Cell Division In Cardiomyocytes Research Articles

MiRNA-519e promotes cardiomyocyte cell division accompanied by pro-angiogenic and anti-apoptotic effects

Abstract Background MicroRNAs have the therapeutic potential for heart repair after myocardial infarction(MI). Using a functional high-throughput screening approach, we identified microRNA-519e(miR-519e) as an inducer of cell-cycling in human-derived iPSC-cardiomyocytes(hiPSC-­CM). Purpose This study examines miR-519e for cell division in hiPSC-­CM, additional beneficial functions in heart-related non-CMs, and direct target identification and validation in vitro and in vivo. Methods MiR-519e-mimics or miR-scrambled(miR-scr) were transfected into hiPSC-­CM, human aortic endothelial cells(HAEC) and human cardiac fibroblasts(cFB). Proliferative markers (EdU(5-Ethynyl-2'- deoxyuridine), H3P(Phospho-Histone H3) and AURKB(Aurora B-kinase)) and apoptotic response using MTT assay after exposure to doxorubicin were assessed in hiPSC­-CMs. Angiogenic capacity of HAEC was evaluated by tube formation assay and fibrotic response of cFBs using EdU and α-SMA(smooth muscle actin) staining. In vivo, a mouse MI model with ligation of the left anterior descending followed by intramyocardial injection of miR-519e or miR-scr was used. Direct target identification of miR-519e was assessed using in silico analysis combined with RNA-Seq of miR-519e-mimic treated hiPSC-CMs and validated using Ago2-RNA immunoprecipitation(RIP) followed by qPCR. Results After transfection of miR-519e-mimics in hiPSC-­CM, a significant proliferative response indicative of cell division was observed in immunofluorescence staining and on protein level as compared with miR-scr treated hiPSC-CMs (EdU, H3P and AURKB, p<0.01). Apoptosis was blunted in miR-519e-treated hiPSC-CMs (p<0.05). MiR-519e-treatment of HAEC resulted in more meshes (p<0.01) and longer tube length (p<0.05), indicating enhanced pro-angiogenic capacity. Importantly, EdU-uptake and α-SMA were similar in miR-519e-treated cFB as compared to miR-scr, suggesting neutral effects of miR-519e on myofibroblast transition. In mice undergoing MI, injection of miR-519e-mimics significantly downregulated CDKN1A/P21(Cyclin dependent kinase inhibitor 1A) (p<0.05 vs. miR-scr), a direct target of miR-519e and negative master regulator of cell-cycling. For further novel target identification of miR-519e, we performed in silico analysis combined with RNA-Seq data from miR-519e-treated hiPSC-CM and validation using Ago2-RIP followed by qPCR. Herein, we identified PTEN(Phosphatase and tensin homolog) and TGFBR2(Transforming growth factor β receptor 2) that are involved in cardiomyocyte proliferation and hypoxic response, as direct targets of miR-519e. Conclusion Collectively, miR-519e is a potent inducer of cell division in human cardiomyocytes, with anti-apoptotic and pro-angiogenic capacity in the absence of pro-fibrotic effects. We show that miR-519e treatment downregulates key negative cell-cycle regulators in vivo and we identified novel direct targets of miR-519e that are involved in regulatory pathways for heart regeneration.

A broadly applicable method for quantifying cardiomyocyte cell division identifies proliferative events following myocardial infarction

Cardiomyocyte proliferation is a challenging metric to assess. Current methodologies have limitations in detecting the generation of new cardiomyocytes and technical challenges that reduce widespread applicability. Here, we describe an improved cell suspension and imaging-based methodology that can be broadly employed to assess cardiomyocyte cell division in standard laboratories across a multitude of model organisms and experimental conditions. We highlight additional metrics that can be gathered from the same cell preparations to enable additional relevant analyses to be performed. We incorporate additional antibody stains to address potential technical concerns of miscounting. Finally, we employ this methodology with a dual-thymidine analog-labeling approach to a post-infarction murine model, which allowed us to robustly identify unique cycling events, such as cardiomyocytes undergoing multiple rounds of cell division.

