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Article

Mitochondrial Transcription Factor A Is Increased but Expression of ATP Synthase ß Subunit and Medium-Chain Acyl-CoA Dehydrogenase Genes Are Decreased in Hearts of Copper-Deficient Rats¹

Department of Human Nutrition and Food Management, The Ohio State University, Columbus, OH 43210 and ^* Center for Cardiovascular Research, Departments of Medicine and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110

²To whom correspondence should be addressed at Department of Human Nutrition, 213 Justin Hall, Kansas State University, Manhattan, KS 66506-1407.

	ABSTRACT

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The mechanism(s) by which impaired mitochondrial respiratory function and the accumulation of lipid droplets and mitochondria in hearts of copper-deficient rats occur remains unclear. It is not known whether specific components of the regulatory pathway involved in mitochondrial biogenesis, such as mitochondrial transcription factor A (mtTFA) and nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2), are activated in copper deficiency. Little is known about gene expression of enzymes involved in fatty acid oxidation (FAO) in hearts of copper-deficient rats. Male weanling rats were fed copper-adequate (CuA), copper-deficient (CuD) or pair-fed (CuP) diets for 5 wk. Mitochondria and lipid droplet volume densities from electron micrographs were greater and there was an elevation in the mtTFA protein level in hearts of copper-deficient rats. DNA binding activities of NRF-1 and NRF-2 did not differ among the groups. Northern blot analysis of cardiac tissue revealed that transcripts of F₁F₀-ATP synthase subunit c were greater, but mRNA levels of ATP synthase ß subunit and the FAO enzyme, medium-chain acyl-CoA dehydrogenase (MCAD), were lower in hearts of copper-deficient rats. Long-chain acyl-CoA dehydrogenase (LCAD) mRNA levels did not differ among treatment groups. These results suggest that certain components of the mitochondrial biogenesis program are activated in hearts of copper-deficient rats. F₁F₀-ATP synthase ß subunit and MCAD transcript levels remain low, which may contribute to impaired mitochondrial respiratory function, decreased fatty acid utilization and lipid droplet accumulation in hearts of copper-deficient rats.

KEY WORDS: • rats • mitochondrial transcription factor A • ATP synthase • copper deficiency • medium-chain acyl-CoA dehydrogenase

	INTRODUCTION

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Dietary copper depletion leads to cardiac hypertrophy characterized by increased cardiac mitochondrial volume density (Medeiros et al. 1991a , Wildman et al. 1994 ). The enlarged myocyte in hearts of copper-deficient rats appear to be due not only to a relative increase but to an increased absolute area occupied by mitochondria but not myofibrils (Mao et al. 1998 ). Moreover, the rate of mitochondrial protein synthesis is increased in hearts of copper-deficient rats (Medeiros et al. 1997 ). These results strongly suggest that cardiac mitochondrial biogenesis is stimulated in copper-deficient animals. Mitochondrial transcription factor A (mtTFA)³ is the major transcription factor governing mitochondrial DNA replication and transcription during mitochondrial biogenesis (Virbasius and Scarpulla 1994 ). In turn, the mtTFA gene promoter can be modulated by nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) (Virbasius and Scarpulla 1994 ). It is not known whether mtTFA is one of the signals that enhances mitochondrial biogenesis in hearts of copper-deficient animals, and whether NRF-1 and/or NRF-2 are involved in the activation of mtTFA gene expression.

Copper is a cofactor for cytochrome c oxidase (CCO), a key enzyme in the mitochondrial oxidative phosphorylation system. Animals fed diets deficient in copper have decreased cardiac CCO activity and impaired mitochondrial respiratory function (Chao et al. 1993 , Matz et al. 1995 ). The levels of nuclear-encoded protein subunits of CCO are decreased, whereas mitochondrially encoded subunits are unaffected in hearts of copper-deficient rats, although mRNA of nuclear subunits are not altered compared with controls (Medeiros et al. 1997 ). Another oxidative phosphorylation enzyme, F₁F₀-ATP synthase, appears to be altered in hearts of copper-deficient rats. The F₁F₀-ATP synthase subunit protein level is decreased in hearts of copper-deficient animals (Chao et al. 1994 ). Respiration studies by Matz et al. (1995) have provided additional evidence that cardiac mitochondrial ATP synthase is influenced by dietary copper deprivation. However, it is not known from these studies what subunits in F₁F₀-ATP synthase, if any, are affected and whether the gene expression of subunits in F₁ and F₀ sectors are regulated differentially.

