Cardiac Myosin ATPase Research Articles

Hypertrophic cardiomyopathy management: a systematic review of the clinical practice guidelines and recommendations

Abstract Aims In light of recent advances in imaging techniques, molecular understanding and therapeutic options in hypertrophic cardiomyopathy (HCM), we performed a systematic review of current guidelines for the diagnosis and management of HCM in order to identify consensus and discrepant areas in the clinical practice guidelines. Methods and results We systematically reviewed the English language guidelines and recommendations for the management of HCM in adults. MEDLINE and EMBASE databases were searched for guidelines published in the last 10 years. Following a systematic search, three guidelines on the diagnosis and management of HCM were identified, all of which were robustly developed (AGREE rigour of development score ≥50%). These guidelines were authored by the major European (European Society of Cardiology; 2023), American (American Heart Association /American College of Cardiology/American Medical Society for Sports Medicine /Heart Rhythm Society/Pediatric and Congenital Electrophysiology Society/Society for Cardiovascular Magnetic Resonance; 2024), and Japanese [Japanese Circulation Society (JCS)/Japanese Heart Failure Society (JHFS); 2018] cardiovascular societies. There was broad consensus on echocardiographic recommendations, the medical and invasive management of HCM, the application of genetic testing and family screening, and exercise and reproductive recommendations in HCM. There were areas of variability in the definition and diagnostic criteria for HCM, cardiovascular magnetic resonance imaging recommendations, and assessment of sudden cardiac death (SCD) risk and prevention strategies. Due to the JCS/JHFS guidelines being older, there are no recommendations on the use of cardiac myosin ATPase inhibitors. Conclusion Contemporary guidelines for HCM achieve consensus across a broad range of criteria and recommendations concerning diagnosis and management. However, variations in the approach towards risk assessment for SCD exist between the guidelines. There are also more subtle differences concerning diagnostic criteria and the utility of late gadolinium enhancement for risk stratification, which will likely evolve as the evidence-base broadens.

Austrian consensus statement on the diagnosis and management of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease that is characterized by left ventricular hypertrophy unexplained by secondary causes. Based on international epidemiological data, around 20,000-40,000 patients are expected to be affected in Austria. Due to the wide variety of clinical and morphological manifestations the diagnosis can be difficult and the disease therefore often goes unrecognized. HCM is associated with asubstantial reduction in quality of life and can lead to sudden cardiac death, especially in younger patients. Early and correct diagnosis, including genetic testing, is essential for comprehensive counselling of patients and their families and for effective treatment. The latter is especially true as an effective treatment of outflow tract obstruction has recently become available in the form of afirst in class cardiac myosin ATPase inhibitor, as anoninvasive alternative to established septal reduction therapies. The aim of this Austrian consensus statement is to summarize the recommendations of international guidelines with respect to the genetic background, pathophysiology, diagnostics and management in the context of the Austrian healthcare system and resources, and to present them in easy to understand algorithms.

Mavacamten: a novel avenue towards hypertrophic obstructive cardiomyopathy

Hypertrophic obstructive cardiomyopathy (HCM) is the most common heterogeneous genetic cardiovascular disorder. Its pathophysiology involves left ventricular hypertrophy, increased fibrosis, hypercontractility, and reduced compliance. The symptomatic obstructive HCM presents as dyspnoea, syncope, chest pain, palpitations, arrhythmias, or sudden death, usually after provocative manoeuvres like exercise. Until April 2022, treatment options were disease non-specific like Beta blockers, Cardio-selective Calcium Channel Blockers, Dipyridamole, and Ranolazine. Mavacamten is a first-in-class, FDA-approved drug molecule for HCM. It works by selective and reversible inhibition of the cardiac myosin ATPase thereby decreasing the formation of actin- myosin cross- bridges in systole and diastole. The excessive actin-myosin cross-bridging is the hallmark of disease and is responsible for the compromised functioning of the heart in both the systole and diastole phase due to left ventricular outflow tract (LVOT) obstruction and increased ventricular filling pressure respectively. Mavacamten acts by producing super-relaxed state of heart which is then translated into decreased LVOT obstruction and improved cardiac filling pressures thereby improving the functional capacity and symptoms in patients with New York Heart Association (NYHA) stage II, and III symptomatic or obstructive heart failure. Mavacamten is administered orally 2.5-15 mg per day with titration guided by lab investigations and clinical symptoms. Its bioavailability is 85%, undergoes metabolism by CYP2C19 and CYP3A4 and is excreted mainly in urine. It is also an enzyme inducer and shows considerable drug interactions. Common adverse effects are dizziness and syncope. Sometimes the drug may worsen heart failure or completely block ventricular function. Mavacamten has raised hopes for the possibility of managing this potentially lethal intrigue disorder with medicines alone.

