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Cardiovascular Research 1998 37(1):141-150; doi:10.1016/S0008-6363(97)00198-3
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Mitochondrial gene expression is impaired by ethanol exposure in cultured chick cardiac myocytes

John M Kennedy*

The University of Illinois at Chicago, Department of Physiology and Biophysics, Mail Code 901, 835 S. Wolcott Ave., Chicago, IL 60612-7342, USA

* Tel.: +1 312 9967992; Fax: +1 312 9961414; E-mail: jkennedy@uic.edu

Received 27 January 1997; accepted 18 June 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: A depression in cytochrome c oxidase (COX) activity occurs following chronic embryonic ethanol exposure in vivo. The aim of this study was to examine the effect of chronic ethanol exposure on COX activity in isolated cardiac cells maintained in vitro. Additionally, the mechanism by which ethanol produces an impairment in COX activity was evaluated by examining mitochondrial gene expression. Methods: Spontaneously beating cardiac myocyte cultures were established from 10-day embryonic chick hearts. Various concentrations of ethanol (0–250 mM) were introduced at the time of plating and cells were harvested over 7 days. COX activity was determined in myocyte homogenates. The levels of nuclear-encoded (COXIV) and mitochondrial-encoded (COXII) subunit proteins were measured by Western blotting. Relative levels of mitochondrial DNA and the mitochondrially-encoded COXIII mRNA were determined by Southern and Northern blotting. Results: A consistent decrease in COX activity in ethanol-exposed cardiac myocytes of approximately 30% was observed with an ethanol concentration of 25 mM. Increasing the ethanol concentration to 250 mM produced only a minor enhancement of this effect, while severely decreasing cellular viability. The content of the mitochondrially-encoded COXII subunit was reduced by ethanol exposure, while that of the nuclear-encoded COXIV subunit was unchanged. The content of the mitochondrially-encoded COXIII mRNA was unchanged by ethanol exposure. However, prolonged ethanol exposure produced an increase in mitochondrial DNA levels in cardiac myocytes. Conclusions: Ethanol exposure of cardiac myocytes produces deficits in COX activity in the absence of systemic variables, indicating that ethanol has a direct effect on cardiac mitochondria. The ethanol-induced decrease in COX activity is correlated with a specific decrease in at least one mitochondrially-encoded gene product, COXII. No changes were observed in the level of the nuclear-encoded COXIV subunit, indicating that expression of this nuclear-encoded gene is not impaired by ethanol exposure.

KEYWORDS Ethanol; Heart; Mitochondria; Myocyte; Culture; Cytochrome oxidase


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Prenatal ethanol exposure has been shown to produce a plethora of abnormalities affecting human growth and development which collectively constitute the fetal alcohol syndrome (FAS) [1, 2]. In addition to the characteristic features of growth retardation, facial dysmorphology, and central nervous system impairment which define FAS [2], literature surveys have revealed that almost half of all FAS cases suffer some degree of heart defect [3–5]. In particular, congenital malformations such as septal defects [5], ultrastructural alterations such as myofibrillar dysgenesis [6], and abnormal EKGs [3]have been reported to occur as a result of fetal alcohol exposure in humans. The contractile force generated by isolated human fetal cardiac papillary muscle is also reduced by exposure to ethanol [7], suggesting that cardiac performance in the ethanol-exposed fetus may be seriously impaired and may contribute to the presentation of congestive heart failure in FAS children [3].

A more detailed examination of the effects of ethanol on contractile mechanisms in embryonic hearts has been conducted using animal models of embryonic ethanol exposure. Acute exposure of fetal lamb hearts to physiologic ethanol concentrations produces a reduction in myocyte shortening velocity [8]and a lengthening of the isometric phase of ventricular contraction [9], indicating that fetal cardiac contractility is impaired immediately following ethanol exposure. Likewise, cardiac contractility [10], stroke volume, end diastolic volume, and cardiac output [11]are depressed in a dose- and time-dependent manner in embryonic chicks exposed to ethanol. Studies utilizing cultured cardiac myocytes have demonstrated an ethanol-related inhibition of growth and division of cardiac myocytes [12, 13]which is accompanied by a disruption in the organization of myofilaments [12]and a specific reduction in the content of sarcomeric proteins such as myosin, actin, and {alpha}-actinin [13]. Furthermore, chronic ethanol exposure in adult rats has been shown to cause a decrease in the synthetic rate of myofibrillar proteins [14], suggesting that the synthesis and/or assembly of sarcomeres may be impaired in cardiac cells chronically exposed to ethanol.

