Cardiomyocyte Cell Cycle Activity Research Articles

Cell-Cycle-Specific Autoencoding Improves Cluster Analysis of Cycling Cardiomyocytes.

Our previous analyses of cardiomyocyte single-nucleus RNA sequencing (snRNAseq) data from the hearts of fetal pigs and pigs that underwent apical resection surgery on postnatal day (P) 1 (ARP1), myocardial infarction (MI) surgery on P28 (MIP28), both ARP1 and MIP28 (ARP1MIP28), or controls (no surgical procedure or CTL) identified 10 cardiomyocyte subpopulations (clusters), one of which appeared to be primed to proliferate in response to MI. However, the clusters composed of primarily proliferating cardiomyocytes still contained noncycling cells, and we were unable to distinguish between cardiomyocytes in different phases of the cell cycle. Here, we improved the precision of our assessments by conducting similar analyses with snRNAseq data for only the 1646 genes included under the Gene Ontology term "cell cycle." Two cardiac snRNAseq datasets, one from mice (GEO dataset number GSE130699) and one from pigs (GEO dataset number GSE185289), were evaluated via our cell-cycle-specific analytical pipeline. Cycling cells were identified via the co-expression of 5 proliferation markers (AURKB, MKI67, INCENP, CDCA8, and BIRC5). The cell-cycle-specific autoencoder (CSA) algorithm identified 7 cardiomyocyte clusters in mouse hearts (mCM1 and mCM3-mCM8), including one prominent cluster of cycling cardiomyocytes in animals that underwent MI or Sham surgery on P1. Five cardiomyocyte clusters (pCM1, pCM3-pCM6) were identified in pig hearts, 2 of which (pCM1 and pCM4) displayed evidence of cell cycle activity; pCM4 was found primarily in hearts from fetal pigs, while pCM1 comprised a small proportion of cardiomyocytes in both fetal hearts and hearts from ARP1MIP28 pigs during the 2 weeks after MI induction, but was nearly undetectable in all other experimental groups and at all other time points. Furthermore, pseudotime trajectory analysis of snRNAseq data from fetal pig cardiomyocytes identified a pathway that began at pCM3, passed through pCM2, and ended at pCM1, whereas pCM3 was enriched for the expression of a cell cycle activator that regulates the G1/S phase transition (cyclin D2), pCM2 was enriched for an S-phase regulator (CCNE2), and pCM1 was enriched for the expression of a gene that regulates the G2M phase transition and mitosis (cyclin B2). We also identified 4 transcription factors (E2F8, FOXM1, GLI3, and RAD51) that were more abundantly expressed in cardiomyocytes from regenerative mouse hearts than from nonregenerative mouse hearts, from the hearts of fetal pigs than from CTL pig hearts, and from ARP1MIP28 pig hearts than from MIP28 pig hearts during the 2 weeks after MI induction. The CSA algorithm improved the precision of our assessments of cell cycle activity in cardiomyocyte subpopulations and enabled us to identify a trajectory across 3 clusters that appeared to track the onset and progression of cell cycle activity in cardiomyocytes from fetal pigs.

Mitochondrial-to-nuclear communications through multiple routes regulate cardiomyocyte proliferation

The regenerative capacity of the adult mammalian heart remains a formidable challenge in biological research. Despite extensive investigations into the loss of regenerative potential during evolution and development, unlocking the mechanisms governing cardiomyocyte proliferation remains elusive. Two recent groundbreaking studies have provided fresh perspectives on mitochondrial-to-nuclear communication, shedding light on novel factors that regulate cardiomyocyte proliferation. The studies identified two mitochondrial processes, fatty acid oxidation and protein translation, as key players in restricting cardiomyocyte proliferation. Inhibition of these processes led to increased cell cycle activity in cardiomyocytes, mediated by reduction in H3k4me3 levels through accumulated α-ketoglutarate (αKG), and activation of the mitochondrial unfolded protein response (UPRmt), respectively. In this research highlight, we discuss the novel insights into mitochondrial-to-nuclear communication presented in these studies, the broad implications in cardiomyocyte biology and cardiovascular diseases, as well as the intriguing scientific questions inspired by the studies that may facilitate future investigations into the detailed molecular mechanisms of cardiomyocyte metabolism, proliferation, and mitochondrial-to-nuclear communications.