Abstract 13955: Real-World Patients With Obstructive Hypertrophic Cardiomyopathy Treated With Mavacamten: A US Single Center Experience

Background: Mavacamten, a first-in-class allosteric reversible inhibitor of cardiac myosin ATPase, was approved by the US Food and Drug Administration for the treatment of symptomatic obstructive hypertrophic cardiomyopathy (HCM) in April 2022. Purpose: To describe the characteristics of real-world patients initiated on mavacamten at a US HCM center of excellence. Methods: All patients started on mavacamten from May 2022 to February 2023 were included. Baseline demographics, clinical characteristics, and echocardiographic data, including left ventricular ejection fraction (LVEF) and left ventricular outflow tract (LVOT) gradients at rest and with Valsalva, were collected. Descriptive statistics were presented as means (standard deviations) and medians (interquartile ranges [IQRs]) or as counts and percentages as appropriate. Results: Of 45 patients initiated on mavacamten in the study period, 47% were female, 13% were non-White, and median age was 63 years (IQR 48, 70). Two-thirds were privately insured. Hypertension was common (56%), and 27% had an implantable cardioverter defibrillator. Median duration of HCM diagnosis was 6 years (IQR 4, 13), and two patients (4%) had undergone septal reduction therapy. At the time of the baseline echocardiogram prior to drug initiation, 42% had NYHA III symptoms, 56% were on background monotherapy with a beta-blocker, 24% with a nondihydropyridine calcium-channel blocker, and 20% were on both. All patients were in either sinus or an atrial-paced rhythm. Median LVEF was 69% (IQR 65, 75), and median septal wall thickness was 19 mm (IQR 18, 22). Median resting LVOT gradient was 57 mmHg (IQR 35, 80), and median Valsalva LVOT gradient was 78 mmHg (IQR 63, 91) ( Table ). Conclusions: In this single center experience of patients with obstructive HCM initiated on mavacamten, clinical characteristics were largely similar to those of patients enrolled in the phase 3 randomized trials of mavacamten, EXPLORER-HCM and VALOR-HCM.

Novel Therapeutic Avenues for Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a complicated, heterogeneous genetic condition that causes left ventricular hypertrophy, fibrosis, hypercontractility, and decreased compliance. Despite the advances made over the past 3decades in understanding the molecular and cellular mechanisms aggravating HCM, the relationship between pathophysiological stress stimuli and distinctive myocyte growth profiles is still imprecise. Currently, mavacamten, a selective and reversible inhibitor of cardiac myosin ATPase, is the only drug approved by the US FDA for the treatment of HCM. Thus, there is an unmet need for developing novel disease-specific therapeutic approaches. This article provides an overview of emerging therapeutic targets for the treatment of HCM based on various molecular pathways and novel developments that are hopefully soon to enter the clinical study. These newly discovered targets include the dual specificity tyrosine-phosphorylation-regulated kinase 1B, the absence of the melanoma 1 inflammasome, the leucine-rich repeat kinase 2 enzyme, and the cluster of differentiation 147.

Mavacamten, a Novel Therapeutic Strategy for Obstructive Hypertrophic Cardiomyopathy.

