Differences in thick filament activation in fast rodent skeletal muscle and slow porcine cardiac muscle.

There is a growing appreciation that regulation of muscle contraction requires both thin filament and thick filament activation in order to fully activate the sarcomere. The prevailing mechano-sensing model for thick filament activation was derived from experiments on fast-twitch muscle. We address the question whether, or to what extent, this mechanism can be extrapolated to the slow muscle in the hearts of large mammals, including humans. We investigated the similarities and differences in structural signatures of thick filament activation in porcine myocardium as compared to fast rat extensor digitorum longus (EDL) skeletal muscle under relaxed conditions and sub-maximal contraction using small angle X-ray diffraction. Thick and thin filaments were found to adopt different structural configurations under relaxing conditions, and myosin heads showed different changes in configuration upon sub-maximal activation, when comparing the two muscle types. Titin was found to have an X-ray diffraction signature distinct from those of the overall thick filament backbone, and its spacing change appeared to be positively correlated to the force exerted on the thick filament. Structural changes in fast EDL muscle were found to be consistent with the mechano-sensing model. In porcine myocardium, however, the structural basis of mechano-sensing is blunted suggesting the need for additional activation mechanism(s) in slow cardiac muscle. These differences in thick filament regulation can be related to their different physiological roles where fast muscle is optimized for rapid, burst-like, contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned, graded response to allow for their substantial functional reserve. KEY POINTS: Both thin filament and thick filament activation are required to fully activate the sarcomere. Thick and thin filaments adopt different structural configurations under relaxing conditions, and myosin heads show different changes in configuration upon sub-maximal activation in fast extensor digitorum longus muscle and slow porcine cardiac muscle. Titin has an X-ray diffraction signature distinct from those of the overall thick filament backbone and this titin reflection spacing change appeared to be directly proportional to the force exerted on the thick filament. Mechano-sensing is blunted in porcine myocardium suggesting the need for additional activation mechanism(s) in slow cardiac muscle. Fast skeletal muscle is optimized for rapid, burst-like contractions, and the slow cardiac muscle in large mammalian hearts adopts a more finely tuned graded response to allow for their substantial functional reserve.

Myosin-binding protein C regulates the sarcomere lattice and stabilizes the OFF states of myosin heads.

Muscle contraction is produced via the interaction of myofilaments and is regulated so that muscle performance matches demand. Myosin-binding protein C (MyBP-C) is a long and flexible protein that is tightly bound to the thick filament at its C-terminal end (MyBP-CC8C10), but may be loosely bound at its middle- and N-terminal end (MyBP-CC1C7) to myosin heads and/or the thin filament. MyBP-C is thought to control muscle contraction via the regulation of myosin motors, as mutations lead to debilitating disease. We use a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-CC1C7 domains of fast MyBP-C in permeabilized skeletal muscle. We show that cleavage leads to alterations in crossbridge kinetics and passive structural signatures of myofilaments that are indicative of a shift of myosin heads towards the ON state, highlighting the importance of MyBP-CC1C7 to myofilament force production and regulation.

Myosin in autoinhibited off state(s), stabilized by mavacamten, can be recruited in response to inotropic interventions.

Mavacamten is a FDA-approved small-molecule therapeutic designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin toward ordered off states close to the thick filament backbone. It remains elusive whether these myosin heads in the off state(s) can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by 1) Ca2+, 2) increased chronotropy [heart rate (HR)], 3) stretch, and 4) ß-adrenergic (ß-AR) stimulation, all known physiological inotropic interventions. At the molecular level, we show that Ca2+ increases myosin ATPase activity by shifting mavacamten-stabilized myosin heads from the inactive super-relaxed state to the active disordered relaxed state. At the myofilament level, both Ca2+ and passive lengthening can shift mavacamten-ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dtmax, an index of inotropy, increased with HR in mavacamten-treated animals. Finally, we show that ß-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are at least partially activable, thus preserving cardiac reserve mechanisms.

The structural OFF and ON states of myosin can be decoupled from the biochemical super- and disordered-relaxed states.