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.

Thrombospondin 1 and Reelin act through Vldlr to regulate cardiac growth and repair.

Adult mammalian cardiomyocytes have minimal cell cycle capacity, which leads to poor regeneration after cardiac injury such as myocardial infarction. Many positive regulators of cardiomyocyte cell cycle and cardioprotective signals have been identified, but extracellular signals that suppress cardiomyocyte proliferation are poorly understood. We profiled receptors enriched in postnatal cardiomyocytes, and found that very-low-density-lipoprotein receptor (Vldlr) inhibits neonatal cardiomyocyte cell cycle. Paradoxically, Reelin, the well-known Vldlr ligand, expressed in cardiac Schwann cells and lymphatic endothelial cells, promotes neonatal cardiomyocyte proliferation. Thrombospondin1 (TSP-1), another ligand of Vldlr highly expressed in adult heart, was then found to inhibit cardiomyocyte proliferation through Vldlr, and may contribute to Vldlr's overall repression on proliferation. Mechanistically, Rac1 and subsequent Yap phosphorylation and nucleus translocation mediate the regulation of the cardiomyocyte cell cycle by TSP-1/Reelin-Vldlr signaling. Importantly, Reln mutant neonatal mice displayed impaired cardiomyocyte proliferation and cardiac regeneration after apical resection, while cardiac-specific Thbs1 deletion and cardiomyocyte-specific Vldlr deletion promote cardiomyocyte proliferation and are cardioprotective after myocardial infarction. Our results identified a novel role of Vldlr in consolidating extracellular signals to regulate cardiomyocyte cell cycle activity and survival, and the overall suppressive TSP-1-Vldlr signal may contribute to the poor cardiac repair capacity of adult mammals.

Abstract P1084: Cardiomyocyte-specific Overexpression Of Ccnd2 Via Modified Mrnafor Remuscularization In Hearts With Myocardial Infarction

Introduction: The adult mammalian heart has limited regenerative capacity due to postnatal cardiomyocyte (CM) cell cyclearrest. With the success of modified mRNA (modRNA) vaccine against SARS-CoV-2, modRNA technology offers efficient, transient, safe, nonimmunogenic, and controlled mRNA delivery without any risk of genomic integration. Using our previously reported cardiomyocyte specific modRNA translation system (CM SMRTs), we investigated the myocardial regenerative potential and therapeutic outcome of transient and specific cardiomyocyte overexpression of cyclin D2 (CCND2) in a pig model of myocardial infarction (MI). Methods: Modified mRNA was synthesized by in vitro transcription. AMI was induced by occlusion of the left anterior descending coronary artery for 60 minutes with reperfusion. Pigs were randomly assigned to vehicle group (1ml sucrose-citrate buffer, n=6), nGFP-CM SMRTs group (7.5mg CM-specific nuclear GFP modRNA, n=7)and CCND2-CM SMRTs group (7.5mg CM-specific CCND2 modRNA, n=8). CM cell cycle activity was measured 3 days after injection by immunostaining for proliferation marker Ki67, M-phase marker phospho-histone H3 (PH3) and cytokinetic marker aurora B kinase (AuB). Cardiac function was evaluated by echo and cardiac MRI (cMRI) on day 28; infarct size was determined via late gadolinium enhancement cMRI; arrhythmia incidence was assessed via implanted loop recorders. Results: The short-term in vivo study demonstrated a highly specific overexpression of targeted genes in CMs. CCND2-CM SMRTs significantly promoted CM cell cycle activation that was evidenced by significant increase of cell cycle markers compared with nGFP-CM SMRTs group: Ki67 (4.21±0.34% vs. 0.81±0.05%), PH3 (0.51±0.06% vs. 0.13±0.03%), and Symmetric AuB (0.035±0.007/mm 2 vs. 0.000±0.000/mm 2 ), 3 days after AMI and modRNA treatment. The long-term follow-up of the animals also showed improved heart function (EF: 50.34±1.94% vs. 38.60±1.18%) and decreased scar size (8.80±1.07% vs. 22.77±2.77%). The frequency of arrhythmic events in animals between all groups were similar. Conclusion: The results in large animal model of AMI demonstrate remuscularization and therapeutic potential of cardiomyocyte-specific overexpression of CCND2 via modRNA.

