Cell Cycle–Mediated Cardiac Regeneration in the Mouse Heart

AbstractRationaleMammals lose the ability to regenerate their hearts within one week after birth. During this regenerative window, cardiac energy metabolism shifts from glycolysis to fatty acid oxidation, and recent evidence suggests that metabolism may participate in controlling cardiomyocyte cell cycle. However, the molecular mechanisms mediating the loss of postnatal cardiac regeneration are not fully understood.ObjectiveThis study aims at providing an integrated resource of mRNA, protein and metabolite changes in the neonatal heart to identify metabolism-related mechanisms associated with the postnatal loss of regenerative capacity.Methods and ResultsMouse ventricular tissue samples taken on postnatal days 1, 4, 9 and 23 (P01, P04, P09 and P23, respectively) were analyzed with RNA sequencing (RNAseq) and global proteomics and metabolomics. Differential expression was observed for 8547 mRNAs and for 1199 of the 2285 quantified proteins. Furthermore, 151 metabolites with significant changes were identified. Gene ontology analysis, KEGG pathway analysis and fuzzy c-means clustering were used to identify biological processes and metabolic pathways either up- or downregulated on all three levels. Among these were branched chain amino acid degradation (upregulated at P23) and production of free saturated and monounsaturated medium- to long-chain fatty acids (upregulated at P04 and P09; downregulated at P23). Moreover, the HMG-CoA synthase (HMGCS)-mediated mevalonate pathway and ketogenesis were transiently activated. Pharmacological inhibition of HMGCS in primary neonatal rat ventricular cardiomyocytes reduced the percentage of BrdU+ cardiomyocytes, providing evidence that the mevalonate and ketogenesis routes may participate in regulating cardiomyocyte cell cycle.ConclusionsThis is the first systems-level resource combining data from genome-wide transcriptomics with global quantitative proteomics and untargeted metabolomics analyses of the mouse heart throughout the early postnatal period. This integrated multi-level data of molecular changes associated with the loss of cardiac regeneration may open up new possibilities for the development of regenerative therapies.

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Abstract P325: The Role Of Runx1 In Cardiomyocyte Cell Cycle And Ploidy

Circulation Research ◽

10.1161/res.129.suppl_1.p325 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Samantha K Swift ◽

Michaela Patterson

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Heart Development ◽

Cardiac Injury ◽

Cardiac Regeneration ◽

Cardiac Conduction ◽

Calcium Handling ◽

Mouse Heart ◽

Disease States ◽

Eccentric Hypertrophy

Adult mammalian cardiomyocytes (CMs) are thought to be post-mitotic and therefore unable to regenerate the myocardium after injury. In recent years various studies have shown that the adult mammalian CM is capable of a small amount of proliferation, potentially restricted to a subset of CMs. One such study demonstrated that having greater percentages of the rare mononuclear diploid cardiomyocyte (MNDCM) is associated with improved outcomes after myocardial infarction (MI). An accompanying genome-wide association analysis identified genetic loci associated with the frequency of the MNDCM population. One candidate to come out of this screen was Runx1. Concurrently, RUNX1 captured the attention of cardiac regeneration researchers due to its increased presence in disease states, with some suggesting it may be a marker for dedifferentiation (fetal gene induction). One recent study demonstrated improved calcium handling and decreased eccentric hypertrophy following RUNX1 ablation after injury, perhaps corroborating the idea that RUNX1 is involved with CM dedifferentiation. We hypothesize that Runx1 influences dedifferentiation in CMs, impacting ploidy, as well as CM cell cycle activity and post-MI outcomes. We found that CM-specific overexpression (OE) of Runx1 results in a doubling of the MNDCM population, thereby validating its influence on the population. Via multiple contexts including postnatal development and adult injury, knocking out Runx1 decreases DNA synthesis while Runx1 OE increases DNA synthesis. Furthermore, an initial analysis of RNAseq data demonstrates that RUNX1 OE in a neonatal mouse hearts demonstrated differential expression in genes related to cardiac conduction, contraction, heart development, regeneration, and regulation of cell differentiation . After MI in the adult mouse heart, the effects of Runx1 OE resulted in transient benefits which included increased cell cycle activity and preservation of function. These data suggest that Runx1 is not simply a marker of CM dedifferentiation, but also a regulator of the process including cell cycle activation. Ongoing work will tease apart this role in more detail and could establish RUNX1 as a prominent therapeutic target for mitigating effects of cardiac injury.

