Cardiomyocyte Cell Cycle Arrest Research Articles

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.

Potential Application of Modified mRNA in Cardiac Regeneration.

Heart failure remains the leading cause of human death worldwide. After a heart attack, the formation of scar tissue due to the massive death of cardiomyocytes leads to heart failure and sudden death in most cases. In addition, the regenerative ability of the adult heart is limited after injury, partly due to cell-cycle arrest in cardiomyocytes. In the current post-COVID-19 era, urgently authorized modified mRNA (modRNA) vaccines have been widely used to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Therefore, modRNA-based protein replacement may act as an alternative strategy for improving heart disease. It is a safe, effective, transient, low-immunogenic, and integration-free strategy for in vivo protein expression, in addition to recombinant protein and stem-cell regenerative therapies. In this review, we provide a summary of various cardiac factors that have been utilized with the modRNA method to enhance cardiovascular regeneration, cardiomyocyte proliferation, fibrosis inhibition, and apoptosis inhibition. We further discuss other cardiac factors, modRNA delivery methods, and injection methods using the modRNA approach to explore their application potential in heart disease. Factors for promoting cardiomyocyte proliferation such as a cocktail of three genes comprising FoxM1, Id1, and Jnk3-shRNA (FIJs), gp130, and melatonin have potential to be applied in the modRNA approach. We also discuss the current challenges with respect to modRNA-based cardiac regenerative medicine that need to be overcome to apply this approach to heart disease. This review provides a short description for investigators interested in the development of alternative cardiac regenerative medicines using the modRNA platform.

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.

RBPMS is 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 the genetic deletion of RNA-binding protein with multiple splicing (Rbpms), an uncharacterized RNA-binding factor, causes perinatal lethality in mice due to congenital cardiovascular defects. The loss of Rbpms causes premature onset of cardiomyocyte binucleation and cell cycle arrest during development. Human iPSC-derived cardiomyocytes with RBPMS gene deletion have a similar blockade to cytokinesis. Sequencing analysis revealed that RBPMS plays a role in RNA splicing and influences RNAs involved in cytoskeletal signaling pathways. We found that RBPMS mediates the isoform switching of the heart-enriched LIM domain protein Pdlim5. The loss of Rbpms leads to an 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.

RBPMS, an RNA-Binding Protein That Mediates Cardiomyocyte Binucleation and Cardiovascular Development

SummaryNoncompaction 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 R NA b inding p rotein with m ultiple s plicing ( Rbpms ), an uncharacterized RNA binding and splicing factor, causes perinatal lethality in mice due to congenital cardiovascular defects. Loss of Rbpms causes premature onset of cardiomyocyte binucleation and cell cycle arrest during development. Human iPSC-derived cardiomyocytes with RBPMS gene deletion have a similar blockade to cytokinesis. 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.

Abstract 9815: Role for Btg1 and Btg2 in Postnatal Cardiomyocyte Cell Cycle Arrest and Maturation