Abnormalities in mitochondrial enzyme activity [15–18]and ultrastructure [19–21]are common features in embryonic heart cells exposed to ethanol. Similarly, mitochondria have long been recognized as targets of ethanol's adverse effects in the liver [22–24]. The effect of ethanol on cytochrome c oxidase (COX) has received particular attention [15, 17, 24, 25]since this enzyme dictates the aerobic capacity of a tissue [26, 27]. In vertebrates the COX enzyme is a multi-subunit complex composed of three large catalytic subunits encoded in the mitochondrial genome and up to ten smaller regulatory subunits encoded in the nuclear genome [26, 27]. As a consequence of this unique genetic structure, the effective regulation of COX gene expression requires a coordination of events controlling the expression of both nuclear-encoded and mitochondrial-encoded genes. Consequently, an understanding of ethanol's effects on mitochondrial biogenesis must also consider the dual nature of mitochondrial genetics.

The mechanisms responsible for ethanol-induced changes in COX activity have not been fully delineated. Ethanol exposure does not change the kinetic characteristics of COX activity in submitochondrial particles isolated from the liver's of ethanol-fed adult rats, but does reduce the total COX activity per unit membrane [30]. Thus the catalytic properties of COX are unchanged, while the COX content is reduced by chronic ethanol exposure. Moreover, ethanol-induced decreases in liver COX activity can be correlated with decreases in the synthesis of COX subunits encoded in the mitochondrial genome [23, 28], suggesting that ethanol reduces the COX content by somehow altering expression of mitochondrial proteins. Similarly, mitochondrial protein synthesis is reduced in the hearts of adult rats chronically exposed to ethanol [31, 32]. However, no changes occur in the steady state levels of mitochondrial-encoded mRNAs in adult rat livers chronically exposed to ethanol [28]. Likewise, ethanol-induced decreases in COX activity in the embryonic chick heart are independent of changes in either mitochondrial DNA or mitochondrial mRNA levels [17]. These studies suggest that ethanol causes decreases in COX activity by decreasing the content of COX subunits encoded in the mitochondrial genome by a post-transcriptional mechanism. However, neither the synthesis of nuclear-encoded COX subunits nor the expression of nuclear-encoded COX mRNAs were examined in these studies. Therefore, it is not known whether ethanol exposure causes a coordinated decrease in COX subunits encoded in separate genomes or influences the transcription of nuclear-encoded COX genes.

The first objective of the present study was to demonstrate an ethanol-induced deficit in COX activity in cultured embryonic cardiac myocytes in order to provide evidence that there is a direct link between ethanol exposure and mitochondrial dysfunction. Although ultrastructural damage to mitochondria in cultured cardiac myocytes [21]or hepG2 cells [33]has been demonstrated, no changes in mitochondrial function or enzyme activity have been reported in cultured cells as a result of ethanol exposure [34]. Secondly, experiments were conducted to examine mitochondrial gene expression. Steady-state levels of COX subunits encoded in the nuclear and mitochondrial genome were examined to determine if ethanol produced coordinated changes in mitochondrial gene expression. The effect of ethanol exposure on the expression of nuclear-encoded COX subunits has not been previously examined. Finally, mitochondrial DNA content and COXIII mRNA levels were measured in order to determine if observed effects on mitochondrial gene expression were organized at the level of mitochondrial DNA replication or transcription. Results presented in this report indicate that an ethanol-induced impairment in mitochondrial gene expression occurs in cultured cardiac myocytes which is independent of both mitochondrial transcription and expression of a nuclear-encoded COX gene.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cardiac myocyte cultures
Embryonic chick cardiac myocyte cultures were established according to methods described by Clark and Zak [35]. Hearts were obtained by sterile dissection from 10-day chick embryos. Individual cardiac myocytes were dissociated by mincing and trypsinization (0.25 mg/ml trypsin for 30 min at 37°C) in phosphate-buffered saline (PBS; 2.7 mM KCl, 1.5 mM potassium phosphate monobasic, 137 mM NaCl, 8.1 mM sodium phosphate dibasic, pH 7.5). Trypsinization was terminated by the addition of an equal volume of horse serum. A suspension of isolated cardiac myocytes was obtained following three successive rounds of pelleting and trituration in serum-free culture media (a 1:1 mixture of F-12 nutrient mixture and Dulbecco's modified Eagle's medium). Between 4x106 and 5x106 cells were plated onto 60-mm Falcon Primaria culture dishes (Becton Dickinson, Lincoln Park, NJ) in the presence or absence of ethanol (0–250 mM) and incubated at 37°C in a 5% CO2 atmosphere. All animal procedures were performed in accordance to protocols published by the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 85-23, revised 1985). All media and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

