Myosin Transducer Mutations Differentially Affect Motor Function, Myofibril Structure, and the Performance of Skeletal and Cardiac Muscles

Abstract

Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc 5 affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc 5 (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc 5 myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc 5 mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders. INTRODUCTIONThe myosin molecular motor of striated muscle is a hexameric molecule composed of two myosin heavy chains (MHCs) and four light chains. The N-terminal globular motor domain is a product inhibited ATPase comprised of several communicating domains and functional units (reviewed by Holmes, 1999, 2005;. Alterations to the various domains dramatically affect the biochemical and mechanical properties of the motor in vitro, and mutations that diminish or enhance the molecular performance of myosin in vivo are associated with the pathogenesis of both skeletal and cardiac myopathies (reviewed by Sellers, 1999;Redowicz, 2002;Oldfors et al., 2004;Chang and Potter, 2005;Laing and Nowak, 2005;Tardiff, 2005;Oldfors, 2007). Central to an understanding of myosinbased myopathies is a fundamental appreciation for how depressed or enhanced molecular motor function differentially affects diverse striated muscles.Among the communicating functional units of myosin is the recently described transducer region (Coureux et al., 2004). Myosin V crystal structures reveal the transducer's central location within the motor domain near the nucleotide binding site. The transducer includes the last three strands of a seven-stranded ␤-sheet, which undergoes distortion essential for rearrangements within the nucleotide pocket during the ATPase cycle, and the loops an...

Drosophila Model of Congenital Heart Diseases

“…Interestingly, these myosin mutants showed different pathologies in the heart. Compared to wild-type, D45 mutant hearts are dilated exhibiting an increased systolic and diastolic diameter, whereas Mhc 5 mutants appear restricted showing a decreased diameter only during diastolic phase (Cammarato et al, 2008a). The depressed motor function and dilation in D45 myosin is evocative of DCM in humans, whereas the increased motor function and reduced diastolic function in Mhc 5 is similar to human restricted cardiomyopathy (RCM, Cammarato et al, 2008a), a rare type of cardiomyopathy in which decreased myocardium elasticity affects the ventricular blood filling during the diastolic phase.…”

“…Ocorr et al, 2007a;Buechling et al, 2009;Choma et al, 2010). In particular, the contractile and electrical properties have been investigated in the Drosophila heart and found to be remarkably similar in fundamental aspects, such as the contribution of ion channels to heart contraction or the effects of mutations of genes of the myofibrillar apparatus (K. Ocorr, unpublished and Lalevée et al, 2006;Wolf et al, 2006;Ocorr et al, 2007b;Cammarato et al, 2008b;Mery et al, 2008). In addition to determining cardiac parameters in vivo and in situ (see below and Fig.…”

“…Two mutant alleles of myosin, D45 (A261T) and Mhc 5 (G200D), have missense mutations occurring close to the ATP catalytic site, and it was postulated that those amino acid substitutions would affect ATPase activity (Kronert et al, 1999). In fact, ATPase activities of both D45 and Mhc 5 mutant myosin were depressed compared to wild-type myosin; however, in vivo motility of F-actin on a myosin coated slide showed a reduced velocity for D45 myosin to almost half of that of wild-type and an increased velocity in Mhc 5 myosin to about 115% of wild-type (Cammarato et al, 2008a). Interestingly, these myosin mutants showed different pathologies in the heart.…”

Fluorescent Labeling of <em>Drosophila</em> Heart Structures

“…These tools permit unparalleled investigative power to study development, structure and function of cardiac muscle and its components. For example, mutational analysis of cytoarchitectural proteins in the heart has helped identify a variety of cardiomyopathy models in Drosophila 8,9 . Fluorescent labeling of the sarcomeric components of these models has revealed that cardiac dilation in flies is accompanied by myofibrillar disorganization and a loss of contractile material.…”

Pathologies et vieillissement cardiaque

“…Par exemple, une myosine hypoactive conduit chez la mouche adulte, comme chez l'homme, à une augmentation de la taille du coeur, une altération de sa fonction de contraction et une augmentation des arythmies avec l'âge caractéristiques d'une CMD. Une étude originale menée chez la drosophile a montré par ailleurs qu'une myosine hyperactive conduit en revanche à une diminution de la lumière cardiaque et une relaxation incomplète entre les contractions typiques d'une CMR, suggérant que chez l'homme, une augmentation de l'activité motrice de la myosine est à l'origine des formes rares de CMR [16]. Un avantage incontournable du modèle drosophile est la possibilité de réaliser des cribles afin d'identifier des gènes et des interactions génétiques jouant un rôle dans le maintien de la structure et de la fonction cardiaque, ou de sélectionner des drogues susceptibles de moduler la sévérité des cardiomyopathies.…”