Pharmacological treatment options for hypertrophic cardiomyopathy (HCM) are currently limited and comprise non-disease specific therapies such as β-blockers, non-dihydropyridine calcium channel blockers, and disopyramide. These agents that offer a variable degree of symptomatic relief, often suboptimal, are often limited by side-effects and fail to address the key molecular abnormalities of the disease. Mavacamten is a novel, first-in-class, allosteric inhibitor of cardiac myosin ATPase, which reduces actin-myosin cross-bridge formation, thereby reducing myocardial contractility and improving myocardial energetic consumption in experimental HCM models. Following a successful Phase 2 study, the recently published phase III, placebo-controlled, randomized EXPLORER-HCM trial demonstrated the efficacy and safety of mavacamten in reducing left ventricular outflow tract obstruction and ameliorating exercise capacity, New York Heart Association functional class and health status in patients with obstructive HCM. Mavacamten represents the first agent specifically developed for HCM successfully tested in a Phase III trial, to be registered soon for clinical use, representing a radical change of paradigm in the pharmacological treatment of HCM.

Emerging Medical Treatment for Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common myocardial disease characterized by otherwise unexplained left ventricular hypertrophy. The main cause of disabling symptoms in patients with HCM is left ventricular outflow tract (LVOT) obstruction. This phenomenon is multifactorial, determined both by anatomical and functional abnormalities: myocardial hypercontractility is believed to represent one of its major determinants. The anatomical anomalies are targeted by surgical interventions, whereas attenuating hypercontractility is the objective of old and new drugs including the novel class of allosteric myosin inhibitors. This review summarizes the current treatment modalities and discusses the emerging therapeutical opportunities focusing on the recently developed cardiac myosin ATPase inhibitors Mavacamten and CK-274. Novel surgical and interventional approaches are also discussed.

Abstract 14390: The Cardiac Myosin Inhibitor, Ck-3772271, Attenuates Cardiac Fibrosis and Diastolic Dysfunction in the Dahl/salt Sensitive Rat Model of Heart Failure With Preserved Ejection Fraction

Introduction: Heart failure with preserved ejection fraction (HFpEF) is characterized by underlying contractile dysfunction and progressive myocardial fibrosis and stiffness. CK-3772271 (CK-271) is a novel small molecule cardiac myosin inhibitor that reduces cardiac myosin ATPase activity and reduces cardiac contractility in unloaded isolated cardiomyocytes in vitro and in healthy rats and dogs in vivo . The effect of chronic CK-271 treatment on cardiac function and morphology was evaluated in the Dahl/Salt Sensitive (DSS) rat hypertension model of HFpEF. Methods: DSS male rats were fed either a control low salt (LS, 0.3% NaCl) or high salt (HS, 4% NaCl) diet to induce a hypertension-driven HFpEF disease phenotype. 6 weeks after HS diet treatment, DSS rats were randomized into two sub-groups: continued HS diet or a HS diet formulated with CK-271 (100 ppm) for an additional 6 weeks. Body mass, systolic blood pressure, and cardiac function were measured longitudinally. After 12 weeks of HS treatment, hearts were collected to assess cardiac fibrosis. Results: HS diet treatment increased systolic blood pressure (LS:132.2 ± 4.7 mm Hg vs. HS: 163.5 ± 4.0 mm Hg, mean ± SEM, p< 0.001) and caused cardiac hypercontractility as evidenced by an increase in the end-systolic pressure-volume relationship (LS: 0.8 ± 0.17 vs. HS: 2.1 ± 0.46 mm Hg/ml). HS diet also increased isovolumic relaxation time (IVRT) (LS: 16.8 ± 0.6 vs. HS: 22.8 ± 0.6 ms, p<0.0001), left atrial area (LS: 30.3 ± 0.8 vs. HS: 42.5 ± 2.2 mm 2 , p<0.0001) and cardiac fibrosis (LS: 3.4 ± 0.4 vs. HS: 5.0 ± 0.6 %). 6 weeks of CK-271 treatment reduced fractional shortening (HS: 53.8 ± 1.4 vs. HS+CK-271: 42.7 ± 1.0 %, p<0.0001) and reduced HS diet-induced diastolic dysfunction, including IVRT (HS: 22.8 ± 0.6 vs. HS+CK-271: 19.5 ± 0.5 ms, p<0.0001) and left atrial area (HS: 42.5 ± 2.2 vs. HS+CK-271: 35.4 ± 0.8 mm 2 , p<0.0001). Furthermore CK-271 reduced the development of cardiac fibrosis (HS: 5.0 ± 0.6 vs. HS+CK-271: 3.5 ± 0.3 %, p<0.05) induced by HS diet. Conclusion: The small molecule cardiac myosin inhibitor, CK-3772271, attenuated the development of cardiac hypercontractility, diastolic dysfunction and fibrosis in the DSS rat model of HFpEF. Cardiac myosin inhibition may be a novel approach to mitigate the development of HFpEF.

