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 microns. 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 mumol/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 (K1/2) (0.62 +/- 0.03 mumol/L). After skinning, the Hill coefficient fell to 2.72 and the K 1/2 shifted rightward to 2.2 mumol/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.(ABSTRACT TRUNCATED AT 250 WORDS)