Lin28a cardiomyocyte-specific modified mRNA translation system induces cardiomyocyte cell division and cardiac repair

The mammalian heart has a limited regenerative capacity. Previous work suggested the heart can regenerate during development and immediately after birth by inducing cardiomyocyte (CM) proliferation; however, this capacity is lost seven days after birth. modRNA gene delivery, the same technology used successfully in the two mRNA vaccines against SARS-CoV-2, can prompt cardiac regeneration, cardiovascular regeneration and cardiac protection. We recently established a novel CM-specific modRNA translational system (SMRTs) that allows modRNA translation only in CMs. We demonstrated that this system delivers potent intracellular genes (e.g., cell cyclepromoting Pkm2), which are beneficial when expressed in one cell type (i.e., CMs) but not others (non-CMs). Here, we identify Lin28a as an important regulator of the CM cell cycle. We show that Lin28a is expressed in CMs during development and immediately after birth, but not during adulthood. We describe that specific delivery of Lin28a into CM, using CM SMRTs, enables CM cell division and proliferation. Further, we determine that this proliferation leads to cardiac repair and better outcome post MI. Moreover, we identify the molecular pathway of Lin28a in CMs. We also demonstrate that Lin28a suppress Let-7 which is vital for CM proliferation, partially due to its suppressive role on cMYC, HMGA2 and K-RAS.

Regulation of Cardiomyocyte Division During Cardiac Regeneration.

This review explores efforts made over the previous three decades to determine mechanisms of cardiomyocyte cell division. Many investigators have explored cell therapy strategies in animal models and clinical trials over the past 2 decades with marginal results thus far in clinical testing. Hence, there is a greater focus now on strategies to induce cardiomyocyte proliferation. Reports to induce reactivation of the cardiomyocyte cell cycle predated the focus on cell therapy, and we summarize the literature on this topic, which began with the very first transgenic mouse studies in cardiovascular science. These earlier studies form the foundation for the use of cell cycle manipulation in cardiac repair and should inform current and future investigations with respect to rigor of assessment in the degree of cardiomyocyte cell division and gold standard measures of cardiac functional improvement.

Cardiomyocyte ploidy is dynamic during postnatal development and varies across genetic backgrounds.

Somatic polyploidization, an adaptation by which cells increase their DNA content to support growth, is observed in many cell types, including cardiomyocytes. Although polyploidization is believed to be beneficial, progression to a polyploid state is often accompanied by loss of proliferative capacity. Recent work suggests that genetics heavily influence cardiomyocyte ploidy. However, the developmental course by which cardiomyocytes reach their final ploidy state has only been investigated in select backgrounds. Here, we assessed cardiomyocyte number, cell cycle activity, and ploidy dynamics across two divergent mouse strains: C57BL/6J and A/J. Both strains are born and reach adulthood with comparable numbers of cardiomyocytes; however, the end composition of ploidy classes and developmental progression to reach the final state differ substantially. We expand on previous findings that identified Tnni3k as a mediator of cardiomyocyte ploidy and uncover a role for Runx1 in ploidy dynamics and cardiomyocyte cell division, in both developmental and injury contexts. These data provide novel insights into the developmental path to cardiomyocyte polyploidization and challenge the paradigm that hypertrophy is the sole mechanism for growth in the postnatal heart.

Telomere shortening in heart failure due to excess neurohormonal activation is associated with loss of nuclear TRF-2 and increased telomeric DNA damage and predicts cardiac recovery

