Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca²⁺ sensitivity of force (pCa₅₀), PKA treatment has been shown to decrease pCa₅₀, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca²⁺-independent force and maximum Ca²⁺-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I_1,1/I_1,0) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d_1,0) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I_1,1/I_1,0 and, as hypothesized, treatment with MLCK also increased I_1,1/I_1,0, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by ∼2 nm (Δ 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca²⁺ sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.