Modulation of the rate of cardiac muscle contraction by troponin C constructs with various calcium binding affinities

Abstract

Norman C, Rall JA, Tikunova SB, Davis JP. Modulation of the rate of cardiac muscle contraction by troponin C constructs with various calcium binding affinities. Am J Physiol Heart Circ Physiol 293: H2580-H2587, 2007. First published August 10, 2007; doi:10.1152/ajpheart.00039.2007.-We investigated whether changing thin filament Ca 2ϩ sensitivity alters the rate of contraction, either during normal cross-bridge cycling or when cross-bridge cycling is increased by inorganic phosphate (P i). We increased or decreased Ca 2ϩ sensitivity of force production by incorporating into rat skinned cardiac trabeculae the troponin C (TnC) mutants V44QTnCF27W and F20QTnCF27W . The rate of isometric contraction was assessed as the rate of force redevelopment (ktr) after a rapid release and restretch to the original length of the muscle. Both in the absence of added Pi and in the presence of 2.5 mM added Pi F27W . This study suggests that TnC Ca 2ϩ binding properties modulate the rate of cardiac muscle contraction at submaximal levels of Ca 2ϩ activation. This result has physiological relevance considering that, on a beat-to-beat basis, the heart contracts at submaximal Ca 2ϩ activation.force; thin filament CARDIAC MUSCLE CONTRACTION is initiated by Ca 2ϩ binding to troponin C (TnC), which triggers conformational changes on the thin filament, exposing myosin-binding sites on actin. After the myosin heads (cross bridges) attach to actin, the thin filaments slide along the thick filaments and the muscle contracts (18). The kinetics of cross-bridge cycling can be studied with several approaches. One approach has been developed by Brenner (3, 4). According to this protocol, a rapid shorteningrestretch maneuver mechanically detaches the cross bridges, and the rate at which the cross bridges reattach and generate force is a measure of the rate of contraction, known as the rate of force (tension) redevelopment (k tr ).In both cardiac and skeletal muscle, it is well established that k tr becomes faster with increasing levels of activation by Ca 2ϩ (5,6,13,28,46). Many studies have investigated the role Ca 2ϩ plays in the activation dependence of k tr (for review, see Ref. 13). It has been proposed that the effects of Ca 2ϩ on k tr occur either as a direct effect of Ca 2ϩ on the cross bridges or indirectly by activation of the thin filament, which subsequently allows cross bridges to cycle from non-force-generating states to force-generating states. Studies in skeletal muscle investigated the hypothesis that Ca 2ϩ has a direct effect on the cross bridge cycle. Caged inorganic phosphate (P i ) experiments suggest that Ca 2ϩ does not regulate the kinetics of P i release but rather the distribution of cross bridges between non-force-generating and force-generating states (25,45). Similarly, in vitro motility assays have shown that Ca 2ϩ , through binding to TnC, controls the number of cross bridges interacting with actin rather than directly controlling the rate of ATP hydrolysis or the filament sliding speed (14, 17). Moreover, Ca 2ϩ does...

Calcium binding kinetics of troponin C strongly modulate cooperative activation and tension kinetics in cardiac muscle

“…L48Q cTn increased pCa 50 by ~0.4 units compared with WT cTn, and I61Q cTn decreased pCa 50 by ~0.5 units. These shifts in Ca 2+ sensitivity were anticipated based on our earlier work with similar skeletal TnC mutants in skeletal fibers [14] and the work of others in trabeculae [17]. However, there was an unexpected large reduction in n H (~4 units) for both L48Q and I61Q cTn (Table 3), indicating an apparent loss of cooperativity of tension generation with both mutants.…”

“…It has been demonstrated in skeletal muscle that the Ca 2+ binding properties of TnC can affect cooperative activation and tension development and relaxation kinetics [11–15]. Previous studies have also examined the relationships between specific point mutations in cardiac TnC (cTnC) and how they alter cTn Ca 2+ binding properties [16] and Ca 2+ sensitivity of tension in cardiac muscle [17]. However, it is not clear how cTnC Ca 2+ binding properties contribute to the complex cooperative interactions between the thin and thick filaments during thin filament activation in the organized structure of the cardiac sarcomere.…”

Effect of Calcium-Sensitizing Mutations on Calcium Binding and Exchange with Troponin C in Increasingly Complex Biochemical Systems

“…Further structural studies might be necessary to decipher why this mutation has detrimental effects on ability of TnC to regulate ATPase. However, even though the F20Q mutation significantly decreased maximal actomyosin ATPase activity, it was previously shown to only moderately decrease the maximal isometric tension development by TnC F27W reconstituted into skinned cardiac trabeculae (13). Similarly, several skeletal TnC F29W mutants significantly reduced maximal ATPase activity without affecting maximal isometric tension development by TnC F29W reconstituted into skinned skeletal muscle (38).…”

“…However, when these TnC F27W mutants were reconstituted into skinned cardiac trabeculae, F20QTnC F27W actually desensitized cardiac muscle to Ca 2+ (13, 14). These results indicate that in muscle additional factors can influence the apparent Ca 2+ binding properties of TnC.…”

Engineered Troponin C Constructs Correct Disease-related Cardiac Myofilament Calcium Sensitivity

“…Rat cardiac trabeculae were harvested and prepared from male LBN-F1 rats (175-200 g) as described previously (39). Briefly, rats were anesthetized via intraperitoneal injection of pentobarbital sodium (Nembutal, 50 mg/kg), and the thoracic cavity was opened.…”