There is a growing awareness that both thick-filament and classical thin-filament regulations play central roles in modulating muscle contraction. Myosin ATPase assays have demonstrated that under relaxed conditions, myosin may reside either in a high-energy-consuming disordered-relaxed (DRX) state available for binding actin to generate force or in an energy-sparing super-relaxed (SRX) state unavailable for actin binding. X-ray diffraction studies have shown that the majority of myosin heads are in a quasi-helically ordered OFF state in a resting muscle and that this helical ordering is lost when myosin heads are turned ON for contraction. It has been assumed that myosin heads in SRX and DRX states are equivalent to the OFF and ON states, respectively, and the terms have been used interchangeably. In this study, we use X-ray diffraction and ATP turnover assays to track the structural and biochemical transitions of myosin heads, respectively, induced with either omecamtiv mecarbil (OM) or piperine in relaxed porcine myocardium. We find that while OM and piperine induce dramatic shifts of myosin heads from the OFF to the ON state, there are no appreciable changes in the population of myosin heads in the SRX and DRX states in both unloaded and loaded preparations. Our results show that biochemically defined SRX and DRX can be decoupled from structurally defined OFF and ON states. In summary, while SRX/DRX and OFF/ON transitions can be correlated in some cases, these two phenomena are measured using different approaches, reflect different properties of the thick filament, and should be investigated and interpreted separately.

Unlocking the Role of sMyBP-C: A Key Player in Skeletal Muscle Development and Growth.

Skeletal muscle is the largest organ in the body, responsible for gross movement and metabolic regulation. Recently, variants in the MYBPC1 gene have been implicated in a variety of developmental muscle diseases, such as distal arthrogryposis. How MYBPC1 variants cause disease is not well understood. Here, through a collection of novel gene-edited mouse models, we define a critical role for slow myosin binding protein-C (sMyBP-C), encoded by MYBPC1, across muscle development, growth, and maintenance during prenatal, perinatal, postnatal and adult stages. Specifically, Mybpc1 knockout mice exhibited early postnatal lethality and impaired skeletal muscle formation and structure, skeletal deformity, and respiratory failure. Moreover, a conditional knockout of Mybpc1 in perinatal, postnatal and adult stages demonstrates impaired postnatal muscle growth and function secondary to disrupted actomyosin interaction and sarcomere structural integrity. These findings confirm the essential role of sMyBP-C in skeletal muscle and reveal specific functions in both prenatal embryonic musculoskeletal development and postnatal muscle growth and function.

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

In striated muscle, some sarcomere proteins regulate crossbridge cycling by varying the propensity of myosin heads to interact with actin. Myosin-binding protein C (MyBP-C) is bound to the myosin thick filament and is predicted to interact and stabilize myosin heads in a docked position against the thick filament and limit crossbridge formation, the so-called OFF state. Via an unknown mechanism, MyBP-C is thought to release heads into the so-called ON state, where they are more likely to form crossbridges. To study this proposed mechanism, we used the C2-/- mouse line to knock down fast-isoform MyBP-C completely and total MyBP-C by ~24%, and conducted mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers presented deficits in force production and reduced calcium sensitivity. Structurally, passive C2-/- fibers presented altered SL-independent and SL-dependent regulation of myosin head ON/OFF states, with a shift of myosin heads towards the ON state. Unexpectedly, at shorter sarcomere lengths, the thin filament was axially extended in C2-/- vs. non-transgenic controls, which we postulate is due to increased low-level crossbridge formation arising from relatively more ON myosins in the passive muscle that elongates the thin filament. The downstream effect of increasing crossbridge formation in a passive muscle on contraction performance is not known. Such widespread structural changes to sarcomere proteins provide testable mechanisms to explain the etiology of debilitating MyBP-C-associated diseases.

Titin-based force regulates cardiac myofilament structures mediating length-dependent activation.

The Frank-Starling law states that the heart's stroke volume increases with greater preload due to increased venous return, allowing the heart to adapt to varying circulatory demands. Molecularly, increasing preload increases sarcomere length (SL), which alters sarcomere structures that are correlated to increased calcium sensitivity upon activation. The titin protein, spanning the half-sarcomere, acts as a spring in the I-band, applying a SL-dependent force suggested to pull against and alter myofilaments in a way that supports the Frank-Starling effect. To evaluate this, we employed the titin cleavage (TC) model, where a tobacco-etch virus protease recognition site is inserted into distal I-band titin and allows for rapid, specific cleavage of titin in an otherwise-healthy sarcomere. Here, we evaluated the atomic-level structures of amyopathic cardiac myofilaments following 50% titin cleavage under passive stretch conditions using small-angle X-ray diffraction, which measures these structures under near-physiological (functional) conditions. We report that titin-based forces in permeabilized papillary muscle regulate both thick and thin myofilament structures clearly supporting titin's role in the Frank-Starling mechanism.