Abstract The mechanisms and clinical significance of telomere shortening in heart failure remain elusive. Mammalian cardiomyocyte (CM) regeneration is limited and CM cell division cannot account for telomere shortening. Whether telomere shortening in turn affects cardiac recovery remains unexplored. We induced heart failure by excess neurohormonal activation (NHA), a universal dysregulation in the failing heart. B6 mice were subjected to AngII-infusion, uninephrectomy and high-salt (AngII++) to induce excess NHA for 5 weeks. Cardiac function was assessed by ultrasound. Human iPSC-derived CMs, mouse CMs and rat ventricular CMs (H9C2) were stimulated with AngII. Human cardiac biopsies from patients with heart failure with reduced ejection fraction (HFrEF) were grouped depending on cardiac recovery. Telomere length was quantified by Q-FISH. ROS-induced DNA/RNA-damage was evaluated after staining for 8-oxo-7,8-dihydro-2'-deoxyguanosine, -guanine and – guanosine-residues (8-oxo-dG). H9C2-cells were co-stained for telomeres, telomere repeat binding factor 2 (TRF-2) and 8-oxo-dG, telomere-specific DNA-damage was quantified by automated colocalization analysis. Superoxide (O2-) was quantified by 2-hydroxyethidium (2-HE) using HPLC analysis. Fibrosis was quantified by Sirius red staining. AngII++-mice exhibited left ventricular (LV) hypertrophy (p<0.0001 vs B6) along with reduced LV ejection fraction (p=0.03 vs B6), reduced global longitudinal strain (p<0.001 vs B6) along with significant telomere shortening in CMs isolated from AngII++-mice (p=0.0082 vs B6). Telomere length showed a significant correlation with LV systolic function (r2=0.64, p=0.006). While 8-oxo-dG-staining revealed significantly increased ROS-induced DNA/RNA damage in AngII++-myocardial sections overall (p<0.0001 vs B6), DNA/RNA-damage was highest in areas of fibrotic adverse remodeling congruent with significant telomere shortening (p<0.0122 fibrotic vs non-fibrotic AngII++-myocardium, p<0.0001 vs B6). In all cells tested, NHA resulted in significant telomere shortening. As a potential explanation, CMs stimulated with AngII revealed significantly increased, dose-dependent O2–production (AngII 10 nM p=0.03, AngII 50 nm p=0.0004 vs H9C2) along with significant loss of nucelar TRF2 (AngII nM p=0.0049, Ang II 50 nM p<0.0001 vs H9C2), while colocalization analysis revealed a significantly increased ROS-induced damage to the telomeric DNA (AngII 10/50 nm p<0.05 vs H9C2). HFrEF-patients that had recovered cardiac function (ΔLVEF +5 – 30%) showed significantly longer myocardial telomeres than patients with no recovery/worsening (ΔLVEF −10 – 0%) (p=0.008), overall the telomere signal correlated significantly with ΔLVEF (r2=0.41, p=0.03). Our data provides insides into how excess NHA via increased ROS, loss of nuclear TRF-2 and increased telomeric DNA-damage causes myocardial telomere shortening in heart failure and further that the extent of telomere shortening determines cardiac recovery. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): BMBF 01EO1003

A deleterious mutation in the ALMS1 gene in a naturally occurring model of hypertrophic cardiomyopathy in the Sphynx cat

BackgroundFamilial hypertrophic cardiomyopathy is a common inherited cardiovascular disorder in people. Many causal mutations have been identified, but about 40% of cases do not have a known causative mutation. Mutations in the ALMS1 gene are associated with the development of Alstrom syndrome, a multisystem familial disease that can include cardiomyopathy (dilated, restrictive). Hypertrophic cardiomyopathy has not been described. The ALMS1 gene is a large gene that encodes for a ubiquitously expressed protein. The function of the protein is not well understood although it is believed to be associated with energy metabolism and homeostasis, cell differentiation and cell cycle control. The ALMS1 protein has also been shown to be involved in the regulation of cell cycle proliferation in perinatal cardiomyocytes. Although cardiomyocyte cell division and replication in mammals generally declines soon after birth, inhibition of ALMS1 expression in mice lead to increased cardiomyocyte proliferation, and deficiency of Alstrom protein has been suggested to impair post-natal cardiomyocyte cell cycle arrest. Here we describe the association of familial hypertrophic cardiomyopathy in Sphynx cats with a novel ALMS1 mutation.ResultsA G/C variant was identified in exon 12 (human exon 13) of the ALMS1 gene in affected cats and was positively associated with the presence of hypertrophic cardiomyopathy in the feline population (p < 0.0001). The variant was predicted to change a highly conserved nonpolar Glycine to a positively charged Arginine. This was predicted to be a deleterious change by three in silico programs. Protein prediction programs indicated that the variant changed the protein structure in this region from a coil to a helix. Light microscopy findings included myofiber disarray with interstitial fibrosis with significantly more nuclear proliferative activity in the affected cats than controls (p < 0.0001).ConclusionThis study demonstrates a novel form of cardiomyopathy associated with ALMS1 in the cat. Familial hypertrophic cardiomyopathy is a disease of genetic heterogeneity; many of the known causative genes encoding for sarcomeric proteins. Our findings suggest that variants in genes involved with cardiac development and cell regulation, like the ALMS1 gene, may deserve further consideration for association with familial hypertrophic cardiomyopathy.

