Heather Fraser

Varespladib (A-002), a secretory phospholipase A2 inhibitor, reduces atherosclerosis and aneurysm formation in ApoE-/- mice.

The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE−/− mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE−/− mouse models.

A Comparison between Ranolazine and CVT-4325, a Novel Inhibitor of Fatty Acid Oxidation, on Cardiac Metabolism and Left Ventricular Function in Rat Isolated Perfused Heart during Ischemia and Reperfusion

Inhibition of fatty acid oxidation has been reported to be cardioprotective against myocardial ischemic injury; however, recent studies have questioned whether the cardioprotection associated with putative fatty acid oxidation inhibitors, such as ranolazine and trimetazidine, are due to changes in substrate oxidation. Therefore, the goals of this study were to compare the effects of ranolazine with a new fatty acid oxidation inhibitor, CVT-4325 [(R)-1-(2-methylbenzo[d]thiazol-5-yloxy)-3-(4-((5-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)methyl)-piperazin-1-yl)propan-2-ol], on carbohydrate and fatty acid oxidation and on left ventricular (LV) function in the response to ischemia/reperfusion in rat isolated perfused hearts. Metabolic fluxes were determined in hearts perfused in an isovolumic Langendorff mode using 13C nuclear magnetic resonance isotopomer analysis or in isolated working hearts using [14C]glucose and [3H]palmitate, with and without 10 μM ranolazine or 3 μM CVT-4325. Isovolumic perfused hearts were also subjected to 30 min of low-flow ischemia (0.3 ml/min) and 60 min of reperfusion, and working hearts were subjected to 15 min of zero-flow ischemia and 60 min of reperfusion. Regardless of the experimental protocol, ranolazine had no effect on carbohydrate or fatty acid oxidation, whereas CVT-4325 significantly reduced fatty acid oxidation up to ∼7-fold with a concomitant increase in carbohydrate oxidation. At these same concentrations, although ranolazine significantly improved LV functional recovery following ischemia/reperfusion, CVT-4325 had no significant protective effect. These results demonstrate that at pharmacologically relevant concentrations, ischemic protection by ranolazine was not mediated by inhibition of fatty acid oxidation and conversely that inhibition of fatty acid oxidation with CVT-4325 was not associated with improved LV functional recovery.

Alteration of glycogen and glucose metabolism in ischaemic and post-ischaemic working rat hearts by adenosine A1 receptor stimulation

Cardioprotection by adenosine A1 receptor activation limits infarct size and improves post‐ischaemic mechanical function. The mechanisms responsible are unclear but may involve alterations in myocardial glucose metabolism. Since glycogen is an important source of glucose during ischaemia, we examined the effects of N6‐cyclohexyladenosine (CHA), an A1 receptor agonist, on glycogen and glucose metabolism during ischaemia as well as reperfusion. Isolated working rat hearts were perfused with Krebs‐Henseleit solution containing dual‐labelled 5‐3H and 14C glucose and palmitate as energy substrates. Rates of glycolysis and glucose oxidation were measured directly from the production of 3H2O and 14CO2. Glycogen turnover was measured from the rate of change of [5‐3H and 14C]glucosyl units in total myocardial glycogen. Following low‐flow (0.5 ml min−1) ischaemia (60 min) and reperfusion (30 min), left ventricular minute work (LV work) recovered to 22% of pre‐ischaemic values. CHA (0.5 μM) improved the recovery of LV work 2 fold. CHA altered glycogen turnover in post‐ischaemic hearts by stimulating glycogen synthesis while having no effects on glycogen degradation. CHA also partially inhibited glycolysis. These changes accelerated the recovery of glycogen in CHA‐treated hearts and reduced proton production. During ischaemia, CHA had no measurable effect on glycogen turnover or glucose metabolism. Glycogen phosphorylase activity, which was elevated after ischaemia, was inhibited by CHA, possibly in response to CHA‐induced inhibition of AMP‐activated protein kinase activity. These results indicate that CHA‐induced cardioprotection is associated with alterations of glycogen turnover during reperfusion as well as improved metabolic coupling of glycolysis to glucose oxidation.