Disruption of Z-Disc Function Promotes Mechanical Dysfunction in Human Myocardium: Evidence for a Dual Myofilament Modulatory Role by Alpha-Actinin 2.

The ACTN2 gene encodes α-actinin 2, located in the Z-disc of the sarcomeres in striated muscle. In this study, we sought to investigate the effects of an ACTN2 missense variant of unknown significance (p.A868T) on cardiac muscle structure and function. Left ventricular free wall samples were obtained at the time of cardiac transplantation from a heart failure patient with the ACTN2 A868T heterozygous variant. This variant is in the EF 3-4 domain known to interact with titin and α-actinin. At the ultrastructural level, ACTN2 A868T cardiac samples presented small structural changes in cardiomyocytes when compared to healthy donor samples. However, contractile mechanics of permeabilized ACTN2 A868T variant cardiac tissue displayed higher myofilament Ca2+ sensitivity of isometric force, reduced sinusoidal stiffness, and faster rates of tension redevelopment at all Ca2+ levels. Small-angle X-ray diffraction indicated increased separation between thick and thin filaments, possibly contributing to changes in muscle kinetics. Molecular dynamics simulations indicated that while the mutation does not significantly impact the structure of α-actinin on its own, it likely alters the conformation associated with titin binding. Our results can be explained by two Z-disc mediated communication pathways: one pathway that involves α-actinin's interaction with actin, affecting thin filament regulation, and the other pathway that involves α-actinin's interaction with titin, affecting thick filament activation. This work establishes the role of α-actinin 2 in modulating cross-bridge kinetics and force development in the human myocardium as well as how it can be involved in the development of cardiac disease.

The structural OFF and ON states of myosin can be decoupled from the biochemical super-relaxed and disordered-relaxed states.

There is a growing awareness that both thick filament and classical thin filament regulation play central roles in modulating muscle contraction. Myosin ATPase assays have demonstrated that under relaxed conditions, myosin may reside in either a high energy-consuming disordered-relaxed (DRX) state available for binding actin to generate force, or in an energy-sparing super-relaxed (SRX) state unavailable for actin binding. X-ray diffraction studies have shown the majority of myosin heads are in a quasi-helically ordered OFF state in a resting muscle and that this helical ordering is lost when myosin heads are turned ON for contraction. It has been assumed that myosin heads in SRX and DRX states are equivalent to the OFF and ON state respectively and the terms have been used interchangeably. Here, we use X-ray diffraction and ATP turnover assays to track the structural and biochemical transitions of myosin heads respectively induced with either omecamtiv mecarbil (OM) or piperine in relaxed porcine myocardium. We find that while OM and piperine induce dramatic shifts of myosin heads from the OFF to ON states, there are no appreciable changes in the population of myosin heads in the SRX and DRX states in both unloaded and loaded preparations. Our results show that biochemically defined SRX and DRX can be decoupled from structurally-defined OFF and ON states. In summary, while SRX/DRX and OFF/ON transitions can be correlated in some cases, these two phenomena are measured using different approaches, do not necessarily reflect the same properties of the thick filament and should be investigated and interpreted separately.

Myosin-binding protein C forms C-links and stabilizes OFF states of myosin.

Contraction force in muscle is produced by the interaction of myosin motors in the thick filaments and actin in the thin filaments and is fine-tuned by other proteins such as myosin-binding protein C (MyBP-C). One form of control is through the regulation of myosin heads between an ON and OFF state in passive sarcomeres, which leads to their ability or inability to interact with the thin filaments during contraction, respectively. MyBP-C is a flexible and long protein that is tightly bound to the thick filament at its C-terminal end but may be loosely bound at its middle- and N-terminal end (MyBP-CC1C7). Under considerable debate is whether the MyBP-CC1C7 domains directly regulate myosin head ON/OFF states, and/or link thin filaments ("C-links"). Here, we used a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-CC1C7 domains of fast MyBP-C in permeabilized skeletal muscle. After cleavage, the thin filaments were significantly shorter, a result consistent with direct interactions of MyBP-C with thin filaments thus confirming C-links. Ca2+ sensitivity was reduced at shorter sarcomere lengths, and crossbridge kinetics were increased across sarcomere lengths at submaximal activation levels, demonstrating a role in crossbridge kinetics. Structural signatures of the thick filaments suggest that cleavage also shifted myosin heads towards the ON state - a marker that typically indicates increased Ca2+ sensitivity but that may account for increased crossbridge kinetics at submaximal Ca2+ and/or a change in the force transmission pathway. Taken together, we conclude that MyBP-CC1C7 domains play an important role in contractile performance which helps explain why mutations in these domains often lead to debilitating diseases.

