Mitochondrial adaptations in denervated relationship to muscle performance

muscle:

KAREN L. WICKS AND DAVID A. HOOD Department of Physical Education, York University, North York, Ontario M3J lP3, Canada

WICKS, KAREN L., AND DAVID A. HOOD. Mitochondrid adaptations in denervated muscle: relationship to muscle performance. Am. J. Physiol. 260 (Cell Physiol. 29): C841-C850, 1991.-We have studied mitochondrial adaptations in muscle subject to chronic denervation, and their relationship to muscle performance, using a model of unilateral sciatic nerve denervation in rats over periods of 2, 5, 8, 14, 21, 28, 35, and 42 days (n = 5-9 rats/day). Time to peak tension (TPT), one-half relaxation time (SRT), and endurance performance were evaluated during in situ stimulation of denervated and contralateral gastrocnemius-plantaris muscles. Denervation led to a 70% decline in muscle mass after 42 days. TPT and 1/2RT increased 17 and 30%, respectively, indicating a transformation toward slower muscle. The activities of the enzymes cytochrome-c oxidase (CYTOX), succinate dehydrogenase, and citrate synthase were decreased by 8-14 days, and by 42 days these were 34-58% of control. The mitochondrial phospholipid cardiolipin was reduced earlier, by 5 days, and gradually decreased to 37% of control. Thus phospholipid removal appears to precede the loss of enzyme activity during decreases in mitochondrial content. Endurance performance was reduced in parallel with decreases in enzyme activity and cardiolipin. Cytochrome c mRNA levels decreased to 52% of control by 5 days. Denervation resulted in coordinated changes in mRNA levels encoding the nuclear-derived CYTOX subunit VIc and the mitochondrially derived CYTOX subunit III. However, changes in CYTOX activity did not always parallel alterations in subunit mRNA levels. Thus transcriptional and translational mechanisms operate in regulating mitochondrial gene expression during denervation.

chondrion the ability to synthesize a number of its own proteins. However, the degree of its autonomy as an organelle is limited, since -90% of its protein constituents are nuclear gene products and must be imported into the mitochondria (37). Mitochondrial biogenesis, therefore, involves the close cooperation of both the nuclear and mitochondrial genomes. Experimental models involving chronic use of skeletal muscle have proven useful in improving our understanding of the processes of mitochondrial biogenesis. For example, mitochondrial content and tissue oxidative capacity can be variably increased 25-100% by endurance training (cf. Ref. 33 for review). Such studies have demonstrated a close relationship between increases in the mitochondrial content of the muscle and improvements in its endurance performance. A more dramatic three- to sixfold increase in mitochondrial content can be induced using chronic, low-frequency, indirect stimulation of fast-twitch skeletal muscle. This results in large phenotypic changes in rabbit muscle resembling a “white-tored” transformation (21). Recent work has extended our understanding of this transformation beyond the protein level to the level of gene expression. Williams et al. (39) first demonstrated increases in cytochrome b and cytochrome-c oxidase subunit VIc transcripts in skeletal muscle. Subsequently, Hood et al. (23) have shown that a coordinated expression exists between mitochondrial(subunit III) and nuclear-encoded (subunit VIc) mRNAs of cytochrome-c oxidase in response to chronic stimulacytochrome-c oxidase; gene expression; contractile properties; tion. cardiolipin; endurance; oxidative enzymes; cytochrome c In contrast to the mechanisms of mitochondrial biogenesis, relatively little attention has been paid to the MITOCHONDRIA are highly labile organelles whose num- processes involved in mitochondrial degradation. The ber, composition, and morphology can be altered in re- question remains as to whether mitochondria are desponse to various physiological demands placed on the graded and replaced as entire units, or sequentially by cell. The mitochondrial content of a tissue, as well as the the action of distinct proteolytic enzyme systems (7). Substantial differences in the half-lives of a number of protein and phospholipid profiles of a given population proteins (14) and phospholipids (ll), along with the of mitochondria, are functions of the balance between rates of synthesis and degradation of constituent mole- discovery of endogenous mitochondrial phospholipases cules. A great deal is known about cellular protein and and proteases (7), support the latter theory. Furtherphospholipid turnover. However, the specific mecha- more, the mechanisms governing the expression of nuclear and mitochondrial genes during organelle degranisms of assembly and dissassembly of mitochondrial components during conditions of enhanced biogenesis or dation are not established. degradation are not yet fully understood. Dramatic decreasesin mitochondrial enzyme activities It is believed that mitochondrial formation occurs have been observed after long-term denervation (16,18). Chronic denervation of skeletal muscle may therefore through growth and division (4), whereby newly synthesized phospholipids and proteins are incorporated into a represent a useful model for the study of hypotheses preexisting lipid bilayer (37). Possession of its own tranregarding mitochondrial degradation. A careful evaluation of the time-dependent changes in specific mitochonscription and translation system confers upon the mito0363-6143/91

$1.50

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drial constituents throughout the course of denervation has not yet been performed. This time course approach has been used in other experimental models (i.e., chronic muscle stimulation) to provide insight into the regulation and coordination of biochemical adaptive processes (23). Therefore, the purpose of this study was to examine the effect of muscle denervation on the chronological pattern of change of mitochondrial proteins, lipids, and mRNAs and to relate these changes to the functional properties of the muscle, including its endurance performance. Knowledge of the temporal changes of these key constituents during decrements in mitochondrial content should provide insight into the mechanisms by which mitochondria are disassembled. METHODS

