Cardiomyocyte Cell Cycle Arrest Research Articles

Induced Cytokinesis Generates Highly Proliferative Mononuclear Cardiomyocytes at the Expense of Contractility.

Cytokinesis is the last step in the eukaryotic cell cycle, which physically separates a mitotic cell into 2 daughter cells. A few days after birth in mouse cardiomyocytes, DNA synthesis occurs without cytokinesis, leading to the majority of cardiomyocytes becoming binucleated instead of generating 2 daughter cells with 1 nucleus each. This results in cell cycle arrest of cardiomyocytes, and the mouse heart is no longer able to regenerate. A longstanding unanswered question is whether binucleation of cardiomyocytes is a result of cytokinesis failure. To address this, we generated several transgenic mouse models to determine whether forced induction of cardiomyocyte cytokinesis generates mononucleated cardiomyocytes and restores the endogenous regenerative properties of the myocardium. We focused on 2 complementary regulators of cytokinesis: Plk1 (polo-like kinase 1) and Ect2 (epithelial cell-transformation sequence 2). We found that cardiomyocyte-specific transgenic overexpression of constitutively active Plk1(T210D) promotes mitosis and cytokinesis in adult hearts, whereas overexpression of Ect2 alone promotes only cytokinesis. Cardiomyocyte-specific overexpression of both Plk1(T210D) and Ect2 concomitantly (double transgenic) prevents binucleation of cardiomyocytes postnatally and results in widespread cardiomyocyte mitosis, cardiac enlargement, contractile failure, and death before 2 weeks of age. Similarly, doxycycline-inducible cardiomyocyte-specific overexpression of both proteins (inducible double transgenic) in the adult heart results in reversible widespread cardiomyocyte mitosis and contractile failure. Transient induction of both genes in adult mice improves left ventricular systolic function after myocardial infarction. These results collectively demonstrate that cytokinesis failure mediates cardiomyocyte multinucleation and cell cycle exit of postnatal cardiomyocytes, but may be a protective mechanism to preserve the contractile function of the myocardium.

GDF10 promotes rodent cardiomyocyte maturation during the postnatal period.

Cardiomyocytes and cardiac fibroblasts undergo coordinated maturation after birth, and cardiac fibroblasts are required for postnatal cardiomyocyte maturation in mice. Here, we investigate the role of cardiac fibroblast-expressed Growth Differentiation Factor 10 (GDF10) in postnatal heart development. In neonatal mice, Gdf10 is expressed specifically in cardiac fibroblasts, with its highest expression coincident with the onset of cardiomyocyte cell cycle arrest and transition to hypertrophic growth. In neonatal rat ventricular myocyte (NRVM) cultures, GDF10 treatment promotes cardiomyocyte maturation indicated by increased binucleation, downregulation of cell cycle progression genes, and upregulation of cell cycle inhibitor genes. GDF10 treatment leads to an increase in cardiomyocyte cell size, together with increased expression of mature sarcomeric protein isoforms and decreased expression of fetal cardiac genes. RNAsequencing of GDF10-treated NRVM shows an increase in the expression of genes related to myocardial maturation, including upregulation of sodium and potassium channel genes. In vivo, loss of Gdf10 leads to a delay in myocardial maturation indicated by decreased cardiomyocyte cell size and binucleation, as well as increased mitotic activity, at postnatal (P) day 7. Further, induction of mature sarcomeric protein isoform gene expression is delayed, and expression of cell cycle progression genes is prolonged. However, by P10, indicators of cardiomyocyte maturation and mitotic activity are normalized in Gdf10-null hearts relative to controls. Together, these results implicate GDF10 as a novel crosstalk mediator between cardiomyocytes and cardiac fibroblasts, which is required for appropriate timing of cardiomyocyte maturation steps including binucleation, hypertrophy, mature sarcomeric isoform gene expression, and cell cycle arrest in the postnatal period.

Redox-dependent purine degradation triggers postnatal loss of cardiac regeneration potential

Postnatal cardiomyocyte cell cycle withdrawal is a critical step wherein the mammalian heart loses regenerative potential after birth. Here, we conducted interspecies multi-omic comparisons between the mouse heart and that of the opossum, which have different postnatal time-windows for cardiomyocyte cell cycle withdrawal. Xanthine metabolism was activated in both postnatal hearts in parallel with cardiomyocyte cell cycle arrest. The pentose phosphate pathway (PPP) which produces NADPH was found to decrease simultaneously. Postnatal myocardial tissues became oxidized accordingly, and administration of antioxidants to neonatal mice altered the PPP and suppressed the postnatal activation of cardiac xanthine metabolism. These results suggest a redox-driven postnatal switch from purine synthesis to degradation in the heart. Importantly, inhibition of xanthine metabolism in the postnatal heart extended postnatal duration of cardiomyocyte proliferation and maintained postnatal heart regeneration potential in mice. These findings highlight a novel role of xanthine metabolism as a redox-dependent metabolic regulator of cardiac regeneration potential.

