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Original Article

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

Wu, Lin MD; Belardinelli, Luiz MD; Fraser, Heather PhD

Author Information
Journal of Cardiovascular Pharmacology: April 2008 - Volume 51 - Issue 4 - p 372-379
doi: 10.1097/FJC.0b013e318166803b
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Abstract

INTRODUCTION

Myocardial ischemia is the result of an inadequate supply of oxygenated blood to the myocardium and/or an increase of myocardial work and oxygen demand (ie, an imbalance in the ratio of supply to demand). Three classes of drugs are used clinically to treat myocardial ischemia: β-adrenergic receptor blockers, calcium channel blockers, and nitrates. These drugs relieve ischemia by either increasing blood flow to the heart (vasodilation) or decreasing cardiac work (negative chronotropic and inotropic effects).1 However, the use of drugs that relieve myocardial ischemia by reducing cardiac output may be contraindicated in patients with congestive heart failure, bradycardia, and hypotension.1 Thus, drugs that decrease myocardial oxygen consumption (MVO2) without decreasing cardiac work or causing hemodynamic changes may be useful to avoid the limitations of traditional drug therapy.1-4 A shift in the proportions of ATP generated from fatty acid oxidation (FOX) and glucose oxidation (GOX) provides the heart with an efficient method to maintain a constant fuel source in diverse physiological, nutritional, and pathological circumstances.5-8 Because GOX can be more energy efficient than palmitate (ie, free fatty acid) oxidation and generate more ATP per unit of oxygen consumed, when the supply of oxygen to the heart is limited, a relative reduction of FOX and increase of GOX may be beneficial.4,5 Inhibition of FOX has been shown to decrease cardiac MVO2 without concomitantly reducing or even improving cardiac function.3,4,7,9-11

CVT-4325 is a piperazine derivative shown to inhibit the rate of FOX in rat heart mitochondria with a high potency (IC50 = 380 nM).12 However, a recent report indicates that the inhibition of FOX by CVT-4325 was not associated with improved LV functional recovery in rat working hearts with low-flow or zero-flow ischemia.13 In contrast, CVT-4325 improved left ventricular function in dogs with heart failure.14

One possible explanation for these controversial research results is the differences of underlying mechanisms between demand15 and low- or zero-flow13 ischemia. In low- or zero-flow ischemia, oxygen and metabolite supply may be insufficient to support normal myocardial function, regardless of the substrate oxidized. In contrast, in demand-induced ischemia, where oxygen and metabolite supply remain constant, an increase of efficiency of ATP production per unit of oxygen consumed may lead to a significant increase of cardiac performance. We hypothesized that CVT-4325 would cause a concentration-dependent shift in metabolism from FOX to GOX that, subsequently, would decrease MVO2 and increase myocardial energy efficiency and pump function in demand- (pacing)-induced ischemia.

In this study, guinea pig isolated hearts were used as a convenient model of demand-(pacing)-induced ischemia, because they have a relatively low spontaneous heart rate and are, thus, easy to pace. Rat isolated working hearts were used as a model to measure the effects of CVT-4325 on FOX and GOX, as previously described.16 Therefore, the primary objectives of this study were to (1) characterize the effects of CVT-4325 on substrate (palmitate and glucose) use (oxidation) in rat isolated working hearts, and (2) measure the effects of CVT-4325 on MVO2 and left ventricular function during normoxia and demand-induced ischemic conditions in guinea pig isolated hearts.

MATERIALS AND METHODS

Materials

Bovine serum albumin (BSA; essentially fatty acid free), palmitate, and lactate analysis reagent kits were purchased from Sigma/Aldrich (St. Louis, Mo). Insulin (Humulin R) was purchased from Eli Lilly (Indianapolis, Ind). Radioisotopes were obtained from NEN (Boston, Mass). CVT-4325 {(S)-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, C25H26F3N5O3S}, provided by the Bioorganic Chemistry Department at CV Therapeutics, was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10-2 M and stored at 4°C. The stock solution was further diluted to 2 × 10−4 M in physiological saline for use in experiments. The final concentration of DMSO in saline during experiments was not more than 0.1%. BSA concentration in all solutions used in this study was 3%. Palmitate was bound to 3% BSA solution and dialyzed overnight at 4°C in K-H solution before use in experiments.