Animals and Tissues Male Sprague-Dawley rats (n = 80,300-450 g; Charles River Canada, St-Constant, Quebec, Canada) were housed individually and given food and water ad libitum. Animals were randomly assigned to groups that were unilaterally denervated for 2, 5, 8, 14, 21, 28, 35, or 42 days (n = 5-9 per time period). Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg). To denervate the hindlimb muscles, the left sciatic nerve was exposed, and a 5-mm section was excised. The incision was closed with metal clips after administration of sterile ampicillin antibiotic (Penbritin, Ayerst, Montreal, Canada). The sham-operated contralateral leg served as an internal control for each animal. In addition, muscles from three other nonoperated animals were used to compare with sham-operated limbs and to evaluate any systematic differences between the right and left hindlimb muscles. After surgery, the hind feet of the animals were painted with saturated picric acid to discourage them from damaging the denervated foot. The day of denervation represented day 0 of the experimental time course. Stimulation After the denervation period, animals were anesthetized as above. A catheter was inserted into the carotid artery and attached to a pressure transducer (Gould RS 3200, Cleveland, OH) to monitor blood pressure. The gastrocnemius-plantaris muscles of both the denervated and control sides were then exposed and prepared for in situ stimulation. The sciatic nerve was tied and cut, providing a nerve stump for indirect stimulation. Platinum electrodes were inserted at each end of the muscle parallel to its length for direct stimulation. The leg was stabilized via a pin inserted through the femur, with the Achilles tendon attached to a strain gauge. The soleus muscle was cut at its insertion in the Achilles tendon and thus did not contribute to the force output measured. The temperature of the muscle was maintained at 37°C with a heat lamp and monitored using a surface thermometer (Yellow Springs). Oxygen (100%) was provided to the animals throughout the experiment. The voltage required to elicit a maximum contraction via direct muscle stimulation was initially established for both dener-

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vated and contralateral hindlimbs. In contrast to the contralateral limb, the denervated muscle was completely unresponsive to indirect nerve stump stimulation. The contractile properties of the muscle (time to peak tension, one-half relaxation time) were evaluated by eliciting a short series of single supramaximal twitches at a high paper speed. The gastrocnemius-plantaris group was then stimulated via the muscle at a twitch frequency of 1 Hz for 5 or 15 min. Tissue Sampling and Preparation After the stimulation protocol using both limbs, all denervated and control muscles (red, white, and mixed gastrocnemius, plantaris, soleus, tibialis anterior, and extensor digitorum longus) were excised, freeze-clamped, and stored at -70°C. Mixed gastrocnemius, white and red quadriceps, soleus, and heart muscles were also obtained from nonoperated animals and treated similarly. Muscle water contents were determined using a small section of the gastrocnemius muscle dried to a constant weight at 70°C. Mixed gastrocnemius muscles were pulverized to a fine powder with a stainless steel mortar cooled to the temperature of liquid N2 (23). Powders were then stored under liquid N2 until analyses of lipids, enzymes, and specific mRNAs were performed. Lipid Extraction Total lipids were extracted as described by Christie (6). This method was initially designed for l-g samples of various tissue types and was therefore modified to permit its application to small skeletal muscle samples weighing 100-500 mg. Briefly, the procedure involved three successive homogenizations of tissue powder in methanol (10 ml/g), chloroform (20 ml/g), and chloroform-methanol (2:1, vol/vol; 30 ml/g). The extract was filtered under vacuum, and the remaining powder was washed again with chloroform and methanol. The lipids were partitioned by mixing the final filtrate first with 0.25 vol potassium chloride (O.SS%), and then with 0.25 vol methanol-water (l:l, vol/vol). The filtrate was centrifuged (10 min, 1,800 g), and the lower phase, containing phospholipids, neutral lipids, and glycolipids, was kept for analysis. To avoid degradation of lipids, butylated hydroxytoluene (O.Ol%, wt/vol) was added to all reagents as an antioxidant. Lipid extracts were used for determination of cardiolipin content immediately after the lipid extraction. In preliminary experiments, we found that avoiding sample storage resulted in superior phospholipid resolution, and maximum cardiolipin recovery. Quantification of Cardiolipin Two-dimensional thin-layer chromatography was employed to separate the components of lipid extracts on Whatman HP-KF plates (10 x 10 cm; Chromatographic Specialties, Brockville, Ontario, Canada). A volume of lipid extract (5-20 pg phosphorus) was applied to the origin and allowed to migrate in the first dimension using chloroform-methanol-Hz0 (65:25:4, vol/vol). Plates were dried in air for 10 min, then run in the second dimension

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using l-butanol-acetic acid-H20 (60:20:20, vol/vol; Ref. 28). Cardiolipin was identified using an authentic standard (bovine heart cardiolipin, Sigma) run in each dimension on the sample plates. A separate plate was spotted with 5 ~1 cardiolipin standard (23 pg) and was concurrently developed in both dimensions to correct all samples for percent recovery. Phospholipids were visualized by spraying with concentrated sulfuric acid-formaldehyde (97:3, vol/vol; Ref. 28) and heated at 100°C (32) for 20 min. The cardiolipin spot was scraped into a tube containing 70% perchloric acid. An area equal in size to the cardiolipin spot was also scraped from each plate and used to correct all samples for background phosphorus inherent to the silica gel. The samples were then heated over a flame for 20 min to digest the cardiolipin, and the phosphorus content was measured photometrically by the formation of phosphomolybdic acid (32). The content of total phosphorus-containing lipid was determined by quantifying the phosphorus content of 5 ~1 of the unfractionated lipid extract in the same manner. To permit an accurate measurement of cardiolipin in samples as small as 100 mg, the sensitivity of the phosphorus assay (32) was increased U-fold by reducing the volume of water required. Enzymes

and Total Protein

Frozen tissue powders were extracted with 100 mM Na-K-phosphate extraction buffer and 2 mM EDTA, pH 7.2 (23). Cytochrome-c oxidase (CYTOX) and succinate dehydrogenase (SDH) were used as marker enzymes representative of the inner mitochondrial membrane, while citrate synthase (CS) was used as a matrix enzyme marker. The maximum activity of CYTOX was measured by the rate of oxidation of fully reduced cytochrome c (19). CS and SDH activity were measured as described by Reichmann et al. (31). Tissue total protein content was measured according to Lowry et al. (26).

