Abstract
Calcium controls the level of muscle activation via interactions with the troponin complex. Replacement of the native, skeletal calcium-binding subunit of troponin, troponin C, with mixtures of functional cardiac and mutant cardiac troponin C insensitive to calcium and permanently inactive provides a novel method to alter the number of myosin cross-bridges capable of binding to the actin filament. Extraction of skeletal troponin C and replacement with functional and mutant cardiac troponin C were used to evaluate the relationship between the extent of thin filament activation (fractional calcium binding), isometric force, and the rate of force generation in muscle fibers independent of the calcium concentration. The experiments showed a direct, linear relationship between force and the number of cross-bridges attaching to the thin filament. Further, above 35% maximal isometric activation, following partial replacement with mixtures of cardiac and mutant troponin C, the rate of force generation was independent of the number of actin sites available for cross-bridge interaction at saturating calcium concentrations. This contrasts with the marked decrease in the rate of force generation when force was reduced by decreasing the calcium concentration. The results are consistent with hypotheses proposing that calcium controls the transition between weakly and strongly bound cross-bridge states.
Highlights
The cyclic interaction of myosin and actin produces force and shortening in contractile cells
In the present study we describe a method to control the fraction of troponin complexes to which calcium is bound, thereby controlling the fraction of the thin filament available for myosin binding while maintaining the free calcium troponin C; CBMII, cardiac binding mutant; REL, relaxing solution; ktr, rate of tension redevelopment; PAGE, polyacrylamide gel electrophoresis; kN, kilonewton; cTnC, cardiac TnC; sTnC, skeletal TnC; a, actin; m, myosin
It was important to determine whether the cardiac TnC or CBMII TnC preferentially bound to the fiber thin filaments under the extraction/replacement procedure
Summary
Tropomyosin; Tn, troponin; TnC, bridge attachment in the absence of calcium by “blocking” cross-bridge access to binding sites on the thin filament [3, 4]. To reconcile these observations with Brenner’s data and hypothesis it was proposed that [Ca2ϩ] controls the transition from weak to strong cross-bridge binding preceding the generation of force [11, 16] To this point, studies have investigated calcium regulation of muscle contraction by adding various compounds, removing proteins, or adjusting the free calcium concentration. In the present study we describe a method to control the fraction of troponin complexes to which calcium is bound, thereby controlling the fraction of the thin filament available for myosin binding while maintaining the free calcium troponin C; CBMII, cardiac binding mutant (site II); REL, relaxing solution; ktr, rate of tension redevelopment; PAGE, polyacrylamide gel electrophoresis; kN, kilonewton; cTnC, cardiac TnC; sTnC, skeletal TnC; a, actin; m, myosin A preliminary report of this work was published previously [22]
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