RELATION BETWEEN STEADY-STATE FORCE AND INTRACELLULAR [CA-2+] IN INTACT HUMAN MYOCARDIUM - INDEX OF MYOFIBRILLAR RESPONSIVENESS TO CA-2+

A novel approach was developed allowing the measurement of steady-state force and intracellular calcium concentration ([Ca2+]i) in tetanized human ventricular trabeculae carneae without pharmacological intervention. We compared and contrasted three methods of assessing calcium sensitivity of the myofilaments: 1) force-[Ca2+] relations in skinned fiber preparations, 2) peak twitch force-peak [Ca2+]i relations, and 3) steady-state force-[Ca2+]i relations in intact muscles. Steady-state contractile activation was achieved rapidly by tetanizing intact human trabeculae, loaded with aequorin, a Ca2+-sensitive bioluminescent protein, at a stimulation frequency of 15-20 Hz. Steady-state force and [Ca2+]i were measured during tetani, and the force versus [Ca2+]i relation was obtained by varying the extracellular calcium concentration ([Ca2+]o). Force-[Ca2+]i relations obtained from control and myopathic hearts were fitted to the Hill equation: %Force=[Ca2+]inh/([Ca2+] inh50%+[Ca2+]i nh), where nh is the Hill coefficient, and [Ca2+]50% is the [Ca2+] required for 50% activation. The curves of tetani had Hill coefficients of 5.21±0.20 (n=6) and 5.61±0.60 (n=10) and [Ca2+]50% of 0.56±0.05 μM (n=6) and 0.54±0.09 μM (n=10) in control and myopathic muscles, respectively. We also constructed peak force-peak [Ca2+]i relations using isometric twitches from the same muscles. These curves were shifted toward higher [Ca2+]i compared with the steady-state force-[Ca2+]i curve derived from tetani. Ryanodine (1 μM), which increased the time course of the Ca2+ and force transients, shifted the peak force-peak [Ca2+]i relation to the left, without affecting the steady-state force-[Ca2+]i relation. Exposure to 10 mM caffeine shifted the steady-state force-[Ca2+]i relation to the left, whereas exposure to 3 μM isoproterenol shifted this relation to the right. Experiments using skinned fiber preparations were performed in parallel with experiments on intact muscles from the same hearts. The force-pCa (-log[Ca2+]) relations in saponin-skinned trabeculae from control and myopathic tissue were superimposable. Ryanodine (1 μM) had no effect on the force-pCa relation in skinned fibers. Maximal tension was evoked by the posttetanic twitch, which was larger than the tetanus. This potentiation was abolished in the presence of ryanodine, a sarcoplasmic reticulum inhibitor. We propose that the changes in the steady-state force-[Ca2+]i relations are correlated with alterations in the sensitivity of the myofilaments to Ca2+, whereas changes in the peak force-peak [Ca2+]i relations represent temporal changes in the twitch transient. In both intact aequorin-loaded trabeculae and saponin-skinned fibers, there were no differences in the force-[Ca2+] relation between control and myopathic hearts, indicating that the Ca2+ sensitivity of the myofilaments is unaltered in the diseased myocardium. This leads us to the conclusion that the steady-state force-[Ca2+]i relation can be used reliably to estimate sensitivity changes at the level of the myofilaments in lieu of the peak force-peak [Ca2+]i relation.