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Hypercontractility of the cardiac sarcomere may be essential for the underlying pathological hypertrophy and fibrosis in genetic hypertrophic cardiomyopathies. Aficamten (CK-274) is a novel cardiac myosin inhibitor that was discovered from the optimization of indoline compound 1. The important advancement of the optimization was discovery of an Indane analogue (12) with a less restrictive structure-activity relationship that allowed for the rapid improvement of drug-like properties. Aficamten was designed to provide a predicted human half-life (t1/2) appropriate for once a day (qd) dosing, to reach steady state within two weeks, to have no substantial cytochrome P450 induction or inhibition, and to have a wide therapeutic window in vivo with a clear pharmacokinetic/pharmacodynamic relationship. In a phase I clinical trial, aficamten demonstrated a human t1/2 similar to predictions and was able to reach steady state concentration within the desired two-week window.

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.

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.

Introduction Diabetes is a significant risk factor for thrombosis. The present study aimed at assessing coagulability, platelet reactivity, and thrombogenicity of the diabetic female Zucker Diabetic Fatty (ZDF) rat model and its relevance in studying antithrombotic mechanisms. Materials and Methods The basal coagulant state in ZDF rats was evaluated by clotting times, thromboelastography, and thrombin generation assay. A 14-day treatment with dapagliflozin in ZDF rats was pursued to investigate if glycemic control can improve coagulability. Thrombus formation in the Arterio-Venous (A-V) shunt model and the FeCl 3 -induced arterial thrombosis model was studied, with the antithrombotic effect of apixaban in the former model further investigated. Results ZDF rats exhibited significantly shortened clotting times, enhanced thrombin generation, and decreased fibrinolysis at baseline. Effective glycemic control achieved with dapagliflozin did not improve any of these parameters. ZDF rats displayed accelerated thrombus formation and were amenable to apixaban treatment in the A-V shunt model albeit with less sensitivity than normal rats. ZDF rats exhibited less platelet aggregation in response to ADP, collagen and PAR-4, and attenuated thrombotic response in the FeCl 3 model. Conclusions ZDF rats are at a chronic hypercoagulable and hypofibrinolytic state yet with compromised platelet reactivity. They display accelerated and attenuated thrombosis in the A-V shunt and FeCl 3 model of thrombosis, respectively. Results shed new light on the pathophysiology of the ZDF rat model and illustrate its potential value in translational research on anticoagulant agents in diabetics. Caution needs to be exerted in utilizing this model in assessing antiplatelet mechanisms in diabetes-associated atherothrombosis.

Endothelial dysfunction is common in patients with GH deficiency who are at increased risk for premature cardiovascular death. GH regulates vascular tone and reactivity in humans.Our objective was to explore the mechanisms underlying the GH's acute vascular effects. DESIGN AND STUDY SETTING: There were 10 healthy, lean and young, volunteers studied after an overnight fast. GH was infused systemically for 6 h at 0.06 microg/kg.min. Biopsy of the vastus lateralis muscle was done in seven subjects before and after GH infusion. Human aortic endothelial cells (HAECs) were incubated with GH in vitro.GH infusion increased plasma GH to 32.9 +/- 1.5 ng/ml and forearm blood flow by 66% (P0.001). GH infusion did not significantly change plasma IGF-I concentrations, muscle IGF-I mRNA expression, and muscle Akt phosphorylation, suggesting a lack of IGF-I action in muscle. Because it was reported that GH exerts an acute vascular effect via a nitric oxide (NO)-dependent mechanism, we performed additional in vitro experiments using HAECs. HAECs express abundant GH receptors. Incubating HAECs with GH at 30 ng/ml for 3 or 6 h did not alter endothelial NO synthase (eNOS) protein content but time dependently increased the phosphorylation and activity of eNOS, thus demonstrating a direct effect of GH on endothelial cells.GH exerts an acute vascular effect independent of both systemic and local IGF-I production, and this effect is likely via direct action on GH receptors and eNOS in the vascular endothelium.

