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Background: Recent advances in single cell sequencing have led to an increased focus on the role of cell-type composition in phenotypic presentation and disease progression. Cell-type composition research in the heart is challenging due to large, frequently multinucleated cardiomyocytes that preclude most single cell approaches from obtaining accurate measurements of cell composition. Our in silico studies reveal that ignoring cell type composition when calculating differentially expressed genes (DEGs) can have significant consequences. For example, a relatively small change in cell abundance of only 10% can result in over 25% of DEGs being false positives. Methods: We have implemented an algorithmic approach that uses snRNAseq datasets as a reference to accurately calculate cell type compositions from bulk RNAseq datasets through robust data cleaning, gene selection, and multi-sample cross-subject and cross-cell-type deconvolution. We applied our approach to cardiomyocyte-specific α1A adrenergic receptor (CM-α1A-AR) knockout mice. 8-12 week-old mice (either WT or CM-α1A-KO) were subjected to permanent left coronary artery (LCA) ligation or sham surgery (n=4 per group). Transcriptomes from the infarct border zones were collected 3 days later and analyzed using our algorithm to determine cell-type abundances, corrected differential expression calculations using DESeq2, and validated these findings using RNAscope. Results: Uncorrected DEGs for the CM-α1A-KO X LCA interaction term featured many cell-type specific genes such as Timp4 (fibroblasts) and Aplnr (cardiomyocytes) and overall GO enrichment for terms pertaining to cardiomyocyte differentiation (P=3.1E-4). Using our algorithm, we observe a striking loss of cardiomyocytes and gain in fibroblasts in the α1A-KO + LCA mice that was not recapitulated in WT + LCA animals, although we did observe a similar increase in macrophage abundance in both conditions. This recapitulates prior results that showed a much more severe heart failure phenotype in CM-α1A-KO + LCA mice. Following correction for cell-type, our DEGs now highlight a novel set of genes enriched for GO terms such as cardiac contraction (P=3.7E-5) and actin filament organization (P=6.3E-5). Conclusions: Our algorithm identifies and corrects for cell-type abundance in bulk RNAseq datasets opening new avenues for research on novel genes and pathways as well as an improved understanding of the role of cardiac cell types in cardiovascular disease.
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Factors responsible for cardiomyocyte proliferation could serve as potential therapeutics to stimulate endogenous myocardial regeneration following insult, such as ischemic injury. A previously published forward genetics approach on cardiomyocyte cell cycle and ploidy led us to the transcription factor, Runx1. Here, we examine the effect of Runx1 on cardiomyocyte cell cycle during postnatal development and cardiac regeneration using cardiomyocyte-specific gain- and loss-of-function mouse models. RUNX1 is expressed in cardiomyocytes during early postnatal life, decreases to negligible levels by 3 wk of age, and increases upon myocardial injury, all consistent with observed rates of cardiomyocyte cell-cycle activity. Loss of Runx1 transiently stymied cardiomyocyte cell-cycle activity during normal postnatal development, a result that corrected itself and did not extend to the context of neonatal heart regeneration. On the other hand, cardiomyocyte-specific Runx1 overexpression resulted in an expansion of diploid cardiomyocytes in uninjured hearts and expansion of 4 N cardiomyocytes in the context of neonatal cardiac injury, suggesting Runx1 overexpression is sufficient to induce cardiomyocyte cell-cycle responses. Persistent overexpression of Runx1 for >1 mo continued to promote cardiomyocyte cell-cycle activity resulting in substantial hyperpolyploidization (≥8 N DNA content). This persistent cell-cycle activation was accompanied by ventricular dilation and adverse remodeling, raising the concern that continued cardiomyocyte cell cycling can have detrimental effects.NEW & NOTEWORTHY Runx1 is sufficient but not required for cardiomyocyte cell cycle.
