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J Biol Chem, Vol. 275, Issue 5, 3343-3347, February 4, 2000

Effects of Aging on Mitochondrial DNA Copy Number and Cytochrome c Oxidase Gene Expression in Rat Skeletal Muscle, Liver, and Heart^*

Rocco Barazzoni, Kevin R. Short, and K. Sreekumaran Nair

From the Endocrine Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota 55905

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mitochondrial DNA (mtDNA) deletions and mutations have been reported to occur with aging in various tissues. To determine the functional impact of these changes, we measured mtDNA copy number, mitochondria-encoded cytochrome c oxidase (COX) subunit I and III transcript levels, and COX enzyme activity in skeletal muscles (medial and lateral gastrocnemius and soleus), liver, and heart in 6- and 27-month-old rats. Substantial age-related reductions of mtDNA copy number occurred in skeletal muscle groups (23-40%, p < 0.03) and liver (50%, p < 0.01) but not in the heart. The decline in mtDNA was not associated with reduced COX transcript levels in tissues with high oxidative capacities such as red soleus muscle or liver, while transcript levels were reduced with aging in the less oxidative mixed fiber gastrocnemius muscle (17-22%, p < 0.05). Consistent with transcript levels, COX activity also remained unchanged in aging liver and heart but declined with age in the lateral gastrocnemius (32%, p < 0.05). Thus, the effects of aging on mitochondrial gene expression are tissue-specific. A substantial age-related decline in mtDNA copy number proportional to tissue oxidative capacities is demonstrated in skeletal muscle and liver. mtDNA levels are in contrast preserved in the aging heart muscle, presumably due to its incessant aerobic activity. Reduced mtDNA copy number has no major effects on mitochondrial encoded transcript levels and enzyme activities in various tissues under these base-line study conditions. In contrast, maintenance of mitochondrial transcript levels that may be linked to oxidative metabolism and energy demand appears to be the main determinant of mitochondrial oxidative capacity in aging tissues.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Aging is associated with deletions and mutations of mitochondrial DNA (mtDNA)¹ resulting from the combined effects of intense oxidative damage (1-4) and low efficiency of mitochondrial DNA repair systems (5). Age-associated mtDNA mutations have been proposed as a possible cause of senescence (1, 2) because of their potential negative impact on mitochondrial gene expression and oxidative capacity. However, whether, to what extent, and in which tissues age-related oxidative alterations affect mitochondrial gene expression remains largely to be defined. In highly oxidative organs such as liver and heart, the effects of aging on specific steps of mitochondrial gene expression and respiratory chain activity are indeed controversial (6-11). Impaired mitochondrial enzyme activity (12) and reduced mitochondrial protein fractional synthetic rate (13) are associated with reduced muscle mass and endurance capacity in aging skeletal muscle (14-16), but the molecular level at which these changes occur is not known. Moreover, skeletal muscle groups are highly heterogeneous with respect to oxidative metabolism and function (17). In particular muscle fiber composition and oxidative metabolism have been shown to influence skeletal muscle mitochondrial gene expression in adult animal models (18). However the impact of these variables on aging mitochondrial gene expression has not been previously investigated.

To address these issues, we measured mitochondrial DNA copy number and mitochondrial DNA-encoded cytochrome c oxidase (COX) subunits I and III mRNA expression as well as COX enzyme activity in tissues of young and old rats. Cytochrome c oxidase was selected because of its key role as a flux-generating enzyme in the electron transfer chain (19). Furthermore, its transcript levels reflect other mitochondria-encoded genes in that all information on mitochondrial DNA is transcribed into single policystrionic products (20, 21). Skeletal muscle as well as liver and heart muscle were studied in order to include tissues with a wide range of oxidative capacities and therefore different potential for age-related mitochondrial oxidative damage (1-4). Finally, the role of different functional and metabolic characteristics in different skeletal muscle groups was further addressed by studying the lateral and medial portions of the moderately oxidative gastrocnemius with mixed type I-II fiber content as compared with the highly oxidative type I soleus muscle (17).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals and Experimental Protocol-- Young (n = 7, age 6 months) and old (n = 9, age 27 months) Fischer 344 male rats were purchased from the NIA, National Institutes of Health. All animals were fed a standard commercial chow diet, and animal care as well as all experiments were carried out in keeping with institutional guidelines. On the study day, rats were injected with an intraperitoneal overdose of sodium pentobarbital. The medial and lateral head of the gastrocnemius muscle, the soleus muscle, liver, and heart were then quickly removed in this order. Tissues were immediately frozen in isopentane, cooled to the temperature of liquid nitrogen, and stored at 80 °C until analysis.