Abstract P1173: Deciphering CITED4-dependent Signaling In The Heart Through Nascent Proteomics And Secretomics

Objective: Neuregulin-1 (Nrg1) promotes cardiomyocyte hypertrophy, survival, and cell cycle activity through ErbB signaling. It is investigated in clinical trials as a cardioprotective agent; however, its therapeutic use might be limited due to its pro-neoplastic potential for non-cardiomyocytes that makes targeted approaches necessary. High-throughput screening of hypertrophic agonists found that Nrg1 induces CITED4 (C4) expression. C4 is also upregulated in both physiological and pathological cardiac growth and necessary to prevent adverse remodeling in vivo. However, how C4 regulates Nrg1-signaling in the heart is yet unknown. Methods: We combined pulsed-SILAC labeling, click-chemistry and mass spectrometry that allows to capture immediate changes in the proteome and secretome after Nrg1 (12h and 24h) stimulation in siRNA-mediated C4-knockdown (C4KD) and control (ctr) NRVM and complemented this with established cell- and molecular biology techniques to validate and investigate cellular function and molecular signaling. Results: We confirmed dose-dependent C4 mRNA upregulation in response to Nrg1 stimulation in NRVM. Nrg1 downstream signaling through AKT was hindered in C4KD. Computational analysis comparing the nascent proteome after Nrg1 stimulation in C4KD and ctr NRVM revealed that C4 significantly regulates proteins responsible for translation, RNA processing and energy derivation. Consistent with previously observed development of cardiac fibrosis in C4 knockout mice in vivo , we found significant upregulation of TGFb2 in C4KD NRVM. We next confirmed the upregulation of TGFb2 and its downstream effectors in C4KD and ctr, while adenoviral overexpression of C4 led to reduced TGFb2 expression. Additionally, we found TGFb2 secretion increased from C4KD NRVM with Nrg1 stimulation in the nascent secretome. Conditioned media from C4KD NRVM led to an increased pro-fibrotic response in cardiac fibroblasts. In conclusion, nascent proteomics and secretomics help to elucidate C4-dependent Nrg1-signaling, which may be an important contribution toward our goal to identify targetable cardioprotective pathways in the heart.

Am80, a retinoic acid receptor agonist, activates the cardiomyocyte cell cycle and enhances engraftment in the heart.

Human induced pluripotent stem cell-derived (hiPSC) cardiomyocytes are a promising source for regenerative therapy. To realize this therapy, however, their engraftment potential after their injection into the host heart should be improved. Here, we established an efficient method to analyze the cell cycle activity of hiPSC cardiomyocytes using a fluorescence ubiquitination-based cell cycle indicator (FUCCI) system. Invitro high-throughput screening using FUCCI identified a retinoic acid receptor (RAR) agonist, Am80, as an effective cell cycle activator in hiPSC cardiomyocytes. The transplantation of hiPSC cardiomyocytes treated with Am80 before the injection significantly enhanced the engraftment in damaged mouse heart for 6months. Finally, we revealed that the activation of endogenous Wnt pathways through both RARA and RARB underlies the Am80-mediated cell cycle activation. Collectively, this study highlights an efficient method to activate cell cycle in hiPSC cardiomyocytes by Am80 as a means to increase the graft size after cell transplantation into a damaged heart.

Systemic Hypoxemia Induces Cardiomyocyte Hypertrophy and Right Ventricular Specific Induction of Proliferation.

