RESUMEN
Abnormalities of cardiomyocyte Ca(2+) homeostasis and excitation-contraction (E-C) coupling are early events in the pathogenesis of hypertrophic cardiomyopathy (HCM) and concomitant determinants of the diastolic dysfunction and arrhythmias typical of the disease. T-tubule remodelling has been reported to occur in HCM but little is known about its role in the E-C coupling alterations of HCM. Here, the role of T-tubule remodelling in the electro-mechanical dysfunction associated to HCM is investigated in the Δ160E cTnT mouse model that expresses a clinically-relevant HCM mutation. Contractile function of intact ventricular trabeculae is assessed in Δ160E mice and wild-type siblings. As compared with wild-type, Δ160E trabeculae show prolonged kinetics of force development and relaxation, blunted force-frequency response with reduced active tension at high stimulation frequency, and increased occurrence of spontaneous contractions. Consistently, prolonged Ca(2+) transient in terms of rise and duration are also observed in Δ160E trabeculae and isolated cardiomyocytes. Confocal imaging in cells isolated from Δ160E mice reveals significant, though modest, remodelling of T-tubular architecture. A two-photon random access microscope is employed to dissect the spatio-temporal relationship between T-tubular electrical activity and local Ca(2+) release in isolated cardiomyocytes. In Δ160E cardiomyocytes, a significant number of T-tubules (>20%) fails to propagate action potentials, with consequent delay of local Ca(2+) release. At variance with wild-type, we also observe significantly increased variability of local Ca(2+) transient rise as well as higher Ca(2+)-spark frequency. Although T-tubule structural remodelling in Δ160E myocytes is modest, T-tubule functional defects determine non-homogeneous Ca(2+) release and delayed myofilament activation that significantly contribute to mechanical dysfunction.
Asunto(s)
Cardiomiopatía Hipertrófica/fisiopatología , Acoplamiento Excitación-Contracción , Contracción Miocárdica , Miocitos Cardíacos/patología , Miofibrillas/patología , Sarcolema/patología , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patología , Citoesqueleto de Actina/ultraestructura , Potenciales de Acción , Animales , Calcio/metabolismo , Señalización del Calcio , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Transporte Iónico , Ratones , Ratones Noqueados , Microscopía Confocal , Mutación , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Miofibrillas/metabolismo , Miofibrillas/ultraestructura , Imagen Óptica , Sarcolema/metabolismo , Sarcolema/ultraestructura , Troponina T/genética , Troponina T/metabolismoRESUMEN
Tropomyosin (Tm) plays a central role in the regulation of muscle contraction and is present in three main isoforms in skeletal and cardiac muscles. In the present work we studied the functional role of α- and ßTm on force development by modifying the isoform composition of rabbit psoas skeletal muscle myofibrils and of regulated thin filaments for in vitro motility measurements. Skeletal myofibril regulatory proteins were extracted (78%) and replaced (98%) with Tm isoforms as homogenous ααTm or ßßTm dimers and the functional effects were measured. Maximal Ca(2+) activated force was the same in ααTm versus ßßTm myofibrils, but ßßTm myofibrils showed a marked slowing of relaxation and an impairment of regulation under resting conditions compared to ααTm and controls. ßßTm myofibrils also showed a significantly shorter slack sarcomere length and a marked increase in resting tension. Both these mechanical features were almost completely abolished by 10 mM 2,3-butanedione 2-monoxime, suggesting the presence of a significant degree of Ca(2+)-independent cross-bridge formation in ßßTm myofibrils. Finally, in motility assay experiments in the absence of Ca(2+) (pCa 9.0), complete regulation of thin filaments required greater ßßTm versus ααTm concentrations, while at full activation (pCa 5.0) no effect was observed on maximal thin filament motility speed. We infer from these observations that high contents of ßßTm in skeletal muscle result in partial Ca(2+)-independent activation of thin filaments at rest, and longer-lasting and less complete tension relaxation following Ca(2+) removal.