DNA Analysis-- To measure mtDNA copy number, cDNA probes for mtDNA-encoded cytochrome c oxidase subunit I (COX I) and nucleus-encoded 28 S rRNA genes were generated by reverse transcription-PCR amplification from skeletal muscle total RNA. Primers for the COX I probe corresponded to nucleotides 6255-6274 (forward) and 6615-6635 (reverse; PCR product of 381 base pairs) of the rat mitochondrial genome (GenBank^TM accession no. X14848). The amplified 28 S rRNA cDNA probe (330 base pairs) was a generous gift of Dr. B. McIver. Amplification products were cloned into the TA-plasmid vector (TA Cloning KIT, Invitrogen, CA) used to transfect competent bacteria (TA Cloning KIT, Invitrogen, CA), isolated (Endo Free Plasmid Maxi Kit, Qiagen, Germany), and sequenced before use. Total DNA was extracted from 30-50 mg of each tissue using the Wizard Genomic DNA Isolation kit (Promega, Madison, WI). Heart specimens for all analyses were taken from the left ventricle. For each tissue, 8 µg of DNA from each animal was digested with the EcoRI restriction endonuclease (Promega), separated on one 0.8% agarose gel, and transferred overnight to nylon membranes (HyBond N+; Amersham Pharmacia Biotech) (22). Blots were subsequently hybridized to the COX I, and the 28 S rRNA probes were radiolabeled with [³²P]CTP (Decaprime KIT; Ambion, Austin, TX) as follows. All membranes were prehybridized at 68 °C for 30 min (ExpressHyb Hybridization Solution; CLONTECH, Palo Alto, CA), hybridized to the radioactive probe for 1 h at 68 °C, and then washed at room temperature three times for 10 min in 2× SSC, 0.05% SDS and at 50 °C two times for 20 min in 0.1× SSC, 0.1% SDS. Membranes were then exposed to films at 80 °C for 3-16 h (Kodak Biomax MR; Eastman Kodak Co.). The resulting images were quantitated by laser densitometry (Ultroscan; Amersham Pharmacia Biotech).

RNA Analysis-- Total RNA was isolated from 30-50 mg of each tissue by the guanidinium method (Tri Reagent; MRC, Inc., Cincinnati, OH). For each tissue, 15 µg of total RNA from each animal were separated on one 1.5% agarose, 2.2 M formaldehyde gel, transferred overnight to nylon membranes (HyBond N+, Amersham Pharmacia Biotech) (22), and hybridized as reported above with COX I, COX III, and 28 S rRNA radioactive probes in this order. The COX I probe was the same used for DNA analysis, whereas different cDNA probes were generated as reported above for COX III and 28 S rRNA. Primers for reverse transcription-PCR amplification of the COX III probe corresponded to nucleotides 8836-8856 (forward) and 9174-9190 (reverse; PCR product of 355 base pairs) of the rat mitochondrial genome (GenBank^TM accession no. X14848). Primers for the 28 S rRNA probe corresponded to nucleotides 4203-4222 (forward) and 4370-4389 (reverse; PCR product of 186 base pairs) of the rat ribosomal RNA genome (GenBank^TM accession no. V01270). Signal detection and quantification following hybridization were carried out as described for DNA analysis.

Cytochrome c Oxidase Enzyme Activity-- COX enzyme activity was measured spectrophotometrically from tissue homogenates as previously reported (13). This measurement was not performed in the soleus muscle due to insufficient tissue availability.

Calculations-- In both Southern and Northern blots, COX I and COX III bands in each tissue were normalized to the corresponding 28 S rRNA band, and individual results were expressed as a percentage of the average value for young animals. mtDNA levels were thus normalized to nuclear DNA content as expressed by the abundance of the 28 S rRNA gene. Although it is theoretically possible that variations in nuclear DNA levels may have contributed to the current results, we consider it unlikely for several reasons. In particular, no studies have shown increased amounts of total DNA from young adult to old age. Several studies have in turn reported stable DNA content in aging skeletal muscle (23) as well as heart (24) and liver (25). Changes in liver ploidy also appear to be complete by 2 months of age in rats, and nuclear content is substantially unchanged thereafter in this tissue (25).

