Cross-bridge Formation Research Articles

New insights into the cellular and molecular mechanisms of skeletal muscle fatigue: the Marion J. Siegman Award Lectureships.

Skeletal muscle fibers need to have mechanisms to decrease energy consumption during intense physical exercise to avoid devastatingly low ATP levels, with the formation of rigor cross bridges and defective ion pumping. These protective mechanisms inevitably lead to declining contractile function in response to intense exercise, characterizing fatigue. Through our work, we have gained insights into cellular and molecular mechanisms underlying the decline in contractile function during acute fatigue. Key mechanistic insights have been gained from studies performed on intact and skinned single muscle fibers and more recently from studies performed and single myosin molecules. Studies on intact single fibers revealed several mechanisms of impaired sarcoplasmic reticulum Ca2+ release and experiments on single myosin molecules provide direct evidence of how putative agents of fatigue impact myosin's ability to generate force and motion. We conclude that changes in metabolites due to an increased dependency on anaerobic metabolism (e.g., accumulation of inorganic phosphate ions and H+) act to directly and indirectly (via decreased Ca2+ activation) inhibit myosin's force and motion-generating capacity. These insights into the acute mechanisms of fatigue may help improve endurance training strategies and reveal potential targets for therapies to attenuate fatigue in chronic diseases.

Abstract Or104: Targeting Tropomyosin Overlap Flexibility via Small Molecule Intervention in a Murine Dilated Cardiomyopathy Model

Familial dilated cardiomyopathy (DCM) is a genetic heart disorder characterized by enlargement of one or both ventricles, rendering the heart unable to pump blood efficiently. Mutations in cardiac thin filament (CTF) proteins have been associated with characterized clinical manifestations of DCM. Genetic segregation identified a mutation in α-Tropomyosin (Tm) at residue 230 (D230N-Tm) in two unrelated, multigenerational families linked to severe, early onset DCM. To meaningfully link genotype to phenotype our group sought to investigate the effects of D230N-Tm on the biophysical structure of Tm filament. We showed that D230N-Tm caused a compaction of the cardiac Troponin T (cTnT)–Tm overlap region accompanied by a decreased in flexibility of the Tm filament. Given the altered biophysical structure we investigated whether we could target this decrease in flexibility via small molecule intervention and improve function. Computational modeling and biophysical experiments identified small molecule 4-Hydroxy Duloxetine β-D-Glucuronide (Z06) shown to improve compaction of the cTnT-Tm overlap and restore flexibility of the Tm filament. We hypothesized that the decrease in flexibility of Tm impairs its ability to shift through its different conformational states, leading to impaired crossbridge formation. To investigate whether Z06 improves myofilament function, we performed an NADH-coupled ATPase assay using isolated myofibrils from our D230N-Tm transgenic mice and compared them to Non-Transgenic (NTg) littermates. ATPase activity was measured at saturating calcium (100uM) in the presence (250uM) and absence (0uM) of Z06. In the absence of Z06, D230N-Tm exhibited a decrease in ATPase activity (1.793±0.203 umol/min) compared to NTg littermates (5.420±0.562 umol/min), a common trend in DCM models. In the presence of 250uM Z06, D230N-Tm ATPase rates improve to levels comparable to NTg mice (4.816±0.544 umol/min). Moving forward, we will perform calcium titrations, allowing us to investigate if Z06 can restore cooperativity of myofilament activation. Additionally, we will test if the restorative effects of Z06 are present at the myocellular level, and if so, we will optimize delivery to test in vivo effects of Z06 in our D230N-Tm mouse model.

Fast myosin binding protein C knockout in skeletal muscle alters length-dependent activation and myofilament structure.

In striated muscle, the sarcomeric protein myosin-binding protein-C (MyBP-C) is bound to the myosin thick filament and is predicted to stabilize myosin heads in a docked position against the thick filament, which limits crossbridge formation. Here, we use the homozygous Mybpc2 knockout (C2-/-) mouse line to remove the fast-isoform MyBP-C from fast skeletal muscle and then conduct mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers present deficits in force production and calcium sensitivity. Structurally, passive C2-/- fibers present altered sarcomere length-independent and -dependent regulation of myosin head conformations, with a shift of myosin heads towards actin. At shorter sarcomere lengths, the thin filament is axially extended in C2-/-, which we hypothesize is due to increased numbers of low-level crossbridges. These findings provide testable mechanisms to explain the etiology of debilitating diseases associated with MyBP-C.