The Decreased Proliferation Capacity of Cardiomyocytes Induced By Androsterone Is Mediated By the Interactions Between Androgen Receptor and Retinoblastoma Protein.

Our previous study has demonstrated that the decline in cardiomyocytes proliferation capacity induced by maternal androgen excess was mainly attributed to the accumulation of androsterone in the heart. However, the underlying mechanism by which androsterone inhibits cardiomyocytes proliferation remains unknown. In this study, pregnant mice were injected subcutaneously daily with dihydrotestosterone (DHT) from gestational day (GD) 16.5 to GD18.5. On GD18.5, fetal heart tissue was dissected and used for analyzing androgen receptor (AR) levels. H9c2 cells and primary cardiomyocytes, isolated from fetal hearts, were applied to investigate the mechanism. H9c2 cells under androsterone treatment were subjected to RNA sequencing analysis and the results showed that genes were primarily enriched in cell cycle and DNA replication pathways. Elevated AR levels were observed in fetal cardiac tissue in the maternal DHT-treated group. Androsterone treatment increased the ratio of nuclear AR and cytoplasmic AR both in H9c2 cells and primary cardiomyocytes. The ablation and overexpression of AR can mildly reverse and aggravate cell cycle arrest induced by androsterone, respectively. ChIP-qPCR analysis suggested that AR can directly repress cell cycle and DNA replication-related gene expression, which was mediated by the recruitment of retinoblastoma protein (Rb). The repression of cell proliferation in response to androsterone was alleviated partly through the downregulation of Rb by siRNA transfection. In conclusion, AR repression to cell cycle and DNA replication-related gene expression, mediated by recruitment of Rb, may be one of the potential mechanisms of cell cycle arrest in cardiomyocytes induced by androsterone.

Cardiomyocyte proliferation is promoted by intracellular Gas6-mediated Yap activity during neonatal heart regeneration

Abstract Purpose The adult mammalian heart has limited regenerative capacity after injury, mostly attributable to postnatal cardiomyocyte (CM) cell cycle arrest. The key to unlocking the regenerative potential of the adult hearts may lie within the developmental transitions occurring during neonatal life. Gas6, a secretory protein, stimulates the proliferation of various type cells through binding to TAM receptor family, such as endothelial progenitor cells, tumor cells, and hair follicle stem-cell. However, no study to date has studied the role of Gas6 during the proliferation of CMs. Herein, our study uncovers that Gas6 is transiently expressed during early postnatal heart and regenerative heart, and functions through intracellular pathway by regulating Yap activity to promote CM proliferation. Methods CM isolation was conducted to assess the change of Gas6 expression in both postnatal development and regenerated stages using the CM-specific Tdtomato mice. Cardiac deletion of Gas6 gene mice was used to evaluate the impaired heart regeneration. CM-specific Gas6 overexpression mice were generated using adeno-associated virus system to evaluate Gas6 pro-proliferate and protect effect after ischemic injury. Secreted signal peptide truncation (ΔSP-Gas6) and predicted functional region truncation (ΔCC-Gas6) were generated to identify the intracellular mechanism of Gas6 via Sav1/p-mst1/p-lats1/ p-yap axis. Results We found that Gas6 expression is enriched in CM4 and the expression profile correlates with the expression of the CM4 marker Acta2, as well as cell-cycle genes Mki67 and Aurora B, in individual CM. Gas6 was highly expressed in the neonatal heart and continued to increase during the neonatal heart regenerative process, but was nearly absent in the adult heart under physiological and ischemia conditions. CM-specific Gas6 deletion resulted in significantly reduced CM proliferation rate and increased CM size in P0 hearts, and congestive heart failure in P28 hearts. Conversely, sustained CM-specific overexpression of Gas6 in postnatal hearts increased CM division, elevated CM numbers, and reduced CM size; and reduced infarct scar area and enhanced cardiac function after ischemia injury. Pre-overexpression of Gas6 in adult CMs protected adult cardiac function against ischemia injury, indicated by the activation of mitosis, angiogenesis and reduced oxidative phosphorylation. In primary neonatal CMs, both Gas6 and ΔSP-Gas6promoted CM proliferation with sarcomere disassembly. Mechanistically, Gas6 interacted with Sav1to inhibit Mst1recruitment and Mst1/Lats1/Yap phosphorylation cascade, therefore, promoted Yap translocation into nucleus. Conclusions We demonstrate that Gas6 is an important intrinsic regulator of neonatal heart regeneration, which acts as a promoter of Yap transcriptional activity to enhance CM proliferation. Supplementing Gas6 in the adult heart represents a potential therapeutic approach for cardiac repair.

