Myocyte Proliferation Research Articles

Tissue-Specific Cell Cycle Indicator Reveals Unexpected Findings for Cardiac Myocyte Proliferation.

Discerning cardiac myocyte cell cycle behavior is challenging owing to commingled cell types with higher proliferative activity. To investigate cardiac myocyte cell cycle activity in development and the early postnatal period. To facilitate studies of cell type-specific proliferation, we have generated tissue-specific cell cycle indicator BAC transgenic mouse lines. Experiments using embryonic fibroblasts from CyclinA2-LacZ-floxed-EGFP, or CyclinA2-EGFP mice, demonstrated that CyclinA2-βgal and CyclinA2-EGFP were expressed from mid-G1 to mid-M phase. Using Troponin T-Cre;CyclinA2-LacZ-EGFP mice, we examined cardiac myocyte cell cycle activity during embryogenesis and in the early postnatal period. Our data demonstrated that right ventricular cardiac myocytes exhibited reduced cell cycle activity relative to left ventricular cardiac myocytes in the immediate perinatal period. Additionally, in contrast to a recent report, we could find no evidence to support a burst of cardiac myocyte cell cycle activity at postnatal day 15. Our data highlight advantages of a cardiac myocyte-specific cell cycle reporter for studies of cardiac myocyte cell cycle regulation.

The exercise-regulated myokine chitinase-3-like protein 1 stimulates human myocyte proliferation.

Chitinase-3-like protein 1 (CHI3L1) is involved in tissue remodelling and inflammatory processes. Plasma levels are elevated in patients with insulin resistance and T2DM. We recently showed that CHI3L1 and its receptor protease-activated receptor 2 (PAR-2) are expressed in skeletal muscle. Activation of PAR-2 by CHI3L1 protects against TNF-α-induced inflammation and insulin resistance. However, the effect of exercise on CHI3L1 and PAR-2 signalling remains unknown. The aim of this work was to study the impact of exercise on CHI3L1 production and the effect of CHI3L1/PAR-2 signalling on skeletal muscle growth and repair. Three human exercise studies were used to measure CHI3L1 plasma levels (n = 32). In addition, muscle and adipose tissue CHI3L1 mRNA expression was measured in response to acute and long-term exercise (n = 24). Primary human skeletal muscle cells were differentiated in vitro, and electrical pulse stimulation was applied. In addition, myoblasts were incubated with CHI3L1 protein and activation of MAP kinase signalling as well as proliferation was measured. Circulating CHI3L1 levels and muscle CHI3L1 mRNA were increased after acute exercise. In addition, CHI3L1 mRNA expression as well as CHI3L1 secretion was enhanced in electrically stimulated cultured myotubes. Incubation of cultured human myoblasts with CHI3L1 protein leads to a strong activation of p44/42, p38 MAPK and Akt as well as enhanced myoblast proliferation. Our findings suggest that CHI3L1 is induced by acute exercise and that CHI3L1/PAR-2 signalling activates myocyte proliferation, which is important for restructuring of skeletal muscle in the response to exercise training.

Integrative Analysis of MicroRNA and mRNA Data Reveals an Orchestrated Function of MicroRNAs in Skeletal Myocyte Differentiation in Response to TNF-α or IGF1.

