Enhanced Ca²⁺ binding of cardiac troponin reduces sarcomere length dependence of contractile activation independently of strong crossbridges

Abstract

Calcium sensitivity of the force-pCa relationship depends strongly on sarcomere length (SL) in cardiac muscle and is considered to be the cellular basis of the Frank-Starling law of the heart. SL dependence may involve changes in myofilament lattice spacing and/or myosin crossbridge orientation to increase probability of binding to actin at longer SLs. We used the L48Q cardiac troponin C (cTnC) variant, which has enhanced Ca(2+) binding affinity, to test the hypotheses that the intrinsic properties of cTnC are important in determining 1) thin filament binding site availability and responsiveness to crossbridge activation and 2) SL dependence of force in cardiac muscle. Trabeculae containing L48Q cTnC-cTn lost SL dependence of the Ca(2+) sensitivity of force. This occurred despite maintaining the typical SL-dependent changes in maximal force (F(max)). Osmotic compression of preparations at SL 2.0 μm with 3% dextran increased F(max) but not pCa(50) in L48Q cTnC-cTn exchanged trabeculae, whereas wild-type (WT)-cTnC-cTn exchanged trabeculae exhibited increases in both F(max) and pCa(50). Furthermore, crossbridge inhibition with 2,3-butanedione monoxime at SL 2.3 μm decreased F(max) and pCa(50) in WT cTnC-cTn trabeculae to levels measured at SL 2.0 μm, whereas only F(max) was decreased with L48Q cTnC-cTn. Overall, these results suggest that L48Q cTnC confers reduced crossbridge dependence of thin filament activation in cardiac muscle and that changes in the Ca(2+) sensitivity of force in response to changes in SL are at least partially dependent on properties of thin filament troponin.

Thin filament incorporation of an engineered cardiac troponin C variant (L48Q) enhances contractility in intact cardiomyocytes from healthy and infarcted hearts

“…This hypothesis was based on previous results, where L48Q cTnC enhanced the Ca 2+ binding properties of cTn and thin filaments in solution, and the Ca 2+ sensitivity of myofilament contraction [23,24]. In this study we found that an ~20% myofilament incorporation of L48Q cTnC increased contractility of cardiomyocytes from both healthy hearts and hearts with systolic dysfunction (post-MI).…”

“…Recent work by our group [21,23,24] suggests that the mechanism of by which L48Q cTnC increases contraction of intact cardiomyocytes is by augmented Ca 2+ binding to cTn that results in increased thin filament activation at any given [Ca 2+ ]. Our solution studies, and work by others [22], have demonstrated that the L48Q point mutation significantly increases the Ca 2+ affinity of cTnC and increases the affinity of cTnC for cTnI in both the presence and absence of Ca 2+ .…”

“…The force-pCa relationship in demembranated right ventricular trabeculae from rats (male Sprague-Dawley, 200–250g) was determined after passive overnight exchange of native cTn with recombinant cTn complexes containing either 100% WT cTnC or a mix of 75% WT and 25% L48Q-cTnC, cmycTnT, cTnI as described [24]. Force-pCa data were fit by the Hill equation as previously described [20].…”

“…Previous work by us [21] and others [22] has demonstrated that a single amino acid substitution of leucine at position 48 with glutamine (L48Q) in cTnC increases the Ca 2+ binding affinity of the troponin complex (cTn). We have also demonstrated that when cTn containing L48Q cTnC (L48Q cTnC-cTn) was exchanged into demembranated cardiac muscle or cardiac myofibrils, the Ca 2+ sensitivity of force and the rate of force development was increased without impairing maximal force generation or relaxation [23,24]. We recently reported that exchange of L48Q cTnC-cTn into demembranated cardiac muscle increased thin filament activation at submaximal Ca 2+ concentrations independent of strong crossbridge binding.…”

“…We recently reported that exchange of L48Q cTnC-cTn into demembranated cardiac muscle increased thin filament activation at submaximal Ca 2+ concentrations independent of strong crossbridge binding. This effectively increased contraction at submaximal intracellular Ca 2+ levels that occur during cardiomyocyte twitch contraction [24]. We have also demonstrated that engraftment of neonatal cardiomyocytes into the infarct zone of adult rat hearts enhances the myofilament Ca 2+ sensitivity of cardiac tissue in the non-infarcted regions and is a major contributor to the improvement in systolic function [9].…”