Cardiomyocyte Cell Cycle Research Articles

RNA-Binding Protein Hnrnpa1 Triggers Daughter Cardiomyocyte Formation by Promoting Cardiomyocyte Dedifferentiation and Cell Cycle Activity in a Post-Transcriptional Manner.

Stimulating cardiomyocyte (CM) dedifferentiation and cell cycle activity (DACCA) is essential for triggering daughter CM formation. In addition to transcriptional processes, RNA-binding proteins (RBPs) are emerging as crucial post-transcriptional players in regulating CM DACCA. However, whether post-transcriptional regulation of CM DACCA by RBPs could effectively trigger daughter CM formation remains unknown. By performing integrated bioinformatic analysis of snRNA-seq data from neonatal and adult hearts, this study identified Hnrnpa1 as a potential RBP regulating CM DACCA. Hnrnpa1 expression decreased significantly during postnatal heart development. With the use of α-MHC-H2B-mCh/CAG-eGFP-anillin transgenic mice, Hnrnpa1 overexpression promoted CM DACCA, thereby triggering daughter CM formation and enhancing cardiac repair after myocardial infarction (MI). In contrast, CRISPR/Cas9 technology is used to generate CM-specific Hnrnpa1 knockout mice. Hnrnpa1 knockout inhibited cardiac regeneration and worsened cardiac function in the neonatal MI model. Nanopore RNA sequencing, RIP assay, IP-MS, MeRIP-qPCR, PAR-CLIP and luciferase reporter experiments showed that Hnrnpa1 induced Mettl3 post-transcriptional splicing to inhibit m6A-dependent Pbx1 and E2F1 degradation, thereby increasing Runx1, Ccne1, Cdk2 and Ccnb2 expression to promote CM DACCA. In conclusion, Hnrnpa1 triggered daughter CM formation by promoting CM DACCA in a post-transcriptional manner, indicating that Hnrnpa1 might serve as a promising target in cardiac repair post-MI.

Abstract 4136030: Fatty acids disturb cardiomyocyte proliferation by increasing PDK4 and HMGCS2 via PPARδ activation

Background: Almost all cardiac diseases are difficult to cure entirely because cardiomyocytes rarely proliferate to recover cardiac damage. In fact, most mammals have a capacity to regenerate cardiomyocytes immediately after birth. Newborn infant drastically changes cardiac metabolism from glycolysis to oxidative phosphorylation using fatty acids. Previous research revealed that lipid metabolism had a negative influence on cardiomyocyte cell cycle but the mechanism has not been clarified completely. Objective: This study was performed to elucidate the way fatty acid metabolism affects cardiomyocyte proliferation. Methods and results: Isolated and cultured neonatal rat cardiomyocytes (NRCM) were treated with the mixture of palmitic acid, oleic acid, and L-carnitine (FA mix) for 24 hours. Realtime RT-PCR revealed that FA mix increased the mRNA expressions of pyruvate dehydrogenase kinase 4 (PDK4), a fatty acid metabolism regulator, and HMG-CoA synthase 2 (HMGCS2), a ketogenic factor, as well as β-oxidation-related enzymes in dose dependent manner. Simultaneously, the results of immunofluorescent staining showed that FA mix decreased cardiomyocytes expressing Ki67, a cell cycle marker. GW501516, a peroxisome proliferator-activated receptor δ (PPARδ) agonist, also increased fatty acid metabolism factors with the reduction of Ki67 positive cardiomyocytes, whereas fenofibrate, a PPARα agonist, or pioglitazone, a PPARγ agonist, caused little or no change in these phenotypes. Moreover, NRCM pretreated with GSK3787, a PPARδ antagonist, before FA mix stimulation suppressed the upregulations of fatty acid metabolism factors and the reduction of Ki67 expressing cardiomyocytes. Finally, the lentiviral vectors which expressed PDK4 or HMGCS2 were produced to investigate these proteins suppressed NRCM cell cycle directly. Either overexpressing PDK4 or HMGCS2 decreased Ki67 positive NRCM without the upregulation of each other or β-oxidation-related enzymes. Conclusion: Fatty acids disturbed NRCM proliferation by upregulating PDK4 and HMGCS2 through the activation of PPARδ independently from β-oxidation.