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Ciclo Celular , Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core , Miócitos Cardíacos , Animais , Miócitos Cardíacos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regeneração , Camundongos , Animais Recém-Nascidos , Poliploidia , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND/PURPOSE: Racism and hidden bias experienced by underrepresented nursing students contribute to a loss of confidence and anxiety. The A-CHARM nursing project developed virtual simulation experiences for nursing students to practice how to address racism. 'Nik's Story' virtual simulation was created as part of the A-CHARM project. The purpose of this study was to examine the effectiveness of an education intervention, that included Nik's story, on cultural humility and cultural diversity awareness. METHOD: This quasi-experimental study included a convenience sample of final year nursing students. After informed consent, participants completed a pre-intervention questionnaire that included the Cultural Humility Scale "context for difference in perspective" subscale, and the Cultural Diversity Awareness questionnaire to assess baseline knowledge. Students participated in an education intervention that included a lecture, Nik's story virtual simulation experience, a debrief and then completed a post-education/simulation questionnaire that included usability/learner engagement questions and the Cultural Humility Scale "context for difference in perspective" subscale, and the Cultural Diversity Awareness questionnaire. RESULTS: Forty-seven students consented and completed the pre/post intervention questionnaire. Participants rated the effectiveness, engagement and usability of the simulation experience highly. There was a significant positive change in cultural humility "context for difference in perspective" subscale (pre-scores = 6.9, SD = 3.3; post-scores = 31.0, SD = 3.8, p < 0.001), and cultural diversity awareness (pre-scores = 95.4, SD = 8.9; post-scores = 103.4, SD = 9.8, p < 0.001). DISCUSSION: This intervention was effective in improving cultural humility and cultural diversity awareness in nursing students. CONCLUSION: Simulation experiences regarding racism in the clinical setting provide a strategy for students to learn how to professionally navigate unwanted experiences.
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The regulation of ploidy in cardiomyocytes is a complex and tightly regulated aspect of cardiac development and function. Cardiomyocyte ploidy can range from diploid (2N) to 8N or even 16N, and these states change during key stages of development and disease progression. Polyploidization has been associated with cellular hypertrophy to support normal growth of the heart, increased contractile capacity, and improved stress tolerance in the heart. Conversely, alterations to ploidy also occur during cardiac pathogenesis of diseases, such as ischemic and non-ischemic heart failure and arrhythmia. Therefore, understanding which genes control and modulate cardiomyocyte ploidy may provide mechanistic insight underlying cardiac growth, regeneration, and disease. This chapter summarizes the current knowledge regarding the genes involved in the regulation of cardiomyocyte ploidy. We discuss genes that have been directly tested for their role in cardiomyocyte polyploidization, as well as methodologies used to identify ploidy alterations. These genes encode cell cycle regulators, transcription factors, metabolic proteins, nuclear scaffolding, and components of the sarcomere, among others. The general physiological and pathological phenotypes in the heart associated with the genetic manipulations described, and how they coincide with the respective cardiomyocyte ploidy alterations, are further discussed in this chapter. In addition to being candidates for genetic-based therapies for various cardiac maladies, these genes and their functions provide insightful evidence regarding the purpose of widespread polyploidization in cardiomyocytes.
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Miócitos Cardíacos , Poliploidia , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proliferação de Células/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Ischemic heart failure continues to be a highly prevalent disease among westernized countries and there is great interest in understanding the mechanisms preventing or exacerbating disease progression. The literature suggests an important role for the activation of interleukin-13 or interleukin-4 signaling in improving ischemic heart failure outcomes after myocardial infarction in mice. Dupilumab, a neutralizing antibody that inhibits the shared IL13/IL4 receptor subunit IL4Rα, is widely used for conditions such as ectopic dermatitis in humans. If global depletion of IL4Rα influences ischemic heart failure, either in mice or in humans taking dupilumab, is unknown. Here, we investigated the pathophysiological effects of global IL4Rα genetic deletion in adult mice after surgically induced myocardial infarction (MI). We also determined heart failure risk in patients with ischemic heart disease and concomitant usage of dupilumab using the collaborative patient data network TriNetX. Global deletion of IL4Rα results in exacerbated cardiac dysfunction associated with reduced capillary size after myocardial infarction in mice. In agreement with our findings in mice, dupilumab treatment significantly increased the risk of heart failure development in patients with preexisting diagnosis of ischemic heart disease. Our results indicate that systemic IL4Rα signaling is protective against heart failure development in adult mice and human patients specifically following an ischemic event. Thus, the compelling evidence presented hereby advocates for the development of a randomized clinical trial specifically investigating heart failure development after myocardial ischemia in patients taking dupilumab for another underlying condition.NEW & NOTEWORTHY A body of literature suggests a protective role for IL4Rα signaling postmyocardial infarction in mice. Here, our observational study demonstrates that humans taking the IL4Rα neutralizing antibody, dupilumab, have increased incidence of heart failure following an ischemic event. Similarly, global IL4Rα deletion in mice exacerbates heart failure postinfarct. To our knowledge, this is the first study reporting an adverse association in humans of dupilumab use with heart failure following a cardiac ischemic event.
