Unger, Steven A PhD; Kennedy, Jennifer A PhD; McFadden-Lewis, Kate BSc Hons; Minerds, Kirsty BSc Hons; Murphy, Geraldine A BA; Horowitz, John D PhD
The oxygen-sparing effect of a shift in metabolic substrate utilization from fatty acid to carbohydrate has been proposed to underlie the beneficial effect during ischemia of inhibitors of myocardial fatty acid oxidation.1,2 Perhexiline is a prophylactic antianginal agent without significant hemodynamic effects in patients.3 We have shown previously in isolated rat cardiac mitochondria, that perhexiline is an inhibitor of the enzyme carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in the transport of long-chain fatty acids into mitochondria.4 We subsequently demonstrated5 in isolated rat hearts, perfused in the Langendorff mode with palmitate in addition to glucose, that a therapeutic concentration of perhexiline (2 μM) protected in vitro against diastolic contracture during low-flow ischemia, but did not reduce the concentration of long chain acylcarnitines (LCAC), products of CPT-1. The failure of cardiac LCAC concentration to decrease in perhexiline-treated hearts was compatible with the finding that perhexiline is a relatively non-selective CPT inhibitor, inhibiting CPT-2 with similar potency to its inhibition of CPT-1. Although simultaneous inhibition of CPT-1 and CPT-2 would be expected to inhibit long-chain fatty acid oxidation, and thereby lead to increased efficiency of myocardial oxygen utilization, we did not assess fatty acid oxidation directly in that study.5 To date, few studies have examined the effects of perhexiline on myocardial fatty acid oxidation either in intact cardiomyocytes or in the intact heart. Whereas Jeffrey et al6 reported in a working rat heart preparation that perhexiline administered acutely in vitro inhibited fatty acid oxidation, our previous studies in humans7 suggest that some biologic effects of perhexiline, including those on platelets, exhibit a considerably delayed onset, being best demonstrated after prolonged treatment. Moreover, delayed onset of perhexiline effects is not restricted to in vivo studies because similar delays in onset of metabolic effects of perhexiline have been reported in isolated hepatocytes.8
The current study was designed to test the hypotheses that: (1) perhexiline in therapeutic concentrations inhibits long-chain fatty acid oxidation, both in cultured cardiomyocytes and in working rat hearts and that (2) these effects of perhexiline exhibit slow onset. Simultaneously, the potential ability of perhexiline to increase cardiac efficiency of oxygen utilization in working rat hearts was compared with that of oxfenicine, a specific inhibitor of CPT-1.
MATERIALS AND METHODS
Adult and neonatal rats were treated in accordance with the ethical guidelines for animal research reviewed and approved by the ethics committees of the University of Adelaide and The Queen Elizabeth Hospital.
Hearts were removed from neonatal Sprague-Dawley rat pups, 1 to 3 days of age, and ventricles (10 per culture) were cut open and rinsed with Hanks Balanced Salt solution (HBSS). Small pieces approximately 1 mm × 1 mm were digested at 4°C overnight with gentle shaking in sterile HBSS containing trypsin 1 mg/mL, followed by digestion in collagenase 0.2 mg/mL at 37°C for 15 minutes with shaking. Following centrifugation and washing, the pellet was resuspended in DMEM with 10% FCS and penicillin/streptomycin and pre-plated into flasks for 1 hour to allow adherence of fibroblasts. The suspended cells were plated in 6-well plates (Falcon) or T25 flasks at 0.75 × 106 cells per well and 2.22 × 106 cells per flask. After 3 days in culture at 37°C with 95% relative humidity, they were washed and replaced with serum-free DMEM containing 1% BSA and perhexiline (as the maleate salt) or vehicle (0.1% ethanol). Cells were exposed to perhexiline, or vehicle, for 48 hours, and then medium was replaced with serum-free DMEM containing 1% BSA, 5 mM glucose, 100 μU/mL insulin, and 1.2 mM palmitate, and incubated at 37°C to determine substrate oxidation. Palmitate was prebound to albumin as described by Lopaschuck and Barr.9 In some cultures the effect on palmitate oxidation of a shorter preincubation time of 1 hour with perhexiline was compared with that of 48 hours preincubation. After preincubation the cells were incubated for 8 hours with 9,10-3H palmitate at 37°C, and then the medium from each well was collected and used to estimate the amount of palmitate oxidized.
