Myofilament Ca2+ sensitivity in intact versus skinned rat ventricular muscle

Abstract

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 (K1/2) (0.62±0.03 μmol/L). After skinning, the Hill coefficient fell to 2.72 and the K1/2 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 (K1/2 =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