Statistical Analysis-- Results in young and old rats were compared using Student's t test for unpaired data. Linear regression analysis was used to study the relationship between different variables. p values of less than 0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mitochondrial DNA and COX I and III mRNA Levels-- mtDNA copy number was significantly reduced in old animals in all skeletal muscle groups and in the liver but not in the heart (Fig. 1). The mtDNA decline was similar in the red and white portions of the gastrocnemius (by 23-25%), whereas it was higher in the more oxidative soleus muscle (40%) and highest in the liver (approximately 50%). In contrast to mtDNA, COX I and III transcript levels were not reduced by aging in the soleus muscle and liver (Figs. 2 and 3), while a significant decline was observed in the gastrocnemius muscle (17-22%, Fig. 2). COX transcript levels relative to mtDNA were comparable in young and old animals in both portions of the gastrocnemius, while they were markedly increased in the soleus and liver (+70-240%; Fig. 2-3). At variance with the other tissues, both cardiac COX mRNA levels and mRNA:mtDNA ratios were unchanged by aging in old animals (Fig. 3).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effects of aging on mitochondrial DNA content in gastrocnemius medial, gastrocnemius lateral, soleus, liver, and heart tissues. Bars represent average ± S.E. of values from seven young and nine old animals. Under each bar are shown representative bands from two animals of each age group. Top bands show signals from the mtDNA fragment (3.0 kilobases), and bottom bands show signals from the nuclear DNA fragment containing the 28 S rRNA gene (6.4 kilobases). *, statistically different results (p < 0.03 or less) using Student's t test for unpaired data comparing young and old rats.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Effects of aging on COX I and COX III mRNA levels and their ratios to mtDNA in skeletal muscle groups. A, COX I and COX III mRNA levels in gastrocnemius medial, gastrocnemius lateral, and soleus muscles. All individual ratios were expressed as a percentage of the average value in young rats. Under each bar, two representative bands from two animals of each age group are shown. Top bands show signals from COX I (~2-kilobase) or COX III (~1.2-kilobase) mRNA, and bottom bands show signals from the 28 S rRNA probe. B, COX I and COX III mRNA:mtDNA ratio in gastrocnemius medial, gastrocnemius lateral, and soleus muscles. mtDNA values used here are the same as reported in Fig. 1. *, statistically different results (p < 0.05) using Student's t test for unpaired data comparing young and old rats.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effects of aging on COX I and COX III mRNA levels and their ratios to mtDNA in liver and heart. A, COX I and COX III mRNA levels in liver and heart tissues. All individual ratios were expressed as a percentage of the average value in young rats. Under each bar, two representative bands from two animals in each age group are shown as in Fig. 2. B, COX I and COX III mRNA:mtDNA ratio in the liver and heart. All individual ratios were expressed as a percentage of the average value in young rats. mtDNA values used here are the same as reported in Fig. 1. *, statistically different results (p < 0.01) using Student's t test for unpaired data comparing young and old rats.

COX Enzyme Activity-- COX enzyme activity was significantly lower in old animals only in the lateral head of the gastrocnemius (32%; Table I). In the medial gastrocnemius as well as liver and heart tissues, COX activities were unaltered in old as compared with young rats. A positive correlation was observed in the lateral and medial portions of the gastrocnemius muscle between COX I (r = 0.36, p = 0.05) and III (r = 0.39, p = 0.04) mRNA and COX enzyme activity. A similar correlation was also found in all tissues between COX I (r = 0.29, p = 0.02) and III (r = 0.24, p = 0.06) mRNA and COX enzyme activity when expressed as percentages of average values in young animals in each tissue.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The current data demonstrate that a substantial mitochondrial DNA depletion occurs with aging in skeletal muscle and liver. Furthermore, such a decline is proportional to tissue oxidative capacities as indicated by cytochrome c enzyme activities. Therefore, the present results indicate that reduced mitochondrial DNA copy number may be a major result of cumulative oxidative damage in aging tissues along with previously reported mutations and deletions (1-4). On the other hand, the preserved mitochondrial DNA as well as COX transcript levels and enzyme activity in the highly oxidative aging heart muscle are intriguing in their implication that template depletion is not an inevitable result of age-associated oxidative alterations. The mechanisms determining the preserved cardiac mitochondrial DNA levels as well as gene expression in old animals are likely to include the unique incessant myocardial contractile activity. Previous studies have indeed shown that long term aerobic exercise in skeletal muscles may increase mitochondrial density in humans (14) as well as mitochondrial DNA content in animal models (18, 26, 27). Left ventricular hypertrophy is also a common feature of the aging myocardium in both humans and rodents (28), and experimentally induced hypertrophy increased cardiac mitochondrial DNA content in adult rats (29, 30). Differential effects of aging on mitochondrial antioxidant defense systems should also be taken into account. Mitochondrial manganese-superoxide dismutase activity indeed appears to be higher in the heart than in skeletal muscle (31, 32), and this difference is enhanced in aging rats by larger age-related increments in heart (31, 32). Interestingly, the high manganese-superoxide dismutase activity in the liver does not increase with age (32), suggesting that a relative deficiency of mitochondrial antioxidants may contribute to the substantial age-associated decline in mitochondrial DNA in this tissue. Of note, the findings in this paper are supported by reports of reduced mitochondrial density in aging skeletal muscle (14) and liver (33) with unchanged mitochondrial number in the aging heart (34).