Engineered Cardiac Tissues as a Platform for CRISPR-Based Mitogen Discovery.

Improved understanding of cardiomyocyte (CM) cell cycle regulation may allow researchers to stimulate pro-regenerative effects in injured hearts or promote maturation of human stem cell-derived CMs. Gene therapies, in particular, hold promise to induce controlled proliferation of endogenous or transplanted CMs via transient activation of mitogenic processes. Methods to identify and characterize candidate cardiac mitogens in vitro can accelerate translational efforts and contribute to the understanding of the complex regulatory landscape of CM proliferation and postnatal maturation. In this study, A CRISPR knockout-based screening strategy using in vitro neonatal rat ventricular myocyte (NRVM) monolayers is established, followed by candidate mitogen validation in mature 3-D engineered cardiac tissues (ECTs). This screen identified knockout of the purine metabolism enzyme adenosine deaminase (ADA-KO) as an effective pro-mitogenic stimulus. RNA-sequencing of ECTs further reveals increased pentose phosphate pathway (PPP) activity as the primary driver of ADA-KO-induced CM cycling. Inhibition of the pathway's rate limiting enzyme, glucose-6-phosphate dehydrogenase (G6PD), prevented ADA-KO induced CM cycling, while increasing PPPactivityviaG6PD overexpression increased CM cycling. Together, this study demonstrates the development and application of a genetic/tissue engineering platform forin vitrodiscovery and validation of new candidate mitogens affecting regenerative or maturation states of cardiomyocytes.

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.

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.

Cell cycle visualization tools to study cardiomyocyte proliferation in real-time.

Cardiomyocytes in the adult human heart are quiescent and those lost following heart injury are not replaced by proliferating survivors. Considerable effort has been made to understand the mechanisms underlying cardiomyocyte cell cycle exit and re-entry, with view to discovering therapeutics that could stimulate cardiomyocyte proliferation and heart regeneration. The advent of large compound libraries and robotic liquid handling platforms has enabled the screening of thousands of conditions in a single experiment but success of these screens depends on the appropriateness and quality of the model used. Quantification of (human) cardiomyocyte proliferation in high throughput has remained problematic because conventional antibody-based staining is costly, technically challenging and does not discriminate between cardiomyocyte division and failure in karyokinesis or cytokinesis. Live cell imaging has provided alternatives that facilitate high-throughput screening but these have other limitations. Here, we (i) review the cell cycle features of cardiomyocytes, (ii) discuss various cell cycle fluorescent reporter systems, and (iii) speculate on what could improve their predictive value in the context of cardiomyocyte proliferation. Finally, we consider how these new methods can be used in combination with state-of-the-art three-dimensional human cardiac organoid platforms to identify pro-proliferative signalling pathways that could stimulate regeneration of the human heart.

Quantitative label-free digital holographic imaging of cardiomyocyte optical volume, nucleation, and cell division

Cardiac regeneration in newborn rodents depends on the ability of pre-existing cardiomyocytes to proliferate and divide. This capacity is lost within the first week of postnatal development when these cells rapidly switch from hyperplasia to hypertrophy, withdraw from the cell cycle, become binucleated, and increase in size. How these dynamic changes in cell size and nucleation impact cardiomyocyte proliferative potential is not well understood. In this study, we innovate the application of a commercially available digital holographic imaging microscope, the Holomonitor M4, to evaluate the proliferative responses of mononucleated and binucleated cardiomyocytes after CHIR99021 treatment, a model proliferative stimulus. This system enables long-term label-free quantitative tracking of primary cardiomyocyte dynamics in real-time with single-cell resolution. Our results confirm that chemical inhibition of glycogen synthase kinase 3 with CHIR99021 promotes complete cell division of both mononucleated and binucleated cardiomyocytes with high frequency. Quantitative tracking of cardiomyocyte volume dynamics during these proliferative events revealed that both mononucleated and binucleated cardiomyocytes reach a similar size-increase threshold prior to attempted cell division. Binucleated cardiomyocytes attempt to divide with lower frequency than mononucleated cardiomyocytes, which may be associated with inadequate increases in cell size. By defining the interrelationship between cardiomyocyte size, nucleation, and cell cycle control, we may better understand the cellular mechanisms that drive the loss of mammalian cardiac regenerative capacity after birth.