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resis (27). Slot blots of total RNA were prepared on Hybond-N nylon membranes (Amersham, Canada) using a vacuum blotting apparatus as described previously (19, 23 . cDNA-RNA

Hybridization

The slot blots were hybridized to radioactively labeled ([““P]dCTP) complementary DNA (cDNA) probes containing 1) the 743-bp sequence for the mitochondrially encoded CYTOX subunit III (19, 23), 2) the 243-bp sequence for the nuclear-encoded subunit VIc (19, 23), and 3) the 960-bp sequence of nuclear-encoded cytochrome c (34). The mitochondrial subunit III plasmid was used directly in the nick-translation reaction. The nuclear subunit VIc and cytochrome c inserts were excised from the plasmid using restriction endonuclease cleavage, separated from the plasmid DNA by gel electrophoresis, electroeluted from the gel, and isolated by phenol-extraction and ethanol precipitation (27). The nick-translation and cDNA-RNA hybridization conditions were as recently described (19, 23). The total poly(A)+ RNA content was evaluated by hybridizing the slot blots with end-labeled oligo(dT)zO (Pharmacia; Ref. 15). These values were used to account for any potential variation in total poly(A)+-containing RNA as a result of the denervation process. Autoradiographs were produced by exposing the slot blots to Hyperfilm-ECL (Amersham). Signal intensities were quantified by laser densitometry and expressed as arbitrary scanner units. Statistical

Analyses

The data were analyzed using Student’s t test and oneway and two-way analyses of variance with repeated measures on one factor (contralateral vs. denervated limbs) where appropriate (alpha level of 0.05). RESULTS

RNA Isolation

Structural

Frozen muscle powders were processed for RNA isolation using lOO- to 3OO-mg samples (22). All operations for RNA isolation were done under sterile conditions. Laboratory glassware and equipment were baked overnight in an oven (13O”C), and nonvolatile solutions were autoclaved. Muscle powders were weighed in 13-ml sterile polystyrene tubes containing 2.5 ml of denaturing solution (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% Sarcosyl, 0.1 M ,&mercaptoethanol). Samples were then homogenized at room temperature with a polytron for -2 min. The remaining steps in the isolation procedure were as previously described (22), except that an additional 30-min incubation with DNase, followed by a phenol extraction (22), was added to the procedure. Samples were stored in 75% ethanol at -20°C until just before use, at which time they were resuspended in sterile water. The quality, stability, and concentration of the RNA samples were evaluated spectrophotometrically at 260 and 280 nm, and also by agarose (1.4%) gel electropho-

Control nondenervated muscle weights for the gastrocnemius, soleus, and plantaris were 2.09 $- 0.03, 0.18 t 0.003, and 0.39 t 0.01 g, respectively (n = 58). Denervation resulted in a time-dependent loss of muscle mass, the rate and degree of which were similar in all denervated hindlimb muscles over the course of the experiment (Fig. I). A rapid reduction in muscle mass to 50-60% of the control value occurred after 14 days of denervation, followed by a more gradual reduction to 25-30% of control in all muscles. The size of the sham-operated contralateral muscles did not differ from left or right hindlimb muscles obtained from other nonoperated animals (data not shown). This indicated that sham-operated muscles did not hypertrophy or atrophy in response to any adjustments in weight-bearing distribution after denervation. The total protein content of the control nondenervated gastrocnemius was 177.6 t 7.5 mg/g muscle. Overall, denervation resulted in a reduced protein content (P < 0.05) that was evident between 14 and 35 days and averaged 75% of control muscle. The percent water con-

and Compositional

Properties

of the Muscle

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100 -

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38 Control

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80-

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30 26 22 18 I’

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10 15 20 25 30 Days of Denervation

1. Mass of denervated gastrocnemius as a function of denervation time. 7-10per day. FIG.

control)

=





35



40

1

45

14 0

and soleus muscles (% of Values are means t SE; n

tent of the denervated gastrocnemius muscles (76.3 t 0.2%) did not differ from that of control muscles (75.3 t 0.2%), nor from the water content of left or right nonoperated muscles (76.7 t 0.3 and 76.2 t 0.2%, respectively).

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B 26 c 0 -l-l

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Muscle Performance Contractile characteristics. Denervated

muscle became progressively slower in its contractile characteristics as time progressed. The one-half relaxation time (SRT) of denervated muscle gradually increased to 30 t 6.5% above control muscle, an effect which became significant by 14 days (Fig. 2A; P < 0.05). This slowing of contractile speed was also evident from an increased time to peak tension (TPT) above control (Fig. 2B). This average increase of 17 t 3.4% occurred later than for SRT, since a significant denervation effect (P < 0.05) was not apparent until 21 days. There was no significant effect of denervation time on the TPT or YzRT of contralateral nondenervated muscle. The voltage required to produce 50% of peak tension was 30 t 1.0 V for the denervated muscle. This was eightfold higher than the 3.8 t 0.3 V required to elicit maximal force via direct stimulation of the contralateral control muscle. This difference did not change as a function of denervation time. As expected, the voltage required for half maximum tension development of the control muscle when stimulated via the nerve was much lower (0.25 V). When the voltage required for 50% activation was reexpressed per gram of contracting muscle, a progressive increase with time was observed in the denervated muscles, ranging from 11.5 V/g after 2 days to 42.4 V/g after 42 days. No change was evident over time in the contralateral sham-operated muscles (1.4 t 0.1 V/g).

Tension output and endurance performance. Initial twitch tension in control muscle was 688 t 46 g (n = 28). The initial twitch tension generated by the denervated muscles declined significantly below this beyond 8 days (P < 0.05) of denervation to 54-56% of control at 14 and 21 days, and as low as 21% by 42 days. When expressed per gram of contracting muscle, the initial tension averaged 232 t 18 g/g in contralateral nondenervated muscle. Analysis of variance revealed that de-

0 -c,

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FIG. 2. One-half relaxation time in ms in denervated and contralateral denervation time. Values are means

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and time to peak tension control muscle as a function 2 SE; n = 3-5 per day.

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nervation resulted in a significant increase in the tension produced per gram of denervated muscle when considered over the entire time course. Initial tension rose from 233 t 13.5 g/g at 2 days to 341 t 26 g/g muscle by 8 days (P < 0.05). This was gradually reduced back to lower values (P < 0.05) by 35 days of denervation. The steady-state tension output at 1 Hz was recorded for 5 min in initial experiments and was later lengthened to 15 min. For uniformity, only the responses during the first 5 min are illustrated in Fig. 3. The contralateral nondenervated gastrocnemius-plantaris muscles were able to maintain 106 t 2.5% of their initial tension over the entire stimulation period. Muscles denervated for as short a time as 2 or 5 days failed to generate >85-91% of their initial tension after 5 min. This decrease was progressive as denervation time proceeded, until 14 days. Thereafter, the response became more variable between 21 and 35 days, and a further significant decrement in tension output was not observed until 42 days, when the percent of initial tension generated after 5 min of stimulation was only 29 t 7% of control. Total Phospholipid and Cardiolipin Contents