Troponin Structural Dynamics in the Native Cardiac Thin Filament Revealed by Cryo Electron Microscopy

Cardiac muscle contraction occurs due to repetitive interactions between myosin thick and actin thin filaments (TF) regulated by Ca2+ levels, active cross-bridges, and cardiac myosin-binding protein C (cMyBP-C). The cardiac TF (cTF) has two nonequivalent strands, each comprised of actin, tropomyosin (Tm), and troponin (Tn). Tn shifts Tm away from myosin-binding sites on actin at elevated Ca2+ levels to allow formation of force-producing actomyosin cross-bridges. The Tn complex is comprised of three distinct polypeptides – Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. The molecular mechanism of their collective action is unresolved due to lack of comprehensive structural information on Tn region of cTF. C1 domain of cMyBP-C activates cTF in the absence of Ca2+ to the same extent as rigor myosin. Here we used cryo-EM of native cTFs to show that cTF Tn core adopts multiple structural conformations at high and low Ca2+ levels and that the two strands are structurally distinct. At high Ca2+ levels, cTF is not entirely activated by Ca2+ but exists in either partially or fully activated state. Complete dissociation of TnI C-terminus is required for full activation. In presence of cMyBP-C C1 domain, Tn core adopts a fully activated conformation, even in absence of Ca2+. Our data provide a structural description for the requirement of myosin to fully activate cTFs and explain increased affinity of TnC to Ca2+ in presence of active cross-bridges. We suggest that allosteric coupling between Tn subunits and Tm is required to control actomyosin interactions.

Analysis of metabolite and strain effects on cardiac cross-bridge dynamics using model linearisation techniques.

Multi-scale models of cardiac energetics are becoming crucial in better understanding the prevalent chronic diseases operating at the intersection of metabolic and cardiovascular dysfunction. Computationally efficient models of cardiac cross-bridge kinetics that are sensitive to changes in metabolite concentrations are necessary to simulate the effects of disease-induced changes in cellular metabolic state on cardiac mechanics across disparate spatial scales. While these models do currently exist, deeper analysis of how the modelling of metabolite effects and the assignment of strain dependence within the cross-bridge cycle affect the properties of the model is required. In this study, model linearisation techniques were used to simulate and interrogate the complex modulus of an ODE-based model of cross-bridge kinetics. Active complex moduli were measured from permeabilised rat cardiac trabeculae under five different metabolite conditions with varying ATP and Pi concentrations. Sensitivity to metabolites was incorporated into an existing three-state cross-bridge model using either a direct dependence or a rapid equilibrium approach. Combining the two metabolite binding methods with all possible locations of strain dependence within the cross-bridge cycle produced 64 permutations of the cross-bridge model. Using linear model analysis, these models were systematically explored to determine the effects of metabolite binding and their interaction with strain dependence on the frequency response of cardiac muscle. The results showed that the experimentally observed effects of ATP and Pi concentrations on the cardiac complex modulus could be attributed to their regulation of cross-bridge detachment rates. Analysis of the cross-bridge models revealed a mechanistic basis for the biochemical schemes which place Pi release following cross-bridge formation and ATP binding prior to cross-bridge detachment. In addition, placing strain dependence on the reverse rate of the cross-bridge power stroke produced the model which most closely matched the experimental data. From these analyses, a well-justified metabolite-sensitive model of rat cardiac cross-bridge kinetics is presented which is suitable for parameterisation with other data sets and integration with multi-scale cardiac models.