Pkm2 Regulates Cardiomyocyte Cell Cycle and Promotes Cardiac Regeneration.

The adult mammalian heart has limited regenerative capacity, mostly attributable to postnatal cardiomyocyte cell cycle arrest. In the last 2 decades, numerous studies have explored cardiomyocyte cell cycle regulatory mechanisms to enhance myocardial regeneration after myocardial infarction. Pkm2 (Pyruvate kinase muscle isoenzyme 2) is an isoenzyme of the glycolytic enzyme pyruvate kinase. The role of Pkm2 in cardiomyocyte proliferation, heart development, and cardiac regeneration is unknown. We investigated the effect of Pkm2 in cardiomyocytes through models of loss (cardiomyocyte-specific Pkm2 deletion during cardiac development) or gain using cardiomyocyte-specific Pkm2 modified mRNA to evaluate Pkm2 function and regenerative affects after acute or chronic myocardial infarction in mice. Here, we identify Pkm2 as an important regulator of the cardiomyocyte cell cycle. We show that Pkm2 is expressed in cardiomyocytes during development and immediately after birth but not during adulthood. Loss of function studies show that cardiomyocyte-specific Pkm2 deletion during cardiac development resulted in significantly reduced cardiomyocyte cell cycle, cardiomyocyte numbers, and myocardial size. In addition, using cardiomyocyte-specific Pkm2 modified RNA, our novel cardiomyocyte-targeted strategy, after acute or chronic myocardial infarction, resulted in increased cardiomyocyte cell division, enhanced cardiac function, and improved long-term survival. We mechanistically show that Pkm2 regulates the cardiomyocyte cell cycle and reduces oxidative stress damage through anabolic pathways and β-catenin. We demonstrate that Pkm2 is an important intrinsic regulator of the cardiomyocyte cell cycle and oxidative stress, and highlight its therapeutic potential using cardiomyocyte-specific Pkm2 modified RNA as a gene delivery platform.

Abstract 132: Imaging-Based Assay for Screening of Cell Cycle Modifying Substances in Postnatal Cardiomyocytes

Cardiovascular diseases are the main cause of death in the industrial world. Especially large myocardial infarctions and the potentially resulting left ventricular dysfunction are chronical diseases with a poor prognosis. Due to limited endogenous cardiac repair capacity, we are investigating the regulation of the cell cycle in cardiomyocytes (CMs) as a potential treatment strategy. We hypothesize that controlled induction of cardiomyocyte proliferation will lead to an increase in muscle mass and improvement of cardiac function. We are using CMs from double transgenic αMHC-H2B-mCherry/CAG-eGFP-anillin mice for the analysis of postnatal CM development. This double transgenic system enables us to unequivocally identify CM nuclei as well as cell cycle variations like acytokinetic mitosis (mitosis without cytokinesis), resulting in binucleated CMs, and endoreplication (mitosis without cytokinesis and karyokinesis), leading to polyploid CMs. With the goal of inducing authentic cell division, we are screening for pro-proliferative substances in P1 and P6 postnatal CMs of αMHC-H2B-mCherry/CAG-eGFP-anillin mice. Here we will present the detailed analysis of several screening hits demonstrating an increase in cell cycle activity leading mainly to binucleation but only to limited cell division in CMs.

