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.
- cardiac muscle
- excitation-contraction coupling
- intracellular calcium
- intracellular magnesium
ASJC Scopus subject areas
- Cardiology and Cardiovascular Medicine