2.2 Ethanol treatment of cultures
Cardiac myocytes were exposed to ethanol from the time they were initially plated (in most cases) to the end of the experimental time course. Over the course of 24 h, media ethanol concentrations were reduced by approximately 60% from culture dishes treated with 50, 100, or 250 mM ethanol (data not shown). Therefore, cultures were fed each day with ethanol-containing media in order to re-establish the peak ethanol concentration. This model of ethanol treatment produced fluctuating levels of ethanol similar to that reported by Ni et al. [13]. Similar results (not shown) to those reported here were obtained if ethanol concentrations were held constant by culturing cardiac myocytes in an enclosed chamber system similar to the model described by Adickes et al. [36]. A model producing fluctuating ethanol levels may more closely mimic the type of exposure experienced in utero by fetal heart muscle cells following maternal binge drinking associated with the fetal alcohol syndrome and was chosen as the model system in the present experiments.

2.3 Biochemical assays
Cardiac myocytes were scraped from culture dishes in ice-cold PBS, pelleted by centrifugation, and homogenized in 0.01 M potassium phosphate buffer (pH 7.4) containing 0.5% Tween-20. Cytochrome c oxidase activity in homogenates was determined as described previously [37, 38]. The protein concentration of homogenates was determined by standard methods [39]and used to normalize COX activity, equalize the loading of gel electrophoresis samples, and determine the total protein per dish.

2.4 Immunocytochemistry
Immunocytochemical procedures were performed in order to examine the possibility that ethanol treatment altered the proportion of non-muscle cells present in the cultures. Glass coverslips were placed in culture dishes and cells were plated onto them as described above. After rinsing with PBS, cells attached to coverslips were processed for immunocytochemistry by successive 5-min treatments at 4°C with: 4% paraformaldehyde, methanol, acetone/methanol (1:1), 70% ethanol, and finally rinsed again with PBS. Myocytes were identified with an antibody which reacts with all chicken sarcomeric myosin heavy chains (Aby1) and a rhodamine-conjugated anti-rat secondary antibody as described previously [40]. DAPI (4',6-diamidino-2-phenylindole) was added along with the secondary antibody at a concentration of 5 µM in order to counter-stain nuclei of both myocytes and non-muscle cells. The proportion of non-muscle cells was determined as the fraction of DAPI-positive, MHC-negative cells. In each experiment counts were made from three separate coverslips and from six random microscopic fields per coverslip (between 1 and 25 cells were typically examined per field) in order to establish the non-muscle cell fraction for each ethanol condition tested.

2.5 COX subunit levels
The relative level of the mitochondrially-encoded COXII subunit and the nuclear-encoded COXIV subunit were determined by densitometric analysis of Western blots. A monoclonal antibody specific for the COXIV subunit and a polyclonal antibody against the COXII subunit were kindly provided by Dr. Roderick A. Capaldi (University of Oregon). Aliquots of cellular homogenates containing identical amounts of protein were subjected to denaturing gel electrophoresis [41–43]in 0.75-mm thick polyacrylamide gels (4% stacking gel; 12% running gel) using a Mini-Protean II gel electrophoresis apparatus (Bio-Rad Laboratories) at 200 V. Separated proteins were transferred to 0.45 nitrocellulose sheets by electroblotting [44]in a buffer containing 25 mM Tris–HCl (pH 8.3), 0.2 M glycine, and 20% methanol. Immunodetection of nitrocellulose-bound COX subunits was performed using anti-COX subunit primary antibodies and reactive bands were visualized by utilization of alkaline phosphatase-conjugated secondary antibodies and a 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium solution (Gibco BRL, Gaithersburg, MD) for the color reaction [45]. The relative intensity of individual bands was measured by densitometry (Ephortec densitometer, Joyce Loebl) and compared to values obtained from a standard curve of a control sample in order to determine the relative level of subunit expression. Densitometric measurements for individual samples were only accepted if the values obtained fell within the linear range of the standard curve and data was expressed as arbitrary scan units.

