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Force production by actin–myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower β-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca 2+ -activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca 2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases β-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of β-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and β-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that β-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms. 

Cardiac performance is tightly regulated at the cardiomyocyte level by sarcomere length, such that increases in sarcomere length lead to sharply enhanced force generation at the same Ca 2+ concentration. Length-dependent activation of myofilaments involves dynamic and complex interactions between a multitude of thick- and thin-filament components. Among these components, troponin, myosin, and the giant protein titin are likely to be key players, but the mechanism by which these proteins are functionally linked has been elusive. Here, we investigate this link in the mouse myocardium using in situ FRET techniques. Our objective was to monitor how length-dependent Ca 2+ -induced conformational changes in the N domain of cardiac troponin C (cTnC) are modulated by myosin–actin cross-bridge (XB) interactions and increased titin compliance. We reconstitute FRET donor- and acceptor-modified cTnC(13C/51C)AEDANS-DDPM into chemically skinned myocardial fibers from wild-type and RBM20-deletion mice. The Ca 2+ -induced conformational changes in cTnC are quantified and characterized using time-resolved FRET measurements as XB state and sarcomere length are varied. The RBM20-deficient mouse expresses a more compliant N2BA titin isoform, leading to reduced passive tension in the myocardium. This provides a molecular tool to investigate how altered titin-based passive tension affects Ca 2+ -troponin regulation in response to mechanical stretch. In wild-type myocardium, we observe a direct association of sarcomere length–dependent enhancement of troponin regulation with both Ca 2+ activation and strongly bound XB states. In comparison, measurements from titin RBM20-deficient animals show blunted sarcomere length–dependent effects. These results suggest that titin-based passive tension contributes to sarcomere length–dependent Ca 2+ -troponin regulation. We also conclude that strong XB binding plays an important role in linking the modulatory effect of titin compliance to Ca 2+ -troponin regulation of the myocardium.