A significant effect of denervation (P < 0.05) on the content of total phosphorus-containing lipids (TPL) was observed (Table 1). The reduction was 16% by 2 days

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12Or ctl 2d 5d 2id 8d 35d 14d 28d 42d

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(minutes)

3. Muscle endurance performance, expressed as % of initial tension generated over the first 5 min of l-Hz in situ stimulation. Values represent means t SE for each group of animals denervated for 2-42 days (d); n = 7-9 per day. Mean tension generated by all contralateral control (ctl) muscles is also shown (n = 64). FIG.

and -32% between 14 and 42 days. Control values were not significantly different over time. Cardiolipin content was dramatically altered by denervation, becoming significantly lower than control by 5 days (P < 0.05; Fig. 4). Between 14 and 42 days, the cardiolipin content averaged 37% of control. Despite the variable response, analysis of variance revealed no significant effect of time on cardiolipin content in control muscle, which averaged 0.872 t 0.035 pmol/g for all control muscles (n = 45). The proportion of cardiolipin relative to TPL (%TPL) averaged 3.6 t 0.1% in control muscle and was also invariant over time (P > 0.05; Table 1). In denervated muscle, this percentage was significantly reduced (P < 0.05) to -60% of control between 8 and 42 days. Enzyme Activities Figure 5 reveals that the enzyme activities of CYTOX, CS, and SDH were reduced as a function of denervation time (P < 0.05) to 34-58% of control between 28 and 42 days. Significant differences between the denervated and control values became apparent by 8 days for CYTOX and by 14 days for CS and SDH (P < 0.05). Despite the apparent variability with respect to control CS and SDH activities, analysis of variance revealed no significant

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differences between contralateral nondenervated values over time (P > 0.05). The decrease in enzyme activity observed in denervated muscles could not be solely attributed to a generalized loss of total muscle protein. When CYTOX activity was reexpressed per milligram of protein, a significant reduction of 38% was still apparent (data not shown). Total RNA, Poly(A)+ RNA, and Specific mRNAs Total RNA was 553 t 13 pg/g muscle in all contralatera1 control muscles measured (n = 37). The RNA content in these muscles was unaffected by denervation time (P > 0.05). Denervated muscle exhibited an -20% increase in total RNA between 14 and 42 days (P < 0.05). The poly(A)+ RNA content of denervated muscle varied from 70 to 165% of nondenervated control muscle (Table 2), but these were not different (P > 0.05) from control at any time. The CYTOX subunits III and VIc and cytochrome c mRNA content of denervated muscle were markedly reduced to -58-72% of control by 8 days and to 29-46% of control by 35 days, when expressed per microgram of total RNA (P c 0.05; Table 2). When specific variations in the poly(A)+ RNA pool size were accounted for, a significant reduction to 52% of control values was apparent as early as 5 days of denervation for cytochrome c mRNA and to 35-38% at 8 days for both the CYTOX subunit mRNAs (P c 0.05; Fig. 6). Between 8 and 35 days all three specific mRNAs were -40% of those found in control muscle. Analysis of variance revealed no significant differences between the mRNAs encoding VIc and subunit III in the denervated muscles as a function of time (P > 0.05). The increases in subunits III and VIc mRNA levels at 42 days (P < 0.05) could be attributed to the tendency toward a reduction to 70% of control of the poly(A)+ RNA pool, as well as a tendency toward an increase in mRNA levels per microgram RNA (Table 2). Characterization of Different Fiber Types The total phospholipid and cardiolipin contents, as well as oxidative enzyme activities, were determined for fast-twitch white (FTW), fast-twitch red (FTR), slowtwitch red (STR), and heart muscle (Fig. 7). This was

1. Total phospholipid and cardiolipin contents in denervated and contralateral sham-operated control muscles TABLE

Total Days of Denervation

2 5 8 14 21 28 35 42 Values a percent

of control

Cardiolipin %TPL

Pg p/g Denervated

Mean

Phospholipid

622.1t89.0 792.7t119.2 660.0k79.6 504.6t79.0 482.8k59.0 462.3k4.0 452.2t5.8 428.9t73.5 side

%Control Control

809.0t57.4 890.2k88.3 897.5t125.2 775.0t65.8 807.1t104.7 649.7t73.7 680.9t47.5 589.1t74.8 784.5t33.2

are means k SE; n = 4-7 experiments per day of denervation, of control. TPL, total phosphorous-containing lipids.

%Control Denervated

83.8214.6 90.6t12.7 SO.lt10.9 64.4t8.1 63.7k11.9 73.9t8.7 67.6k5.7 71.7t5.3 mean

of control

3.0t0.4 2.5k0.3 2.3t0.2 1.81kO.3 2.3k0.3 2.5k0.2 2.5t0.3 2.0t0.4 side represents

Control

3.0t0.3 3.4t0.4 3.320.4 3.5t0.2 4.5t0.6 4.0t0.3 3.4kO.5 4.1kO.5 3.6tO.l n = 43. %Control,

103k17 77klO 72k7 54k13 52t3 59t3 75t13 50t9 denervated

value

as

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FIG. 4. Cardiolipin muscles as a function = 4-7 per day.

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15 20 25 30 Days of Denervation

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content in denervated of denervation time.

