyocytes, via immunostaining and qPCR analysis.

Results:
Our studies showed similar expression of different cardiac progentior markers (Nkx2.5, GATA4, KFL-4, ISL-1, BRY-T, MESP-1, MYH-6, CTnT) in the two hiPSC-CPC populations. However, while the iCell hiPSC-CPCs predominantly expressed the cell surface marker KDR, the ISX-9-hiPSC-CPCs predominantly expressed SSEA-1. The iCell-hiPSC-CPCs showed higher proliferation rates when compared to the ISX-9-hiPSC-CPCs. However, >95% of both the cell populations expressed the proliferation marker, ki67. Finally, the while both the CPC populations could differentiate into cardiomyocytes (cTNT
+
), only a few of these differentiated cardiomyocytes showed spontaneous contractility in vitro.

Conclusion:
In our study, we have compared and characterized two distinct populations of hiPSC-CPCs to identify a promising candidate for myocardial regeneration therapies.
Abstract We046: Building Mass or Strength: Stage-specific Effects of Insulin and Wnt on Cell Fate Decisions in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Authors: Yuan Qianliang , Schoger Eric , Kim Rosa , Kuster Diederik , Boon Reinier , Zelarayan Laura , van der Velden Jolanda , Buikema Jan
Published: October 2024

Background:
Reactivation of the so-called fetal gene programs and pathways have been reported in failing hearts, however, it remains unclear whether this phenomenon is beneficial or detrimental to the remaining functional myocardium. During cardiac development, the orchestration of these embryonic growth pathways, facilitate temporal and stage-specific cell fate decisions such as required for specification and growth of the various myocardial compartments. Previously, we demonstrated that molecular activation of the Wnt pathway resulted in massive expansion of immature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Yet, it is unknown what exact cues guide the cell fate decisions of immature hiPSC-CMs to either proliferate or terminally differentiate into more mature cardiomyocytes.

Purpose:
This work uncovers the embryonic growth pathways that control the cues for proliferation versus terminal differentiation in functional immature hiPSC-CMs.

Methods:
We utilised spontaneously beating hiPSC-CMs at day 11 of differentiation and performed a combinatory screen for various dosages of CHIR99021/Wnt and Insulin/Akt pathway. Proliferation was determined by quantification of cell number and the proportion of Ki67 positive hiPSC-CMs. Terminal differentiation was evaluated by presence of more mature sarcomere markers and cell cycle exit.

Results:
Our combinatory screen for Insulin/Akt and CHIR99021/Wnt demonstrates synergistic effects on proliferation of immature hiPSC-CMs, whereas the absence of these pathway activators leads to rapid cell cycle exit and terminal differentiation of hiPSC-CMs. Detailed characterization reveals that CHIR99021/Wnt also importantly regulates sarcomere homeostasis and architecture in hiPSC-CMs. Moreover, we identify a novel temporal interplay between CHIR99021/Wnt via TCF and Insulin/Akt via FOXO as regulators of cell-fate decision in immature hiPSC-CMs.

Conclusions:
Molecular targeting of the embryonic growth pathways in acute and chronic forms of heart failure requires a detailed understanding of the specific effects on cardiomyocyte function and sarcomere architecture. This work provides a starting point to develop future strategies to target acute and chronic forms of heart failure via modulation of embryonic growth pathways.
Abstract We049: Cell-Specific Metabolic Labelling Identifies hiPSC-CM Proteome In Vitro and In Vivo Post-Transplantation
Authors: Sridharan Divya , Dougherty Julie , Ahmed Uzair , Sanghvi Shridhar , Baseeruddin Alvi Syed , Park Ki Ho , Knoblaugh Sue , Singh Harpreet , Kirby Elizabeth , Khan Mahmood
Published: October 2024

Background:
Human induced pluripotent stem cells (hiPSCs) and their derived cardiomyocytes (hiPSC-CMs) have been used for cell-based therapies for myocardial regeneration. Underlying paracrine signals derived from these hiPSC-CMs have been shown to play a critical role in improving the function of the ischemic myocardium. However, characterization of the transplanted cell proteome in vivo has been challenging due to the lack of tools to distinguish cell-specific proteome in a complex multicellular microenvironment.

Methods:
To establish cell-specific proteome labelling, we adopted the biorthogonal non-canonical amino acid tagging (BONCAT) system in hiPSCs. We expressed the mutant mouse tRNA synthetase gene, “L274GmMetRS” in hiPSCs (L274G-hiPSCs) using lentiviral transduction to enable incorporation of the non-canonical amino acid azidonorleucine (Anl) in these cells. We assessed the pluripotency and trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo (teratoma formation assay). Furthermore, we validated the cell-specific Anl incorporation in the L274G-hiPSC-CMs both in vitro (co-culture) and in vivo (post-transplantation).

Results:
Our studies showed that the L274G-hiPSCs could efficiently differentiate into all the three germ lineages and can be used to track the cell-specific proteome in their differentiated progenies, including L274G-hiPSC-CMs. Furthermore, immunostaining and western blots showed cell-specific BONCAT in L274G-hiPSC-CMs both in co-cultures (in vitro) and post-transplantation (in vivo).

Conclusion:
The newly-established L274G-hiPSC line can be used to track cell-specific proteome of hiPSC-CMs in vitro and in vivo, to characterize cell-specific proteomic responses and delineate mechanisms underlying cell therapies.