Dynamic upregulation of retinoic acid signal in the early postnatal murine heart promotes cardiomyocyte cell cycle exit and maturation

The adult mammalian heart has extremely limited cardiac regenerative capacity. Most cardiomyocytes live in a state of permanent cell-cycle arrest and are unable to re-enter the cycle. Cardiomyocytes switch from cell proliferation to a maturation state during neonatal development. Although several signaling pathways are involved in this transition, the molecular mechanisms by which these inputs coordinately regulate cardiomyocyte maturation are not fully understood. Retinoic acid (RA) plays a pivotal role in development, morphogenesis, and regeneration. Despite the importance of RA signaling in embryo heart development, little is known about its function in the early postnatal period. We found that mRNA expression of aldehyde dehydrogenase 1 family member A2 (Aldh1a2), which encodes the key enzyme for synthesizing all-trans retinoic acid (ATRA) and is an important regulator for RA signaling, was transiently upregulated in neonatal mouse ventricles. Single-cell transcriptome analysis and immunohistochemistry revealed that Aldh1a2 expression was enriched in cardiac fibroblasts during the early postnatal period. Administration of ATRA inhibited cardiomyocyte proliferation in cultured neonatal rat cardiomyocytes and human cardiomyocytes. RNA-seq analysis indicated that cell proliferation-related genes were downregulated in prenatal rat ventricular cardiomyocytes treated with ATRA, while cardiomyocyte maturation-related genes were upregulated. These findings suggest that RA signaling derived from cardiac fibroblasts is one of the key regulators of cardiomyocyte proliferation and maturation during neonatal heart development.

Cardiomyocyte proliferation and regeneration in congenital heart disease.

Despite advances in prenatal screening and a notable decrease in mortality rates, congenital heart disease (CHD) remains the most prevalent congenital disorder in newborns globally. Current therapeutic surgical approaches face challenges due to the significant rise in complications and disabilities. Emerging cardiac regenerative therapies offer promising adjuncts for CHD treatment. One novel avenue involves investigating methods to stimulate cardiomyocyte proliferation. However, the mechanism of altered cardiomyocyte proliferation in CHD is not fully understood, and there are few feasible approaches to stimulate cardiomyocyte cell cycling for optimal healing in CHD patients. In this review, we explore recent progress in understanding genetic and epigenetic mechanisms underlying defective cardiomyocyte proliferation in CHD from development through birth. Targeting cell cycle pathways shows promise for enhancing cardiomyocyte cytokinesis, division, and regeneration to repair heart defects. Advancements in human disease modeling techniques, CRISPR-based genome and epigenome editing, and next-generation sequencing technologies will expedite the exploration of abnormal machinery governing cardiomyocyte differentiation, proliferation, and maturation across diverse genetic backgrounds of CHD. Ongoing studies on screening drugs that regulate cell cycling are poised to translate this nascent technology of enhancing cardiomyocyte proliferation into a new therapeutic paradigm for CHD surgical interventions.

Effect of Intramyocardial Administration of Baculovirus Encoding the Transcription Factor Tbx20 in Sheep With Experimental Acute Myocardial Infarction.