2.6 Mitochondrial DNA and mRNA levels
Prior to DNA extraction, myocyte cultures were washed extensively with ice-cold Tris-buffered saline (25 mM Tris–HCl, 137 mM NaCl, 2.5 mM KCl, and 0.015 mg/ml phenol red, pH 7.4), scraped from the culture dish, and collected by centrifugation at 1500xg. Cells from 5 culture dishes were pooled for each sample. Cells were resuspended in TE-buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). Total cellular DNA was isolated from this suspension by successive steps of RNAse digestion, proteinase K digestion, organic extraction, and ethanol precipitation as described previously [17, 37, 42]. The final DNA pellet was dissolved in TE-buffer and stored at –80°C. Total cellular RNA was extracted directly from culture dishes. Cells from 4 culture dishes were pooled for each sample. Cells were washed extensively with ice-cold PBS and RNA was extracted by the single-step acid guanidium thiocyanate–phenol–chloroform procedure [17, 37, 46]. The concentration of RNA and DNA in samples was determined by spectrophotometry at 260 nm.

A cDNA insert encoding 654 base pairs of the chicken COXIII mitochondrial gene was isolated from pCO3.10 by treatment with PstI restriction endonuclease in order to generate a probe for hybridization analysis. This cDNA probe is used to measure both mitochondrial DNA and COXIII mRNA levels by Northern, Southern, and slot blotting techniques described in detail previously [17, 37, 47]. RNA and DNA samples were size fractionated by electrophoresis and transferred or serially diluted and directly loaded onto Zeta-probe nitrocellulose membranes (Bio-Rad, Richmond CA). The probe was labeled to a high specific activity using a random primers labeling system (Gibco-BRL) and [{alpha}-32P]dATP (Amersham). Hybridization and washing conditions were as described previously [17, 37, 47]. Autoradiography was performed at –80°C using Kodak XRP-5 film. The relative hybridization intensity was determined by densitometry of autoradiograms. For quantitative slot blots, data were normalized to densitometric measurements of nucleic acid staining intensity with methylene blue (for mitochondrial DNA) or the hybridization intensity of a cDNA probe for the rat 18S rRNA (for COXIII mRNA) to correct for any errors in sample loading.

2.7 Statistical analysis
Data are expressed as the mean±standard error and analyzed for statistical significance using a two-way ANOVA and Neuman–Keuls post-hoc test. Differences between control and ethanol-treated conditions were accepted as statistically significant if P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Cytochrome c oxidase activity
The effect of 3 days of ethanol exposure on cytochrome c oxidase activity was evaluated in 7 independent cell culture experiments using different ethanol concentrations. Data from a single representative experiment is shown in Fig. 1A. A statistical analysis was performed on pooled data from all experiments. A concentration-dependent decrease in COX activity was observed. There were no significant differences observed with ethanol concentrations of 5 mM, 10 mM, or 15 mM. No attempts were made to determine if longer exposure times would produce decreases in COX activity with these lower ethanol concentrations. Treatment with 25 mM ethanol produced a significant reduction in COX activity to 72.8±2.9% of control values. Treatment with higher ethanol concentrations produced little or no additional reduction in COX activity. COX activity was reduced to 75.3±6.3%, 72.3±2.9%, 70.9±5.6%, 70.0±4.8%, 70.1±1.4%, or 56.8±5.4% of control (0 mM) values following 3 days of exposure to 25, 50, 75, 100, 150, 200, or 250 mM ethanol, respectively (P<0.05).


Figure 1
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  		Fig. 1 Concentration-dependent effect of ethanol on COX activity and protein content in cultured cardiac myocytes. Embryonic chick cardiac myocytes were cultured in the absence or presence of ethanol (0–250 mM) and analyzed on day 3. COX activity (A) is reported in nmol cytochrome c oxidized/min per mg protein, and the protein recovery is reported as the total cellular protein in µg per culture dish. Data are presented as the mean±s.e. from n = 4 culture dishes per treatment. *Significant decrease (P<0.05) compared to 0 mM control culture dishes for this particular experiment.

 
Ethanol exposure also resulted in a reduction in the protein content of cultures. However, higher concentrations of ethanol were required to produce deficits in the protein content. Data in Fig. 1B is from the same representative experiment as shown in Fig. 1A in which a significant decrease in protein content was seen with 250 mM ethanol only. Statistical analysis of pooled data from all experiments revealed significant decreases in protein content with the 4 highest ethanol concentrations tested. Protein content was reduced to 77.5±2.5%, 82.1±13.3%, 70.0±1.4%, or 59.0±3.4% of control (0 mM) values following exposure to 100 mM, 150 mM, 200 mM, or 250 mM, respectively (P<0.05). No attempt was made to count the number of cells per dish. Therefore, no determination can be made as to whether the changes in protein content of cultures was due to changes in cell number or cell size.

