Abstract 527: Role of IGFBP3 in Neonatal Heart Regeneration

Background: The adult mammalian heart is unable to regenerate after an injury. However, newborn mice are able to fully regenerate the heart after myocardial infarction (MI). The neonatal MI model therefore is a potential blueprint for regenerating the adult heart that could offer novel therapies for patients suffering from heart disease. To investigate the mechanism by which neonatal heart regeneration occurs, we screened for secreted proteins that are upregulated after neonatal MI but not after adult MI. We hypothesized that such a protein could be a cardiomyocyte mitogen that underlies the cardiomyocyte proliferation that occurs after neonatal MI. Methods: We performed microarrays on neonatal and adult MI (and sham) heart tissue samples: we identified IGFBP3 (Insulin Growth Factor Binding Protein 3), which canonically transports IGF ligands in circulation, as a secreted protein that is uniquely upregulated after neonatal MI. We used in situ hybridization, reporter mice, and immunostaining to validate the findings from the microarray. Single cell RNA-seq data revealed that IGFBP3 is expressed in vascular cells. Results: We first tested whether IGFBP3 is necessary during neonatal heart regeneration: we performed neonatal (P1) MI in global Igfbp3 knockout mice and wild-type mice, and found that knockout mice have more fibrosis and worse ejection fraction (EF) one month after P1 MI. To determine if IGFBP3 is sufficient to promote cardiomyocyte proliferation, we injected recombinant IGFBP3 protein into the heart of one week-old mice (P7) and saw more cycling myocytes. We generated novel transgenic vascular-Igfbp3-overexpression mice, which exhibit less fibrosis as well as improved EF after P7 MI compared to controls. Conclusions: IGFBP3 is a secreted protein that is necessary for complete regeneration after a neonatal MI. Its ectopic expression can cause cardiomyocyte proliferation and can improve systolic function, and it prolongs the window of neonatal heart regeneration. Therefore, IGFBP3 may represent a cardiomyocyte mitogen with potential therapeutic value for adult heart disease.

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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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Abstract 214: TLR3 Mediates Neonatal Heart Repair and Regeneration Through Glycolysis-dependent YAP/TAZ Mediated miR-152 Expression

Circulation Research ◽

10.1161/res.119.suppl_1.214 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Xiaohui Wang ◽

Yuanping Hu ◽

Tuanzhu Ha ◽

John Kalbfleisch ◽

Race Kao ◽

...

Keyword(s):

Cardiac Metabolism ◽

Poly I:C ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Neonatal Cardiomyocytes ◽

Neonatal Heart ◽

Heart Repair ◽

Inhibition Of Glycolysis ◽

Neonatal Cardiomyocyte

The neonatal heart possesses the capability of regenerating and repairing damaged myocardium which is lost when cardiac metabolism switches from predominate glycolysis to oxidative phosphorylation seven days after birth. We have observed that Toll-like receptor 3 (TLR3) deficient neonatal hearts exhibit impaired cardiac function and larger infarct size after myocardial infarction (MI). We also found that stimulation of neonatal cardiomyocytes with the TLR3 ligand, poly (I:C) significantly enhances glycolytic capacity. Our observation suggests that TLR3 is required for neonatal heart repair and regeneration of damaged myocardium. This study investigated the mechanisms by which TLR3 mediates neonatal heart regeneration and repair. Neonatal cardiomyocytes were isolated from one day old WT mice and treated with poly (I:C) (1μg/ml) for 12-36 hours. We observed that poly (I:C) treatment: i) significantly enhances glycolytic metabolism; ii) increases YAP/TAZ activation: iii) increases miR-152 expression; iv) suppresses expression of DNMT1 and p27kip1, and v) promotes cardiomyocyte proliferation. However, inhibition of glycolysis with 2-Deoxyglucose (2-DG) prevented poly (I:C)-induced YAP/TAZ activation and miR-152 expression in neonatal cardiomyocytes. Similarly, inhibition of YAP/TAZ activation with Verteprofin (VP) abolished poly (I:C) induced miR-152 expression and neonatal cardiomyocyte proliferation. To investigate the role of miR-152 in neonatal cardiomyocyte proliferation, we transfected neonatal cardiomyocytes with miR-152 mimics and observed that increased miR-152 levels significantly promotes neonatal cardiomyocyte proliferation. We also observed that transfection of neonatal cardiomyocytes with miR-152 mimics markedly suppresses the expression of DNMT1 and p27kip1. Inhibition of DNMT1 with 5Azcytidine significantly promotes neonatal cardiomyocyte proliferation. Finally, we observed that treatment of neonatal mice (n=6) with 2-DG abolished cardiac functional recovery 3 weeks after MI. We conclude that TLR3 is required for neonatal heart regeneration and repair after MI. The mechanisms involve glycolytic dependent activation of YAP/TAZ mediated by miR-152 which represses DNMT1/p27kip1 expression.

