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.