Assessment of glycogen turnover in aerobic, ischemic, and reperfused working rat hearts

Glycogen and its turnover are important components of myocardial glucose metabolism that significantly impact on postischemic recovery. We developed a method to measure glycogen turnover (rates of glycogen synthesis and degradation) in isolated working rat hearts using [3H]- and [14C]glucose. In aerobic hearts perfused with 11 mM glucose, 1.2 mM palmitate, and 100 μU/ml insulin, rates of glycogen synthesis and degradation were 1.24 ± 0.3 and 0.53 ± 0.25 μmol ⋅ min-1 ⋅ g dry wt-1, respectively. Low-flow ischemia (0.5 ml/min, 60 min) elicited a marked glycogenolysis; rates of glycogen synthesis and degradation were 0.54 ± 0.16 and 2.12 ± 0.14 μmol ⋅ min-1 ⋅ g dry wt-1, respectively. During reperfusion (30 min), mechanical function recovered to 20% of preischemic values. Rates of synthesis and degradation were 1.66 ± 0.16 and 1.55 ± 0.21 μmol ⋅ min-1 ⋅ g dry wt-1, respectively, and glycogen content remained unchanged (25 ± 3 μmol/g dry wt). The assessment of glycogen metabolism needs to take into account the simultaneous synthesis and degradation of glycogen. With this approach, a substantial turnover of glycogen was detectable not only during aerobic conditions but also during ischemia as well as reperfusion.Glycogen and its turnover are important components of myocardial glucose metabolism that significantly impact on postischemic recovery. We developed a method to measure glycogen turnover (rates of glycogen synthesis and degradation) in isolated working rat hearts using [3H]- and [14C]glucose. In aerobic hearts perfused with 11 mM glucose, 1.2 mM palmitate, and 100 microU/ml insulin, rates of glycogen synthesis and degradation were 1.24 +/- 0.3 and 0.53 +/- 0. 25 micromol. min-1. g dry wt-1, respectively. Low-flow ischemia (0.5 ml/min, 60 min) elicited a marked glycogenolysis; rates of glycogen synthesis and degradation were 0.54 +/- 0.16 and 2.12 +/- 0.14 micromol. min-1. g dry wt-1, respectively. During reperfusion (30 min), mechanical function recovered to 20% of preischemic values. Rates of synthesis and degradation were 1.66 +/- 0.16 and 1.55 +/- 0. 21 micromol. min-1. g dry wt-1, respectively, and glycogen content remained unchanged (25 +/- 3 micromol/g dry wt). The assessment of glycogen metabolism needs to take into account the simultaneous synthesis and degradation of glycogen. With this approach, a substantial turnover of glycogen was detectable not only during aerobic conditions but also during ischemia as well as reperfusion.

Activation of Ca2+-independent nitric oxide synthase by 17β-estradiol in post-ischemic rat heart

BACKGROUND Nitric oxide (NO) donors or facilitation of endogenous NO production is cardioprotective. This study sought to determine whether enhanced myocardial NO production might contribute to estrogen-induced cardioprotection. METHODS Ca(2+)-dependent and Ca(2+)-independent NOS activities (pmol min(-1) mg(-1) protein), NOS protein expression (quantitative immunoblot), cGMP content (pmol mg(-1) protein) and LV work (Joules) were measured in hearts isolated from ovariectomized rats that were either untreated or treated chronically with 17beta-estradiol (0.25 mg, 21 day release formulation). RESULTS After 14 days, serum levels of 17beta-estradiol were 6+/-1 and 135+/-16 pg ml(-1) in untreated and 17beta-estradiol-treated animals, respectively. After 60 min aerobic working mode perfusion, Ca(2+)-dependent NOS (untreated, 1.47+/-0.36; 17beta-estradiol 1.13+/-0.25) and Ca(2+)-independent NOS (untreated, 0.45+/-0.24; 17beta-estradiol, 0.41+/-0.21) activities, eNOS and iNOS proteins and cGMP content (untreated, 0.64+/-0.08; 17beta-estradiol, 0.76+/-0.12) were not different in the two groups. After 60 min low-flow (0.5 ml min(-1)) ischemia and 30 min reperfusion, Ca(2+)-dependent NOS activities were again similar (untreated, 1.25+/-0.23; 17beta-estradiol, 0.78+/-0.27). However, after reperfusion, Ca(2+)-independent NOS activity (untreated, 0. 39+/-0.10; 17beta-estradiol, 1.36+/-0.36) was 3.5-fold higher (P=0. 008) and cGMP content (untreated, 0.30+/-0.03; 17beta-estradiol, 0. 49+/-0.07) was 1.6-fold higher (P=0.017) in hearts from 17beta-estradiol-treated animals. Although pre-ischemic function was similar, recovery of post-ischemic LV work was 2-fold greater (P=0.024) in the 17beta-estradiol group. CONCLUSION The ability of ischemia and reperfusion in combination with chronic 17beta-estradiol to increase Ca(2+)-independent NOS activity and cGMP content supports a role for enhanced myocardial NO signaling in 17beta-estradiol-induced cardioprotection.

