Adult Cardiomyocyte Proliferation Research Articles

Tead1 is required for perinatal cardiomyocyte proliferation.

Adult heart size is determined predominantly by the cardiomyocyte number and size. The cardiomyocyte number is determined primarily in the embryonic and perinatal period, as adult cardiomyocyte proliferation is restricted in comparison to that seen during the perinatal period. Recent evidence has implicated the mammalian Hippo kinase pathway as being critical in cardiomyocyte proliferation. Though the transcription factor, Tead1, is the canonical downstream transcriptional factor of the hippo kinase pathway in cardiomyocytes, the specific role of Tead1 in cardiomyocyte proliferation in the perinatal period has not been determined. Here, we report the generation of a cardiomyocyte specific perinatal deletion of Tead1, using Myh6-Cre deletor mice (Tead1-cKO). Perinatal Tead1 deletion was lethal by postnatal day 9 in Tead1-cKO mice due to dilated cardiomyopathy. Tead1-deficient cardiomyocytes have significantly decreased proliferation during the immediate postnatal period, when proliferation rate is normally high. Deletion of Tead1 in HL-1 cardiac cell line confirmed that cell-autonomous Tead1 function is required for normal cardiomyocyte proliferation. This was secondary to significant decrease in levels of many proteins, in vivo, that normally promote cell cycle in cardiomyocytes. Taken together this demonstrates the non-redundant critical requirement for Tead1 in regulating cell cycle proteins and proliferation in cardiomyocytes in the perinatal heart.

Molecular switch model for cardiomyocyte proliferation.

This review deals with the human adult cardiomyocyte proliferation as a potential source for heart repair after injury. The mechanism to regain the proliferative capacity of adult cardiomyocytes is a challenge. However, recent studies are promising in showing that the ‘locked’ cell cycle of adult cardiomyocytes could be released through modulation of cell cycle checkpoints. In support of this are the signaling pathways of Notch, Hippo, Wnt, Akt and Jak/Stat that facilitate or inhibit the transition at cell cycle checkpoints. Cyclins and cyclin dependant kinases (CDKs) facilitate this transition which in turn is regulated by inhibitory action of pocket protein e.g. p21, p27 and p57. Transcription factors e.g. E2F, GATA4, TBx20 up regulate Cyclin A, A2, D, E, and CDK4 as promoters of cell cycle and Meis-1 and HIF-1 alpha down regulate cyclin D and E to inhibit the cell cycle. Paracrine factors like Neuregulin-1, IGF-1 and Oncostatin M and Extracellular Matrix proteins like Agrin have been involved in cardiomyocyte proliferation and dedifferentiation processes.A molecular switch model is proposed that transforms the post mitotic cell into an actively dividing cell. This model shows how the cell cycle is regulated through on- and off switch mechanisms through interaction of transcription factors and signaling pathways with proteins of the cell cycle checkpoints. Signals triggered by injury may activate the right combination of the various pathways that can ‘switch on’ the proliferation signals leading to myocardial regeneration.

A conserved HH-Gli1-Mycn network regulates heart regeneration from newt to human

The mammalian heart has a limited regenerative capacity and typically progresses to heart failure following injury. Here, we defined a hedgehog (HH)-Gli1-Mycn network for cardiomyocyte proliferation and heart regeneration from amphibians to mammals. Using a genome-wide screen, we verified that HH signaling was essential for heart regeneration in the injured newt. Next, pharmacological and genetic loss- and gain-of-function of HH signaling demonstrated the essential requirement for HH signaling in the neonatal, adolescent, and adult mouse heart regeneration, and in the proliferation of hiPSC-derived cardiomyocytes. Fate-mapping and molecular biological studies revealed that HH signaling, via a HH-Gli1-Mycn network, contributed to heart regeneration by inducing proliferation of pre-existing cardiomyocytes and not by de novo cardiomyogenesis. Further, Mycn mRNA transfection experiments recapitulated the effects of HH signaling and promoted adult cardiomyocyte proliferation. These studies defined an evolutionarily conserved function of HH signaling that may serve as a platform for human regenerative therapies.

