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Myocardial Cell Damage by Fatty Acid Ethyl Esters

Bora, Puran; Farrar, Melody; Miller, D.; Chaitman, Bernard; Guruge, Bandula

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Journal of Cardiovascular Pharmacology: January 1996 - Volume 27 - Issue 1 - p 1-6
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Abstract

Alcohol-induced heart disease is common in the United States and is similar to idiopathic cardiomyopathies, with the defining difference being the chronic consumption of ethanol (1). The disease causes arrhythmias (2), cardiomegaly, and congestive heart failure (CHF) (3-6).

The chronic administration of ethanol to laboratory animals (7,8) and alcohol abuse by humans (9,10) leads to gross morphological changes in myocardium. Ultrastructural studies show changes in mitochondria, including swelling, disruption of the cristae, and the formation of dense inclusion bodies (9-12). Ethanol consumption leads to an increase in the basal metabolic rate of some subjects, demonstrated by a 5-7% change in O2 consumption (13), which could reflect an alteration in mitochondrial function. These changes include diminished phosphate/O2 ratios and respiratory control ratios (14-17). The lack of any demonstrable myocardial metabolism of ethanol (18,19) makes it difficult to establish a link between exposure to ethanol and the observed metabolic changes.

Fatty acid ethyl esters (FAEE), metabolic products of ethanol, have been detected in concentrations as high as 115 μM in human hearts obtained at autopsy of persons who were either acutely intoxicated or were chronic ethanol abusers (20). FAEE are synthesized [biological half-life (t½) 20-24 h] and accumulated nonoxidatively at high rates in myocardium and are potentially toxic (21). FAEE synthase, the enzyme or enzymes that catalyze synthesis of these esters, exists in three forms in human myocardium (22-28). In salt gradient elution, these three forms elute from diethylamino ethyl (DEAE)-cellulose at conductivities of 5, 7, and 11 ms and are designated synthase-I, synthase-II, and synthase-III, respectively. All three synthases (I, II, and III) have been purified to homogeneity from human myocardium (22-28).

FAEE were shown to bind to mitochondria in vivo and to cause a concentration-dependent reduction in the respiratory control ratio index of oxidative phosphorylation (21). These FAEE could be the metabolic link between ethanol abuse and changes in mitochondrial function. Therefore, we investigated the effects of FAEE on rat myocardium.

MATERIALS AND METHODS

All reagents were of the highest grade commercially available. Oleic acid ethyl ester and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical, St. Louis, MO, U.S.A. Radiolabeled compounds were purchased from Amersham, Arlington Heights, IL, U.S.A.

Preparation of mitochondria

Fresh rat hearts were used to isolate mitochondria by a standard method (22). Rats were killed by cervical dislocation, and their ventricles were removed and placed in homogenizing medium (HM): 0.25 M sucrose, 10 mM EDTA, and 0.1% bovine serum albumin (BSA) at 0 °-4 °C. The mitochondria were isolated as rapidly as possible. The ventricles were homogenized with a polytron. The suspension was centrifuged at 2,500 g for 10 min. The fat on the surface of supernatant was removed with a cotton swab, the pellet was discarded, and the supernatant fraction was centrifuged at 9,000 g for 10 min. The resulting mitochondria pellet was washed three times with 30 ml HM each and repelleted at 9,000 g for 10 min. The final pellet was gently resuspended in 2 ml HM.

The mitochondrial preparation was characterized microscopically and enzymatically. Electron microscopy of the final mitochondrial pellet after fixation, staining, and processing as described previously (22) demonstrated that in low-power views (×6,000), essentially all formed elements were mitochondria. Only a small amount of disrupted mitochondrial components was present. Assay of an aliquot of mitochondria showed almost no enzymatic activity for cleavage of cholesterol [14C]oleate (lysosomal marker) or synthesis of triglyceride from [14C]oleoyl coenzyme A (microsomal marker).

Determination of lipids from mitochondria

To examine the effect of ethyl oleate on isolated mitochondria, incubations of ethyl [3H] oleate and isolated mitochondria were performed at 37 °C in the medium containing pyruvate 21 mM, malate 21 mM, and ADP 350 μM, to ensure stability of the mitochondria. Control incubations were performed without ethyl esters. After preincubation for 15-60 min, suspensions containing isolated mitochondria with or without ethyl ester were centrifuged at 9,000 g for 10 min at 4 °C. The remittant mitochondrial pellet was resuspended in fresh incubation medium. The resuspended mitochondria were extracted into lipid solvents, and the amount of bound ethyl [3H]oleate and [3H]oleate per milligram of mitochondrial protein was determined after thin-layer chromatography (TLC) on silica gel was developed with petroleum ether/diethyl ether/acetic acid (75:5:1) (22). Mitochondria incubated with ethyl [3H]oleate vesicles were extracted before centrifugation. The amount of [3H]oleate released was quantified and expressed as nanomoles of [3H]oleate release per minute per milligram of protein. Recoveries of 65-70% were corrected for extraction, and chromatographic losses were assessed in parallel procedures with labeled oleate ranging from 30 to 35%.