Varespladib methyl in cardiovascular disease.

Importance of the field: The high risk of recurrent cardiovascular events amongst patients with cardiovascular disease receiving evidence-based therapies has prompted investigations into complimentary treatments that may reduce residual risk. Analyses of clinical trials in statin-treated patients demonstrate that elevated lipid levels and an activated systemic inflammatory state are associated with a higher risk of recurrent cardiovascular events. Areas covered in this review: This article reviews evidence supporting the causal role for secretory phospholipase A2 (sPLA2) in experimental atherosclerosis, the involvement of various sPLA2 isozymes as mediators of pro-atherogenic lipoprotein remodeling and participants in vascular and systemic inflammatory responses, and the evidence that sPLA2 inhibition reduces atherosclerosis in experimental models and biomarkers associated with cardiovascular events in coronary heart disease (CHD) patients. What the reader will gain: The experimental basis for sPLA2 inhibition with varespladib methyl as a potential candidate for lowering recurrent cardiovascular events particularly in acute coronary syndrome patients is discussed. Take home message: Varespladib methyl therapy reduces atherogenic lipoprotein concentrations and systemic inflammatory markers in CHD patients. The future role of varespladib methyl in CHD patients awaits the results of ongoing clinical trials.

Fatty acid uptake in diabetic rat adipocytes

The effect of diabetic status and insulin on adipocyte plasma membrane properties and fatty acid uptake was examined. Studies with inhibitors and isolated adipocyte ghost plasma membranes indicated 9Z, 11E, 13E, 15Z-octatetraenoic acid (cis-parinaric acid) uptake was protein mediated. Cis-parinaric acid uptake was inhibited by trypsin treatment or incubation with phloretin, and competed with stearic acid. The initial rate, but not maximal uptake, of cis-parinaric acid uptake was enhanced two-fold in adipocytes from diabetic rats. Concomitantly, the structure and lipid composition of adipocyte ghost membranes was dramatically altered. However, the increased initial rate of cis-parinaric acid uptake in the diabetic adipocytes was not explained by membrane alterations or by a two-fold decrease in cytosolic adipocyte fatty acid binding protein (ALBP), unless ALBP stimulated fatty acid efflux. Thus, diabetic status dramatically altered adipocyte fatty acid uptake, plasma membrane structu re, lipid composition, and cytosolic fatty acid binding protein. (Mol Cell Biochem 167: 51-60, 1997)

A Novel Partial Fatty Acid Oxidation Inhibitor Decreases Myocardial Oxygen Consumption and Improves Cardiac Efficiency in Demand-induced Ischemic Heart

The benefits of inhibition of fatty acid oxidation (FOX) and stimulation of glucose oxidation (GOX) in ischemia are controversial. The objective of this study was to evaluate the effect of the FOX inhibitor CVT-4325 on the rates of FOX, GOX, myocardial oxygen consumption (MVO2), and cardiac efficiency in the absence and presence of palmitate during demand-induced ischemia of the rodent isolated hearts. Palmitate concentration-dependently increased FOX, decreased GOX, and increased MVO2. CVT-4325 inhibited FOX and increased GOX in the presence (but not the absence) of 1.2 mM palmitate, with EC50 values of 0.9 and 5.8 μM, respectively. The potency for CVT-4325 to inhibit FOX was 10-fold greater (0.9 versus 9.7 μM) in the presence of 1.2 mM compared with 0.4 mM palmitate. The increase in MVO2 caused by 1.2 mM palmitate was significantly reduced by 3 to 10 μM CVT-4325 in guinea pig hearts. In the presence of 1.2 mM palmitate, an increase in pacing rate of the guinea pig heart from 3.5 to 6.5 Hz caused a significant 50% increase in MVO2, a decrease in cardiac efficiency, and an increase in lactate concentration in the cardiac effluent from 0.04 ± 0.01 to 0.10 ± 0.02 mM (P < 0.01). CVT-4325 (3 μM) attenuated the increase (P < 0.05) in MVO2 while maintaining cardiac contractility, and decreased the lactate production to 0.05 ± 0.01 mM (P < 0.01). Thus, the FOX inhibitor CVT-4325 decreased MVO2 and increased myocardial efficiency during demand-(pacing)-induced ischemia in the presence of palmitate in the rodent isolated hearts.