Components of the respiratory chain appear compromised in the hearts of copper-deficient rats, but it is unclear whether energy metabolism may be altered similarly. Energy substrate utilization in the hearts of copper-deficient animals may be affected, particularly fatty acids, because mitochondrial fatty acid ß-oxidation provides up to 60% of the ATP requirement of the heart (Lopaschuk et al. 1994 ). Excessive lipid droplets accumulate in the hearts of copper-deficient rats (Wildman et al. 1994 ). How the enzymes involved in fatty acid ß-oxidation in the heart are affected by the compromised mitochondrial function in copper deficiency has not been investigated.

In this report, we describe the results of two studies. The objective of study 1 was to determine whether specific regulatory components of the mitochondrial biogenesis are upregulated in the hearts of copper-deficient rats, and if so, whether NRF-1 and/or NRF-2 is involved in upregulating mtTFA gene expression. Study 2 was designed to determine whether copper deficiency would result in altered gene expression of key proteins involved in oxidative phosphorylation (e.g., F₁F₀-ATP synthase) and fatty acid oxidation [medium- (MCAD) and long-chain acyl-CoA dehydrogenase (LCAD)].

	MATERIALS AND METHODS

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Study 1

    Animals and diets. Male weanling Long-Evans rats (n = 35) were purchased from Harlan Sprague Dawley (Indianapolis, IN). Upon arrival, the rats were weighed and assigned randomly to the following three dietary treatment groups: copper-adequate (CuA, n = 12), copper-deficient (CuD, n = 13) and pair-fed (CuP, n = 10). The initial body weights of the dietary treatment groups were not different.

All rats were fed a modified AIN-76 diet for 5 wk (AIN 1980 ). The semipurified diets were purchased from Research Diet (New Brunswick, NJ). Diets for copper-adequate and pair-fed groups contained copper at 94 µmol Cu/kg diet. Copper was omitted in the diet of the copper-deficient group (<15 µmol Cu/kg). The dietary copper levels were verified by flame atomic absorption spectrophotometry (Varian Spectra AA-5, Victoria, Australia). The rats were housed singly in stainless steel cages in a room with a 12-h light:dark cycle at constant room temperature (21.7°C). Rats in the CuA and CuD groups had free access to the appropriate diet and deionized-distilled water. Daily food intake for rats in the copper-deficient group was measured. Rats in the pair-fed group were given the average amount of food consumed by the copper-deficient group on the previous day. Body weights were obtained weekly. This animal protocol was reviewed and approved by the Ohio State University Institutional Animal Welfare Committee.

    Sample collection. At the end of the study, rats were anesthetized with carbon dioxide inhalation. A midline incision was made to open the thoracic and abdominal cavities. Hearts from a subgroup of four rats in each dietary group were processed as described below for electron microscopy examination to verify the presence of increased mitochondrial volume density in the copper-deficient group. Hearts from the remaining rats were quickly removed, weighed, snap frozen in liquid nitrogen and stored at -80 ^oC for later analysis. Liver sample from all rats were removed and stored at -20 ^oC for later analysis of Cu,Zn-superoxide dismutase (Cu, Zn-SOD) activity.

    Liver Cu,Zn-SOD activity assay. Liver Cu,Zn-SOD activity was measured spectrophotometrically on the basis of the ability of liver homogenate to inhibit the autoxidation of pyrogallol as described by Marklund and Marklund (1974) and modified by Prohaska (1983) . One unit of Cu,Zn-SOD activity was defined as the amount of activity that inhibited the autoxidation of pyrogallol by 50%, expressed as units/g wet tissue.