Animals

Male Sprague-Dawley rats (300-350 g) and Hartley guinea pigs (250-300 g) were purchased from Simonsen (Gilroy, Calif). This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication no. 85-23, revised 1996). Animal use for this project was approved by the institutional animal care and use committee of CV Therapeutics (Palo Alto, Calif).

Pressure-Controlled Working Rat Heart Model

Rats were anesthetized with a ketamine and xylazine mixture (150 mg/kg ketamine and 30 mg/kg xylazine, administered intraperitoneally). The aorta was rapidly cannulated, and hearts were perfused in Langendorff mode at a constant pressure of 60 mm Hg. The hearts were perfused in working mode under aerobic conditions and at a constant left atrial preload of 11.5 mm Hg and aortic afterload of 80 mm Hg. The working heart perfusate consisted of a Krebs-Henseleit (K-H) solution containing 3% BSA with either 0.4 or 1.2 mM palmitate (prebound to BSA), and insulin (100 μU/mL). The K-H solution contained (in mM) 118 NaCl, 2.8 KCl, 1.2 KH2PO4, 2.5 CaCl2, 0.5 MgSO4, 5.5 glucose, and 25 NaHCO3. The solution was continuously gassed with 95% O2 and 5% CO2 and warmed to 36.5 to 37°C. CVT-4325 (0-30 μM) was added directly into the heart perfusate after 5 minutes of stabilization of hearts in working mode. Hearts were paced at 300 beats/min, and left ventricular minute work [cardiac work (CW)], calculated as cardiac output × left ventricular developed pressure, was recorded throughout the experiment and used to normalize values of FOX and GOX.

Guinea Pig Isolated, Perfused (Constant Flow) Heart Model

Guinea pigs (Hartley) of either sex, weighing 250 to 350 g, were anaesthetized by inhalation of isofluorane and killed by cervical dislocation. The hearts were isolated and perfused at a constant rate of 10 mL/min with K-H solution (see above) containing 2.0 mM pyruvate and 0.57 mM EDTA (pH was adjusted to 7.4), using a roller pump (Gilson Minipuls3, Middleton, Wisc).17 Freshly isolated hearts were perfused briefly with K-H solution without BSA until coronary perfusion pressure was stable, then with K-H solution containing 3% BSA alone for 10 to 15 min, then with K-H solution containing BSA without or with bound palmitate, and with insulin (100 μU/mL). All solutions were equilibrated with 95% O2 and 5% CO2, as described below. To facilitate exit of fluid from the left ventricle (LV), the leaflets of the mitral valve were trimmed with fine, spring-handled scissors. The right atrial wall was partially removed, and a bipolar, teflon-coated electrode was placed on the atrial septum to pace the heart. Electrical stimuli, 3 ms in width and threefold threshold amplitude, were delivered to the pacing bipolar electrode at a basic frequency of 3.5 Hz (Grass S48 stimulator, W. Warwick, RI). The His bundle electrogram and coronary perfusion pressure were monitored continuously throughout each experiment.17 Coronary conductance (mL·min−1·mm Hg−1) was calculated as the ratio of coronary flow (10 mL/min) to perfusion pressure (mm Hg). An increase in heart rate to create demand-induced ischemia was obtained by changing the rate of stimulation in a stepwise manner from 3.5 to 6.5 Hz. The total duration of the experimental protocol was limited to 2 h, the time during which the preparation exhibited good stability. Steady-state responses to control and interventions are reported.