The structural and functional integrities of porcine myocardium are mostly preserved by cryopreservation.

Structural and functional studies of heart muscle are important to gain insights into the physiological bases of cardiac muscle contraction and the pathological bases of heart disease. While fresh muscle tissue works best for these kinds of studies, this is not always practical to obtain, especially for heart tissue from large animal models and humans. Conversely, tissue banks of frozen human hearts are available and could be a tremendous resource for translational research. It is not well understood, however, how liquid nitrogen freezing and cryostorage may impact the structural integrity of myocardium from large mammals. In this study, we directly compared the structural and functional integrity of never-frozen to previously frozen porcine myocardium to investigate the consequences of freezing and cryostorage. X-ray diffraction measurements from hydrated tissue under near-physiological conditions and electron microscope images from chemically fixed porcine myocardium showed that prior freezing has only minor effects on structural integrity of the muscle. Furthermore, mechanical studies similarly showed no significant differences in contractile capabilities of porcine myocardium with and without freezing and cryostorage. These results demonstrate that liquid nitrogen preservation is a practical approach for structural and functional studies of myocardium.

Danicamtiv Increases Myosin Recruitment and Alters Cross-Bridge Cycling in Cardiac Muscle.

BACKGROUND: Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. METHODS: Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficit. RESULTS: Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the ON state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv corrected decreased calcium sensitivity in demembranated tissue, abnormal twitch magnitude and kinetics in intact cardiac tissue, and reduced ejection fraction in the whole organ. CONCLUSIONS: As demonstrated by the detailed studies of Danicamtiv, increasing myosin recruitment and altering cross-bridge cycling are 2 mechanisms to increase force and calcium sensitivity in cardiac muscle. Myosin activators such as Danicamtiv can treat the causative hypocontractile phenotype in genetic dilated cardiomyopathy.

Right Ventricular Sarcomere Contractile Depression and the Role of Thick Filament Activation in Human Heart Failure With Pulmonary Hypertension.

BACKGROUND: Right ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in patients with heart failure with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. We thus sought to characterize RV myocyte contractile depression in HFrEF-PH, identify those components reflected by clinical RV indices, and uncover underlying biophysical mechanisms. METHODS: Resting, calcium-, and load-dependent mechanics were prospectively studied in permeabilized RV cardiomyocytes isolated from explanted hearts from 23 patients with HFrEF-PH undergoing cardiac transplantation and 9 organ donor controls. RESULTS: Unsupervised machine learning using myocyte mechanical data with the highest variance yielded 2 HFrEF-PH subgroups that in turn mapped to patients with decompensated or compensated clinical RV function. This correspondence was driven by reduced calcium-activated isometric tension in decompensated clinical RV function, whereas surprisingly, many other major myocyte contractile measures including peak power and myocyte active stiffness were similarly depressed in both groups. Similar results were obtained when subgroups were first defined by clinical indices, and then myocyte mechanical properties in each group compared. To test the role of thick filament defects, myofibrillar structure was assessed by x-ray diffraction of muscle fibers. This revealed more myosin heads associated with the thick filament backbone in decompensated clinical RV function, but not compensated clinical RV function, as compared with controls. This corresponded to reduced myosin ATP turnover in decompensated clinical RV function myocytes, indicating less myosin in a crossbridge-ready disordered-relaxed (DRX) state. Altering DRX proportion (%DRX) affected peak calcium-activated tension in the patient groups differently, depending on their basal %DRX, highlighting potential roles for precision-guided therapeutics. Last, increasing myocyte preload (sarcomere length) increased %DRX 1.5-fold in controls but only 1.2-fold in both HFrEF-PH groups, revealing a novel mechanism for reduced myocyte active stiffness and by extension Frank-Starling reserve in human heart failure. CONCLUSIONS: Although there are many RV myocyte contractile deficits in HFrEF-PH, commonly used clinical indices only detect reduced isometric calcium-stimulated force, which is related to deficits in basal and recruitable %DRX myosin. Our results support use of therapies to increase %DRX and enhance length-dependent recruitment of DRX myosin heads in such patients.

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

Myosin in autoinhibited off state(s), stabilized by mavacamten, can be recruited via inotropic effectors.

