The beating of the heart is driven by sliding between myosin-containing thick filaments and actin-containing thin filaments and at the single-molecule level by the ‘powerstroke’ in the lever arm of the myosin head, which tilts while its catalytic domain is attached to actin. This tilting lever-arm paradigm was developed before the molecular structure of the thick filament had been determined, and excluded the interactions between the myosin heads and the thick filaments that are now known to stabilise an OFF state of myosin. Here we re-examine the paradigm using measurements of the orientation of two components of the lever arm, the N- and C-lobes of the myosin regulatory light chain (RLC) in heart muscle cells by fluorescence for in situ structure (FISS), comparing them with those in cryo-electron microscopy (cryo-EM) structures of myosin fragments and of the C zone of thick filaments in the OFF state. We show that these FISS and cryo-EM results, combined with those of other structural studies on myosin fragments and muscle cells, can be explained by a modified tilting lever-arm paradigm that includes interactions between the myosin heads and the thick filament during the contractile cycle. In the new model, one head of each myosin dimer remains docked on the surface of the thick filament while its partner ‘working’ head executes the powerstroke and hydrolyses ATP. The interaction between the docked and working heads of the dimer both primes the working head to attach to an appropriate actin monomer in the pre-powerstroke state and re-captures the working head into the dimer after it has hydrolysed ATP.

Slow skeletal muscles maintain posture and produce graded movement at low metabolic cost. ATP utilization during fixed-end contractions is typically five times slower in slow muscles than in fast muscles from the same species. Mechanical measurements previously suggested that more myosin motors are attached to thin filaments during contraction of slow muscle, which seems incompatible with its high efficiency. We therefore used small-angle X-ray diffraction to provide a structural estimate of the fraction of myosin motors attached to thin filaments in slow muscle. The X-ray signals associated with myosin binding to actin indicate that only ∼10% of myosin motors are actin bound during fixed-end tetani of rat soleus slow muscles, compared with ∼25% in mouse extensor digitorum longus fast muscle. Moreover, X-ray signals associated with the helical organization of OFF myosin motors in the thick filaments show that ∼70% of myosin motors remain in the OFF conformation during tetanic contraction of rat soleus muscle, compared with only 30% in mouse extensor digitorum longus muscle. The much slower force development in soleus muscle also allowed clear separation of early structural changes in thick filaments on activation, some of which are distinct from those reported previously in fast muscles. Moreover, the early structural changes in soleus muscle have about the same amplitude in a twitch and a tetanus, suggesting that they are triggered by thin filament activation rather than thick filament stress and implying a fast signalling pathway between thin and thick filaments. KEY POINTS: The interaction between myosin motors and actin filaments in slow skeletal muscles maintains posture and produces graded movement at low metabolic cost. Mechanical studies have suggested that more myosin motors are attached to actin filaments during isometric contraction of slow than fast muscle, but this seems incompatible with its high efficiency. We used X-ray diffraction to show that there are fewer myosin motors attached to actin in slow muscle than in fast muscle because more motors are sequestered on the myosin filament. The slower force development in slow muscle also allowed us to isolate and characterize fast changes in myosin motor conformation associated with activation of the actin filaments.

Muscle contraction is driven by myosin motors from the thick filaments pulling on the actin-containing thin filaments of the sarcomere, and it is regulated by structural changes in both filaments. Thin filaments are activated by an increase in intracellular calcium concentration [Ca2+]i and by myosin binding to actin. Thick filaments are activated by direct sensing of the filament load. However, these mechanisms cannot explain muscle relaxation when [Ca2+]i decreases at high load and myosin motors are attached to actin. There is, therefore, a fundamental gap in our understanding of muscle relaxation, despite its importance for muscle function in vivo, for example, for rapid eye movements or, on slower timescales, for the efficient control of posture. Here, we used time-resolved small-angle X-ray diffraction (SAXD) to determine how muscle thin and thick filaments switch OFF in extensor digitorum longus (EDL) muscles of the mouse in response to decreases in either [Ca2+]i or muscle load and to describe the distribution of muscle sarcomere lengths (SLs) during relaxation. We show that reducing load at high [Ca2+]i is more effective in switching OFF both the thick and thin filaments than reducing [Ca2+]i at high load in normal relaxation. In the latter case, the thick filaments initially remain fully ON, although the number of myosin motors bound to actin decreases and the force per attached motor increases. That initial slow phase of relaxation is abruptly terminated by yielding of one population of sarcomeres, triggering a redistribution of SLs that leads to the rapid completion of mechanical relaxation.