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Cardiopatias , Insuficiência Cardíaca , Infarto do Miocárdio , Isquemia Miocárdica , Animais , Humanos , Camundongos , Anticorpos Neutralizantes/efeitos adversos , Anticorpos Neutralizantes/imunologia , Infarto do Miocárdio/genética , Isquemia Miocárdica/genéticaRESUMO
There is great interest in identifying signaling pathways that promote cardiac repair after myocardial infarction (MI). Prior studies suggest a beneficial role for IL-13 signaling in neonatal heart regeneration; however, the cell types mediating cardiac regeneration and the extent of IL-13 signaling in the adult heart after injury are unknown. We identified an abundant source of IL-13 and the related cytokine, IL-4, in neonatal cardiac type 2 innate lymphoid cells, but this phenomenon declined precipitously in adult hearts. Moreover, IL-13 receptor deletion in macrophages impaired cardiac function and resulted in larger scars early after neonatal MI. By using a combination of recombinant IL-13 administration and cell-specific IL-13 receptor genetic deletion models, we found that IL-13 signaling specifically to macrophages mediated cardiac functional recovery after MI in adult mice. Single transcriptomics revealed a subpopulation of cardiac macrophages in response to IL-13 administration. These IL-13-induced macrophages were highly efferocytotic and were identified by high IL-1R2 expression. Collectively, we elucidated a strongly proreparative role for IL-13 signaling directly to macrophages following cardiac injury. While this pathway is active in proregenerative neonatal stages, reactivation of macrophage IL-13 signaling is required to promote cardiac functional recovery in adults.
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Interleucina-13 , Infarto do Miocárdio , Camundongos , Animais , Interleucina-13/metabolismo , Imunidade Inata , Linfócitos/metabolismo , Macrófagos/metabolismo , Receptores de Interleucina-13/metabolismoRESUMO
Cardiomyocyte proliferation is a difficult phenomenon to capture and prove. Here we employ a retrospective analysis of single cell ventricular suspensions to definitively identify cardiomyocytes that have completed cell division. Through this analysis we determined that the capacity of cardiomyocytes to re-enter the cell cycle and complete cell division after injury are separate and variable traits. Further, we provide evidence that Tnni3k definitively influences both early and final stages of the cell cycle.
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Coração , Miócitos Cardíacos , Ciclo Celular , Divisão Celular , Proliferação de Células , Ventrículos do Coração , Miócitos Cardíacos/metabolismo , Estudos Retrospectivos , Animais , CamundongosRESUMO
Somatic polyploidization, an adaptation by which cells increase their DNA content to support growth, is observed in many cell types, including cardiomyocytes. Although polyploidization is believed to be beneficial, progression to a polyploid state is often accompanied by loss of proliferative capacity. Recent work suggests that genetics heavily influence cardiomyocyte ploidy. However, the developmental course by which cardiomyocytes reach their final ploidy state has only been investigated in select backgrounds. Here, we assessed cardiomyocyte number, cell cycle activity, and ploidy dynamics across two divergent mouse strains: C57BL/6J and A/J. Both strains are born and reach adulthood with comparable numbers of cardiomyocytes; however, the end composition of ploidy classes and developmental progression to reach the final state differ substantially. We expand on previous findings that identified Tnni3k as a mediator of cardiomyocyte ploidy and uncover a role for Runx1 in ploidy dynamics and cardiomyocyte cell division, in both developmental and injury contexts. These data provide novel insights into the developmental path to cardiomyocyte polyploidization and challenge the paradigm that hypertrophy is the sole mechanism for growth in the postnatal heart.
