Significance Myosin binding protein-C (MyBP-C) is a regulatory protein of heart muscle. Mutations in MyBP-C are frequently associated with disease, but the mechanism action poorly understood. By characterizing effects its N-terminal domains on structures thin and thick filaments contracting muscle cells, we showed that stabilizes ON state OFF filaments. The results lead to model for control contraction which functions coordinated by MyBP-C, providing an integrated framework design...

Significance Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated structural changes in myosin-containing thick filaments. We showed that phosphorylation myosin regulatory light chain generates a signal transmitted between molecules filament and from filaments, altering their sensitivity. A closely related dual-filament signaling pathway underlies enhanced contractility when it stretched. These coordinated cooperative structure are...

Key points Omecamtiv mecarbil and blebbistatin perturb the regulatory state of thick filament in heart muscle. increases contractility at low levels activation by stabilizing ON filament. decreases high disrupting acto‐myosin ATPase cycle. Blebbistatin reduces OFF inhibiting ATPase. Thick regulation is a promising target for novel therapeutics disease. Abstract Contraction muscle triggered transient rise intracellular free calcium concentration linked to change structure actin‐containing...

The heart's response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator myocardial contractility, little known about its mechanism action. Here, we used kinase A (PKA) and Cε (PKCε), well ribosomal S6 II (RSK2), have different specificities for cMyBP-C's multiple sites, show individual sites are not...

Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters muscle mechanics is important because it often altered in disease. The effect this protein has on during a physiological range of shortening velocities, which the heart generates power and performs work, not been addressed. We have expressed phosphorylated recombinant Rattus norvegicus left ventricular RLC. In vitro we these species with kinase zipper-interacting kinase. compare rat permeabilized trabeculae,...

Highlights•The orientation of the phosphorylated cRLC was measured by polarized fluorescence.•Phosphorylated myosin heads are not disordered on level region.•cRLC phosphorylation induces a new conformational state myosin.•cRLC controls contractility at head–backbone interface.AbstractThe effect conformation regulatory light chain (cRLC) region in ventricular trabeculae from rat heart determined fluorescence thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than...

The Frank-Starling relation is a fundamental auto-regulatory property of the heart that ensures volume blood ejected in each heartbeat matched to extent venous filling. At cellular level, muscle cells generate higher force when stretched, but despite intense efforts underlying molecular mechanism remains unknown. We applied fluorescence-based method, which reports structural changes separately thick and thin filaments rat cardiac muscle, elucidate mechanism. distinct troponin C myosin...

Significance The efficiency of the heart as a pump depends on an autoregulatory mechanism, Frank–Starling law heart, that potentiates strength contraction in response to increase ventricular filling. Disruption this mechanism compromises ability blood, potentially leading failure. We used fluorescent probes myosin muscle cells investigate molecular basis mechanism. Our results show stronger at longer lengths is due calcium-dependent interfilament signaling pathway links stress sensing...

The cardiac isoform of myosin-binding protein C (cMyBP-C) is a key regulatory found in myofilaments that can control the activation state both actin-containing thin and myosin-containing thick filaments. However, contrast to filament–based mechanisms regulation, mechanism myosin-based regulation by cMyBP-C has yet be defined detail. To clarify its function this process, we used microscale thermophoresis build an extensive interaction map between isolated fragments β-cardiac myosin. We show...

Abstract Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence phosphatases on cMyBP-C’s multiple sites remained largely obscure. Here we provide detailed biochemical characterization dephosphorylation 1 and 2 A (PP1 PP2A), develop an integrated kinetic model for using data both PP1, PP2A known to phosphorylate cMyBP-C. We find strong...

Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been studied extensively in vitro vivo, the precise role cardiac kinase (cMLCK), primary acting upon RLC, regulation cardiomyocyte remains poorly understood. In this study, using recombinantly expressed purified proteins, various analytical methods, situ assays, mechanical measurements isolated ventricular...

GelBox is open-source software that was developed with the goal of enhancing rigor, reproducibility, and transparency when analyzing gels immunoblots. It combines image adjustments (cropping, rotation, brightness, contrast), background correction, band-fitting in a single application. Users can also associate each lane an metadata (for example, sample type). data files integrate raw data, supplied metadata, adjustments, band-level analyses file to improve traceability. has user-friendly...

The heart can adapt its performance in response to changing metabolic demands of the rest body. A central mechanism intrinsic is modulate function cardiac contractile proteins via post-translational modifications. Although phosphorylation myosin motor-associated regulatory light chain (RLC) by kinase (cMLCK) has been recognized as a key signalling pathway increase myocardial function, little known about molecular action. Here, we show that RLC not stochastic process but spatially tightly...

Novel bifunctional probes for α-helix labelling is described. These both solve an existing problem with current of this class, as well introduce new orthogonality that will assist the future study dynamic proteins in situ .

Heart muscle is activated by Ca(2+) to generate force and shortening, the signaling pathway involves allosteric mechanisms in thin filament. Knowledge about structure-function relationship among proteins filament critical understanding physiology pathology of cardiac function, but remains obscure. We investigate conformation troponin (Tn) on its response activation propose a molecular mechanism for regulation contraction Tn based uniquely information from situ protein domain orientation....

Hypertrophic cardiomyopathy (HCM) is frequently linked to mutations in the protein components of myosin-containing thick filaments leading contractile dysfunction and ultimately heart failure. However, molecular structure-function relationships that underlie these pathological effects remain largely obscure. Here we chose an example mutation (R58Q) myosin regulatory light chain (RLC) associated with a severe HCM phenotype combined results from wide range vitro situ structural functional...

Direct modulation of cardiac myosin function has emerged as a therapeutic target for both heart disease and failure. However, the development myosin-based therapeutics been hampered by lack targeted in vitro screening assays. In this study we use Artificial Intelligence-based virtual high throughput (vHTS) to identify novel small molecule effectors human β-cardiac myosin. We test top scoring compounds from vHTS biochemical counter-screens chemical scaffold called 'F10' cardiac-specific...