RESUMO
Transient receptor potential canonical type 6 (TRPC6) is a nonselective receptor-operated cation channel that regulates reactive fibrosis and growth signaling. Increased TRPC6 activity from enhanced gene expression or gain-of-function mutations contribute to cardiac and/or renal disease. Despite evidence supporting a pathophysiological role, no orally bioavailable selective TRPC6 inhibitor has yet been developed and tested in vivo in disease models. Here, we report an orally bioavailable TRPC6 antagonist (BI 749327; IC50 13 nM against mouse TRPC6, t1/2 8.5-13.5 hours) with 85- and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration â¼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis.
Assuntos
Cardiomegalia/tratamento farmacológico , Nefroesclerose/tratamento farmacológico , Canal de Cátion TRPC6/antagonistas & inibidores , Animais , Avaliação Pré-Clínica de Medicamentos , Fibrose , Células HEK293 , Coração/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , CamundongosRESUMO
Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.
Assuntos
Proteínas de Transporte/genética , Contração Miocárdica/genética , Miocárdio/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Coração/diagnóstico por imagem , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Fosforilação , Sarcômeros/metabolismoRESUMO
A limitation in the application of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. The mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, remain unclear. Here, we generate a single-cell RNA sequencing (scRNA-seq) reference of mouse in vivo CM maturation with extensive sampling of previously difficult-to-isolate perinatal time periods. We subsequently generate isogenic embryonic stem cells to create an in vitro scRNA-seq reference of PSC-CM-directed differentiation. Through trajectory reconstruction, we identify an endogenous perinatal maturation program that is poorly recapitulated in vitro. By comparison with published human datasets, we identify a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. Our study can be leveraged toward improving the clinical viability of PSC-CMs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Animais , Camundongos , Miócitos Cardíacos , Diferenciação Celular , Células-Tronco Embrionárias , Fatores de Transcrição/genéticaRESUMO
Neurons can regulate the development, pathogenesis, and regeneration of target organs. However, the role of neurons during heart development and regeneration remains unclear. We genetically inhibited sympathetic innervation in vivo, which resulted in heart enlargement with an increase in cardiomyocyte number. Transcriptomic and protein analysis showed down-regulation of the two clock gene homologs Period1/Period2 (Per1/Per2) accompanied by up-regulation of cell cycle genes. Per1/Per2 deletion increased heart size and cardiomyocyte proliferation, recapitulating sympathetic neurondeficient hearts. Conversely, increasing sympathetic activity by norepinephrine treatment induced Per1/Per2 and suppressed cardiomyocyte proliferation. We further found that the two clock genes negatively regulate myocyte mitosis entry through the Wee1 kinase pathway. Our findings demonstrate a previously unknown link between cardiac neurons and clock genes in regulation of cardiomyocyte proliferation and heart size and provide mechanistic insights for developing neuromodulation strategies for cardiac regen5eration.
RESUMO
Cardiomyocytes undergo significant structural and functional changes after birth, and these fundamental processes are essential for the heart to pump blood to the growing body. However, due to the challenges of isolating single postnatal/adult myocytes, how individual newborn cardiomyocytes acquire multiple aspects of the mature phenotype remains poorly understood. Here we implement large-particle sorting and analyze single myocytes from neonatal to adult hearts. Early myocytes exhibit wide-ranging transcriptomic and size heterogeneity that is maintained until adulthood with a continuous transcriptomic shift. Gene regulatory network analysis followed by mosaic gene deletion reveals that peroxisome proliferator-activated receptor coactivator-1 signaling, which is active in vivo but inactive in pluripotent stem cell-derived cardiomyocytes, mediates the shift. This signaling simultaneously regulates key aspects of cardiomyocyte maturation through previously unrecognized proteins, including YAP1 and SF3B2. Our study provides a single-cell roadmap of heterogeneous transitions coupled to cellular features and identifies a multifaceted regulator controlling cardiomyocyte maturation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Fatores de Processamento de RNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Redes Reguladoras de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Receptores Ativados por Proliferador de Peroxissomo/genética , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Transcriptoma , Proteínas de Sinalização YAPRESUMO
Muscle contraction, which is initiated by Ca2+, results in precise sliding of myosin-based thick and actin-based thin filament contractile proteins. The interactions between myosin and actin are finely tuned by three isoforms of myosin binding protein-C (MyBP-C): slow-skeletal, fast-skeletal, and cardiac (ssMyBP-C, fsMyBP-C and cMyBP-C, respectively), each with distinct N-terminal regulatory regions. The skeletal MyBP-C isoforms are conditionally coexpressed in cardiac muscle, but little is known about their function. Therefore, to characterize the functional differences and regulatory mechanisms among these three isoforms, we expressed recombinant N-terminal fragments and examined their effect on contractile properties in biophysical assays. Addition of the fragments to in vitro motility assays demonstrated that ssMyBP-C and cMyBP-C activate thin filament sliding at low Ca2+. Corresponding 3D electron microscopy reconstructions of native thin filaments suggest that graded shifts of tropomyosin on actin are responsible for this activation (cardiac > slow-skeletal > fast-skeletal). Conversely, at higher Ca2+, addition of fsMyBP-C and cMyBP-C fragments reduced sliding velocities in the in vitro motility assays and increased force production in cardiac muscle fibers. We conclude that due to the high frequency of Ca2+ cycling in cardiac muscle, cardiac MyBP-C may play dual roles at both low and high Ca2+. However, skeletal MyBP-C isoforms may be tuned to meet the needs of specific skeletal muscles.
Assuntos
Proteínas de Transporte/fisiologia , Músculo Esquelético/fisiologia , Contração Miocárdica , Miocárdio , Actinas/metabolismo , Animais , Cálcio/metabolismo , Masculino , Contração Muscular , Isoformas de Proteínas/fisiologia , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Tropomiosina/metabolismoRESUMO
Striated cardiac and skeletal muscles play very different roles in the body, but they are similar at the molecular level. In particular, contraction, regardless of the type of muscle, is a precise and complex process involving the integral protein myofilaments and their associated regulatory components. The smallest functional unit of muscle contraction is the sarcomere. Within the sarcomere can be found a sophisticated ensemble of proteins associated with the thick filaments (myosin, myosin binding protein-C, titin, and obscurin) and thin myofilaments (actin, troponin, tropomyosin, nebulin, and nebulette). These parallel thick and thin filaments slide across one another, pulling the two ends of the sarcomere together to regulate contraction. More specifically, the regulation of both timing and force of contraction is accomplished through an intricate network of intra- and interfilament interactions belonging to each myofilament. This review introduces the sarcomere proteins involved in striated muscle contraction and places greater emphasis on the more recently identified and less well-characterized myofilaments: cardiac myosin binding protein-C, titin, nebulin, and obscurin. © 2017 American Physiological Society. Compr Physiol 7:675-692, 2017.