and contralateral Values are means

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41

control t SE; n

done to evaluate the utility of cardiolipin as an index of tissue mitochondrial content, as suggested by Okano et al. (29). These tissues possess up to a l&fold range in mitochondrial contents and therefore present a good comparison to test this hypothesis. As expected, cardiac muscle had the highest cardiolipin content among these muscle fiber types. It was 6.1-fold greater than FTW muscle, and 1.8-fold higher than FTR muscle (P < 0.05). STR was intermediate in cardiolipin content between the two fast-twitch muscles and was Q-fold less than heart. When expressed as a percent of the TPL (Table 3), cardiolipin content ranged from 2.8% in FTW to 7.4% in cardiac muscle (P < 0.05). FTR and heart muscle were equal in this regard (P > 0.05), and were both significantly greater than FTW muscle (P < 0.05). In contrast to the sixfold range in cardiolipin among the different fiber types, the TPL content varied by only threefold between FTW and heart muscle (P < 0.05). The TPL content in FTR muscle was 84% higher than FTW, whereas that of cardiac muscle was at least 60% greater than all other fiber types (P < 0.05). CYTOX, CS, and SDH displayed similar differences among the fiber types (Fig. 7). A 9- to 15-fold difference was observed between enzyme activities in cardiac muscle vs. those in the least oxidative fiber type, FTW. This difference was 2.8- to U-fold between FTR and heart muscle and 5- to 6.6-fold between STR and heart. The relationship illustrated in Fig. 7 suggested that the density of oxidative enzymes per unit of mitochondrial membrane was higher in cardiac muscle. We evaluated this by expressing enzyme activities per micromole of cardiolipin (Table 3). The results reveal that the CYTOX activity per micromole of cardiolipin was greater by 90% (P < 0.05) for heart muscle in comparison with FTW muscle. The three skeletal muscles were not significantly different from each other. Similarly, with respect to SDH, cardiac muscle was 70% greater than in FTR (P < 0.05) and FTW muscle; however, the variability in FTW muscle precluded attaining statistical significance for this fiber type. Activity of CS, a matrix enzyme, did not differ among the fiber types when expressed per micromole of cardiolipin. These data indicate that cardiac muscle possesses a higher density of inner membrane proteins than does skeletal muscle. When the activities of SDH and CYTOX were compared per micromole of cardiolipin. CYTOX was significantlv greater



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10 15 20 25 30 35 40 45 Days of Denervation of cytochrome-c oxidase (A), succinate dehydro-

5

FIG. 5. Activities genase (B), and citrate synthase (C) in denervated and contralateral, sham-operated, control muscles as a function of denervation time. Values are means k SE; n = 5-9 per day. Activities at day 0 represent means k SE of left and right nonoperated, control gastrocnemius muscles.

than SDH in all fiber types (P < 0.05), indicating a greater density of CYTOX enzyme per unit of inner mitochondrial membrane. DISCUSSION

It is well established that denervation results in profound structural and biochemical changes within skeletal muscle (cf. Ref. 12, for review). We have studied the

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2. Poly(A)+ RNA and specific mRNA levels as a ratio of denervated over contralateral nondenervated muscleper microgram of total RNA

TABLE

2 1.57k0.26 1.12t0.17 1.21t0.19 0.91kO.24

Poly(A)+ RNA Subunit III mRNA Subunit VIc mRNA Cytochrome c mRNA Values

are means 1.4

5

1

l

1.33kO.15 0.89t0.20 0.86kO.17 0.70t0.10

1.65t0.73 0.62t0.13 0.58kO.15 0.72t0.18

CYTOX III

1.2-

A CYTOX VIc

g

l.O-

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0.8-

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0.4-

Cytochrome

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’ 35

’ 40

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FIG. 6. Expression of mRNAs encoding the nuclear-derived cytochrome-c oxidase subunit VIc (CYTOX VIc) and cytochrome c and the mitochondrially derived cytochrome-c oxidase subunit III (CYTOX III) in denervated muscle as a function of time. Values are means t SE; n = 3-5 per day. Data represent ratio of denervated over control arbitrary scanner units and have been corrected for variations in the tissue poly(A)+ RNA content.

G

90 80

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s :;

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0.0

21

1.19t0.20 0.4620.12 0.37kO.08 0.45kO.08

1.09t0.20 0.38t0.21 0.71kO.19 0.59t0.20

28

35

1.14kO.21 0.38t0.15 0.28t0.06 0.54kO.20

42

1.04kO.19 0.4OkO.06 0.29t0.11 0.46t0.14

0.7OkO.21 0.63kO.13 0.73t0.18 0.58t0.28

~fr SE; n = 3-5 experiments.

g

LII

14

8

A Citrate n

Cytochrome

l

Succinate

0.5 Cardiolipin

Heart

Synthase c Oxidase Dehydrogenase

1.0

1.5 Content

2.0 (umole/g)

2.5

FIG. 7. Enzyme activity as a function of cardiolipin content in fasttwitch white (FTW), fast-twitch red (FTR), slow-twitch red (STR), and heart muscles. Values are means t SE; n = 5-7 per fiber type.

effects of denervation by using a time course approach in an attempt to determine the interrelationships between changes in various mitochondrial constituents. Given sufficient time resolution, this should provide information regarding mitochondrial disassembly. We

have described simultaneous patterns of change of three important mitochondrial constituents: lipids, proteins, and mRNAs. The mRNAs selected for this study were chosen because they are representative of both the nuclear (CYTOX subunit VIc, cytochrome c) and the mitochondrial (CYTOX subunit III) genomes. This permitted an evaluation of inter-, as well as intragenomic mRNA expression. Cardiolipin was selected as a representative mitochondrial phospholipid, since it is synthesized and localized exclusively in the mitochondria and has been used as an index of mitochondrial inner membrane lipids (cf. Ref. 2 for review). The changes in enzyme activity that have been observed in the present study are in agreement with the results of others (16, 18), and in combination with morphological findings (8), indicate that the mitochondrial content of denervated muscle diminishes as a function of denervation time. These changes are specific to the affected muscle, since enzyme activities measured in the contralateral nondenervated limbs were not different from normal unoperated animals. Thus the results observed are not likely brought about by any humorally mediated substance, but are a direct result of a loss of innervation and/or muscular activity. The enzyme changes are undoubtedly a reflection of a general decrease in muscle protein synthesis, in combination with an accelerated rate of protein degradation (10). Nonetheless, it is evident from the enzyme data expressed per milligram of protein that the loss of specific mitochondrial proteins occurs at a rate which exceeds that of the total cellular protein. Furthermore, the data indicate that the degradation of a specific mitochondrial phospholipid, cardiolipin, exceeds its rate of synthesis during the course of denervation. The specificity of this response is evident from the fact that the percent of total cellular phospholipid consisting of cardiolipin diminishes significantly during the course of denervation. Thus the decreases

3. Total phospholipid content, cardiolipin content as a percent of total phospholipid, cytochrome-c oxidase, citrate synthase, and succinate dehydrogenase activities per micromole cardiolipin in heart and skeletal muscle fiber types

TABLE

Tissue FTW

Total phospholipid, pg P/g Cardiolipin, %TPL Cytochrome-c oxidase, U/pm01 cardiolipin Citrate synthase, U/pm01 cardiolipin Succinate dehydrogenase, U/pm01 cardiolipin Values containing

are means lipids.

k SE; n = 4-7 experiments.

