We are investigating the biophysical, biochemical, and structural properties of the cardiac isoforms of Drosophila melanogaster myosin. We identified two major isoforms expressed in the cardiac tube, “CardM1” and “CardM2,” which differ at the lip of the ATP-binding region, the relay domain, and the converter domain, suggesting unique mechanical and/or biochemical properties (PMID: 39799399). We expressed each of these MHC isoforms in the indirect flight muscles (IFMs) and tergal depressor of the trochanter (TDT or jump) muscles via transformation with the corresponding cDNA transgene. Ultrastructural analyses revealed that IFMs and TDT muscles expressing CardM1 or CardM2 assemble normal myofibrils; however, flies expressing CardM1 or CardM2 exhibited impaired jump and flight abilities. Mechanical assays of TDT muscle fibers demonstrated that CardM2 produces significantly greater stretch activation (the delayed increase in force generation following muscle stretch) relative to CardM1 and the TDT muscle control. CardM1 and CardM2 exhibit decreased actin-activated ATPase V max values and decreased in vitro actin filament sliding velocities compared to TDT muscle and IFM myosin isoforms. AlphaFold 3 3D modeling of S1 fragments of CardM1 and CardM2 bound to actin predicts differences in the coordination of loop 1, a region involved in phosphate release, with the lip of the ATP-binding region, suggesting a role for isoform-specific nucleotide affinity in myosin-based stretch activation. Ongoing efforts include introducing R237W and S532P mutations, which are linked to hereditary dilated cardiomyopathy (DCM) into CardM1 and CardM2 to determine molecular mechanisms behind DCM. 3D modeling predicts that these disease mutations alter the orientation of the relay domain, suggesting that the function of this key intermolecular communication domain might be altered.
Camillo et al. (Sun,) studied this question.