Mononuclear diploid cardiomyocytes support neonatal mouse heart regeneration in response to paracrine IGF2 signaling

Injury to the newborn mouse heart is efficiently regenerated, but this capacity is lost by one week after birth. We found that IGF2, an important mitogen in heart development, is required for neonatal heart regeneration. IGF2 originates from the endocardium/endothelium and is transduced in cardiomyocytes by the insulin receptor. Following injury on postnatal day 1, absence of IGF2 abolished injury-induced cell cycle entry during the early part of the first postnatal week. Consequently, regeneration failed despite the later presence of additional cell cycle-inducing activities 7 days following injury. Most cardiomyocytes transition from mononuclear diploid to polyploid during the first postnatal week. Regeneration was rescued in Igf2-deficient neonates in three different contexts that elevate the percentage of mononuclear diploid cardiomyocytes beyond postnatal day 7. Thus, IGF2 is a paracrine-acting mitogen for heart regeneration during the early postnatal period, and IGF2-deficiency unmasks the dependence of this process on proliferation-competent mononuclear diploid cardiomyocytes.

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Malonate Promotes Adult Cardiomyocyte Proliferation and Heart Regeneration

Circulation ◽

10.1161/circulationaha.120.049952 ◽

2021 ◽

Author(s):

Jiyoung Bae ◽

Rebecca J. Salamon ◽

Emma B. Brandt ◽

Wyatt G. Paltzer ◽

Ziheng Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Succinate Dehydrogenase ◽

Familial Cancer ◽

Metabolic Reprogramming ◽

Current Evidence ◽

Mouse Heart ◽

Heart Regeneration ◽

Ros Production ◽

Cardiomyocyte Proliferation ◽

Metabolic Switch

Background: Neonatal mouse cardiomyocytes undergo a metabolic switch from glycolysis to oxidative phosphorylation, which results in a significant increase in reactive oxygen species (ROS) production that induces DNA damage. These cellular changes contribute to cardiomyocyte cell cycle exit and loss of the capacity for cardiac regeneration. The mechanisms that regulate this metabolic switch and the increase in ROS production have been relatively unexplored. Current evidence suggests that elevated ROS production in ischemic tissues occurs due to accumulation of the mitochondrial metabolite succinate during ischemia via succinate dehydrogenase (SDH), and this succinate is rapidly oxidized at reperfusion. Interestingly, mutations in SDH in familial cancer syndromes have been demonstrated to promote a metabolic shift into glycolytic metabolism, suggesting a potential role for SDH in regulating cellular metabolism. Whether succinate and SDH regulate cardiomyocyte cell cycle activity and the cardiac metabolic state remains unclear. Methods: Here, we investigated the role of succinate and succinate dehydrogenase (SDH) inhibition in regulation of postnatal cardiomyocyte cell cycle activity and heart regeneration. Results: Our results demonstrate that injection of succinate in neonatal mice results in inhibition of cardiomyocyte proliferation and regeneration. Our evidence also shows that inhibition of SDH by malonate treatment after birth extends the window of cardiomyocyte proliferation and regeneration in juvenile mice. Remarkably, extending malonate treatment to the adult mouse heart following myocardial infarction injury results in a robust regenerative response within 4 weeks following injury via promoting adult cardiomyocyte proliferation and revascularization. Our metabolite analysis following SDH inhibition by malonate induces dynamic changes in adult cardiac metabolism. Conclusions: Inhibition of SDH by malonate promotes adult cardiomyocyte proliferation, revascularization, and heart regeneration via metabolic reprogramming. These findings support a potentially important new therapeutic approach for human heart failure.

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Molecular atlas of postnatal mouse heart development

10.1101/302802 ◽

2018 ◽

Author(s):

Virpi Talman ◽

Jaakko Teppo ◽

Päivi Pöhö ◽

Parisa Movahedi ◽

Anu Vaikkinen ◽

...

Keyword(s):

Cell Cycle ◽

Heart Development ◽

Molecular Mechanisms ◽

Mevalonate Pathway ◽

Cardiac Regeneration ◽

Neonatal Rat ◽

Acid Oxidation ◽

Mouse Heart ◽

Tissue Samples ◽

Combining Data

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 348: Interleukin 13 is Required for Neonatal Heart Regeneration

Circulation Research ◽

10.1161/res.121.suppl_1.348 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Caitlin C O’Meara ◽

Dana Murphy ◽

Angela Lemke ◽

Michael J Flister

Keyword(s):

Cell Cycle ◽

Knockout Mice ◽

Heart Development ◽

Contradictory Result ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Rat Cardiomyocytes ◽