MicroRNAs regulating Meis1 expression and inducing cardiomyocyte proliferation

Cardiovascular disease has been the biggest killer in the United States for decades, with almost a million new cases each year. Even though mammalian rodent neonatal cardiomyocytes show proliferative potential for up to 5 days, adult cardiomyocytes lose this ability. Insufficient cardiomyocyte proliferation is one of the major reasons for the lack of regeneration of myocardial tissue, post injury. Several studies have looked at the mechanisms responsible for the arrest in proliferation at an adult stage. Following up on a recent study by Eulalio et al's study on functional screening of 875 miRNAs for neonatal cardiomyocyte proliferation, we recently identified several miRNAs that induce proliferation in naturally senescent adult cardiomyocytes. Additional studies by Mahmood et al 2013 have identified Meis1 as the major regulator of cardiomyocyte cell cycle. In our present study we have identified three of the adult cardiomyocyte proliferation inducing miRNAs to have binding sites on the 3'UTR of Meis1 gene by in-silico analysis and luciferase assay. Additionally we found these miRNAs; miR-548c-3p, miR-509-3p, and miR-23b-3p to induce significant proliferation in adult cardiomyocytes through translational inhibition of Meis1. We found a significant increase in the number of ACMs with each miRNA, in combination, and with siRNA mediated inhibition of Meis1 gene. We confirmed that these microRNAs, through inhibition of Meis1, affect its downstream targets and thereby regulate cell-cycle progression. Further investigating of the mechanism of action of these miRNAs can identify other treatment options for abnormalities associated with the lack of cardiac regeneration post myocardial injury.

FGF10: A potential target for heart regeneration

In mammals, cardiomyocyte proliferation decreases rapidly after birth resulting in limited regenerative capacities in the adult heart. Deciphering developmental mechanisms controlling cardiomyocyte proliferation may thus identify new therapeutic targets to promote adult cardiomyocyte cell cycle re-entry and therefore, cardiac regeneration. We recently uncovered a role for the Fibroblast Growth Factor 10 (FGF10) signalling in regulating both fetal and adult cardiomyocyte proliferation. Our study aims to characterize the role of FGF10 in pathological conditions and to determine the relevance of FGF10 as a potential target for heart regeneration. We used an experimental mouse model of Myocardial Infarction (MI), together with FGF10 gain and loss of function mouse models. We first showed that MI leads to increased FGF10 expression levels in cardiomyocytes of the injured ventricle suggesting a potential role for FGF10 in pathological conditions. Adult transgenic mice with reduced FGF10 expression were subjected to MI. Our results revealed that while reduced FGF10 expression has no impact on cardiomyocyte hypertrophy and apoptosis, it significantly impairs cardiomyocyte proliferative capacities and exacerbates cardiac fibrosis infiltration post-MI. Moreover, reduced FGF10 expression leads to a worsened cardiac function and remodelling post-MI, strongly demonstrating the protective role of FGF10 in pathological conditions. To evaluate FGF10 regenerative capacities, conditional overexpression of FGF10 was achieved in adult mice subjected to MI. By promoting cardiomyocyte proliferation and preventing cardiac fibrosis infiltration, increased FGF10 expression levels post-MI preserves cardiac remodelling and function. Altogether, this study thus identifies FGF10 as a potential target to improve the limited innate regenerative capacities of the myocardium after injury, of direct clinical relevance for heart regeneration.

Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration

Human diseases are often caused by loss of somatic cells that are incapable of re-entering the cell cycle for regenerative repair. Here, we report a combination of cell-cycle regulators that induce stable cytokinesis in adult post-mitotic cells. We screened cell-cycle regulators expressed in proliferating fetal cardiomyocytes and found that overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 efficiently induced cell division in post-mitotic mouse, rat, and human cardiomyocytes. Overexpression of the cell-cycle regulators was self-limiting through proteasome-mediated degradation of the protein products. Invivo lineage tracing revealed that 15%-20% of adult cardiomyocytes expressing the four factors underwent stable cell division, with significant improvement in cardiac function after acute or subacute myocardial infarction. Chemical inhibition of Tgf-β and Wee1 made CDK1 and cyclin B dispensable. These findings reveal a discrete combination of genes that can efficiently unlock the proliferative potential in cells that have terminally exited the cell cycle.

GW28-e1093 Features of Adult Mammalian Cardiomyocyte Proliferation with Complete Cytokinesis

GW28-e1093 Features of Adult Mammalian Cardiomyocyte Proliferation with Complete Cytokinesis

Hippo pathway deficiency reverses systolic heart failure after infarction.

Mammalian organs vary widely in regenerative capacity. Poorly regenerative organs, such as the heart are particularly vulnerable to organ failure. Once established, heart failure (HF) commonly results in mortality1. The Hippo pathway, a kinase cascade that prevents adult cardiomyocyte proliferation and regeneration2, is upregulated in human HF. We show that deletion of the Hippo pathway component Salvador (Salv) in mouse hearts with established ischemic HF after myocardial infarction (MI) induced a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function compared to controls. Using TRAP (translating ribosomal affinity purification), we isolated cardiomyocyte specific translating mRNA. Hippo deficient cardiomyocytes had increased expression of proliferative genes and stress response genes, such as the mitochondrial quality control (MQC) gene, Park2. Genetic studies indicated that Park2 was essential for heart repair suggesting a requirement for MQC in regenerating myocardium. Gene therapy with a virus encoding Salv shRNA improved heart function when delivered at the time of infarct or after ischemic HF post-MI was established. Our findings indicate that the failing heart has a previously unrecognized reparative capacity involving more than cardiomyocyte renewal.

Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury.

Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. β-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size. In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca2+-dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia-resistant connexin 43 mutant enhanced the redifferentiation of ACM-derived new cardiomyocytes after MI and improved cardiac function. Mature ACMs can reenter the cell cycle and form new cardiomyocytes through a 3-step process: dedifferentiation, proliferation, and redifferentiation. Intercellular Ca2+ signal from neighboring functioning cardiomyocytes through gap junctions induces the redifferentiation process. This novel mechanism contributes to new cardiomyocyte formation in post-MI hearts in mammals.

Live cell screening platform identifies PPAR\u03b4 as a regulator of cardiomyocyte proliferation and cardiac repair

Zebrafish can efficiently regenerate their heart through cardiomyocyte proliferation. In contrast, mammalian cardiomyocytes stop proliferating shortly after birth, limiting the regenerative capacity of the postnatal mammalian heart. Therefore, if the endogenous potential of postnatal cardiomyocyte proliferation could be enhanced, it could offer a promising future therapy for heart failure patients. Here, we set out to systematically identify small molecules triggering postnatal cardiomyocyte proliferation. By screening chemical compound libraries utilizing a Fucci-based system for assessing cell cycle stages, we identified carbacyclin as an inducer of postnatal cardiomyocyte proliferation. In vitro, carbacyclin induced proliferation of neonatal and adult mononuclear rat cardiomyocytes via a peroxisome proliferator-activated receptor δ (PPARδ)/PDK1/p308Akt/GSK3β/β-catenin pathway. Inhibition of PPARδ reduced cardiomyocyte proliferation during zebrafish heart regeneration. Notably, inducible cardiomyocyte-specific overexpression of constitutively active PPARδ as well as treatment with PPARδ agonist after myocardial infarction in mice induced cell cycle progression in cardiomyocytes, reduced scarring, and improved cardiac function. Collectively, we established a cardiomyocyte proliferation screening system and present a new drugable target with promise for the treatment of cardiac pathologies caused by cardiomyocyte loss.