Identification of diphenylalkylisoxazol-5-amine scaffold as novel activator of cardiac myosin

To identify novel potent cardiac myosin activator, a series of diphenylalkylisoxazol-5-amine compounds 4–7 have been synthesized and evaluated for cardiac myosin ATPase activation. Among the 37 compounds, 4a (CMA at 10 µM = 81.6%), 4w (CMA at 10 µM = 71.2%) and 6b (CMA at 10 µM = 67.4%) showed potent cardiac myosin activation at a single concentration of 10 µM. These results suggested that the introduction of the amino-isoxazole ring as a bioisostere for urea group is acceptable for the cardiac myosin activation. Additional structure–activity relationship (SAR) studies were conducted. Para substitution (-Cl, –OCH3, -SO2N(CH3)2) to the phenyl rings or replacement of a phenyl ring with a heterocycle (pyridine, piperidine and tetrahydropyran) appeared to attenuate cardiac myosin activation at 10 µM. Additional hydrogen bonding acceptor next to the amino group of the isoxazoles did not enhance the activity. The potent isoxazole compounds showed selectivity for cardiac myosin activation over skeletal and smooth muscle myosin, and therefore these potent and selective isoxazole compounds could be considered as a new series of cardiac myosin ATPase activators for the treatment of systolic heart failure.

Mavacamten: treatment aspirations in hypertrophic cardiomyopathy

Managing dynamic left ventricular outflow tract (LVOT) obstruction remains one of the most challenging therapeutic aspects of hypertrophic cardiomyopathy. Individuals who are affected frequently present with disabling symptoms, which can be ameliorated following reduction of LVOT obstruction. Pharmacological treatment currently comprises non-disease specific therapies such as β blockers, negatively inotropic calcium channel blockers, and disopyramide, which offer a variable degree of symptomatic relief and are often limited by side-effects. 1 Ammirati E Contri R Coppini R Cecchi F Frigerio M Olivotto I Pharmacological treatment of hypertrophic cardiomyopathy: current practice and novel perspectives. Eur J Heart Fail. 2016; 18: 1106-1118 Crossref PubMed Scopus (57) Google Scholar Enhanced myocardial contractility is a key factor in the pathophysiology of LVOT obstruction. 2 Anderson RL Trivedi DV Sarkar SS et al. Deciphering the super relaxed state of human β-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers. Proc Natl Acad Sci USA. 2018; 115: e8143-e8152 Crossref PubMed Scopus (93) Google Scholar Mavacamten is a first-in-class, selective allosteric inhibitor of cardiac myosin ATPase, which reduces actin-myosin cross-bridge formation, thereby reducing myocardial contractility and improving myocardial energetics. 3 Rohde JA Roopnarine O Thomas DD Muretta JM Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin. Proc Natl Acad Sci USA. 2018; 115: e7486-e7494 Crossref PubMed Google Scholar Animal studies showed that mavacamten reduced myocardial contraction in a dose-dependent manner and relieved LVOT obstruction. 4 Stern JA Markova S Ueda Y et al. A small molecule inhibitor of sarcomere contractility acutely relieves left ventricular outflow tract obstruction in feline hypertrophic cardiomyopathy. PLoS One. 2016; 11e0168407 Crossref PubMed Scopus (46) Google Scholar These findings were replicated in a phase 2 trial (PIONEER-HCM), in which mavacamten reduced the post-exercise LVOT gradient, increased peak oxygen consumption (pVO2), and improved symptoms in patients with the condition. 5 Heitner SB Jacoby D Lester SJ et al. Mavacamten treatment for obstructive hypertrophic cardiomyopathy: a clinical trial. Ann Intern Med. 2019; 170: 741-748 Crossref PubMed Scopus (67) Google Scholar Mavacamten for treatment of symptomatic obstructive hypertrophic cardiomyopathy (EXPLORER-HCM): a randomised, double-blind, placebo-controlled, phase 3 trialTreatment with mavacamten improved exercise capacity, LVOT obstruction, NYHA functional class, and health status in patients with obstructive hypertrophic cardiomyopathy. The results of this pivotal trial highlight the benefits of disease-specific treatment for this condition. Full-Text PDF

