Measurement of myofilament calcium sensitivity at physiological temperature in intact cardiac trabeculae

Abstract

Cardiac contraction-relaxation coupling is determined by both the free intracellular calcium concentration ([Ca2+]i) and myofilament properties. We set out to develop a technique where we could assess these parameters (twitch and steady-state force [Ca2+]i) under near physiological conditions. Bis-fura-2 was iontophorically introduced into ultrathin rat trabeculae preparations to monitor the [Ca2+]i, and steady-state contractures were achieved by using a modified Krebs-Henseleit solution containing high K+. During K+ contractures, the very slow changes in [Ca2+]i and force development were in equilibrium and allowed for the construction of a steady-state, force-[Ca2+]i relationship. Twitch contractions before and after this myofilament calcium sensitivity assessment were unaltered, and this protocol could be repeated several times. For the first time, this novel protocol allows us to measure myofilament calcium sensitivity under physiological temperature. Not only do the data so obtained allow us to assess myofilament calcium sensitivity, the data also will allow us, in the same preparation under nearly identical conditions, to compare the dynamic to the steady-state, force-calcium relationship. To test whether the steady-state relationship between force and calcium in our novel protocol reproduces expected changes, we determined this relationship in the presence of isoproterenol and under acidosis and alkalosis. As expected, beta-adrenergic stimulation resulted in an increase of calcium amplitude and twitch force and a desensitization of the myofilaments as indicated by a rightward shift of the obtained steady-state, force-calcium relationship. An increase in pH shifted the curve leftward, whereas a decrease in pH resulted in the expected rightward shift.