IntroductionSkeletal muscle cell differentiation is impaired by elevated levels of the inflammatory cytokine tumor necrosis factor-α (TNF-α) with pathological significance in chronic diseases or inherited muscle disorders. Insulin like growth factor-1 (IGF1) positively regulates muscle cell differentiation. Both, TNF-α and IGF1 affect gene and microRNA (miRNA) expression in this process. However, computational prediction of miRNA-mRNA relations is challenged by false positives and targets which might be irrelevant in the respective cellular transcriptome context. Thus, this study is focused on functional information about miRNA affected target transcripts by integrating miRNA and mRNA expression profiling data.Methodology/Principal FindingsMurine skeletal myocytes PMI28 were differentiated for 24 hours with concomitant TNF-α or IGF1 treatment. Both, mRNA and miRNA expression profiling was performed. The data-driven integration of target prediction and paired mRNA/miRNA expression profiling data revealed that i) the quantity of predicted miRNA-mRNA relations was reduced, ii) miRNA targets with a function in cell cycle and axon guidance were enriched, iii) differential regulation of anti-differentiation miR-155-5p and miR-29b-3p as well as pro-differentiation miR-335-3p, miR-335-5p, miR-322-3p, and miR-322-5p seemed to be of primary importance during skeletal myoblast differentiation compared to the other miRNAs, iv) the abundance of targets and affected biological processes was miRNA specific, and v) subsets of miRNAs may collectively regulate gene expression.ConclusionsJoint analysis of mRNA and miRNA profiling data increased the process-specificity and quality of predicted relations by statistically selecting miRNA-target interactions. Moreover, this study revealed miRNA-specific predominant biological implications in skeletal muscle cell differentiation and in response to TNF-α or IGF1 treatment. Furthermore, myoblast differentiation-associated miRNAs are suggested to collectively regulate gene clusters and targets associated with enriched specific gene ontology terms or pathways. Predicted miRNA functions of this study provide novel insights into defective regulation at the transcriptomic level during myocyte proliferation and differentiation due to inflammatory stimuli.

Nerves Regulate Cardiomyocyte Proliferation and Heart Regeneration

Some organisms, such as adult zebrafish and newborn mice, have the capacity to regenerate heart tissue following injury. Unraveling the mechanisms of heart regeneration is fundamental to understanding why regeneration fails in adult humans. Numerous studies have revealed that nerves are crucial for organ regeneration, thus we aimed to determine whether nerves guide heart regeneration. Here, we show using transgenic zebrafish that inhibition of cardiac innervation leads to reduction of myocyte proliferation following injury. Specifically, pharmacological inhibition of cholinergic nerve function reduces cardiomyocyte proliferation in the injured hearts of both zebrafish and neonatal mice. Direct mechanical denervation impairs heart regeneration in neonatal mice, which was rescued by the administration of neuregulin 1 (NRG1) and nerve growth factor (NGF) recombinant proteins. Transcriptional analysis of mechanically denervated hearts revealed a blunted inflammatory and immune response following injury. These findings demonstrate that nerve function is required for both zebrafish and mouse heart regeneration.

Abstract 154: Allogeneic Cardiosphere-derived Cells From an Aged Donor Elicit Long-term Improvements in Left Ventricular Function and Myocyte Proliferation in a Mini-swine Model of Chronic Myocardial Ischemia

Objective: Virtually all large animal studies of cell-based therapy have employed young donor-recipient pairs. Since aging may impact the reparative ability of allogeneic cardiosphere-derived cells (CDCs), we tested whether CDCs from an aged donor promote functional repair of ischemic myocardium using a mini-swine model that enabled serial assessment of left ventricular (LV) function over an extended follow-up period. Methods: Immunosuppressed mini-swine (cyclosporine 100 mg/day) with a chronic (4-months) LAD stenosis were untreated (n=8) or received 20 million allogeneic aged CDCs (n=10). Cells were cultivated from a 9-year old mini-pig and infused into the 3 major coronary arteries under continuous flow (1 million cells/min). LV function was assessed by echocardiography at baseline and over a 3-month follow-up period, at which time histological assessment of myocyte morphometry, proliferation (Ki67), and cell retention (Y-FISH) was performed. Results: Wall thickening (WT) of the ischemic LAD region was impaired at baseline (LAD: 42.7 ± 2.1% vs. Remote: 81.4 ± 4.8%, p<0.01) and not different between treatment groups. One month later, LAD WT improved in aged CDC-treated animals (from 41.0 ± 3.1 to 53.0 ± 1.7%, p=0.01) but remained depressed in untreated animals (from 45.1 ± 2.8% to 44.9 ± 2.6%, p=0.97). Extended follow-up revealed that LAD WT continued to improve, reaching 60.2 ± 1.6% (p=0.01) 3-months after CDC treatment. Histological analyses demonstrated morphometric changes consistent with myocyte regeneration, including an increase in myocyte nuclear density (1231 ± 34 vs. 1094 ± 34 nuclei/mm 2 , p=0.02) and reduction in myocyte diameter (13.9 ± 0.2 vs. 14.5 ± 0.3 μm, p=0.05). Increases in Ki67 + myocytes (468 ± 92 vs. 199 ± 32 nuclei/10 6 myocyte nuclei, p=0.02) and rare Y + cells in sex-mismatched recipients (0.5 ± 0.2 cells/cm 2 ) supported endogenous myocyte proliferation as the primary source of new myocytes. Conclusion: CDCs from an aged donor retain their reparative capacity to promote functional improvement and increase myocyte number following global intracoronary infusion. The progressive improvement in LV function 3-months after treatment with ongoing myocyte proliferation supports a long-lasting benefit after a single infusion.

