Ablation of myosin-binding protein-C accelerates force development in mouse myocardium

Abstract

Myosin-binding protein-C (MyBP-C) is a thick filament-associated protein that binds tightly to myosin. Given that cMyBP-C may act to modulate cooperative activation of the thin filament by constraining the availability of myosin cross-bridges for binding to actin, we investigated the role of MyBP-C in the regulation of cardiac muscle contraction. We assessed the Ca(2+) sensitivity of force (pCa(50)) and the activation dependence of the rate of force redevelopment (k(tr)) in skinned myocardium isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) mice. Mechanical measurements were performed at 22 degrees C in the absence and presence of a strong-binding, nonforce-generating analog of myosin subfragment-1 (NEM-S1). In the absence of NEM-S1, maximal force and k(tr) and the pCa(50) of isometric force did not differ between WT and cMyBP-C(-/-) myocardium; however, ablation of cMyBP-C-accelerated k(tr) at each submaximal force. Treatment of WT and cMyBP-C(-/-) myocardium with 3 muM NEM-S1 elicited similar increases in pCa(50,) but the effects of NEM-S1 to increase k(tr) at submaximal forces and thereby markedly reduce the activation dependence of k(tr) occurred to a greater degree in cMyBP-C(-/-) myocardium. Together, these results support the idea that cMyBP-C normally acts to constrain the interaction between myosin and actin, which in turn limits steady-state force development and the kinetics of cross-bridge interaction.

FIGURE 1

Effect of NEM-S1 on force-pCa relationships in skinned myocardium from WT and cMyBP-C^−/−. (A) All values are mean ± SE. Smooth lines were generated by fitting the mean data to a Hill equation: P/P_o = [Ca²⁺]ⁿ/(kⁿ + [Ca²⁺]ⁿ), where n is the Hill coefficient and k is the [Ca²⁺] required for half-maximal activation (pCa₅₀). The relationships between Ca²⁺-activated force and pCa were determined in the absence (open symbols) and presence (solid symbols) of 3 μM NEM-S1. pCa₅₀ values for WT were control (○), 5.73 ± 0.01; 3 μM NEM-S1 (•), 5.79 ± 0.01, whereas pCa₅₀ values for cMyBP-C^−/− were control (Δ), 5.72 ± 0.01; 3 μM NEM-S1 (▴), 5.82 ± 0.01. (B) Hill plot transformations of the force-pCa data were generated using the following equation: log[P_rel/(1 − P_rel)] = n(log[Ca²⁺] + k), where P_rel is force as a fraction of P_o, n is the Hill coefficient, and k is the [Ca²⁺] required for half-maximal activation (pCa₅₀). All values are mean ± SE. Fiber characteristics are listed in Table 1.

FIGURE 2

Rate of force redevelopment in skinned myocardium from WT and cMyBP-C^−/−. Differences in the rate constant of force redevelopment (k_tr) were expressed relative to respective peak forces. (A) The rates of force redevelopment measured in the absence of NEM-S1 from two representative skinned myocardial preparations from either WT (left trace: pCa 4.5, P/P_o = 1.0, k_tr = 34.7 s⁻¹; right trace: pCa 5.8, P/P_o = 0.40, k_tr = 5.1 s⁻¹) or cMyBP-C^−/− (middle trace: pCa 5.8, P/P_o = 0.40, k_tr = 11.4 s⁻¹). (B) The rates of force redevelopment measured in the presence of NEM-S1 from two representative skinned myocardial preparations from either WT (left trace: pCa 4.5, P/P_o = 1.0, k_tr = 33.0 s⁻¹; right trace: pCa 5.8, P/P_o = 0.55, k_tr = 16.1 s⁻¹) or cMyBP-C^−/− (middle trace: pCa 5.8, P/P_o = 0.57, k_tr = 23.5 s⁻¹).

FIGURE 3

Effect of NEM-S1 on the Ca²⁺ dependence of the rate of force redevelopment. Force redevelopment after rapid release and restretch was measured as a function of pCa in skinned myocardium from WT (○,•) and cMyBP-C^−/− (Δ,▴) in the absence (open symbols) and presence (solid symbols) of 3 μM NEM-S1. All values are mean ± SE. Fiber characteristics are listed in Table 2.

FIGURE 4

Effect of NEM-S1 on the activation dependence of the rate of force redevelopment. Force redevelopment after rapid release and restretch was measured as a function of relative steady-state isometric force (P/P_o) in skinned myocardium from WT (○,•) and cMyBP-C^−/− (Δ,▴) in the absence (open symbols) and presence (solid symbols) of 3 μM NEM-S1. All values are mean ± SE. Fiber characteristics are listed in Table 2.