Screening lncRNAs essential for cardiomyocyte proliferation by integrative profiling of lncRNAs and mRNAs associated with heart development

BackgroundThe proliferation potential of mammalian cardiomyocytes declines markedly shortly after birth. Both long non-coding RNAs (lncRNAs) and mRNAs demonstrate altered expression patterns during cardiac development. However, the role of lncRNAs in the cell cycle arrest of cardiomyocytes remains inadequately understood. MethodThe expression pattern of lncRNAs and mRNAs was analyzed in mouse hearts exhibiting varying regenerative potentials on postnatal days (P) 1, 7, and 28. Weighted correlation network analysis (WGCNA) was employed to elucidate the co-expression relationship between lncRNAs and mRNAs. Protein-protein interaction (PPI) network was built using the STRING database, and hub lncRNAs were identified by CytoHubba. Molecular Complex Detection (MCODE) was used to screen core modules of the PPI network in Cytoscape. Upstream lncRNAs and miRNAs which may regulate mRNAs were predicted using miRTarBase and AnnoLnc2, respectively. Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery. ResultsCompared with the P1 heart, 618 mRNAs and 414 lncRNAs displayed.transcriptional changes in the P7 heart, while 2358 mRNAs and 1290 lncRNAs showed from P7 to P28. Gene Ontology (GO) analysis revealed that module 1 in the both comparisons was enriched in the mitotic cell cycle process. 2810408I11Rik and 2010110K18Rik were identified as hub lncRNAs and their effects on the proliferation of cardiomyocytes were verified in vitro. Additionally, four lncRNA-miRNA-mRNA regulatory axes were predicted to explain the mechanism by which 2810408I11Rik and 2010110K18Rik regulate cardiomyocyte proliferation. Notably, the overexpression of 2810408I11Rik enhances cardiomyocyte proliferation and heart regeneration in the adult heart following MI. ConclusionThis study systematically analyzed the landscape of lncRNAs and mRNAs at P1, P7, and P28. These findings may enhance our understanding of the framework for heart development and could have significant implications for heart regeneration.

Metabolic Reprogramming: A Byproduct or a Driver of Cardiomyocyte Proliferation?

Defining mechanisms of cardiomyocyte proliferation should guide the understanding of endogenous cardiac regeneration and could lead to novel treatments for diseases such as myocardial infarction. In the neonatal heart, energy metabolic reprogramming (phenotypic alteration of glucose, fatty acid, and amino acid metabolism) parallels cell cycle arrest of cardiomyocytes. The metabolic reprogramming occurring shortly after birth is associated with alterations in blood oxygen levels, metabolic substrate availability, hemodynamic stress, and hormone release. In the adult heart, myocardial infarction causes metabolic reprogramming but these changes cannot stimulate sufficient cardiomyocyte proliferation to replace those lost by the ischemic injury. Some putative pro-proliferative interventions can induce the metabolic reprogramming. Recent data show that altering the metabolic enzymes PKM2 [pyruvate kinase 2], LDHA [lactate dehydrogenase A], PDK4 [pyruvate dehydrogenase kinase 4], SDH [succinate dehydrogenase], CPT1b [carnitine palmitoyl transferase 1b], or HMGCS2 [3-hydroxy-3-methylglutaryl-CoA synthase 2] is sufficient to partially reverse metabolic reprogramming and promotes adult cardiomyocyte proliferation. How metabolic reprogramming regulates cardiomyocyte proliferation is not clearly defined. The possible mechanisms involve biosynthetic pathways from the glycolysis shunts and the epigenetic regulation induced by metabolic intermediates. Metabolic manipulation could represent a new approach to stimulate cardiac regeneration; however, the efficacy of these manipulations requires optimization, and novel molecular targets need to be defined. In this review, we summarize the features, triggers, and molecular regulatory networks responsible for metabolic reprogramming and discuss the current understanding of metabolic reprogramming as a critical determinant of cardiomyocyte proliferation.

