A Troponin-T Mutation Initiates Cardiac and Skeletal Myopathy due to Impaired Inhibition of Contraction in Drosophila Melanogaster

Abstract

Muscle contraction results from a series of molecular events that involve transient interactions between myosin-containing thick and actin-containing thin filaments. The high affinity of myosin for actin suggests that without regulation, muscle would remain in a continuous state of contraction. Regulation of striated muscle contraction is primarily achieved by Ca2+-dependent modulation of myosin crossbridge cycling on actin by the thin filament (TF) troponin-tropomyosin complex. Alterations in various subunits of the complex trigger contractile dysregulation and myopathy. For example, point mutations located over a span of ten amino acids (130-39) of human cardiac troponin T (cTnT) are associated with distinct cardiomyopathic responses. The Drosophila up101 (E88K) mutation localizes to the end of this well-conserved region of TnT. Here we examined the consequences of the lesion on distinct muscle types from the fly. Cardiac performance and morphology were assessed using direct immersion DIC optics, high-speed video imaging and motion analysis. Relative to controls, up101 hearts displayed a phenotype reminiscent of human restrictive cardiomyopathy. End-diastolic and end-systolic dimensions and percent fractional shortening were significantly reduced. Furthermore systolic intervals were significantly prolonged. This suggests TF dysregulation initiates excessive periods of force production and diastolic dysfunction. To characterize the fundamental cause of cardiac remodeling, we isolated TFs from the indirect flight muscles (IFM). The constitutively expressed up101 mutation results in flightlessness due to severe IFM hypercontraction. Electron microscopy and three-dimensional reconstruction of IFM TFs revealed Ca+2-dependent tropomyosin movement, typical of control filaments, was not a general feature of the TnT mutants. The vast majority of Ca+2-free mutant TFs exhibited tropomyosin associated with successive actin monomers over actin subdomains 3 and 4, distal to known myosin binding sites. Thus, up101 TnT likely promotes TF dysinhibition and ubiquitous myopathic remodeling.