A two-step, custom-made oxygenation system was developed for the oxygenation of K-H solution containing either 3% BSA alone or 3% BSA with bound palmitate. The primary oxygenator was a length of coiled silastic tubing (Fisher Scientific, Pittsburgh, Pa) suspended in the K-H solution and flushed with a 95% O2 and 5% CO2 mixture. All solutions then went through a secondary oxygenator to balance the difference of O2 contents among solutions before they were perfused to the heart.

Measurement of MVO2

The oxygen tensions in the cardiac perfusate and effluent solutions were continuously monitored throughout the experiments by two flow-through oxygen electrodes (Microelectrodes, Bedford, NH). The difference of the oxygen tension in the perfusate (PAO2) and effluent (PVO2) solutions was calculated and recorded in real time throughout each experiment. MVO2 was calculated using the following equation: MVO2 = (PAO2−PVO2) × coronary flow×c/760/dry heart weight. Coronary flow is the rate of perfusion (normal flow was 10 mL/min); c is the solubility of oxygen in buffer at 37°C, which is 0.022 in the presence of plasma. To determine dry heart weight, the hearts were dried in air at room temperature before weighing.

Measurement of Ventricular Contractility

A latex balloon connected to a pressure transducer (AD Instruments, Castle, Australia) was inserted into the left ventricle from a small incision in the left atrium. After closing the left atrium with a prepared suture around the area of the incision, the balloon was filled with normal saline to achieve a diastolic pressure of 0 to 5 mm Hg. The left ventricular developed pressure (LVDP) and the maximal rate of pressure development (dP/dtmax) were monitored and recorded continuously. Cardiac efficiency was defined as the ratio between energy output (work) and oxygen consumption (MVO2) for the heart. In this study, it was calculated as heart rate × (either LVDP or dP/dtmax)/MVO2.

Measurement of Glucose and Fatty Acid Oxidation

Glucose and fatty acid oxidation rates of rat isolated working hearts were measured simultaneously, using dual-labeled substrates (14C-glucose and 3H-palmitate) as previously described by Saddik and Lopaschuk.18 Average rates of FOX and GOX were calculated from linear time courses of product accumulation between 15 and 60 min of perfusion. Rates of FOX and GOX are expressed as nanomoles of substrate metabolized per milligram of mercury of developed pressure per liter of cardiac output, after normalization to heart weight. Estimates of the concentration of CVT-4325 necessary to cause a half-maximal effect (i.e., the value of EC50) to decrease the rate of FOX and increase the rate of GOX were calculated by least squares nonlinear fitting of concentration-response curves.

Measurement of Lactate Concentration in the Cardiac Effluent

Samples of cardiac effluent solution from the guinea pig pulmonary artery before and after interventions at pacing rates of 3.5, 5, and 6.5 Hz were collected and frozen at −20°C for lactate measurement. Lactate was measured using a rapid enzymatic method (Sigma/Aldrich, St. Louis, Mo).

Exclusion Criteria

Criteria for the exclusion of hearts from the study were a coronary perfusion pressure of <50 mm Hg, an unstable perfusion pressure during the equilibration period, an inability to pace a heart at a constant rate, or an inability to record stable values of either PO2 or left ventricular pressure for the duration of an experiment.

Statistical Analyses

All data are reported as means ± SEM. The concentration-response curves were analyzed using Prism version 4 (GraphPad Software Inc., San Diego, Calif). The statistical significance of differences in values of measurements obtained from the same hearts before and after an intervention (eg, Fig. 3) was determined by repeated-measure one-way analysis of variance, followed by Student-Newman-Keuls test. The statistical significance of differences in values of measured parameters among groups of hearts treated with different interventions (eg, Figs. 1, 5B, and 6) was determined using a two-way mixed-design analysis of variance. The differences of MVO2 among groups of hearts paced at different rates (Fig. 5A) were compared using pairwise Welch t tests.

