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.

Determinants of frequency-dependent contraction and relaxation of mammalian myocardium

“…In isolated rabbit trabeculae, contracting isometrically at body temperature within the in vivo range of the rabbit, myofilament calcium sensitivity greatly decreased when stimulation frequency was increased [48]. This frequency dependent myofilament desensitization correlated with TnI phosphorylation, which is known to de-sensitize the myofilaments for activator Ca 2+ in skinned fibers [13] as well as in intact myocardium [49]. These results draw a significant parallel to IDAR.…”

Pulmonary artery banding alters the expression of Ca2+transport proteins in the right atrium in rabbits

“…Exposure to BDM for a short time has been shown to be reversible (28). Muscles were mounted in the setup as previously described (44,45) and stimulated at 1 Hz, which is slightly below their physiological resting rates, while perfused with an oxygenated Krebs-Henseleit solution, now without BDM and containing 2 mM Ca 2ϩ . The muscles were stretched until an increase in passive (diastolic) force was no longer accompanied by a substantial increase in developed force.…”

Energetics of stress production in isolated cardiac trabeculae from the rat

“…As shown in Fig. 9A We investigated these phenomena by examining dynamic stress-fluorescence phase-plane loops, the relaxation limbs of which index myofilament Ca 2ϩ sensitivity (46,47). Figure 9B shows rightward shifts of the relaxation trajectories of the dynamic phase-plane loops between the early and late bouts of stimulation at 0.2 Hz (a shifts to l) and 4 Hz (b shifts to k).…”