Glucose oxidation was estimated after 48 hours preincubation only. Rate of glucose oxidation was estimated from 14CO2 produced by cells incubated with U-14C glucose in T-25 flasks. Cells were incubated for 8 hours at 37°C with palmitate- and glucose-containing medium as described above for palmitate oxidation. 14C glucose containing medium was introduced to the flasks for the last 4 hours of incubation only, to prevent saturation of the 14CO2 capture solution. 14CO2 evolved during incubation was captured with 2M sodium hydroxide solution attached to each flask via a flow through system (5% CO2 in air) to enable gas capture.
At the end of incubation with radiolabeled substrates, the cells were washed with phosphate buffered saline (PBS) and then PBS containing EDTA 2 mM added. After scraping from the wells or flasks, the cellular DNA content was estimated fluorometrically as described by Labarca and Paigen.10 Metabolic data were normalized to DNA content.
Working Rat Heart
Albino Wistar rats, 400 g body weight, were anaesthetized with halothane. Hearts were removed rapidly and placed in ice-cold calcium-free Krebs bicarbonate buffer. The hearts were cannulated via the aorta onto the perfusion apparatus and perfused initially in the Langendorff-mode with Krebs bicarbonate buffer (in mM: NaCl 118, KCl 4.7, KH2PO4 1.18, NaHCO3 25, MgCl2 1.05, CaCl2 2.5). The left atrium and the pulmonary artery were then cannulated and the heart switched to working mode with a preload on the left atrium of 11 mm Hg and an afterload on the aortic outflow of 80 mm Hg. The oxygen concentration of perfusate entering the heart, and that exiting the heart in the pulmonary artery outflow, was estimated via a flow-through Clarke type oxygen electrode (Rank Bros). Flow rate in the aortic outflow and on the inflow to the atrium was monitored using electromagnetic flow probes (Zepeda instruments). Aortic pressure was monitored using a pressure transducer (Omeda). Physiological data were recorded continuously using an 8-channel Maclab (AD Instruments) connected to a PowerMac computer. Cardiac work was estimated from mean aortic pressure and cardiac output, converted as per Grossman11 to g.mH2O/min/g dry weight (ie, CW = MAP (mm Hg) × CO (mL/min) × 0.0136/g dry weight). Efficiency (g.m/μmol) was estimated from the ratio of cardiac work (in g.m/min/g dry weight) to oxygen consumption (in μmol/min/g dry weight). Perfusate consisted of Krebs bicarbonate containing 3% BSA, 0.4 mM palmitic acid (pre-bound to albumin), 11 mM glucose, and 100 μU/mL of insulin oxygenated with 95%O2/5%CO2 maintained at 37°C and was recycled. 9,10-3H palmitate or U- 14C-glucose was added to the perfusate to estimate rates of oxidation of palmitate and glucose respectively. Hearts were perfused for 60 minutes in normal flow conditions, with sampling of perfusate at 10-minute intervals to estimate substrate metabolism. Perhexiline 2 μM, oxfenicine 2 mM, or vehicle were added at the start of perfusion in the working mode. At the end of perfusion hearts were frozen in an aluminium clamp cooled with liquid nitrogen, and stored at -80°C until further analysis. The hearts were pulverized under liquid nitrogen and aliquots used for subsequent analysis, one aliquot being used to estimate dry weights of tissue. Data were then expressed relative to dry weight of tissue.
