A small-scale procedure for preparing tightly coupled intact skeletal muscle mitochondria from myodystrophic (myd/myd) mice is described. Mitochondrial preparations derived from heart, liver, and skeletal muscle of mydhyd and their littermate (+/?) controls are characterized with respect to their cytochrome content and their oxidative and phosphorylativecapacities. Our data indicate that there is an impairment in the NADH CoQ region of the respiratory chain of myodystrophic skeletal muscle mitochondria. Both heart and liver mitochondria of myd/myd exhibited normal activities of respiratory chain-linked oxidative phosphorylation. MUSCLE & NERVE
2:340-348
1979
DEFECTIVE OXIDATIVE METABOLISM OF MYODYSTROPHIC SKELETAL MUSCLE MITOCHONDRIA C. P. LEE, PhD, M. E. MARTENS, BA, L. JANKULOVSKA, BS, and M. A. NEYMARK, PhD
T h e pioneering study of I.uft et a]'' on isolated skeletal muscle mitochondria] preparations of a patient with severe hypermetabolism of nonthyroid origin constituted the first report of' altered mitochondrial structure and function associated with a neuromuscular disease. This and subsequent studies revealed that in Luft disease the skeletal muscle mitochondria exhibited loosely coilpled respiration with virtually no respiratory c o n t r ~ l ~ and - . ' ~possessed ~~~ a relatively high basal A T P ~ S ~and ~ * an ~ " abnormal energy-lin ked Ca2+ transport s y ~ t e mSince . ~ the original report of Lmft disease, reports of altered mitochondrial structUre2,15,16,19,31.34.39,42 and func~~on~,8-14.1fi.17.24.3"'"6.3R.40.4" associated with human neuromuscular disease have appeared. These studies have shown that in addition to altered rates of- respiration, loose coupling, and high basal ATPase activity, muscle mitochon-
From the Department of Biochemistry Wayne State University School of Medicine, Detroit, MI. Acknowledgments. This work has been supported by grants from the Muscular Dystrophy Association and the National Institutes of Health. Address reprint requests to Dr. Lee at the Department of Biochemistry, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, Received for publication March 2, 1979; revised manuscript accepted for publication May 7, 1979. 0148-639)(/0205/0340$OO.OO/O 1979 Houghton Mifflin Professional Publishers
340
Mitochondria1 Disorder
dria in various neuromuscular diseases have also exhibited decreases in phosphorylation efficiencies, expressed as P/O ratio^,^*'^.'^ cytochrome content14.40 and ATPase activity"" as well as in fatty acid metabolism.'~R~'0~'2.*4~~' Such abnormalities in mitochondrial structure and function have also been reported in certain animal models of muscular dystrophy, such as the strain 129 dystrophic mouse (dy/dy)'X,23,50,41 and the dystrophic hamSter.26,32.43,47-49 Mouse myodystrophy (myd/myd), a new animal model for muscular dystrophy resulting from a spontaneous recessive mutation on chromosome 8, was first identified clinically and histopathologically by Lane et alZ1in 1976. Mydimyd is characterized by the variation in fiber size, loss of striation, and central migration of nuclei of skeletal muscle seen in dy/dy, an animal model of muscular dystrophy since 1955.33In contrast to dy/dy, no abnormal accumulation of fat and connective tissue is found with mydlmyd. A systematic investigation of the cellular energy metabolism of myd/ myd and their normal littermate controls has recently been initiated in our laboratory. In this article, we report results concerning the electron transfer and energy transfer capacities of mitochondria] preparations isolated from liver, heart, and skeletal muscle of myd/myd and their normal littermate controls. Our data indicate an impairment in the NADH CoQ region of the respiratory
MUSCLE & NERVE
Sep/Oct 1979
chain of skeletal muscle of mydimyd. Part of this investigation has recently been briefly reportcd.2’ MATERIALS AND METHODS
