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

## TL;DR

This paper reveals how phosphorylation of a heart protein called RLC helps the heart adjust its strength by controlling how muscle proteins respond to force.

## Contribution

The study shows that RLC phosphorylation is spatially controlled and targets specific regions of heart muscle proteins.

## Key findings

- cMLCK preferentially phosphorylates myosin heads near cMyBP-C on thick filaments.
- RLC phosphorylation increases force-dependent recruitment of myosin motors during activation.
- This mechanism modulates mechanosignaling between regions of the thick filament.

## Abstract

Phosphorylation of the myosin-heavy chain associated regulatory light chain (RLC) by cardiac myosin light chain kinase (cMLCK) is a key regulatory pathway to modulate contractility in the heart. However, the molecular mechanisms underlying cardiac contractile regulation via RLC phosphorylation have remained elusive. We show that cMLCK treatment leads to preferential phosphorylation of myosin heads in the region of the thick filament associated with cardiac myosin binding protein-C, which increases their force-dependent recruitment during myofilament activation. The combined results lead to a model of regulation by RLC phosphorylation via modulation of the mechanosignaling between different regions of the thick filament. Impairment of this mechanism is likely the mechanistic basis for the functional effects of cardiomyopathy-linked mutations in sarcomeric proteins.

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.