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Abstract 17185: Sympathetic Reinnervation is Required for Mammalian Cardiac Regeneration

Circulation ◽

10.1161/circ.132.suppl_3.17185 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Ian A White ◽

Julie Gordon ◽

Wayne Balkan ◽

Joshua M Hare

Keyword(s):

Sympathetic Innervation ◽

Cardiac Regeneration ◽

Ventricular Myocardium ◽

Left Ventricular ◽

Neonatal Mouse ◽

Left Ventricular Myocardium ◽

Autonomic Nerves ◽

Mouse Heart ◽

Minimal Scarring ◽

Age Related

Rationale: Established animal models of tissue and limb regeneration demonstrate a critical dependence on concurrent reinnervation by the peripheral nervous system. The abundance of autonomic nerves in the mammalian heart suggests they play a similar role in the response to cardiac injury. Objective: To test the hypothesis that reinnervation is required for innate neonatal cardiac regeneration. Methods and Results: Crossing Wnt1:cre transgenic mice with a double-tandem (td) tomato reporter strain identified all neural crest-derived cell lineages including the peripheral autonomic nerves in the heart. Whole mount epi-fluorescence microscopy facilitated the clear resolution of subepicardial autonomic nerves in the mouse ventricles providing unprecedented detail of the subepicardial neuroanatomy of the mouse heart. We confirmed that sympathetic nerve structures envelop the entire heart, and importantly, exhibit robust re-growth into the regenerating myocardium following resection of the left ventricular apex in neonatal mice. While innervated hearts regenerate with minimal scarring to the left ventricular myocardium, we report that innate cardiac regeneration was inhibited following sympathectomy, as determined by cross-sectional percentage of viable LV myocardium (n=9, 0.87±1.4% vs. n=6, 14.05±4.4% ; p<0.01). Conclusions: Ablation of post-ganglionic sympathetic nerves blocks the innate regenerative capacity of neonatal mouse hearts. Therefore, the innate ability of the neonatal mouse heart to undergo regeneration in response to injury is dependent on sympathetic innervation of the ventricular myocardium. This finding has significant implications for adult regeneration following myocardial infarction where nerve growth is hindered by age related influences and scar tissue.

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Hypoxia-induced myocardial regeneration

Journal of Applied Physiology ◽

10.1152/japplphysiol.00328.2017 ◽

2017 ◽

Vol 123 (6) ◽

pp. 1676-1681 ◽

Cited By ~ 15

Author(s):

Wataru Kimura ◽

Yuji Nakada ◽

Hesham A. Sadek

Keyword(s):

Oxidative Stress ◽

Cell Cycle ◽

Systolic Heart Failure ◽

Cardiac Regeneration ◽

Oxygen Metabolism ◽

Functional Improvement ◽

Cardiomyocyte Proliferation ◽

Mammalian Heart ◽

Cell Cycle Reentry ◽

And Oxidative Stress

The underlying cause of systolic heart failure is the inability of the adult mammalian heart to regenerate damaged myocardium. In contrast, some vertebrate species and immature mammals are capable of full cardiac regeneration following multiple types of injury through cardiomyocyte proliferation. Little is known about what distinguishes proliferative cardiomyocytes from terminally differentiated, nonproliferative cardiomyocytes. Recently, several reports have suggested that oxygen metabolism and oxidative stress play a pivotal role in regulating the proliferative capacity of mammalian cardiomyocytes. Moreover, reducing oxygen metabolism in the adult mammalian heart can induce cardiomyocyte cell cycle reentry through blunting oxidative damage, which is sufficient for functional improvement following myocardial infarction. Here we concisely summarize recent findings that highlight the role of oxygen metabolism and oxidative stress in cardiomyocyte cell cycle regulation, and discuss future therapeutic approaches targeting oxidative metabolism to induce cardiac regeneration.

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Abstract 20: Resident Cardiac Stem Cell Growth Determines the Number of Ventricular Cardiomyocytes in the Mouse Heart

Circulation Research ◽

10.1161/res.111.suppl_1.a20 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Barbara Ogórek ◽

João Ferreira-Martins ◽

Donato Cappetta ◽

Alex Matsuda ◽

Sergio Signore ◽

...

