Abstract
1. We investigated the effects of acidosis, inorganic phosphate (Pi) and caffeine on the Ca2+ affinity of isolated fast-twitch skeletal and cardiac troponin C (TnC), labelled with fluorescent probes to report Ca2+ binding to the regulatory sites. We also measured the effects of these interventions on the maximum force development and the Ca2+ sensitivity of skinned fibres from fast-twitch skeletal muscle and cardiac muscle, as has been done previously. The two types of experiment were carried out under similar solution conditions, so that we could assess the contribution of any direct actions on TnC to the modulation of Ca2+ sensitivity in the skinned muscle fibres. 2. In skinned fibres, acidosis (decreasing pH from 7.0 to 6.2) and Pi (20 mM) suppressed maximum force to the same extent within a given muscle type, but had greater effects on cardiac fibres compared with skeletal fibres. Caffeine (20 mM) depressed maximum force equally in cardiac and skeletal muscle. Thus, the fall of force induced by acidosis or Pi may involve a different mechanism from that induced by caffeine. 3. Skinned skeletal fibres were more Ca2+ sensitive than cardiac fibres by 0.29 pCa units (pCa = -log10[Ca2+]). Isolated skeletal TnC also had a greater Ca2+ affinity than cardiac TnC, by 0.20 pCa units. These results suggest that the Ca2+ sensitivity of skinned fibres is at least partly determined by the type of TnC present. 4. Acidosis reduced the Ca2+ sensitivity of force in skinned fibres profoundly and had a 2-fold greater effect in cardiac muscle than skeletal muscle (falls in pCa for 50% activation, pCa50, were 1.09 and 0.55, respectively). Acidosis also reduced the Ca2+ affinity of TnC, again having double the effect on the pCa50 for cardiac TnC (0.58) as on that for skeletal TnC (0.28). The greater effect of acidosis on cardiac skinned fibres, compared with skeletal, may be partly explained, therefore, by the type of TnC present, and one-half of the effect on fibres may be attributed to the direct effect of H+ on TnC. 5. Pi reduced the Ca2+ sensitivity of force in skeletal and cardiac skinned fibres by 0.30 and 0.19 pCa units, respectively. However, the Ca2+ affinity of isolated cardiac and skeletal TnC was unaffected by Pi, indicating that the decrease in muscle Ca2+ sensitivity is not mediated by a direct action of Pi on TnC. 6. Caffeine increased the Ca2+ sensitivity of cardiac skinned fibres by 0.31 pCa units, which was 3 times greater than for the skeletal fibres (0.09 pCa units).(ABSTRACT TRUNCATED AT 400 WORDS)