FTR

335k26 2.8kO.7 12.6k3.1 27.4k5.8

7.9k2.4 FTW,

fast-twitch

white;

FTR,

617k50 6.8t1.0 15.4t3.0 25.6k3.0 8.1-t-1.1

fast-twitch

red; STR,

STR

Heart

495k48 3.8t0.5 22.6t3.8 29.7t5.0 9.1t1.5

993t43 7.4t0.9 24.4t3.1 38.0t2.8 13.4t1.5

slow-twitch

red; TPL,

total

phosphorous-

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C848

MITOCHONDRIAL

ADAPTATIONS

observed in mitochondrial constituents are proportionally greater than the decreases in the components of the entire cell. The enzymatic alterations during chronic denervation appear to be highly specific, depending on the metabolic pathway involved. For example, glycolytic enzymes have been shown to decrease to a greater degree than mitochondrial en .zymes (18)) while no change has been observed in creatine phosphokinase activity (39, and increases in enzymes of the pentose-phosphate pathway have been demonstrated (38). Our results show a similar pattern of change of three mitochondrial enzymes as a function of denervation time, supporting the concept that enzymes of the citric acid cycle and the respiratory chain behave as a “constant-proportion group” (30) in denervated muscle. Th .e similar patterns observed for the three enzymes in the muscle fiber types provide additional support for this. Muscle endurance performance was eval uated using lHz isometric contractions, whit h result in a 45fold increase in muscle oxygen consumption (20). We chose this moderate contractile effort recognizing that too severe a contraction protocol would not allow us to distinguish a graded pattern of tension loss during progressive decreases in mitochondrial content that were likelv to occur as a function of denervation time. A numbed of factors probably contributed to the decline in endurance performance observed, including changes in tissue oxidative capacity and blood flow. It is remarkable that a clear description of the alterations in muscle blood flow which occur as a function of denervation time remains to be characterized. The present study has addressed the issue of mitochondria 1 content and its constituents and suggests that, as with the process of mitochondrial synthesis, endurance performance closely parallels changes in the muscle mitochondrial content (Ref. 33; Fig. 8). Morphological studies have documented a large decrease in mitochondrial volume, as well as a large increase in the number of abnormal mitochondria found in denervated muscle (8). This indicates a progressive disassembly of mitochondria, the pattern of which remains speculative. Are mitochondria degraded as a whole, or m l/2 FIT l TPT x % Initial Tension A CYTOX Activity l Cardiolipin

- 120

0

5

10

15 20 25 30 Days of Denervation

35

40

-100

: --h 7

-80

g

-60

;

-40 -20

:. 0 =

45

FIG. 8. Relationship between biochemical [cardiolipin content and cytochrome-c oxidase (CYTOX) activity] and functional adaptations (l/z RT, TPT, and endurance performance) in muscle as a function of denervation time. All values are expressed as a percent of the contralateral nondenervated value, except endurance performance, which is expressed as the percent of initial tension after 5 min of l-Hz stimulation. Values are means k SE; n = 3-9 per day.

IN

DENERVATED

MUSCLE 1.4 1.2 1.0 -

III VIC

0.8 0.6 0.4 n3, VI

0.0

I

0.2

0.4

I

0.6

I

0.8

I

1.0

I

1.2

I

1.4

mf?NA (Den/M) 9. Relationship between decreases in cytochrome-c oxidase (CYTOX) enzyme activity and in mitochondrially (subunit III) and nuclear-encoded (subunit VIc) mRNAs in denervated rat gastrocnemius muscle. Values are means t SE; n = 3-9 per day. 2d, 2 day; 5d, 5 day; 42d, &-day denervated muscle. FIG.

are individual component proteins and phospholipids removed individually while maintaining general structural integrity (7)? If the latter case is true, then one would expect sequential functional changes to occur as denervation time progressed. In support of this, Joffe et al. (24) have documented selective decrements in NADH oxidase and state 3 respiration, but not in cytochrome-c oxidase when expressed per milligram mitochondrial protein in 28-day denervated rat muscle. Our data indicate that the pattern of mitochondrial degradation occurs via the initial removal of membrane phospholipids, followed subsequently by the loss of protein constituents. Significant decreases in cardiolipin content were observed as early as 5 days after denervation, whereas both CYTOX and SDH activities were not significantly diminished until 8 and 14 days, respectively. Support for this pattern of temporal change, first involving alterations in the phospholipid content of mitochondria, followed by changes in specific protein constituents, have been documented during the process of organelle biogenesis. During development, liver mitochondria exhibit an increase in density during the latter stages of gestation (2), and the formation of cardiolipin appears to precede that of cytochrome aa3 (13). Furthermore, there is evidence that accretion of the mitochondrial membrane occurs before insertion of specific protein constituents during the adaptation of yeast from an anaerobic to an aerobic environment (1). The synthesis and degradation of mitochondria may simply be a function of the rate of turnover of individual components, as reflected by their half-lives (&). These appear to range widely among mitochondrial constituents, from a few minutes to 7-10 days (7). Prior work using radioactive tracers (11) has shown that mitochondrial phospholipids have tl/, values that are variable and dependent on the tissue measured. Although skeletal muscle was not among those studied, the minimum value for the tl/, of cardiolipin found in liver and kidney was -11 days. With respect to protein constituents, a similar turnover time of 5-6 days for heme a and cytochromes b and c has been documented (3). An estimate of the tl/, of proteins can also be obtained from the change in concentration during the transition from one steady state to another (21, 36). In the present study, estimates for