Abstract 15822: Cardiac Effects of EDG-7500, a Novel Cardiac Sarcomere Regulator: In vitro and in vivo Evidence for Slowing Isovolumic Contraction and Improved Ventricular Compliance

Background: Hypertrophic Cardiomyopathy (HCM) is a disease where excess acto-myosin crossbridge formation results in hyperdynamic contraction, LV hypertrophy, and diastolic dysfunction due to increased myocardial stiffness. Conventional negative inotropes alleviate, to some degree, the systolic alteration in HCM, but do not improve LV filling. EDG-7500 is a small molecule that preferentially decreases diastolic tension and slows the velocity of myocardial force generation by regulating, but not inhibiting, cardiac myosin. This study assessed pharmacodynamic responses to EDG-7500. Methods: Myofilament biomechanics and sarcomere structure were evaluated on LV permeabilized pig fibers. In vivo, dogs were instrumented for arterial pressure and LV pressure-volume recordings. Data were obtained with vehicle, EDG-7500 (n = 5; 0.3 mg/kg IV), or metoprolol (2-4 mg/kg IV, n = 3); systemic and LV hemodynamics and load-independent function were examined. *:P<0.05 for EDG-7500 vs. vehicle Results: X-ray diffraction studies showed EDG-7500 preserved the myosin head population competent to form crossbridges as well as the recruitment of reserve myosin-heads during calcium activation. EDG-7500 (1μM) slowed crossbridge formation, with a limited reduction in maximal force (-6±2%) and decreased diastolic tension. At 0.3 mg/kg, EDG-7500 slowed early contraction (PEP: +10±1%*) and reduced the peak rate of LV pressure development (+dP/dtmax: -8 ± 1%*) with minimal reductions in peak end systolic elastance (Emax: -4±1%*). Systemic mean arterial pressure and cardiac output were unchanged. EDG-7500 increased LV end-diastolic volume (+5 ± 1%*) and preserved LV end-diastolic pressure (EDP: -8 ± 3%, 9 ± 1 to 8 ± 1 mmHg). A downward and rightward shift of the pressure-volume relationship indicated improved LV compliance. β-AR blockade did not elicit this pro-compliant effect (EDP: +26 ± 12%). Conclusions: Cardiac sarcomere regulation with EDG-7500 has a unique cardiovascular profile characterized by the overall preservation of the myosin motor-head population and a slowing of early cardiac contraction and improved LV distensibility. This cardiovascular profile could have salutary effects in patients with HCM or patients with impaired diastolic function.

Abstract 15612: EDG-7500, a Novel Cardiac Sarcomere Regulator That Preserves Intrinsic Myosin-Motor Function, Improves Cardiac Function and Reserve in a Minipig Model of Hypertrophic Cardiomyopathy

Introduction: Hypertrophic Cardiomyopathy (HCM) is a myocardial disease characterized by LV hypertrophy, hyperdynamic contraction, and diastolic dysfunction that results in impaired exercise capacity. In HCM, excess acto-myosin crossbridge formation during diastole increases myocardial stiffness leading to hindered ventricular filling and limited cardiac reserve. EDG-7500, a novel sarcomere regulator that slows the rate of contraction but does not directly inhibit the myosin motor head, may offer salutary effects in HCM with minimal reduction in systolic performance. Here, this hypothesis was tested in the setting of the pathological HCM beta-myosin heavy chain (MYH7) R403Q mutation. Methods: In vitro, force dynamics were evaluated in human IPSC-derived engineered heart tissues (EHT) with the MYH7 R403Q mutation compared to isogenic controls (WT). The biomechanical effects of EDG-7500 were evaluated in permeabilized fibers from tissues isolated from minipigs carrying the MYH7 R403Q mutation. The in vivo cardiac responses to EDG-7500 were studied via echocardiography and invasive hemodynamics in the R403Q minipigs. In a subset of pigs, β-adrenergic receptor (β-AR) dependent cardiac output reserve was studied. Results: R403Q mutant EHT preserved peak force (PF) relative to WT but impaired relaxation kinetics (RT90: +19±4 ms, 171±4 vs. 152±2 ms in WT, P<0.05). EDG-7500 (0.3uM) accelerated relaxation kinetics in R403Q EHTs (RT90: -8±3ms, 180±4 to 172±2 ms, P<0.05) without affecting PF. RT90 improvement occurred at 6-fold lower concentrations than systolic inhibition (PF IC20: 1.95 ± 0.70 μM). In vivo, EDG-7500 improved early LV filling, accelerating e’ (6.9 ± 0.8 to 8.6 ± 0.6 cm/s, P < 0.05) and decreased atrial volume (-22%, P < 0.05) while enhancing β-AR dependent cardiac output recruitment; in R403Q pigs, EDG-7500 plus dobutamine led to higher cardiac outputs relative to only dobutamine (3.1±0.3 vs. 2.6±0.3 L/min, P<0.05). Conclusions: EDG-7500, a novel cardiac sarcomere regulator, improved diastolic function in EHT and animals with the HCM-pathogenic MYH7 R403Q mutation, improving β-AR dependent cardiac output reserve. This novel profile could be valuable for the treatment of patients with HCM and diseases of diastolic dysfunction.

