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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, p2 , p2 , p 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.

Introduction: Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular (LV) hypertrophy in the absence of known causes. HCM can be due to genetic mutations that affect sarcomeric proteins, leading to myocardial hypercontractility. HCM is often complicated by left ventricular outflow tract obstruction (LVOTO) and patients may be at greater risk of cardiovascular death. Medications targeted to address the pathophysiology of HCM and ameliorate LVOTO are needed. CK-3773274 (CK-274) is a novel small molecule cardiac myosin inhibitor that reduces myocardial contractility in vitro and in vivo . Cats have naturally occurring HCM commonly complicated by LVOTO and are an excellent large animal model for investigation of HCM therapeutics. In this study, we aim to characterize the pharmacodynamic effect of a single oral dose of CK-274 on cardiac function in cats with hypertrophic cardiomyopathy and LVOTO. Methods: Eight purpose-bred cats with naturally occurring HCM and LVOTO due to the A31P mutation in cardiac myosin binding protein C (cMyBP-C) were included in a randomized, controlled, crossover study. Cats were randomized to receive vehicle, 0.3 mg/kg, or 1 mg/kg CK-274 treatment, and received echocardiograms at baseline, 6, 24, and 48 hours post-treatment. All cats were crossed over to all treatment groups. Results: CK-274 (1 mg/kg) reduced mean LV fractional shortening at 6, 24 and 48 hours post-treatment (mean reduction 13.6%, p=0.03; 15.4%, p=0.01; 11.6% p=0.02, respectively). CK-274 (1 mg/kg) increased LV systolic internal dimension at 6 and 24-hours post-treatment (mean increase 0.21 cm, p=0.046; 0.25 cm, p=0.03), and did not affect LV diastolic internal dimension. LVOT peak pressure gradient was reduced with CK-274 (0.3 mg/kg) treatment [median pressure gradient at baseline 27.1 mmHg (IQR 18.3-33.3) vs 24 hours post-drug, 7.3 mmHg (IQR 14.2-19.7) p=0.01]. Heart rate did not change for any treatment group over time. No differences were noted following vehicle administration at any time point. Conclusion: In HCM affected cats with the cMyBP-C A31P mutation, the cardiac myosin inhibitor CK-274 is well tolerated at the studied doses and caused dose-related changes in LV systolic function and reductions in LVOT peak pressure gradient.

Cardiac sarcomere hypercontractility appears to underlie pathological hypertrophy and fibrosis in select genetic hypertrophic cardiomyopathies. The small molecule, CK-3773274, is a novel cardiac myosin inhibitor that decreases contractility in vitro and in healthy animals in vivo . The objective of this study was to evaluate the effect of CK-3773274 in the genetic R403Q mouse model of hypertrophic cardiomyopathy. At approximately 40 weeks of age, left ventricular wall dimensions were determined by echocardiography in male wild type (WT) and heterozygous R403Q mice. As an indicator of cardiac hypertrophy, R403Q mice had significantly greater septal and posterior wall thickness than WT mice (septal wall WT: 0.93 ± 0.03 mm vs. R403Q: 1.22 ± 0.08 mm; posterior wall WT: 0.84 ± 0.04 mm vs. R403Q: 1.09 ± 0.04 mm; mean ± SEM, p< 0.05). R403Q mice were treated with single oral doses of CK-3773274 ranging from 0.25 to 1.5 mg/kg and fractional shortening (FS) and heart rate were assessed at select time points over a 24-hour period. One hour after dose administration, CK-3773274 significantly reduced FS in a dose-related fashion relative to pre-dose baseline values (FS % R403Q baseline: 55.5 ± 2%; 0.25 mg/kg: 43.9 ± 2%; 1 mg/kg: : 27.3 ± 2%; 1.5 mg/kg: 13.7 ± 1%; mean ±SEM, p10 and IC 50 ) was 0.11 and 0.78 μM, respectively. In summary, single oral dose administration of CK-3773274 reduced fractional shortening in a dose and concentration-dependent manner in the genetic R403Q mouse model of hypertrophic cardiomyopathy. Cardiac myosin inhibition may be a viable approach to reduce underlying hypercontractility of the cardiac sarcomere in hypertrophic cardiomyopathies.

