| First Author | Kawai M | Year | 2017 |
| Journal | Biophys J | Volume | 112 |
| Issue | 8 | Pages | 1726-1736 |
| PubMed ID | 28445763 | Mgi Jnum | J:307871 |
| Mgi Id | MGI:6725998 | Doi | 10.1016/j.bpj.2017.02.045 |
| Citation | Kawai M, et al. (2017) Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model. Biophys J 112(8):1726-1736 |
| abstractText | 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 degrees C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3x increase in Ca(2+)-sensitivity and a decrease (0.8x) in cooperativity (nH) 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 (K1) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k2) and its equilibrium constant (K2) increased (1.5x). The phosphate study revealed that rate constant of the force-generation step (k4) decreased (0.5x), reversal step (k-4) increased (2x), and the phosphate-release step (1/K5) increased (2x). 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 alpha-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. |