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Miócitos Cardíacos , Ploidias , Animais , Camundongos , Miócitos Cardíacos/metabolismo , Camundongos Endogâmicos C57BL , Poliploidia , Patrimônio Genético , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Introduction: While Yap and Wwtr1 regulate resident cardiac fibroblast to myofibroblast differentiation following cardiac injury, their role specifically in activated myofibroblasts remains unexplored. Methods: We assessed the pathophysiological and cellular consequence of genetic depletion of Yap alone (Yap fl/fl ;Postn MCM ) or Yap and Wwtr1 (Yap fl/fl ;Wwtr1 fl/+ ;Postn MCM ) in adult mouse myofibroblasts following myocardial infarction and identify and validate novel downstream factors specifically in cardiac myofibroblasts that mediate pathological remodeling. Results: Following myocardial infarction, depletion of Yap in myofibroblasts had minimal effect on heart function while depletion of Yap/Wwtr1 resulted in smaller scars, reduced interstitial fibrosis, and improved ejection fraction and fractional shortening. Single cell RNA sequencing of interstitial cardiac cells 7 days post infarction showed suppression of pro-fibrotic genes in fibroblasts derived from Yap fl/fl ,Wwtr1 fl/+ ;Postn MCM hearts. In vivo myofibroblast depletion of Yap/Wwtr1 as well in vitro knockdown of Yap/Wwtr1 dramatically decreased RNA and protein expression of the matricellular factor Ccn3. Administration of recombinant CCN3 to adult mice following myocardial infarction remarkably aggravated cardiac function and scarring. CCN3 administration drove myocardial gene expression of pro-fibrotic genes in infarcted left ventricles implicating CCN3 as a novel driver of cardiac fibrotic processes following myocardial infarction. Discussion: Yap/Wwtr1 depletion in myofibroblasts attenuates fibrosis and significantly improves cardiac outcomes after myocardial infarction and we identify Ccn3 as a factor downstream of Yap/Wwtr1 that contributes to adverse cardiac remodeling post MI. Myofibroblast expression of Yap, Wwtr1, and Ccn3 could be further explored as potential therapeutic targets for modulating adverse cardiac remodeling post injury.
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During the past two decades, the field of mammalian myocardial regeneration has grown dramatically, and with this expanded interest comes increasing claims of experimental manipulations that mediate bona fide proliferation of cardiomyocytes. Too often, however, insufficient evidence or improper controls are provided to support claims that cardiomyocytes have definitively proliferated, a process that should be strictly defined as the generation of two de novo functional cardiomyocytes from one original cardiomyocyte. Throughout the literature, one finds inconsistent levels of experimental rigor applied, and frequently the specific data supplied as evidence of cardiomyocyte proliferation simply indicate cell-cycle activation or DNA synthesis, which do not necessarily lead to the generation of new cardiomyocytes. In this review, we highlight potential problems and limitations faced when characterizing cardiomyocyte proliferation in the mammalian heart, and summarize tools and experimental standards, which should be used to support claims of proliferation-based remuscularization. In the end, definitive establishment of de novo cardiomyogenesis can be difficult to prove; therefore, rigorous experimental strategies should be used for such claims.
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Miócitos Cardíacos , Regeneração , Animais , Ciclo Celular , Proliferação de Células , Coração/fisiologia , Mamíferos , Miócitos Cardíacos/fisiologiaRESUMO
Injury from myocardial infarction (MI) and consequent post-MI remodeling is accompanied by massive loss of cardiomyocytes (CM), a cell type critical for contractile function that is for all practical purposes non-regenerable due to its profound state of proliferative senescence. Identification of factors that limit CM survival and/or constrain CM renewal provides potential therapeutic targets. Tip60, a pan-acetyltransferase encoded by the Kat5 gene, has been reported to activate apoptosis as well as multiple anti-proliferative pathways in non-cardiac cells; however, its role in CMs, wherein it is abundantly expressed, remains unknown. Here, using mice containing floxed Kat5 alleles and a tamoxifen-activated Myh6-MerCreMer recombinase transgene, we report that conditional depletion of Tip60 in CMs three days after MI induced by permanent coronary artery ligation greatly improves functional recovery for up to 28 days. This is accompanied by diminished scarring, activation of cell-cycle transit markers in CMs within the infarct border and remote zones, reduced expression of cell-cycle inhibitors pAtm and p27, and reduced apoptosis in the remote regions. These findings implicate Tip60 as a novel, multifactorial target for limiting the damaging effects of ischemic heart disease.