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Mechanistic basis of neonatal heart regeneration revealed by transcriptome and histone modification profiling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905824116 ◽

2019 ◽

Vol 116 (37) ◽

pp. 18455-18465 ◽

Cited By ~ 13

Author(s):

Zhaoning Wang ◽

Miao Cui ◽

Akansha M. Shah ◽

Wenduo Ye ◽

Wei Tan ◽

...

Keyword(s):

Molecular Mechanisms ◽

Rna Binding ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cardiac Regeneration ◽

Later Life ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Neonatal Heart ◽

Short Period

The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. To uncover the molecular mechanisms underlying neonatal heart regeneration, we compared the transcriptomes and epigenomes of regenerative and nonregenerative mouse hearts over a 7-d time period following myocardial infarction injury. By integrating gene expression profiles with histone marks associated with active or repressed chromatin, we identified transcriptional programs underlying neonatal heart regeneration, and the blockade to regeneration in later life. Our results reveal a unique immune response in regenerative hearts and a retained embryonic cardiogenic gene program that is active during neonatal heart regeneration. Among the unique immune factors and embryonic genes associated with cardiac regeneration, we identified Ccl24, which encodes a cytokine, and Igf2bp3, which encodes an RNA-binding protein, as previously unrecognized regulators of cardiomyocyte proliferation. Our data provide insights into the molecular basis of neonatal heart regeneration and identify genes that can be modulated to promote heart regeneration.

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Nrg1 is an injury-induced cardiomyocyte mitogen for the endogenous heart regeneration program in zebrafish

eLife ◽

10.7554/elife.05871 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 111

Author(s):

Matthew Gemberling ◽

Ravi Karra ◽

Amy L Dickson ◽

Kenneth D Poss

Keyword(s):

Cardiac Injury ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Adult Zebrafish ◽

Perivascular Cells ◽

Adult Heart ◽

Muscle Hyperplasia ◽

Zebrafish Heart

Heart regeneration is limited in adult mammals but occurs naturally in adult zebrafish through the activation of cardiomyocyte division. Several components of the cardiac injury microenvironment have been identified, yet no factor on its own is known to stimulate overt myocardial hyperplasia in a mature, uninjured animal. In this study, we find evidence that Neuregulin1 (Nrg1), previously shown to have mitogenic effects on mammalian cardiomyocytes, is sharply induced in perivascular cells after injury to the adult zebrafish heart. Inhibition of Erbb2, an Nrg1 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial Nrg1 overexpression enhances this proliferation. In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium. Our findings identify Nrg1 as a potent, induced mitogen for the endogenous adult heart regeneration program.

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Abstract MP161: A Calcineurin-hoxb13 Axis Regulates Growth Mode of Mammalian Cardiomyocytes

Circulation Research ◽

10.1161/res.127.suppl_1.mp161 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Ngoc Uyen Nhi Nguyen ◽

Diana C Canseco ◽

Feng Xiao ◽

Yuji Nakada ◽

Shujuan Li ◽

...

Keyword(s):

Cell Cycle ◽

Systolic Function ◽

Left Ventricular Systolic Function ◽

Left Ventricular ◽

Cardiomyocyte Proliferation ◽

Double Knockout ◽

Adult Heart ◽

Hypertrophic Growth ◽

Cycle Arrest ◽

Specific Deletion

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. Our group recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2. We recently also showed that Meis1, a TALE family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a Meis1 cofactor in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can both extend the postnatal window of cardiomyocyte proliferation and reactivate cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1/Hoxb13 double knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and an improvement in left ventricular systolic function following myocardial infarction both by echocardiography and MRI. ChIP-seq analysis demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204 (S204), resulting in its nuclear localization and cell cycle arrest. Collectively, these results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

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gp130 Controls Cardiomyocyte Proliferation and Heart Regeneration

Circulation ◽

10.1161/circulationaha.119.044484 ◽

2020 ◽

Vol 142 (10) ◽

pp. 967-982

Author(s):

Yandong Li ◽

Jie Feng ◽

Shen Song ◽

Haotong Li ◽

Huijun Yang ◽

...

Keyword(s):

Myocardial Infarction ◽

Gene Therapy ◽

Rna Sequencing ◽

Cardiac Injury ◽

Oncostatin M ◽

Functional Screening ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Adult Mice ◽