A recent study suggests that systemic hypoxemia in adult male mice can induce cardiac myocytes to proliferate. The goal of the present experiments was to confirm these results, provide new insights on the mechanisms that induce adult cardiomyocyte cell cycle reentry, and to determine if hypoxemia also induces cardiomyocyte proliferation in female mice. EdU-containing mini pumps were implanted in 3-month-old, male and female C57BL/6 mice. Mice were placed in a hypoxia chamber, and the oxygen was lowered by 1% every day for 14 days to reach 7% oxygen. The animals remained in 7% oxygen for 2 weeks before terminal studies. Myocyte proliferation was also studied with a mosaic analysis with double markers mouse model. Hypoxia induced cardiac hypertrophy in both left ventricular (LV) and right ventricular (RV) myocytes, with LV myocytes lengthening and RV myocytes widening and lengthening. Hypoxia induced an increase (0.01±0.01% in normoxia to 0.11±0.09% in hypoxia) in the number of EdU+ RV cardiomyocytes, with no effect on LV myocytes in male C57BL/6 mice. Similar results were observed in female mice. Furthermore, in mosaic analysis with double markers mice, hypoxia induced a significant increase in RV myocyte proliferation (0.03±0.03% in normoxia to 0.32±0.15% in hypoxia of RFP+ myocytes), with no significant change in LV myocyte proliferation. RNA sequencing showed upregulation of mitotic cell cycle genes and a downregulation of Cullin genes, which promote the G1 to S phase transition in hypoxic mice. There was significant proliferation of nonmyocytes and mild cardiac fibrosis in hypoxic mice that did not disrupt cardiac function. Male and female mice exhibited similar gene expression following hypoxia. Systemic hypoxia induces a global hypertrophic stress response that was associated with increased RV proliferation, and while LV myocytes did not show increased proliferation, our results minimally confirm previous reports that hypoxia can induce cardiomyocyte cell cycle activity in vivo.

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.

DOT1L regulates chamber-specific transcriptional networks during cardiogenesis and mediates postnatal cell cycle withdrawal

Mechanisms by which specific histone modifications regulate distinct gene networks remain little understood. We investigated how H3K79me2, a modification catalyzed by DOT1L and previously considered a general transcriptional activation mark, regulates gene expression during cardiogenesis. Embryonic cardiomyocyte ablation of Dot1l revealed that H3K79me2 does not act as a general transcriptional activator, but rather regulates highly specific transcriptional networks at two critical cardiogenic junctures: embryonic cardiogenesis, where it was particularly important for left ventricle-specific genes, and postnatal cardiomyocyte cell cycle withdrawal, with Dot1L mutants having more mononuclear cardiomyocytes and prolonged cardiomyocyte cell cycle activity. Mechanistic analyses revealed that H3K79me2 in two distinct domains, gene bodies and regulatory elements, synergized to promote expression of genes activated by DOT1L. Surprisingly, H3K79me2 in specific regulatory elements also contributed to silencing genes usually not expressed in cardiomyocytes. These results reveal mechanisms by which DOT1L successively regulates left ventricle specification and cardiomyocyte cell cycle withdrawal.

Cardiac Troponin I-Interacting Kinase Affects Cardiomyocyte S-Phase Activity but Not Cardiomyocyte Proliferation.