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Acetiltransferases , Infarto do Miocárdio , Acetiltransferases/metabolismo , Acetiltransferases/farmacologia , Acetiltransferases/uso terapêutico , Animais , Apoptose/genética , Ciclo Celular , Lisina Acetiltransferase 5 , Camundongos , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , TransativadoresRESUMO
Neonatal heart regeneration depends on proliferation of pre-existing cardiomyocytes, yet the mechanisms driving regeneration and cardiomyocyte proliferation are not comprehensively understood. We recently reported that the anti-inflammatory cytokine, interleukin 13 (IL13), promotes neonatal cardiac regeneration; however, the signaling pathway and cell types mediating this regenerative response remain unknown. Here, we hypothesized that expression of the type II heterodimer receptor for IL13, comprised of IL4Rα and IL13Rα1, expressed directly on cardiomyocytes mediates cardiomyocyte cell cycle and heart regeneration in neonatal mice. Our data demonstrate that indeed global deletion of one critical subunit of the type II receptor, IL4Rα (IL4Rα-/-), decreases cardiomyocyte proliferation during early postnatal development and significantly impairs cardiac regeneration following injury in neonatal mice. While multiple myocardial cell types express IL4Rα, we demonstrate that IL4Rα deletion specifically in cardiomyocytes mediates cell cycle activity and neonatal cardiac regeneration. This demonstrates for the first time a functional role for IL4Rα signaling directly on cardiomyocytes in vivo. Reciprocally, we examined the therapeutic benefit of activating the IL4Rα receptor in non-regenerative hearts via IL13 administration. Following myocardial infarction, administration of IL13 reduced scar size and promoted cardiomyocyte DNA synthesis and karyokinesis, but not complete cytokinesis, in 6-day old non-regenerative mice. Our data demonstrate a novel role for IL4Rα signaling directly on cardiomyocytes during heart regeneration and suggest the potential for type II receptor activation as one potential therapeutic target for promoting myocardial repair.
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Coração/fisiologia , Miócitos Cardíacos/citologia , Receptores de Superfície Celular/metabolismo , Animais , Animais Recém-Nascidos , Ciclo Celular , Células Cultivadas , Feminino , Coração/crescimento & desenvolvimento , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Ratos , Receptores de Superfície Celular/genética , Regeneração , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Tip60, a pan-acetyltransferase encoded by the Kat5 gene, is enriched in the myocardium; however, its function in the heart is unknown. In cancer cells, Tip60 acetylates Atm (Ataxia-telangiectasia mutated), enabling its auto-phosphorylation (pAtm), which activates the DNA damage response (DDR). It was recently reported that activation of pAtm at the time of birth induces the DDR in cardiomyocytes (CMs), resulting in proliferative senescence. We therefore hypothesized that Tip60 initiates this process, and that depletion of Tip60 accordingly diminishes the DDR while extending the duration of CM cell-cycle activation. To test this hypothesis, an experimental model was used wherein a Myh6-driven Cre-recombinase transgene was activated on postnatal day 0 (P0) to recombine floxed Kat5 alleles and induce Tip60 depletion in neonatal CMs, without causing pathogenesis. Depletion of Tip60 resulted in reduced numbers of pAtm-positive CMs during the neonatal period, which correlated with reduced numbers of pH2A.X-positive CMs and decreased expression of genes encoding markers of the DDR as well as inflammation. This was accompanied by decreased expression of the cell-cycle inhibitors Meis1 and p27, activation of the cell-cycle in CMs, reduced CM size, and increased numbers of mononuclear/diploid CMs. Increased expression of fetal markers suggested that Tip60 depletion promotes a fetal-like proliferative state. Finally, infarction of Tip60-depleted hearts at P7 revealed improved cardiac function at P39 accompanied by reduced fibrosis, increased CM cell-cycle activation, and reduced apoptosis in the remote zone. These findings indicate that, among its pleiotropic functions, Tip60 induces the DDR in CMs, contributing to proliferative senescence.