Hypertrophic Growth ◽

Interleukin 13 ◽

Neonatal Heart

Shortly after birth neonatal mice can fully regenerate their hearts, but this potential is lost in the first week of life. Cell cycle entry of existing cardiomyocytes is thought to be an essential mechanism enabling neonatal mouse heart regeneration. In previous studies we found that the cytokine interleukin 13 (IL13) was a an upstream regulator of differentially expressed gene networks during neonatal heart regeneration and stimulated cell cycle activity of cultured rat cardiomyocytes, suggesting that this factor might be important in neonatal heart regeneration in vivo . In the present study, we subjected wildtype and IL13 knockout mice to ventricular apical resection at one day of age and assessed heart regeneration 21 days post resection (dpr). Compared to wildtype controls, IL13 knockout mice failed to regenerate their hearts as determined by extensive scar formation at the ventricular apex. To gain insight into the mechanism of impaired regeneration, we quantified cardiomyocyte proliferation and expression of macrophage markers at 7 dpr. We found no difference in gene expression of macrophage markers in IL13 knockout mice compared to wildtype. Interestingly, IL13 knockout mice demonstrate a significant increase cardiomyocyte cell cycle activity as determined by phosphorylated Histone H3 (pH3) staining. This seemingly contradictory result appears to be due to an underlying developmental defect in IL13 knockout hearts. Cardiomyocytes in IL13 knockout mice appeared large and disorganized. Cardiomyocytes from IL13 knockout unoperated mice showed decreased pH3 staining and had increased expression marker of hypertrophic growth such as Nppb and Nppa. Histologically, hearts from IL13 knockout mice appeared to have a dilated cardiomyopathy phenotype. Collectively our data suggests that during heart development IL13 influences proliferative versus hypertrophic growth. We surmise that following neonatal apical resection in IL13 knockout mice the significant increase in cardiomyocyte proliferation is a compensatory attempt to repair the injury, but the underlying cardiomyocyte phenotype inhibits complete regeneration. These data are the first to report a role for IL13 in normal heart development and neonatal heart regeneration.

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Yap is required for scar formation but not myocyte proliferation during heart regeneration in zebrafish

Cardiovascular Research ◽

10.1093/cvr/cvy243 ◽

2018 ◽

Vol 115 (3) ◽

pp. 570-577 ◽

Cited By ~ 9

Author(s):

Michael A Flinn ◽

Brooke E Jeffery ◽

Caitlin C O’Meara ◽

Brian A Link

Keyword(s):

Heart Development ◽

Critical Role ◽

Scar Formation ◽

Cardiac Cells ◽

Mouse Heart ◽

Heart Regeneration ◽

Post Injury ◽

Myocyte Proliferation ◽

The Impact ◽

Zebrafish Heart

Abstract Aims The Hippo signalling pathway regulates multiple cellular processes during organ development and maintenance by modulating activity of the transcriptional cofactor Yap. Core components of this pathway are required for neonatal mouse heart regeneration, however, investigations to date have typically focused on expression and activity in cardiomyocytes. Due to the regenerative capacity of zebrafish and the fact that global loss of Yap is not fully embryonic lethal in zebrafish, we leveraged a yap null mutant to investigate the impact of constitutive Yap deletion during zebrafish heart regeneration. Methods and results Following cryoinjury in adult hearts, myocyte proliferation was not decreased in yap mutants, contrary to expectations based on mouse data. Experiments in larval zebrafish (Danio rerio) revealed that deletion of either Yap or Taz had a modest effect on heart growth, reducing gross organ size, while their combined deletion was synergistic; thus, Yap and Taz share some overlapping roles in zebrafish heart development. Surprisingly, adult yap mutants exhibited decreased collagen composition at 7 days post-injury, suggesting a critical role for Yap in scar formation during heart regeneration. siRNA-mediated Yap knockdown in primary rat (Rattus norvegicus) cardiac cells revealed a fibroblast-specific role for Yap in controlling the expression of cytoskeletal and myofibroblast activation genes, as well as pro-inflammatory cyto/chemokines. Corroborating these RNAseq data, we observed increased macrophage infiltration in the scars of yap mutants at 7 days post-injury. Conclusion These results suggest that Yap deletion has minimal effect on myocyte proliferation in adults, but significantly influences scar formation and immune cell infiltration during zebrafish heart regeneration. Collectively, these data suggest an unexpected role for Yap in matrix formation and macrophage recruitment during heart regeneration.

