Load-dependence of the activation of myosin filaments in heart muscle.

Contraction of heart muscle requires activation of both the actin and myosin filaments. The mechanism of myosin filament activation is unknown, but the leading candidate hypothesis is direct mechano-sensing by the filaments. Here, we tested this hypothesis by activating intact trabeculae from rat heart by electrical stimulation under different loads and measuring myosin filament activation by X-ray diffraction.

Functional control of myosin motors in the cardiac cycle.

Contraction of the heart is driven by cyclical interactions between myosin and actin filaments powered by ATP hydrolysis. The modular structure of heart muscle and the organ-level synchrony of the heartbeat ensure tight reciprocal coupling between this myosin ATPase cycle and the macroscopic cardiac cycle. The myosin motors respond to the cyclical activation of the actin and myosin filaments to drive the pressure changes that control the inflow and outflow valves of the heart chambers.

Dependence of myosin filament structure on intracellular calcium concentration in skeletal muscle.

Contraction of skeletal muscle is triggered by an increase in intracellular calcium concentration that relieves the structural block on actin-binding sites in resting muscle, potentially allowing myosin motors to bind and generate force. However, most myosin motors are not available for actin binding because they are stabilized in folded helical tracks on the surface of myosin-containing thick filaments. High-force contraction depends on the release of the folded motors, which can be triggered by stress in the thick filament backbone, but additional mechanisms may link the activation of the thick filaments to that of the thin filaments or to intracellular calcium concentration.

Activation of skeletal muscle is controlled by a dual-filament mechano-sensing mechanism.

Contraction of skeletal muscle is triggered by a transient rise in intracellular calcium concentration leading to a structural change in the actin-containing thin filaments that allows binding of myosin motors from the thick filaments. Most myosin motors are unavailable for actin binding in resting muscle because they are folded back against the thick filament backbone. Release of the folded motors is triggered by thick filament stress, implying a positive feedback loop in the thick filaments.

In situ FRET-based localization of the N terminus of myosin binding protein-C in heart muscle cells.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated regulatory protein frequently found mutated in patients suffering from hypertrophic cardiomyopathy (HCM). Recent in vitro experiments have highlighted the functional significance of its N-terminal region (NcMyBP-C) for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. To better understand the interactions of cMyBP-C in its native sarcomere environment, in situ Foerster resonance energy transfer-fluorescence lifetime imaging (FRET-FLIM) assays were developed to determine the spatial relationship between the NcMyBP-C and the thick and thin filaments in isolated neonatal rat cardiomyocytes (NRCs).

Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A.

PKA-mediated phosphorylation of sarcomeric proteins enhances heart muscle performance in response to β-adrenergic stimulation and is associated with accelerated relaxation and increased cardiac output for a given preload. At the cellular level, the latter translates to a greater dependence of Ca sensitivity and maximum force on sarcomere length (SL), that is, enhanced length-dependent activation. However, the mechanisms by which PKA phosphorylation of the most notable sarcomeric PKA targets, troponin I (cTnI) and myosin-binding protein C (cMyBP-C), lead to these effects remain elusive.

Activation of the myosin motors in fast-twitch muscle of the mouse is controlled by mechano-sensing in the myosin filaments.

Myosin motors in resting muscle are inactivated by folding against the backbone of the myosin filament in an ordered helical array and must be released from that conformation to engage in force generation. Time-resolved X-ray diffraction from single fibres of amphibian muscle showed that myosin filament activation could be inhibited by imposing unloaded shortening at the start of stimulation, suggesting that filaments were activated by mechanical stress. Here we improved the signal-to-noise ratio of that approach using whole extensor digitorum longus muscles of the mouse contracting tetanically at 28°C.

Cooling intact and demembranated trabeculae from rat heart releases myosin motors from their inhibited conformation.

Myosin filament-based regulation supplements actin filament-based regulation to control the strength and speed of contraction in heart muscle. In diastole, myosin motors form a folded helical array that inhibits actin interaction; during contraction, they are released from that array. A similar structural transition has been observed in mammalian skeletal muscle, in which cooling below physiological temperature has been shown to reproduce some of the structural features of the activation of myosin filaments during active contraction.

Myosin motors that cannot bind actin leave their folded OFF state on activation of skeletal muscle.

The myosin motors in resting skeletal muscle are folded back against their tails in the thick filament in a conformation that makes them unavailable for binding to actin. When muscles are activated, calcium binding to troponin leads to a rapid change in the structure of the actin-containing thin filaments that uncovers the myosin binding sites on actin. Almost as quickly, myosin motors leave the folded state and move away from the surface of the thick filament.

The regulatory light chain mediates inactivation of myosin motors during active shortening of cardiac muscle.

The normal function of heart muscle depends on its ability to contract more strongly at longer length. Increased venous filling stretches relaxed heart muscle cells, triggering a stronger contraction in the next beat- the Frank-Starling relation. Conversely, heart muscle cells are inactivated when they shorten during ejection, accelerating relaxation to facilitate refilling before the next beat.

Myosin-based regulation of twitch and tetanic contractions in mammalian skeletal muscle.