Keyword(s):

Cell Cycle ◽

Late Gestation ◽

Primary Data ◽

Mouse Heart ◽

Lineage Specification ◽

Exponential Equation ◽

Cell Organization ◽

Parenchymal Cells ◽

Best Fit

The objective of this study was to determine the role of c-kit-positive cardiac stem cells (CSCs) in the formation of the heart during prenatal life, and immediately after birth. Mice in which EGFP is under the control of the c-kit-promoter were employed to measure the number of CSCs (Ns), the fraction of cycling MCM5-positive CSCs (f) and the length of the cell cycle (Ts) in CSCs. The number of CSCs committed to the myocyte lineage (LCC: lineage committed cells) included myocyte progenitors (c-kit-positive, Nkx2.5-positive cells), myocyte precursors (c-kit-positive, Nkx2.5-positive, and α-sarcomeric actin-positive cells) and replicating amplifying myocytes (c-kit-negative, Nkx2.5-positive, α-sarcomeric actin-positive, and MCM5-positive cells). These variables derived from CSC growth and lineage specification were evaluated to define the rate of formation of terminally differentiated myocytes (r). Based on a hierarchically structured cell organization, the rate of entry (Rs) of CSCs into the cell cycle was computed from Rs = f x (Ns/Ts), and the rate of generation of mature myocytes, r, was obtained from r = Rs x 2 Gt = ((f x Ns)/Ts) x 2 Gt . The exponent Gt defines the number of transit generations, i.e., the number of divisions that one CSC has to go through before it acquires the terminally differentiated myocyte phenotype. To validate this scenario and establish the number of post-mitotic myocytes formed, the primary data listed above were collected at E9, E14, E19 and P1. The number of mature cardiomyocytes generated by 1 CSC in 1 day was 1.1 x 10 3 , 20 x 10 3 , 501 x 10 3 , and 440 x 10 3 at E9, E14, E19 and P1, respectively. The total number of myocytes (Nm) formed from E9 to E14, E19 and P1 was derived from an exponential equation with the best fit to the experimental data: Nm = exp (0.69 x t) where Nm is the number of myocytes and t is time in days. Accordingly, CSCs generated 1 x 10 5 , 1 x 10 6 and 1.8 x 10 6 myocytes at from E9 to E14, E19 and P1, respectively. These values accounted for all parenchymal cells present at mid and late gestation and in the neonatal heart measured morphometrically. Thus, the expansion of the myocyte mass during embryonic, fetal and immediate postnatal development is controlled by activation, growth and differentiation of resident c-kit-positive CSCs.

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Abstract 246: Acetylation of Vgll4 Regulates Hippo-yap Signaling and Postnatal Cardiac Growth

Circulation Research ◽

10.1161/res.119.suppl_1.246 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Zhiqiang Lin ◽

Haidong Guo ◽

Sylvia Zohrabian ◽

Yuan Cao ◽

William T. Pu

Keyword(s):

Heart Failure ◽

Cell Cycle ◽

Transcription Factor ◽

Signaling Pathways ◽

Binding Protein ◽

Cardiac Regeneration ◽

Cardiac Growth ◽

Kinase Cascade

Binding of the transcription co-activator YAP with the transcription factor TEAD stimulates growth of the heart and other organs. Many signaling pathways, including the Hippo kinase cascade, converge to regulate YAP activity. However, less in known about the mechanisms that govern TEAD. YAP overexpression potently stimulates fetal cardiomyocyte (CM) proliferation, but YAP’s mitogenic potency declines postnatally, when mammalian cardiomyocytes largely exit the cell cycle. Here, we show that VGLL4, a CM-enriched TEAD1 binding protein, inhibits CM proliferation by limiting its binding to YAP and by targeting TEAD1 for degradation. VGLL4 antagonism of TEAD1 was governed by its acetylation at K225. Overexpression of VGLL4-K225R, an acetylation-refractory mutant, enhanced TEAD1 degradation, limited neonatal CM proliferation, and caused CM necrosis and heart failure. Our study defines an acetylation-mediated, VGLL4-dependent switch that regulates YAP-TEAD1 activity and restrains CM proliferation. These insights may enable more effective regulation of TEAD-YAP activity in applications ranging from cardiac regeneration to restraining cancer.

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Abstract 67: Metabolic Programming Regulates Proliferation and Binucleation of Postnatal Cardiomyocytes

Circulation Research ◽

10.1161/res.119.suppl_1.67 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Daniela Liccardo ◽

Ryan LaCanna ◽

Ying Tian

Keyword(s):