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MITOCHONDRIAL

ADAPTATIONS

CYTOX, SDH, and CS were -15 days. The tr/, for cardiolipin appears to be somewh .at shorter, on the order of 8-10 days (Fig. 8). However, more accurate estimates using nonreutilizable tracers are warranted in skeletal muscle. Mitochondria are synthesized in a cooperative fashion, utilizing gene products of both the nuclear and mitochondrial genomes. However, only 10% of the proteins required for organelle synthesis are derived from the mitochondrial genome (4). The remaining 90% include proteins required for the 1transcription of mtDNA , as well as for th .e translation of those gene products into protein subunits. This has led to the view that mitochondrial gene expression is controlled by nuclear-derived factors (9). The transcription of such factors should naturally precede the transcription of mitochondrial genes during organelle biogenesis. However, this may not be true for genes encoding some structural components of the mitochondria. We have recently documented that muscle mRNA levels encoding subunits III and VIc of CYTOX are coordinately expressed during mitochondrial synthesis induced by chronic muscle use (23). Furthermore, these transcripts closely parallel each other across the 13-fold range of m itochon drial con .tents found in selected rat tissues (19). A major purpose of the present study was to evaluate the expression of nuclear and mitochondrial genes during conditions of diminished mitochondrial synthesis and/or enhanced degradation. The results indicate that the coordinated change in CYTOX subunit mRNA levels is preserved during the progressive reduction of mitochondrial content induced by denervation. A coordinated expression may indicate that a common signal, or common regulatory mechanisms, operates in controlling the levels of these mRNAs in muscle. This intergenomic coordinated expression appears to hold for the formation of a single holoenzyme (CYTOX) but was not apparent when mRNAs of more distantly related proteins (e.g., cytochrome b and CYTOX subunit VIc) were measured during mitochondrial biogenesis (39). The cytochrome c mRNA levels obtained in the present study (at 8 days) closely resemble those found by Babij and Booth (5) in 7-day denervated rat gastrocnemius muscle. However, the pattern of change of cytochrome c mRNA does not exactly parallel that of the other nuclear gene product, CYTOX subunit VIc. For example, cytochrome c mRNA levels were depressed earlier during the course of denervation than were CYTOX subunit VIc levels. Furthermore, cytochrome c mRNA remained low at 42 days when subunit VIc mRNA had returned toward control levels. Thus, within the nuclear genome, the expression of mRNAs encoding closely related proteins also differs. The relationship between the expression of the CYTOX subunits and the enzyme protein content (estimated by enzyme activity) is illustrated in Fig. 9. Data points that fall on the line of identity indicate parallel changes in mRNA and protein levels within the cell. This can be interpreted as indicative of transcriptional regulation of CYTOX protein level within the cell (19, 23). Deviations from the line of identity as seen at 2 and 5 days illustrate that low mRNA levels are not accompanied by equal changes in protein content. This could

IN

DENERVATED

C849

MUSCLE

indicate a reduction in transcription rate or reduced mRNA stability, along with little change in protein turnover. In contrast, at 42 days the return of subunit mRNA levels toward control in the absence of increases in enzyme activity could be interpreted as translational control of CYTOX in long-term denervated muscle. It is evident from our data, and that of others (US), that both the soleus (89% type slow oxidative fibers) and the mixed gastrocnemius (94% type fast oxidative-glycolytic and fast glycolytic fibers; Ref. 21) muscles exhibited identical patterns of weight loss as a function of time. Although these data do not provide histological evidence within a given muscle, they nonetheless do not support the concept that fiber types atrophy at different rates (12). It therefore seems unlikely that the change in contractile properties toward those of slow muscle that we and others (25) have observed is due to a preferential atrophy of fast fibers, allowing for a greater dominance of slow-twitch fibers in the contractile response. A more likely possibility is that a population of fast-twitch fibers is being transformed toward a slower phenotype via alterations in contractile proteins, or modifications in the Ca” + regulatory system of the sarcoplasmic reticulum. It may be that two temporally separate events occur, since the time courses of change in TPT and 1/2RT were different, with the onset of a significant increase in TPT occurring -1 wk before the change in 1/2RT. Finally, our experimental design has not allowed us to conclude whether or not the absence of the nerve or muscular activity per se is primarily responsible for the marked biochemical alterations observed. However, the results obtained by Babij and Booth (5) using hindlimb immobilization and suspension and denervation models point to the role of muscular activity as a dominating influence on muscle gene expression. Furthermore, it is well known that increases in mitochondrial enzymes can occur in denervated muscle subject to chronic contractile activity (17). These results implicate muscle use, in contrast to neurotrophic factors, in influencing the pattern of mitochondrial gene expression within muscle. We thank Angelo Karaiskakis for excellent technical assistance. In addition, we gratefully acknowledge Dr. Ray Wu for generously providing the cytochrome c cDNA probe. This work was supported by the Natural Science and Engineering Council of Canada. Address reprint requests to D. A. Hood. Received

27 August

1990; accepted

in final

form

27 November

1990.

REFERENCES 1. AITHAL, H. N., AND E. R. TUSTANOFF. Assembly of complex III into newly developing mitochondrial membranes. Can. J. Biochem. 53: 1278-1281, 1975. 2. APRILLE, J. R. Perinatal development of mitochondria in rat liver. In: Mitochondrial Physiology and Pathology, edited by G. Fiskum. New York: Van Nostrand Reinhold, 1986, p. 66-99. 3. ASCHENBRENNER, V., R. DRUYAN, R. ALBIN, AND M. RABINOWITZ. Haem a, cytochrome c and total protein turnover in mitochondria from rat heart and liver. Biochem. J. 119: X7-160, 1970. 4. ATTARDI, G., AND G. SCHATZ. Biogenesis of mitochondria. Annu. Rev. Cell Biol. 4: 289-333, 1988. 5. BABIJ, P., AND F. BOOTH. cu-Actin and cytochrome c mRNAs in atrophied adult rat skeletal muscle. Am. J. Physiol. 254 (Cell Physiol. 23): C6514656, 1988.