Gene therapy has been proposed as a strategy to induce cardiac regeneration following acute myocardial infarction (AMI). Given that Tbx20, a transcription factor of the T-box subfamily, stimulates cell proliferation and angiogenesis, we designed a baculovirus overexpressing Tbx20 (Bv-Tbx20) and evaluated its effects in cultured cardiomyocytes and in an ovine model of AMI. Cell proliferation and angiogenesis were measured in cardiomyocytes transduced with Bv-Tbx20 or Bv-Null (control). Subsequently, in sheep with AMI, Bv-Tbx20 or Bv-Null was injected in the infarct border. Cardiomyocyte cell cycle activity, angioarteriogenesis, left ventricular function, and infarct size were assessed. Cardiomyocytes transduced with BvTbx20 increased cell proliferation, cell cycle regulatory and angiogenic gene expression, and tubulogenesis. At 7 days posttreatment, sheep treated with Bv-Tbx20 showed increased Tbx20, promitotic and angiogenic gene expression, decreased levels of P21, increased Ki67- (17.09±5.73 versus 7.77±7.24 cardiomyocytes/mm2, P<0.05) and PHH3 (phospho-histone H3)-labeled cardiomyocytes (10.10±3.51 versus 5.23±2.87 cardiomyocytes/mm2, P<0.05), and increased capillary (2302.68±353.58 versus 1694.52±211.36 capillaries/mm2, P<0.001) and arteriolar (146.95±53.14 versus 84.06±16.84 arterioles/mm2, P<0.05) densities. At 30 days, Bv-Tbx20 decreased infarct size (9.89±1.92% versus 12.62±1.33%, P<0.05) and slightly improved left ventricular function. Baculoviral gene transfer-mediated Tbx20 overexpression exerted angiogenic and cardiomyogenic effects invitro. In sheep with AMI, Bv-Tbx20 induced angioarteriogenesis, cardiomyocyte cell cycle activity, infarct size limitation, and a slight recovery of left ventricular function, suggesting that Bv-Tbx20 gene therapy may contribute to cardiac regeneration following AMI.

Abstract We039: Adducin Promotes Cardiomyocyte Sarcomere Disassembly

Introduction: Recent interest in understanding the cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. Despite advancements, the intricacies of cardiomyocyte mitosis, especially the breakdown of sarcomeres to enable daughter cardiomyocytes' division, remain largely unexplored. Hypothesis: This study investigates the role of Adducin in the disassembly of sarcomeres during cardiomyocyte mitosis, hypothesizing that Adducin serves as a crucial regulator in this process. Goals: Our objective is to elucidate the function of Adducin in sarcomere disassembly during the mitosis of mammalian cardiomyocytes and understand how this process is regulated. Methods: We examined the expression patterns of α/γ-Adducins, particularly in neonatal cardiomyocytes undergoing mitosis. Subsequently, we experimented with the overexpression of various α-Adducin splices and phosphorus in vitro and in vivo, with a keen interest in the phosphorylation effects of α-Adducin on sarcomere disassembly. Results: Here, we identify Adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-Adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phosphoforms of α-Adducin in vitro and in vivo identified Thr445/Thr480 phosphorylation of a short isoform of α-Adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Additionally, co-overexpressing this α-Adducin variant with γ-Adducin in adult mice stabilizes the Adducin complex and maintains sarcomere disassembly, facilitated through interactions with α-Actinin. Conclusion: The study highlights Adducin, particularly α-Adducin phosphorylated at Thr445/Thr480, as a pivotal regulator of sarcomere disassembly during neonatal cardiomyocyte mitosis. This mechanism is essential for coordinating the cytoskeletal changes necessary for the mitotic process in cardiomyocytes, offering insights into potential therapeutic targets for cardiomyopathy.

