Phosphorylation of RLC by cMLCK in cardiac myofibrils preferentially occurs in the thick filament C-zone, increasing calcium sensitivity by up to 0.12 pCa and isometric force, and selectively activating the blocked head of myosin leading to enhanced force-dependent recruitment and mechanosensing.
RLC phosphorylation exerts functional effects by increasing the gain of mechanosignaling between different zones of the thick filament, providing mechanistic insights for potential heart failure therapies.
Effect estimate: Increase in RLC phosphorylation level from ~0.05 to >0.8 mol Pi/mol RLC; Calcium sensitivity increase by ~0.08 to 0.12 pCa; Increase in force redevelopment rate ktr from ~13 to ~16 s−1; Increase in isometric force and recruitment rate of myosin heads
Absolute Event Rate: 0.8% vs 0.05%
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.
Squarci et al. (2026) studied Cardiac muscle tissue and cardiac myofibrils from rat and human myocardium, ex vivo experimental model. Cardiac myosin light chain kinase (cMLCK) treatment to phosphorylate regulatory light chain (RLC) vs. Baseline unphosphorylated RLC or BDM-treated myofibrils was evaluated on Level and spatial distribution of RLC phosphorylation and functional changes in cardiac contractile force and myosin head activation (Increase in RLC phosphorylation level from ~0.05 to >0.8 mol Pi/mol RLC; Calcium sensitivity increase by ~0.08 to 0.12 pCa; Increase in force redevelopment rate ktr from ~13 to ~16 s−1; Increase in isometric force and recruitment rate of myosin heads). Phosphorylation of RLC by cMLCK in cardiac myofibrils preferentially occurs in the thick filament C-zone, increasing calcium sensitivity by up to 0.12 pCa and isometric force, and selectively activating the blocked head of myosin leading to enhanced force-dependent recruitment and mechanosensing.
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