The R146N and R249Q Myosin Mutations Disrupt Motor Function and Myofibrillar Structure and cause Cardiomyopathy in Drosophila

Abstract

Numerous myosin-based myopathies have been identified without a detailed understanding of their molecular basis. To pursue mechanistic investigations, we generated transgenic Drosophila expressing the R146N or R249Q myosin mutations that cause hypertrophic cardiomyopathy in humans. Our studies show severely compromised flight ability in homozygotes, with an age-dependent worsening of muscle function and myofibrillar disarray in the indirect flight muscle ultrastructure. The R146N mutation caused an increase in basal Ca-ATPase and Mg-ATPase activities, with a significant decrease in actin motility. Semi-automated optical heartbeat analysis performed using high-speed movies of semi-intact mutant heterozygous hearts indicated restrictive cardiac physiology and diastolic dysfunction. Based upon molecular modeling, we predict 1) the R146N mutation alters ionic interaction of the N-terminal motor domain with E774 of the lever arm, resulting in failure of the lever arm to cock prior to the power-stroke; and 2) the R249Q mutation disrupts electrostatic interaction with D262 of the central β-sheet domain, interrupting communication between the ATP-binding and actin-binding sites. Thus, the models imply that these conserved charge interactions are critical for myosin function. To test these predictions, lines expressing R146E, E774R, R249D and D262R mutations, as well as putative suppressor lines R146E+E774R and R249D+D262R, will be studied using an integrative biochemical, biophysical and physiological approach. Transgenic Drosophila expressing either R146E or R249D myosin revealed more severe phenotypes relative to cardiomyopathy mutations. Both homozygous mutants displayed flightless phenotypes and highly compromised myofibrillar ultrastructure, with the R146E mutants showing increased basal Ca-ATPase and Mg-ATPase activities, and no actin motility. Heterozygotes present with severe restricted cardiac physiology and diastolic dysfunction. Overall our studies indicate that the Drosophila is a valuable tool to test significant myosin interactions during health and disease. Supported by NIH R01GM32443 to SIB.