FIGURE 1
FIGURE 1:
Effects of CVT-4325 on fatty acid oxidation (FOX, A) and glucose oxidation (GOX, B) in the absence (0 mM) and presence (0.4 and 1.2 mM) of palmitate in rat isolated working hearts. Rates of FOX and GOX are normalized for cardiac work. The numbers of experiments are shown in parentheses. *P < 0.05-0.01 compared with 0 mM palmitate, ▿, P < 0.01 compared with palmitate alone.

RESULTS

Effect of CVT-4325 on Rates of FOX and GOX in Rat Isolated Working Hearts

Palmitate concentration-dependently increased FOX and decreased GOX (Fig. 1). The rates of FOX and GOX were 0 ± 0 and 102 ± 9.2 nmol·mm Hg−1·L−1, respectively, in hearts perfused with 3% BSA and insulin (100 μU/mL) alone (control, n = 5). The addition of BSA-bound palmitate (0.4 and 1.2 mM) to the cardiac perfusate caused a significant increase in the rate of FOX to 22 ± 2 and 48 ± 2 nmol·mm Hg−1·L−1 (n = 9 and 11, P < 0.01) and a decrease in the rate of GOX to 60 ± 3 and 17 ± 2 nmol·mm Hg−1·L−1 (P < 0.05), respectively. CVT-4325 (1 μM) had no effect on the rates of FOX or GOX in the absence of palmitate (n = 6, P > 0.05, Fig. 1A and B). In the presence of 0.4 mM palmitate, the rates of FOX and GOX were decreased by 23% and increased by 22% by 1 μM CVT-4325 (from 22 ± 2 to 17 ± 1 and from 60 ± 3 to 73 ± 4 nmol·mm Hg−1·L−1, respectively, n = 14). In the presence of 1.2 mM palmitate, the rates of FOX and GOX were decreased by 35% and increased by 165% by 1 μM CVT-4325 (from 48 ± 2 to 31 ± 3 and from 17 ± 2 to 45 ± 7 nmol·mm Hg−1·L−1, n = 7, respectively, P < 0.01) (Fig. 1).

The effect of CVT-4325 to decrease FOX and increase GOX in the presence of 1.2 mM palmitate was concentration dependent (Fig. 2). The concentrations of CVT-4325 to cause half-maximal (EC50, potency of CVT-4325) changes in the rates of FOX and GOX were 0.9 and 5.8 μM, respectively (Fig. 2A). In the presence of a higher concentration (1.2 versus 0.4 mM) of palmitate, the concentration-response curve of CVT-4325 to inhibit FOX was shifted to the left (Fig. 2B). The EC50 for CVT-4325 to inhibit FOX was 0.9 μM in presence of 1.2 mM palmitate, which was 10 times lower than that observed in hearts perfused with 0.4 mM palmitate (9.7 μM, n = 4-11). These data indicate that the metabolic shift in the rates of use of fatty acids caused by CVT-4325 was greater at higher concentrations of palmitate.

FIGURE 2
FIGURE 2:
Concentration-response curves for CVT-4325 on fatty acid oxidation (FOX) and glucose oxidation (GOX) in rat isolated working hearts. A, CVT-4325 decreased FOX and increased GOX (n = 4-11) in a concentration-dependent manner in hearts perfused with 1.2 mM palmitate. Rates of FOX and GOX are normalized for cardiac work. Control values for GOX and FOX were 0.41 ± 0.05 and 1.13 ± 0.05 nmol·mm Hg−1·min−1, respectively (n = 11). B, Concentration-response curve for CVT-4325 to inhibit FOX in the presence of 0.4 and 1.2 mM palmitate. Control values of FOX were 17 ± 2 and 48 ± 2 nmol·mm Hg−1·L−1 for 0.4 and 1.2 mM palmitate.