Load sharing between synergistic muscles characterized by a ligand-binding approach and elastography

The skeletal muscle contraction is determined by cross-bridge formation between the myosin heads and the actin active sites. When the muscle contracts, it shortens, increasing its longitudinal shear elastic modulus ({mu }_{L}). Structurally, skeletal muscle can be considered analogous to the molecular receptors that form receptor–ligand complexes and exhibit specific ligand-binding dynamics. In this context, this work aims to apply elastography and the ligand-binding framework to approach the possible intrinsic mechanisms behind muscle synergism. Based on the short-range stiffness principle and the acoustic–elasticity theory, we define the coefficient C, which is directly related to the fraction saturation of molecular receptors and links the relative longitudinal deformation of the muscle to its {mu }_{L}. We show that such a coefficient can be obtained directly from {mu }_{L} estimates, thus calculating it for the biceps brachii, brachioradialis, and brachialis muscles during isometric elbow flexion torque (τ) ramps. The resulting Cleft(tau right) curves were analyzed by conventional characterization methods of receptor–ligand systems to study the dynamical behavior of each muscle. The results showed that, depending on muscle, Cleft(tau right) exhibits typical ligand-binding dynamics during joint torque production. Therefore, the above indicates that these different behaviors describe the longitudinal shortening pattern of each muscle during load sharing. As a plausible interpretation, we suggested that this could be related to the binding kinetics of the cross-bridges during their synergistic action as torque increases. Likewise, it shows that elastography could be useful to assess contractile processes at different scales related to the change in the mechanical properties of skeletal muscle.

Mouse Models of Cardiomyopathies Caused by Mutations in Troponin C.

Cardiac muscle contraction is regulated via Ca2+ exchange with the hetero-trimeric troponin complex located on the thin filament. Binding of Ca2+ to cardiac troponin C, a Ca2+ sensing subunit within the troponin complex, results in a series of conformational re-arrangements among the thin filament components, leading to an increase in the formation of actomyosin cross-bridges and muscle contraction. Ultimately, a decline in intracellular Ca2+ leads to the dissociation of Ca2+ from troponin C, inhibiting cross-bridge cycling and initiating muscle relaxation. Therefore, troponin C plays a crucial role in the regulation of cardiac muscle contraction and relaxation. Naturally occurring and engineered mutations in troponin C can lead to altered interactions among components of the thin filament and to aberrant Ca2+ binding and exchange with the thin filament. Mutations in troponin C have been associated with various forms of cardiac disease, including hypertrophic, restrictive, dilated, and left ventricular noncompaction cardiomyopathies. Despite progress made to date, more information from human studies, biophysical characterizations, and animal models is required for a clearer understanding of disease drivers that lead to cardiomyopathies. The unique use of engineered cardiac troponin C with the L48Q mutation that had been thoroughly characterized and genetically introduced into mouse myocardium clearly demonstrates that Ca2+ sensitization in and of itself should not necessarily be considered a disease driver. This opens the door for small molecule and protein engineering strategies to help boost impaired systolic function. On the other hand, the engineered troponin C mutants (I61Q and D73N), genetically introduced into mouse myocardium, demonstrate that Ca2+ desensitization under basal conditions may be a driving factor for dilated cardiomyopathy. In addition to enhancing our knowledge of molecular mechanisms that trigger hypertrophy, dilation, morbidity, and mortality, these cardiomyopathy mouse models could be used to test novel treatment strategies for cardiovascular diseases. In this review, we will discuss (1) the various ways mutations in cardiac troponin C might lead to disease; (2) relevant data on mutations in cardiac troponin C linked to human disease, and (3) all currently existing mouse models containing cardiac troponin C mutations (disease-associated and engineered).

