Alterations in Ca2+ sensitive tension due to partial extraction of C-protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers.
C-protein, a substantial component of muscle thick filaments, has been postulated to have various functions, based mainly on results from biochemical studies. In the present study, effects on Ca(2+)-activated tension due to partial removal of C-protein were investigated in skinned single myocytes from rat ventricle and rabbit psoas muscle. Isometric tension was measured at pCa values of 7.0 to 4.5: (a) in untreated myocytes, (b) in the same myocytes after partial extraction of C-protein, and (c) in some myocytes, after readdition of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO2, 124 mM NaH2PO4, pH 5.9 (Offer et al., 1973; Hartzell and Glass, 1984). In addition, the extracting solution contained 0.2 mg/ml troponin and, for skeletal muscle, 0.2 mg/ml myosin light chain-2 in order to minimize loss of these proteins during the extraction procedure. Between 60 and 70% of endogenous C-protein was extracted from cardiac myocytes by a 1-h soak in extracting solution at 20-23 degrees C; a similar amount was extracted from psoas fibers during a 3-h soak at 25 degrees C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca2+ activation of tension was decreased. Readdition of C-protein to previously extracted myocytes resulted in recovery of both tension and slope to near their control values. The effects on tension did not appear to be due to disruption of cooperative activation of the thin filament, since C-protein extraction from cardiac myocytes that were 40-60% troponin-C (TnC) deficient produced effects similar to those observed in cells that were TnC replete. Measurements of the tension-pCa relationship in skeletal muscle fibers were also made at a sarcomere length of 3.5 microns which, because of the distribution of C-protein on the thick filament, should eliminate any interaction between C-protein and actin. The effects of C-protein extraction were similar at long and short sarcomere lengths. These data are consistent with a model in which C-protein modulates the range of movement of myosin, such that the probability of myosin binding to actin is increased after its extraction.
Department of Physiology, University of Wisconsin School of Medicine, Madison 53706.
P A
PA
Hofmann
H C
HC
Hartzell
R L
RL
Moss
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D002457:Cell Extracts; D002469:Cell Separation; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006321:Heart / Q000502:physiology*; D007537:Isometric Contraction / Q000187:drug effects / Q000502:physiology*; D009132:Muscles / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D009206:Myocardium / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D014336:Troponin / Q000494:pharmacology; D019209:Troponin C
D002352:Carrier Proteins; D002457:Cell Extracts; D014336:Troponin; D019209:Troponin C; C020723:citrate-binding transport protein; D002118:Calcium