Cell Cycle ◽

Natriuretic Peptides ◽

Metabolic Programming ◽

Postnatal Life ◽

Mouse Heart ◽

Cardiomyocyte Proliferation ◽

Beta Oxidation ◽

Neonatal Cardiomyocytes ◽

Short Period

In contrast to adult, neonatal cardiomyocytes are able to proliferate and lose this capacity soon after birth when they withdraw from the cell cycle, become binucleated and differentiate. The arrest of cardiomyocytes cell cycle can be reversible for a short period, conferring the neonatal heart a regenerative potential within the first week of postnatal life. In the timeframe surrounding birth, heart maturation is also characterized by a change in energy metabolism, switching from glycolysis to beta-oxidation. However little is known about how metabolic programming in postnatal cardiomyocytes regulates their ability to proliferate, become binucleated and differentiate. In this study, we show that blocking beta-oxidation in mouse neonatal cardiomyocytes with etomoxir treatment promotes glycolysis and cell cycle re-entry, while increasing fatty acid beta-oxidation but reducing glycolysis leads to a decrease of the number of proliferating cardiomyocytes. In neonatal mice our data demonstrate that cardiomyocytes undergo binucleation and differentiation during the first week after birth and this process is correlated with the upregulation of the natriuretic peptides, ANP and BNP expression. Notably, in the postnatal mouse heart, beta-oxidation blockade through in vivo etomoxir injections, increases ventricular cardiomyocytes number, decreases natriuretic peptides expression and reduces the conversion of cardiomyocytes from a mononucleated to a binucleated phenotype. These findings highlight the importance of metabolic programming in regulating cardiomyocyte proliferation and suggest a potential therapeutic target for heart regeneration by modulating energy metabolic programming.

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Abstract 20134: Transcriptional Profiling Identifies Candidate Regulators of Neonatal Heart Regeneration

Circulation ◽

10.1161/circ.130.suppl_2.20134 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Caitlin O’Meara ◽

Joseph Wamstad ◽

Laurie Boyer ◽

Richard T Lee

Keyword(s):

Cell Cycle ◽

Transcriptional Profiling ◽

Cardiac Regeneration ◽

Heart Regeneration ◽

Transcriptional Signature ◽

Adult Mice ◽

Neonatal Heart ◽

Sirna Knockdown

Some higher organisms, such as zebrafish and neonatal mice, are capable of complete and sufficient regeneration of the myocardium following injury, which is thought to occur primarily by proliferation of pre-existing cardiomyocytes. Although adult humans and adult mice lack this cardiac regeneration potential, there is great interest in understanding how regeneration can occur in the heart so that we can activate this process in humans suffering from heart failure. The aim of our study was to identify mechanisms by which mature, post-mitotic adult cardiomyocytes can re-enter the cell cycle to ultimately facilitate heart regeneration following injury. We derived a core transcriptional signature of injury-induced cardiomyocyte regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo cardiomyocyte differentiation and in an in vitro cardiomyocyte explant model, as well as a neonatal heart resection model. We identified a panel of transcription factors, growth factors, and cytokines, whose expression significantly correlated with the differentiated state of the cell in all datasets examined, suggesting that these factors play a role in regulating cardiomyocyte cell state. Furthermore, potential upstream regulators of core differentially expressed networks were identified using Ingenuity Pathway Analysis and we found that one predicted regulator, interleukin-13 (IL13), significantly induced cardiomyocyte cell cycle activity and STAT6/STAT3 signaling in vitro. siRNA knockdown experiments demonstrated that STAT3/periostin and STAT6 signaling are critical for cardiomyocyte cell cycle activity in response to IL13. These data reveal novel insights into the transcriptional regulation of mammalian heart regeneration and provide the founding circuitry for identifying potential regulators for stimulating cardiomyocyte cell cycle activity.

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Phenotypic Screen with the Human Secretome Identifies FGF16 as Inducing Proliferation of iPSC-Derived Cardiac Progenitor Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20236037 ◽

2019 ◽

Vol 20 (23) ◽

pp. 6037 ◽

Cited By ~ 3

Author(s):

Karin Jennbacken ◽

Fredrik Wågberg ◽

Ulla Karlsson ◽

Jerry Eriksson ◽

Lisa Magnusson ◽

...

Keyword(s):

Progenitor Cells ◽

Cardiac Fibroblasts ◽

Cardiac Regeneration ◽

Cardiac Cells ◽

Mouse Heart ◽

Cardiac Progenitor Cells ◽

High Sequence Identity ◽

Paracrine Factors ◽

Human Cardiomyocytes ◽

Phenotypic Screen

Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count. From this screen, we identified FGF1, FGF4, FGF9, FGF16, FGF18, and seven additional proteins that induce proliferation of cardiac progenitor cells. FGF9 and FGF16 belong to the same FGF subfamily, share high sequence identity, and are described to have similar receptor preferences. Interestingly, FGF16 was shown to be specific for proliferation of cardiac progenitor cells, whereas FGF9 also proliferated human cardiac fibroblasts. Biosensor analysis of receptor preferences and quantification of receptor abundances suggested that FGF16 and FGF9 bind to different FGF receptors on the cardiac progenitor cells and cardiac fibroblasts. FGF16 also proliferated naïve cardiac progenitor cells isolated from mouse heart and human cardiomyocytes derived from induced pluripotent cells. Taken together, the data suggest that FGF16 could be a suitable paracrine factor to induce cardiac regeneration and repair.

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