A dynamic calcium-force relationship model for sag behavior in fast skeletal muscle.

In vitro studies using isolated or skinned muscle fibers suggest that the sigmoidal relationship between the intracellular calcium concentration and force production may depend upon muscle type and activity. The goal of this study was to investigate whether and how the calcium-force relationship changes during force production under physiological conditions of muscle excitation and length in fast skeletal muscles. A computational framework was developed to identify the dynamic variation in the calcium-force relationship during force generation over a full physiological range of stimulation frequencies and muscle lengths in cat gastrocnemius muscles. In contrast to the situation in slow muscles such as the soleus, the calcium concentration for the half-maximal force needed to drift rightward to reproduce the progressive force decline, or sag behavior, observed during unfused isometric contractions at the intermediate length under low-frequency stimulation (i.e., 20 Hz). The slope at the calcium concentration for the half-maximal force was required to drift upward for force enhancement during unfused isometric contractions at the intermediate length under high-frequency stimulation (i.e., 40 Hz). The slope variation in the calcium-force relationship played a crucial role in shaping sag behavior across different muscle lengths. The muscle model with dynamic variations in the calcium-force relationship also accounted for the length-force and velocity-force properties measured under full excitation. These results imply that the calcium sensitivity and cooperativity of force-inducing crossbridge formation between actin and myosin filaments may be operationally altered in accordance with the mode of neural excitation and muscle movement in intact fast muscles.

Estimation of crossbridge-state during cardiomyocyte beating using second harmonic generation.

Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and others. Here, we established an assay system to dynamically measure second harmonic generation (SHG) anisotropy derived from myosin filaments depended on their crossbridge status in pulsating cardiomyocytes. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost the force-generating ability upon myocardial differentiation. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a Drosophila disease model. Thus, we successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on cardiomyocytes. Because genomic inspection alone may not catch the risk of cardiomyopathy in some cases, our study demonstrated herein would be of help in the risk assessment of future heart failure.

Myosin S2 stabilizer peptide decreases recovery rates of contraction.

A peptide, called the Stabilizer, designed to increase the energy required to unzip the myosin S2 coiled coil was tested on myofibrils, skinned trabeculae, and live cardiomyocytes. The Stabilizer bound to myosin S2 with submicromolar affinity and positive cooperativity measured by both TIRF and MST, which suggests a modification in the myosin S2 structure. Myofibril ATPase activity was activated at pCa 9 and inhibited at pCa 4.5, consistent with an inhibition of actin-activated ATPase activity. In vitro motility assays of actin sliding velocity were partially depressed in the presence of the Stabilizer. Both force development rates and regulatory light chain orientation (measured by fluorescence polarization) were also slowed by the Stabilizer following a slack-re-stretch maneuver in skinned trabeculae. To test perturbations of living cardiomyocytes, the Stabilizer peptide was chemically esterified with a cell penetrating molecule to facilitate translocation across the cell membrane and removal of the targeting molecule once inside the cell. Confocal microscopy imaging showed sarcomere labeling with fluorescently tagged targeted Stabilzer in live cardiomyocytes. The Stabilizer decreased sarcomere length shortening in living cardiomyocytes. These data are consistent with a reduced contractility by the Stabilizer, which may result from the reinforcement of the myosin S2 coiled coil structure mitigating myosin's flexibility and probability of cross-bridge formation. (Supported by NIH/NHLBI R01HL149164.)

Thermogenic regulation of the simple harmonic motion of tropomyosin byweak-binding myosin cross-bridges explains spontaneous oscillatory contractions.

