Structural and functional impact of troponin C-mediated Ca2+ sensitization on myofilament lattice spacing and cross-bridge mechanics in mouse cardiac muscle

Abstract

Acto-myosin cross-bridge kinetics are important for beat-to-beat regulation of cardiac contractility; however, physiological and pathophysiological mechanisms for regulation of contractile kinetics are incompletely understood. Here we explored whether thin filament-mediated Ca sensitization influences cross-bridge kinetics in permeabilized, osmotically compressed cardiac muscle preparations. We used a murine model of hypertrophic cardiomyopathy (HCM) harboring a cardiac troponin C (cTnC) Ca-sensitizing mutation, Ala8Val in the regulatory N-domain. We also treated wild-type murine muscle with bepridil, a cTnC-targeting Ca sensitizer. Our findings suggest that both methods of increasing myofilament Ca sensitivity increase cross-bridge cycling rate measured by the rate of tension redevelopment (k); force per cross-bridge was also enhanced as measured by sinusoidal stiffness and I/I ratio from X-ray diffraction. Computational modeling suggests that Ca sensitization through this cTnC mutation or bepridil accelerates k primarily by promoting faster cross-bridge detachment. To elucidate if myofilament structural rearrangements are associated with changes in k, we used small angle X-ray diffraction to simultaneously measure myofilament lattice spacing and isometric force during steady-state Ca activations. Within in vivo lattice dimensions, lattice spacing and steady-state isometric force increased significantly at submaximal activation. We conclude that the cTnC N-domain controls force by modulating both the number and rate of cycling cross-bridges, and that the both methods of Ca sensitization may act through stabilization of cTnC's D-helix. Furthermore, we propose that the transient expansion of the myofilament lattice during Ca activation may be an additional factor that could increase the rate of cross-bridge cycling in cardiac muscle. These findings may have implications for the pathophysiology of HCM.

Familial Dilated Cardiomyopathy Associated With a Novel Combination of Compound Heterozygous TNNC1 Variants

“…Kinetics of Tension Redevelopment (k TR ): After force reached steady-state in each pCa solution, measurement of the rate of tension redevelopment was obtained by shortening CMPs by 20% L 0 , followed by rapid, 25% re-stretch, then release back to L 0 (Gonzalez-Martinez et al, 2018). The apparent rate constant (k TR ) was obtained from each tension recovery time course as described previously (Chase et al, 1994;Regnier et al, 1999).…”

“…Conditions where steady-state, isometric force was below 15% of the maximal force were excluded from the k TR analysis because of the relatively low signal-to-noise at the lowest levels of isometric force. Estimations of the 3-state model parameters (f, g, and k OFF ) were computed in MatLab as previously described (Gonzalez-Martinez et al, 2018) with the exceptions that force for each condition was assumed to be 1.0, and also because there are no direct measurements of Ca 2+ binding to or dissocation from the mutant cTnCs, the value for k ON for all simulations was assumed to be that derived from measurements on WT, 1.84 × 10 8 (Pinto et al, 2011a). In addition to estimates for parameters f, g, and k OFF , the modeling also provides an estimate for pCa 50 ; the modified least-squares criterion is weighted such that the best fit, predicted pCa 50 is essentially the same (well within experimental error) as that measured experimentally for the same condition.…”

“…Sinusoidal stiffness (SS) was obtained by oscillating CMP length ∼0.2% L 0 peak-to-peak at 100 Hz, and recording the length and force signals at a sampling rate of 1 kHz. SS measurements were performed and the data were analyzed as described previously (Gonzalez-Martinez et al, 2018).…”

“…This result informs us that, according to Brenner (1988), the sum of the sum of the rates of cross-bridge attachment and detachment (f +g) is approximately constant for all four conditions. To explore the possibility that the variants may have altered cross-bridge attachment (f ) and detachment (g) rates even though the sum remained constant, we used a 3-state model of muscle regulation to evaluate the Ca 2+ -dependence of k TR (Landesberg and Sideman, 1994;Hancock et al, 1997;Loong et al, 2013;Gonzalez-Martinez et al, 2018). The force-k TR data for CMPs containing the mutants (WT/D132N, WT/D145E or D132N/D145E) appear to be less curvilinear than for WT ( Figure 7B).…”

Sexual dimorphism in cardiac transcriptome associated with a troponin C murine model of hypertrophic cardiomyopathy

“…The encoded mutant cTnC proteins have been studied for their pathogenic effects in vitro and in vivo (Landstrom et al, 2008; Martins et al, 2015). The A8V variant of cTnC, caused by a point mutation in TNNC1 , markedly increases myofilament Ca 2+ sensitivity and kinetics of actomyosin cross‐bridge cycling (Gonzalez‐Martinez et al, 2018; Pinto et al, 2009). Furthermore, the cTnC‐A8V knock‐in mouse model exhibits cardiac morphology similar to human HCM/RCM (Martins et al, 2015).…”