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. Opening and closing of the valves in turn switches the myosin motors between roughly isometric and roughly isotonic contraction modes. Peak filament stress in the heart is much smaller than in fully activated skeletal muscle, although the myosin filaments in the two muscle types have the same number of myosin motors. Calculations indicate that only 5

Calcium binding to troponin triggers the contraction of skeletal and heart muscle through structural changes in the thin filaments that allow myosin motors from the thick filaments to bind to actin and drive filament sliding. Here, we review studies in which those changes were determined in demembranated fibres of skeletal and heart muscle using fluorescence for in situ structure (FISS), which determines domain orientations using polarised fluorescence from bifunctional rhodamine attached to cysteine pairs in the target domain. We describe the changes in the orientations of the N-terminal lobe of troponin C (TnCN) and the troponin IT arm in skeletal and cardiac muscle cells associated with contraction and compare the orientations with those determined in isolated cardiac thin filaments by cryo-electron microscopy. We show that the orientations of the IT arm determined by the two approaches are essentially the same and that this region acts as an almost rigid scaffold for regulatory changes in the more mobile regions of troponin. However, the TnCN orientations determined by the two methods are clearly distinct in both low- and high-calcium conditions. We discuss the implications of these results for the role of TnCN in mediating the multiple signalling pathways acting through troponin in heart muscle cells and the general advantages and limitations of FISS and cryo-EM for determining protein domain orientations in cells and multiprotein complexes.

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. Here, we used x-ray diffraction in combination with temperature-jump activation to determine the steady-state calcium dependence of thick filament structure and myosin motor conformation in near-physiological conditions. We found that x-ray signals associated with the perpendicular motors characteristic of isometric force generation had almost the same calcium sensitivity as force, but x-ray signals associated with perturbations in the folded myosin helix had a much higher calcium sensitivity. Moreover, a new population of myosin motors with a longer axial periodicity became prominent at low levels of calcium activation and may represent an intermediate regulatory state of the myosin motors in the physiological pathway of filament activation.

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 under-stand 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). In vitro studies showed that ligation of genetically encoded fluorophores to NcMyBP-C had no or little effect on its binding to thick and thin filament proteins. Using this assay, FRET between mTFP conjugated to NcMyBP-C and Phalloidin-iFluor 514 labeling the actin filaments in NRCs was detected by time-domain FLIM. The measured FRET efficiencies were intermediate between those observed when the donor was attached to the cardiac myosin regulatory light chain in the thick filaments and troponin T in the thin filaments. These results are consistent with the coexistence of multiple conformations of cMyBP-C, some with their N-terminal domains binding to the thin filament and others binding to the thick filament, supporting the hypothesis that the dynamic interchange between these conformations mediates interfilament signaling in the regulation of contractility. Moreover, stimulation of NRCs with beta-adrenergic agonists reduces FRET between NcMyBP-C and actin-bound Phalloidin, suggesting that cMyBP-C phosphorylation reduces its interaction with the thin filament.

Our understanding of thin filament structure has been greatly enhanced by the publication of a number of models derived from cryo-electron microscopy studies (Yamada et al. (2020) and then later confirmed by Risi et al. (2021)). These studies elucidated atomic level details of the calcium-dependent activation of the thin filament leading to force development in cardiac muscle. However, while cryo-EM reconstruction is best suited to capture the high-resolution organization of static structures, troponin and tropomyosin are dynamic components of the thin filament and corresponding disorder is not always easily recorded and classified by the method. Thus, in order to better model these structures, it may be necessary to generate small ensembles of structures to incorporate known sources of disorder as guided by information derived from other biophysical techniques. One example is for troponin subunit C, which binds calcium and the troponin subunit I switch peptide during thin filament activation. While the cryo-EM structure has offered a single computed conformation of troponin C, polarized fluorescence of the troponin core domain labeled with bifunctional rhodamine indicates that in cardiac muscle troponin C is very dynamic, and its N-terminal lobe takes up multiple distinct orientations (Sevrieva et al., 2014). In this study, we incorporate data derived both from structural methodologies to build a set of unique models that satisfy the cryo-EM and the fluorescence data. Analysis of these structures shows the details of how the dynamic nature of the N-lobe of troponin C is important for its function in thin filament regulation.