In This Issue

HomeCirculation ResearchVol. 123, No. 9In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published11 Oct 2018https://doi.org/10.1161/RES.0000000000000232Circulation Research. 2018;123:1009is related toLactobacillus plantarum 299v Supplementation Improves Vascular Endothelial Function and Reduces Inflammatory Biomarkers in Men With Stable Coronary Artery DiseaseRestoring Endothelial Function by Targeting Desert Hedgehog Downstream of Klf2 Improves Critical Limb Ischemia in Adultsis related toMidbody Positioning and Distance Between Daughter Nuclei Enable Unequivocal Identification of Cardiomyocyte Cell Division in MiceUnequivocal Identification of Cardiomyocyte Division (p 1039)Download figureDownload PowerPointHesse et al devise a new method for assessing cardiomyocyte proliferation.A myocardial infarction can lead to the death and destruction of a significant number of heart cells that, due to the limited regenerative capacity of the heart, do not regrow. Scientists are, therefore, vigorously searching for pharmacological agents that can induce the proliferation of cardiomyocytes and regrowth of the myocardium. The gold-standard marker of cell proliferation in vitro is Aurora B-kinase, but Hesse and colleagues argue that this poses a problem in the case of cardiac myocytes, many of which are binucleated. Indeed, Aurora B-kinase-based assays do not differentiate between binucleation and true cell division, the researchers show. The team, therefore, proposes an alternative marker—the positioning of the midbody and the distance between daughter nuclei. Using time-lapse microscopy to image cells from mice expressing fluorescently labeled anillin (a midbody protein), they observed that while true division resulted in symmetrical positioning of the midbody between daughter nuclei, binucleation was marked by randomly located and fragmented midbodies. Furthermore, in binucleation, but not true division, the daughter nuclei remained close together, almost touching. The authors suggest adopting this new strategy could provide a more accurate indication of real proliferation.Dhh Agonists Ameliorate Endothelial Function (p 1053)Download figureDownload PowerPointA Dhh agonist improves vascular function in mice with critical limb ischemia, report Caradu et al.Dysfunction of the vascular endothelium, which can result in leaky, inflamed blood vessels, is often associated with hypertension, diabetes, obesity, and advanced age. Although the pathways leading to endothelial dysfunction are largely unknown, it has been shown that the transcription factor Klf2, essential for endothelial health, is down-regulated in the context of vascular inflammation, high glucose, and disturbed blood flow. And recently, it was shown that morphogen Dhh is suppressed under similar conditions. Intrigued by this apparent coregulation, Caradu and colleagues tested whether Klf2 and Dhh might work in the same pathway. Indeed, they found that Klf2 directly activates the Dhh promoter. Moreover, knock-down of Dhh produced effects similar to those of Klf2 suppression—namely vessel leakiness and increased expression of proinflammatory factors. The team also found that critical limb ischemia is associated with capillary leakage and inflammation in both human patients and mice, and that treatment of the animals with a Dhh agonist could improve endothelial function. Together, the results imply that boosting the Klf2-Dhh pathway might be an effective approach for treating ischemic vascular disease.L Plantarum 299v and Endothelial Function (p 1091)Download figureDownload PowerPointProbiotic treatment improves vascular health in patients with coronary artery disease, report Malik et al.Atherosclerosis and plaque instability have been found to be associated with differences in the microbial composition of the human gut—both in terms of specific species and their diversity. Furthermore, in mice, fecal transfers, antibiotics, and high-fiber diets—all of which can alter the gut microbiome—have been shown to influence vascular health, systemic inflammation, and atherogenesis. Despite this evidence, it is unclear whether treatments that target the microbiome improve human vascular health. To find out, Malik et al gave Lactobacillus plantarum 299v (Lp299v)—a gut microbe shown to have positive cardiovascular effects in animals and human smokers—to 20 men with coronary artery disease. They found that daily treatments for 6 weeks reduced the levels of proinflammatory cytokines in the blood and improved vasodilation in the brachial arteries. Additionally, plasma taken from the subjects after treatment was shown to improve acetylcholine-induced dilation in CAD patient arterioles in tissue culture. The findings suggest that probiotic supplementation might be an additional tool for reducing cardiovascular disease risk, and open the door for further large-scale probiotic trials. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesLactobacillus plantarum 299v Supplementation Improves Vascular Endothelial Function and Reduces Inflammatory Biomarkers in Men With Stable Coronary Artery DiseaseMobin Malik, et al. Circulation Research. 2018;123:1091-1102Midbody Positioning and Distance Between Daughter Nuclei Enable Unequivocal Identification of Cardiomyocyte Cell Division in MiceMichael Hesse, et al. Circulation Research. 2018;123:1039-1052Restoring Endothelial Function by Targeting Desert Hedgehog Downstream of Klf2 Improves Critical Limb Ischemia in AdultsCaroline Caradu, et al. Circulation Research. 2018;123:1053-1065 October 12, 2018Vol 123, Issue 9 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000232PMID: 30355170 Originally publishedOctober 11, 2018 PDF download Advertisement