Pretreatment of Rats
A subset of Albino Wistar rats were pretreated for 24 hours with perhexiline gluconate (200 mg) applied in a paste with sorbolene cream to the dorsal skin after removing the hair with cold wax strips under light anesthetic with halothane, nitrous oxide, and oxygen. Perhexiline gluconate was used because of its greater solubility than the maleate salt. Transdermal dosing was used after preliminary studies indicated that gavage with perhexiline resulted in gastric irritation to the rats. Preliminary studies indicated that 200 mg applied in a paste delivered sufficient perhexiline to achieve a plasma level of 2 μM (plasma concentration; perhexiline, 2.02 ± 0.29 μM; hydroxy-perhexiline 0.86 ± 0.21 μM, n = 8). Control rats were treated in an equivalent manner with the vehicle. The paste was covered by a protective layer of Tegaderm. Following pretreatment of the animals, the hearts were set up in the isolated working heart preparation as described above. Perhexiline was present in the perfusate for perhexiline pretreatment group at 2 μM to prevent any washout of the drug from these hearts during in vitro assessment.
Measurement of Substrate Oxidation
Palmitate oxidation was estimated from the rate of formation of 3H2O from 9,10-3H palmitate. 3H2O was separated from 3H palmitate in the heart perfusate or the cell culture medium by solvent extraction essentially as described by Lopaschuk and Barr.9 Palmitate oxidation was linear over the incubation period and less than 5% of the substrate was removed during the incubation.
Evolved 14CO2 was captured continuously via a gas outflow tube from the perfusion apparatus, which was otherwise closed. This was sampled at 10-minute intervals; 1 mL aliquots of the perfusate were removed at 10-minute intervals and injected under mineral oil to prevent loss of CO2. Perfusate samples were processed as below to evolve and capture the dissolved CO2.
At the end of incubation dissolved 14CO2 in the medium was estimated by incubating the media with 9M sulphuric acid for 1 hour at room temperature, and capturing the evolved 14CO2 with 2M sodium hydroxide essentially as described previously by Lopaschuk and Barr.9 Glucose oxidation was linear over the incubation period and less than 5% of the substrate was removed from the incubation medium.
An aliquot of powdered tissue was extracted in perchloric acid (0.6 M). The resulting extract was neutralized and assayed for ATP by the spectrophotometric method of Bergmeyer.12
A second aliquot of tissue powder was extracted by hydrolysis with 30% KOH, precipitation with ethanol, and enzymatic digestion with amyloglucosidase to form glucose, which was then assayed via the glucose oxidase method (Sigma).
Powdered tissue was extracted with 0.6M perchloric acid, neutralized, and then assayed as per the method described by Buttery et al.13
Lipids were extracted from the powdered tissue using the method originally described by Folch et al,14 and triglycerides were subsequently hydrolyzed enzymatically with determination of the liberated glycerol (Boehringer Mannheim triglyceride GPO-PAP kit), using a Precimat glycerol standard (Boehringer Mannheim, Germany).
Perhexiline maleate, oxfenicine, fatty acid free BSA, and trypsin were obtained from Sigma Chemical Co (St. Louis, MO); DMEM and fetal calf serum were from GibcoBRL (Grand Island, NY) and collagenase type 2 was obtained from Worthington Biochemical Corp (Lakewood, NJ). (9,10-3H)-palmitic acid, 30-60 Ci/mmol, was obtained from NEN Life Science (Boston, MA) and D(U-14C) glucose, 310 mCi/mmol, was obtained from Amersham Biosciences (Buckinghamshire, UK). Perhexiline gluconate was synthesized from perhexiline maleate by Dr. M. Campbell, Victorian College of Pharmacy, Australia.
The data are presented as mean and SEM. Effects of drug treatments on cardiac efficiency and substrate metabolism over time were analyzed by two-way repeat measures ANOVA, with Tukey-Kramer post hoc test. Multiple comparisons of hemodynamic parameters between treatments were analyzed by ANOVA with Dunnett's or Tukey-Kramer post hoc tests, and single comparisons by unpaired t test. Cell culture experiments were carried out at least in duplicate in each culture. The effect of perhexiline was estimated as % inhibition of oxidation rate in the corresponding control cultures. The effect of increasing concentrations of perhexiline was analyzed by one-way ANOVA with Dunnett's post hoc test. The effect of time of exposure was assessed in paired samples by paired t test. A critical P value of 0.05 was adopted.
