Biology subjects

Kampourakis, T.

Publications and source records attributed to Kampourakis, T..

5 recordsLinked to original sources

bioRxiv2026

Biomolecular condensation of cMLCK enables myosin motor phosphorylation in the heart

The heart needs to adapt its output to the metabolic demands of the organism. Phosphorylation of the myosin motors by cardiac myosin light chain kinase (cMLCK) increases heart muscle contractile function, yet its regulation and mechanism of action have remained unclear. Here, we show that cMLCK undergoes liquid-liquid phase separation and forms biomolecular condensates associated with the sarcoplasmic reticulum of cardiac muscle cells. Condensates selectively enrich enzymatic cofactors and substrates, which increases the catalytic activity of cMLCK. Our study reveals that cMLCK is fine-tuned to work in the molecular environment of condensates, enabling physiologically relevant levels of cardiac myosin motor phosphorylation. These findings establish a condensate-based mechanism for the spatial and temporal regulation of cardiac thick filament contractile function.

biochemistry↗

bioRxiv2026

Hearts may grow concentrically to balance ATP supply and demand and eccentrically to stabilize titin-based stress

Hearts change their wall thickness (concentric growth) and chamber size (eccentric growth) as they adapt to circulatory demands and the intrinsic function of their contractile cells. Factors associated with wall thickening include variants of sarcomeric proteins that enhance contractility, mitochondrial dysfunction, and hypertension. Chambers can dilate due to many factors including sarcomeric variants that depress contractility and aortic and / or mitral valve insufficiency. Despite intensive study, the mechanisms that regulate cardiac growth remain unclear. It is also uncertain whether inherited variants induce growth via the same mechanisms as more common clinical pathologies, such as hypertension. Here we show that computer simulations of a beating left ventricle reproduce both variant and non-variant-related growth patterns when myocytes grow concentrically to regulate intracellular ATP concentration and eccentrically to maintain titin-based intracellular stress. The simulations support the hypothesis that cardiac growth reflects homeostatic feedback through three interacting systems whereby myocytes add or remove mitochondria and sarcomeres (1) in parallel to match ATP generation to myocardial energy demand, and (2) in series to regulate passive tension, while (3) the autonomic nervous system regulates cardiac power, and thus myocardial ATPase, via baroreflex control. The new framework provides a mechanistic basis for the patterns of eccentric and concentric growth induced by a wide range of clinically-relevant conditions and could facilitate in silico testing of potential therapies for cardiac disease. Significance statementHearts grow in response to both physiological and pathological stimuli. The patterns of concentric (wall thickening / thinning) and eccentric (chamber dilation / constriction) induced by different challenges are well recognized but the underlying mechanisms remain unclear. This work presents simulations of a beating left ventricle where (1) concentric growth is regulated by myocytes attempting to stabilize the intracellular ATP concentration and (2) eccentric growth is regulated by titin-mediated stress. The calculations reproduce the growth associated with inherited variants of sarcomeric proteins, mitochondrial dysfunction, hypertension, and both mitral and aortic valve insufficiency. The new ability to predict cardiac growth and its potential modification by treatments, including myotropes, brings the field closer to in silico optimization of therapy for cardiovascular disease.

biophysics↗

bioRxiv2025

Direct measurement of mavacamten and deoxyATP perturbation of the SRX/DRX ratio in porcine cardiac myofibrils using a simple, accessible and multiplexed approach

Cardiac muscle adapts to varying physiological demands by modulating the number of active myosin II motors available for contraction. These motors are organized into thick filaments in the sarcomere and generate force through an ATP-dependent interaction with thin filaments that contain actin. To conserve energy, when demand is low myosin can occupy the super-relaxed (SRX) state, which acts as a reserve. Here, we build upon the earlier studies from the Cooke lab to quantify the size of this cardiac reserve using fluorescence imaging of Cy3-ATP directly in myofibrils. This approach employs a pulse-chase method and exploits the high permeability of isolated myofibrils to monitor nucleotide release in situ. By preserving sarcomeric architecture while enabling rapid reagent exchange, this method bridges the gap between complex single-molecule imaging and traditional stopped-flow bulk assays using MANT-ATP. Using this approach we have studied biochemical perturbation of the SRX reserve with deoxyATP and mavacamten. DeoxyATP caused large depletion of the cardiac reserve, and mavacamten increased its size, consistent with its clinical application. Our results demonstrate the utility of this technique and the potential for further enhancement using multiplexing, holding promise for future applications in health and disease.

biophysics↗

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↗

bioRxiv2023

The cardiac myosin binding protein-C phosphorylation state as a function of multiple protein kinase and phosphatase activities

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a crucial determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-Cs multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2A (PP1 and PP2A) and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and protein kinases A (PKA), C and RSK2. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by PKA. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results emphasize the importance of phosphatases for cMyBP-C regulation and prompt us to propose reciprocal relationships between cMyBP-C m-motif conformation, phosphorylation state and myofilament function.

biochemistry↗