Elucidating the mechanism of Danicamtiv on force, kinetics, and myosin structure and function

Myosin modulators are a novel class of small molecules that alter cardiac contractility. Omecamtiv mecarbil, the first identified myosin activator, showed only modest clinical benefits in systolic heart failure patients. Thus, there is an urgency to develop alternative myosin activators. Danicamtiv (Dani) has emerged as a potential candidate; however, a detailed mechanism is not known. Here, we aim to elucidate the mechanism of Dani on contractile function in pig cardiac muscle. Demembranated ventricular tissues show a significant 0.1 pCa unit increase in calcium sensitivity and 10% increase in maximal force after incubation in 1 µM Dani. The most potent effects occur in submaximal calcium concentrations, leading to a flattening of the force-calcium relationship, suggesting decreased cooperativity. Maximal rates of tension redevelopment are decreased by approximately 60% with Dani. Isolated cardiac myofibrils provide details about contractile kinetics. Experiments with 1 µM Dani show a 49% decrease in fast-phase relaxation kinetics. Slow-phase isometric relaxation exhibits 47% slower crossbridge detachment rate and 34% longer thin filament deactivation. Next, we assess ATP utilization in the crossbridge cycle. Filament sliding velocity slows 55% on addition of 0.5 µM Dani, similar to the effect of ADP on velocity. The effects of Dani and ADP are not additive suggesting a similar mode of action. ATP binding is unaltered up to 10 µM Dani using stopped flow spectroscopy. Results of X-ray diffraction studies of porcine myocardium at rest show an increase in equatorial intensity ratio (I1,1/I1,0) in response to 50 µM Dani, reflecting an increased proximity of myosin heads to the thin filament. In conclusion, we hypothesize that Dani primes the thick filament for activation and alters relaxation through inhibited ATP hydrolysis product release. Future studies will test these hypotheses

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 deficient. 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 crossbridge 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 cardiomyopath

The effect of small molecule myosin modulators on ATP turnover in pig cardiac HMM using stopped flow spectroscopy

Myosin modulators are a novel class of pharmaceutical agents designed to treat patients with cardiomyopathies by directly modulating cardiac myosin function in the sarcomere. Compounds including omecamtiv mecarbil (OM), danicamtiv (Dani), and deoxy-ATP (dATP) have previously been shown to increase myofibril ATPase activity while mavacamten (Mava) reduced ATPase activity. In the absence of actin, ATPase activity is a combination of the direct effects of nucleotide binding and the equilibrium between the high activity (DRX) and low activity (SRX) states of myosin. In this study, we investigate how these small molecules affect the single turnover kinetics of pig cardiac heavy meromyosin (pcHMM) in the absence of actin. pcHMM bound to fluorescent mant.ATP or mant.dATP is rapidly mixed with a high concentration of unlabeled ATP. The rate constant for replacement of mant.ADP by ATP defines the turnover of mant.ATP. Preliminary titration experiments demonstrate that OM, Dani, and Mava all inhibit ATP turnover with an IC50 of 0.59 µM, 3.5 µM, and 0.276 µM, respectively. 100% dATP increases the ATP turnover by 100%. These experiments indicate that each myosin modulator differentially alters cardiac HMM activity. We will discuss how each modulator affects ATP turnover by a direct effect on catalytic activity vs an effect on the amount of HMM in the super-relaxed population

Modulating the tension-time integral of the cardiac twitch prevents dilated cardiomyopathy in murine hearts

Dilated cardiomyopathy (DCM) is often associated with sarcomere protein mutations that confer reduced myofilament tension-generating capacity. We demonstrated that cardiac twitch tensiontime integrals can be targeted and tuned to prevent DCM remodeling in hearts with contractile dysfunction. We employed a transgenic murine model of DCM caused by the D230N-tropomyosin (Tm) mutation and designed a sarcomere-based intervention specifically targeting the twitch tension-time integral of D230N-Tm hearts using mu ltisca le computational models of intramolecular and intermolecular interactions in the thin filament and cell-level contractile simulations. Our models predicted that increasing the calcium sensitivity of thin filament activation using the cardiac troponin C (cTnC) variant L480 can sufficiently augment twitch tension-time integrals of D230N-Tm hearts. Indeed, cardiac muscle isolated from double-transgenic hearts expressing D230N-Tm and L480 cTnC had increased calcium sensitivity of tension development and increased twitch tension-time integrals compared with preparations from hearts with D230N-Tm alone. Longitudinal echocardiographic measurements revealed that DTG hearts retained normal cardiac morphology and function, whereas D230N-Tm hearts developed progressive DCM. We present a computational and experimental framework for targeting molecular mechanisms governing the twitch tension of cardiomyopathic hearts to counteract putative mechanical drivers of adverse remodeling and open possibilities for tension-based treatments of genetic cardiomyopathies.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Modulating the tension-time integral of the cardiac twitch prevents dilated cardiomyopathy in murine hearts