Ethanol-induced changes in COX activity and protein recovery were also examined over a 7-day time course. Results from a representative experiment are shown in Fig. 2. Although the magnitude and timing of the changes varied in the 5 independent culture experiments used to assess time course effects, the pattern was very similar to that depicted in Fig. 2. The COX activity in dissociated cardiac myocytes (Fig. 2A, time 0) was similar to that reported for 11 day embryonic chick ventricular muscle [17]. However, during the initial 48 h in culture the COX activity decreased rapidly to values only 30% of that measured in dissociated cardiac cells on day 0. Over the next 4 days in culture, COX activity in control cardiac myocyte cultures increased to attain levels similar to the initial time point. Statistical analysis of data from all time course experiments indicated that a significant reduction in COX activity was seen by day 2 in culture with 75 mM ethanol, and this deficit was maintained throughout the 7-day time course examined. COX activity was reduced to 74.6±1.6%, 68.9±3.1%, 72.1±2.7%, 64.9±7.6%, or 74.4±3.1% of control (0 mM) values by exposure to 75 mM ethanol for 2, 3, 4, 5, 6, or 7 days, respectively (P<0.05). In control cultures maintained for a prolonged period, COX activity decreased as the viability of the cultures began to deteriorate.


Figure 2
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  		Fig. 2 Time course effect of ethanol on COX activity and protein content in cultured cardiac myocytes. Embryonic chick cardiac myocytes were cultured in the absence or presence of ethanol (75 mM) and the COX activity (A) and protein content (B) were determined as described in Section 2over a 7-day time course in culture. Prior to plating, aliquots of dissociated cardiac cells were assayed to represent the 0 time point. Both COX activity and protein content decreased dramatically over the initial 24 h in culture. Statistical analysis of this particular experiment demonstrated significant differences (*P<0.05) in COX activity between control and ethanol-treated cultures starting on day 2 of culture, while significant differences in the protein content were not observed until day 4 of the culture (n = 4 culture dishes for each treatment at each time point).

 
The effect of 75 mM ethanol on the protein content was also examined over a 7-day time course. Data shown in Fig. 2B is from the same representative experiment depicted in Fig. 2A. Significant differences were observed by 4 days in culture with 75 mM ethanol. The protein content was reduced to 88.4±1.1%, 84.8±2.1%, 81.4±2.3%, or 79.5±2.2% of control (0 mM) values by exposure to 75 mM ethanol for 4, 5, 6, or 7 days, respectively (P<0.05). Thus, while effects on the protein content were seen only with ethanol concentrations of 100 mM or greater on day 3 of culture (Fig. 1B), longer exposure times may produce deficits in the protein content with lower ethanol concentrations.

Since the COX activity of 0 mM control cardiac myocyte cultures were shown to vary with the time in culture (Fig. 2A), an additional set of experiments was conducted to evaluate the effect of ethanol exposure initiated at different time points during culture. Results from a representative experiment are shown in Fig. 3. In these experiments ethanol exposure (50 mM) was initiated either on day 0, day 1, day 2, or day 3 and COX activity was measured in control and ethanol-exposed cultures 3, 4, or 5 days after the initiation of ethanol exposure (only results from 4 days of exposure are shown in Fig. 3). Ethanol exposure produced deficits in COX activity which were independent of the time point at which ethanol exposure was initiated. In addition, the magnitude of this effect was similar for each group and was independent of the magnitude of COX activity at the initiation of exposure. The COX activity of cultures treated with 50 mM ethanol for 4 days was reduced to 71.6±1.3%, 73.1±3.6%, 75.6±1.4%, or 69.3±2.1% of control (0 mM) values when ethanol exposure was initiated on day 0, day 1, day 2, or day 3, respectively (P<0.05). Thus, the effects of ethanol exposure override the time course effects shown in Fig. 2 for control cultures.


Figure 3
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  		Fig. 3 Ethanol effects on COX activity are indepent of the time point at which ethanol administration is administered. Cardiac myocyte cultures were established and ethanol treatment (50 mM) was initiated on either day 0 (as in all other experiments described), day 1, day 2, or day 3. COX activity was analyzed 4 days after the initiation of ethanol treatment. Data are presented as the mean±s.e. for n = 4 culture dishes for each treatment group. *Significant difference (P<0.05) in this particular experiment between ethanol-treated and 0 mM control groups of the same age in culture.