Identifying genetic variants that affect the level of cell cycle reentry and establishing the degree of cell cycle progression in those variants could help guide development of therapeutic interventions aimed at effecting cardiac regeneration. We observed that C57Bl6/NCR (B6N) mice have a marked increase in cardiomyocyte S-phase activity after permanent coronary artery ligation compared with infarcted DBA/2J (D2J) mice. Cardiomyocyte cell cycle activity after infarction was monitored in D2J, (D2J×B6N)-F1, and (D2J×B6N)-F1×D2J backcross mice by means of bromodeoxyuridine or 5-ethynyl-2'-deoxyuridine incorporation using a nuclear-localized transgenic reporter to identify cardiomyocyte nuclei. Genome-wide quantitative trait locus analysis, fine scale genetic mapping, whole exome sequencing, and RNA sequencing analyses of the backcross mice were performed to identify the gene responsible for the elevated cardiomyocyte S-phase phenotype. (D2J×B6N)-F1 mice exhibited a 14-fold increase in cardiomyocyte S-phase activity in ventricular regions remote from infarct scar compared with D2J mice (0.798±0.09% versus 0.056±0.004%; P<0.001). Quantitative trait locus analysis of (D2J×B6N)-F1×D2J backcross mice revealed that the gene responsible for differential S-phase activity was located on the distal arm of chromosome 3 (logarithm of the odds score=6.38; P<0.001). Additional genetic and molecular analyses identified 3 potential candidates. Of these, Tnni3k (troponin I-interacting kinase) is expressed in B6N hearts but not in D2J hearts. Transgenic expression of TNNI3K in a D2J genetic background results in elevated cardiomyocyte S-phase activity after injury. Cardiomyocyte S-phase activity in both Tnni3k-expressing and Tnni3k-nonexpressing mice results in the formation of polyploid nuclei. These data indicate that Tnni3k expression increases the level of cardiomyocyte S-phase activity after injury.

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 15113: C-cbl E3 Ubiquitin Ligase Regulates Cardiomyocyte Proliferation and Cardiac Regeneration

Background: Neonatal mammalian hearts display considerable regenerative capacity following injury through cardiomyocyte proliferation. However, this regenerative capacity is lost by post-natal day 7. Previous studies have shown the role of receptor tyrosine kinases (RTKs) on cardiomyocyte proliferation and cardiac regeneration. However, the mechanisms that control RTK turnover and signaling during and beyond the regenerative window of the heart remain largely unknown. Hypothesis: This study investigates the role of the proto-oncogene Casitas b-lineage lymphoma (c-Cbl), an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets RTKs, on cardiomyocyte proliferation and cardiac regeneration following myocardial infarction (MI). Methods and Results: c-Cbl expression was downregulated during post-natal development and re-expressed in the adult heart following MI. RNA-sequencing studies reveal a distinct transcriptional profile in c-Cbl-deficient mouse hearts, of which c-Cbl deletion substantially delayed cardiomyocyte maturation and enhanced cardiomyocyte proliferation. c-Cbl deletion also extended the regenerative window for post-natal mouse hearts following MI and enhanced the ability to recover following MI with improved myocardial function, reduced scar formation, and increased cardiomyocyte cell-cycle activity. Mechanistically, c-Cbl E3 ubiquitin ligase activity was markedly enhanced in the adult than neonatal heart following MI, along with increased ubiquitination and downregulation of key RTKs involved in cardiomyocyte proliferation. Intriguingly, adenoviral expression of a c-Cbl mutant deficient in E3 ligase enhanced RTK signaling and promoted cardiomyocyte proliferation in vitro. Conclusions: These results reveal c-Cbl as a critical regulator of cardiomyocyte proliferation and a potential therapeutic target for the maintenance of cardiac regeneration and function following MI.

RNA-Binding Protein LIN28a Regulates New Myocyte Formation in the Heart Through Long Noncoding RNA-H19.