Myocardial ischemia-reperfusion injury contributes significantly to morbidity and mortality in patients with diabetes. Insulin decreases myocardial infarct size in animals and the rate of apoptosis in cultured cells. Ischemia-reperfusion activates p38 mitogen-activated protein kinase (MAPK), which regulates cellular apoptosis. To examine whether p38 MAPK affects insulin's cardioprotection against ischemia-reperfusion injury, we studied overnight-fasted adult male rats by use of an in vivo rat model of myocardial ischemia-reperfusion. A euglycemic clamp (3 mU·min−1·kg−1) was begun either 10 min before ischemia (InsulinBI), 5 min before reperfusion (InsulinBR), or 30 min after the onset of reperfusion (InsulinAR), and continued until the end of the study. Compared with saline control, insulin decreased the infarct size in both InsulinBI ( P < 0.001) and InsulinBR ( P < 0.02) rats but not in InsulinAR rats. The ischemic area showed markedly increased phosphorylation of p38 MAPK compared with the nonischemic area in saline animals. Acute activation of p38 MAPK with anisomycin (2 mg/kg iv 10 min before ischemia) had no effect on infarct size in saline rats. However, it completely abolished insulin's protective effect in InsulinBI and InsulinBR rats. Activation of p38 MAPK by anisomycin was associated with marked and persistent elevation in IRS-1 serine phosphorylation. Treatment of animals with SB-239063, a potent and specific inhibitor of p38 MAPK, 10 min before reperfusion enabled insulin-mediated myocardial protection in InsulinAR rats. We conclude that insulin protects myocardium against ischemia-reperfusion injury when given prior to ischemia or reperfusion, and activation of p38 MAPK abolishes insulin's cardioprotective effect.

Insulin stimulates muscle glucose disposal via both glycolysis and glycogen synthesis. Insulin activates glycogen synthase (GS) in skeletal muscle by phosphorylating PKB (or Akt), which in turn phosphorylates and inactivates glycogen synthase kinase 3 (GSK-3), with subsequent activation of GS. A rapamycin-sensitive pathway, most likely acting via ribosomal 70-kDa protein S6 kinase (p70S6K), has also been implicated in the regulation of GSK-3 and GS by insulin. Amino acids potently stimulate p70S6K, and recent studies on cultured muscle cells suggest that amino acids also inactivate GSK-3 and/or activate GS via activating p70S6K. To assess the physiological relevance of these findings to normal human physiology, we compared the effects of amino acids and insulin on whole body glucose disposal, p70S6K, and GSK-3 phosphorylation, and on the activity of GS in vivo in skeletal muscle of 24 healthy human volunteers. After an overnight fast, subjects received intravenously either a mixed amino acid solution (1.26 μmol·kg-1·min-1× 6 h, n = 9), a physiological dose of insulin (1 mU·kg-1·min-1euglycemic hyperinsulinemic clamp × 2 h, n = 6), or a pharmacological dose of insulin (20 mU·kg-1·min-1euglycemic hyperinsulinemic clamp × 2 h, n = 9). Whole body glucose disposal rates were assessed by calculating the steady-state glucose infusion rates, and vastus lateralis muscle was biopsied before and at the end of the infusion. Both amino acid infusion and physiological hyperinsulinemia enhanced p70S6Kphosphorylation without affecting GSK-3 phosphorylation, but only physiological hyperinsulinemia also increased whole body glucose disposal and GS activity. In contrast, a pharmacological dose of insulin significantly increased whole body glucose disposal, p70S6K, GSK-3 phosphorylation, and GS activity. We conclude that amino acids at physiological concentrations mediate p70S6Kbut, unlike insulin, do not regulate GSK-3 and GS phosphorylation/activity in human skeletal muscle.