Sphingolipid metabolism controls mammalian heart regeneration

Utilization of lipids as energy substrates after birth causes cardiomyocyte (CM) cell-cycle arrest and loss of regenerative capacity in mammalian hearts. Beyond energy provision, proper management of lipid composition is crucial for cellular and organismal health, but its role in heart regeneration remains unclear. Here, we demonstrate widespread sphingolipid metabolism remodeling in neonatal hearts after injury and find that SphK1 and SphK2, isoenzymes producing the same sphingolipid metabolite sphingosine-1-phosphate (S1P), differently regulate cardiac regeneration. SphK2 is downregulated during heart development and determines CM proliferation via nuclear S1P-dependent modulation of histone acetylation. Reactivation of SphK2 induces adult CM cell-cycle re-entry and cytokinesis, thereby enhancing regeneration. Conversely, SphK1 is upregulated during development and promotes fibrosis through an S1P autocrine mechanism in cardiac fibroblasts. By fine-tuning the activity of each SphK isoform, we develop a therapy that simultaneously promotes myocardial repair and restricts fibrotic scarring to regenerate the infarcted adult hearts.

Abstract P1086: Induced Pluripotent Stem Cell-based Cardiac Tissue Modeling Of Mitogenic Cardiomyopathy In Alström Syndrome

Introduction: Cardiomyopathies are a prominent cause of heart failure and affect millions worldwide. Mutation in the Alström syndrome 1 (ALMS1) gene causing Alström syndrome (ALMS) , reported an extremely rare type of dilated cardiomyopathy - called mitogenic cardiomyopathy - leading to neonatal heart failure and death in early infancy due to delayed postnatal cardiomyocyte (CM) cell cycle arrest. Methods: Induced pluripotent stem cells (iPSCs) from ALMS patients with or without mitogenic cardiomyopathy were used to generate ALMS patient-specific cardiomyocytes (iPSC-CMs) . The iPSC-CMs were divided into two main groups based on the presence or absence of the mitogenic phenotype. After transcriptome sequencing, differentially expressed genes (FDR < 0.05, logFC > 0.5 | < -0.5) were found by testing (QL F-test) a robust fitted linear model (edgeR). Harnessing recent advances in stem cell differentiation and tissue engineering, we aimed at creating a human engineered heart tissue model as an unprecedented in vitro disease system to study mechanisms responsible for persistent CM proliferation. Results: ALMS1-deficient mitogenic iPSC-CMs exhibited an impaired ability to undergo cell cycle arrest, evidenced by a higher cell percentage in the G2/M phase (19.3% in mitogenic vs . 7.5% in non-mitogenic iPSC-CMs). Furthermore, increased extracellular matrix levels of the fibroblast-derived protein periostin (POSTN) were observed in co-cultures of ALMS1-deficient iPSC-CMs and ALMS1 fibroblasts. Finally, we found preliminary evidence of dysregulation of the Hippo signaling pathway, observing altered cellular localization of its key downstream effector Yes-associated protein (YAP) and dysregulated ratio between phosphor and total YAP protein levels that in combination with transcript alteration of PPIC, SH3BP4, NTN4, LRIG3 suggest perturbations in YAP signaling in ALMS patients. Conclusion: ALMS1-deficient mitogenic iPSC-CMs recapitulate postnatal proliferation, observed in ALMS patients with mitogenic cardiomyopathy. Preliminary molecular analyses in these in vitro (multicellular) models point to an important role for altered YAP signaling and provide novel cues to enhance CM proliferation, a much-sought objective in cardiac repair.

Omentin-1 drives cardiomyocyte cell cycle arrest and metabolic maturation by interacting with BMP7.

Mammalian cardiomyocytes (CMs) undergo maturation during postnatal heart development to meet the increased demands of growth. Here, we found that omentin-1, an adipokine, facilitates CM cell cycle arrest and metabolic maturation. Deletion of omentin-1 causes mouse heart enlargement and dysfunction in adulthood and CM maturation retardation in juveniles, including delayed cell cycle arrest and reduced fatty acid oxidation. Through RNA sequencing, molecular docking analysis, and proximity ligation assays, we found that omentin-1 regulates CM maturation by interacting directly with bone morphogenetic protein 7 (BMP7). Omentin-1 prevents BMP7 from binding to activin type II receptor B (ActRIIB), subsequently decreasing the downstream pathways mothers against DPP homolog 1 (SMAD1)/Yes-associated protein (YAP) and p38 mitogen-activated protein kinase (p38 MAPK). In addition, omentin-1 is required and sufficient for the maturation of human embryonic stem cell-derived CMs. Together, our findings reveal that omentin-1 is a pro-maturation factor for CMs that is essential for postnatal heart development and cardiac function maintenance.