Control cells oxidized palmitate at the rate of 0.73 ± 0.13 nmol. μg DNA−1.h−1(n = 7) Perhexiline exposure for 56 hours (48 hours preincubation plus 8 hours incubation with palmitate) produced a concentration-dependent inhibition of palmitate oxidation from 2 μM to 10 μM (Fig. 1). There was no significant inhibition of palmitate oxidation at 1 μM perhexiline. The effect of 2 μM perhexiline increased with the duration of preincubation, being significant for 48 hours compared with 1 hour preincubation (20 ± 4 compared with 6 ± 4% inhibition respectively, P < 0.05, paired t test, n = 6). The inhibition of palmitate oxidation was not due to decreased viability of the cells, because Trypan Blue exclusion indicated 99.5% viability and washout experiments indicated that the inhibitory effect was reversible after 24 hours washout of the drug (data not shown).
Control cells oxidized glucose at a rate of 0.17 ± 0.06 nmol. (μg DNA)−1.h−1(n = 4). The effect of 48 hours preincubation with perhexiline, 2 μM and 5 μM is shown in Figure 2. Although the inhibitory effect of 2 μM perhexiline was specific for palmitate oxidation, producing no significant effect on glucose oxidation, 5 μM perhexiline produced a significant inhibitory effect on glucose oxidation compared with control cultures (Fig. 2).
Working Heart Preparation
Cardiac efficiency was relatively stable over the 60 minutes of normal flow in isolated working rat hearts (Figs. 3A and 3B). Acute in vitro exposure to either perhexiline, 2 μM, or oxfenicine, 2 mM, had no significant effect on cardiac efficiency during the 60-minute period of normal flow (Fig. 3A). Similarly these drug treatments had no effect on other hemodynamic parameters measured (Table 1). In contrast, pretreatment with perhexiline for 24 hours resulted in a significant increase in heart rate (by 20%, P < 0.05), cardiac output (by 31%, P < 0.01), and cardiac work (by 29%, P < 0.05) without any corresponding increase in oxygen consumption compared with control hearts from pretreated animals (Table 1). These changes were reflected in an increase in cardiac efficiency of approximately 30% (P < 0.02) in hearts from perhexiline-pretreated rats relative to that in hearts from control treated rats (Fig. 3B).
Heart rates in the pretreated control rat hearts (186 ± 11 beats/min) were somewhat lower than for other subgroups, but this difference was not significant (F = 1.69, P = 0.18, one-way ANOVA). However pretreatment of rats (control or perhexiline) appeared to increase the oxygen consumption of hearts significantly when compared with those from non-pretreated rats (duration of treatment, F = 11.5, P = 0.002; drug treatment, F = 1.84, P = 0.20, two-way ANOVA).
Neither acute treatment with perhexiline in vitro, nor perhexiline pretreatment in vivo, had any significant effect on palmitate oxidation in isolated rat hearts when compared with the relative controls (Fig. 4A and Fig. 5). However a metabolic shift in substrate utilization was demonstrable with acute oxfenicine treatment in vitro, characterized by a marked, significant decrease in palmitate oxidation relative to both control and perhexiline treated hearts (Fig. 4A), and a more modest but significant increase in glucose oxidation relative to control and perhexiline treated hearts (Fig. 4B). The lack of effect of acute treatment with perhexiline was not a function of the palmitate concentration (0.4 mM) of the perfusate, because 1 hour of exposure to perhexiline 2 μM also failed to affect palmitate oxidation during normal flow in a separate group of Langendorff-perfused rat hearts exposed to 1.2 mM palmitate (control 0.92 ± 0.28 versus perhexiline 1.08 ± 0.16 μmol/g dry wt/min, n = 8).
The increased cardiac work following perhexiline pretreatment did not alter the cardiac concentrations of ATP, glycogen, lactate, or triglyceride relative to those of hearts from control pretreated animals (Table 2).