Abstract 106: Pluripotent Stem Cell Microrna-294 Induces Cardiomyocyte Proliferation and Augments Cardiac Function After Myocardial Infarction

Rationale: Embryonic heart is characteristic of rapidly dividing cardiomyocytes that give rise to sufficient numbers required to build a working myocardium. In contrast, cardiomyocytes retain some proliferative capacity in the neonates but lose most of it in adulthood. Embryonic stem cell cycle (ESCC) miRs are a class of microRNAs regulating the unique cell cycle of ESCs and their characteristic pluripotency. Nevertheless, expression of miR-294, a member of the ESCC miRs is lost during developmental transitions from the ESCs to mature cells. Effect of miR-294 to induce cardiac proliferation and heart function has not been previously studied. Objective: To determine whether miR-294 drives cardiomyocyte cell cycle reentry leading to augmentation of cardiac function after myocardial infarction. Methods and Results: miR expression analysis in the heart during development revealed elevated levels of miR-294 in the prenatal stages while the expression was lost in the neonates and adults as confirmed by qRT-PCR. Neonatal ventricular cardiomyocytes (NRVMs) were treated with miR-294 mimic to determine the effect on proliferation and cell cycle. Elevated mRNA levels of cyclins A2, E1, CDK2 together with c-myc, E2F1 and E2F3 was observed in NRVMs treated with 25nM mimic for miR-294. Additionally, miR-294 treated NRVMs showed in AKT phosphorylation along with enhanced protein levels of cyclin D1 and E2F1. Increased expression of p-histone 3, Ki67 and Aurora B kinase (G2/M) was confirmed by immunocytochemistry in NRVMs after miR-294 treatment compared to control cells. Administration of miR-294 in mice subjected to myocardial infarction demonstrated augmentation of cardiac function in mice receiving miR-294 8 weeks after injury. Increase myocyte proliferation was observed in the heart after miR-294 treatment as analyzed by BrdU uptake, p-Histone 3 and Aurora B expression by immunostaining. Concurrently, a decrease in infarct size along with decreased apoptosis was observed in the miR-294 hearts compared to the control. Conclusion: Ectopic expression of miR-294 recapitulates embryonic signaling and enhances cardiomyocyte ability to proliferate and reenter the cell cycle leading to augmented cardiac function in mice after myocardial infarction.