Abstract We003: Identification of CircRNA-miRNA-mRNA Regulatory Network and Crucial Signaling Pathways Related to Cardiomyocyte Proliferation in Neonatal Mice

Introduction: Neonatal pig hearts exhibit remarkable regenerative capacity within two days post-birth. In this study, we propose that a comprehensive multiomics analysis encompassing circRNA, miRNA, and mRNA data from neonatal mouse hearts can unveil intricate circRNA-miRNA-mRNA relationships associated with cardiac regeneration. Our aim is to identify new novel circRNAs and miRNAs implicated in cardiomyocyte proliferation through leveraging multiomics analysis. Methods: Utilizing neonatal mouse hearts, we conducted circRNA, miRNA, and mRNA sequencing across four different postnatal days (P1, P3, P7, and P28; n=6, encompassing both males and females). We employed integrative bioinformatics analyses across distinct experimental designs (P1 vs P3, P1 vs P7, and P1 vs P28). Back splice junctions generated by CIRCexplorer2’s parse command was utilized to annotate sequenced circRNAs as known or novel. Validation of circRNA, miRNA, and mRNA expression levels was achieved through qRT-PCR, while protein expression levels were assessed via western blotting. Fluorescence in situ hybridization (FISH) technology was employed to determine circRNA localization. The functional significance of key genes involved in cardiomyocyte proliferation was assessed via siRNA-mediated knockdown of circRNAs and miRNAs, accompanied by cell cycle assessment using bioluminescence assay and immunostaining. Results: Intersection of differentially expressed target genes from circRNA, miRNA, and mRNA analyses at each experimental design facilitated the construction of circRNA-miRNA-mRNA regulatory networks, aiding in the development of cardiac regeneration-related regulatory networks and inference of novel relationships between circRNA, miRNA, and mRNA. Integrative bioinformatics analysis revealed significant alterations in circRNA-miRNA-mRNA expression profiles during postnatal cardiomyocyte maturation in mice. Pathway enrichment analysis identified several differentially expressed circRNAs enriched in cell division and proliferation-related pathways. qPCR and cell cycle studies identified 17 potential circRNAs associated with cardiomyocyte proliferation in mice, with knockdown of these circRNAs significantly impacting cardiomyocyte cell cycle. Conclusion: Our findings suggest a crucial role for circRNAs in the regulation of neonatal myocardial regeneration.

Runx1 is sufficient but not required for cardiomyocyte cell-cycle activation.

Factors responsible for cardiomyocyte proliferation could serve as potential therapeutics to stimulate endogenous myocardial regeneration following insult, such as ischemic injury. A previously published forward genetics approach on cardiomyocyte cell cycle and ploidy led us to the transcription factor, Runx1. Here, we examine the effect of Runx1 on cardiomyocyte cell cycle during postnatal development and cardiac regeneration using cardiomyocyte-specific gain- and loss-of-function mouse models. RUNX1 is expressed in cardiomyocytes during early postnatal life, decreases to negligible levels by 3 wk of age, and increases upon myocardial injury, all consistent with observed rates of cardiomyocyte cell-cycle activity. Loss of Runx1 transiently stymied cardiomyocyte cell-cycle activity during normal postnatal development, a result that corrected itself and did not extend to the context of neonatal heart regeneration. On the other hand, cardiomyocyte-specific Runx1 overexpression resulted in an expansion of diploid cardiomyocytes in uninjured hearts and expansion of 4 N cardiomyocytes in the context of neonatal cardiac injury, suggesting Runx1 overexpression is sufficient to induce cardiomyocyte cell-cycle responses. Persistent overexpression of Runx1 for >1 mo continued to promote cardiomyocyte cell-cycle activity resulting in substantial hyperpolyploidization (≥8 N DNA content). This persistent cell-cycle activation was accompanied by ventricular dilation and adverse remodeling, raising the concern that continued cardiomyocyte cell cycling can have detrimental effects.NEW & NOTEWORTHY Runx1 is sufficient but not required for cardiomyocyte cell cycle.

Identification of circular RNAs regulating cardiomyocyte proliferation in neonatal pig hearts.