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 Ca2+ 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. Here, we specifically altered the phosphorylation level of cTnI in heart muscle cells and characterized the structural and functional effects at different levels of background phosphorylation of cMyBP-C and with two different SLs. We found Ser22/23 bisphosphorylation of cTnI was indispensable for the enhancement of length-dependent activation by PKA, as was cMyBP-C phosphorylation. This high level of coordination between cTnI and cMyBP-C may suggest coupling between their regulatory mechanisms. Further evidence for this was provided by our finding that cardiac troponin (cTn) can directly interact with cMyBP-C in vitro, in a phosphorylation- and Ca2+-dependent manner. In addition, bisphosphorylation at Ser22/Ser23 increased Ca2+ sensitivity at long SL in the presence of endogenously phosphorylated cMyBP-C. When cMyBP-C was dephosphorylated, bisphosphorylation of cTnI increased Ca2+ sensitivity and decreased cooperativity at both SLs, which may translate to deleterious effects in physiological settings. Our results could have clinical relevance for disease pathways, where PKA phosphorylation of cTnI may be functionally uncoupled from cMyBP-C phosphorylation due to mutations or haploinsufficiency.

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 degrees C. Changes in X-ray signals associated with myosin filament activation, including the decrease in the first-order myosin layer line associated with the helical motor array, increase in the spacing of a myosin-based retlection associated with packing of myosin tails in the filament backbone, and increase in the ratio of the 1,1 and 1,0 equatorial reflections associated with movement of motors away from the backbone, were delayed by imposing 10-ms unloaded shortening at the start of stimulation. These results show that myosin filaments are predominantly activated by filament stress, as in amphibian muscle. However, a small component of filament activation at zero load was detected, implying an independent mechanism of partial filament activation. X-ray interference measurements indicated a switch-like change in myosin motor conformation at the start of force development, accompanied by transient disordering of motors in the regions of the myosin filament near its midpoint, suggesting that filament zonal dynamics also play a role in its activation.

The contractility of cardiac muscle is controlled by a dual-filament mechanism in which calcium-dependent structural changes in the thin filament gate the access of the myosin motors to the actin-binding sites, whereas regulatory structural changes in the thick filament modulate the number of motors available for the actomyosin interaction. Here we investigated the role of those structural changes in determining the kinetics of force generation during maximal activation by photolysis of caged calcium (NP-EGTA) in demembranated rat cardiac trabeculae at 27°C with lattice compression by Dextran, conditions in which the native OFF state of the thick filament is preserved in the absence of calcium.

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. Here, we used small-angle x-ray diffraction to characterize the structural changes in the myosin filaments associated with cooling of resting and relaxed trabeculae from the right ventricle of rat hearts from 39°C to 7°C. In intact quiescent trabeculae, cooling disrupted the folded helical conformation of the myosin motors and induced extension of the filament backbone, as observed in the transition from diastole to peak systolic force at 27°C. Demembranation of trabeculae in relaxing conditions induced expansion of the filament lattice, but the structure of the myosin filaments was mostly preserved at 39°C. Cooling of relaxed demembranated trabeculae induced changes in motor conformation and filament structure similar to those observed in intact quiescent trabeculae. Osmotic compression of the filament lattice to restore its spacing to that of intact trabeculae at 39°C stabilized the helical folded state against disruption by cooling. The myosin filament structure and motor conformation of intact trabeculae at 39°C were largely preserved in demembranated trabeculae at 27°C or above in the presence of Dextran, allowing the physiological mechanisms of myosin filament-based regulation to be studied in those conditions.