On the Problem of Cardiogenic Differentiation: Extracellular Matrix as an Emerging Clue

It is well known that myocardium fails to regenerate due to the lack of adult cardiomyocyte proliferation. In this regard, a lot of methods for cardiogenic differentiation of different cells are currently developed to provide an alternative source of functionally active cardiomyocytes, including embryonic stem cells, cardiac stem cells, induced pluripotent stem cells and fibroblasts. However, the approaches available show quite a poor result in the efficiency as well as estimation criteria of cardiac differentiation. Many studies are intended to reveal the fundamental mechanisms of cardiogenesis to mimic them in vitro, but significant advances in studying of intracellular signaling pathways during heart development are insufficient to produce high efficient methods. Extracellular matrix is another component which is becoming acknowledged as a key player in heart differentiation. However, investigation of its spatiotemporal effects on cardiomyocyte maturation is limited due to the challenges in cell-matrix handling within the heart tissue. We consider that cardiomyocyte primary culture may be a good model for studying the role of extracellular matrix in cardiogenesis, being an easy to manipulate homogenous culture of cardiomyocytes, which synchronically follow rearrangements similar to their embryonic developmental stages. The better understanding of the cell-matrix interactions underlying cardiomyocyte maturation may bring researchers closer to the efficient cardiogenic differentiation.

Redox Paradox: Can Hypoxia Heal Ischemic Hearts?

Adult cardiomyocytes are largely thought to lack proliferative and therefore regenerative potential. Reporting in Nature, Nakada etal. (2016) find that a hypoxic regime reduces mitochondrial metabolism and promotes proliferation in adult mouse cardiomyocytes, resulting in increased regeneration following myocardial infarction. These findings suggest the potential to transform post-MI care.

Towards regenerating the mammalian heart: challenges in evaluating experimentally induced adult mammalian cardiomyocyte proliferation

In recent years, there has been a dramatic increase in research aimed at regenerating the mammalian heart by promoting endogenous cardiomyocyte proliferation. Despite many encouraging successes, it remains unclear if we are any closer to achieving levels of mammalian cardiomyocyte proliferation for regeneration as seen during zebrafish regeneration. Furthermore, current cardiac regenerative approaches do not clarify whether the induced cardiomyocyte proliferation is an epiphenomena or responsible for the observed improvement in cardiac function. Moreover, due to the lack of standardized protocols to determine cardiomyocyte proliferation in vivo, it remains unclear if one mammalian regenerative factor is more effective than another. Here, we discuss current methods to identify and evaluate factors for the induction of cardiomyocyte proliferation and challenges therein. Addressing challenges in evaluating adult cardiomyocyte proliferation will assist in determining 1) which regenerative factors should be pursued in large animal studies; 2) if a particular level of cell cycle regulation presents a better therapeutic target than another (e.g., mitogenic receptors vs. cyclins); and 3) which combinatorial approaches offer the greatest likelihood of success. As more and more regenerative studies come to pass, progress will require a system that not only can evaluate efficacy in an objective manner but can also consolidate observations in a meaningful way.

Overexpression of Tbx20 in Adult Cardiomyocytes Promotes Proliferation and Improves Cardiac Function After Myocardial Infarction.

Adult mammalian cardiomyocytes (CMs) have the potential to proliferate, but this is not sufficient to generate adequate CMs after myocardial infarction (MI). The transcription factor Tbx20 is required for CM proliferation during development and adult CM homeostasis. The ability of Tbx20 overexpression (Tbx20(OE)) to promote adult CM proliferation and to improve cardiac function after MI was examined. Tbx20(OE) was induced specifically in adult mouse differentiated CMs. Increased CM proliferation and fetal-like characteristics were found in Tbx20(OE) hearts compared with controls without causing pathology 4 weeks after Tbx20(OE) at baseline. Moreover, Tbx20(OE) in adult CM after MI significantly improved survival, cardiac function, and infarct size 4 weeks after MI. Improved cardiac repair, as indicated by increased CM proliferation and capillary density, was observed in the MI border zone of Tbx20(OE) hearts compared with controls. Expression of proliferation activator (cyclin D1, E1, and IGF1) and fetal contractile protein (ssTNI, βMHC) mRNA was increased whereas negative cell-cycle regulators (p21, Meis1) were decreased in Tbx20(OE) hearts compared with controls under both baseline and MI conditions. Tbx20(OE) in adult hearts activates multiple proproliferation pathways, including Akt, YAP and BMP. Interestingly, p21, Meis1, and a novel cell-cycle inhibitory gene, Btg2, are directly bound and repressed by Tbx20 with induction of proliferation in neonatal CM. Tbx20(OE), specifically in adult CM, activates multiple cardiac proliferative pathways, directly represses cell-cycle inhibitory genes p21, Meis1, and Btg2, promotes adult CM proliferation; and preserves cardiac performance after MI.