Abstract 17067: MAVERICK-HCM: Phase 2 Randomized, Multi-Center, Double-Blind, Placebo-Controlled Concentration-Guided Study to Evaluate Mavacamten (MYK-461) in Adults With Symptomatic Non-Obstructive Hypertrophic Cardiomyopathy

Introduction: Hypertrophic cardiomyopathy (HCM) carries substantial lifelong morbidity. Mutations in sarcomeric proteins that lead to HCM cause excess contractility, poor relaxation, and inefficient energy utilization. Although obstructive physiology is a prominent feature of disease, approximately 30% of HCM patients experience symptoms in the context of non-obstructive HCM (nHCM). Management options are extremely limited with little other than cardiac transplantation to offer severely affected patients. Mavacamten (MAVA), a first-in-class small molecule, is a selective modulator of cardiac myosin that inhibits the cardiac myosin ATPase to reversibly attenuate cardiac contractility. In addition to its effects on contractility, MAVA has been shown to improve LV compliance in preclinical and clinical studies. The Phase 2 MAVERICK-HCM study is designed to evaluate safety and tolerability, and to provide pilot data on the effects of MAVA on biomarkers, cardiac function, and exercise capacity in nHCM. Methods: This double-blind study will enroll 60 individuals with nHCM (Left ventricular outflow tract gradient <30 mmHg; resting or provoked), NYHA Class II or III, and LVEF ≥55%. Subjects will be randomized 1:1:1 to one of two target plasma drug concentrations (Group 1: ~200 ng/mL and Group 2: ~500 ng/mL) or placebo for 16 weeks. The starting dose of MAVA is 5 mg, with one-step dose titration at Week 6 based on plasma drug concentration. Predefined criteria, including LVEF, will guide study drug discontinuation if indicated. Cardiopulmonary exercise testing will be performed at baseline and Week 16 to assess the impact on exercise capacity. The primary endpoint is the frequency and severity of treatment-emergent adverse events. The study design also includes multiple secondary endpoints including echocardiographic measures of diastolic function, NT-proBNP levels, patient reported outcomes, and physical activity by wearable accelerometer. An independent data monitoring committee will oversee safety. Conclusions: MAVERICK-HCM is testing a novel therapy to improve symptom burden in nHCM; a patient cohort currently lacking effective treatment. By targeting a core molecular mechanism of disease, MAVA has the potential to alter natural history.

Exploration of diphenylalkyloxadiazoles as novel cardiac myosin activator

To explore novel cardiac myosin activator, a series of diphenylalkyl substituted 1,3,4-oxadiazoles and 1,2,4-oxadiazoles have been prepared and tested for cardiac myosin ATPase activation in vitro. In all cases, three carbon spacer between the oxadiazole core and one of the phenyl ring was considered crucial. In case of 1,3,4-oxadiazole, zero to two carbon spacer between oxadiazole core and other phenyl ring are favorable. Phenyl ring can be replaced by cyclohexyl moiety. In case of 1,2,4-oxadiazole, zero or one carbon spacer between the oxadiazole and other phenyl ring are favorable. Introduction of hydrogen bonding donor (NH) group at the 2nd position of the 1,3,4-oxadiazole enhances the activity. Substitutions on either of the phenyl rings or change of phenyl ring to other heterocycle are not tolerated for both the oxadiazoles. The prepared oxadiazoles showed selective activation for cardiac muscle over smooth and skeleton muscles.

