Myofilament Ca²⁺ sensitivity in intact versus skinned rat ventricular muscle

The Ca²⁺ sensitivity of myofilaments was compared before and after skinning in the same rat trabeculae at a diastolic sarcomere length of 2.2 to 2.3 μm. Trabeculae from rat right ventricle were loaded with fura-2 salt by iontophoretic microinjection, and [Ca²⁺](i) was determined from the epifluorescence at 510 nm when excited at 340 and 380 nm. Steady-state activation was achieved by stimulating the muscle at 10 Hz after 10 to 20 minutes of application of ryanodine (5 μmol/L). The muscles were then skinned with Triton X-100 (1%) for 15 to 25 minutes and subsequently activated with solutions containing varied [Ca²⁺]. The intact force- [Ca²⁺] relation was highly cooperative (Hill coefficient, 4.87±0.35; n=10), with a low [Ca²⁺](i) required for half-maximal activation (K( 1/2 )) (0.62±0.03 μmol/l). After skinning, the Hill coefficient fell to 2.72 and the K( 1/2 ) shifted rightward to 2.2 μmol/L in the presence of 1.2 mmol/L free Mg²⁺. Because of uncertainty regarding the appropriate [Mg²⁺], we measured [Mg²⁺](i) at 0.72±0.06 mmol/L (n=11) with Mg-fura-2 salt. When activating solutions were modified to contain [Mg²⁺]=0.5 mmol/L, the force- [Ca²⁺] relation was shifted to the left (K( 1/2 )=0.93±0.1, n=10) with a Hill coefficient of 3.75±0.37, but the changes were not sufficient to superimpose with the intact force-[Ca²⁺] relation (P<.05 versus intact). These results suggest that, despite the significant effect of Mg²⁺ on the force-[Ca²⁺] relation in skinned muscles, the Ca²⁺ responsiveness of the myofilaments is still altered by skinning. Possible factors that might underlie the observed discrepancy in the force-[Ca²⁺] relation between intact and skinned muscles include decreased Ca²⁺ binding to troponin (either by loss of natural calcium sensitizers or inadvertent proteolysis), alterations in myosin light chain phosphorylation, and changes in cross-bridge kinetics as a result of skinning.