    Tissue preparation for electron microscopy and morphometric analysis. Four hearts from each group in study 1 were prepared for transmission electron microscopy using a modified procedure as described by Medeiros et al. (1991b) . Briefly, hearts were perfused by injecting KCl (1.0 mol/L) into left ventricles and removed by severing the great vessels. Hearts were then quickly perfused with 2% glutaraldehyde in 0.1 mol/L Sorenson’s PBS and 0.1 mol/L sucrose via the aorta. Hearts were blotted and weighed, and samples were cut from the left ventricle, tangentially to the outer wall to obtain cardiac muscle fibers at the longitudinal plane. Samples were fixed in the glutaraldehyde perfusion buffer followed by postfixation in osmium tetraoxide. Samples were then dehydrated by a gradient of increasing alcohol concentration and embedded with Spur resin.

All electron microscopic processing and morphometric analyses were performed in a blind manner. Embedded sample blocks were orientated to obtain longitudinal views of cardiac muscle. Transmission electron micrographs were printed at 20,000x for the morphometric analysis to determine volume densities of various intracellular components using a point system as described by Weibel (1979) and Steer (1981) . A 20 x 25 cm² transparent grid with 100 reference points per electron micrograph was used. Other intracellular material was defined as those substances that were not identified as either mitochondria or myofibril, such as sarcoplasm, other organelle and lipid droplets. For volume density of lipid droplets, 1000 reference points were used. Mean values were calculated by averaging four prints for each rat.

    Western blot analysis for mitochondrial transcription factor A. Cardiac nonmyofibrillar proteins were obtained as described by McCormick et al. (1989) . Left ventricle (0.2 g) from each rat was homogenized in 2 mL of 0.1 mol/L KCl in 1.5% Triton X-100, followed by centrifugation for 20 min at 1100 x g. The supernatant containing the nonmyofibrillar protein fraction (predominantly mitochondrial proteins) was used for Western immunoblotting of mtTFA as described by Parisi et al. (1993) . Nonmyofibrillar protein (150 µg) was separated on a 12% denaturing polyacrylamide gel. Purified human mtTFA protein (a gift from Dr. David A. Clayton, Stanford University) and molecular weight markers were also loaded as standards. Proteins were transferred to a nitrocellulose membrane by a Trans Blot apparatus (Bio-Rad Laboratories, Richmond, CA). The blots were stained with Ponceau S to confirm uniform protein loading and blocked with 5% nonfat milk in PBS (136.7 mmol/L NaCl, 26.9 mmol/L KCl, 10 mmol/L Na₂HPO₄, 1.7 mmol/L KH₂PO₄) for at least 2 h at 24°C. The membranes were then incubated with a polyclonal affinity purified rabbit antiserum to mouse-mtTFA(a gift provided by Dr. David A. Clayton, Stanford University) at 1:10,000 dilution for 2–16 h with gentle shaking. After washing, the blots were incubated with biotinylated secondary antibody against rabbit as instructed (Vectastain ABC-AP kit, Vector Laboratories, Burlingame, CA) and the antibody signal was detected with an alkaline phosphatase substrate kit (Vector Laboratories). For six rats from each group, antibody signal was quantitated using the IS-1000 Digital Imaging System (Alpha Innotech, San Leansdro, CA). The ratio of signal density was obtained using the copper-adequate group as control.

    Nuclear extract preparation and gel mobility shift assays. Nuclear extracts were prepared from cardiac tissue as described by Deryckere and Gannon (1994) . The protein concentration of nuclear extracts was determined using Protein DC assay kits (Bio-Rad Laboratories).

Synthetic double-stranded oligonucleotide probes for NRF-1 and NRF-2 were synthesized at Washington University School of Medicine (St. Louis, MO). The NRF-1 and NRF-2 oligonucleotide sequences represent their binding sites (underlined) from the rat cytochrome c (RC4) and human mitochondrial transcription factor A (h-mtTFA), respectively:

RC4 (-172/-147): 5'-GATCCTGCTAGCCCGCATGCGCGCGCACCTTA-3'

3'-GACGATCGGGCGTACGCGCGCGTGGAATTCGA-5'

h-mtTFA(-34/-13): 5'-GATCCTCTACCGACCGGATGTTAGCAGAG-3'

3'-GATCCTCTGCTAACATCCGGTCGGTAGAG-5'

The double-stranded oligonucleotides were 5' end-labeled with [-³²P]-ATP and subsequently purified using a G-25 Sephadex column (Boehringer, Indianapolis, IN).

