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The R249Q mutation in human β-cardiac myosin results in hypertrophic cardiomyopathy. We previously showed that inserting this mutation into Drosophila melanogaster indirect flight muscle myosin yields mechanical and locomotory defects. Here, we use transgenic Drosophila mutants to demonstrate that residue R249 serves as a critical communication link within myosin that controls both ATPase activity and myofibril integrity. R249 is located on a β-strand of the central transducer of myosin, and our molecular modeling shows that it interacts via a salt bridge with D262 on the adjacent β-strand. We find that disrupting this interaction via R249Q, R249D or D262R mutations reduces basal and actin-activated ATPase activity, actin in vitro motility and flight muscle function. Further, the R249D mutation dramatically affects myofibril assembly, yielding abnormalities in sarcomere lengths, increased Z-line thickness and split myofibrils. These defects are exacerbated during aging. Re-establishing the β-strand interaction via a R249D/D262R double mutation restores both basal ATPase activity and myofibril assembly, indicating that these properties are dependent upon transducer inter-strand communication. Thus, the transducer plays an important role in myosin function and myofibril architecture.

The myosin molecular motor interacts with actin filaments in an ATP-dependent manner to yield muscle contraction. Myosin heavy chain residue R369 is located within loop 4 at the actin-tropomyosin interface of myosin’s upper 50 kDa subdomain. To probe the importance of R369, we introduced a histidine mutation of that residue into Drosophila myosin and implemented an integrative approach to determine effects at the biochemical, cellular, and whole organism levels. Substituting the similarly charged but bulkier histidine residue reduces maximal actin binding in vitro without affecting myosin ATPase activity. R369H mutants exhibit impaired flight ability that is dominant in heterozygotes and progressive with age in homozygotes. Indirect flight muscle ultrastructure is normal in mutant homozygotes, suggesting that assembly defects or structural deterioration of myofibrils are not causative of reduced flight. Jump ability is also reduced in homozygotes. In contrast to these skeletal muscle defects, R369H mutants show normal heart ultrastructure and function, suggesting that this residue is differentially sensitive to perturbation in different myosin isoforms or muscle types. Overall, our findings indicate that R369 is an actin binding residue that is critical for myosin function in skeletal muscles, and suggest that more severe perturbations at this residue may cause human myopathies through a similar mechanism.

Valosin-containing protein (VCP) is a highly conserved mechanoenzyme that helps maintain protein homeostasis in all cells and serves specialized functions in distinct cell types. In skeletal muscle, it is critical for myofibrillogenesis and atrophy. However, little is known about VCP’s role(s) in the heart. Its functional diversity is determined by differential binding of distinct cofactors/adapters, which is likely disrupted during disease. VCP mutations cause multisystem proteinopathy (MSP), a pleiotropic degenerative disorder that involves inclusion body myopathy. MSP patients display progressive muscle weakness. They also exhibit cardiomyopathy and die from cardiac and respiratory failure, which are consistent with critical myocardial roles for the enzyme. Nonetheless, efficient models to interrogate VCP in cardiac muscle remain underdeveloped and poorly studied. Here, we investigated the significance of VCP and mutant VCP in the Drosophila heart. Cardiac-restricted RNAi-mediated knockdown of TER94, the Drosophila VCP homolog, severely perturbed myofibrillar organization and heart function in adult flies. Furthermore, expression of MSP disease-causing alleles engendered cardiomyopathy in adults and structural defects in embryonic hearts. Drosophila may therefore serve as a valuable model for examining role(s) of VCP in cardiogenesis and for identifying novel heart-specific VCP interactions, which when disrupted via mutation, contribute to or elicit cardiac pathology.