A role for C-protein in the regulation of contraction and intracellular Ca2+ in intact rat ventricular myocytes.
1. C-protein is a major component of muscle thick filaments whose function is unknown. We have examined for the first time the role of the regulatory binding domain of C-protein in modulating contraction and intracellular Ca2+ concentration ([Ca2+]i) in intact cardiac myocytes. 2. Rat ventricular myocytes were reversibly permeabilised with the pore-forming toxin streptolysin O. Myosin S2 (which binds to the regulatory domain of C-protein) was introduced into cells during permeabilisation to compete with the endogenous C-protein-thick filament interaction. 3. Introduction of S2 into myocytes increased contractility by approximately 30%, significantly lengthened the time to peak of the contraction and the time to half-relaxation, but had no effect on [Ca2+]i transient amplitude. 4. Our data are consistent with increased myofilament Ca2+ sensitivity when there is reduced binding of C-protein to myosin near the head-tail junction. 5. We propose that the effects of introducing S2 into intact cardiac cells can be equated with the consequences of selectively phosphorylating C-protein in vivo, and that the regulation of contraction by C-protein is mediated by the effects of crossbridge cycling on the Ca2+ affinity of troponin C.
Calaghan
S C
SC
School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK. s.c.calaghan@leeds.ac.uk
Trinick
J
J
Knight
P J
PJ
White
E
E
D000818:Animals; D001426:Bacterial Proteins; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins; D002463:Cell Membrane Permeability / Q000187:drug effects; D005456:Fluorescent Dyes; D016257:Fura-2; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D008297:Male; D008955:Models, Cardiovascular; D009124:Muscle Proteins / Q000378:metabolism* / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000737:chemistry; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D015879:Myosin Subfragments / Q000032:analysis; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D012235:Rhodamines; D013301:Streptolysins / Q000494:pharmacology; D000099163:Myosin Binding Protein C
D001426:Bacterial Proteins; D002118:Calcium; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D016257:Fura-2; D009124:Muscle Proteins; D015879:Myosin Subfragments; D012235:Rhodamines; D013301:Streptolysins; D000099163:Myosin Binding Protein C; C025964:streptolysin O; C009434:tetramethylrhodamine isothiocyanate