Cardiac myosin binding protein C regulates postnatal myocyte cytokinesis

Homozygous cardiac myosin binding protein C-deficient (Mybpc(t/t)) mice develop dramatic cardiac dilation shortly after birth; heart size increases almost twofold. We have investigated the mechanism of cardiac enlargement in these hearts. Throughout embryogenesis myocytes undergo cell division while maintaining the capacity to pump blood by rapidly disassembling and reforming myofibrillar components of the sarcomere throughout cell cycle progression. Shortly after birth, myocyte cell division ceases. Cardiac MYBPC is a thick filament protein that regulates sarcomere organization and rigidity. We demonstrate that many Mybpc(t/t) myocytes undergo an additional round of cell division within 10 d postbirth compared with their wild-type counterparts, leading to increased numbers of mononuclear myocytes. Short-hairpin RNA knockdown of Mybpc3 mRNA in wild-type mice similarly extended the postnatal window of myocyte proliferation. However, adult Mybpc(t/t) myocytes are unable to fully regenerate the myocardium after injury. MYBPC has unexpected inhibitory functions during postnatal myocyte cytokinesis and cell cycle progression. We suggest that human patients with homozygous MYBPC3-null mutations develop dilated cardiomyopathy, coupled with myocyte hyperplasia (increased cell number), as observed in Mybpc(t/t) mice. Human patients, with heterozygous truncating MYBPC3 mutations, like mice with similar mutations, have hypertrophic cardiomyopathy. However, the mechanism leading to hypertrophic cardiomyopathy in heterozygous MYBPC3(+/-) individuals is myocyte hypertrophy (increased cell size), whereas the mechanism leading to cardiac dilation in homozygous Mybpc3(-/-) mice is primarily myocyte hyperplasia.

Executioner Caspase-3 and 7 Deficiency Reduces Myocyte Number in the Developing Mouse Heart.

Executioner caspase-3 and -7 are proteases promoting cell death but non-apoptotic roles are being discovered. The heart expresses caspases only during development, suggesting they contribute to the organ maturation process. Therefore, we aimed at identifying novel functions of caspases in heart development. We induced simultaneous deletion of executioner caspase-3 and -7 in the mouse myocardium and studied its effects. Caspase knockout hearts are hypoplastic at birth, reaching normal weight progressively through myocyte hypertrophy. To identify the molecular pathways involved in these effects, we used microarray-based transcriptomics and multiplexed quantitative proteomics to compare wild type and executioner caspase-deficient myocardium at different developmental stages. Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult null myocardium. Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase-null myocardium. Correlation between mRNA expression and protein abundance of relevant genes was confirmed, but transcriptomics and proteomics indentified complementary molecular pathways influenced by caspases in the developing heart. Forced expression of wild type or proteolytically inactive caspases in cultured cardiomyocytes induced expression of genes promoting cell division. The results reveal that executioner caspases can modulate heart’s cellularity and maturation during development, contributing novel information about caspase biology and heart development.

RNA Mimics as Therapeutics for Cardiac Regeneration: A Paradigm Shift

RNA Mimics as Therapeutics for Cardiac Regeneration: A Paradigm Shift

Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications.

The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2'-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner.

Engineering Cardiovascular Regeneration

The human heart has very limited regenerative capacity, making the loss of heart muscle tissue during myocardial infarction essentially irreversible. Regenerative medicine aims at promoting the formation of new functional heart muscle in an injured heart, either by stimulating myocyte proliferation, formation of myocytes from preexisting stem cells, direct reprogramming of fibroblasts into myocytes, or adding new muscle tissue from exogenous stem cell sources. Recent advances in stem cell research make these goals more realistic. This review provides a short overview about different approaches for cardiovascular regeneration, stem cell sources, and modes of application, focusing on current cardiac tissue engineering techniques and their way towards clinical application.

Revascularization of Chronic Hibernating Myocardium Stimulates Myocyte Proliferation and Partially Reverses Chronic Adaptations to Ischemia