Midbody Positioning and Distance Between Daughter Nuclei Enable Unequivocal Identification of Cardiomyocyte Cell Division in Mice.

New strategies in the field of cardiac regeneration are directed at identifying proliferation-inducing substances to induce regrowth of myocardium. Current screening assays utilize neonatal cardiomyocytes and markers for cytokinesis, such as Aurora B-kinase. However, detection of cardiomyocyte division is complicated because of cell cycle variants, in particular, binucleation. To analyze the process of cardiomyocyte binucleation to identify definitive discriminators for cell cycle variants and authentic cardiomyocyte division. Herein, we demonstrate by direct visualization of the contractile ring and midbody in Myh6 (myosin, heavy chain 6)-eGFP (enhanced green fluorescent protein)-anillin transgenic mice that cardiomyocyte binucleation starts by formation of a contractile ring. This is followed by irregular positioning of the midbody and movement of the 2 nuclei into close proximity to each other. In addition, the widespread used marker Aurora B-kinase was found to also label binucleating cardiomyocytes, complicating the interpretation of existing screening assays. Instead, atypical midbody positioning and the distance of daughter nuclei on karyokinesis are bona fide markers for cardiomyocyte binucleation enabling to unequivocally discern such events from cardiomyocyte division in vitro and in vivo. The 2 criteria provide a new method for identifying cardiomyocyte division and should be considered in future studies investigating cardiomyocyte turnover and regeneration after injury, in particular in the postnatal heart to prevent the assignment of false positive proliferation events.

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Functionally Conserved Noncoding Regulators of Cardiomyocyte Proliferation and Regeneration in Mouse and Human

The adult mammalian heart has little regenerative capacity after myocardial infarction (MI), whereas neonatal mouse heart regenerates without scarring or dysfunction. However, the underlying pathways are poorly defined. We sought to derive insights into the pathways regulating neonatal development of the mouse heart and cardiac regeneration post-MI. Total RNA-seq of mouse heart through the first 10 days of postnatal life (referred to as P3, P5, P10) revealed a previously unobserved transition in microRNA (miRNA) expression between P3 and P5 associated specifically with altered expression of protein-coding genes on the focal adhesion pathway and cessation of cardiomyocyte cell division. We found profound changes in the coding and noncoding transcriptome after neonatal MI, with evidence of essentially complete healing by P10. Over two-thirds of each of the messenger RNAs, long noncoding RNAs, and miRNAs that were differentially expressed in the post-MI heart were differentially expressed during normal postnatal development, suggesting a common regulatory pathway for normal cardiac development and post-MI cardiac regeneration. We selected exemplars of miRNAs implicated in our data set as regulators of cardiomyocyte proliferation. Several of these showed evidence of a functional influence on mouse cardiomyocyte cell division. In addition, a subset of these miRNAs, miR-144-3p, miR-195a-5p, miR-451a, and miR-6240 showed evidence of functional conservation in human cardiomyocytes. The sets of messenger RNAs, miRNAs, and long noncoding RNAs that we report here merit further investigation as gatekeepers of cell division in the postnatal heart and as targets for extension of the period of cardiac regeneration beyond the neonatal period.

