RESUMO
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.
Assuntos
Miócitos Cardíacos/fisiologia , Poliploidia , Regeneração/fisiologia , Proliferação de Células , Humanos , Miocárdio/citologiaRESUMO
TNNI3K expression worsens disease progression in several mouse heart pathology models. TNNI3K expression also reduces the number of diploid cardiomyocytes, which may be detrimental to adult heart regeneration. However, the gene is evolutionarily conserved, suggesting a beneficial function that has remained obscure. Here, we show that C57BL/6J-inbred Tnni3k mutant mice develop concentric remodeling, characterized by ventricular wall thickening and substantial reduction of cardiomyocyte aspect ratio. This pathology occurs in mice carrying a Tnni3k null allele, a K489R point mutation rendering the protein kinase-dead, or an allele corresponding to human I686T, the most common human non-synonymous TNNI3K variant, which is hypomorphic for kinase activity. Mutant mice develop these conditions in the absence of fibrosis or hypertension, implying a primary cardiomyocyte etiology. In culture, mutant cardiomyocytes were impaired in contractility and calcium dynamics and in protein kinase A signaling in response to isoproterenol, indicating diminished contractile reserve. These results demonstrate a beneficial function of TNNI3K in the adult heart that might explain its evolutionary conservation and imply that human TNNI3K variants, in particular the widespread I686T allele, may convey elevated risk for altered heart geometry and hypertrophy.
Assuntos
Cardiopatias/patologia , Contração Muscular , Mutação , Miócitos Cardíacos/patologia , Proteínas Serina-Treonina Quinases/genética , Remodelação Vascular , Animais , Cardiopatias/etiologia , Cardiopatias/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismoRESUMO
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.
Assuntos
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
BACKGROUND: The embryonic day E10-13 period of mouse heart development is characterized by robust cardiomyocyte proliferation that creates the compact zone of thickened ventricular wall myocardium. This process is initiated by the formation of the epicardium on the outer heart surface, which releases insulin-like growth factor 2 (IGF2) as the primary cardiomyocyte mitogen. Two receptors mediate IGF2 signaling, the IGF1R and the insulin receptor (INSR). RESULTS: In this study, we addressed the relative roles of the two IGF2 receptors in mouse heart development. We find that both receptors are expressed in the mouse heart during the E10-13 period, although IGF1R is much more prominently activated by IGF2 than INSR. Genetic manipulation indicates that only Igf1r is required for embryonic ventricular wall morphogenesis. INSR is not hyperactivated in the absence of IGF1R, and INSR does not compensate functionally for IGF1R in the absence of the latter. CONCLUSIONS: These results define the molecular components that are responsible for a major burst of cardiomyocyte proliferation during heart development. These results may also be relevant to understanding the efficiency of regeneration of the mammalian heart after neonatal and adult injury.
Assuntos
Coração/embriologia , Fator de Crescimento Insulin-Like II/metabolismo , Pericárdio/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animais , Proliferação de Células/fisiologia , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Organogênese , Pericárdio/crescimento & desenvolvimentoRESUMO
AIMS: RNA binding proteins play essential roles in mediating RNA splicing and are key post-transcriptional regulators in the heart. Our recent study demonstrated that RBPMS (RNA binding protein with multiple splicing) is crucial for cardiac development through modulating mRNA splicing, but little is known about its functions in the adult heart. In this study, we aim to characterize the post-natal cardiac function of Rbpms and its mechanism of action. METHODS AND RESULTS: We generated a cardiac-specific knockout mouse line and found that cardiac-specific loss of Rbpms caused severe cardiomyocyte contractile defects, leading to dilated cardiomyopathy and early lethality in adult mice. We showed by proximity-dependent biotin identification assay and mass spectrometry that RBPMS associates with spliceosome factors and other RNA binding proteins, such as RBM20, that are important in cardiac function. We performed paired-end RNA sequencing and RT-PCR and found that RBPMS regulates mRNA alternative splicing of genes associated with sarcomere structure and function, such as Ttn, Pdlim5, and Nexn, generating new protein isoforms. Using a minigene splicing reporter assay, we determined that RBPMS regulates target gene splicing through recognizing tandem intronic CAC motifs. We also showed that RBPMS knockdown in human induced pluripotent stem cell-derived cardiomyocytes impaired cardiomyocyte contraction. CONCLUSION: This study identifies RBPMS as an important regulator of cardiomyocyte contraction and cardiac function by modulating sarcomeric gene alternative splicing.
Assuntos
Processamento Alternativo , Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Camundongos , Conectina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Knockout , Miócitos Cardíacos/metabolismo , RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
During development, cells make switch-like decisions to activate new gene programs specifying cell lineage. The mechanisms underlying these decisive choices remain unclear. Here, we show that the cardiovascular transcriptional coactivator myocardin (MYOCD) activates cell identity genes by concentration-dependent and switch-like formation of transcriptional condensates. MYOCD forms such condensates and activates cell identity genes at critical concentration thresholds achieved during smooth muscle cell and cardiomyocyte differentiation. The carboxyl-terminal disordered region of MYOCD is necessary and sufficient for condensate formation. Disrupting this region's ability to form condensates disrupts gene activation and smooth muscle cell reprogramming. Rescuing condensate formation by replacing this region with disordered regions from functionally unrelated proteins rescues gene activation and smooth muscle cell reprogramming. Our findings demonstrate that MYOCD condensate formation is required for gene activation during cardiovascular differentiation. We propose that the formation of transcriptional condensates at critical concentrations of cell type-specific regulators provides a molecular switch underlying the activation of key cell identity genes during development.