BackgroundThe time course and extent of recovery after revascularization of viable dysfunctional myocardium are variable. Although fibrosis is a major determinant, myocyte structural and molecular remodeling may also play important roles. ObjectivesThis study sought to determine whether persistent myocyte loss and/or irreversibility of protein changes that develop in hibernating myocardium have an impact on functional recovery in the absence of infarction. MethodsSwine implanted with a chronic left anterior descending artery (LAD) stenosis to produce hibernating myocardium underwent percutaneous revascularization, with serial functional recovery evaluated for 1 month (n = 12). Myocardial tissue was evaluated to assess myocyte size, nuclear density, and proliferation indexes in comparison with those of normal animals and nonrevascularized controls. Proteomic analysis by 2-dimensional differential in-gel electrophoresis was used to determine the reversibility of molecular adaptations of hibernating myocytes. ResultsAt 3 months, physiological features of hibernating myocardium were confirmed, with depressed LAD wall thickening and no significant infarction. Revascularization normalized LAD flow reserve, with no immediate change in LAD wall thickening. Regional LAD wall thickening slowly improved but remained depressed 1 month post–percutaneous coronary intervention. Surprisingly, revascularization was associated with histological evidence of myocytes re-entering the growth phase of the cell cycle and increases in the number of c-Kit+ cells. Myocyte nuclear density returned to normal, whereas regional myocyte hypertrophy regressed. Proteomic analysis demonstrated heterogeneous effects of revascularization. Up-regulated stress and cytoskeletal proteins normalized, whereas reduced contractile and metabolic proteins persisted. ConclusionsDelayed recovery of hibernating myocardium in the absence of scar may reflect persistent reductions in the amounts of contractile and metabolic proteins. Although revascularization appeared to stimulate myocyte proliferation, the persistence of small immature myocytes may have contributed to delayed functional recovery.

Gene expression changes controlling distinct adaptations in the heart and skeletal muscle of a hibernating mammal.

Throughout the hibernation season, the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) experiences extreme fluctuations in heart rate, metabolism, oxygen consumption, and body temperature, along with prolonged fasting and immobility. These conditions necessitate different functional requirements for the heart, which maintains contractile function throughout hibernation, and the skeletal muscle, which remains largely inactive. The adaptations used to maintain these contractile organs under such variable conditions serves as a natural model to study a variety of medically relevant conditions including heart failure and disuse atrophy. To better understand how two different muscle tissues maintain function throughout the extreme fluctuations of hibernation we performed Illumina HiSeq 2000 sequencing of cDNAs to compare the transcriptome of heart and skeletal muscle across the circannual cycle. This analysis resulted in the identification of 1,076 and 1,466 differentially expressed genes in heart and skeletal muscle, respectively. In both heart and skeletal muscle we identified a distinct cold-tolerant mechanism utilizing peroxisomal metabolism to make use of elevated levels of unsaturated depot fats. The skeletal muscle transcriptome also shows an early increase in oxidative capacity necessary for the altered fuel utilization and increased oxygen demand of shivering. Expression of the fetal gene expression profile is used to maintain cardiac tissue, either through increasing myocyte size or proliferation of resident cardiomyocytes, while skeletal muscle function and mass are protected through transcriptional regulation of pathways involved in protein turnover. This study provides insight into how two functionally distinct muscles maintain function under the extreme conditions of mammalian hibernation.

Protective function of tocilizumab in human cardiac myocytes ischemia reperfusion injury.

To investigate the protective function of tocilizumab in human cardiac myocytes ischemia-reperfusion injury. The human cardiac myocytes were treated by tocilizumab with different concentrations(1.0 mg/mL, 3.0 mg/mL, 5.0 mg/mL) for 24 h, then cells were cultured in ischemia environment for 24 h and reperfusion environment for 1 h. The MTT and flow cytometry were used to detect the proliferation and apoptosis of human cardiac myocytes, respectively. The mRNA and protein expressions of Bcl-2 and Bax were measured by qRT-PCR and western blot, respectively. Compared to the negative group, pretreated by tocilizumab could significantly enhance the proliferation viability and suppress apoptosis of human cardiac myocytes after suffering ischemia reperfusion injury (P<0.05). The expression of Bcl-2 in tocilizumab treated group were higher than NC group (P<0.05), while the Bax expression were lower (P<0.05). Tocilizumab could significantly inhibit apoptosis and keep the proliferation viability of human cardiac myocytes after suffering ischemia reperfusion injury. Tocilizumab may obtain a widely application in the protection of ischemia reperfusion injury.

Chronic Hypoxia Increases Intracellular Ca(2+) Concentration via Enhanced Ca(2+) Entry Through Receptor-Operated Ca(2+) Channels in Pulmonary Venous Smooth Muscle Cells.