Neonatal Heart

Background: A key cause of the high mortality of cardiovascular diseases is the cardiomyocyte inability to renew after cardiac injury. As a promising strategy to supplement functional myocytes for cardiac repair, there is a pressing need to understand the cellular and molecular mechanisms of heart regeneration. Methods: Seven genetic mouse lines were used: global OSM (oncostatin M) knockout, monocyte-/macrophage-specific OSM deletion, cardiomyocyte-specific lines, including OSM receptor deletion, gp130 (glycoprotein 130) deletion, gp130 activation, and Yap (yes-associated protein) ablation with gp130 activation mice. A series of molecular signaling experiments, including RNA sequencing, immunostaining, coimmunoprecipitation, and imaging flow cytometry, were conducted. Two models of cardiac injury, apical resection and myocardial infarction operation, were performed in neonatal, juvenile, and adult mice. Heart regeneration and cardiac function were evaluated by Masson staining and echocardiography, respectively. Gene recombinant adenovirus-associated virus was constructed and infected myocardial-infarcted mice as a gene therapy. Results: OSM was identified by RNA sequencing as a key upstream regulator of cardiomyocyte proliferation during neonatal heart regeneration in mice. Cardiomyocyte proliferation and heart regeneration were suspended in neonatal mice after cardiac injury when OSM was conditionally knockout in macrophages. The cardiomyocyte-specific deficiency of the OSM receptor heterodimers, OSM receptor and gp130, individually in cardiomyocytes reduced myocyte proliferation and neonatal heart regeneration. Conditional activation of gp130 in cardiomyocytes promoted cardiomyocyte proliferation and heart regeneration in juvenile and adult mice. Using RNA sequencing and functional screening, we found that Src mediated gp130-triggered cardiomyocyte proliferation by activating Yap (yes-associated protein) with Y357 phosphorylation independently of the Hippo pathway. Cardiomyocyte-specific deletion of Yap in Myh6-gp130 ACT mice blocked the effect of gp130 activation–induced heart regeneration in juvenile mice. Gene therapy with adenovirus-associated virus encoding constitutively activated gp130 promoted cardiomyocyte proliferation and heart regeneration in adult mice after myocardial infarction. Conclusions: Macrophage recruitment is essential for heart regeneration through the secretion of OSM, which promotes cardiomyocyte proliferation. As the coreceptor of OSM, gp130 activation is sufficient to promote cardiomyocyte proliferation by activating Yap through Src during heart regeneration. gp130 is a potential therapeutic target to improve heart regeneration after cardiac injury.

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Metformin accelerates zebrafish heart regeneration by inducing autophagy

npj Regenerative Medicine ◽

10.1038/s41536-021-00172-w ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Fangjing Xie ◽

Shisan Xu ◽

Yingying Lu ◽

Kin Fung Wong ◽

Lei Sun ◽

...

Keyword(s):

Myocardial Infarction ◽

Systolic Function ◽

Cell Types ◽

Underlying Mechanism ◽

Systematic Evaluation ◽

Autophagic Flux ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Clinical Value ◽

Zebrafish Model

AbstractMetformin is one of the most widely used drugs for type 2 diabetes and it also exhibits cardiovascular protective activity. However, the underlying mechanism of its action is not well understood. Here, we used an adult zebrafish model of heart cryoinjury, which mimics myocardial infarction in humans, and demonstrated that autophagy was significantly induced in the injured area. Through a systematic evaluation of the multiple cell types related to cardiac regeneration, we found that metformin enhanced the autophagic flux and improved epicardial, endocardial and vascular endothelial regeneration, accelerated transient collagen deposition and resolution, and induced cardiomyocyte proliferation. Whereas, when the autophagic flux was blocked, then all these processes were delayed. We also showed that metformin transiently enhanced the systolic function of the heart. Taken together, our results indicate that autophagy is positively involved in the metformin-induced acceleration of heart regeneration in zebrafish and suggest that this well-known diabetic drug has clinical value for the prevention and amelioration of myocardial infarction.

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Thyroid hormone-dependent regulation of metabolism and heart regeneration

Journal of Endocrinology ◽

10.1530/joe-21-0335 ◽

2021 ◽

Author(s):

Ines Ross ◽

Denzel B Omengan ◽

Guo N Huang ◽

Alexander Y Payumo

Keyword(s):

Thyroid Hormones ◽

Scientific Evidence ◽

Heart Regeneration ◽

Direct Effects ◽

Cardiomyocyte Proliferation ◽

Animal Kingdom ◽

Regenerative Capacity ◽

Newborn Mice ◽

Regulation Of Metabolism ◽

Animal Metabolism

While adult zebrafish and newborn mice possess a robust capacity to regenerate their hearts, this ability is generally lost in adult mammals. The logic behind the diversity of cardiac regenerative capacity across the animal kingdom is not well understood. We have recently reported that animal metabolism is inversely correlated to the abundance of mononucleated diploid cardiomyocytes in the heart, which retain proliferative and regenerative potential. Thyroid hormones are classical regulators of animal metabolism, mitochondrial function, and thermogenesis and a growing body of scientific evidence demonstrates that these hormonal regulators also have direct effects on cardiomyocyte proliferation and maturation. We propose that thyroid hormones dually control animal metabolism and cardiac regenerative potential through distinct mechanisms, which may represent an evolutionary tradeoff for the acquisition of endothermy and loss of heart regenerative capacity. In this review, we describe the effects of thyroid hormones on animal metabolism and cardiomyocyte regeneration, and highlight recent reports linking the loss of mammalian cardiac regenerative capacity to metabolic shifts occurring after birth.

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