The binding reactions for NRF-1 were performed as described by Chau et al. (1992) . Nuclear extract (25 µg) was incubated in 25 µL buffer containing 25 mmol/L Tris, pH 7.9, 6.25 mmol/L MgCl₂, 0.5 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 50 mmol/L KCl, 10% (v/v) glycerol, 5 µg of poly(dI-dC)-poly(dI-dC) and 15 fmol of radiolabeled probes. After incubation at room temperature for 15 min, the binding reactions were electrophoresed on a 5% polyacrylamide gel. Competition reactions were performed by adding 100-fold excess of unlabeled oligonucleotide to the binding reaction before the addition of labeled oligonucleotide. For the supershift assay, 1 µL goat NRF-1 antiserum was added to the binding reactions and incubated for an additional 15 min before electrophoresis (Virbasius et al. 1993 ). Gels were then dried and visualized by autoradiography. For five rats from each group, signal density for NRF-1 DNA complex was quantitated using IS-1000 Digital Imaging System (Alpha Innotech).

The binding reactions for the NRF-2 mobility shift assay were performed in 20 µL binding buffer consisting of 20 mmol/L HEPES-KOH, pH 7.9, 50 mmol/L KCl, 1 mmol/L benzamidine, 20% (v/v) glycerol, and 4 µg of poly(dI-dC)-poly(dI-dC) (Martin et al. 1996 ). Crude nuclear extract (15 µg) and 15 fmol of ³²P end-labeled NRF-2 oligonucleotide were added and the reactions were incubated for 15 min at room temperature. A 100-fold excess of unlabeled oligonucleotides was added to the competition reactions before the addition of labeled oligonucleotide. The samples were electrophoresed and quantitated as for NRF-1.

Study 2

The feeding protocol in study 2 was similar to study 1 except that the pair-fed group was omitted. Male weanling Long-Evans rats (n = 14) were divided into copper-adequate (CuA, n = 7) and copper-deficient (CuD, n = 7) groups. The rats were fed their respective diets as outlined above. Rats were given free access to the diets and deionized-distilled water. After 5 wk, the animals were killed by carbon dioxide inhalation and hearts quickly removed, weighed, snap frozen in liquid nitrogen and stored at -80°C until RNA isolation. Liver samples were removed to assess Cu,Zn-SOD activity as described in study 1.

    Northern blot analysis. Total RNA was extracted from heart tissue using RNAzol (Tel-test, Friendswood, TX) and purity determined at 260 nm and 280 nm absorbance. Total RNA (15 µg) was loaded into each well of agarose gel and electrophoresed. The RNA was transferred to Gene Screen (New England Nuclear-Dupont, Boston, MA) by capillary blotting. Hybridization of RNA to cDNA probes was completed using Quick-hyb (Stratagene, La Jolla, CA). The following probes were used: ß subunit of ATP synthase, subunit c of ATP synthase, MCAD and LCAD. ß-Actin was used to normalize signals. The sequences of all probes were determined to verify the accuracy of the cDNA probes.

    Statistical analysis. Data from study 1 were analyzed by one-way ANOVA using the General Linear Models procedure of Statistical Analysis System (SAS Institute, Cary, NC). When significant F-values existed, a least significant difference post-hoc test was used to determine which of the group means differed from each other. For the variables in the morphometric analysis of electron micrographs, a one-sided test was performed because of the small sample sizes. Data from study 2 were analyzed by student’s t test. Differences were considered significantly different at P < 0.05. All values are expressed as means ± SEM.

	RESULTS

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Study 1

All of the rats completed the 5-wk study. The rats fed the copper-adequate diet had final body weights greater than those rats fed the copper-deficient and pair-fed diets (Table 1 ). Daily food intake measured at wk 4 was 16.3 ± 0.27 g for the CuD group; this was less than the intake of 18.7 ± 0.28 g for the CuA group (P < 0.01). The heart weight in the CuD group was significantly greater than in the CuA group, leading to a much greater ratio of heart:body weight (Table 1) . The heart weight of the CuD group was also greater than that of the CuP group despite the similar body weights. No differences in the absolute or relative heart weights were found between the CuA and CuP groups. Liver Cu,Zn-SOD activities for rats in the CuA and CuP groups were not different but were significantly higher than that of rats fed the copper-deficient diet. Results of the above measurements were not different between the subgroup of rats used for EM analysis and the rest of their groups; therefore, the results were reported collectively.