Impact of preterm birth on the developing myocardium of the neonate.

BackgroundGlobally, ∼10% of infants are born before full term. Preterm birth exposes the heart to the demands of postnatal cardiovascular function before cardiac development is complete. Our aim was to examine, in hearts collected from infants at autopsy, the effects of preterm birth on myocardial structure and on cardiomyocyte development.Methods and resultsHeart tissue was collected at perinatal autopsies of 16 infants who died following preterm birth between 23 and 36 weeks of gestation, and survived for 1-42 days; the hearts of 37 appropriately grown stillborn infants, aged 20-40 weeks of gestation, were used for comparison. Using confocal microscopy and image analysis, cardiomyocyte proliferation, maturation, ploidy, and size were quantified, and interstitial collagen and myocardial capillarization were measured. Preterm birth resulted in a marked reduction in the proliferation of cardiomyocytes relative to age-matched stillborn infant controls (preterm vs. control P<0.0001). In contrast, preterm birth did not affect heart weight, capillarization, interstitial collagen or cardiomyocyte maturation, ploidy, and size.ConclusionsPreterm birth appears to lead to an abrupt reduction in cardiomyocyte cell division. This reduced cardiomyocyte proliferation in preterm infants may adversely impact upon the final number of cardiomyocytes which may reduce cardiac functional reserve, and impair the reparative capacity of the myocardium.

Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognise homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at two weeks postnatal compared to wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

Abstract 19780: Mammalian Cardiac Renewal by Preexisting Cardiomyocytes

Although recent studies have revealed that adult heart cells are generated after birth in mammals, the frequency and source of these new heart cells is unclear. Here we combined two different pulse-chase approaches--genetic fate-mapping and stable isotope labeling with Multi-isotope Imaging Mass Spectrometry (MIMS)--to determine the origin of cardiomyocytes in adult mice. MIMS allows quantitative imaging of stable isotope reporters in domains smaller than a micron cubed. The nuclear incorporation of a 15N stable isotope-enriched tracer here as 15N-thymidine is detectable with high precision by an increase in the 15N/14N ratio above natural abundance (0.37%) in a pattern resembling chromatin evident in cells having divided during a labeling period. We administered 15N-thymidine for 8 weeks to three age groups of C57BL6 mice starting at day-4 (neonate), at 10-weeks (young adult) and at 22-months (old adult) and extrapolated DNA synthesis yields a yearly rate of 5.5% in the young adult that declines further to 2.6% in advanced aged mice. Fig1a. To evaluate the origin of newly generated cardiomyocytes, we performed 15N-thymidine labeling of double-transgenic MerCreMer/ZEG mice, which we previously developed for genetic lineage mapping. Fig1b. After treatment with 4OH-tamoxifen, we show that DNA synthesis occurs at a low rate in predominantly pre-existing cardiomyocytes [77% expressed GFP, a frequency essentially identical to that of surrounding 15N- cardiomyocytes (15N, 77% vs 15N-, 84%; Fisher’s exact=n.s.)]. Fig1c. Cell cycle activity in cardiomyocytes led to polyploidy and multinucleation, and to new diploid, mononucleated cardiomyocytes [17% of total; predominantly GFP+ (83% vs 82%, p=n.s.)]. Fig1d. Our results support a low rate of cell division of cardiomyocytes as the origin of mammalian adult cardiogenesis and are inconsistent with a high rate of stem cell activity in the normal mammalian myocardium.