Effects of CVT-4325 on MVO2 in the Presence of Palmitate in the Normoxic Guinea Pig Heart

Increasing the perfusate palmitate concentration (from 0 to 0.1, 0.4, and 1.2 mM) significantly increased MVO2 of the guinea pig isolated heart paced at a rate of 3.5 Hz in a concentration-dependent manner by 30% from 39 ± 3 to 51 ± 3 mL·min−1·100 g−1 dry weight (n = 9, P < 0.01, Fig. 3A). This increase was partially reversible when the cardiac perfusate containing palmitate was replaced with a palmitate-free solution.

FIGURE 3
FIGURE 3:
Effects of palmitate and CVT-4325 on MVO2 in guinea pig isolated hearts. A, Concentration-response relationship for palmitate to increase MVO2. B, Concentration-response relationship for CVT-4325 to decrease MVO2 in the absence (BSA alone) and presence of 1.2 mM palmitate. *Compared with control, P ≤ 0.001. Numbers in parenthesis are the percent change of MVO2 above BSA (panel A) or below 1.2 mM palmitate (panel B).

CVT-4325 reduced MVO2 in the presence but not in the absence of palmitate (Figs. 3 and 4). In the absence of palmitate, CVT-4325 (0.3-10 μM) did not change MVO2 (n = 7, Figs. 3B and 4A, P > 0.05). In contrast, 5 μM diltiazem (a calcium channel blocker) significantly decreased MVO2 in the absence of palmitate from 41.4 ± 3.5 to 32.2 ± 2.8 mL·min−1·100 g−1 (n = 6, P < 0.001, Fig. 4A) and prolonged the S-H interval from 43 ± 1 to 69 ± 4 milliseconds (n = 6, P < 0.01). In the presence of 1.2 mM palmitate, CVT-4325 (3 and 10 μM) significantly decreased MVO2 from 48 ± 3 to 42 ± 3 and 39 ± 3 mL·min−1·100 g−1 (n = 9, Figs. 3B and 4B, P < 0.01), respectively. Neither palmitate (0.1-1.2 mM) nor CVT-4325 (3 μM) caused a significant change in S-H interval, coronary conductance, LVDP, or dP/dtmax (n = 9 and 7, P > 0.05, not shown).

FIGURE 4
FIGURE 4:
Effects of CVT-4325 on MVO2 in absence and presence of palmitate in guinea pig isolated normoxic hearts. A, PAO2-PVO2 recorded from a heart serially exposed to 0.3-10 μM CVT-4325 and 5 μM diltiazem in the absence of palmitate (BSA alone). B, PAO2-PVO2 recorded from a heart serially exposed to 0.3-10 μM CVT-4325 in the presence of 1.2 mM palmitate.

Reversal by CVT-4325 of the Palmitate-induced Increase in MVO2 During Demand (Pacing)-induced Ischemia

In guinea pig isolated hearts perfused with BSA alone (no palmitate), increasing the pacing rate in a stepwise fashion from 3.5 to 5 to 6.5 Hz resulted in a linear increase of 26% in MVO2 (n = 13, P < 0.001, Fig. 5A). The increase in MVO2 caused by pacing was greater in the presence of 1.2 mM palmitate than in its absence (P < 0.001, Fig. 5A). In hearts paced at a rate of 3.5 Hz, 1.2 mM palmitate significantly increased the MVO2 by 33% from 30 ± 1 mL·min−1·100 g−1 (BSA alone, n = 35) to 40 ± 2 mL·min−1·100 g−1 (n = 20, P < 0.0001, Fig. 5A). MVO2 values at 6.5 Hz were 50% greater in the presence of 1.2 mM palmitate than in the presence of BSA alone (n = 7, P < 0.001) (Fig. 5A). In the presence of 1.2 mM palmitate, 3 μM CVT-4325 caused a significant (n = 9, P = 0.042, Fig. 5A) downward shift in the relationship between heart rate and MVO2, and this effect became greater at higher heart rates (5.5, 6, and 6.5 Hz; P ≤ 0.028 versus palmitate). In contrast, 3 μM CVT-4325 had no effect on MVO2 in hearts perfused with BSA alone (absence of palmitate, n = 4, P > 0.05), which indicates that the effect of CVT-4325 on MVO2 was palmitate dependent.