 
In order to ascertain that the effect of ethanol represents a direct action of the drug on cardiac myocytes it was important to establish that ethanol did not produce a change in the proportion of non-muscle cells and that there were no density dependent differences in COX activity. Non-muscle cells such as fibroblast are likely to have less COX activity than myocytes. Consequently, the effect of ethanol exposure on the proportion of non-muscle cells in these cultures was determined by immunocytochemical methods following exposure to different concentrations of ethanol for various lengths of time (Table 1). Although the proportion of fibroblasts in the untreated cultures varied from experiment to experiment, there was no difference with ethanol treatment within a given experiment. In addition, decreases in the protein content of cultures treated with high concentrations of ethanol (Fig. 1B) suggests the possibility that the cell density was less in ethanol-treated cultures. It is not known whether cell density influences mitochondrial biogenesis or the accumulation of cytochrome c oxidase in cardiac myocyte cultures. Therefore, COX activity was examined at various plating densities. Although the plating density was correlated with the protein content of cultures three days after plating (Fig. 4A), there was no relationship between the protein content and the COX activity of myocyte cultures (Fig. 4B). Therefore, the ethanol-induced decrease in COX activity of myocyte cultures was attributed to a direct effect on cultured cells rather than to changes in cell density or proportion of non-muscle cells in the culture.


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  		Table 1 Percent of non-muscle cells following ethanol exposure

 

Figure 4
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  		Fig. 4 The effect of cell density on COX activity in cultured cardiac myocytes. COX activity and protein content were measured in homogenates of cardiac myocyte culture dishes plated at different densities (0.5–6.25x106 cells per 60-mm culture dish). There was a linear relationship (P<0.01) between the number of cells plated and the protein recovery (panel A). The relationship between protein recovery and the COX activity for each culture dish is shown in panel B. There was no significant correlation between protein recovery and COX activity. Taken together, the data in A and B indicate that COX activity was independent of cell density in cardiac myocyte cultures.

 
3.2 Cytochrome c oxidase subunit levels
Experiments were conducted to determine whether ethanol's effects on cytochrome c oxidase activity were a result of changes in the expression of genes encoded in either the nuclear or mitochondrial genome. The relative levels of nuclear-encoded (COXIV) and mitochondrial-encoded (COXII) subunits for cytochrome c oxidase were determined by densitometric analysis of Western blots probed with anti-COX subunit specific antibodies. No changes in the relative level of the nuclear-encoded COXIV subunit were observed with ethanol concentrations up to 200 mM. In contrast, ethanol exposure produced decreases in the relative level of the mitochondrial-encoded COXII subunit (Fig. 5). Significant decreases were observed in the COXII subunit content with ethanol concentrations of 50 mM and greater. Both the dose-dependency and the magnitude of this effect were similar to that demonstrated for the effect of ethanol on COX activity (Fig. 1A), suggesting that the effects of ethanol on COX activity are exerted at the level of mitochondrial gene expression.


Figure 5
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  		Fig. 5 The effect of ethanol exposure on COXIV and COXII subunit level in cardiac myocyte cultures. The relative levels of the nuclear-encoded COXIV subunit and mitochondrial-encoded COXII subunit were determined by densitometry of Western blots as described in Section 2. Cardiac myocyte cultures were exposed to ethanol for 4 days prior to analysis. Subunit levels were determined for n = 3 (10 mM, 75 mM, 200 mM) or n = 4 (0 mM, 25 mM, 50 mM, 100 mM) samples per treatment obtained from three separate culture experiments. Values were expressed relative to a standard curve and are presented as the mean±s.e. in arbitrary densitometric scan units. *Significant difference (P<0.05) between ethanol-treated groups and the 0 mM control group.

 
3.3 Mitochondrial DNA and mRNA levels
Mitochondrial DNA and COXIII mRNA levels were measured in order to determine if the observed changes in the expression of mitochondrial genes for COX were correlated with changes in mitochondrial transcription or mitochondrial gene dosage. Results were obtained from cultures maintained for 3 days, 5 days, or 7 days (Fig. 6A). No significant differences were observed in the relative mitochondrial DNA content on day 3. However, increases in the relative mitochondrial DNA content in excess of 2-fold were observed with either 5 or 7 days of treatment with 200 mM ethanol. Seven days of treatment also produced a significant increase in the mitochondrial DNA content with 50 mM ethanol (Fig. 6A).