Btg1 and Btg2 regulate neonatal cardiomyocyte cell cycle arrest

Rodent cardiomyocytes undergo mitotic arrest in the first postnatal week. Here, we investigate the role of transcriptional co-regulator Btg2 (B-cell translocation gene 2) and functionally-similar homolog Btg1 in postnatal cardiomyocyte cell cycling and maturation. Btg1 and Btg2 (Btg1/2) are expressed in neonatal C57BL/6 mouse left ventricles coincident with cardiomyocyte cell cycle arrest. Btg1/2 constitutive double knockout (DKO) mouse hearts exhibit increased pHH3+ mitotic cardiomyocytes compared to Wildtype at postnatal day (P)7, but not at P30. Similarly, neonatal AAV9-mediated Btg1/2 double knockdown (DKD) mouse hearts exhibit increased EdU+ mitotic cardiomyocytes compared to Scramble AAV9-shRNA controls at P7, but not at P14. In neonatal rat ventricular myocyte (NRVM) cultures, siRNA-mediated Btg1/2 single and double knockdown cohorts showed increased EdU+ cardiomyocytes compared to Scramble siRNA controls, without increase in binucleation or nuclear DNA content. RNAseq analyses of Btg1/2-depleted NRVMs support a role for Btg1/2 in inhibiting cell proliferation, and in modulating reactive oxygen species response pathways, implicated in neonatal cardiomyocyte cell cycle arrest. Together, these data identify Btg1 and Btg2 as novel contributing factors in mammalian cardiomyocyte cell cycle arrest after birth.

Actomyosin-mediated cellular tension promotes Yap nuclear translocation and myocardial proliferation through α5 integrin signaling.

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state results in the loss of regenerative potential of the mammalian heart shortly after birth. Nonmuscle myosin IIB (NM IIB)-mediated actomyosin contractility regulates cardiomyocyte cytokinesis in the embryonic heart, and NM IIB levels decline after birth, suggesting a role for cellular tension in the regulation of cardiomyocyte cell cycle activity in the postnatal heart. To investigate the role of actomyosin contractility in cardiomyocyte cell cycle arrest, we conditionally activated ROCK2 kinase domain (ROCK2:ER) in the murine postnatal heart. Here, we show that α5/β1 integrin and fibronectin matrix increase in response to actomyosin-mediated tension. Moreover, activation of ROCK2:ER promotes nuclear translocation of Yap, a mechanosensitive transcriptional co-activator, and enhances cardiomyocyte proliferation. Finally, we show that reduction of myocardial α5 integrin rescues the myocardial proliferation phenotype in ROCK2:ER hearts. These data demonstrate that cardiomyocytes respond to increased intracellular tension by altering their intercellular contacts in favor of cell-matrix interactions, leading to Yap nuclear translocation, thus uncovering a function for nonmuscle myosin contractility in promoting cardiomyocyte proliferation in the postnatal heart.

Abstract 15792: Unchecked Cytokinesis Generates Highly Proliferative Mononuclear Cardiomyocytes at the Expense of Contractility

Introduction: A few days after birth in mouse cardiomyocytes, DNA synthesis occurs without cytokinesis leading to the majority of cardiomyocytes becoming binuclear instead of generating 2 daughter cells with one nucleus each. This results in cell cycle arrest of cardiomyocytes and the mouse heart is no longer able to regenerate. Hypothesis: A long-standing unanswered question in the field is whether multinucleation of cardiomyocytes is a result of cytokinesis failure. Methods and Results: To address this, we generated several transgenic mouse models to determine whether forced induction of cardiomyocyte cytokinesis generates mononuclear cardiomyocytes and restores the endogenous regenerative properties of the myocardium. We focused on two complementary regulators of cytokinesis, namely Polo-like kinase 1 (Plk1) and epithelial cell-transformation sequence 2 (Ect2), Here we report that cardiomyocyte-specific transgenic overexpression of constitutively active Plk1(T210D) alone promotes mitosis and cytokinesis in adult hearts, while overexpression of Ect2 alone promotes cytokinesis. Intriguingly, cardiomyocyte-specific overexpression of both Plk1(T210D) and Ect2 concomitantly (double transgenic) prevents binucleation of cardiomyocytes postnatally and results in widespread cardiomyocyte mitosis, cardiac enlargement, contractile failure and death before two weeks of age. Similarly, high-dose doxycycline inducible cardiomyocyte-specific overexpression of both proteins (inducible double transgenic) in the adult heart results in reversible widespread cardiomyocyte mitosis, cardiac enlargement, and contractile failure, while low-dose transient induction also results in significant cardiomyocyte proliferation and lower ejection fraction that is reversible after doxycycline is removed. Finally, we show that transient low-dose induction of both genes in adults improves left ventricular systolic function following myocardial infarction. Conclusion: Collectively, these results demonstrate that cytokinesis failure mediates cardiomyocyte multinucleation and cell cycle exit of postnatal cardiomyocytes but may be a protective mechanism to preserve the contractile function of the myocardium.

