Biology subjects

Squarci, C.

Publications and source records attributed to Squarci, C..

3 recordsLinked to original sources

bioRxiv2025

Spatial control of myosin regulatory light chain phosphorylation modulates cardiac thick filament mechano-sensing

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 post-translational 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 signalling 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. 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, phosphorylation of RLC likely only leads to activation of one of the two myosin motors of the cardiac myosin molecule and increases their force-dependent recruitment. We propose that RLC phosphorylation exerts its functional effects via increasing the gain of the mechano-signalling 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.

biochemistry↗

bioRxiv2024

Danicamtiv reduces myosin's working stroke but enhances contraction by activating the thin filament

Heart failure is a leading cause of death worldwide, and even with current treatments, the 5-year transplant-free survival rate is only [~]50-70%. As such, there is a need to develop new treatments for patients that improve survival and quality of life. Recently, there have been efforts to develop small molecules for heart failure that directly target components of the sarcomere, including cardiac myosin. One such molecule, danicamtiv, recently entered phase II clinical trials; however, its mechanism of action and direct effects on myosins mechanics and kinetics are not well understood. Using optical trapping techniques, stopped flow transient kinetics, and in vitro reconstitution assays, we found that danicamtiv reduces the size of cardiac myosins working stroke, and in contrast to studies in muscle fibers, we found that it does not affect actomyosin detachment kinetics at the level of individual crossbridges. We demonstrate that danicamtiv accelerates actomyosin association kinetics, leading to increased recruitment of myosin crossbridges and subsequent thin filament activation at physiologically-relevant calcium concentrations. Finally, we computationally model how the observed changes in mechanics and kinetics at the level of single crossbridges contribute to increased cardiac contraction and improved diastolic function compared to the related myotrope, omecamtiv mecarbil. Taken together, our results have important implications for the design of new sarcomeric-targeting compounds for heart failure. SIGNIFICANCE STATEMENTHeart failure is a leading cause of death worldwide, and there is a need to develop new treatments that improve outcomes for patients. Recently, the myosin-binding small molecule danicamtiv entered clinical trials for heart failure; however, its mechanism at the level of single myosin crossbridges is not well understood. We determined the molecular mechanism of danicamtiv and showed how drug-induced molecular changes can mechanistically increase heart contraction. Moreover, we demonstrate fundamental differences between danicamtiv and the related myosin-binding small molecule omecamtiv mecarbil that explain the improved diastolic function seen with danicamtiv. Our results have important implications for the design of new therapeutics for heart failure.

biophysics↗

bioRxiv2021

Titin switches from an extensible spring to a mechanical rectifier upon muscle activation

In contracting striated muscle titin acts as a spring in parallel with the array of myosin motors in each half-sarcomere and could prevent the intrinsic instability of thousands of serially linked half-sarcomeres, if its stiffness, at physiological sarcomere lengths (SL), were ten times larger than reported. Here we define titin mechanical properties during tetanic stimulation of single fibres of frog muscle by suppressing myosin motor responses with Para-Nitro-Blebbistatin, which is able to freeze thick filament in the resting state. We discover that thin filament activation switches I-band titin spring from the large SL-dependent extensibility of the OFF-state to an ON-state in which titin acts as a SL-independent mechanical rectifier, allowing free shortening while opposing stretch with an effective stiffness 4 pN nm-1 per half-thick filament. In this way during contraction titin limits weak half-sarcomere elongation to a few % and, also, provides an efficient link for mechanosensing-based thick filament activation.

physiology↗