Mavacamten is a novel, FDA-approved, small molecule therapeutic designed to regulate cardiac function by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin towards ordered off states close to the thick filament backbone. It remains unresolved whether mavacamten permanently sequesters these myosin heads in the off state(s) or whether these heads can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by Ca2+, increased heart rate, stretch, and ß-adrenergic (ß-AR) stimulation, all known physiological inotropic effectors. At the molecular level, we show that, in presence of mavacamten, Ca2+ increases myosin ATPase activity by shifting myosin heads from the reserve super-relaxed (SRX) state to the active disordered relaxed (DRX) state. At the myofilament level, both Ca2+ and passive lengthening can shift ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments in the presence of mavacamten. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dtmax, an index of inotropy, increased with heart rate in mavacamten treated animals. Finally, we show that ß-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are activable, at least partially, thus leading to preservation of cardiac reserve mechanisms.

The distinctive mechanical and structural signatures of residual force enhancement in myofibers.

In muscle, titin proteins connect myofilaments together and are thought to be critical for contraction, especially during residual force enhancement (RFE) when force is elevated after an active stretch. We investigated titin's function during contraction using small-angle X-ray diffraction to track structural changes before and after 50% titin cleavage and in the RFE-deficient, mdm titin mutant. We report that the RFE state is structurally distinct from pure isometric contractions, with increased thick filament strain and decreased lattice spacing, most likely caused by elevated titin-based forces. Furthermore, no RFE structural state was detected in mdm muscle. We posit that decreased lattice spacing, increased thick filament stiffness, and increased non-crossbridge forces are the major contributors to RFE. We conclude that titin directly contributes to RFE.

Right Ventricular Sarcomere Contractile Depression and the Role of Thick Filament Activation in Human Heart Failure with Pulmonary Hypertension.

Rationale: Right ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in heart failure patients with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. Objective: To determine components of myocyte contractile depression in HFrEF-PH, identify those reflected by clinical RV indices, and elucidate their underlying biophysical mechanisms. Methods and Results: Resting, calcium- and load-dependent mechanics were measured in permeabilized RV cardiomyocytes isolated from explanted hearts from 23 HFrEF-PH patients undergoing cardiac transplantation and 9 organ-donor controls. Unsupervised machine learning using myocyte mechanical data with the highest variance yielded two HFrEF-PH subgroups that in turn mapped to patients with depressed (RVd) or compensated (RVc) clinical RV function. This correspondence was driven by reduced calcium-activated isometric tension in RVd, while surprisingly, many other major myocyte contractile measures including peak power, maximum unloaded shortening velocity, and myocyte active stiffness were similarly depressed in both groups. Similar results were obtained when subgroups were first defined by clinical indices, and then myocyte mechanical properties in each group compared. To test the role of thick-filament defects, myofibrillar structure was assessed by X-ray diffraction of muscle fibers. This revealed more myosin heads associated with the thick filament backbone in RVd but not RVc, as compared to controls. This corresponded to reduced myosin ATP turnover in RVd myocytes, indicating less myosin in a cross-bridge ready disordered-relaxed (DRX) state. Altering DRX proportion (%DRX) affected peak calcium-activated tension in the patient groups differently, depending on their basal %DRX, highlighting potential roles for precision-guided therapeutics. Lastly, increasing myocyte preload (sarcomere length) increased %DRX 1.5-fold in controls but only 1.2-fold in both HFrEF-PH groups, revealing a novel mechanism for reduced myocyte active stiffness and by extension Frank-Starling reserve in human HF. Conclusions: While there are multiple RV myocyte contractile deficits In HFrEF-PH, clinical indices primarily detect reduced isometric calcium-stimulated force related to deficits in basal and recruitable %DRX myosin. Our results support use of therapies to increase %DRX and enhance length-dependent recruitment of DRX myosin heads in such patients.

Danicamtiv increases myosin recruitment and alters the chemomechanical cross bridge cycle in cardiac muscle.

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the "on" state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Danicamtiv corrected calcium sensitivity in demembranated and abnormal twitch magnitude and kinetics in intact cardiac tissue. Significance Statement: Directly augmenting sarcomere function has potential to overcome limitations of currently used inotropic agents to improve cardiac contractility. Myosin modulation is a novel mechanism for increased contraction in cardiomyopathies. Danicamtiv is a myosin activator that is currently under investigation for use in cardiomyopathy patients. Our study is the first detailed mechanism of how Danicamtiv increases force and alters kinetics of cardiac activation and relaxation. This new understanding of the mechanism of action of Danicamtiv can be used to help identify patients that could benefit most from this treatment.