Abstract 15035: LIN28a Extends Regenerative Window of the Postnatal Heart and Promotes New Myocyte Formation in the Adult Heart After Injury via Lncrna-h19

The neonatal heart holds a remarkable regenerative capacity that is lost within the first week of life coinciding with a profound shift in cardiomyocyte (CM) metabolism, and CM cell cycle arrest. Whether reprogramming metabolism reactivates CM cell cycle enhancing cardiac function and repair after injury is unknown. Here, we identify a novel role for the RNA-binding protein LIN28a, a master regulator of cellular metabolism, in cardiac development and repair following injury. LIN28a was found as primarily active during cardiac development and rapidly decreases after birth. Injury to the neonatal heart, but not in adults, reactivates LIN28a leading to regenerative processes while LIN28a inhibition with small molecules attenuated pro-reparative effects of the postnatal heart. LIN28a reintroduction at P1, P3, P5, P7, and P14 decreased maturation-associated polyploidization, nucleation, and cell size, enhancing CM cell cycle activity and number of CMs per hearts in LIN28a transgenic pups compared to WT littermates. Moreover, LIN28a overexpression extended CM cell cycle activity and regenerative processes beyond P7 resulting in increased cardiac function 30 days after apical resection. In the adult heart, LIN28a overexpression promoted formation of new CMs and enhanced cardiac function, and survival in mice 12 weeks after myocardial infarction compared to WT littermate controls. Furthermore, the percentage of mononucleated CMs in the LIN28a overexpressing hearts was positively correlated to better cardiac functional recovery after injury. Mechanistically, CMs overexpressing LIN28a showed increased glycolytic metabolism while its attenuation by 2-DG reverted LIN28a-induced enhancement in CM cell cycle activity in adult hearts 4 days after injury. LIN28a immunoprecipitation followed by RNA sequencing in CMs isolated from LIN28a injured hearts identified lncRNA-H19 as its most significantly altered target. Ablation of lncRNA-H19 blunted LIN28a-induced enhancement on CM metabolism and cell cycle activity. Collectively, LIN28a reprograms CM metabolism enhancing cell cycle activity in the injured heart and pro-reparative processes thereby linking CM metabolism to the regulation of ploidy/nucleation and repair in the heart.

Abstract 11817: Induced Pluripotent Stem Cell-Derived Cardiac Disease Modeling for Studying Mitogenic Cardiomyopathy in Alström Syndrome

Introduction: Cardiomyopathies are a prominent cause of heart failure and affect millions of people worldwide. We reported two case studies describing siblings with a mutation in the Alström 1 (ALMS1) gene causing Alström syndrome (ALMS), who developed mitogenic cardiomyopathy, characterized by delayed postnatal cardiomyocyte (CM) cell cycle arrest resulting in neonatal heart failure and death in early infancy. OBJECTIVES : To generate induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from ALMS patients and investigate molecular mechanisms of persistent CM proliferation in co-culture with ALMS1 fibroblasts. METHODS : We reprogrammed somatic cells from ALMS patients with mitogenic cardiomyopathy into iPSCs using Sendai virus technology and generated ALMS-specific iPSC-CMs using established in vitro differentiation protocols. We analyzed cell cycle progression by determining CM percentages in the G0/G1, S and G2/M phase using flow cytometry, and measured expression and secretion of periostin (POSTN) and collagens from ALMS1 fibroblasts in the extracellular matrix (ECM) using RT-QPCR and western blot. Finally, we investigated Hippo signaling by measuring expression and nuclear translocation of its downstream effectors YAP/TAZ using western blot and immunostaining. RESULTS : iPSC-CMs were successfully generated from ALMS patients and controls. ALMS1-deficient iPSC-CMs exhibited impaired ability to undergo cell cycle arrest, evidenced by a higher cell percentage in the G2/M phase. Furthermore, increased ECM levels of POSTN, a potent regulator of CM proliferation, were observed in co-cultures of ALMS1-deficient iPSC-CMs and ALMS1 fibroblasts. Finally, we found preliminary evidence of dysregulation of the Hippo pathway with altered cellular localization of YAP, and dysregulated total YAP and phosphorylated YAP. CONCLUSION : ALMS1-deficient iPSC-CMs recapitulate persistent postnatal proliferation observed in ALMS patients with mitogenic cardiomyopathy. Increased POSTN in the ECM and altered downstream Hippo signaling contribute to the mitogenic phenotype and suggest critical cues for future therapies aiming at stimulating CM turnover, a much-sought objective in cardiac repair to improve contractile function.