Developmental cardiac tissue holds remarkable capacity to regenerate after injury and consists of regenerative mononuclear diploid cardiomyocytes. On maturation, mononuclear diploid cardiomyocytes become binucleated or polyploid and exit the cell cycle. Cardiomyocyte metabolism undergoes a profound shift that coincides with cessation of regeneration in the postnatal heart. However, whether reprogramming metabolism promotes persistence of regenerative mononuclear diploid cardiomyocytes enhancing cardiac function and repair after injury is unknown. Here, we identify a novel role for RNA-binding protein LIN28a, a master regulator of cellular metabolism in cardiac repair after injury. LIN28a overexpression was tested using mouse transgenesis on postnatal cardiomyocyte numbers, cell cycle, and response to apical resection injury. With the use of neonatal and adult cell culture systems and adult and Mosaic Analysis with Double Markers myocardial injury models in mice, the effect of LIN28a overexpression on cardiomyocyte cell cycle and metabolism was tested. Last, isolated adult cardiomyocytes from LIN28a and wild-type mice 4 days after myocardial injury were used for RNA-immunoprecipitation sequencing. LIN28a was found to be active primarily during cardiac development and rapidly decreases after birth. LIN28a reintroduction at postnatal day (P) 1, P3, P5, and P7 decreased maturation-associated polyploidization, nucleation, and cell size, enhancing cardiomyocyte cell cycle activity in LIN28a transgenic pups compared with wild-type littermates. Moreover, LIN28a overexpression extended cardiomyocyte cell cycle activity beyond P7 concurrent with increased cardiac function 30 days after apical resection. In the adult heart, LIN28a overexpression attenuated cardiomyocyte apoptosis, enhanced cell cycle activity, cardiac function, and survival in mice 12 weeks after myocardial infarction compared with wild-type littermate controls. Instead, LIN28a small molecule inhibitor attenuated the proreparative effects of LIN28a on the heart. Neonatal rat ventricular myocytes overexpressing LIN28a mechanistically showed increased glycolysis, ATP production, and levels of metabolic enzymes compared with control. LIN28a immunoprecipitation followed by RNA-immunoprecipitation sequencing in cardiomyocytes isolated from LIN28a-overexpressing hearts after injury identified long noncoding RNA-H19 as its most significantly altered target. Ablation of long noncoding RNA-H19 blunted LIN28a-induced enhancement on cardiomyocyte metabolism and cell cycle activity. Collectively, LIN28a reprograms cardiomyocyte metabolism and promotes persistence of mononuclear diploid cardiomyocytes in the injured heart, enhancing proreparative processes, thereby linking cardiomyocyte metabolism to regulation of ploidy/nucleation and repair in the heart.

Live cell screening identifies glycosides as enhancers of cardiomyocyte cell cycle activity.

Promoting cardiomyocyte proliferation is a promising strategy to regenerate the heart. Yet, so far, it is poorly understood how cardiomyocyte proliferation is regulated, and no factor identified to promote mammalian cardiomyocyte proliferation has been translated into medical practice. Therefore, finding a novel factor will be vital. Here, we established a live cell screening based on mouse embryonic stem cell-derived cardiomyocytes expressing a non-functional human geminin deletion mutant fused to Azami Green (CM7/1-hgem-derived cardiomyocytes). We screened for a subset of compounds of the small molecule library Spectrum Collection and identified 19 potential inducers of stem cell-derived cardiomyocyte proliferation. Furthermore, the pro-proliferative potential of identified candidate compounds was validated in neonatal and adult rat cardiomyocytes as well as human induced pluripotent stem cell-derived cardiomyocytes. 18 of these compounds promoted mitosis and cytokinesis in neonatal rat cardiomyocytes. Among the top four candidates were two cardiac glycosides, peruvoside and convallatoxin, the flavonoid osajin, and the selective α-adrenoceptor antagonist and imidazoline I1 receptor ligand efaroxan hydrochloride. Inhibition of PTEN and GSK-3β enhanced cell cycle re-entry and progression upon stimulation with cardiac glycosides and osajin, while inhibition of IP3 receptors inhibited the cell cycle-promoting effect of cardiac glycosides. Collectively, we established a screening system and identified potential compounds to promote cardiomyocyte proliferation. Our data suggest that modulation of calcium handling and metabolism promotes cardiomyocyte proliferation, and cardiac glycosides might, besides increasing myocardial contraction force, contribute to cardiac repair by inducing cardiomyocyte proliferation.

UCP2 modulates cardiomyocyte cell cycle activity, acetyl-CoA, and histone acetylation in response to moderate hypoxia.