In contrast with the age effect on mitochondrial DNA copy number, the effects of aging on mitochondrial transcript levels were limited to the mixed gastrocnemius muscle, with more pronounced changes observed in the lateral than in the medial less oxidative portion (17). Most importantly, no changes were detected in the aging soleus muscle and liver despite marked reductions of mitochondrial template availability. Moreover, transcript levels relative to mitochondrial DNA were either preserved or markedly increased in all aging tissues. Finally, transcript levels were positively related to oxidative capacities in all tissues as well as in the two portions of the gastrocnemius. The above observations support several important conclusions. First, the impact of template depletion on mitochondrial gene expression may vary in different organs and in skeletal muscle groups with different fiber composition. Furthermore, transcript levels can be maintained despite reduced template availability in aging skeletal muscle and liver, thereby implying compensatory age-related changes of transcription efficiency, transcript stability, or both. Finally, the maintenance of transcript levels appears to be the most important step in regulating mitochondrial oxidative capacity in aging rat tissues.

The current findings in skeletal muscle indicate that different molecular mechanisms may regulate mitochondrial gene expression in type I and mixed type II-type I muscle groups in aging animals, and this conclusion is consistent with previous studies in growing or adult animal models (18, 26, 27). In particular, increased oxidative capacities following exercise training or chronic electrical stimulation were primarily associated at the molecular level with increased mitochondrial DNA content in moderately oxidative type II (18, 26) but with increased mitochondrial transcripts in highly oxidative type I muscle groups (27). These differential results are likely to be due at least in part to the different mitochondrial density (17) as well as mitochondrial DNA content (18) in different fiber types. More importantly, they suggest that reduced mitochondrial DNA availability may be a limiting factor of mitochondrial gene expression in type II but not in type I fibers as indeed confirmed by the current investigation.

In the presence of reduced mitochondrial DNA copy number, the transcript:template ratios were higher in the more oxidative tissues in this study. Both the COX I (r = 0.69; p < 0.001) and COX III (r = 0.59; p = 0.001) mRNA:mtDNA ratios were positively related with oxidative capacity COX activity in skeletal muscles and liver in old animals. These observations suggest that oxidative metabolism itself might enhance mitochondrial transcript levels in aging tissues in the presence of reduced mitochondrial DNA. Oxygen consumption and organ perfusion are highest in the liver and heart, and they are also substantially higher in type I postural compared with type II exercise-related skeletal muscles (35). A stimulatory drive of energy demand on mitochondrial transcription and respiratory activity could be therefore mediated by oxidative substrate availability. In vitro studies have indeed shown that fatty acid supply is directly related to its mitochondrial oxidation rate (36), and increased mitochondrial transcript:template ratio was recently reported in diabetic human skeletal muscle in which glucose and fat availability are substantially enhanced (37).

