Mechanical and kinetic effects of shortened tropomyosin reconstituted into myofibrils.
The effects of tropomyosin on muscle mechanics and kinetics were examined in skeletal myofibrils using a novel method to remove tropomyosin (Tm) and troponin (Tn) and then replace these proteins with altered versions. Extraction employed a low ionic strength rigor solution, followed by sequential reconstitution at physiological ionic strength with Tm then Tn. SDS-PAGE analysis was consistent with full reconstitution, and fluorescence imaging after reconstitution using Oregon-green-labeled Tm indicated the expected localization. Myofibrils remained mechanically viable: maximum isometric forces of myofibrils after sTm/sTn reconstitution (control) were comparable (~84%) to the forces generated by non-reconstituted preparations, and the reconstitution minimally affected the rate of isometric activation (k (act)), calcium sensitivity (pCa(50)), and cooperativity (n (H)). Reconstitutions using various combinations of cardiac and skeletal Tm and Tn indicated that isoforms of both Tm and Tn influence calcium sensitivity of force development in opposite directions, but the isoforms do not otherwise alter cross-bridge kinetics. Myofibrils reconstituted with Delta23Tm, a deletion mutant lacking the second and third of Tm's seven quasi-repeats, exhibited greatly depressed maximal force, moderately slower k (act) rates and reduced n (H). Delta23Tm similarly decreased the cooperativity of calcium binding to the troponin regulatory sites of isolated thin filaments in solution. The mechanisms behind these effects of Delta23Tm also were investigated using P ( i ) and ADP jumps. P ( i ) and ADP kinetics were indistinguishable in Delta23Tm myofibrils compared to controls. The results suggest that the deleted region of tropomyosin is important for cooperative thin filament activation by calcium.
Siththanandan
V B
VB
Physiology Department, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA. siththanandanvb@nhlbi.nih.gov
Tobacman
L S
LS
Van Gorder
N
N
Homsher
E
E
D000818:Animals; D002478:Cells, Cultured; D005260:Female; D007700:Kinetics; D019563:Mechanics; D009119:Muscle Contraction / Q000502:physiology*; D009210:Myofibrils / Q000737:chemistry* / Q000502:physiology*; D011817:Rabbits; D013314:Stress, Mechanical; D014335:Tropomyosin / Q000737:chemistry* / Q000502:physiology*
D014335:Tropomyosin