Design and synthesis of sulfonamidophenylethylureas as novel cardiac myosin activator

To optimize the lead urea scaffold 1 and 2 as selective cardiac myosin ATPase activator, a series of urea derivatives have been synthesized to explore its structure activity relationship. Among them N,N-dimethyl-4-(2-(3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (13, CMA = 91.6%, FS = 17.62%; EF = 11.55%), N,N-dimethyl-4-(2-(1-methyl-3-(3-phenylpropyl)ureido)ethyl)benzene sulfonamide (40, CMA = 52.3%, FS = 38.96%; EF = 24.19%) and N,N-dimethyl-4-(2-(3-methyl-3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (41, CMA = 47.6%, FS = 23.19%; EF = 15.47%) proved to be efficient to activate the cardiac myosin in vitro and in vivo. Further the % change in ventricular cell contractility at 5 μM of 13 (47.9 ± 3.2), 40 (45.5 ± 2.4) and 41 (63.5 ± 2.2) showed positive inotropic effect in isolated rat ventricular myocytes. The potent compounds 13, 40, 41 were highly selective for cardiac myosin over skeletal and smooth muscle myosin, thus proving them these new urea derivatives is a novel scaffold for discovery of cardiac myosin activators for the treatment of systolic heart failure.

Exploration of flexible phenylpropylurea scaffold as novel cardiac myosin activators for the treatment of systolic heart failure

A series of flexible urea derivatives have been synthesized and demonstrated as selective cardiac myosin ATPase activator. Among them 1-phenethyl-3-(3-phenylpropyl)urea (1, cardiac myosin ATPase activation at 10 μM = 51.1%; FS = 18.90; EF = 12.15) and 1-benzyl-3-(3-phenylpropyl)urea (9, cardiac myosin ATPase activation = 53.3%; FS = 30.04; EF = 18.27) showed significant activity in vitro and in vivo. The change of phenyl ring with tetrahydropyran-4-yl moiety viz., 1-(3-phenylpropyl)-3-((tetrahydro-2H-pyran-4-yl)methyl)urea (14, cardiac myosin ATPase activation = 81.4%; FS = 20.50; EF = 13.10), and morpholine moiety viz., 1-(2-morpholinoethyl)-3-(3-phenylpropyl)urea (21, cardiac myosin ATPase activation = 44.0%; FS = 24.79; EF = 15.65), proved to be efficient to activate the cardiac myosin. The potent compounds 1, 9, 14 and 21 were found to be selective for cardiac myosin over skeletal and smooth myosins. Thus, these urea derivatives are potent scaffold to develop as a newer cardiac myosin activator for the treatment of systolic heart failure.

Detection of Small-Molecule Modulators of Actin-Myosin Structure and Function using High-Throughput Time-Resolved FRET

We have used a time-resolved FRET assay to develop a system for detecting small-molecule modulators of actin-myosin structure and function. The fluorescent donor was fluorescein attached to actin C374, and the non-fluorescent acceptor was Dabcyl attached to the N-terminus of a peptide containing the 12 N-terminal amino acids of the A1 essential light chain (DNT). FRET was measured from the decrease in fluorescence lifetime, measured by time-resolved fluorescence in a novel fluorescence plate-reader, resolving structural distributions of the bound complex. The DNT binding site on actin overlaps with that of myosin, as indicated by (a) a similar distance observed in the actin-DNT complex as in the actin-myosin complex and (b) a significant decrease in actin-DNT FRET upon binding skeletal and cardiac myosin. A high-throughput FRET screen of a small-molecule library (NCC) showed that actin-DNT FRET is significantly affected by some compounds. All of these compounds inhibit actin-activated skeletal and cardiac myosin ATPase and affect the microsecond dynamics of actin, as detected by transient phosphorescence anisotropy. We concluded that the actin-DNT system allows detection of the effects of pharmacologically active compounds on actin structural states and consequently on functional properties of actomyosin. These results set the stage for a drug-discovery campaign, seeking therapies for cardiomyopathy and other disorders of involving actin-myosin dysfunction.