Little is known about the expression patterns and functions of circular RNAs (circRNAs) in the heart of large mammals. In this study, we examined the expression profiles of circRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in neonatal pig hearts. Pig heart samples collected on postnatal days 1 (P1), 3 (P3), 7 (P7), and 28 (P28) were sent for total RNA sequencing. Our data revealed a total of 7,000 circRNAs in the 24 pig hearts. Pathway enrichment analysis of hallmark gene sets demonstrated that differentially expressed circRNAs were engaged in different pathways. The most significant difference was observed between P1 and the other 3 groups (P3, P7, and P28) in pathways related to cell cycle and muscle development. Out of the 10 circRNAs that were validated through real-time quantitative PCR to verify their expression, 6 exhibited significant effects on cell cycle activity in human induced pluripotent stem cell-derived cardiomyocytes following small interfering RNA-mediated knockdown. circRNA-miRNA-mRNA networks were constructed to understand the potential mechanisms of circRNAs in the heart. In conclusion, our study provided a data set for exploring the roles of circRNAs in pig hearts. In addition, we identified several circRNAs that regulate cardiomyocyte cell cycle.

Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis.

Recent interest in understanding cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow the abscission of daughter cardiomyocytes. Here, we use a proteomics screen to identify adducin, an actin capping protein previously not studied in cardiomyocytes, as a regulator of sarcomere disassembly. We generated many adeno-associated viruses and cardiomyocyte-specific genetic gain-of-function models to examine the role of adducin in neonatal and adult cardiomyocytes in vitro and in vivo. We identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phospho-isoforms of α-adducin in vitro and in vivo identified Thr445/Thr480 phosphorylation of a short isoform of α-adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this α-adducin variant along with γ-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with α-actinin. These results highlight an important mechanism for coordinating cytoskeletal morphological changes during cardiomyocyte mitosis.

Inhibition of cyclin-dependent kinase 7 mitigates doxorubicin cardiotoxicity and enhances anticancer efficacy.

The anthracycline family of anticancer agents such as doxorubicin (DOX) can induce apoptotic death of cardiomyocytes and cause cardiotoxicity. We previously reported that DOX-induced apoptosis is accompanied by cardiomyocyte cell cycle re-entry. Cell cycle progression requires cyclin-dependent kinase 7 (CDK7)-mediated activation of downstream cell cycle CDKs. This study aims to determine whether CDK7 can be targeted for cardioprotection during anthracycline chemotherapy. DOX exposure induced CDK7 activation in mouse heart and isolated cardiomyocytes. Cardiac-specific ablation of Cdk7 attenuated DOX-induced cardiac dysfunction and fibrosis. Treatment with the covalent CDK7 inhibitor THZ1 also protected against DOX-induced cardiomyopathy and apoptosis. DOX treatment induced activation of the proapoptotic CDK2-FOXO1-Bim axis in a CDK7-dependent manner. In response to DOX, endogenous CDK7 directly bound and phosphorylated CDK2 at Thr160 in cardiomyocytes, leading to full CDK2 kinase activation. Importantly, inhibition of CDK7 further suppressed tumour growth when used in combination with DOX in an immunocompetent mouse model of breast cancer. Activation of CDK7 is necessary for DOX-induced cardiomyocyte apoptosis and cardiomyopathy. Our findings uncover a novel proapoptotic role for CDK7 in cardiomyocytes. Moreover, this study suggests that inhibition of CDK7 attenuates DOX-induced cardiotoxicity but augments the anticancer efficacy of DOX. Therefore, combined administration of CDK7 inhibitor and DOX may exhibit diminished cardiotoxicity but superior anticancer activity.

Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation.

Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

MBNL1 Regulates Programmed Postnatal Switching Between Regenerative and Differentiated Cardiac States.

Discovering determinants of cardiomyocyte maturity is critical for deeply understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration, but elucidation of endogenous developmental regulators of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. MBNL1 (muscleblind-like 1) regulates fibroblast, thymocyte, and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation. MBNL1 gain- and loss-of-function mouse models were studied at several developmental time points and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro assays to determine the mechanisms through which MBNL1 exerts its effects. MBNL1 is coexpressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, whereas loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deficiency was insufficient to promote regeneration in the adult heart because of cell cycle checkpoint activation. Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Targeting loss of cardiomyocyte maturity and downregulation of cell cycle inhibitors through MBNL1 deletion was not sufficient to promote adult regeneration.

