RESUMO
Centromere-binding protein F (CENP-F) is a very large and complex protein with many and varied binding partners including components of the microtubule network. Numerous CENP-F functions impacting diverse cellular behaviors have been identified. Importantly, emerging data have shown that CENP-F loss- or gain-of-function has critical effects on human development and disease. Still, it must be noted that data at the single cardiac myocyte level examining the impact of CENP-F loss-of-function on fundamental cellular behavior is missing. To address this gap in our knowledge, we analyzed basic cell structure and function in cardiac myocytes devoid of CENP-F. We found many diverse structural abnormalities including disruption of the microtubule network impacting critical characteristics of the cardiac myocyte. This is the first report linking microtubule network malfunction to cardiomyopathy. Importantly, we also present data demonstrating a direct link between a CENP-F single nucleotide polymorphism (snp) and human cardiac disease. In a proximate sense, these data examining CENP-F function explain the cellular basis underlying heart disease in this genetic model and, in a larger sense, they will hopefully provide a platform upon which the field can explore diverse cellular outcomes in wide-ranging areas of research on this critical protein.
Assuntos
Cardiomiopatia Dilatada/genética , Proteínas Cromossômicas não Histona/genética , Insuficiência Cardíaca/genética , Mutação com Perda de Função , Proteínas dos Microfilamentos/genética , Miócitos Cardíacos/patologia , Polimorfismo de Nucleotídeo Único , Animais , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Proteínas Cromossômicas não Histona/metabolismo , Modelos Animais de Doenças , Estudos de Associação Genética , Predisposição Genética para Doença , Insuficiência Cardíaca/fisiopatologia , Humanos , Junções Intercelulares/patologia , Camundongos , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/patologia , Miócitos Cardíacos/metabolismo , Volume SistólicoRESUMO
The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 µm long filaments that then 'stitch' together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.