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6. CHRISTIE, W. W. Lipid Analysis (2nd ed.). New York: Pergamon, 1982. 7. DESAUTELS, M. Mitochondrial proteolysis. In: Mitochondrial Physiology and Pathology, edited by G. Fiskum. New York: Van Nostrand Reinhold, 1986, p. 40-65. 8. ENGEL, A. G., AND H. H. STONNINGTON. Morphological effects of denervation of muscle. A quantitative ultrastructural study. Ann. NYAcad. Sci. 228: 68-88, 1974. 9. FOX, T. D. Nuclear gene products required for translation of specific mitochondrially coded mRNAs in yeast. Trends Genet. 2: 97-100,1986. 10. GOLDSPINK, D. F. The effects of denervation on protein turnover of rat skeletal muscle. Biochem. J. 156: 71-80, 1976. 11. GROSS, N. J., G. S. GETZ, AND M. RABINOWITZ. Apparent turnover of mitochondrial deoxyribonucleic acid and mitochondrial phospholipids in the tissues of the rat. J. BioZ. Chem. 244: 1552-1562, 1969. 12. GUTMANN, E. Neurotrophic relations. Annu. Reu. Physiol. 38: 177216, 1976. 13. HALLMAN, M., AND P. KANKARE. Cardiolipin and cytochrome aa in intact liver mitochondria of rats. Evidence of successive formation of inner membrane components. Biochem. Biophys. Res. Commun. 45: 1004-1010, 1971. 14. HARE, J. F., AND R. HODGES. Turnover of mitochondrial inner membrane proteins in hepatoma monolayer cultures. J. Biol. Chem. 257: 3575-3580,1982. 15. HARLEY, C. B. Hybridization of oligo (dT) to RNA on nitrocellulose. Gene Anal. Tech. 4: 17-22, 1987. 16. HEARN, G. R. Succinate-cytochrome c reductase, cytochrome oxidase and aldolase activities of denervated rat skeletal muscle. Am. J. PhysioZ. 196: 465-466, 1959. 17. HENRIKSSON, J., H. GALBO, AND E. BLOMSTRAND. Role of the motor nerve in activity-induced enzymatic adaptation in skeletal muscle. Am. J. Physiol. 242 (Cell Physiol. 11): C272-C277, 1982. 18. HOGAN, E. L., D. M. DAWSON, AND F. C. A. ROMANUL. Enzymatic changes in denervated muscle. Arch. Neural. 13: 274-282, 1965. 19. HOOD, D. A. Co-ordinate expression of cytochrome c oxidase subunit III and VIc mRNAs in rat tissues. Biochem. J. 269: 503506, 1990. 20. HOOD, D. A., J. GORSKI, AND R. L. TERJUNG. Oxygen cost of twitch and tetanic isometric contractions of rat skeletal muscle. Am. J. Physiol. 250 (Endocrinol. Metab. 13): E636-E647, 1986. 21. HOOD, D. A., AND D. PETTE. Chronic long-term electrostimulation creates a unique metabolic enzyme profile in rabbit fast-twitch muscle. FEBS Lett. 247: 471-474, 1989. 22. HOOD, D. A., AND J. A. SIMONEAU. Rapid isolation of total RNA from small mammal and human skeletal muscle. Am. J. Physiol. 258 (Cell Physiol. 27): C1092-C1096, 1989. 23. HOOD, D. A., R. ZAK, AND D. PETTE. Chronic stimulation of rat skeletal muscle induces coordinate increases in mitochondrial and nuclear mRNAs of cytochrome-c-oxidase subunits. Eur. J.

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Biochem. 179: 275-280, 1989. 24. JOFFE, M., N. SAVAGE, AND H. ISAACS. Biochemical functioning of mitochondria in normal and denervated mammalian skeletal muscle. Muscle Nerve 4: 514-519, 1981. 25. LEWIS, D. M., C. J. C. KEAN, AND J. D. MCGARRICK. Dynamic properties of slow and fast muscle and their trophic regulation. Ann. NY Acad. Sci. 228: 105-120,1974. 26. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275,195l. 27. MANIATIS, T., E. F. FRITSCH, AND J. SAMBROOK. MoZecular Cloning: A Laboratory Handbook. Cold Spring Harbor, NY: Cold Spring Harbor, 1982. 28. MRAK, R. E., AND S. FLEISCHER. Lipid composition of sarcoplasmic reticulum from mice with muscular dystrophy. Muscle Nerue 5: 439-446,1982. 29. OKANO, G., H. MATSUZAKA, AND T. SHIMOJO. A comparative study of the lipid composition of white, intermediate, red and heart muscle in rats. Biochim. Biophys. Acta 619: 167-175, 1980. 30. PETTE, D. Mitochondrial enzyme activities. In: Regulation of Metabolic Processes in Mitochondria, edited by J. M. Tager, S. Papa, E. Quagliariello, and E. C. Slater. Amsterdam: Elsevier, 1966. p. 30-50. 31. REICHMANN, H., H. HOPPELER, 0. MATHIEU-COSTELLO, F. VON BERGEN, AND D. PETTE. Biochemical and ultrastructural changes of skeletal muscle mitochondria after chronic electrical stimulation in rabbits. Pfluegers Arch. 404: l-9, 1985. 32. ROUSER, G., AND S. FLEISCHER. Isolation, characterization, and determination of polar lipids of mitochondria. Methods Enzymol. 10: 385-406, 1969. 33. SALTIN, B., AND P. D. GOLLNICK. Skeletal muscle adaptability: significance for metabolism and performance. In: Handbook of Physiology. Skeletal MuscZe. Bethesda, MD: Am. Physiol. Sot., 1983, sect. 10, chapt. 19, p. 555-631. 34. SCARPULLA, R. C., K. M. AGNE, AND R. Wu. Isolation and structure of a rat cytochrome c gene. J. Biol. Chem. 256: 6480-6486, 1981. 35. SHACKELFORD, J. E., AND H. G. LEBHERZ. Effect of denervation on the levels and rates of synthesis of specific enzymes in “fasttwitch” (breast) muscle fibres of the chicken. J. Biol. Chem. 256: 6423-6429,1982. 36. TERJUNG, R. L. The turnover of cytochrome c in different skeletalmuscle fiber types of the rat. Biochem. J. 178: 569-574, 1979. 37. TZAGOLOFF, A. Mitochondria. New York: Plenum, 1982. 38. WAGNER, K. R., AND S. R. MAX. Neurotrophic regulation of glucose 6-phosphate dehydrogenase in rat skeletal muscle. Brain Res. 170: 572-576,1979. 39. WILLIAMS, R. S., M. GARCIA-M• LL, J. MELLOR, S. SALMONS, AND W. HARLAN. Adaptation of skeletal muscle to increased contractile activity: expression of nuclear genes encoding mitochondrial proteins. J. Biol. Chem. 262: 2764-2767, 1987.

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Mitochondrial adaptations in denervated muscle: relationship to muscle performance.

We have studied mitochondrial adaptations in muscle subject to chronic denervation, and their relationship to muscle performance, using a model of uni...
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