Hypoxic pulmonary hypertension (HPH) is characterized by pulmonary vascular remodeling. Intracellular Ca(2+)concentration ([Ca(2+)]i) is an essential signal for myocyte proliferation. Whether chronic hypoxia (CH) affects the basal [Ca(2+)]I and Ca(2+)entry through store- and/or receptor-operated calcium channels (SOCC, ROCC), and whether canonical transient receptor potential (TRPC) proteins are involved in CH-induced Ca(2+)influx and proliferation in pulmonary venous smooth muscle cells (PVSMCs) is examined. Rats were exposed to CH. PVSMCs were isolated from distal pulmonary veins. In freshly isolated PVSMCs, CH increased the basal [Ca(2+)]i; removal of Ca(2+)or application of SKF-96365 reversed the elevated [Ca(2+)]i, whereas nifedipine had no effect. Receptor-operated Ca(2+)entry (ROCE) was expressed in PVSMCs. In freshly isolated PVSMCs from CH rats, ROCE was enhanced, whereas store-operated Ca(2+)entry had no alteration. Furthermore, real-time polymerase chain reaction and western blotting showed that mRNA and protein expression level of TRPC6, but neither TRPC1 nor TRPC3, in pulmonary venous smooth muscle (PV) from CH rats and PVSMCs exposed to CH was greater than in normal PV and PVSMCs. The knockdown of TRPC6 in hypoxic PVSMCs with siRNA inhibited the enhanced ROCE and attenuated CH-induced PVSMCs proliferation. The enhanced Ca(2+)entry through ROCC, due to upregulated TRPC6, is a novel pathogenic mechanism contributing to the increased basal [Ca(2+)]iin PVSMCs and excessive PVSMC proliferation during the development of HPH.

Abstract 15610: The Infusion of Mesenchymal Stromal Cells Post Myocardial Infarction Increases Myocardial S100A6, Attenuates Myocyte Hypertrophy, Reduces Myocyte Apoptosis and Improves Cardiac Function

The beneficial effects originally attributed to the ability of bone-marrow derived mesenchymal stromal cells (BM-MSCs) to differentiate into cardiomyocytes have been questioned due to the transient presence of donor cells at injury site following myocardial infarction (MI) suggesting that the MSC-induced improvement in hemodynamic function may be attributable to paracrine effects. We showed that S100A6, a 20 kDa EF-hand calcium-binding dimer, is upregulated and secreted following MI and forced expression post-MI was beneficial to the preservation of cardiac function. The aim of this study was to determine whether the beneficial effects of infused BM-MSCs may be related to the autocrine secretion of S100A6. Balb/c murine cultured green fluorescence protein (GFP)-marked BM-MSCs express S100A6 at baseline and in response to hypoxia (5%C02/95% N2) for 1 hr increase S100A6 mRNA and protein (2-3 fold, and release S100A6 (1 nM) in the culture media, responses inhibited in BM-MSCs transfected with S100A6 siRNA. Treatment of neonatal Balb/c cardiac myocytes with human recombinant S100A6 (1nM) for 1-24 hrs attenuated baseline apoptosis (30 per cent decrease in BAX/BCL2 ratio), induced cyclin-dependent kinase 1(CDK1) mRNA 1.5 fold, miR199a 2 fold and myocyte proliferation 2.5 fold, the latter inhibited by anti-miR 199a. In 12 week old Balb/c mice, saline or GFP-marked BM-MSCs transfected with either a scrambled or S100A6 siRNA were infused intravenously 3-4 hrs post coronary artery ligation. After 3-4 days the GFP-marked cells were confined to ischemic areas and represented approximately 10% of total cellularity and co-expressed collagen type IV and myosin heavy chain, characteristic of MSCs and cardiomyocytes, respectively, and were CD45(-). Despite the absence of donor cells in the infarcted myocardium 21 days after infusion, mice that have received MSCs alone compared to MSCs transfected with an S100A6 siRNA or saline alone showed a 6-fold increase in S100A6 mRNA and protein, 3-fold increase in miR199a in peri-infarcted myocardium, attenuated myocyte hypertrophy, decreased fibrosis and apoptosis, and preservation of cardiac function. In conclusion, the secretion of S100A6 by infused BM-MSCs may contribute in limiting adverse LV remodeling post-MI.