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Pontos de Checagem do Ciclo Celular , Dano ao DNA , Lisina Acetiltransferase 5/metabolismo , Miócitos Cardíacos/metabolismo , Transativadores/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/genética , Biomarcadores , Modelos Animais de Doenças , Ecocardiografia , Expressão Gênica , Imuno-Histoquímica , Lisina Acetiltransferase 5/genética , Camundongos , Camundongos Transgênicos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Ploidias , Transativadores/genética , CicatrizaçãoRESUMO
Most mouse cardiomyocytes (CMs) become multinucleated shortly after birth via endoreplication and interrupted mitosis, which persists through adulthood. The very closely related inbred mouse strains BALB/cJ and BALB/cByJ differ substantially (6.6% vs. 14.3%) in adult mononuclear CM level. This difference is the likely outcome of a single X-linked polymorphic gene that functions in a CM-nonautonomous manner, and for which the BALB/cByJ allele is recessive to that of BALB/cJ. From whole exome sequence we identified two new X-linked protein coding variants that arose de novo in BALB/cByJ, in the genes Gdi1 (R276C) and Irs4 (L683F), but show that neither affects mononuclear CM level individually. No BALB/cJ-specific X-linked protein coding variants were found, implicating instead a variant that influences gene expression rather than encoded protein function. A substantially higher percentage of mononuclear CMs in BALB/cByJ are tetraploid (66.7% vs. 37.6% in BALB/cJ), such that the overall level of mononuclear diploid CMs between the two strains is similar. The difference in nuclear ploidy is the likely result of an autosomal polymorphism, for which the BALB/cByJ allele is recessive to that of BALB/cJ. The X-linked and autosomal genes independently influence mitosis such that their phenotypic consequences can be combined or segregated by appropriate breeding, implying distinct functions in karyokinesis and cytokinesis.
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Alelos , Núcleo Celular/genética , Miócitos Cardíacos/metabolismo , Ploidias , Animais , Feminino , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Proteínas Substratos do Receptor de Insulina/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
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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Traumatismos Cardíacos/terapia , Coração/fisiologia , Fator de Crescimento Insulin-Like II/metabolismo , Miócitos Cardíacos/fisiologia , Regeneração/fisiologia , Animais , Animais Recém-Nascidos , Diploide , Regulação da Expressão Gênica , Genótipo , Traumatismos Cardíacos/etiologia , Fator de Crescimento Insulin-Like II/genética , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
In mammals, most cardiomyocytes (CMs) become polyploid (they have more than two complete sets of chromosomes). The purpose of this review is to evaluate assumptions about CM ploidy that are commonly discussed, even if not experimentally demonstrated, and to highlight key issues that are still to be resolved. Topics discussed here include (a) technical and conceptual difficulties in defining a polyploid CM, (b) the candidate role of reactive oxygen as a proximal trigger for the onset of polyploidy, (c) the relationship between polyploidization and other aspects of CM maturation, (d) recent insights related to the regenerative role of the subpopulation of CMs that are not polyploid, and (e) speculations as to why CMs become polyploid at all. New approaches to experimentally manipulate CM ploidy may resolve some of these long-standing and fundamental questions.
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Miócitos Cardíacos/fisiologia , Poliploidia , Regeneração/fisiologia , Proliferação de Células , Humanos , Miocárdio/citologiaRESUMO
A major objective of modern biomedical research aims to promote tissue self-regeneration after injury, obviating the need for whole organ transplantation and avoiding mortality due to organ failure. Identifying the population of cells capable of regeneration, alongside understanding the molecular mechanisms that activate that population to re-enter the cell cycle, are two important steps to advancing the field of endogenous tissue regeneration toward the clinic. In recent years, an emerging trend has been observed, whereby polyploidy of relevant parenchymal cells, arising from alternative cell cycles as part of a normal developmental process, is linked to restricted proliferative capacity of those cells. An accompanying hypothesis, therefore, is that a residual subpopulation of diploid parenchymal cells retains proliferative competence and is the major driver for any detected postnatal cell turnover. In this perspective review, we examine the emerging literature on residual diploid parenchymal cells and the possible link of this population to endogenous tissue regeneration, in the context of both heart and liver. We speculate on additional cell types that may play a similar role in their respective tissues and discuss outstanding questions for the field.