Time-resolved X-ray diffraction of isolated fast-twitch muscles of mice was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the 'twitch' response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments.

Stress-dependent activation of myosin in the heart requires thin filament activation and thick filament mechanosensing.

Myosin-based regulation in the heart muscle modulates the number of myosin motors available for interaction with calcium-regulated thin filaments, but the signaling pathways mediating the stronger contraction triggered by stretch between heartbeats or by phosphorylation of the myosin regulatory light chain (RLC) remain unclear. Here, we used RLC probes in demembranated cardiac trabeculae to investigate the molecular structural basis of these regulatory pathways. We show that in relaxed trabeculae at near-physiological temperature and filament lattice spacing, the RLC-lobe orientations are consistent with a subset of myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments.

Dependence of thick filament structure in relaxed mammalian skeletal muscle on temperature and interfilament spacing.

Contraction of skeletal muscle is regulated by structural changes in both actin-containing thin filaments and myosin-containing thick filaments, but myosin-based regulation is unlikely to be preserved after thick filament isolation, and its structural basis remains poorly characterized. Here, we describe the periodic features of the thick filament structure in situ by high-resolution small-angle x-ray diffraction and interference. We used both relaxed demembranated fibers and resting intact muscle preparations to assess whether thick filament regulation is preserved in demembranated fibers, which have been widely used for previous studies.

Myosin filament-based regulation of the dynamics of contraction in heart muscle.

Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the "C-zone.

Cardiac myosin regulatory light chain kinase modulates cardiac contractility by phosphorylating both myosin regulatory light chain and troponin I.

Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been studied extensively and , the precise role of cardiac myosin light chain kinase (cMLCK), the primary kinase acting upon RLC, in the regulation of cardiomyocyte contractility remains poorly understood. In this study, using recombinantly expressed and purified proteins, various analytical methods, and kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions.

Low temperature traps myosin motors of mammalian muscle in a refractory state that prevents activation.

Myosin motors in the thick filament of resting striated (skeletal and cardiac) muscle are trapped in an OFF state, in which the motors are packed in helical tracks on the filament surface, inhibiting their interactions with actin and utilization of ATP. To investigate the structural changes induced in the thick filament of mammalian skeletal muscle by changes in temperature, we collected x-ray diffraction patterns from the fast skeletal muscle extensor digitorum longus of the mouse in the temperature range from near physiological (35°C) to 10°C, in which the maximal isometric force ( ) shows a threefold decrease. In resting muscle, x-ray reflections signaling the OFF state of the thick filament indicate that cooling produces a progressive disruption of the OFF state with motors moving away from the ordered helical tracks on the surface of the thick filament.

Site-specific phosphorylation of myosin binding protein-C coordinates thin and thick filament activation in cardiac muscle.

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 of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and Cε (PKCε), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C's multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites.

Structural and functional effects of myosin-binding protein-C phosphorylation in heart muscle are not mimicked by serine-to-aspartate substitutions.

Myosin-binding protein-C (cMyBP-C) is a key regulator of contractility in heart muscle, and its regulatory function is controlled in turn by phosphorylation of multiple serines in its m-domain. The structural and functional effects of m-domain phosphorylation have often been inferred from those of the corresponding serine-to-aspartate (Ser-Asp) substitutions, in both and studies. Here, using a combination of binding assays and structural and functional assays in ventricular trabeculae of rat heart and the expressed C1mC2 region of cMyBP-C, containing the m-domain flanked by domains C1 and C2, we tested whether these substitutions do in fact mimic the effects of phosphorylation.

Reversible Covalent Reaction of Levosimendan with Cardiac Troponin C in Vitro and in Situ.

The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties, including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target because cardiac troponin C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan.

Hypertrophic cardiomyopathy mutation R58Q in the myosin regulatory light chain perturbs thick filament-based regulation in cardiac muscle.

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

Regulation of Contraction by the Thick Filaments in Skeletal Muscle.

Contraction of skeletal muscle cells is initiated by a well-known signaling pathway. An action potential in a motor nerve triggers an action potential in a muscle cell membrane, a transient increase of intracellular calcium concentration, binding of calcium to troponin in the actin-containing thin filaments, and a structural change in the thin filaments that allows myosin motors from the thick filaments to bind to actin and generate force. This calcium/thin filament mediated pathway provides the "START" signal for contraction, but it is argued that the functional response of the muscle cell, including the speed of its contraction and relaxation, adaptation to the external load, and the metabolic cost of contraction is largely determined by additional mechanisms.

Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament.

Key Points: Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle. Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament. Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto-myosin ATPase cycle.

Distinct contributions of the thin and thick filaments to length-dependent activation in heart muscle.

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

Reversible Covalent Binding to Cardiac Troponin C by the Ca-Sensitizer Levosimendan.

The binding of Ca to cardiac troponin C (cTnC) triggers contraction in heart muscle. In the diseased heart, the myocardium is often desensitized to Ca, which leads to impaired contractility. Therefore, compounds that sensitize cardiac muscle to Ca (Ca-sensitizers) have therapeutic promise.