Assuntos
Miócitos de Músculo Liso , Fatores de Transcrição , Linhagem da Célula/genética , Diferenciação Celular/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Miócitos de Músculo Liso/metabolismo , Ativação TranscricionalRESUMO
Light-sheet microscopy (LSM) enables us to strengthen the understanding of cardiac development, injury, and regeneration in mammalian models. This emerging technique decouples laser illumination and fluorescence detection to investigate cardiac micro-structure and function with a high spatial resolution while minimizing photodamage and maximizing penetration depth. To unravel the potential of volumetric imaging in cardiac development and repair, we sought to integrate our in-house LSM, Adipo-Clear, and virtual reality (VR) with neonatal mouse hearts. We demonstrate the use of Adipo-Clear to render mouse hearts transparent, the development of our in-house LSM to capture the myocardial architecture within the intact heart, and the integration of VR to explore, measure, and assess regions of interests in an interactive manner. Collectively, we have established an innovative and holistic strategy for image acquisition and interpretation, providing an entry point to assess myocardial micro-architecture throughout the entire mammalian heart for the understanding of cardiac morphogenesis.
Assuntos
Coração , Miocárdio , Animais , Camundongos , Animais Recém-Nascidos , Microscopia de Fluorescência/métodos , Coração/diagnóstico por imagem , MamíferosRESUMO
Adult hearts are characterized by inefficient regeneration after injury, thus, the features that support or prevent cardiomyocyte (CM) proliferation are important to clarify. Diploid CMs are a candidate cell type that may have unique proliferative and regenerative competence, but no molecular markers are yet known that selectively identify all or subpopulations of diploid CMs. Here, using the conduction system expression marker Cntn2-GFP and the conduction system lineage marker Etv1CreERT2, we demonstrate that Purkinje CMs that comprise the adult ventricular conduction system are disproportionately diploid (33%, vs. 4% of bulk ventricular CMs). These, however, represent only a small proportion (3%) of the total diploid CM population. Using EdU incorporation during the first postnatal week, we demonstrate that bulk diploid CMs found in the later heart enter and complete the cell cycle during the neonatal period. In contrast, a significant fraction of conduction CMs persist as diploid cells from fetal life and avoid neonatal cell cycle activity. Despite their high degree of diploidy, the Purkinje lineage had no enhanced competence to support regeneration after adult heart infarction.
RESUMO
Mutations in nuclear envelope proteins (NEPs) cause devastating genetic diseases, known as envelopathies, that primarily affect the heart and skeletal muscle. A mutation in the NEP LEM domain-containing protein 2 (LEMD2) causes severe cardiomyopathy in humans. However, the roles of LEMD2 in the heart and the pathological mechanisms responsible for its association with cardiac disease are unknown. We generated knockin (KI) mice carrying the human c.T38>G Lemd2 mutation, which causes a missense amino acid exchange (p.L13>R) in the LEM domain of the protein. These mice represent a preclinical model that phenocopies the human disease, as they developed severe dilated cardiomyopathy and cardiac fibrosis leading to premature death. At the cellular level, KI/KI cardiomyocytes exhibited disorganization of the transcriptionally silent heterochromatin associated with the nuclear envelope. Moreover, mice with cardiac-specific deletion of Lemd2 also died shortly after birth due to heart abnormalities. Cardiomyocytes lacking Lemd2 displayed nuclear envelope deformations and extensive DNA damage and apoptosis linked to p53 activation. Importantly, cardiomyocyte-specific Lemd2 gene therapy via adeno-associated virus rescued cardiac function in KI/KI mice. Together, our results reveal the essentiality of LEMD2 for genome stability and cardiac function and unveil its mechanistic association with human disease.
Assuntos
Cardiomiopatias , Membrana Nuclear , Humanos , Camundongos , Animais , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Dano ao DNA , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
Noncompaction cardiomyopathy is a common congenital cardiac disorder associated with abnormal ventricular cardiomyocyte trabeculation and impaired pump function. The genetic basis and underlying mechanisms of this disorder remain elusive. We show that the genetic deletion of RNA-binding protein with multiple splicing (Rbpms), an uncharacterized RNA-binding factor, causes perinatal lethality in mice due to congenital cardiovascular defects. The loss of Rbpms causes premature onset of cardiomyocyte binucleation and cell cycle arrest during development. Human iPSC-derived cardiomyocytes with RBPMS gene deletion have a similar blockade to cytokinesis. Sequencing analysis revealed that RBPMS plays a role in RNA splicing and influences RNAs involved in cytoskeletal signaling pathways. We found that RBPMS mediates the isoform switching of the heart-enriched LIM domain protein Pdlim5. The loss of Rbpms leads to an abnormal accumulation of Pdlim5-short isoforms, disrupting cardiomyocyte cytokinesis. Our findings connect premature cardiomyocyte binucleation to noncompaction cardiomyopathy and highlight the role of RBPMS in this process.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Proteínas de Ligação a RNA , Animais , Citocinese , Ventrículos do Coração/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
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.
Assuntos
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.
Assuntos
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
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.