The measurement of COX activities may reflect mitochondrial oxidative capacities and is therefore an important index of mitochondrial function. Consistent with changes in mitochondrial transcript levels, this was not affected by aging in the liver and heart, while it was reduced in the lateral but not in the medial portion of the gastrocnemius muscle. The current data therefore indicate that reduced mitochondrial function may contribute to the age-related dysfunction (12-16) of exercise-related mixed fiber muscles with particular regard to muscle fatigability, which is inversely related to ATP availability. On the other hand these results also suggest that mitochondrial alterations are unlikely to influence aging liver and heart function. With respect to the heart tissue, this observation is in good agreement with previous studies that show no effects of aging on the myocardial potential for energy production in vivo (28). It is, however, possible that under conditions of acute stress demanding short term increments of oxidative phosphorylation, the reduced mitochondrial DNA copy number may become a limiting factor in the liver and possibly enhance the observed age-related impairment of skeletal muscle oxidative capacity. Possible differential effects of aging on other respiratory enzymes as previously reported should also be considered (38). These possible differences are, however, likely to be due to post-transcriptional events. Available evidence suggests that mitochondrial genes are transcribed in a parallel fashion (20, 21), and the observed changes in COX transcript levels should therefore be representative of the directional changes of other mitochondrial transcripts. Thus, possible differential age-related alterations of other enzymes do not contradict the current conclusions on the age effect on mitochondrial gene expression and COX activity.

A previous study in humans undergoing orthopedic surgery presumably for leg injury or joint replacement reported a positive correlation between age and mitochondrial DNA levels in the quadriceps muscle (39). The population selected is, however, unlikely to be representative of healthy young and elderly subjects. In particular, disease and related immobilization may have had major effects on skeletal muscle metabolism and thus influenced the study results. Species differences as well as technical differences related to the use of dot blot as opposed to Southern blot might also contribute to the observed discrepancies. Conflicting results were observed in previous animal studies with respect to age-related changes in mitochondrial DNA in heart or liver (6-11). In these reports, general technical differences from the current study are again represented by the use of the dot blot technique to measure mtDNA copy number. Furthermore, except for one study (11), all other groups used isolated mitochondrial preparations (6-10) as opposed to whole tissue. Published data indicate that mitochondria isolation results in selection of specific mitochondrial subpopulations (40), and this technical issue might at least partially explain the conflicting literature data (6-11). Following mitochondrial isolation and quantitative hybridization, one group reported contradictory results with either unchanged (7) or increased (8) mitochondrial DNA levels in both aging heart and liver in similar groups of rats. Another study reported decreased mitochondrial DNA in the heart but not in the liver in aging rats (9). In the latter study, DNA was also extracted from isolated mitochondria and was normalized for tissue protein content, which is known to decrease in aging animal liver (41). Two of the above studies also reported reduced cardiac mitochondrial transcript levels (7, 11). RNA was however isolated from tissue collected at least 15 min after death of the animal in one study (11) or following mitochondria isolation procedures (7). In our experience, both experimental conditions may cause significant amounts of RNA degradation, which might in turn explain at least in part the differences with the current results. The current data are thus the first to indicate a consistent age-related decline of mitochondrial DNA content proportional to oxidative capacities in different skeletal muscle groups and liver with preserved mitochondrial template as well as transcript and enzyme levels in the aging heart.

In conclusion, we demonstrate that the effects of aging on mitochondrial gene expression are tissue-specific. Reduced mitochondrial DNA copy number is a major age-related alteration in skeletal muscle and liver, where it appears to be a direct result of oxidative damage. In contrast, DNA levels are preserved in the aging heart muscle presumably because of its unique aerobic workload. Age-related changes in mitochondrial DNA copy number have, however, limited impact on further steps of mitochondrial gene expression under base-line conditions. In contrast, maintenance of transcript levels appears to be a key factor in the regulation of mitochondrial oxidative capacity in old animals, and the current results suggest that it may be linked to oxidative metabolism and energy demand.

	ACKNOWLEDGEMENTS

We thank D. Morse and J. Schimke for skillful technical assistance and Drs. B. McIver and L. Hofbauer for helpful comments and suggestions.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants RO AG 09531 and RR 00585.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Mayo Clinic, Endocrine Research Unit, Joseph 5-194, Rochester, MN 55905. Tel.: 507-255-2949; Fax: 507-255-4828; E-mail: nair.sree@mayo.edu.

	ABBREVIATIONS

The abbreviations used are: mtDNA, mitochondrial DNA; mtDNA-encoded COX, cytochrome c oxidase; COX I and III, mtDNA-encoded cytochrome c oxidase subunit I and III, respectively; PCR, polymerase chain reaction.

	REFERENCES

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