Abstract
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.
Highlights
The rate-limiting step of cardiac muscle relaxation is not completely understood
Measuring Ca2ϩ Dissociation—We have previously shown that TnCT53C labeled with IAANS reported the Ca2ϩ binding
In the absence of ADP, ATP-induced detachment of myosin from reconstituted skeletal and cardiac actomyosin is extremely fast (Ͼ500/s) [15, 47]. This may suggest that crossbridge detachment observed by TnCITA5N3CBD is sensed and limited by movement of another myofilament protein (tropomyosin (Tm), TnI, or TnT) or that the rate of cross-bridge detachment from the nucleotide free myofibrils is slower than that measured from reconstituted actomyosin
Summary
The rate-limiting step of cardiac muscle relaxation is not completely understood. Results: We were able to measure two proposed rate-limiting steps of relaxation in ventricular myofibrils. The rate-limiting step of cardiac muscle relaxation has been proposed to reside in the myofilament Both the rates of crossbridge detachment and Ca2؉ dissociation from troponin C (TnC) have been hypothesized to rate-limit myofilament inactivation. At subphysiological temperatures (
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