Genetic and Epigenetic Regulation of Human Cardiac Reprogramming and Differentiation in Regenerative Medicine.

Regeneration or replacement of lost cardiomyocytes within the heart has the potential to revolutionize cardiovascular medicine. Numerous methodologies have been used to achieve this aim, including the engraftment of bone marrow- and heart-derived cells as well as the identification of modulators of adult cardiomyocyte proliferation. Recently, the conversion of human somatic cells into induced pluripotent stem cells and induced cardiomyocyte-like cells has transformed potential approaches toward this goal, and the engraftment of cardiac progenitors derived from human embryonic stem cells into patients is now feasible. Here we review recent advances in our understanding of the genetic and epigenetic control of human cardiogenesis, cardiac differentiation, and the induced reprogramming of somatic cells to cardiomyocytes. We also cover genetic programs for inducing the proliferation of endogenous cardiomyocytes and discuss the genetic state of cells used in cardiac regenerative medicine.

Abstract 17262: MicroRNA-23b Mediated Inhibition of Gsk-3β and Nischarin Promotes Proliferation of Cardiomyocytes

Background: Promoting proliferation of cardiomyocytes (CMs) following myocardial infarction is a promising approach for cardiac regeneration. Recent studies from our lab identified a panel of microRNAs inducing proliferation of adult CMs; of these miR-23b was one of the most robust CM proliferation-inducing miR and is conserved across species. In silico analysis identified binding sites for miR-23b on the 3’UTRs of both GSK-3β (GSK) and nischarin (NISCH). Here, we hypothesize that miR-23b promotes both in vitro and in vivo proliferation of CMs by regulating the expression of cell cycle inhibitors GSK and NISCH. Methods and Results: Adult CMs isolated from 8-12 week old female rats were transfected with hsa-miR-23b, cel-miR-67 (control), siGSK, siNISCH, siGSK + siNISCH (si=siRNA). EdU was added (5 μM) each day post-transfection and cells were fixed on day 6 for IF staining (Troponin-I, EdU, pH3, and DAPI), protein, and RNA analysis. A significant increase in EdU+ CMs was found in cells transfected with miR-23b (8.51%±0.78), siGSK (8.30%±1.31) and siNISCH (5.73%±1.52) when compared to control miR cel-67 (3.13%±0.84). Interestingly, simultaneous inhibition of both GSK and NISCH exhibited robust proliferation (13.88%±1.15) similar to miR-23b transfection. Luciferase assay confirmed binding sites for miR-23b on the 3’UTR of GSK with a 0.85±0.04 fold reduction of chemiluminescent activity. Compared to control, western blotting following miR-23b transfection revealed an increase in pERK (2.46±0.69 fold), a decrease in GSK (0.18±0.12 fold), and an increase in β-catenin (4.17±2.03 fold), indicating miR-23b mediated inhibition of GSK and subsequent increase in β-catenin. For in vivo analysis, 6 day old Fischer rats received intraperitoneal adenoviral miR-23b (10^11 viral particles) followed by EdU injections on alternate days. On day 6, rats were sacrificed, and the hearts were fixed/sectioned for IF staining. Rats injected with miR-23b expressed higher levels of EdU (2.42±0.46 fold), and decreased fold of GSK (0.32±0.10) and NISCH (0.29±0.17) compared to control as confirmed via IHC staining. Conclusion: MiR-23b promotes proliferation of CMs by simultaneous inhibition of both GSK and NISCH and upregulation of downstream targets pERK and β-catenin.