Myodystrophic mice and their normal littermate controls were purchased from Jackson Laboratory, Bar Harbor, Maine. All of the mice were from 10 to 16 weeks old at the time of the experiments. Each assay was made with pooled specimens from two t o four mice. Preparation of Mitochondria. Skeletal muscle mitochondria were isolated from hind leg and back muscle of mydimyd o r control mice by the procedure of Makinen and Lee,29 with slight modification. We have previously shown that the conditions of incubation of skeletal muscle from canine, human, and rat with proteinase are critical in order to achieve good-quality mitochondrial preparations with optimal yield.‘g We therefore conducted a systematic investigation to determine the optimal conditions for the isolation of intact skeletal muscle mitochondria from mydimyd. Thr-ee parameters-the mitochondrial protein recovery, thc state-3 respiratory rate with pyruvate + malate as substrates, and the accompanying respiratory control-were analyzed as a function of t h e pr-oteinase concentration in the isolation process. Muscle tissue derived from two to four mice (approximately 1.8-2.5 g of muscle per mouse) was freed of all connective and fatty tissue, minced finely with scissors, and rinsed thoroughly with isotonic KCI followed by several rinsings with Chappell-Perry7 medium. This medium consisted of 100 mM KC1, 50 m M Tris-HC1, pH 7.5, 1 mM ATP, 5 mM MgCl, and 1 mM EDTA. All procedures were carried out on ice. The minced tissue was suspended in the Chappell-Perry medium (10 mlig tissue), to which was added varying. amounts of Nagarse. l’he mixture was stirred frequently, and the pH was kept constant at 7.2. After 5 miri of’ incubation, additional Chappell-Perry medium (10 mlig tissue) was added to the mixture which was homogenized with a Tekmar Tissuemizer (Tekmar Company, Cincinnati, OH) for 15 sec. The pH was readjusted to 7.3 with 0.1 N HC1 and the homogenate was centrifuged at 600 x g for 10 min. The supernatant was decanted and filtered through one layer of cheesecloth and centrifuged at 14,000 x g for 10 min. The resulting supernatant was discarded and the pellet was resuspended in the following medium (volume equal to the volumc of the original homogenate): 100 m M KCI, 50 m M Tris-HCI, pH 7.5, 0.2 mM ATP,
Mitochondria1 Disorder
1 mM MgCL 0.2 mM EDTA, and 1% defatted bovine serum albumin (BSA). T h e suspension was centrifuged at 7,000 x g for 10 min; then the resulting pellet was resuspended in one-half the volume of the above-modified Chappell-Perry medium but without BSA. This was centrifuged at 3,500 x g for I0 min. Occasionally, a pale “fluffy” layer appeared which could be easily removed by gentle shaking of the centrifuge tube. The pellets were rinsed lightly with 0.25 AMsucrose and resuspended in 0.25 M sucrose to give an approximate content of 10-25 mg mitochondrial protein per milliliter. It is apparent from the data shown in figure 1 that 3 mg proteinase per gram of tissue with an incubation period of 5 min was the optimal condition. The mitochondrial yield per gram of muscle of mydimyd was cornparablc to that of controls. All subsequent experiments were performed with mitochondrial preparations isolated under these conditions. Heart muscle mitochondria were prepared by the method of Tyler and G ~ n z using e ~ ~5 mg proteinase per gram of heart tissue. Two to four hearts ranging in weight from 80 to 100 mg each were used for each assay. Liver mitochondria were isolated essentially according to the method of Johnson and Lardy.” We found that the quality of the mitochondrial preparations could be improved considerably if a medium consisting of 0.25 M sucrose plus 0.5 mM EDTA was used for the initial homogenization step. The homogenate was centrifuged at 600 x g for 10 min; the resulting supernatant was then centrifuged at 15,000 x g for 5 min. T h e mitochondria] pellet was washed twice with the sucroseEDTA medium and then once with 0.25 M sucrose. The pellet was finally resuspended in 0.25 M sucrose to give approximately 20-30 mg mitochondrial protein per milliliter. Two to four livers ranging in weight from 0.8- 1.2 g each were pooled for mitochondrial isolation. There was no significant difference in muscle or organ weight between myd/myd and their normal littermate controls. Statistical differences were assessed using the unpaired Student’s t test.
Substratc oxidation rates and phosphorylating capacities (ADP/O ratios) were determined p~larographically~ by means of a Clark oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH) fitted into a thermostatted plexiglass chamber with a capacity of 1.0 ml. T h e reaction mixture consisted of 150 mM sucrose, 25 m M Tris-
Assays.