FIGURE 5
FIGURE 5:
Effect of CVT-4325 on MVO2 and lactate production during demand-induced ischemia in guinea pig hearts. A, Rate-dependent relationships of MVO2 in absence (BSA alone, open marks) and presence of 1.2 mM palmitate (solid marks) and 3 μM CVT-4325 (square marks). Each point represents mean ± SEM of 13, 7, 9, and 4 hearts for BSA alone, 1.2 mM palmitate, palmitate+CVT-4325, and BSA+CVT-4325, respectively. Values in parenthesis indicate the percent increase in MVO2 above BSA alone. B, Lactate release in the coronary effluent in absence (open bars, n = 10) and presence of CVT-4325 (solid bars, n = 12) in guinea pig hearts perfused with palmitate. *Compared with control at 3.5 Hz, P < 0.01; ** compared with palmitate alone, P < 0.05.

The slope of the heart rate-MVO2 curve for 1.2 mM palmitate was greater than the similar slopes for BSA alone or 1.2 mM palmitate + CVT-4325. For every 1 Hz (60 beats/min) increase in heart rate, MVO2 increased by 3.5 and 3.7 mL·min−1·100 g−1 in hearts perfused with BSA alone or with 1.2 mM palmitate + CVT-4325, but by 6.1 mL·min−1·100 g−1 in presence of 1.2 mM palmitate.

Effect of CVT-4325 on Lactate Production During Demand (Pacing)-induced Ischemia

In guinea pig isolated hearts perfused with 1.2 mM palmitate, lactate concentration in the cardiac effluent was increased from 0.04 ± 0.01 to 0.10 ± 0.02 mM (n = 12, P < 0.01, Fig. 5B) when the heart rate was increased from 3.5 to 6.5 Hz (Fig. 5B). In hearts treated with 1.2 mM palmitate and 3 μM CVT-4325, lactate concentration at 6.5 Hz was 0.05 ± 0.01 mM (n = 10, Fig. 5B), which was significantly lower (P < 0.01) than in hearts perfused with 1.2 mM palmitate alone (0.10 ± 0.02 mM).

Effect of CVT-4325 on Cardiac Efficiency in the Presence of Palmitate During Demand (Pacing)-induced Ischemia of the Guinea Pig Isolated Heart

Cardiac efficiency in the presence of 1.2 mM palmitate was significantly increased by 3 μM CVT-4325 during pacing-induced ischemia (Figs. 6A and B). In the presence of 1.2 mM palmitate, a stepwise increase in pacing rate from 3.5 to 5.5, 6, and 6.5 Hz resulted in a decrease in cardiac efficiency, calculated as (heart rate × LVDP)/MVO2 (n = 12, P < 0.01-0.001, Fig. 6A), or as a biphasic increase and decrease when cardiac efficiency was calculated as (HR × dP/dtmax)/MVO2 (n = 12, Fig. 6B). Because the LVDP was not significantly changed by palmitate (not shown), the palmitate-induced decrease of cardiac efficiency was attributable to an increase of MVO2. In the presence of 3 μM CVT-4325, cardiac efficiency was significantly increased when hearts were perfused with 1.2 mM palmitate at pacing rates of 5.5, 6, and 6.5 Hz (n = 12, P < 0.05-0.01, Fig. 6A and B). The increase of cardiac efficiency caused by CVT-4325 in the presence of palmitate was attributable to a decrease of MVO2, without changes in LVDP or dP/dtmax.