Abstract 17229: Synergistic Inhibition of Retinoblastoma1 and Meis2 Promotes Robust Proliferation of Adult Cardiomyocytes

Background: Cardiac remodeling and heart failure post myocardial Infarction (MI) is mainly due to inadequate regenerative capacity of mammalian heart, which is attributed to limited proliferative potential of adult cardiomyocytes (ACMs). In our recent study, we identified miR-1825 as a master regulator, which induces ACM proliferation through recruiting other proliferation inducing miRs, including miR-199a. In this study, we hypothesize that miR-1825 promotes proliferation through miR-199a mediated inhibition of cell cycle inhibitors Retinoblastoma1 (Rb1) and Meis2. Methods and Results: ACMs were isolated from 8-12 week old male rats and were transfected with cel-miR-67 (control), miR-1825, miR-199a, siRNA against Rb1 and Meis2 both individually and in combination (siRb1, siMeis2, siRb1+siMeis2). To label proliferating ACMs, EdU (5 μM) was added each day for 5 days and cells were harvested/fixed for staining, protein, and RNA analysis. Immuno-staining was performed for EdU, pH3 (mitotic marker), and Troponin-I. Similar to our previous reports, miR-1825 induced 23±0.4% and miR-199a induced 14.8±0.5% proliferation, as shown by EdU+ ACMs. In addition, we identified siRNA mediated silencing of Rb1 and Meis2 promoted 8.7±2.8% and 3.8±1.4% increase in proliferation, whereas simultaneous inhibition of both Rb1 and Meis2 induced robust (23±5.0%) proliferation of ACMs. Mitosis, as identified by p-H3+ACMs, following individual and combined silencing of Rb1 and Meis2 was observed to be 2.6%, 2.2%, and 7.6%, respectively. Western blotting confirmed silencing of Meis1/2 and Rb1 (0.14±0.02 fold) in miR-1825 transfected ACMs. Computational prediction and luciferase assay revealed a direct binding site for miR-199a on the 3’UTRs of both Rb1 and Meis2. Following miR-199a transfection, we found a 0.29±0.06 fold and 0.09±0.07 fold reduction of chemiluminescent activity with Rb1-3’UTR and Meis2-3’UTR, respectively. In addition, binding sites for miR-199a on both Rb1-3’UTR and Meis2-3’UTR are conserved across species as indicated by in-silico analysis. Conclusion: miR-1825 and miR-199a mediated simultaneous silencing of cell cycle inhibitors Rb1 and Meis2 promotes robust proliferation of ACMs. This can be used to regenerate ischemic myocardium.

Abstract 12497: Regulation of Mitochondrial Biogenesis and Oxidative Phosphorylation by miR-1825 Promotes Proliferation and Survival of Quiescent Adult Cardiomyocytes