Figure 6
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  		Fig. 6 The effect of ethanol exposure on mitochondrial DNA and COXIII mRNA levels in cardiac myocyte cultures. Cardiac myocyte cultures were maintained in media containing either 0 mM, 25 mM, 50 mM, or 200 mM ethanol. Five culture dishes were pooled for each DNA sample and four culture dishes were pooled for each RNA sample (n = 4 samples for the determination of mitochondrial DNA and COXIII mRNA for each ethanol concentration in each experiment). Each time point (3, 5, or 7 days) represents a separate cell culture experiment. The relative levels of mitochondrial DNA (A) and COXIII mRNA (B) were determined by slot blotting techniques as described in Section 2. Data for each ethanol treatment are presented as the percent of the 0 mM control (±s.e.) for that particular experiment. *Significantly greater (P<0.05) than the 0 mM control for that experiment. Although no differences were observed in the COXIII mRNA content (B), significant elevations were seen in the mitochondrial DNA content (A) of cultures treated with 50 mM (day 7) or 200 mM (day 5 and 7) ethanol.

 
There were no differences in the relative level of COXIII mRNA between control cultures and those exposed to ethanol. Neither increasing the ethanol concentration nor increasing the length of exposure resulted in alterations in the COXIII mRNA levels (Fig. 6B). These data clearly demonstrate that the mitochondrial DNA copy number and the content of the mitochondrial-encoded COXIII mRNA can be dissociated by ethanol exposure.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In this study we report a decrease in COX activity in cultured myocytes chronically exposed to ethanol. Since indirect effects of ethanol on either cell type or cell density (Table 1, Fig. 4) were effectively eliminated as causative, the defect in COX activity and expression of the mitochondrial-encoded COXII gene reported here demonstrate a direct effect of ethanol on embryonic cardiac myocytes. These effects were obtained with media ethanol concentrations (25–50 mM) which are similar to plasma concentrations achieved in vivo and support previous findings in which a deficit in COX activity was detected in chicks [16, 17]or mice [15]during embryonic development. An advantage of this in vitro model of ethanol exposure is that the effect of ethanol can be evaluated in the absence of systemic, neuronal, hormonal, or vascular variables. Therefore, the data presented here demonstrate that ethanol has a direct effect on cardiac mitochondria.

Protein accumulation was less in cultures treated with high concentrations of ethanol. No attempt was made in the present report to distinguish between specific decreases in cellular protein synthesis and the selective loss of cells. However, other laboratories have confirmed that both total cellular protein synthesis and the cell number per culture dish decline with chronic ethanol exposure of cardiac myocytes [12, 13]. In the experiments reported here, a decrease in the relative content of the mitochondrial-encoded COXII subunit was demonstrated (Fig. 5). These data were normalized relative to the protein content per sample so that the reduction in COXII content is not simply a result of the overall reduction in protein content, but reflects a genuine decrease in the COXII content per myocyte. The decrease in the steady state level of COXII indicates that either COXII synthesis was reduced or its degradation was accelerated. In the adult rat, decreases in mitochondrial protein synthesis are clearly exhibited in both the heart and liver following chronic ethanol consumption [31, 32]. Therefore, a reduction in mitochondrial protein synthesis may also be expected in the myocyte cultures described here. In addition, if the decrease in the level of COXII is explained by a reduction in the rate of COXII synthesis, the reduction in COXII synthesis must be even greater than the decrease in total protein synthesis per cell. For example, if the total cellular protein synthesis were to decrease 2-fold and the level of COXII (relative to the protein content) were to also decrease 2-fold, then the synthesis rate for COXII would be expected to be 4-fold lower.

