The Ca2+/Mg2+ sites of troponin C modulate crossbridge-mediated thin filament activation in cardiac myofibrils

► Strong binding myosin crossbridges were used to activate cardiac myofibrillar ATPase. ► No Ca2+ was bound at the regulatory site II in the N-domain of troponin C. ► Activation was maximal when the high-affinity sites of troponin C were occupied by Mg2+. ► Activation was reduced when Ca2+ displaced Mg2+ from these sites. ► Ca2+/Mg2+ exchange in troponin C may modulate crossbridge kinetics in cardiac muscle.