In vivo effect of FAEE

To demonstrate the in vivo effect of FAEE we performed thoracotomies and injected rats with an oleic acid ethyl ester (OAEE) solution directly into the left ventricular myocardium using a 28-gauge needle. The rats received injections of 30 and 50 μl OAEE. Control rats (n = 12) received injections of the same solution without the OAEE in the same volumes.

After 2 days (n = 10), 4 days (n = 35), and 30 days (n = 10), the rats were killed. Each rat received an overdose (twice normal) of anesthetic cocktail (35 mg/kg ketamine HCl, 4 mg/kg xylazine, and 0.1 mg/kg acepromazine in intramuscular injections). The chest cavity was opened, and the heart was removed while still beating. A 2-mm cross-section of the ventricles was placed in 10% buffered formalin (Baxter S/P) for histological staining with Masson trichrome.

Histology

A 2-mm cross-section of the rat heart was fixed in 10% buffered formalin for a minimum of 24 h. The section was then embedded in paraffin and stained according to standard procedures with Masson trichrome. This stain differentiates between normal cardiac muscle, fibrous connective tissue, and damaged myocardium.

RESULTS

Ethyl oleate binding to mitochondria

Binding of ethyl [3H]oleate to mitochondria increased linearly with time (Fig. 1) with 9 μM ethyl oleate bound/1mg mitochondrial protein after exposure for 60 min. Therefore, the results of in vitro experiments demonstrated that [3H]oleate derived from ethyl [3H]oleate was bound to mitochondria.

Hydrolysis of ethyl oleate by mitochondria

The hydrolysis of ethyl oleate to free fatty acid (FFA) by mitochondria was further evaluated in vitro. Incubation of isolated mitochondria (1 mg/ml) with ethyl [3H]oleate 60 μM, at 37 °C resulted in release of [3H] oleate into the incubation medium (Fig. 2). The rate of release was linear as a function of time for 120 min. In the absence of added mitochondria, very little [3H]oleate formed (Fig. 2). These results demonstrated that the mitochondria may have an esterase that can hydrolyze ethyl oleate at concentrations of FAEE attained in vivo and at rates sufficient to generate micromolar concentrations of FFA (29,30).

Bound mitochondrial oleate and ethyl oleate

To determine the amount of ethyl oleate that had actually entered the mitochondria, 50 μl of 50 μM ethyl [3H]oleate was injected in rat myocardium by the procedure described in the Materials and Methods section. After 12 h of ethyl [3H]oleate injection, the mitochondria of the myocardium were isolated and extracted into lipid solvents; the amount of bound ethyl [3H]oleate and [3H]oleate to mitochondria was determined by TLC. Of the total ethyl oleate injected in the myocardium, 8 μM [3H]oleate and 1 μM ethyl [3H]oleate was bound to mitochondria.

Light microscopy

Histopathological changes induced by FAEE were investigated by injecting low concentrations of cold FAEE in the rat myocardium. Control myocardium (injected with 50 μl of 0.01% DMSO + 0.1% methylene blue dye) showed no damage; however, the myocardium 4 and 30 days after injection of OAEE showed significant cell damage (Fig. 3A-C and Table 1). The cell damage was greatly increased on day 30, with the cells showing gross deformation and enlargement (Fig. 3D and Table 1). The area of cell damage was slightly larger in the myocardium that received the 50-μl injection as compared with those which only 30 μl (Table 1). Myocardium harvested 2 days after OAEE injection showed very little cell damage (Fig. 3B and Table 1).

DISCUSSION

We demonstrated that FAEE can cause cell damage to myocardium without resulting in subsequent fibrosis. The dysfunction arises instead from mitochondrial damage. Human hearts exposed to chronic alcohol consumption or exposed to large quantities of ethanol acutely have been shown to accumulate ≤115 μM FAEE (20,25). Our results show that ethyl oleate binds to rat mitochondria, which can hydrolyze the ester to form oleate. Uncoupling of oxidative phosphorylation has been shown to correlate with binding of ethyl oleate to mitochondria and with hydrolysis of the ester by mitochondria to form oleate, a known uncoupler of oxidative phosphorylation in concentrations as low as 5 μM(21,31,32). Incubation of rabbit myocardial mitochondria with ethyl oleate, which had previously been added directly to the incubation medium in concentrations ≤50 μM without incorporation into vesicles, resulted in a significant uncoupling of oxidative phosphorylation (21). FAEE have been suggested to act in a toxic fatty acid shuttle to induce mitochondrial dysfunction in vivo after prolonged alcohol abuse (21).

