RESUMO
Precise regulation of thin filament length is essential for optimal force generation during muscle contraction. The thin filament capping protein tropomodulin (Tmod) contributes to thin filament length uniformity by regulating elongation and depolymerization at thin filament ends. The leiomodins (Lmod1-3) are structurally related to Tmod1-4 and also localize to actin filament pointed ends, but in vitro biochemical studies indicate that Lmods act instead as robust nucleators. Here, we examined the roles of Tmod4 and Lmod3 during Xenopus skeletal myofibrillogenesis. Loss of Tmod4 or Lmod3 resulted in severe disruption of sarcomere assembly and impaired embryonic movement. Remarkably, when Tmod4-deficient embryos were supplemented with additional Lmod3, and Lmod3-deficient embryos were supplemented with additional Tmod4, sarcomere assembly was rescued and embryonic locomotion improved. These results demonstrate for the first time that appropriate levels of both Tmod4 and Lmod3 are required for embryonic myofibrillogenesis and, unexpectedly, both proteins can function redundantly during in vivo skeletal muscle thin filament assembly. Furthermore, these studies demonstrate the value of Xenopus for the analysis of contractile protein function during de novo myofibril assembly.
Assuntos
Embrião não Mamífero , Desenvolvimento Muscular/genética , Proteínas Musculares/biossíntese , Tropomodulina/biossíntese , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteínas dos Microfilamentos , Contração Muscular/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Sarcômeros/genética , Sarcômeros/ultraestrutura , Tropomodulina/genética , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
In vitro cultures of cardiomyocytes have proven to be a useful tool for toxicological, pharmacological, and developmental studies, as well as for the study of the cellular and molecular mechanisms responsible for proper myocyte function. One deficient area of research is that of myocyte proliferation. Cardiomyocyte proliferation dramatically diminishes soon after birth and has a very limited occurrence within the adult heart, thus limiting the use of adult cells for proliferation studies. An improved understanding of the requirements for myocyte proliferation will allow for the development of better approaches to repair damaged heart tissue. Here, we provide a protocol for the reliable isolation of embryonic mouse myocytes. These myocytes behave similarly to those in vivo, including their ability to proliferate, providing an ideal system for the study of cardiomyocyte proliferation.