Exploration of Pharmacophore in Chrysosplenol C as Activator in Ventricular Myocyte Contraction

Chrysosplenol C (4',5,6-trihydroxy-3,3',7-trimethoxyflavone) isolated from Miliusa balansae has unique structural features as a reversible inotropic agent independent of β-adrenergic signaling and with selective activation of cardiac myosin ATPase. Hence, a series of chrysosplenol analogues were synthesized and explored for identification of pharmacophore that is essential for the increasing contractility in rat ventricular myocytes. Analogue 7-chloro-2-(3-hydroxyphenyl)-3-methoxy-4H-chromen-4-one showed highly potent contractility (54.8% at 10 μM) through activating cardiac myosin ATPase (38.7% at 10 μM). Our systematic structure-activity relationship study revealed that flavonoid nucleus of chrososplenol C appears to be an essential basic skeleton and hydrophobic substituent at position 7 of chromenone such as methoxy or chloro enhances the activity. Additionally, our ATPase study suggested that these chrysosplenol analogues have selectivity toward cardiac myosin activation. Thus, the novel flavonone with 3-/7-hydrophobic substituent and 3'-hydrogen bonding donor function is a novel scaffold for discovery of a new positive inotropic agent.

Myocyte contractility can be maintained by storing cells with the myosin ATPase inhibitor 2,3 butanedione monoxime.

Isolated intact myocytes can be used to investigate contractile mechanisms and to screen new therapeutic compounds. These experiments typically require euthanizing an animal and isolating fresh cells each day or analyzing cultured myocytes, which quickly lose their rod-shaped morphology. Recent data suggest that the viability of canine myocytes can be prolonged using low temperature and N-benzyl-p-toluene sulfonamide (an inhibitor of skeletal myosin ATPase). We performed similar studies in rat myocytes in order to test whether the cardiac myosin ATPase inhibitors 2,3-Butanedione monoxime (BDM) and blebbistatin help to maintain cell-level function over multiple days. Myocytes were isolated from rats and separated into batches that were stored at 4°C in a HEPES-buffered solution that contained 0.5 mmol L−1 Ca2+ and (1) no myosin ATPase inhibitors; (2) 10 mmol L−1 BDM; or (3) 3 μmol L−1 blebbistatin. Functional viability of myocytes was assessed up to 3 days after the isolation by measuring calcium transients and unloaded shortening profiles induced by electrical stimuli in inhibitor-free Tyrode's solution. Cells stored without myosin ATPase inhibitors had altered morphology (fewer rod-shaped cells, shorter diastolic sarcomere lengths, and membrane blebbing) and were not viable for contractile assays after 24 h. Cells stored in BDM maintained morphology and contractile function for 48 h. Storage in blebbistatin maintained cell morphology for 72 h but inhibited contractility. These data show that storing cells with myosin ATPase inhibitors can extend the viability of myocytes that will be used for functional assays. This may help to refine and reduce the use of animals in experiments.

Structural Thermodynamics and Kinetics of the Cardiac Myosin/Omecamtiv Mecarbil Complex Revealed by Time-Resolved FRET