Developmental cardiac tissue is regenerative while operating under low oxygen. After birth, ambient oxygen is associated with cardiomyocyte cell cycle exit and regeneration. Likewise, cardiac metabolism undergoes a shift with cardiac maturation. Whether there are common regulators of cardiomyocyte cell cycle linking metabolism to oxygen tension remains unknown. The objective of the study is to determine whether mitochondrial UCP2 is a metabolic oxygen sensor regulating cardiomyocyte cell cycle. Neonatal rat ventricular myocytes (NRVMs) under moderate hypoxia showed increased cell cycle activity and UCP2 expression. NRVMs exhibited a metabolic shift toward glycolysis, reducing citrate synthase, mtDNA, mitochondrial membrane potential (ΔΨm), and DNA damage/oxidative stress, while loss of UCP2 reversed this phenotype. Next, WT and mice from a global UCP2-KO mouse line (UCP2KO) kept under hypoxia for 4 weeks showed significant decline in cardiac function that was more pronounced in UCP2KO animals. Cardiomyocyte cell cycle activity was reduced, while fibrosis and DNA damage was significantly increased in UCP2KO animals compared with WT under hypoxia. Mechanistically, UCP2 increased acetyl-CoA levels and histone acetylation, and it altered chromatin modifiers linking metabolism to cardiomyocyte cell cycle under hypoxia. Here, we show a potentially novel role for mitochondrial UCP2 as an oxygen sensor regulating cardiomyocyte cell cycle activity, acetyl-CoA levels, and histone acetylation in response to moderate hypoxia.

Abstract P2029: The Role Of Runx1 In Cardiomyocyte Cell Cycle Activity And Its Impact On Cardiac Regeneration

Factors responsible for cardiomyocyte proliferation may serve as a potential therapeutic to stimulate endogenous myocardial regeneration following insult, such as ischemic injury. A previously published forward genetics approach assessing the frequency of a rare and presumed proliferation-competent subpopulation of cardiomyocytes, mononuclear diploid cardiomyocytes (MNDCMs), led us to the transcription factor RUNX1. It is established that RUNX1 induction in cardiomyocytes increases after injury. Here, we examine the effect of RUNX1 on cardiomyocyte cell cycle and establishment of the MNDCM population during postnatal development and cardiac regeneration using both cardiomyocyte-specific gain-and loss of function mouse models. We hypothesize that RUNX1 overexpression (OE) increases cardiomyocyte cell cycle activity with expansion of the MNDCM population, thereby extending the neonatal regenerative window and positively impacting adult cardiac remodeling post injury. During postnatal development, RUNX1 KO decreased postnatal cardiomyocyte cell cycle activity, while RUNX1 OE extended the period of cell cycle activation. This extension observed in RUNX1 OE mice is complete with cytokinesis resulting in an expansion of the MNDCM population and total cardiomyocyte endowment. To determine whether RUNX1 could similarly regulate cardiomyocyte cell cycle in a regenerative and non-regenerative model, we induce P6 and 8-week MIs to measure cell cycle activity and cardiac function post injury. RUNX1 OE neonatal mice with a P6 MI displayed no difference in cardiomyocyte cell cycle activity 7 post injury compared to control littermates. However, RUNX1 OE in adult mice with an 8-week MI showed increased cardiomyocyte cell cycle activity with completion of cytokinesis 2 weeks post injury and limited improvement in cardiac function 28 days post injury. RUNX1 influences cardiomyocyte cell cycle activation in the context of normal postnatal development and adult MI. Conversely, this phenomenon does not appear to translate to the neonatal injury context. We are examining this possible discrepancy with further experiments to fully understand .

The generation of a lactate-rich environment stimulates cell cycle progression and modulates gene expression on neonatal and hiPSC-derived cardiomyocytes.

In situ tissue engineering strategies are a promising approach to activate the endogenous regenerative potential of the cardiac tissue helping the heart to heal itself after an injury. However, the current use of complex reprogramming vectors for the activation of reparative pathways challenges the easy translation of these therapies into the clinic. Here, we evaluated the response of mouse neonatal and human induced pluripotent stem cell-derived cardiomyocytes to the presence of exogenous lactate, thus mimicking the metabolic environment of the fetal heart. An increase in cardiomyocyte cell cycle activity was observed in the presence of lactate, as determined through Ki67 and Aurora-B kinase. Gene expression and RNA-sequencing data revealed that cardiomyocytes incubated with lactate showed upregulation of BMP10, LIN28 or TCIM in tandem with downregulation of GRIK1 or DGKK among others. Lactate also demonstrated a capability to modulate the production of inflammatory cytokines on cardiac fibroblasts, reducing the production of Fas, Fraktalkine or IL-12p40, while stimulating IL-13 and SDF1a. In addition, the generation of a lactate-rich environment improved ex vivo neonatal heart culture, by affecting the contractile activity and sarcomeric structures and inhibiting epicardial cell spreading. Our results also suggested a common link between the effect of lactate and the activation of hypoxia signaling pathways. These findings support a novel use of lactate in cardiac tissue engineering, modulating the metabolic environment of the heart and thus paving the way to the development of lactate-releasing platforms for in situ cardiac regeneration.

