RNA seq analysis of regenerating hearts from three models of successful cardiac regeneration
Ontology highlight
ABSTRACT: Defining conserved molecular pathways in animal models of successful cardiac regeneration could yield insight into why adult mammals have inadequate cardiac regeneration after injury. Here we describe a cross-species transcriptomic screen to identify evolutionarily conserved pathways in the early events of cardiac regeneration in three species that can regenerate myocardium after a major injury. In this study, we performed RNA-seq on regenerating hearts from three model organisms - axolotl, zebrafish and mouse. Apical resection was performed to amputate ~10 - 20% of the left ventricle in all three model organisms. Following resection, hearts were harvested at 12, 24 and 48 hours post-resection and subjected to RNA-seq. RNA-seq on sham controls (no ventricular amputation) was used as interanal control. This approach revealed upregulation of inflammatory genes in all three organisms during regeneration. Furthermore, upregulation of Complement 5a receptor1 (C5aR1) expression in the regenerating hearts of zebrafish, axolotls and mice was observed.
Project description:The adult vertebrate red spotted newt is a champion of regeneration, demonstrating an amazing ability to regenerate damaged organs and tissues back to an uninjured state without the formation of scar or reduction in function. By developing a novel cardiac resection strategy, our group recently demonstrated that newt hearts could morphologically and functionally regenerate, without scarring, within a period of 2-3 months following injury. MicroRNAs (miRs) have been widely publicized as essential post-transcriptional gene regulators in a variety of biological processes, including regeneration. We have conducted a microarray screen for vertebrate miRs, with several candidate miRs showing significant differential expression at important time-points following injury to the newt heart. The newt microRNA expression between uninjured hearts and regenerating hearts, 7 and 21 days post-injury (dpi), was compared by microarray analysis. Three paired samples were analyzed: Uninjured, 7dpi and 21dpi newt hearts. Three arrays were hybridized comparing two-paired samples each time.
Project description:Rationale: Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level. Objective: The objectives of our study were to determine if myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process. Methods and Results: We derived a core transcriptional signature of injury-induced cardiac myocyte regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo cardiac myocyte differentiation, in vitro cardiac myocyte explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of cardiac myocyte differentiation processes including reactivation of latent developmental programs similar to those observed during de-stabilization of a mature cardiac myocyte phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13 (IL13), which induced cardiac myocyte cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of IL13 signaling in cardiac myocytes. These downstream signaling molecules are also modulated in the regenerating mouse heart. Conclusions: Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration. Comparison of transcriptional programs of primary myocardial tissues sampled from neonatal mice and murine hearts undergoing post-injury regeneration, along with in vitro ESC-differentiated cardiomyocytes
Project description:Zebrafish have a high capacity to regenerate their hearts post-injury.Several studies have surveyed transcriptional enhancers to understand how the dynamics of gene expression are controlled during heart regeneration. We have identified a cardiac transcriptional enhancer that is activated at the site of injury called the runx1 enhancer or REN since it regulates the expression of nearby gene runx1. REN is usually active in cardiomyocytes (CMs) and epicardial tissues surrounding the cardiac valves of uninjured zebrafish hearts. However, when REN is activated in regenerating CM and epicardial tissues at the injury site, it is concurrently downregulated around the distal heart valve. Deletion of REN (ΔREN) results in excess Collagen around uninjured zebrafish cardiac valves. This ΔREN Collagen phenotype is rescued with a runx1 deletion (Δrunx1), suggesting that REN and runx1 function in different genetic pathways in uninjured hearts. A different nearby gene that does change expression around valves in ΔREN mutant hearts is adamts1 which encodes a metalloproteinase that degrades Collagen. Taken together, this suggests that in uninjured hearts REN regulates adamts1 independently of runx1. However, during regeneration, CM proliferation at the site of injury is enhanced in both ΔREN and Δrunx1 mutants, suggesting that REN is rewired to runx1 to stimulate its transcription. There are two previous descriptions for how enhancers are activated during regeneration. First, cis-regulatory sequences are reactivated from embryogenesis and second that active adult enhancers are further invigorated during regeneration. Our data point to a third and previously unappreciated mechanism for gene control during zebrafish heart regeneration. We report that an enhancer is repurposed both from one gene and cardiac domain to activate a different nearby gene in regenerating cardiac tissue.
Project description:The adult vertebrate red spotted newt is a champion of regeneration, demonstrating an amazing ability to regenerate damaged organs and tissues back to an uninjured state without the formation of scar or reduction in function. By developing a novel cardiac resection strategy, our group recently demonstrated that newt hearts could morphologically and functionally regenerate, without scarring, within a period of 2-3 months following injury. MicroRNAs (miRs) have been widely publicized as essential post-transcriptional gene regulators in a variety of biological processes, including regeneration. We have conducted a microarray screen for vertebrate miRs, with several candidate miRs showing significant differential expression at important time-points following injury to the newt heart. The newt microRNA expression between uninjured hearts and regenerating hearts, 7 and 21 days post-injury (dpi), was compared by microarray analysis.