Light microscopic changes in mitochondrial structure and function have been documented in myocardium from animals chronically administered alcohol and in alcoholic humans (7-10). To demonstrate the damage caused by low concentrations of FAEE, we injected 30 μl and 50 μl of 50 μM OAEE directly in rat myocardium. Cellular and mitochondrial damage was evident in the myocardium of experimental animals. Four and 30 days after injection of the OAEE, the damage was evident in the myocardium histologically as swollen and deformed cells. Because the t½ of FAEE is ≤24 h, the myocardial cell damage observed on day 4 may be due to a toxic effect of FAEEs however, progressive cell damage on day 30 may be cytotoxicity caused by myocardial cell damage. Although histopathologically some fibrosis was associated with the needle injury, this was common to both the experimental and control animals. However, cellular damage was not common. Dye (methylene blue 0.1%) was used to identify the injection sites, thus assuring us that any procedural damage observed could be differentiated from damage due to the injection of FAEE. When ethyl [3H]oleate was injected in the myocardium, only 8 μM oleate and 1 μM ethyl oleate was recovered from mitochondria; the rest of the ethyl oleate injected was probably hydrolyzed in the cytosol before entering the mitochondria. That 8 μM of [3H]oleate was bound to mitochondria suggests that ethyl [3H]oleate was hydrolyzed in the mitochondria to form fatty acid.

Our study further shows that FAEE can cause myocardial cell damage and that the effectiveness of our in vivo model provides another means to study the physiological and morphological effects of FAEE in the myocardium. Because as much as 115 μM FAEE can be accumulated in the myocardium of alcoholics over several years (20), our results indicate that the micromolar concentration of FAEE (which relates to the amount of FAEE accumulated in the myocardium of alcoholics) can cause myocardial cell damage. To our knowledge, ours is the first report to document the in vivo effect of FAEE (myocardial metabolites of ethanol) in the myocardium of any species. Relating ethanol abuse to cardiac disease has been difficult owing to the lack of an intermediary metabolic link. Our results demonstrate that FAEE may be a link between ethanol abuse and cardiac cell damage.

Acknowledgment: This work was supported by an NIH grant and a grant from the Alcoholic Beverage Medical Research Foundation. We thank Dr. Nalini Bora for critical review of the manuscript and Mary Streif and Jennifer Marlotte for typing the manuscript.

FIG. 1
FIG. 1:
. Ethyl (3H)oleate binding to isolated mitochondria. Mitochondria (1 mg/ml) were incubated with ethyl [3H]oleate (60 μM, 600 dpm/nmol). At the intervals indicated, the mitochondria were sedimented at 9,000 g for 10 min at 4 °C and washed three times with incubation medium (until no background radioactivity appeared in the last wash). They were then extracted with acetone, the lipids were thin-layer chromatographed on silica gel, and developed with petroleum ether/diethyl ether/acetic acid (75:5:1) (23), and the amount of labeled ethyl [3H]oleate or derived [3H]oleate was quantified by liquid scintillation spectrometry with yields corrected for recovery of total radioactivity. Curve points represent data from three independent experiments with triplicate determinations ± SE.
FIG. 2
FIG. 2:
. Hydrolysis of ethyl [3H]oleate by rat mitochondria. Mitochondria (1 mg/ml) were incubated with ethyl [3H]oleate 60 μM at 25 °C (top curve) and aliquots of the suspension were removed at the indicated times. [3H]oleate in these aliquots was quantified as described in the legend to Fig. 1. Control incubations (bottom curve) omitted mitochondria. Curve points (top and bottom) represent data from three independent experiments with triplicate determinations ± SE.
FIG. 3. A:
FIG. 3. A::
Histopathological analyses of control rat myocardium injected with 50 μl phosphate-buffered saline (PBS) + 0.01% dimethylsulfoxide. Sections stained with Masson trichrome. No cell damage is observed. (Original magnification ×400.) B: Histopathological analyses of rat myocardium injected with 50 μl 50 μM fatty acid ethyl ester (FAEE). Sections stained with Masson trichrome. The cell damage after 2 days is shown by light pink stain and by arrow. (Original magnification ×300.) C: Histopathological analyses of rat myocardium injected with 50 μl 50 μM FAEE. Sections stained with Masson trichrome. The significant cell damage after 4 days is shown by light pink stain. (Original magnification ×400.) D: Histopathological analyses of rat myocardium injected with 50 μl 50 μM FAEE. Sections stained with Masson trichrome. The significant cell damage after 30 days is shown by light pink stain. The cells are enlarged and irregular in shape (Original magnification ×400.)

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Keywords:

Fatty acid ethyl esters; Mitochondria; Uncoupling; Oleic acid; Myocardium

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