Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model

Abstract The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca 2+ -binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca 2+ -sensitivity and a decrease (0.8×) in cooperativity ( n H ) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP ( K 1 ) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate ( k 2 ) and its equilibrium constant ( K 2 ) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step ( k 4 ) decreased (0.5×), reversal step ( k −4 ) increased (2×), and the phosphate-release step (1/ K 5 ) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics—possibly caused by changes in the α -myosin heavy chain phosphorylation profile—as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.