FIGURE 6
FIGURE 6:
Effects of CVT-4325 on cardiac efficiency calculated with either LVDP (A) or dP/dt (B) in guinea pig hearts subjected to low and high pacing rates. Control values of cardiac efficiency in absence and presence of 3 μM CVT-4325 were 7.0 ± 0.5 and 7.1 ± 0.6 mm Hg·Hz−1·MVO2−1 calculated with LVDP, and 111.6 ± 9.6 and 124.9 ± 10.0 mm Hg·Hz−1·s−1·MVO2−1 calculated with dP/dt, respectively. Compared with the control: *P < 0.05; **P < 0.01. ▿, compared with the values in absence of CVT-4325 at same rates, P < 0.01.

DISCUSSION

The results in this study indicate that (1) a progressive increase in palmitate concentration resulted in an increase in FOX and a decrease in GOX in rat heart, and an increase in MVO2 in guinea pig heart; (2) CVT-4325 reversed the changes in metabolism of FOX and GOX and in MOV2 in a concentration- and palmitate-dependent manner; and (3) CVT-4325 significantly decreased lactate production and improved cardiac efficiency in demand- (pacing)-induced cardiac ischemia, secondary to a decrease in MVO2.

The energy needs of the heart are met by the generation of ATP from the metabolism of a number of substrates including fatty acids, glucose, lactate, and ketone bodies. Under aerobic (nonischemic) conditions, the heart preferentially uses fatty acids, rather than glucose, to generate ATP.18-20 However, FOX is less efficient than GOX in that more oxygen is used to generate an equivalent amount of ATP from fatty acids than from glucose.5 Although sparing oxygen under aerobic conditions may be of little significance, under ischemic conditions, the consumption of less oxygen (ie, increase in efficiency) should be beneficial.21,22 Thus, compounds that can induce a shift in substrate use from fatty acids to glucose may be useful clinically.3 Recent studies have shown that in heart failure, diabetic, and ischemic heart disease, inhibition of either transportation of fatty acid (CPT 1) or FOX is beneficial.14,23-25 In contrast to the reported beneficial effect of a metabolic shift, however, are the data of Wang et al.13 In that study, CVT-4235 (3 μM) caused a shift from FOX to GOX in both isovolumic and working heart preparations, consistent with the results observed in the present study. However, this relative shift in oxidative metabolism did not result in a beneficial effect on left ventricular functional recovery after low- or no-flow ischemia/reperfusion.13

CVT-4325 decreased the rate of FOX in rat isolated working hearts and concomitantly increased the rate of GOX. The decrease of the rate of FOX by CVT-4325 was dependent on the concentration of fatty acid (palmitate) in the perfusate. In the absence of exogenously added fatty acid, CVT-4325 had no effect on the rate of either FOX or GOX. Furthermore, the EC50 values for the decrease of FOX by CVT-4325 in the presence of normal (0.4 mM) or elevated (1.2 mM) palmitate were approximately 10-fold different (9.7 versus 0.9 μM, respectively). This suggests that the action of FOX inhibitors such as CVT-4325 depends on the concentration of circulating free fatty acids. This is particularly significant in that many patients undergoing acute myocardial ischemia19,26,27 or heart surgery28 are reported to have elevated free fatty acid levels. During diabetes, there is also an increase in FOX in the heart, which reduces metabolic flexibility and reduces cardiac efficiency.7

The beneficial effects of a shift from fatty acid to glucose metabolism were further confirmed in the guinea pig isolated heart in this study. The guinea pig isolated heart is a model with a heart rate controlled by an electric stimulator and a constant coronary perfusion rate.17 In normoxic hearts, CVT-4325 reversed the increase in MVO2 caused by a high concentration (1.2 mM) of palmitate in a concentration-dependent manner, without changing coronary conductance or electrophysiological parameters.