View larger version (167K): [in this window] [in a new window]   Figure 1. Transmission electron micrographs of cardiac muscle from the (A) copper-adequate, (B, C) copper-deficient and (D) pair-fed rats. The mitochondria in the hearts of copper-deficient rats appeared swollen and vacuolated. The mitochondrial cristae were sparse and fragmented, and myofibrils were separated by mitochondria intrusion (B). Excessive lipid droplet accumulation was observed in the heart from the Cu-deficient group (C). Abbreviations: m, mitochondria; M, myofibril; O, other intracellular material; L, lipid droplets; Bar = 1 µm.

View larger version (35K): [in this window] [in a new window]   Figure 2. Mitochondrial transcription factor A (mtTFA) protein level in rats fed adequate or deficient amounts of copper. (A) Comparison of the mtTFA protein levels among the copper-adequate (lanes 1–3), copper-deficient (lanes 4–6) and pair-fed (lanes 7–9) rats in a representative Western blot. Nonmyofibrillar proteins (150 µg) were probed with antibody to mouse mtTFA. The migration of purified human mtTFA (lane 10) was used to identify mtTFA in the samples. mtTFA protein level was higher in rat hearts from the copper-deficient group compared with hearts from the copper-adequate and pair-fed groups. Each lane represents a different rat. (B) Results of densitometric analysis of mtTFA protein level from the copper-adequate, copper-deficient and pair-fed rats. Data are expressed as means ± SEM, n = 6. Means with different letters are different, P < 0.05. For six rats in each group, the density of the signal was quantitated using IS-1000 Digital Imaging System (Alpha Innotech, San Leandro, CA). The copper-adequate group was assigned a value of 1 relative density unit, and the values of relative density unit for the other two groups were obtained using the copper-adequate group as control.

View larger version (95K): [in this window] [in a new window]   Figure 3. Gel mobility shift assay of nuclear respiratory factor (NRF)-1 in the hearts of copper-adequate, copper-deficient and pair-fed rats. HeLa nuclear extract was used as positive (lane 1) and negative controls (specific competitor, lane 2). The intensity of the shifted band was similar in the copper-adequate (CuA; lanes 3–6), copper-deficient (CuD; lanes 7–10) and pair-fed (CuP; lanes 11–13) groups. Addition of NRF-1 antiserum to the reaction in lane 5 produced a supershift band (lane 14). The NRF-1 band from the reaction in lane 5 was also eliminated in a competitor reaction using unlabeled probe (lane 15). Each lane represents one heart sample from a different rat.

View larger version (89K): [in this window] [in a new window]   Figure 4. Gel mobility shift assay of nuclear respiratory factor (NRF)-2 in the hearts of copper-adequate (CuA), copper-deficient (CuD) and pair-fed (CuP) rats. The positions of the NRF-2 shifted complexes are indicated as bands 1 and 2. Free probe ran to the bottom (lane 1). HeLa nuclear extract (lane 2) showed the same pattern as samples (lanes 3–12). Competitive reaction containing unlabeled probe eliminated both bands (lanes 3 and 13). There was no consistent change in signal intensity from the CuA (lanes 4–6), CuD (7–9), and CuP (10–12) groups. Each lane represents a sample from a different rat.

After 5 wk of dietary treatment, the CuD group was indeed copper deficient as demonstrated by direct and indirect indices for copper status (Table 1) . Liver Cu,Zn-SOD activity and body weight in the CuD group were significantly lower than in the CuA group, and the CuD group had significantly greater absolute and relative heart weights.

The results of Northern blot analysis are shown in Figure 5A and B . The CuD group had lower transcript levels of ATP synthase ß subunit and MCAD, two nuclear-encoded mitochondrial proteins. In contrast, the abundance of ATP synthase subunit c mRNA was markedly greater in the CuD group. There was no apparent difference in LCAD transcripts due to treatment. Dietary Cu treatment did not affect the ß-actin mRNA level.