Abstract 15656: Global Intracoronary Infusion of Allogeneic Cardiosphere-Derived Cells (CDCs) Immediately After Reperfusion Stimulates Myocyte Regeneration in Remote Viable Myocardium in Swine With Acute Myocardial Infarction

Introduction: Previous studies evaluating regenerative strategies have focused on administering stem cells to the infarct related artery. Unfortunately, this does not stimulate cardiac regeneration nor prevent myocyte loss in viable remote myocardium that becomes dysfunctional due to LV remodeling. Hypothesis: We hypothesized that administrating allogeneic CDCs to the entire heart (global CDC infusion) at the time of reperfusion could improve function, stimulate myocyte regeneration and prevent myocyte loss throughout the LV. Methods: Yorkshire swine (n=12) underwent a one hour LAD occlusion. Ejection fraction (EF) decreased from 63 ± 2% at baseline to 44 ± 2% after infarction (p&lt;0.05 vs. baseline). Immediately after reperfusion animals were randomized to allogeneic intracoronary CDCs or vehicle in a blinded fashion (n=6 each). Allogeneic CDCs (20 x 10^6 CDCs, cyclosporine 100 mg/day) were infused into the 3 major coronary distributions without interrupting flow. Four weeks later we assessed myocardial function (echo), myocyte proliferation (Ki67), infarct size (TTC), myocyte nuclear density and cell size in comparison to vehicle. Results: After 4 weeks, global CDC infusion increased wall thickening and ejection fraction despite the fact that infarct volume was not different (Table). CDCs stimulated myocyte proliferation (Ki67) in border and remote regions by 3-fold. As a result, myocyte number (nuclear density) increased by 34 % and cell size decreased by 7% in viable infarct border and remote regions. Conclusion: Global infusion of allogeneic CDCs immediately after reperfusion 1.) stimulates myocyte regeneration in viable myocardium remote from the infarct and 2.) improves regional and global LV function without altering infarct size. Thus, treating viable remote myocardium as well as the infarct related artery with allogeneic CDCs may afford a widely available approach to reverse LV dysfunction in patients with acute myocardial infarction.

Abstract 16788: Delivery of Pluripotent Stem Cell Specific Microrna-294 Induces Cardiomyocyte Proliferation Augmenting Cardiac Function After Myocardial Infarction

Rationale: Embryonic heart is characteristic of rapidly dividing cardiomyocytes that give rise to sufficient numbers required to build a working myocardium. In contrast, cardiomyocytes retain some proliferative capacity in the neonates but lose most of it in adulthood. Embryonic stem cell cycle (ESCC) miRs are a class of microRNAs regulating the unique cell cycle of ESCs and their characteristic pluripotency. Nevertheless, expression of miR-294, a member of the ESCC miRs is lost during developmental transitions from the ESCs to mature cells. Effect of miR-294 to induce cardiac proliferation and heart function has not been previously studied. Objective: To determine whether miR-294 drives cardiomyocyte cell cycle reentry leading to augmentation of cardiac function after myocardial infarction. Methods and Results: miR expression analysis was carried out in the heart during development to determine levels of miR-294. Elevated level of miR-294 was observed in the prenatal heart confirmed by qRT-PCR. In contrast, miR-294 expression is lost in the neonates and adult mice. To further assess the effect of miR-294, neonatal ventricular cardiomyocytes (NRVMs) were treated with miR-294 mimic to determine the effect on proliferation and cell cycle. Elevated mRNA levels of cyclins A2 and E1 together with CDK2 was observed in NRVMs treated with 25nM mimic for miR-294. Increased expression of p-histone 3, PCNA (S-phase), Aurora B kinase (G2/M) and Ki67 was confirmed by immunocytochemistry in NRVMs after miR-294 treatment compared to control cells. Intramyocardial administration of miR-294 was carried out in mice at the time of myocardial infarction to determine whether miR-294 delivery increases cardiomyocyte proliferation and survival in response to injury. Enhance cardiac function was observed in mice receiving miR-294 3 weeks after myocardial infarction. Concurrently, increase myocyte proliferation was observed in the heart after miR-294 treatment as analyzed by BrdU uptake, PCNA and Aurora B expression by immunostaining. Conclusion: Ectopic expression of miR-294 recapitulates embryonic signaling and enhances cardiomyocyte ability to proliferate and reenter the cell cycle leading to augmented cardiac function in mice after myocardial infarction.