Myosin filament-based regulation has become recognised as supplementing actin filament-based regulation in both cardiac and skeletal muscle. Here we studied the structural basis of the regulatory transition in the cardiac myosin filament induced by cooling from 39°C to 7°C, using X-ray diffraction from intact quiescent and relaxed demembranated trabeculae isolated from rat hearts. Myosin filament structure and inter-filament spacing in demembranated trabeculae in relaxing solution and in the presence of 3% dextran at 27-37°C was similar to that in intact quiescent trabeculae and in the diastolic phase between beats (Brunello et al., 2020 PNAS 117:8177). Cooling of intact trabeculae induced a decrease in the intensity of the first myosin layer line and the meridional myosin-based reflections indicating disruption of the OFF state of the myosin filament. Cooling also increased the spacing of the M6 reflection (SM6) and produced a biphasic change in that of the M3 reflection (SM3) associated with the axial periodicity of the filament backbone and the myosin motors respectively. These changes were accompanied by increase in the equatorial intensity ratio (I11/I10) associated with movement of motors towards the actin filaments. Cooling of demembranated trabeculae in relaxing solution produced larger changes in the intensity and spacing of the myosin-based reflections, and SM3, SM6 and I11/I10 in relaxing solution at 9°C were close to their respective ON values, measured in steady-state contractions at maximal calcium concentration at 27°C. These results show that cooling of cardiac trabeculae induced a transition in the structure of the myosin filament from the OFF- towards the ON-state in the absence of calcium activation and filament stress. Supported by British Heart Foundation, UK and ESRF, France.

Regulation of contraction in skeletal muscle is mediated by structural changes in both the actin-containing thin filaments and the myosin-containing thick filaments, and contractile dynamics are determined by interactions between motor and regulatory mechanisms. However, those interactions have not been characterised in the physiological context of the twitch response to single action potential stimulation. To do so, we recorded X-ray diffraction patterns using a Pilatus-2M detector at the I22 beamline of the Diamond Light Source in 5ms-frames during the twitch response of isolated intact mouse EDL muscles (starting sarcomere length 2.4μm, 28°C) and compared the results to full activation and relaxation in a 100-ms tetanus. Although peak force in the twitch was only 25% of that in the tetanus, X-ray signals associated with thick filament activation, like the decrease in the intensity of the myosin layer line (ML1) associated with the loss of the folded helical state of myosin motors in the OFF state of the thick filaments, were largely complete in the twitch. The difference reflects the substantial lag between myosin filament activation and motor attachment to actin and force development. Switching off the myosin filament tracked mechanical relaxation in the twitch, but did not start until the end of the 20-ms sarcomere-isometric phase of slow force relaxation following the tetanus, although other X-ray signals showed that myosin motors were detaching from actin during that phase. Moreover, the OFF state of the myosin filament had still not fully recovered at the end of mechanical relaxation, likely reflecting phosphorylation of the regulatory light chain of myosin associated with post-tetanic potentiation. Supported by Medical Research Council and Diamond Light Source, UK.

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. We show that the thick filaments in both preparations exhibit two closely spaced axial periodicities, 43.1 nm and 45.5 nm, at near-physiological temperature. The shorter periodicity matches that of the myosin helix, and x-ray interference between the two arrays of myosin in the bipolar filament shows that all zones of the filament follow this periodicity. The 45.5-nm repeat has no helical component and originates from myosin layers closer to the filament midpoint associated with the titin super-repeat in that region. Cooling relaxed or resting muscle, which partially mimics the effects of calcium activation on thick filament structure, disrupts the helical order of the myosin motors, and they move out from the filament backbone. Compression of the filament lattice of demembranated fibers by 5% Dextran, which restores interfilament spacing to that in intact muscle, stabilizes the higher-temperature structure. The axial periodicity of the filament backbone increases on cooling, but in lattice-compressed fibers the periodicity of the myosin heads does not follow the extension of the backbone. Thick filament structure in lattice-compressed demembranated fibers at near-physiological temperature is similar to that in intact resting muscle, suggesting that the native structure of the thick filament is largely preserved after demembranation in these conditions, although not in the conditions used for most previous studies with this preparation.

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. Muscle relaxation after repetitive stimulation is accompanied by a complete recovery of the folded motor conformation on the filament backbone but by incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscles.

