The rates of Ca ²⁺ dissociation and cross-bridge detachment from ventricular myofibrils as reported by a fluorescent cardiac troponin C

The rate-limiting step of cardiac muscle relaxation has been proposed to reside in the myofilament. Both the rates of cross-bridge detachment and Ca ²⁺ dissociation from troponin C (TnC) have been hypothesized to rate-limit myofilament inactivation. In this study we used a fluorescent TnC to measure both the rate of Ca ²⁺ dissociation from TnC and the rate of cross-bridge detachment from several different species of ventricular myofibrils. The fluorescently labeled TnC was sensitive to both Ca ²⁺ dissociation and cross-bridge detachment at low Ca ²⁺ (presence of EGTA), allowing for a direct comparison between the two proposed rates of myofilament inactivation. Unlike Ca ²⁺ dissociation from TnC, cross-bridge detachment varied in myofibrils from different species and was rate-limited by ADP release. At subphysiological temperatures (<20°C), the rate of Ca ²⁺ dissociation from TnC was faster than the rate of cross-bridge detachment in the presence of ADP. These results support the hypothesis that cross-bridge detachment rate-limits relaxation. However, Ca ²⁺ dissociation from TnC was not as temperature-sensitive as cross-bridge detachment. At a near physiological temperature (35°C) and ADP, the rate of cross-bridge detachment may actually be faster than the rate of Ca ²⁺ dissociation. This provides evidence that there may not be a simple, single rate-limiting step of myofilament inactivation.