We are investigating the structural thermodynamics and kinetics of cardiac myosin in the presence of omecamtiv mecarbil (OM), a small molecule that is currently in clinical trials for treating heart failure. AM has been shown to increase thin-filament activated cardiac myosin ATPase activity, the apparent rate constant for actin-activated phosphate release from cardiac myosin, and contractility in the heart (Malik et al, Science 2011). How OM modulates theses activities remains poorly understood. Based on a published Biophysical Society Annual Meeting poster abstract (Liu et al. Biophys. J. 2013) and on published studies (Malik et al, Science 2011), we hypothesized that OM stabilizes the pre-powerstroke structural state in the presence of ATP analogs and increases the observed rate constant for the actin-activated powerstroke during actin-activated single ATP turnover. To test these predictions, we used a time-resolved FRET biosensor of β-cardiac myosin, which detects the mole faction of pre and post-powerstroke structural states of the myosin light-chain binding domain, to determine the apparent equilibrium constant for the recovery-stroke under saturating ADP, ADP.BeF, ADP.AlF and ADP.V nucleotide states in the presence of saturating OM or DMSO. We varied the temperatures of these experiments to determine whether OM changes the structural thermodynamics of the pre-powerstroke state. In a second set of experiments, we directly measure light-chain domain rotation during ATP binding to myosin and during actin binding to myosin in the presence of ATP. Taken together, these results address two of the key phenotypes that the drug exhibits in vitro, increased actin-activated phosphate release, and increased thin-filament activated steady-state ATPase cycling.

Novel drug targets in clinical development for heart failure

Heart Failure continues to be a leading cause of mortality and morbidity worldwide. The dismal prognosis of patients in acute heart failure (AHF) can be altered only by exploring novel drug molecules. Although the treatment for chronic heart failure (CHF) has seen remarkable progress, there is still a need to develop molecules that could improve the long-term survival outcomes. To review the drug targets for acute and chronic heart failure and the molecules acting on these targets. The article discusses on the mechanism of how the potential drug targets can be modulated to alter the pathophysiological processes in heart failure. Current evidence on molecules acting on these targets has also been described from published literature using the PubMed and Clinical Trials.gov databases. Some of the molecules that are currently being explored for AHF includeomecamtiv mecarbil which activates cardiac myosin ATPase, istaroxime an ionotropicagent that activates SERCA2a pump activity, ularitide and carperitide which are ANP(atrial natriuretic peptide) analogues and recombinant relaxin. Some of the targets forCHF include stabilization of ryanodine receptors, renin inhibition, neprilysin inhibition, neuregulins and SERCA2a gene therapy. Clinical trials in heart failure must be designed to minimize the risk of "drug failures." Nevertheless, it is hoped that in the days to come, drugs with superior efficacy and safety will eventually be produced from the surge of molecules that are in the pipeline.

Modulation of the Cardiac Sarcomere by a Small Molecule Agent MYK0000461: A Potential Therapeutic for the Treatment of Genetic Hypertrophic Cardiomyopathies

We have identified a small molecule inhibitor, MY461, of the cardiac myosin ATPase. This agent was characterized in steady state and transient kinetic assays to understand its mechanism of action. MY461 decreases the steady-state rate of the ATPase activity of purified bovine β-cardiac myosin subfragment-1 (S1) as well as that of bovine cardiac myofibrils, wild type and the mutant R453C of recombinant human β-cardiac myosin S1. We also find that MY461 inhibits cardiac myosin selectively as compared to systems containing rabbit skeletal or chicken smooth muscle myosins Analysis of the individual steps of the chemo-mechanical cycle of cardiac myosin suggests that MY461 exerts its effect by inhibiting the actin-stimulated release of phosphate, presumably by stabilizing the detached state of cardiac myosin prior to the release of phosphate. We find no evidence to suggest that MYK000461 inhibits cardiac myosin in a strongly bound state and no other steps in the chemo-mechanical cycle are affected by MY461. Thus, the enzymatic step governing the weak to strong transition of S1 binding to actin is inhibited without affecting the release from the strongly bound states. This decrease in the rate of transition from the weak to strongly bound state should decrease force production and may underlie its ability to decrease cardiac contractility in cellular and in vivo models of cardiac function. An agent such as MY461 could potentially be used to treat cardiac disorders that stem from hyper contractility such as the genetic hypertrophic cardiomyopathies (HCM). By decreasing the net force of contraction and restoring it back to normal level could potentially be useful in treating patients that suffer from this disease.