View larger version (44K): [in this window] [in a new window]   Figure 5. Results of Northern blot analysis of total RNA extracted from heart tissue of copper-adequate and copper-deficient rats. (A) Representative Northern hybridization. The different DNA probes used are indicated to the right side of the panels. Dietary copper restriction decreased the abundance of mRNA transcripts for ATP synthase ß subunit and medium-chain acyl-CoA dehydrogenase (MCAD), but increased mRNA level of ATP synthase subunit c in the heart. Long-chain acyl-CoA dehydrogenase (LCAD) mRNA levels were not altered by dietary copper treatment. (B) Histogram summarizing the results of Northern hybridization. Densitometric units of signal intensity obtained from autoradiograms were normalized to ß-actin. Data are presented as means ± SEM, n = 7. Means with different letters are different, P < 0.05.

	DISCUSSION

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In addition to increased mitochondrial volume density (Wildman et al. 1994 ), Matz et al. (1995) reported a greater amount of mitochondrial protein in hearts from copper-deficient rats. We showed previously that mitochondrial protein synthesis rates are higher in the hearts of copper-deficient rats (Medeiros et al. 1997 ). Taken together, these findings suggest enhanced mitochondrial biogenesis in the hearts of copper-deficient animals. The classical explanation for the impaired mitochondrial function in copper deficiency has been the reduced activity of cuproenzyme CCO, the enzyme complex IV of the oxidative phosphorylation system. CCO generates a proton gradient across the mitochondrial inner membrane, which is eventually used to produce ATP by F₁F₀-ATP synthase, the enzyme complex V of the oxidative phosphorylation system. In the hearts of copper-deficient animals, the mitochondrial respiratory function is compromised as a result of the reduction in CCO activity and possibly alterations in other components of the respiratory chain (Levenson et al. 1999 , Medeiros and Wildman 1997 ). Increased oxidative stress as indicated by elevated Mn-SOD activity in the mitochondria of hearts of copper-deficient animals may cause structural and functional damage to the mitochondria (Lai et al. 1994 ). Therefore, it may be a compensatory mechanism for the hearts of copper-deficient animals to synthesize more mitochondria to sustain normal ATP production.

Mitochondrial DNA replication during biogenesis is under the control of mitochondrial transcription factor A (mtTFA). mtTFA is nuclear encoded, synthesized in the cytosol and imported into mitochondria, where it plays crucial roles in both mitochondrial transcription and replication by binding the D-loop promoter areas of mitochondrial DNA (Clayton 1998 ). Parisi et al. (1993) demonstrated that addition of either h-mtTFA or Saccharomyces cerevisiae mtTFA to plasmid DNA template could enhance mitochondrial transcript production in vitro. Suppression of mtTFA mRNA by interferons and low mtTFA protein level are associated with a reduction in mitochondrial transcripts in HeLa cells (Inagaki et al. 1997 ). These findings suggest a potential role for mtTFA in stimulating mitochondrial biogenesis in the hearts of copper-deficient animals. Our data demonstrating increased mtTFA protein level in the CuD group support this hypothesis. This increase of mtTFA may stimulate overall mitochondrial biogenesis, leading to a greater mitochondrial volume density in the hearts of copper-deficient rats.

The promoters of the mtTFA gene contain recognition sites for several nuclear-encoded regulatory elements, including NRF-1 and NRF-2. Increased NRF-1/NRF-2 DNA binding activities and their mRNA levels have often been associated with elevated mtTFA mRNA or protein level (Wu et al. 1999 ). Miranda et al. (1999) reported increased NRF-1 and mtTFA transcript levels in DNA-depleted HeLa cells, and mitochondrial biogenesis was stimulated. In light of the elevated mtTFA protein level, NRF-1 and/or NRF-2 DNA binding activities were evaluated in the hearts of copper-deficient rats. However, no difference in NRF-1 or NRF-2 DNA binding activities was detected. This finding is consistent with the observation that mRNA levels of nuclear-encoded CCO subunits were not elevated in the hearts of copper-deficient rats (Medeiros et al. 1997 ). Many of the nuclear-encoded CCO subunits, such as subunit IV and VIc are regulated by NRF-1 or NRF-2 (Gugneja et al. 1995 , Virbasius et al. 1993 ).