Aims Thoracic spinal cord stimulation (SCS) has been shown to improve left ventricular ejection fraction (LVEF) in heart failure (HF). Nevertheless, the optimal duration (intermittent vs. continuous) of stimulation and the mechanisms of action remain unclear. Methods and results We performed chronic thoracic SCS at the level of T1–T3 (50 Hz, pulse width 0.2 ms) in 30 adult pigs with HF induced by myocardial infarction and rapid ventricular pacing for 4 weeks. All the animals were treated with daily oral metoprolol succinate (25 mg) plus ramipril (2.5 mg), and randomized to a control group ( n = 10), intermittent SCS (4 h ×3, n = 10) or continuous SCS (24 h, n = 10) for 10 weeks. Serial measurements of LVEF and +d P /d t and serum levels of norepinephrine and B-type natriuretic peptide (BNP) were measured. After sacrifice, immunohistological studies of myocardial sympathetic and parasympathetic nerve sprouting and innervation were performed. Echocardiogram revealed a significant increase in LVEF and +d P /d t at 10 weeks in both the intermittent and continuous SCS group compared with controls ( P < 0.05). In both SCS groups, there was diffuse sympathetic nerve sprouting over the infarct, peri-infarct, and normal regions compared with only the peri-infarct and infarct regions in the control group. In addition, sympathetic innervation at the peri-infarct and infarct regions was increased following SCS, but decreased in the control group. Myocardium norepinephrine spillover and serum BNP at 10 weeks was significantly decreased only in the continuous SCS group ( P < 0.05). Conclusions In a porcine model of HF, SCS induces significant remodelling of cardiac sympathetic innervation over the peri-infarct and infarct regions and is associated with improved LV function and reduced myocardial norepinephrine spillover.

Obstructive sleep apnea is characterized by intermittent obstruction of the upper airway, which leads to intermittent hypoxia. Myocardial glycogen is a major energy resource for heart during hypoxia. Previous studies have demonstrated that intermittent hypoxia rapidly degrades myocardial glycogen and activates glycogen synthase (GS). However, the underlying mechanisms remain undefined. Because sleep apnea/intermittent hypoxia usually happens at night, whether intermittent hypoxia leads to GS activation in the postabsorptive state is not known. In the present study, male adult rats were studied after either an overnight fast or ad libitum feeding with or without intermittent ventilatory arrest (3 90-s periods at 10-min intervals). Hearts were quickly excised and freeze-clamped. Intermittent hypoxia induced a significant decrease in myocardial glycogen content in fed rats and stimulated GS in both fasted and fed rats. However, the portion of GS in the active form increased by approximately 38% in fasted rats compared with a larger, approximately 130% increase in fed rats. The basal G-6-P content was comparable in fasted and fed animals and increased approximately threefold after hypoxia. The basal phosphorylation states of Akt and GSK-3beta and the activity of protein phosphatase 1 (PP1) were comparable between fasted and fed control rats. Hypoxia significantly increased Akt phosphorylation and PP1 activity only in fed rats. In contrast, hypoxia did not induce significant change in GSK-3beta phosphorylation in either fasted or fed rats. We conclude that hypoxia activates GS in fed rat myocardium through a combination of rapid glycogenolysis, elevated local G-6-P content, and increased PP1 activity, and fasting attenuates this action independent of local G-6-P content.