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. The folded conformation of myosin is disrupted by cooling relaxed trabeculae, similar to the effect induced by maximal calcium activation. Stretch or increased RLC phosphorylation in the physiological range have almost no effect on RLC conformation at a calcium concentration corresponding to that between beats. These results indicate that in near-physiological conditions, the folded myosin motors are not directly switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in the absence of thin filament activation. However, at the higher calcium concentrations that activate the thin filaments, stretch produces a delayed activation of folded myosin motors and force increase that is potentiated by RLC phosphorylation. We conclude that the increased contractility of the heart induced by RLC phosphorylation and stretch can be explained by a calcium-dependent interfilament signaling pathway involving both thin filament sensitization and thick filament mechanosensing.

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. Although both effects are essential for the efficient function of the heart, the underlying mechanisms were unknown. Using bifunctional fluorescent probes on the regulatory light chain of the myosin motor we show that its N-terminal domain may be captured in the folded OFF state of the myosin dimer at the end of the working-stroke of the actin-attached motor, whilst its C-terminal domain joins the OFF state only after motor detachment from actin. We propose that sequential folding of myosin motors onto the filament backbone may be responsible for shortening-induced de-activation in the heart.

The contraction of the heart is modulated by regulatory structural changes in the myosin filament, in addition to the classical calcium-dependent regulation of the actin filament. Dual-filament regulation of cardiac muscle is best studied in intact force-generating sarcomeres, because it involves inter-filament signalling and mechano-sensing via signalling pathways that are still poorly understood. We have recently characterised by X-ray diffraction the structural dynamics of the thick filament associated with contraction and relaxation of electrically-paced intact trabeculae from rat hearts (Brunello et al., 2020 PNAS 117:8177). Here we complemented that approach using demembranated trabeculae to measure the structural changes in the myosin filament associated with the steady-state force-pCa relation. Trabeculae were activated at different [Ca2+] by temperature-jump from 2°C to 27°C in the presence of 3% Dextran to recover the physiological inter-filament lattice spacing. The calcium-dependence of the spacing of the M6 reflection, associated with the axial periodicity of the thick filament backbone, was similar to that of force, consistent with the idea of a stress-sensing mechanism in the filament backbone. Increasing [Ca2+] from the diastolic value (pCa 7.0) to pCa 6.0 induced a decrease in the intensities of the first myosin layer line and of the forbidden myosin-based reflections, that are signatures of the diastolic structure of the thick filament, by ∼50% and ∼80% respectively, with respect to the change observed at maximal [Ca2+] (pCa 4.7). In contrast, active force at pCa 6 was only ∼20% of that at maximal [Ca2+]. These results indicate that the regulatory transition in the myosin filament from the diastolic to the ON state is more sensitive to calcium than force generation. Supported by British Heart Foundation, Wellcome Trust, UK and ESRF, France.

The contractility of cardiac muscle is potentiated by phosphorylation of the myosin regulatory light chain (RLC) and by length-dependent activation, but the molecular pathways mediating these effects remain unclear. Here we studied the role of structural changes in the thick filament in the activation of cardiac muscle by stretch and RLC phosphorylation. We used fluorescence polarization from bifunctional sulphorhodamine probes on the N- and C-lobes of the myosin RLC to monitor changes in the orientation of the myosin motors induced by increasing the sarcomere length in the range 2.0-2.3 µm in relaxed and partially calcium-activated demembranated cardiac trabeculae, at different levels of RLC phosphorylation. We show that under relaxing or diastolic conditions at near physiological temperature and lattice spacing the RLC lobe orientations are consistent with about one third of the myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. This folded conformation is disrupted by cooling relaxed trabecula, but not by RLC phosphorylation or stretch at diastolic [Ca2+]. These results indicate that in near-physiological conditions the myosin filament is not switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in diastole, as suggested previously. However, stretching cardiac trabecula at near-systolic [Ca2+] triggers a stress-dependent activation of the thick filament and a delayed increase in active force. Physiological levels of RLC phosphorylation potentiate both the stress-dependent release of myosin motors from the folded state and the stretch-activated force. We conclude that the increase in heart contractility induced by RLC phosphorylation and stretch can be explained by an inter-filament signaling pathway involving both thin filament sensitization and thick filament mechano-sensing (Supported by Wellcome Trust, BHF, UK).