Post-transcriptional regulation of mtTFA gene expression may exist. On the other hand, the possibility that NRF-1 and/or NRF-2 is involved in the activation of mtTFA gene expression and mitochondrial biosynthesis in the hearts of copper-deficient animals cannot be excluded. One or more additional regulatory factor(s) may be required for the maximal activation of mtTFA gene expression by NRF-1 and/or NRF-2. Peroxisome proliferator-activated receptor- coactivator (PGC-1), a cold inducible transcription coactivator of nuclear receptors, may fall into this category. PGC-1 can potentiate the effect of NRF-1 in inducing mtTFA gene expression (Wu et al. 1999 ). It is possible that other transcription factors identified as regulators of the mtTFA promoter, such as Sp-1, may participate in the regulation of mtTFA gene expression. Another explanation for the lack of difference in NRF-1 and NRF-2 DNA binding activities in this study could be the time point of sample collection. The heart samples for the measurement of DNA binding activity were obtained after 5 wk of dietary treatment, but cardiac hypertrophy in copper-deficient rats has been observed as early as after 3 wk of dietary copper depletion (Jalili et al. 1997 , Medeiros and Wildman 1997 ). Increased mtTFA protein and mitochondrial biogenesis are expected to occur before the development of cardiac hypertrophy. A preliminary study in our laboratory showed elevated mtTFA protein level but unaltered DNA binding activities of NRF-1 and NRF-2 in the hearts of rats fed a copper-deficient diet for 3 wk (unpublished data). If NRF-1 and/or NRF-2 are indeed involved in the activation of mtTFA gene expression, this event is likely to precede the elevation of the mtTFA protein level. Measurement of DNA binding activities for NRF-1 and -2 at different time points before 3 wk may provide insight into their roles in the enhanced mitochondrial biogenesis in the hearts of copper-deficient rats.

Although CCO is the only known enzyme in the mitochondrial respiratory system that requires copper for its functional activity, copper deficiency appears to affect other components of respiratory chain enzyme complexes (Johnson and Thomas 1999 ). Matz et al. (1995) demonstrated that mitochondria from the hearts of copper-deficient rats are more resistant to oligomycin blockade of proton flow, suggesting altered function of F₁F₀-ATP synthase. This functional abnormality could be due to structural alterations. A previous study in our laboratory showed decreased peptide level of the subunit of mitochondrial F₁F₀-ATP synthase in the hearts of copper-deficient rats (Chao et al. 1994 ). Levenson et al. (1999) recently reported diminished cytochrome b₁ mRNA levels in the livers of copper-deficient rats. In this study, we detected an increased mRNA level of F₁F₀-ATP synthase subunit c in the hearts of copper-deficient rats. Multiple copies of subunit c form a ring structure in the F₀ sector, which functions as a proton channel in the mitochondrial inner membrane (Saraste 1999 ). As an integral part of mitochondrial inner membrane structure, the subunit c gene expression may be regulated coordinately by factors governing mitochondrial biogenesis. Molecular signals stimulating mitochondrial biogenesis may simultaneously upregulate gene expression of F₁F₀-ATP synthase subunit c. This notion is supported by a previous finding in our laboratory that subunit e mRNA of the F₀ sector was greater in the hearts of copper-deficient rats (n = 5) compared with controls (n = 5; unpublished data). The increase in the gene expression of F₀ subunits may explain in part the greater resistance of mitochondria isolated from the hearts of copper-deficient animals to the oligomycin blocking of proton flow.

The mRNA content of mitochondrial F₁F₀-ATP synthase ß subunit was depressed in the hearts of copper-deficient rats in this study. The ß subunit catalyzes the ATP synthesis in the catalytic sector F_1, but subunit is important for the structural stability of the F₁ sector (Giraud and Velours 1997 , Pan et al. 1998 ). The lower mRNA of the ß subunit could be the result of adaptation to the decreased peptide of the subunit in the hearts of copper-deficient animals. Another explanation is the potential influence of thyroid hormone on mammalian ß subunit gene promoter (Martin et al. 1996 ). The decreased thyroid hormone levels observed in copper-deficient rats may decrease the ß subunit gene expression indirectly (Lukaski et al. 1995 ). In addition, the reduced CCO activity may provide a feed-forward mechanism to reduce ß subunit gene expression and conserve ATP.

