Ca2+measurements in skinned cardiac fibers: effects of Mg2+on Ca2+activation of force and fiber ATPase

Abstract

In contrast to previous studies, a new fluorescent method was used to accurately determine the Ca²⁺concentration in test solutions used to activate skinned rat cardiac cells. This method used the calcium green-2 fluorescent indicator, which is shown to change its fluorescence over the Ca²⁺range responsible for Ca²⁺activation of force and ATPase. The dissociation constant ( K_d) of calcium green-2 for Ca²⁺was determined for three different Mg²⁺concentrations in solutions similar to those used in the experiment. Increasing Mg²⁺concentration from 1.0 to 8.0 mM had no significant effect on the Ca²⁺sensitivity of either force or actomyosin ATPase activity, in contrast to previous reported studies on force. The ATPase activity was activated at lower Ca²⁺concentration than the force. The ratio (ATPase/force) is proportional to the dissociation rate of force-generating myosin cross bridges and decreased during Ca²⁺activation. These findings are consistent with the hypothesis that cardiac muscle contraction is activated by a single Ca²⁺-specific binding site on troponin C.