Background: Generation of reactive oxygen species (ROS) by mitochondrial oxidative phosphorylation is known to induce DNA Damage Response (DDR) pathway resulting in cell cycle arrest and apoptosis in a number of cell types including Adult Cardiomyocytes (ACMs). In our recent studies, we have identified miR-1825 to be the most robust ACM proliferation inducing miR. Here we hypothesize that miR-1825 promotes proliferation by regulating mitochondrial biogenesis and ROS production. Methods and Results: ACMs isolated from 10 week old male rats were transfected with hsa-miR-1825 and cel-miR-67 (control). EdU was added (5μM) each day for 6 days and cells were used for immuno-staining, RNA and protein analysis. In addition to EdU, TnI, and DAPI staining, Dihydro-rhodamine 123 was used as a marker for mitochondrial ROS production, VDAC immuno-staining was used to detect mitochondrial number. We identified 25% less mitochondrial ROS production in ACMs transfected with miR-1825. Moreover, when ACMs were administered H2O2 (10μM), along with miR-1825 a significant decrease in apoptosis was detected (TUNEL+ ACM 0.53±0.10 fold less than control). In addition to reduction of mitochondrial ROS, miR-1825 transfected ACMs also showed a significant reduction in total ROS. VDAC immuno-staining showed over 85% decrease in mitochondrial number in miR-1825 transfected ACMs compared to control. Our whole transcriptome micro-array analysis of ACMs transfected with miR-1825 identified down regulation of genes involved in oxidative phosphorylation and TCA cycle; while cell-cycle genes like Cyclin-D and Cyclin-B were up regulated. Our RNA sequencing data and in-silico analysis reveal miR-1825 regulates mitochondrial biogenesis indirectly through up-regulating miR-199a expression and by subsequent down regulation of miR-199a target SIRTUIN1 which is a known inhibitor of PGC-1α and mitochondrial biogenesis. Conclusion: We identified that the increase in proliferation of ACMs observed with miR-1825 is through miR-199a mediated inhibition of PGC-1α expression which inhibits mitochondrial biogenesis and ROS production. This is a novel mechanism and has translational potential for inducing proliferation of ACM post myocardial infarction.

Cardiomyocyte proliferation in cardiac development and regeneration: a guide to methodologies and interpretations.

The newt and the zebrafish have the ability to regenerate many of their tissues and organs including the heart. Thus, a major goal in experimental medicine is to elucidate the molecular mechanisms underlying the regenerative capacity of these species. A wide variety of experiments have demonstrated that naturally occurring heart regeneration relies on cardiomyocyte proliferation. Thus, major efforts have been invested to induce proliferation of mammalian cardiomyocytes in order to improve cardiac function after injury or to protect the heart from further functional deterioration. In this review, we describe and analyze methods currently used to evaluate cardiomyocyte proliferation. In addition, we summarize the literature on naturally occurring heart regeneration. Our analysis highlights that newt and zebrafish heart regeneration relies on factors that are also utilized in cardiomyocyte proliferation during mammalian fetal development. Most of these factors have, however, failed to induce adult mammalian cardiomyocyte proliferation. Finally, our analysis of mammalian neonatal heart regeneration indicates experiments that could resolve conflicting results in the literature, such as binucleation assays and clonal analysis. Collectively, cardiac regeneration based on cardiomyocyte proliferation is a promising approach for improving adult human cardiac function after injury, but it is important to elucidate the mechanisms arresting mammalian cardiomyocyte proliferation after birth and to utilize better assays to determine formation of new muscle mass.

Abstract 8: Reprogramming of Adult Mammalian Cardiomyocytes Towards Proliferation and Regeneration Through Comparative Analyses of Epicardial and Myocardial Cells

Heart development and regeneration require an elegant balance of cell proliferation and differentiation. In both adult zebrafish and neonatal mouse cardiac regeneration, new cardiomyocytes are shown to originate from pre-existing cardiomyocytes through de-differentiation of mature cells to the immature progenitor cell state, with developmental gene program reactivation and cell cycle reentry. Interestingly, after adult mammalian cardiac injury, both cardiac muscle cells and the epicardial cells that envelope the heart reinitiate developmental gene programs. However, adult epicardial cells are able to proliferate following injury but adult cardiomyocytes are not capable. We investigated the genetic circuitry of mouse epicardial cells and myocardial cells in different developmental stages and in response to injury. Such comparative analyses revealed a group of ~50 candidate genes that may be responsible for the permanent cell cycle arrest of cardiomyocytes. We generated adenoviruses that express these candidate genes individually, and demonstrated that the mixed viral pool possessed a robust activity to promote proliferation of adult mouse cardiomyocytes. Our comparative approach and functional screens may lead to identification of the dormant genetic circuitry in adult mammalian heart that can be reactivated to drive robust cardiomyocyte proliferation and regeneration.