Spatial control of myosin regulatory light chain phosphorylation modulates cardiac thick filament mechanosensing.

The heart can adapt its performance in response to changing metabolic demands of the rest of the body. A central mechanism intrinsic to the heart is to modulate the function of the cardiac contractile proteins via posttranslational modifications. Although phosphorylation of the cardiac myosin motor-associated regulatory light chain (RLC) by cardiac myosin light chain kinase (cMLCK) has been recognized as a key signaling pathway to increase myocardial contractile function, little is known about its molecular mechanism of action. Here, we show that phosphorylation of RLC is not a stochastic process but a spatially tightly controlled mechanism in the cardiac sarcomere. Myosin motors in the region of the thick filament associated with cardiac myosin binding protein-C (cMyBP-C) are the primary target for phosphorylation by cMLCK. Moreover, we show that phosphorylation of RLC likely only leads to activation of one of the two myosin motors of the dimeric cardiac myosin molecule. Using a combination of structural measurements using bifunctional fluorescent probes on the RLC and spatially explicit modeling we show that RLC phosphorylation increases the force-dependent recruitment of the myosin motors. We propose that RLC phosphorylation exerts its functional effects via increasing the gain of the mechanosignaling between different zones of the thick filament. A better mechanistic understanding of the role of RLC phosphorylation likely underpins the development of therapeutic interventions for both heart disease and heart failure.