A sarcomeric regulatory role for weak-binding, disordered cross-bridges (dXbs) of the myosin disordered-relaxed state is described which explains spontaneous oscillatory contractions (SPOCs). The simple harmonic (SH) theory proposes that as tropomyosin (Tm) interacts with thin filament the random vibrational motion begins to vibrate harmonically with the actin repeat interval (i.e., simple harmonic motion, SHM). This interaction increases as the thick filament electrostatic force increases as lattice spacing decreases by stretch or osmotic compression, causing the SHM to increase with displacement of Tm from thin filament OFF to ON position, exposing sites for cross-bridge formation and force generation. Troponin inhibits this process with decreasing calcium. A thermogenic mechanism is implicit in the SH theory whereby the random/Brownian motion of molecules that interact with Tm modulate the random motion of Tm which, in turn, is regulated harmonically. Heat and internal work from external sources and hydrolysis of ATP are primary sources for Brownian motion of sarcomeric molecules including dXbs which are likely a major source for modulating the SHM thermogenicly. Initially, at partial activation, by calcium or ADP, the increase in dXbs causes thermogenic vibrational energy and SHM of Tm to increase. Thermogenic activation continues throughout the slow contraction phase of SPOCs. Subsequent rapid elongation/relaxation phase occurs as the thermogenic process reaches its limit, as 1) dXbs decrease as force generating cross-bridges increase, 2) contractile efficiency increases (i.e., less heat/internal work) as shortening velocity decreases, and 3) thermogenic excitation by dXbs decreases as lattice spacing increases. The inhibitory force of troponin now overcomes the SHM of Tm which rapidly returns to initial partial OFF/ON conditions and the cycle repeats. Propagation of SPOC waves and implications for the regulatory mechanism of MyBP-C are discussed.

Pharmacological interventions improve myosin-actin cross-bridge function in mice with advanced chronic kidney disease.

Skeletal muscle fiber force production in adult mice with late-stage chronic kidney disease (CKD) is significantly impaired, although by different myofilament and myosin-actin cross-bridge disease responses in the various fiber types. To reverse some of the CKD-induced contractile deficits, we examined the effects of the ATP analog, 2-deoxyadenosine triphosphate (dATP), to enhance cross-bridge kinetics and the troponin sensitizer, Tirasemtiv (2 µM), to increase cross-bridge formation. Single fiber force production, myosin-actin interactions and myofilament properties were measured at 25°C in fibers from soleus and extensor digitorum longus muscles of 16-week-old CKD mice (N=5) and controls (N=5). Late-stage CKD was induced by 0.2% adenine diet for 8 weeks. dATP in myosin heavy chain (MHC) I and IIA fibers with CKD increased cross-bridge kinetics, returning them to control values or faster, leading to more strongly bound cross-bridges for maximal Ca2+-activation (pCa 4.5, pH 7.0, 5 mM Phosphate) and fatiguing (pCa 4.5, pH 6.2, 30 mM Phosphate) conditions. Specific tensions in CKD were also improved 12-21% under maximal and fatiguing conditions, except in maximally Ca2+-activated MHC I fibers, but fell short of control values. Notably, other studies have found that improvements of 13-25% in knee extensor strength in CKD patients improved their physical function, suggesting changes of this magnitude are clinically meaningful. At submaximal Ca2+-activation (pCa 6.0), Tirasemtiv in MHC IIB fibers with CKD increased myofilament stiffness 520%, number of strongly bound cross-bridges 705%, and specific tension 370%, returning specific tension back to control values. This study provides evidence that cellular and molecular contractile function in CKD may be enhanced through use of dATP or Tirasemtiv, which should be tested to potentially improve physical function and overall quality of life in late-stage CKD patients.

