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 Ca2+-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 Na2HPO~, 124 mM NaH2PO4, pH 5.9 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°C; a similar amount was extracted from psoas fibers during a 3-h soak at 25°C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca 2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca 2+ 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 I~m 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 Address reprint requests to Dr.

Mitogen-activated protein kinases (MAPKs) play different regulatory roles in signaling oxidative stress-induced apoptosis in cardiac ventricular myocytes. The regulation and functional role of cross-talk between p38 MAPK and extracellular signal-regulated kinase (ERK) pathways were investigated in cardiac ventricular myocytes in the present study. We demonstrated that inhibition of p38 MAPK with SB-203580 and SB-239063 enhanced H(2)O(2)-stimulated ERK phosphorylation, whereas preactivation of p38 MAPK with sodium arsenite reduced H(2)O(2)-stimulated ERK phosphorylation. In addition, pretreatment of cells with the protein phosphatase 2A (PP2A) inhibitors okadaic acid and fostriecin increased basal and H(2)O(2)-stimulated ERK phosphorylation. We also found that PP2A coimmunoprecipitated with ERK and MAPK/ERK (MEK) in cardiac ventricular myocytes, and H(2)O(2) increased the ERK-associated PP2A activity that was blocked by inhibition of p38 MAPK. Finally, H(2)O(2)-induced apoptosis was attenuated by p38 MAPK or PP2A inhibition, whereas it was enhanced by MEK inhibition. Thus the present study demonstrated that p38 MAPK activation decreases H(2)O(2)-induced ERK activation through a PP2A-dependent mechanism in cardiac ventricular myocytes. This represents a novel cellular mechanism that allows for interaction of two opposing MAPK pathways and fine modulation of apoptosis during oxidative stress.

SUMMARY1. Effects on maximum shortening velocity (Vmax) due to partial extraction of Cprotein were investigated in skinned fibres from rabbit psoas muscles. Up to 80 % of endogenous C-protein was extracted, as assessed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) of fibre segments obtained before and after the extraction protocol. Vmax was obtained at 15°C by measuring the times required to take up various amounts of slack imposed at one end of the fibre.2. During maximal activation with Ca2+, Vmax in control fibres was 4'26 +0-16 (mean+ S.E.M., n = 7) muscle lengths per second (ML/s). Following extraction of approximately 40 % of endogenous C-protein, Vmax in these same fibres was 4-41 + 0-24 ML/s.3. At sufficiently low levels of submaximal activation, high-and low-velocity phases of unloaded shortening were observed. Partial extraction of C-protein significantly increased Vmax in the low-velocity phase but had no effect on the highvelocity phase. The effect on low-velocity Vmax was fully reversed by re-addition of purified C-protein. 4. At low levels of activation, the amount of shortening to the break-point between the high-and low-velocity phases was not significantly affected by C-protein extraction. Under control conditions the average break-point was 85-6 + 3-1 nm/halfsarcomere, while 84-1 + 3-1 nm/half-sarcomere was obtained following partial extraction of C-protein.5. These results are considered in terms of a model in which an internal load slows Vmax at low levels of activation once a given amount of active shortening has occurred. C-protein may contribute to this internal load either by binding to actin and myosin or by influencing mechanical properties of myosin cross-bridges.

The sensitivity of skinned cardiac muscle bundles to Ca2+ is a function of sarcomere length. Ca2+ sensitivity is increased as fiber length is extended along the ascending limb of the force-length curve and it has been suggested that this phenomenon makes a major contribution to the steep force-length relationship that exists in living cardiac muscle. To gain greater insight into the mechanism behind the length dependence of Ca2+ sensitivity isotopic measurements of Ca2+ binding to detergent-extracted bovine, ventricular muscle bundles were made under conditions in which troponin C was the only major Ca2+ binding species. Experiments were designed to determine whether 1) Ca2+-troponin C affinity varies in the sarcomere length range corresponding to the ascending limb of the force-length curve, and 2) Ca2+ binding correlates with length per se or with changes in the number of length-dependent cross-bridge attachments. Measurements were made of Ca2+ binding in the rigor and relaxed states. The latter state was produced by suppressing actin-myosin interaction with the phosphate analogue, sodium vanadate. After vanadate treatment it is possible to obtain a complete Ca2+ saturation curve in the presence of physiological MgATP concentrations and at constant sarcomere length. The results show that the binding of Ca2+ to the regulatory site of cardiac troponin C is length dependent but this length dependence is actually a dependence on the number of attached cross bridges.

The duration of activation in cardiac muscle is a function of the load. On the basis of studies of Ca2+ transients in muscles subjected to quick release, it has been suggested that force or shortening-mediated changes in Ca2+-troponin C affinity may provide a mechanism for a contraction-activation feedback. This study was designed to test the hypothesis that the formation of force-generating complexes between actin and myosin enhances the affinity of cardiac troponin C for Ca2+. This was done by first establishing the normal relationship between Ca2+ binding and force development in chemically skinned bovine ventricular muscle bundles and then comparing the Ca2+-saturation curves obtained with relaxed and contracting muscle bundles. A double isotope technique was used to measure Ca2+ binding during ATP-induced force generation and during relaxation maintained by the phosphate analogue vanadate. The results showed that the generation of force was associated with an enhanced binding of Ca2+ to the Ca2+-specific regulatory site of cardiac troponin C. These data provide direct evidence that feedback between force and activation in the heart may be mediated by the Ca2+-regulatory site of troponin C.