The results of the present study indicate that acute exposure to perhexiline for 1 hour at a therapeutic concentration (2 μM) does not inhibit palmitate oxidation in either isolated working rat hearts or in neonatal rat cardiomyocytes, nor does it stimulate glucose oxidation; 24 hours in vivo pretreatment with perhexiline sufficient to achieve plasma levels of 2 μM also failed to inhibit palmitate oxidation in isolated working rat hearts. In contrast pre-exposure to perhexiline (in a range of concentrations that overlaps the therapeutic plasma concentrations of the drug) for 48 hours produced a significant inhibition of palmitate oxidation in isolated rat cardiomyocytes. Thus this “metabolic” effect increased with increasing concentration and exposure time. The absence of acute effect of perhexiline on substrate selection in the working heart is in contrast to the shift from palmitate to glucose utilization observed for the selective CPT-1 inhibitor oxfenicine in parallel experiments.
In addition to effects on palmitate oxidation, a lag phase was also observed for the onset of effects of perhexiline on cardiac efficiency in working rat hearts. Pretreatment of rats for 24 hours with perhexiline increased cardiac efficiency by approximately 30% compared with controls. However, this effect was not demonstrable with acute in vitro exposure to perhexiline for 1 hour. Moreover the increased cardiac efficiency following 24 hours pretreatment with perhexiline was not attributable to a reduction in fatty acid oxidation because palmitate oxidation was unaffected in these hearts. Furthermore oxfenicine, which produced a marked decrease in palmitate oxidation and an increase in glucose oxidation, had no effect on cardiac efficiency in these non-ischemic hearts.
With respect to the time course of effects on both cardiac efficiency and metabolism, the reason(s) for a lag phase in onset is not clear, but potentially could be due to slow accumulation of perhexiline by the tissue particularly at its site(s) of action. Deschamps et al8 have proposed this slow accumulation as an explanation for the slow onset of effects on fatty acid metabolism by hepatocytes. Alternatively, effects on efficiency might also reflect some indirect action in vivo. Not all effects of perhexiline demonstrate a lag phase because we have previously demonstrated that 2 μM perhexiline exerts some acute effects in vitro including protection against diastolic dysfunction in Langendorff perfused rat hearts under conditions of ischemia/reperfusion,5 and inhibitory effects on superoxide generation by isolated neutrophils.7 In the Langendorff perfused rat heart the effect of perhexiline occurred in the absence of changes in long-chain acylcarnitine concentrations.5 We interpreted this as due to simultaneous inhibition of CPT-1 and -2. However, in light of the current data, a longer period of exposure is necessary for the metabolic effect of perhexiline to be fully manifest.
Although the lag phase for metabolic effects of perhexiline is consistent with the observations of Deschamps et al,8 who found that 5 μM perhexiline inhibited palmitate oxidation by hepatocytes after 72 hours exposure but not after acute exposure, the current data on working rat hearts are at odds with those of Jeffrey et al.6 These authors reported increased cardiac output for no change in oxygen consumption during 1 hour of in vitro exposure to 2 μM perhexiline in normoxic working rat hearts, accompanied by a switch from fatty acid to carbohydrate utilization. The reasons for the apparent discrepancy between our data and those of Jeffrey et al6 are unclear. Failure to demonstrate an acute effect of perhexiline in the current study was not a function of the palmitate concentration of the perfusate, because separate experiments in Langendorff perfused hearts with 1.2 mM palmitate also demonstrated that acute treatment with perhexiline had no effect on metabolism. Moreover the previously described metabolic effect of oxfenicine was detected under the conditions of the current experiments. Furthermore, the absence of an acute effect of perhexiline on metabolism is consistent with the lag phase on palmitate metabolism in cardiomyocytes. There are a number of methodological differences between the two studies. In the experiment of Jeffrey et al,6 neither control nor perhexiline treated hearts used exogenous glucose, increased carbohydrate utilization being accounted for by increased lactate metabolism. These experiments were conducted without the presence of insulin and at a lower glucose concentration than used in our experiments. Moreover Jeffrey et al estimated substrate utilization by NMR analysis of the hearts at the end of perfusion. This method estimates the contribution of the different 13C-labeled substrates as estimated from the distribution of 13C in glutamate at the end of perfusion. Although no direct comparison of these two methods appears to have been made, discrepant results have previously been reported between studies using these two methods of estimating palmitate utilization in the presence of trimetazidine.15,16
Recently, it has been found that perhexiline improves symptomatic status in patients with decompensated aortic stenosis17 and also in those with stable congestive heart failure, irrespective of the presence or absence of significant coronary artery disease.18 Thus the clinical effects of perhexiline extend to circumstances in which myocardial ischemia may be absent. The magnitude of the increase in cardiac efficiency by perhexiline pretreatment under normal flow conditions in the current study was quite substantial (approximately 30%), and is consistent with these clinical data. This relatively large effect on cardiac efficiency under conditions of normal flow distinguishes perhexiline from other metabolic anti-anginal drugs. Although an increase in glucose concentration has been shown to increase efficiency under aerobic conditions in one study,19 the effects of “metabolic” agents such as oxfenicine, ranolazine, and trimetazidine on efficiency have in general been manifest in ischemic models.16,20,21 Greater effects of metabolic agents might be expected in ischemia during which uncoupling of glycolysis and glucose oxidation lead to detrimental accumulation of lactate. Metabolic effects of perhexiline remain of relevance in ischemia but would be expected to occur after longer-term treatment.