Inhibition of DYRK1A, via histone modification, promotes cardiomyocyte cell cycle activation and cardiac repair after myocardial infarction.

SummaryBackgroundWhile the adult mammalian heart undergoes only modest renewal through cardiomyocyte proliferation, boosting this process is considered a promising therapeutic strategy to repair cardiac injury. This study explored the role and mechanism of dual-specificity tyrosine regulated kinase 1A (DYRK1A) in regulating cardiomyocyte cell cycle activation and cardiac repair after myocardial infarction (MI).MethodsDYRK1A-knockout mice and DYRK1A inhibitors were used to investigate the role of DYRK1A in cardiomyocyte cell cycle activation and cardiac repair following MI. Additionally, we explored the underlying mechanisms by combining genome-wide transcriptomic, epigenomic, and proteomic analyses.FindingsIn adult mice subjected to MI, both conditional deletion and pharmacological inhibition of DYRK1A induced cardiomyocyte cell cycle activation and cardiac repair with improved cardiac function. Combining genome-wide transcriptomic and epigenomic analyses revealed that DYRK1A knockdown resulted in robust cardiomyocyte cell cycle activation (shown by the enhanced expression of many genes governing cell proliferation) associated with increased deposition of trimethylated histone 3 Lys4 (H3K4me3) and acetylated histone 3 Lys27 (H3K27ac) on the promoter regions of these genes. Mechanistically, via unbiased mass spectrometry, we discovered that WD repeat-containing protein 82 and lysine acetyltransferase 6A were key mediators in the epigenetic modification of H3K4me3 and H3K27ac and subsequent pro-proliferative transcriptome and cardiomyocyte cell cycle activation.InterpretationOur results reveal a significant role of DYRK1A in cardiac repair and suggest a drug target with translational potential for treating cardiomyopathy.FundingThis study was supported in part by grants from the National Natural Science Foundation of China (81930008, 82022005, 82070296, 82102834), National Key R&D Program of China (2018YFC1312700), Program of Innovative Research Team by the National Natural Science Foundation (81721001), and National Institutes of Health (5R01DK039308-31, 7R37HL023081-37, 5P01HL074940-11).

Stimulation of Cardiomyocyte Proliferation Is Dependent on Species and Level of Maturation.

The heart is one of the least regenerative organs. This is in large part due to the inability of adult mammalian cardiomyocytes to proliferate and divide. In recent years, a number of small molecules and molecular targets have been identified to stimulate cardiomyocyte proliferation, including p38 inhibition, YAP-Tead activation, fibroblast growth factor 1 and Neuregulin 1. Despite these exciting initial findings, a therapeutic approach to enhance cardiomyocyte proliferation in vivo is still lacking. We hypothesized that a more comprehensive in vitro validation using live-cell imaging and assessment of the proliferative effects on various cardiomyocyte sources might identify the most potent proliferative stimuli. Here, we used previously published stimuli to determine their proliferative effect on cardiomyocytes from different species and isolated from different developmental timepoints. Although all stimuli enhanced DNA synthesis and Histone H3 phosphorylation in neonatal rat ventricular cardiomyocytes to similar degrees, these effects varied substantially in mouse cardiomyocytes and human iPSC-derived cardiomyocytes. Our results highlight p21 inhibition and Yap-Tead activation as potent proliferative strategies to induce cultured cardiomyocyte cell cycle activity across mouse, rat and human cardiomyocytes.