Abstract 10618: Modified Rna Ccnd2 Promotes Myocardial Remuscularizationin Lv Infarct by Regulating Cardiomyocyte Cell Cycle

The adult mammalian heart has limited capacity to regenerate, due to postnatal cardiomyocyte cell cycle arrest. This study examined whether the cardiomyocyte cell-cycle reactivation can enhance myocardial remuscularization in hearts with acute myocardial infarction (AMI). Because the G1-to-S phase transition of the cell cycle is critically regulated by cyclin D proteins, and the expression of cyclin D2 (CCND2) could regenerate injured myocardium, we hypothesized that a transient and exclusive overexpression of CCND2 in cardiomyocytes can result in myocytes proliferation and therefore, remuscularize myocardial infarct. The transient and exclusive myocyte overexpression of CCND2 was achieved by Specific ModRNA Translation system (SMRTs) 1 . In vitro assessments were performed in hiPSC-derived cardiomyocytes (hiPSC-CMs) by immunofluorescence staining of cell proliferation markers (Ki67, BrdU, PH3, and Aurora B) to evaluate whether cardiomyocyte-specific modified RNA CCND2 ( CMS modRNA-CCND2) could reactivate cell cycle. Using a mouse model of AMI, normal and infarcted mice were compared 4 weeks after AMI to assess the structural and functional outcomes of myocardial injection 100μg CMS modRNA-CCND2. In vitro , the, CMS modRNA-CCND2 resulted in significantly higher CMs cell cycle activity as evidenced by significantly higher percentage of hiPSC-CMs that expressed Ki67 (8.68 ± 1.64 VS 36.68 ± 2.61 , p<0.01), BrdU (8.44 ± 1.14% VS 17.59 ± 0.84%, p<0.01), PH3 (0.51 ± 0.04% VS 1.68 ± 0.09%, p<0.01), and Aurora B (0.47 ± 0.03% VS 1.59 ± 0.07%, P<0.01), CMS modRNA-Luciferase vs CMS modRNA-CCND2, respectively. In vivo, CMs cell cycle activity was significantly increased in SMRT treated hearts 4 weeks post AMI as evidenced by: Ki67 (0.41 ± 0.04% VS 2.00 ± 0.17%, p<0.01), PH3 (0.039 ± 0.004% VS 0.30 ± 0.006%, p<0.01), AMI VS AMI + SMRT, respectively. LV Infarct size also decreased significantly 4 weeks after AMI in AMI + SMRT group of hearts (50.04 ± 1.42% VS 31.28 ± 1.28%, p<0.01). Thus, CMS modRNA-CCND2 could induce myocardial remuscularization by re-activating CM cell- cycle in hearts with AMI. *, equal contribution first author

Abstract P1034: Neurohormonal Pathways Drive Postnatal Thermogenesis And Loss Of Cardiac Regenerative Potential

Why do mammalian organs including the heart lose regenerative potential during the perinatal window remains enigmatic. Through phylogenetic analysis of vertebrate cardiomyocyte ploidy as a proxy for cardiac regenerative potential, we uncover that certain monotreme, edentate, cetacean, chiropteran species have unusually high percentages of diploid cardiomyocytes in the adult heart. Cardiomyocyte abundance across 41 vertebrate species conforms to Kleiber's law, the 3/4-power law scaling of metabolism with bodyweight, and decreases when the standard metabolic rate and body temperature increase during the ectotherm-to-endotherm transition. Recently, following the fractal feature of the cardiovascular system, we further strengthen the link between endothermy and heart physiology. Thermogenesis increases by more than 10-fold during the ectotherm-to-endotherm transition requiring similar increases in blood flow and cardiac function that may impede heart regenerative potential. Moreover, we report sympathetic nerve-adrenergic receptor and thyroid hormone signaling as two major pathways promoting both thermogenesis and cardiomyocyte cell cycle arrest. Combined inhibition of postnatal adrenergic and thyroid hormone signaling results in juvenile mice with strikingly low body temperatures (~25 ° C), significantly elevated abundances of diploid cardiomyocytes (~60%), dramatically increased cardiomyocyte proliferation, and enhanced cardiac regenerative abilities when analyzed at postnatal day 14. Intriguingly, both neurohormonal pathways seem to be inactive in adult zebrafish hearts. Altogether, our findings support critical roles of two major thermogenic pathways in suppressing heart regenerative capacity, and implicate that the limited regenerative capacity in various adult mammalian tissues may be a tradeoff for the acquisition of endothermy in ontogeny and phylogeny.