Global intracoronary infusion of allogeneic cardiosphere-derived cells improves ventricular function and stimulates endogenous myocyte regeneration throughout the heart in swine with hibernating myocardium.

BackgroundCardiosphere-derived cells (CDCs) improve ventricular function and reduce fibrotic volume when administered via an infarct-related artery using the “stop-flow” technique. Unfortunately, myocyte loss and dysfunction occur globally in many patients with ischemic and non-ischemic cardiomyopathy, necessitating an approach to distribute CDCs throughout the entire heart. We therefore determined whether global intracoronary infusion of CDCs under continuous flow improves contractile function and stimulates new myocyte formation.Methods and ResultsSwine with hibernating myocardium from a chronic LAD occlusion were studied 3-months after instrumentation (n = 25). CDCs isolated from myocardial biopsies were infused into each major coronary artery (∼33×106 icCDCs). Global icCDC infusion was safe and while ∼3% of injected CDCs were retained, they did not affect ventricular function or myocyte proliferation in normal animals. In contrast, four-weeks after icCDCs were administered to animals with hibernating myocardium, %LADWT increased from 23±6 to 51±5% (p<0.01). In diseased hearts, myocyte proliferation (phospho-histone-H3) increased in hibernating and remote regions with a concomitant increase in myocyte nuclear density. These effects were accompanied by reductions in myocyte diameter consistent with new myocyte formation. Only rare myocytes arose from sex-mismatched donor CDCs.ConclusionsGlobal icCDC infusion under continuous flow is feasible and improves contractile function, regresses myocyte cellular hypertrophy and increases myocyte proliferation in diseased but not normal hearts. New myocytes arising via differentiation of injected cells are rare, implicating stimulation of endogenous myocyte regeneration as the primary mechanism of repair.

FGF10 promotes regional foetal cardiomyocyte proliferation and adult cardiomyocyte cell-cycle re-entry.

Cardiomyocyte proliferation gradually declines during embryogenesis resulting in severely limited regenerative capacities in the adult heart. Understanding the developmental processes controlling cardiomyocyte proliferation may thus identify new therapeutic targets to modulate the cell-cycle activity of cardiomyocytes in the adult heart. This study aims to determine the mechanism by which fibroblast growth factor 10 (FGF10) controls foetal cardiomyocyte proliferation and to test the hypothesis that FGF10 promotes the proliferative capacity of adult cardiomyocytes. Analysis of Fgf10(-/-) hearts and primary cardiomyocyte cultures reveals that altered ventricular morphology is associated with impaired proliferation of right but not left-ventricular myocytes. Decreased FOXO3 phosphorylation associated with up-regulated p27(kip) (1) levels was observed specifically in the right ventricle of Fgf10(-/-) hearts. In addition, cell-type-specific expression analysis revealed that Fgf10 and its receptor, Fgfr2b, are expressed in cardiomyocytes and not cardiac fibroblasts, consistent with a cell-type autonomous role of FGF10 in regulating regional specific myocyte proliferation in the foetal heart. Furthermore, we demonstrate that in vivo overexpression of Fgf10 in adult mice promotes cardiomyocyte but not cardiac fibroblast cell-cycle re-entry. FGF10 regulates regional cardiomyocyte proliferation in the foetal heart through a FOXO3/p27(kip1) pathway. In addition, FGF10 triggers cell-cycle re-entry of adult cardiomyocytes and is thus a potential target for cardiac repair.