Different effects of cardiac versus skeletal muscle regulatory proteins on in vitro measures of actin filament speed and force.
Mammalian cardiac and skeletal muscle express unique isoforms of the thin filament regulatory proteins, troponin (Tn) and tropomyosin (Tm), and the significance of these different isoforms in thin filament regulation has not been clearly identified. Both in vitro and skinned cellular studies investigating the mechanism of thin filament regulation in striated muscle have often used heterogeneous mixtures of Tn, Tm and myosin isoforms, and variability in reported results might be explained by different combinations of these proteins. Here we used in vitro motility and force (microneedle) assays to investigate the influence of cardiac versus skeletal Tn and Tm isoforms on actin-heavy meromyosin (HMM) mechanics. When interacting with skeletal HMM, thin filaments reconstituted with cardiac Tn/Tm or skeletal Tn/Tm exhibited similar speed-calcium relationships and significantly increased maximum speed and force per filament length (F/l) at pCa 5 (versus unregulated actin filaments). However, augmentation of F/l was greater with skeletal regulatory proteins. Reconstitution of thin filaments with the heterogeneous combination of skeletal Tn and cardiac Tm decreased sliding speeds at all [Ca2+] relative to thin filaments with skeletal Tn/Tm. Finally, for filaments reconstituted with any heterogeneous mix of Tn and Tm isoforms, force was not potentiated over that of unregulated actin filaments. Combined the results suggest (1) that cardiac regulatory proteins limit the allosteric enhancement of force, and (2) that Tn and Tm isoform homogeneity is important when studying Ca2+ regulation of crossbridge binding and kinetics as well as mechanistic differences between cardiac and skeletal muscle.
Clemmens
Emilie Warner
EW
Entezari
Michelle
M
Martyn
Donald A
DA
Regnier
Michael
M
D000199:Actins / Q000737:chemistry*; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D008297:Male; D020409:Molecular Motor Proteins / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000737:chemistry*; D009206:Myocardium / Q000737:chemistry*; D051381:Rats; D013314:Stress, Mechanical; D014335:Tropomyosin / Q000737:chemistry*; D014336:Troponin / Q000737:chemistry*
D000199:Actins; D020409:Molecular Motor Proteins; D014335:Tropomyosin; D014336:Troponin; D002118:Calcium