An increase of the ventricular pacing rate of hearts perfused with palmitate-containing buffer caused the lactate concentration in the cardiac effluent to increase, which indicates demand-induced ischemia.15 In this study, during demand- (pacing)-induced ischemia in which lactate production was increased (Fig. 5B), CVT-4325 reduced the increases in MVO2 and lactate release caused by 1.2 mM palmitate, especially at faster pacing rates, and improved cardiac efficiency (Fig. 6). Thus, the beneficial effects of CVT-4325 on cardiac efficiency during demand-induced ischemia seem secondary to the decrease in MVO2. The data in this study are consistent with a recent publication in which an inhibition of malonyl-CoA decarboxylase suppressed FOX and reduced lactate production during demand-induced ischemia.15

The cardioprotective effects of CVT-4325 observed in the present study, as indicated by improved cardiac efficiency during demand-induced ischemia in the presence of palmitate, are in contrast to the results from Wang et al,13 who have reported no improvement by CVT-4325 (3 μM), in left ventricular functional recovery after ischemia/reperfusion in either low- or no-flow ischemia models. The model of moderate, demand-induced ischemia used in the present study is different from that of low- or zero-flow ischemia, because oxygen and metabolite supply remain constant in the former, but not the latter, model. MVO2 was moderately increased by 50% in this study during demand-(pacing)-induced ischemia, and this modest increase of MVO2 was reduced by inhibition of FOX with CVT-4325. However, if the imbalance between oxygen supply and demand is great (ie, low- or no-flow ischemia), the relatively small gain in cardiac efficiency attributable to conversion from FOX to GOX may not be significant.13 Furthermore, low-flow ischemia might cause an endothelial injury during either ischemia or ischemia-reperfusion, generation of oxidizing free radicals, and sodium/calcium overloading of myocardium cells that cannot be reduced or reversed by inhibition of FOX.29 Regardless, the improvement in cardiac efficiency by CVT-4325 during demand-induced ischemia may be best demonstrated in situations wherein the levels of plasma free fatty acids are elevated.

The clinical situation of myocardial ischemia is represented by a patient who has underlying coronary artery disease and, thus, a reduced coronary flow reserve, in combination with demand-induced ischemia. The guinea pig hearts used in this study to evaluate the effects of palmitate and CVT-4325 on MVO2 and cardiac efficiency were perfused at a constant flow of 10 mL/min, and, thus, an increase of cardiac oxygen demand secondary to an increase of heart rate could not be met with an increase of coronary flow and oxygen supply. In the clinical setting, and the experimental setting used in the present study, it may be expected that both cardiac efficiency and the balance of oxygen demand and oxygen supply would be improved when glucose rather than free fatty acid (palmitate) is the primary energy substrate.

Study Limitations

The limitations of this study include the facts that (1) the rodent isolated heart lacks innervation, and, thus, autonomic influences on the cardiac response to ischemia are not represented in this model; (2) the guinea pig isolated perfused hearts in this study were not studied in a “working” mode (with adjustable pre- and afterloads), and, thus, the effect of demand-induced ischemia on these parameters was not assessed and did not contribute to changes in MVO2; and (3) in this study, the interpretation of changes in dP/dtmax, which was used as an index of contractility in this study and, thus, cardiac efficiency, was limited, because we did not account for the load- and rate dependence of values of dP/dtmax.

In summary, the results of this study support the hypothesis that increasing GOX relative to FOX may improve cardiac efficiency during demand-induced ischemia.14,30 CVT-4325 decreased the rate of FOX in a palmitate- and concentration-dependent manner. It did so in association with a concomitant increase in the rate of GOX. CVT-4325 improves myocardial efficiency by decreasing MVO2 and lactate release.

ACKNOWLEDGMENTS

The authors thank Dr. John C. Shryock for his thoughtful review and careful editing of this manuscript. We also thank Mr. Jeffrey J. McVeigh for experimental work and expertise, and Dr. Ann W. Olmsted for assistance with the statistical analysis of data.

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Keywords:

fatty acid oxidation; CVT-4325; myocardial oxygen consumption; myocardial ischemia; cardiac efficiency

© 2008 Lippincott Williams & Wilkins, Inc.