Hypercontractility of the cardiac sarcomere appears to underlie pathological hypertrophy and fibrosis in select genetic hypertrophic cardiomyopathies. Here, we characterize the small molecule, CK-3773274, as a novel cardiac myosin inhibitor that decreases contractility in vitro and in vivo . In bovine cardiac myofibrils, CK-3773274 decreased myosin ATPase activity in a concentration-dependent fashion (IC 50 :1.26 μM). CK-3773274 specifically inhibited myosin activity, as it reduced myosin ATPase activity in a concentration-dependent manner in the absence of other sarcomere proteins, including actin, troponin, and tropomyosin. CK-3773274 (10 μM) reduced fractional shortening by 84% in electrically paced, isolated adult rat cardiomyocytes relative to control without any effect on the calcium transient. The effect of CK-3773274 on cardiac contractility in vivo was assessed in healthy male Sprague Dawley (SD) rats using single oral doses ranging from 0.5 to 4 mg/kg. Fractional shortening (FS) and left ventricular dimensions were determined by echocardiography at select time points over a 24-hour period. One hour after dose administration, CK-3773274 significantly reduced fractional shortening in a dose-related fashion by 20-70% relative to vehicle treatment (FS %: vehicle: 47.9± 1%; 0.5 mg/kg: 39 ± 2%; 4 mg/kg: 15 ± 4%; mean ±SEM, pin vitro and in vivo . Cardiac myosin inhibition may be a viable approach to treat the underlying hypercontractility of the cardiac sarcomere in hypertrophic cardiomyopathies.

Limited neural input results in muscle weakness in neuromuscular disease because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission. We developed a small-molecule fast-skeletal-troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neural input is otherwise diminished secondary to neuromuscular disease. Binding selectively to the fast-skeletal-troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards, as does the force-frequency relationship of a nerve-muscle pair, so that CK-2017357 increases the production of muscle force in situ at sub-maximal nerve stimulation rates. Notably, we show that sensitization of the fast-skeletal-troponin complex to calcium improves muscle force and grip strength immediately after administration of single doses of CK-2017357 in a model of the neuromuscular disease myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

Decreased cardiac contractility is a central feature of systolic heart failure and yet we have no effective drugs to improve cardiac contractility. Existing drugs that increase cardiac contractility do so indirectly through signaling cascades and their use is limited by their mechanism-related adverse effects. Direct activation of the cardiac sarcomere to increase cardiac contractility may provide a means to avoid these limitations. Using a reconstituted version of the cardiac sarcomere, we screened a small molecule library and identified several chemical classes that directly activate cardiac myosin. One compound class has been optimized extensively using an iterative process; omecamtiv mecarbil, a small-molecule, selective, cardiac myosin activator is the most advanced exemplar of this novel mechanistic class. It accelerates the transition of myosin into the force-generating state without affecting cardiac myocyte calcium homeostasis. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Initial clinical studies have demonstrated the translation of this mechanism into humans, and further clinical studies of its use in acute and chronic heart failure are planned. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure."

CK-1909178 is a member of a class of fast skeletal troponin activators that sensitize skinned skeletal muscle fibers to calcium. In rat muscle preparations in vitro and in situ, CK-1909178 increased sub-tetanic force without altering maximum force. Given that a major cause of muscle fatigue during repeated muscle contraction is reduced myoplasmic Ca2+ due to impaired sarcoplasmic reticulum Ca2+ release, we tested whether increased calcium sensitivity with CK-1909178 would slow the development of fatigue. Rat flexor digitorum brevis muscle was pretreated in vitro with CK-1909178 and stimulated every 3 seconds at a frequency sufficient to achieve 50% of maximum force for 6 min at 30°C. CK-1909178 diminished the extent of fatigue as compared to control (terminal force 29.5±8% vs. 12.7±4%, p

Previously, we have discovered small molecules that increase cardiac contractility by directly activating the cardiac sarcomere; this mechanism is now being investigated as a therapy for treating systolic heart failure. Using this precedent, we have focused on the identification of compounds that directly increase skeletal muscle contractility for the potential therapy of diseases that result in muscle weakness and fatigue.CK-1909178 is a member of a class of fast skeletal troponin activators that were identified by high throughput screening of skeletal sarcomere preparations. We sought to understand how this compound altered force development in isometric skinned and live muscle fibers. Treatment of skinned rabbit psoas fibers with 0.1 μM CK-1909178 caused a dose-dependent left-shift of the force-pCa relationship without altering the Hill slope or maximum force, consistent with a calcium sensitizing effect on force production. In living rat flexor digitorum brevis (FDB) preparations, CK-1909178 (10 μM) caused significant increases in sub-tetanic force (150% of control at 10 Hz) without altering maximum force. Similar experiments were then performed using a rat extensor digitorum longus (EDL) preparation in-situ, where nervous and vascular connections were left intact and the muscle was stimulated via the peroneal nerve. Infusions of CK-1909178 up to 10 mg/kg rapidly increased sub-tetanic isometric force (190% of control at 30 Hz). In summary, we have identified a skeletal troponin activator that sensitizes the sarcomere to calcium and results in increased sub-maximal muscle force development in-vitro and in-situ. We believe that this mechanism may translate to improved physical power and strength in diseases where muscle function is compromised due to injury, disease or age.