Given the dissociation in the present study between metabolic effects of oxfenicine and perhexiline and changes in efficiency, alternative mechanisms should be considered. None were suggested by the results obtained. Mitochondrial uncoupling proteins (UCPs) may decrease efficiency of cardiac ATP production in normoxic myocardium.22 In addition, endogenous peroxynitrite formation has been shown to limit cardiac efficiency in isolated working rat hearts even under conditions of normal flow.23 However, whether perhexiline can alter levels of UCPs or peroxynitrite in myocardium is unknown. Alternatively, there is some evidence to suggest that calcium sensitization at the level of the troponin/tropomyosin complex may modulate cardiac efficiency.24 Perhexiline, in high concentrations, has been shown to increase calcium sensitivity in cardiac myofibrils,25 and to increase calcium binding to troponin C in skinned skeletal muscle.26 However, there is no information available to indicate whether it has a similar effect on intact cardiac muscle at lower concentrations.
Neonatal cardiomyoctes were used in the isolated cell studies. Although neonatal cardiomyocytes have been shown to express the hepatic isoform of CPT-1,27 and therefore are not identical to adult cardiomyocytes, we have shown previously that perhexiline inhibits the hepatic isoform of CPT-1 in a manner similar to that of the muscle isoform but is a little less potent on the former.4
At 2 μM, perhexiline appeared to inhibit palmitate oxidation in cardiomyocytes without any secondary stimulant effect on glucose oxidation. In light of the smaller numbers of cultures used for glucose oxidation it is possible that a small effect of 2 μM perhexiline on glucose oxidation may have gone undetected. However, 5 μM perhexiline significantly inhibited glucose oxidation to approximately the same degree as it inhibited palmitate oxidation. This lack of specificity with increasing concentration may be related to the known calcium l-channel blocking effect of the drug at higher concentrations, which has been demonstrated in chicken embryonic cardiomyocytes.28 Consistent with this possibility, we have previously observed a small negative inotropic effect of perhexiline at 5 μM in isolated rat hearts (unpublished).
Control rats in the pretreated group tended to have somewhat lower heart rates than in other subgroups, while hearts from both pretreated groups had higher oxygen consumption than those that were not pretreated. This might in theory reflect the impact of prior anesthesia and/or the vehicle used for percutaneous application of perhexiline. While these results were unexpected, they do not invalidate the comparison between the two pretreated groups.
In conclusion, in the non-ischemic working rat heart, perhexiline increases myocardial efficiency by a mechanism(s) that is largely, or entirely, independent of its effects on CPT. The study results also suggest that experiments in normoxic myocardium may fail to demonstrate the effects of “metabolic” anti-anginal agents on cardiac efficiency. Further studies are required to compare the effects of perhexiline with those of agents such as oxfenicine as regards myocardial efficiency and metabolism in severe global ischemia, a condition under which the metabolic effect of these agents may become more important.
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