Oxidative stress-induced formation of a positive-feedback loop for the sustained activation of p38 MAPK leading to the loss of cell division in cardiomyocytes soon after birth

Shortly after birth, mammalian cardiomyocytes irreversibly exit from the cell cycle and become terminally differentiated. The cellular mechanisms responsible for the cessation of cell division and terminal differentiation of cardiomyocytes soon after birth have intrigued developmental biologists as well as cardiovascular physicians, but the genetic cues for the irreversible exit from the cell cycle soon after birth remain largely unknown. We examined whether and if so how oxidative stress to mammalian hearts during fetal-neonatal transition produces changes in the proliferative activity and terminal differentiation of cardiomyocytes. Scavenging of reactive oxygen species (ROS) during fetal-neonatal transition, especially after birth, resulted in an increase in the proliferative activity and a decrease in the ratio of binucleated cardiomyocytes. Exposure to ROS in cultured cardiomyocytes increased the activity of p38 MAPK and the expression of connexin 43 (Cx43). Not only knockdown of Cx43 using siRNA but also the inhibition of p38 MAPK activity resulted in a significant decrease in the production of ROS in cardiomyocytes, suggesting that the signaling pathway ROS-p38 MAPK-Cx43 (especially, Cx43 at mitochondria, mtCx43) constituted a closed regulatory system with positive feedback. In addition, continuous scavenging of ROS or suppression of p38 MAPK activity for 4days after birth resulted in a significant decrease in the expression of mtCx43 and in the number of binucleated cardiomyocytes. This study demonstrated that the ROS-induced formation of a positive-feedback loop ROS-p38 MAPK-mtCx43 for the sustained activation of p38 MAPK soon after birth possibly contributes to the loss of cell division and binucleation in mammalian cardiomyocytes.

Proteins secreted by embryonic stem cells activate cardiomyocytes through ligand binding pathways

Human embryonic stem cells (hESC) underlie embryogenesis but paracrine signals associated with the process are unknown. This study was designed to 1) profile native proteins secreted by undifferentiated hESC and 2) determine their biological effects on primary neonatal cardiomyocytes. We utilized multi-analyte, immunochemical assays to characterize media conditioned by undifferentiated hESC versus unconditioned media. Expression profiling was performed on cardiomyocytes subjected to these different media conditions and altered transcripts were mapped to critical pathways. Thirty-two of 109 proteins were significantly elevated in conditioned media ranging in concentration from thrombospondin (57.2+/-5.0 ng/ml) to nerve growth factor (7.4+/-1.2pg/ml) and comprising chemokines, cytokines, growth factors, and proteins involved in cell adhesion and extracellular matrix remodeling. Conditioned media induced karyokinesis, cytokinesis and proliferation in mono- and binucleate cardiomyocytes. Pathway analysis revealed comprehensive activation of the ROCK 1 and 2 G-protein coupled receptor (GPCR) pathway associated with cytokinesis, and the RAS/RAF/MEK/ERK receptor tyrosine kinase (RTK) and JAK/STAT-cytokine pathway involved in cell cycle progression. These results provide a partial database of proteins secreted by pluripotent hESC that potentiate cell division in cardiomyocytes via a paracrine mechanism suggesting a potential role for these stem cell factors in cardiogenesis and cardiac repair.

Divergent Siblings

Proliferation of mammalian cardiomyocytes ceases around birth when a transition from hyperplastic to hypertrophic myocardial growth occurs. Previous studies demonstrated that directed expression of the transcription factor E2F1 induces S-phase entry in cardiomyocytes along with stimulation of programmed cell death. Here, we show that directed expression of E2F2 and E2F4 by adenovirus mediated gene transfer in neonatal cardiomyocytes induced S-phase entry but did not result in an onset of apoptosis whereas directed expression of E2F1 and E2F3 strongly evoked programmed cell death concomitant with cell cycle progression. Although both E2F2 and E2F4 induced S-phase entry only directed expression of E2F2 resulted in mitotic cell division of cardiomyocytes. Expression of E2F5 or a control LacZ-Adenovirus had no effects on cell cycle progression. Quantitative real time PCR revealed that E2F1, E2F2, E2F3, and E2F4 alleviate G0 arrest by induction of cyclinA and E cyclins. Furthermore, directed expression of E2F1, E2F3, and E2F5 led to a transcriptional activation of several proapoptotic genes, which were mitigated by E2F2 and E2F4. Our finding that expression of E2F2 induces cell division of cardiomyocytes along with a suppression of proapoptotic genes might open a new access to improve the regenerative capacity of cardiomyocytes.