The relative level of the COXIV subunit was unchanged, while that of the COXII subunit was reduced in a concentration-dependent manner by ethanol exposure. This result indicates that the effect of ethanol on the expression of COX subunits is not coordinated between nuclear and mitochondrial genomes. Ethanol appears to exert an effect which is specific to the expression of the mitochondrial genome. This contrasts distinctly with reports in the literature investigating the regulation of COX gene expression by chronic stimulation of skeletal muscle [48, 49]where changes in the levels of mitochondrial- and nuclear-encoded genes are coordinately upregulated. However, a temporal dissociation in the expression of mitochondrial and nuclear genes for COX is exhibited in liver and skeletal muscle following thyroid hormone treatment [50]or embryonic cardiac hypertrophy [37]. Moreover, an uncoupling of the coordinated response of nuclear- and mitochondrial-encoded genes [48, 49]to chronic electrical stimulation has been demonstrated in skeletal muscle of rats exposed to therapeutic levels of the anti-HIV drug, AZT [47]. In addition, Coleman et al. [29]have reported that the expression of mitochondrial-encoded genes, but not nuclear-encoded genes for ATP synthase are reduced by chronic ethanol exposure in adult rat livers. Thus, the results presented here support the argument that expression of the mitochondrial genome can be regulated independently of the nuclear genome. The relative levels of the remaining two mitochondrial subunits and the remaining 9 nuclear-encoded subunits were not examined in the present study. Therefore, the possibility remains that the level of these subunits could be unchanged, decreased, or even increased by chronic ethanol exposure. Likewise, changes in the tissue-specific isoforms of nuclear-encoded COX subunits [25]were not examined but could potentially influence COX activity in ethanol-exposed cardiac myocytes.

Steady-state levels of the COXIII mRNA and mitochondrial DNA were examined in order to determine if an ethanol-induced deficit in mitochondrial transcription or replication could explain the observed reduction in COXII subunit levels. Although COXI, COXII, and COXIII are separate genes, all three are encoded within the same DNA strand of the mitochondrial genome [51]. Since the mitochondrial genome has a single transcription start site, this localization would restrict these three COX subunits to a single primary transcript. Thus, a determination of the relative level of the COXIII mRNA most likely reflects values for COXII and COXI mRNA as well. In this study, COXIII mRNA levels were found to be unaffected by chronic in vitro ethanol exposure (Fig. 6B), suggesting that ethanol-induced decreases in mitochondrially-encoded COX subunit levels in cultured cardiac myocytes is organized by a post-transcriptional mechanism. This confirms previous observations in embryonic chick hearts exposed to ethanol in ovo [17], and is similar to findings in the adult rat liver where the content of all three mitochondrial-encoded COX subunits were coordinately reduced [32]with no changes in their corresponding mRNAs [28].

The relative mitochondrial DNA content was elevated by ethanol exposure in cultured cardiac myocytes (Fig. 6A). This effect was not as readily demonstrated as the effect on COX activity since a significant elevation in mitochondrial DNA levels were not observed until 7 days with 50 mM ethanol exposure. However, these results do confirm a dissociation of mitochondrial DNA replication and transcription which has been reported in embryonic chick hearts following ethanol exposure in ovo [17]or during embryonic cardiac hypertrophy induced by hypothermia [37]. Moreover, the fact that there was no change in the COXIII mRNA level with an increase in the mitochondrial DNA level suggests that transcriptional efficiency is reduced in the ethanol-exposed cardiac myocytes. The mechanism by which the mitochondrial DNA copy number is elevated is unknown. It is possible that ethanol directly stimulates mitochondrial DNA polymerase {gamma} or reduces the degradation of mitochondrial DNA. Alternatively, mitochondrial DNA may accumulate as a result of some indirect action of ethanol. For example, the reduction in COX activity or some signal produced as a result of this reduction in aerobic capacity may trigger a series of events culminating with the activation of mitochondrial DNA replication.

In this report we present data demonstrating a deficit in COX activity in cultured embryonic cardiac myocytes exposed to ethanol. Other reports have shown ethanol-induced ultrastructural abnormalities in mitochondria of cultured cardiac cells [21], but this is the first demonstration of a defect at the biochemical level. Decreases in the content of the COXII subunit which parallel the decreases in COX activity suggest that a defect in mitochondrial gene expression may explain the failure of ethanol-treated cells to accumulate COX. In addition, the effect of chronic ethanol exposure on expression of a nuclear-encoded COX subunit, COXIV, was examined for the first time. There were no changes in the level of COXIV, indicating that the ethanol-induced defect in COX activity is independent of expression of this gene and suggests that the defect in COX activity can be entirely explained by an impairment in the expression of mitochondrial-encoded genes.

Time for primary review 18 days.


   Acknowledgements
 
I would like to thank Roderick Capaldi for the supply of anti-COX antibodies used in this project. I would also like to thank Susan W. Kelley and Maureen A. McElvain for their most excellent technical assistance in the completion of this project. This work was supported by grants from the NIH (AA08716 and AA00179).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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