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Abstract 358: Neonatal Mouse Heart Maturation: Transcriptome-Wide Analysis Reveals Chamber-Specific Exon Enrichment of Cell Cycle Genes

Circulation Research ◽

10.1161/res.115.suppl_1.358 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Marlin Touma ◽

Xuedong Kang ◽

Jae-Hyung Lee ◽

Xinshu Xiao ◽

Yibin Wang

Keyword(s):

Cell Cycle ◽

Rna Splicing ◽

Cellular Proliferation ◽

P Value ◽

Mouse Heart ◽

Newborn Mouse ◽

Postnatal Maturation ◽

Functional Studies ◽

Neonatal Cardiomyocytes ◽

Splicing Regulators

Background: During postnatal maturation of mammalian heart the neonatal cardiomyocytes undergo dramatic changes including complete maturation, proliferation arrest, and terminal exit from the cell cycle (CC). However, transcriptome-wide analysis of CC programs has not been performed in perinatal stages among different cardiac chambers. In particular, the contribution of alternative RNA splicing to the chamber-specific CC activities is unexplored Design/Methods: To achieve comprehensive analysis of differential expression (DE) and alternative splicing (AS) of CC-related genes in left ventricle (LV) versus right ventricle (RV) during maturation deep RNA-seq was performed on male newborn mouse LV and RV at 3 time points of perinatal transition: P0, P3 and P7. Reads were mapped to mouse Transcriptome, and to mouse Genome. Transcriptome-Wide difference in inclusion of individual exons was performed using MATS. DE genes and AS variants were defined as those with fold change ≥2, at expression level ≥3 RBKM and a false discovery rate ≤0.05. Significant gene ontology (GO) terms were determined at P-value ≤0.05. Levels of expression were validated using qRT-PCR Results: Altogether, 2116 DE genes and 1162 AS events were observed. Among them, 109 CC-related genes were further analyzed. Distinct temporal patterns of DE and GO enrichment of CC genes in LV vs. RV during maturation were identified. Chamber -specific induction of genes involved in mitosis, karyokinesis, and cytokinesis was found at P7. RNA Splicing analysis of CC genes revealed 77 AS events. Skipping exon accounted for nearly half splicing events. Among 30 spliced exon variants, significant chamber-and temporal-specific inclusion were observed. Interestingly, the majority of AS variants exhibited opposing patterns of exon usage in RV vs. LV at p7 Conclusions: Our findings suggest novel molecular basis for chamber-specific programming of cellular proliferation and maturation in neonatal heart, including potential splicing regulation of dynamic exon enrichment of cell cycle related genes. Further functional studies to decipher putative splicing regulators of CC programming in LV vs. RV during maturation will likely lead to novel chamber-specific regenerative and therapeutic targets.

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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 264: TNFR2-Bmx Signaling in Cardiac Stem Cells and Cardiac Repair

Circulation Research ◽

10.1161/res.115.suppl_1.264 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Huanjiao J Zhou ◽

Qunhua Huang ◽

Wang Min

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Wild Type Mouse ◽

Cardiac Repair ◽

Mouse Heart ◽

Coronary Artery Ligation ◽

Cardiac Stem Cells ◽

Organ Cultures ◽

Cell Cycle Entry ◽

And Migration

While cytokine TNF via TNFR1 induces inflammation and apoptosis, it through its second receptor TNFR2 induces cell survival and migration by activating bone marrow non-receptor tyrosine kinase Bmx. Since Bmx has been implicated in self-renewal of stem cells, we hypothesize that TNF via TNFR2 activates Bmx in cardiac stem cells (CSCs) to mediate cardiac repair. We show that in human cardiac tissue affected by ischemia heart disease (IHD), TNFR2 is expressed on intrinsic CSCs, identified as c-kit(+) /CD45(-) /VEGFR2(-) interstitial round cells, which are activated as determined by entry to cell cycle and expression of Lin-28. Wild-type mouse heart organ cultures subjected to hypoxic conditions both increase cardiac TNF expression and show induced TNFR2 and Lin-28 expression in c-kit(+) CSCs that have entered cell cycle. These CSC responses are enhanced by exogenous TNF. TNFR2(-/-) mouse heart organ cultures subjected to hypoxia increase cardiac TNF but fail to induce CSC activation. Similarly, c-kit(+) CSCs isolated from mouse hearts exposed to hypoxia or TNF show induction of Lin-28, TNFR2, cell cycle entry, and cardiogenic marker, α-sarcomeric actin (α-SA), responses more pronounced by hypoxia in combination with TNF. Knockdown of Lin-28 by siRNA results in reduced levels of TNFR2 expression, cell cycle entry, and diminished expression of α-SA (references: Stem Cells 2013;31:1881-1892). In the present study, we detect the c-kit(+)Lin28(+) CSCs populations in a mouse coronary artery ligation ischemic model. Furthermore, the c-kit(+) CSCs are reduced in TNFR2-KO and Bmx-KO mice. Mechanistically, we show a crosstalk between the TNFR2-Bmx and the c-Kit signaling pathways to mediate CSC proliferation, survival and migration. These observations suggest that TNFR2-Bmx signaling in c-kit(+) CSCs induces cardiac repair, providing a potential strategy to stimulate cardiac regeneration by TNFR2-specific agonists.

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