The abnormal mitochondrial morphology and altered structural components observed in the hearts of copper-deficient rats apparently affect cardiac energy metabolism and substrate utilization. Excessive lipid droplet accumulation in the hearts of copper-deficient rats has been reported in several previous studies (Medeiros and Wildman 1997 , Wildman et al. 1994 ). In this report, we showed that increased lipid droplet volume density in the hearts of copper-deficient rats was accompanied by a decreased level of MCAD mRNA encoding the ß-oxidation cycle enzyme. MCAD catalyzes a rate-limiting step of fatty acid ß-oxidation in the mitochondria. Fatty acids are the major metabolic fuel for healthy hearts. However, cardiac energy substrates shift from fatty acids to glucose during cardiac hypertrophy (Sack et al. 1997 ). It has been demonstrated that MCAD gene expression is downregulated in hypertrophied hearts induced by pressure overload (Sack et al. 1997 ). It is possible that copper deficiency and pressure overload–induced cardiac hypertrophy share a common regulatory pathway for energy metabolism. It is not known from this study whether decreased mRNA level in the hearts of copper-deficient rats is due to transcription or mRNA stability. Nuclear run-on assays would be of value to evaluate the latter possibility. However, it has been shown that copper deficiency stimulates lipogenesis by inducing fatty acid synthase gene expression at the transcriptional level (Wilson et al. 1997 ). Transcriptional regulation in the hearts of copper-deficient animals with impaired mitochondrial respiratory function may contribute to the switch of energy substrate by blocking the normal metabolic pathway of fatty acid oxidation and affect the enzymes involved in the process. The accumulation of lipid droplets in the hearts of copper-deficient animals could be a result of reduced MCAD activity. Copper deficiency appeared to affect gene expression of MCAD more than that of LCAD in the heart, because mRNA levels of LCAD were not significantly different between the dietary treatment groups. One possible explanation is that medium-chain fatty acids are oxidized preferentially in the mitochondria, whereas an alternative metabolic pathway for long-chain fatty acids is peroxisomal ß-oxidation (Osmundsen et al. 1990 ). As a result, MCAD may be affected to a greater extent by abnormal mitochondrial respiratory function than LCAD. Despite the greater mitochondrial volume density and mtTFA protein level, a derangement in energy metabolism in the hearts of copper-deficient rats is suggested by the decreased transcripts of ATP synthase ß subunit as well as MCAD. Measurements of the actual protein levels would be useful.

In conclusion, the hearts of copper-deficient rats had increased mitochondrial volume density and mtTFA protein, suggesting that mtTFA protein may have stimulated certain components of the cardiac mitochondrial biogenic response. The alterations of gene expression in F₁F₀-ATP synthase subunits indicate that subunits in the F₁ and F₀ sectors may be regulated by differential mechanisms in the hearts of copper-deficient rats. The downregulation of MCAD gene expression could contribute to the disturbed fatty acid metabolism and result in excessive lipid droplet accumulation in the hearts of copper-deficient rats and possibly to the observed hypertrophic growth response. Further studies to measure NRF-1 and NRF-2 DNA binding activities as well as some other transcription factors over the course of dietary copper restriction will be required to unravel the complex regulatory pathway activated in the hearts of copper-deficient animals.

	ACKNOWLEDGMENTS

 
We are grateful to David A. Clayton, Department of Developmental Biology, Stanford University School of Medicine, for generously providing the purified human mtTFA protein and anti-mouse mtTFA antiserum.

	FOOTNOTES

 
¹ Supported by grant R01HL56771 from the National Institutes of Health.

³ Abbreviations used: CCO, cytochrome c oxidase; Cu, Zn-SOD, Cu, Zn-superoxide dismutase; FAO, fatty acid oxidation; h-mtTFA, human mitochondrial transcription factor A; LCAD, long-chain acyl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; mtTFA, mitochondrial transcription factor A; NRF, nuclear respiratory factor; PGC-1, peroxisome proliferator-activated receptor- coactivator.

Manuscript received February 7, 2000. Initial review completed February 29, 2000. Revision accepted April 7, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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