Insulin and protein kinase B (or Akt) play critical roles in cardiomyocytic growth and survival. High concentrations of glucocorticoids antagonize insulin's action. To examine whether endogenous glucocorticoids modulate insulin's effect on Akt signaling in the protein and glycogen synthetic pathways in myocardium, we studied three groups of rats (n = 12 each) 4 d after either a bilateral adrenalectomy (ADX), ADX with physiological stress dose dexamethasone treatment (ADX + DEX), or a sham operation. Rats received either a saline infusion or a 3 mU/kg.min euglycemic insulin clamp for 3 h. ADX had no effect on myocardial Akt or GSK-3 [glycogen synthase (GS) kinase 3] phosphorylation, but it decreased the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (p70(S6K)) (P0.003 for both). Insulin enhanced the phosphorylation of Akt (P0.04), 4E-BP1 (P0.002), and p70(S6K) (P0.0001) in ADX, but not in sham rats. Dexamethasone restored the levels of 4E-BP1 and p70(S6K) phosphorylation and abrogated the insulin-stimulated Akt, 4E-BP1, and p70(S6K) phosphorylation. ADX rats had higher GS activity (P = 0.058) and lower glycogen content (P0.0001) than sham rats. GSK-3 phosphorylation after insulin infusion was greater in ADX rats. Insulin did not alter GS activity. Although insulin did not change the glycogen content in sham or ADX rats, it increased glycogen content by approximately 50% in ADX + DEX rats (P0.02). We conclude that endogenous glucocorticoids differentially modulate the regulation of Akt-4E-BP1/p70(S6K) and Akt-GSK-3-GS signaling pathways in heart by physiologic hyperinsulinemia over a range from deficiency to physiological stress concentrations.

Abstract 2261 Atherothrombosis is the leading cause of death in the western world. Multiple risk factors including hypertension, heart failure, diabetes, obesity, and smoking among others have been attributed to this increased mortality. Currently, most drug discovery efforts utilize normal, healthy animals for evaluating novel antithrombotic drugs. In the present study we investigated the potential utility of using disease animal models. We investigated if hypercoagulability and platelet hyperreactivity exist in the rat models of hypertension (the Spontaneous Hypertensive Rats (SHR)) and diabetes (the Zucker Diabetic Fatty rats (ZDF)). Prothrombin time (PT), activated partial thrombin time (aPTT), thrombin generation, thromboelastography, and plasma TAT levels were measured for assessing coagulation. Platelet P-selectin expression was measured by flow cytometry for assessing platelet activity. For the SHR model, we compared the coagulation parameters and platelet activation in rats at the age of 2- and 8-month old with their respective age-matched WKY rats. For the ZDF model, the female rats were fed on a high fat and high carbohydrate diet from the age of 7–8 weeks for 3–4 weeks, and compared to their aged-matched Fa/+ lean rats fed on a chow diet. There was no significant difference in coagulability between the SHR and the WKY rats at the age of either 2 or 8 months. The SHR rats demonstrated age-dependent progression of increased P-selectin expression on platelets accompanied by increased platelet number and increased mean platelet volume, suggesting their platelets are in a more activated state than the control rats. In contrast, the ZDF rats demonstrated hypercoagulability with significantly shortened PT (by 12%) and aPTT (by 28%). In the thrombin generation assay, the plasma samples from the ZDF rats displayed significantly shortened lag time (by 27%, p=0.012), robustly enhanced thrombin peak (by 169%, p=7.7e-5), and strikingly increased thrombin generation slope (by 264%, p=2.8e-4) under the condition of 1 pM tissue factor and 4 uM phospholipid, similar to the observed hypercoagulability in patients with type 2 diabetes as detected by a thrombin generation assay by others. Additionally, the ZDF rat samples also demonstrated increased clot strength and decreased fibrinolysis as measured by thromboelastography. The ZDF model also exhibited a 40% increase in the P-selectin expression on platelets. These results suggest that the ZDF rats are in a hypercoagulant state whereas the SHR rats are not. Both disease models have increased platelet reactivity. The ZDF rats may represent a good model for studying the diabetes-associated increased risk of thrombosis. Disclosures: Shang: Merck: Employment. Wang:Merck: Employment. Feng:Merck: Employment. Wu:Merck: Employment. Wu:Merck: Employment. Chen:Merck Research Laboratories: Employment. Li:Merck: Employment. Kurowski:Merck: Employment. Chintala:Merck: Employment.