Cryo-Electron Microscopy Reveals Cardiac Myosin Binding Protein-C M-Domain Interactions with the Thin Filament

Cardiac myosin binding protein C (cMyBP-C) modulates cardiac contraction via direct interactions with cardiac thick (myosin) and thin (actin) filaments (cTFs). While its C-terminal domains (e.g. C8-C10) anchor cMyBP-C to the backbone of the thick filament, its N-terminal domains (NTDs) (e.g. C0, C1, M, and C2) bind to both myosin and actin to accomplish its dual roles of inhibiting thick filaments and activating cTFs. While the positions of C0, C1 and C2 on cTF have been reported, the binding site of the M-domain on the surface of the cTF is unknown. Here, we used cryo-EM to reveal that the M-domain interacts with actin via helix 3 of its ordered tri-helix bundle region, while the unstructured part of the M-domain does not maintain extensive interactions with actin. We combined the recently obtained structure of the cTF with the positions of all the four NTDs on its surface to propose a complete model of the NTD binding to the cTF. The model predicts that the interactions of the NTDs with the cTF depend on the activation state of the cTF. At the peak of systole, when bound to the extensively activated cTF, NTDs would inhibit actomyosin interactions. In contrast, at falling Ca2+ levels, NTDs would not compete with the myosin heads for binding to the cTF, but would rather promote formation of active cross-bridges at the adjacent regulatory units located at the opposite cTF strand. Our structural data provides a testable model of the cTF regulation by the cMyBP-C.

Muscle LIM Protein Force-Sensing Mediates Sarcomeric Biomechanical Signaling in Human Familial Hypertrophic Cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics. Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell-derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations. Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain (MYH7) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin-nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein-W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations. Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex.

Evidence for novel phosphorylation sites in the central C4-C5 domains of murine cardiac myosin binding protein C

Cardiac myosin binding protein C (cMyBPC) is a key multi-domain protein in myosin cross bridge formation and cardiac contractility. It is principally regulated by phosphorylation of the residues within the M-domain in the N-terminus, which is essential for proper function. In fact, cardiac hypertrophy and heart failure have been associated with reduced overall cMyBPC phosphorylation levels. However, not much is known about the phosphorylation landscape of the enigmatic central C4-C5 domains of cMyBPC.

Myosin off-on transition modulates the simple harmonic motion of tropomyosin coupling length-dependent activation and the actomyosin chemomechanical cycle

Striated muscle contraction is modulated by structural changes in both myosin thick filaments and actin thin filaments. Yet, the signaling pathway between myofilaments is unknown. The thick filament mechanism involves movement of myosin heads (likely force/strain-dependent) from a folded, horizontal position on thick filament backbone (OFF) to a more perpendicular orientation (ON), closer to thin filament. Upon binding of calcium by troponin, the thin filament mechanism involves movement of tropomyosin from periphery of thin filament to the actin groove, enabling myosin crossbridge formation and force generation.

Thick and thin filament effectors of cardiac myofibril contraction detected in a troponin c-based high-throughput fluorescence lifetime assay

Cardiac muscle contraction is initiated by Ca2+-induced structural changes of the thin filaments to permit cross-bridge formation. Ca2+ binding to cardiac troponin C (TnC) on actin permits myosin binding and ATP binding to myosin allows for cross-bridge cycling during contraction. Site-directed spectroscopic probes attached to TnC at position T53C have been useful to determine structure-function relationships and Ca2+ binding kinetics in thin filaments and myofibrils by monitoring fluorescence intensity.

Actomyosin Complex.

Formation of cross-bridges between actin and myosin occurs ubiquitously in eukaryotic cells and mediates muscle contraction, intracellular cargo transport, and cytoskeletal remodeling. Myosin motors repeatedly bind to and dissociate from actin filaments in a cycle that transduces the chemical energy from ATP hydrolysis into mechanical force generation. While the general layout of surface elements within the actin-binding interface is conserved among myosin classes, sequence divergence within these motifs alters the specific contacts involved in the actomyosin interaction as well as the kinetics of mechanochemical cycle phases. Additionally, diverse lever arm structures influence the motility and force production of myosin molecules during their actin interactions. The structural differences generated by myosin's molecular evolution have fine-tuned the kinetics of its isoforms and adapted them for their individual cellular roles. In this chapter, we will characterize the structural and biochemical basis of the actin-myosin interaction and explain its relationship with myosin's cellular roles, with emphasis on the structural variation among myosin isoforms that enables their functional specialization. We will also discuss the impact of accessory proteins, such as the troponin-tropomyosin complex and myosin-binding protein C, on the formation and regulation of actomyosin cross-bridges.