Project description:Heart failure is a leading cause of mortality and morbidity in the developed world, partly because mammals lack the ability to regenerate heart tissue. Whether this is due to evolutionary loss of regenerative mechanisms present in other organisms or to an inability to activate such mechanisms is currently unclear. Here, we decipher mechanisms underlying heart regeneration in adult zebrafish and show that the molecular regulators of this response are conserved in mammals. We identified miR-99/100 and Let-7a/c, and their protein targets smarca5 and fntb, as critical regulators of cardiomyocyte dedifferentiation and heart regeneration in zebrafish. Although human and murine adult cardiomyocytes fail to elicit an endogenous regenerative response following myocardial infarction, we show that in vivo manipulation of this molecular machinery in mice results in cardiomyocyte dedifferentiation and improved heart functionality after injury. These data provide a proof-of-concept for identifying and activating conserved molecular programs to regenerate the damaged heart. Analysis of miRNA levels in regenerating zebrafish hearts
Project description:Adult zebrafish are capable of regenerating cardiac tissue following ventricular resection within 30 days. We profiled both small RNA and mRNA expression in uninjured (0dpa), 1, 3, 7, 14, 21 and 30 days post amputation to study biological processes orchestrate each stage of regeneration.
Project description:Rationale: Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level. Objective: The objectives of our study were to determine if myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process. Methods and Results: We derived a core transcriptional signature of injury-induced cardiac myocyte regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo cardiac myocyte differentiation, in vitro cardiac myocyte explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of cardiac myocyte differentiation processes including reactivation of latent developmental programs similar to those observed during de-stabilization of a mature cardiac myocyte phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13 (IL13), which induced cardiac myocyte cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of IL13 signaling in cardiac myocytes. These downstream signaling molecules are also modulated in the regenerating mouse heart. Conclusions: Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration.
Project description:The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in the case of zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with 5 of 6 elements directing injury-induced epicardial expression but not ontogenetic expression in hearts of larval animals. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration.
Project description:Unlike the adult mammalian heart, which has limited regenerative capacity, the zebrafish heart can fully regenerate following injury. Reactivation of cardiac developmental programmes is considered key to successfully regenerating the heart, yet the regulatory elements underlying the response triggered upon injury and during development remain elusive. Organ-wide activation of the epicardium is essential for zebrafish heart regeneration and is considered a potential regenerative source to target in the mammalian heart. Here we compared the transcriptome and epigenome of the developing and regenerating zebrafish epicardium by integrating gene expression profiles with open chromatin ATAC-seq data. We identified epicardial enhancer elements with specific activity during development or during adult heart regeneration. By generating gene regulatory networks associated with epicardial development and regeneration, we inferred genetic programmes driving each of these processes, which were largely distinct. We identified Wt1a, Wt1b, and the AP-1 subunits Junbb, Fosab and Fosb as central regulators of the developing network, whereas Hif1ab, Nrf1, Tbx2b and Zbtb7a featured as putative central regulators of the regenerating epicardial network. Targeting hif1ab, nrf1, tbx2b and zbtb7a using CRISPR/Cas9 in injured hearts resulted in elevated epicardial cell numbers infiltrating the wound and excess fibrosis after cryoinjury, illustrating the functional importance of these regulatory factors during zebrafish heart regeneration. Our work reveals striking differences between the regulatory blueprint deployed during epicardial development and regeneration. These findings underline that heart regeneration goes beyond the reactivation of developmental programmes and provide important insights into epicardial regulation.
Project description:Adult humans respond to heart injury by forming a permanent scar, yet other vertebrates are capable of robust and complete cardiac regeneration. Despite progress towards characterizing the mechanisms of cardiac regeneration in fish and amphibians, the large evolutionary gulf between mammals and regenerating vertebrates complicates deciphering which cellular and molecular features truly enable regeneration. To better define these features, we compared cardiac injury responses in zebrafish and medaka, two fish species that share similar heart anatomy and common teleost ancestry but differ in regenerative capability. We used single-cell transcriptional profiling to create a time-resolved comparative cell atlas of injury responses in all major cardiac cell types across both species. With this approach, we identified several key features that distinguish cardiac injury response in the non-regenerating medaka heart. By comparing immune responses to injury, we found altered cell recruitment and a distinct pro-inflammatory gene program in medaka leukocytes, and an absence of the injury-induced interferon response seen in zebrafish. In addition, we found a lack of pro-regenerative signals, including nrg1 and retinoic acid, from medaka endothelial and epicardial cells. Finally, we identified alterations in the myocardial structure in medaka, where they lack primordial layer cardiomyocytes and fail to employ a cardioprotective gene program shared by regenerating vertebrates. Our findings reveal notable variation in injury response across nearly all major cardiac cell types in zebrafish and medaka, demonstrating how evolutionary divergence influences the hidden cellular features underpinning regenerative potential in these seemingly similar vertebrates.