Abstract EC102: RBPMS, An RNA-binding Protein That Mediates Cardiomyocyte Binucleation And Cardiovascular Development

Noncompaction cardiomyopathy is a common congenital cardiac disorder associated with abnormal ventricular cardiomyocyte trabeculation and impaired pump function. The genetic basis and underlying mechanisms of this disorder remain elusive. We show that genetic deletion of RNA binding protein with multiple splicing (Rbpms), an uncharacterized RNA binding and splicing factor, causes perinatal lethality in mice due to congenital cardiovascular defects, including noncompaction cardiomyopathy, ventricular septal defects, and patent ductus arteriosus. Loss of Rbpms causes premature onset of cardiomyocyte binucleation and cell cycle arrest during development, thus disturbing embryonic heart growth. Human iPSC-derived cardiomyocytes with RBPMS gene deletion have a similar blockade to cytokinesis, and Rbpms expression is decreased in human patients with congenital cardiomyopathy, implicating an essential role of Rbpms in human heart development. Paired-end RNA sequencing analysis revealed that RBPMS plays a role in RNA splicing and influences RNAs involved in cytoskeletal signaling pathways. We found that RBPMS mediates isoform switching of the heart-specific LIM domain protein Pdlim5. Loss of Rbpms leads to abnormal accumulation of Pdlim5-short isoforms, disrupting cardiomyocyte cytokinesis. Our findings connect premature cardiomyocyte binucleation to noncompaction cardiomyopathy and highlight the role of Rbpms in this process.

CircRNA Samd4 induces cardiac repair after myocardial infarction by blocking mitochondria-derived ROS output

Reactive oxygen species (ROS) derived from oxygen-dependent mitochondrial metabolism are the essential drivers of cardiomyocyte (CM) cell-cycle arrest in adulthood. Mitochondria-localized circular RNAs (circRNAs) play important roles in regulating mitochondria-derived ROS production, but their functions in cardiac regeneration are still unknown. Herein, we investigated the functions and underlying mechanism of mitochondria-localized circSamd4 in cardiac regeneration. We found that circSamd4 was selectively expressed in fetal and neonatal CMs. The transcription factor Nrf2 controlled circSamd4 expression by binding to the promoter of circSamd4 host gene. CircSamd4 overexpression reduced while circSamd4 silenced increased mitochondrial oxidative stress and subsequent oxidative DNA damage. Moreover, circSamd4 overexpression induced CM proliferation and prevented CM apoptosis, which reduced the size of the fibrotic area and improved cardiac function after myocardial infarction (MI). Mechanistically, circSamd4 reduced oxidative stress generation and maintained mitochondrial dynamics by inducing the mitochondrial translocation of the Vcp protein, which downregulated Vdac1 expression and prevented the mitochondrial permeability transition pore (mPTP) from opening. Our findings suggest that circSamd4 is a novel therapeutic target for heart failure after MI.

Regulation of extracellular matrix composition by fibroblasts during perinatal cardiac maturation

BackgroundCardiac fibroblasts are the main non-myocyte population responsible for extracellular matrix (ECM) production. During perinatal development, fibroblast expansion coincides with the transition from hyperplastic to hypertrophic myocardial growth. Therefore, we investigated the consequences of fibroblast loss at the time of cardiomyocyte maturation by depleting fibroblasts in the perinatal mouse. Methods and resultsWe evaluated the microenvironment of the perinatal heart in the absence of fibroblasts and the potential functional impact of fibroblast loss in regulation of cardiomyocyte cell cycle arrest and binucleation. Cre-mediated expression of diphtheria toxin A in PDGFRα expressing cells immediately after birth eliminated 70–80% of the cardiac fibroblasts. At postnatal day 5, hearts lacking fibroblasts appeared similar to controls with normal morphology and comparable numbers of endothelial and smooth muscle cells, despite a pronounced reduction in fibrillar collagen. Immunoblotting and proteomic analysis of control and fibroblast-deficient hearts identified differential abundance of several ECM proteins. In addition, fibroblast loss decreased tissue stiffness and resulted in increased cardiomyocyte mitotic index, DNA synthesis, and cytokinesis. Moreover, decellularized matrix from fibroblast-deficient hearts promoted cardiomyocyte DNA replication. While cardiac architecture was not overtly affected by fibroblast reduction, few pups survived past postnatal day 11, suggesting an overall requirement for PDGFRα expressing fibroblasts. ConclusionsThese studies demonstrate the key role of fibroblasts in matrix production and cardiomyocyte cross-talk during mouse perinatal heart maturation and revealed that fibroblast-derived ECM may modulate cardiomyocyte maturation in vivo. Neonatal depletion of fibroblasts demonstrated that although hearts can tolerate reduced ECM composition, fibroblast loss eventually leads to perinatal death as the approach simultaneously reduced fibroblast populations in other organs.