Dilated cardiomyopathy (DCM) is often associated with sarcomere protein mutations that confer reduced myofilament tension-generating capacity. We demonstrated that cardiac twitch tensiontime integrals can be targeted and tuned to prevent DCM remodeling in hearts with contractile dysfunction. We employed a transgenic murine model of DCM caused by the D230N-tropomyosin (Tm) mutation and designed a sarcomere-based intervention specifically targeting the twitch tension-time integral of D230N-Tm hearts using mu ltisca le computational models of intramolecular and intermolecular interactions in the thin filament and cell-level contractile simulations. Our models predicted that increasing the calcium sensitivity of thin filament activation using the cardiac troponin C (cTnC) variant L480 can sufficiently augment twitch tension-time integrals of D230N-Tm hearts. Indeed, cardiac muscle isolated from double-transgenic hearts expressing D230N-Tm and L480 cTnC had increased calcium sensitivity of tension development and increased twitch tension-time integrals compared with preparations from hearts with D230N-Tm alone. Longitudinal echocardiographic measurements revealed that DTG hearts retained normal cardiac morphology and function, whereas D230N-Tm hearts developed progressive DCM. We present a computational and experimental framework for targeting molecular mechanisms governing the twitch tension of cardiomyopathic hearts to counteract putative mechanical drivers of adverse remodeling and open possibilities for tension-based treatments of genetic cardiomyopathies.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

MYBPC3-c.772G>A mutation results in haploinsufficiency and altered myosin cycling kinetics in a patient induced stem cell derived cardiomyocyte model of hypertrophic cardiomyopathy

Approximately 40% of hypertrophic cardiomyopathy (HCM) mutations are linked to the sarcomere protein cardiac myosin binding protein-C (cMyBP-C). These mutations are either classified as missense mutations or truncation mutations. One mutation whose nature has been inconsistently reported in the literature is the MYBPC3-c.772G > A mutation. Using patient-derived human induced pluripotent stem cells differentiated to cardiomyocytes (hiPSC-CMs), we have performed a mechanistic study of the structure-function relationship for this MYBPC3-c.772G > A mutation versus a mutation corrected, isogenic cell line. Our results confirm that this mutation leads to exon skipping and mRNA truncation that ultimately suggests ∼20% less cMyBP-C protein (i.e., haploinsufficiency). This, in turn, results in increased myosin recruitment and accelerated myofibril cycling kinetics. Our mechanistic studies suggest that faster ADP release from myosin is a primary cause of accelerated myofibril cross-bridge cycling due to this mutation. Additionally, the reduction in force generating heads expected from faster ADP release during isometric contractions is outweighed by a cMyBP-C phosphorylation mediated increase in myosin recruitment that leads to a net increase of myofibril force, primarily at submaximal calcium activations. These results match well with our previous report on contractile properties from myectomy samples of the patients from whom the hiPSC-CMs were generated, demonstrating that these cell lines are a good model to study this pathological mutation and extends our understanding of the mechanisms of altered contractile properties of this HCM MYBPC3-c.772G > A mutation

Altered Cardiac Energetics and Mitochondrial Dysfunction in Hypertrophic Cardiomyopathy

BackgroundHypertrophic cardiomyopathy (HCM) is a complex disease partly explained by the effects of individual gene variants on sarcomeric protein biomechanics. At the cellular level, HCM mutations most commonly enhance force production, leading to higher energy demands. Despite significant advances in elucidating sarcomeric structure-function relationships, there is still much to be learned about the mechanisms that link altered cardiac energetics to HCM phenotypes. In this work, we test the hypothesis that changes in cardiac energetics represent a common pathophysiologic pathway in HCM.MethodsWe performed a comprehensive multiomics profile of the molecular (transcripts, metabolites, and complex lipids), ultrastructural, and functional components of HCM energetics using myocardial samples from 27 HCM patients and 13 normal controls (donor hearts).ResultsIntegrated omics analysis revealed alterations in a wide array of biochemical pathways with major dysregulation in fatty acid metabolism, reduction of acylcarnitines, and accumulation of free fatty acids. HCM hearts showed evidence of global energetic decompensation manifested by a decrease in high energy phosphate metabolites (ATP, ADP, and phosphocreatine) and a reduction in mitochondrial genes involved in creatine kinase and ATP synthesis. Accompanying these metabolic derangements, electron microscopy showed an increased fraction of severely damaged mitochondria with reduced cristae density, coinciding with reduced citrate synthase activity and mitochondrial oxidative respiration. These mitochondrial abnormalities were associated with elevated reactive oxygen species and reduced antioxidant defenses. However, despite significant mitochondrial injury, HCM hearts failed to upregulate mitophagic clearance.ConclusionsOverall, our findings suggest that perturbed metabolic signaling and mitochondrial dysfunction are common pathogenic mechanisms in patients with HCM. These results highlight potential new drug targets for attenuation of the clinical disease through improving metabolic function and reducing mitochondrial injury