Background: Adult mammalian cardiomyocytes (CM) are predominantly post-mitotic and cannot proliferatively repair the heart following myocardial infarction (MI). Overexpression of the transcription factor Tbx20 in adult mouse CMs promotes proliferative cardiac repair post-MI, via mechanisms including direct repression of anti-proliferative genes p21 , Meis1 , and Btg2 . Btg2 (B-cell translocation gene 2), a tumor suppressor and transcriptional co-regulator, exhibits high structural and functional similarity with Btg1. However, both Btg1 and Btg2 (Btg1/2) are virtually uncharacterized in the heart. Here, we investigate the role of Btg1/2 in postnatal cardiac maturation. Methods and Results: By immunostaining in embryonic, neonatal, and adult C57BL/6 mouse hearts, the highest expression of Btg1/2 was observed in late fetal and early neonatal ventricles, concurrent with upregulation of other CM cell cycle inhibitors. In neonatal mouse CMs in vitro, siRNA-mediated loss of Btg2 leads to increased CM proliferation. In vivo , Btg1/2 constitutive single- and double- knockout (SKO and DKO respectively) mice exhibit normal heart weight-to-body weight ratios compared to age-matched wildtype (WT) controls, at postnatal day (P)7, P30, and 1 year after birth. Interestingly, at P7, DKO mice have significantly higher CM mitotic activity, as indicated by pHH3 staining, compared to WT. In addition, DKO mice also exhibit significantly smaller CM cross-sectional area at P7 compared to WT. However, by P15, CM mitotic activity and cell size are comparable between WT and Btg1/2 KO mice. Currently, siRNA-mediated knockdown of Btg1/2 in neonatal rat ventricular cardiomyocyte cultures and RNAseq studies are being performed, to assess the transcriptional regulatory roles of Btg1/2 in rodent CMs. Conclusions: Here, we highlight two novel regulators of postnatal CM maturation, Btg1 and Btg2, which are upregulated coincident with CM mitotic arrest in mice. Similar to p21 and Meis1, Btg1/2 depletion in mice induces a brief period of increased CM proliferative activity before onset of CM cell cycle arrest. Our results provide evidence for Btg1/2 working in tandem with other cardiac transcription factors and cell cycle regulators, to control CM mitotic arrest after birth.

A microRNA program regulates the balance between cardiomyocyte hyperplasia and hypertrophy and stimulates cardiac regeneration

Myocardial regeneration is restricted to early postnatal life, when mammalian cardiomyocytes still retain the ability to proliferate. The molecular cues that induce cell cycle arrest of neonatal cardiomyocytes towards terminally differentiated adult heart muscle cells remain obscure. Here we report that the miR-106b~25 cluster is higher expressed in the early postnatal myocardium and decreases in expression towards adulthood, especially under conditions of overload, and orchestrates the transition of cardiomyocyte hyperplasia towards cell cycle arrest and hypertrophy by virtue of its targetome. In line, gene delivery of miR-106b~25 to the mouse heart provokes cardiomyocyte proliferation by targeting a network of negative cell cycle regulators including E2f5, Cdkn1c, Ccne1 and Wee1. Conversely, gene-targeted miR-106b~25 null mice display spontaneous hypertrophic remodeling and exaggerated remodeling to overload by derepression of the prohypertrophic transcription factors Hand2 and Mef2d. Taking advantage of the regulatory function of miR-106b~25 on cardiomyocyte hyperplasia and hypertrophy, viral gene delivery of miR-106b~25 provokes nearly complete regeneration of the adult myocardium after ischemic injury. Our data demonstrate that exploitation of conserved molecular programs can enhance the regenerative capacity of the injured heart.

Alternative Splicing of Pericentrin Contributes to Cell Cycle Control in Cardiomyocytes.

Induction of cardiomyocyte proliferation is a promising option to regenerate the heart. Thus, it is important to elucidate mechanisms that contribute to the cell cycle arrest of mammalian cardiomyocytes. Here, we assessed the contribution of the pericentrin (Pcnt) S isoform to cell cycle arrest in postnatal cardiomyocytes. Immunofluorescence staining of Pcnt isoforms combined with SiRNA-mediated depletion indicates that Pcnt S preferentially localizes to the nuclear envelope, while the Pcnt B isoform is enriched at centrosomes. This is further supported by the localization of ectopically expressed FLAG-tagged Pcnt S and Pcnt B in postnatal cardiomyocytes. Analysis of centriole configuration upon Pcnt depletion revealed that Pcnt B but not Pcnt S is required for centriole cohesion. Importantly, ectopic expression of Pcnt S induced centriole splitting in a heterologous system, ARPE-19 cells, and was sufficient to impair DNA synthesis in C2C12 myoblasts. Moreover, Pcnt S depletion enhanced serum-induced cell cycle re-entry in postnatal cardiomyocytes. Analysis of mitosis, binucleation rate, and cell number suggests that Pcnt S depletion enhances serum-induced progression of postnatal cardiomyocytes through the cell cycle resulting in cell division. Collectively, our data indicate that alternative splicing of Pcnt contributes to the establishment of cardiomyocyte cell cycle arrest shortly after birth.