Magnesium lithospermate B enhances the potential of human-induced pluripotent stem cell-derived cardiomyocytes for myocardial repair.

We previously reported that activation of the cell cycle in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enhances their remuscularization capacity after human cardiac muscle patch transplantation in infarcted mouse hearts. Herein, we sought to identify the effect of magnesium lithospermate B (MLB) on hiPSC-CMs during myocardial repair using a myocardial infarction (MI) mouse model. In C57BL/6 mice, MI was surgically induced by ligating the left anterior descending coronary artery. The mice were randomly divided into five groups ( n = 10 per group); a MI group (treated with phosphate-buffered saline only), a hiPSC-CMs group, a MLB group, a hiPSC-CMs + MLB group, and a Sham operation group. Cardiac function and MLB therapeutic efficacy were evaluated by echocardiography and histochemical staining 4 weeks after surgery. To identify the associated mechanism, nuclear factor (NF)-κB p65 and intercellular cell adhesion molecule-1 (ICAM1) signals, cell adhesion ability, generation of reactive oxygen species, and rates of apoptosis were detected in human umbilical vein endothelial cells (HUVECs) and hiPSC-CMs. After 4 weeks of transplantation, the number of cells that engrafted in the hiPSC-CMs + MLB group was about five times higher than those in the hiPSC-CMs group. Additionally, MLB treatment significantly reduced tohoku hospital pediatrics-1 (THP-1) cell adhesion, ICAM1 expression, NF-κB nuclear translocation, reactive oxygen species production, NF-κB p65 phosphorylation, and cell apoptosis in HUVECs cultured under hypoxia. Similarly, treatment with MLB significantly inhibited the apoptosis of hiPSC-CMs via enhancing signal transducer and activator of transcription 3 (STAT3) phosphorylation and B-cell lymphoma-2 (BCL2) expression, promoting STAT3 nuclear translocation, and downregulating BCL2-Associated X, dual specificity phosphatase 2 (DUSP2), and cleaved-caspase-3 expression under hypoxia. Furthermore, MLB significantly suppressed the production of malondialdehyde and lactate dehydrogenase and the reduction in glutathione content induced by hypoxia in both HUVECs and hiPSC-CMs in vitro . MLB significantly enhanced the potential of hiPSC-CMs in repairing injured myocardium by improving endothelial cell function via the NF-κB/ICAM1 pathway and inhibiting hiPSC-CMs apoptosis via the DUSP2/STAT3 pathway.

Morroniside induces cardiomyocyte cell cycle activity and promotes cardiac repair after myocardial infarction in adult rats.

Introduction: Acute myocardial infarction (AMI) is characterized by the loss of cardiomyocytes, which impairs cardiac function and eventually leads to heart failure. The induction of cardiomyocyte cell cycle activity provides a new treatment strategy for the repair of heart damage. Our previous study demonstrated that morroniside exerts cardioprotective effects. This study investigated the effects and underlying mechanisms of action of morroniside on cardiomyocyte cell cycle activity and cardiac repair following AMI. Methods: Neonatal rat cardiomyocytes (NRCMs) were isolated and exposed to oxygen-glucose deprivation (OGD) in vitro. A rat model of AMI was established by ligation of the left anterior descending coronary artery (LAD) in vivo. Immunofluorescence staining was performed to detect newly generated cardiomyocytes. Western blotting was performed to assess the expression of cell cycle-related proteins. Electrocardiography (ECG) was used to examine pathological Q waves. Masson's trichrome and wheat germ agglutinin (WGA) staining assessed myocardial fibrosis and hypertrophy. Results: The results showed that morroniside induced cardiomyocyte cell cycle activity and increased the levels of cell cycle proteins, including cyclin D1, CDK4, cyclin A2, and cyclin B1, both in vitro and in vivo. Moreover, morroniside reduced myocardial fibrosis and remodeling. Discussion: In conclusion, our study demonstrated that morroniside stimulates cardiomyocyte cell cycle activity and cardiac repair in adult rats, and that these effects may be related to the upregulation of cell cycle proteins.