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Poliploidia , Regeneração/genética , Engenharia Tecidual , Diploide , Coração/crescimento & desenvolvimento , Humanos , Fígado/crescimento & desenvolvimentoRESUMO
Recent evidence implicates mononuclear diploid cardiomyocytes as a proliferative and regenerative subpopulation of the postnatal heart. The number of these cardiomyocytes is a complex trait showing substantial natural variation among inbred mouse strains based on the combined influences of multiple polymorphic genes. One gene confirmed to influence this parameter is the cardiomyocyte-specific kinase Tnni3k. Here, we have studied Tnni3k alleles across a number of species. Using a newly-generated kinase-dead allele in mice, we show that Tnni3k function is dependent on its kinase activity. In an in vitro kinase assay, we show that several common human TNNI3K kinase domain variants substantially compromise kinase activity, suggesting that TNNI3K may influence human heart regenerative capacity and potentially also other aspects of human heart disease. We show that two kinase domain frameshift mutations in mice cause loss-of-function consequences by nonsense-mediated decay. We further show that the Tnni3k gene in two species of mole-rat has independently devolved into a pseudogene, presumably associated with the transition of these species to a low metabolism and hypoxic subterranean life. This may be explained by the observation that Tnni3k function in mice converges with oxidative stress to regulate mononuclear diploid cardiomyocyte frequency. Unlike other studied rodents, naked mole-rats have a surprisingly high (30%) mononuclear cardiomyocyte level but most of their mononuclear cardiomyocytes are polyploid; their mononuclear diploid cardiomyocyte level (7%) is within the known range (2-10%) of inbred mouse strains. Naked mole-rats provide further insight on a recent proposal that cardiomyocyte polyploidy is associated with evolutionary acquisition of endothermy.
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Evolução Molecular , Cardiopatias/genética , Proteínas Serina-Treonina Quinases/genética , Alelos , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Proliferação de Células/genética , Cardiopatias/metabolismo , Ventrículos do Coração/crescimento & desenvolvimento , Ventrículos do Coração/metabolismo , Humanos , Mutação com Perda de Função/genética , Camundongos , Ratos-Toupeira/genética , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/genética , Poliploidia , Regeneração/genéticaRESUMO
Adult mammalian cardiomyocyte regeneration after injury is thought to be minimal. Mononuclear diploid cardiomyocytes (MNDCMs), a relatively small subpopulation in the adult heart, may account for the observed degree of regeneration, but this has not been tested. We surveyed 120 inbred mouse strains and found that the frequency of adult mononuclear cardiomyocytes was surprisingly variable (>7-fold). Cardiomyocyte proliferation and heart functional recovery after coronary artery ligation both correlated with pre-injury MNDCM content. Using genome-wide association, we identified Tnni3k as one gene that influences variation in this composition and demonstrated that Tnni3k knockout resulted in elevated MNDCM content and increased cardiomyocyte proliferation after injury. Reciprocally, overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. Our results corroborate the relevance of MNDCMs in heart regeneration. Moreover, they imply that intrinsic heart regeneration is not limited nor uniform in all individuals, but rather is a variable trait influenced by multiple genes.
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Diploide , Coração/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Regeneração/fisiologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Immunoblotting , Hibridização in Situ Fluorescente , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Miocárdio/citologia , Miócitos Cardíacos/citologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Regeneração/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The hearts of many mammalian species are surrounded by an extensive layer of fat called epicardial adipose tissue (EAT). The lineage origins and determinative mechanisms of EAT development are unclear, in part because mice and other experimentally tractable model organisms are thought to not have this tissue. In this study, we show that mouse hearts have EAT, localized to a specific region in the atrial-ventricular groove. Lineage analysis indicates that this adipose tissue originates from the epicardium, a multipotent epithelium that until now is only established to normally generate cardiac fibroblasts and coronary smooth muscle cells. We show that adoption of the adipocyte fate in vivo requires activation of the peroxisome proliferator activated receptor gamma (PPARγ) pathway, and that this fate can be ectopically induced in mouse ventricular epicardium, either in embryonic or adult stages, by expression and activation of PPARγ at times of epicardium-mesenchymal transformation. Human embryonic ventricular epicardial cells natively express PPARγ, which explains the abundant presence of fat seen in human hearts at birth and throughout life.