MUSCLE & NERVE
SepIOct 1979
341
.---A -
-
Protein Recovery
4 .O
250
c
=
Control
=
Dystrophic
Respiratory Control Index -
Respiratory Rate State 3 (Pyr.tMal.1 0.0
$ 3.0
6.0
4-
b \
4.0
q i
n
100
I .o
2 .o
1
0
2.0
I
4.0
0
2.0
4.0
I 2.0
I
I 4.0
Proteinose (mg/g tissue)
I
Figure 7. Hfect of proteinase on skeletal muscle miiochondrfal preparations isolated from myodystrophic mice. Experimental conditions are as described in Materials and Methods. Respiratory control index is the ratio of the state 3 to state 4 respiratory rates. State 3 = presence of ADP; state 4 = absence of ADP.
C1 (pH 7.5), and 10 mM phosphate (pH 7 . 5 ) . Additions were as indicated in figure 2. In the case of succinate oxidation, 2.5 p M rotenone was also included. Acetyl carnitine, when added, was present at a concentration of 5 mM. The final volume was 1.0 ml, and the assay temperature was 30°C. NADH oxidase activity was determined spectrophotometrically at 340 nm. Skeletal muscle mitochondria (1 50-300 pg protein) were suspended in 3.0 ml of medium consisting of 150 mM sucrose, 25 m M Tris-C1, and 10 mM phosphate buffer (pH 7.5), and were sonicated for 50 sec at 70 watts with a Branson Sonifer, Model 185 (Branson Sonic Power Co., Danbury, CT). T h e sonicates were assayed for NADH oxidase activity directly without further fractionation. The assays were performed at 25°C. NADH (200 p M ) was used to initiate the reaction. ATPase activity was determined by the methods of Lindberg and E r n ~ t e r . 'The ~ reaction mixture consisted of 150 mM sucrose, 50 mM TrisHC1 (pH 7.5), and 0.1-0.4 mg mitochondrial protein. The reaction was initiated by the addition of 5 mM ATP. When indicated, 4 mM MgS04,0.6 mM
342
Mitochondria1 Disorder
dinitrophenol (DNP), and/or 2 p g oligoniycin were also added. Final volume was 1.0 ml; assay ternperature, 37"C, and incubation time, 10 min. Protein content was determined by the method of Lowry et aIz7using crystalline BSA as standard. The respiratory chain pigment content of mitochondrial preparations was calculated from reduced rninws oxidized difference spectra recorded at room temperature by means of an Aminco DW-2 spectrophotometer (American instrument Company, Silver Spring. MD). Mitochondria were suspended to a protein concentration of 1.5 mg/ml in a medium that consisted of 150 mM sucrose, 20 mM Tris-C1, 10 mM potassium phosphate (pH 7.5): and 1.7 mM ADP. The mitochondrial suspension was placed in reference arid sample cuvettes, and the oxidized minus oxidized spectrum was recorded to obtain the baseline. After the addition of 5 mM succinate to the sample cuvette, the reaction mixture was incubated at room temperature until cytochrome reduction was complete (approximately 2-3 rnin). The reduced minus oxidized spectrum was then recorded. Concentrations of respiratory chain components were calculated
MUSCLE & NERVE
SepiOct 1979
Figure 2 Poiarographrc tracings of myodystrophrc skeietai muscle mitochondria oxidizmg various substrates Experimental conditions are as described in Materials and Methods
using the fdlowing millimolar extinction coefficients: cytochrome n (605-630 nm), 24.0;45cytochrome u 3 (445-455 nm), 80.0;4" cytochrome 6 (562-575 rim), 20.0:6 cytochrome c + c , (551-540 nm), l Y . l : 3and flavoprotein (465-510 nm), 1 1.0.3 Coenzyme Q was determined as described by Redusing paired mitochondria1 preparations derived from mydirnyd and their normal littermate controls in each set of determinations. Crystalline R nhtilis proteinase (Nagarse) was obtained from Teikoku Chemical Co., Osaka, Japan. Carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) was a gift from Dr. Peter Hytler, E. I . DuPont de Neniours and Co., Wilmington, DE. Other chemicals of the purest grades available were obtained commercially. Glassredistilled water was used throughout the investigation.
Reagents.
RESULTS AND DISCUSSION
Figure 2 shows typical polarographic tracings exhibited by skeletal muscle mitochondria1 preparations derived from mydimyd with pyruvate + malate (A), palniityl carnitine malate (B), and succinate rotenone (C) as substrates. These results show that the rate of respiration of all substrates tested is dependent on the presence (state 3) and absence Respiratory and Phosphorylating Activities.
+
Mitochondria1 Disorder
+
(state 4) of added ADP. The state 3 to state 4 cycle can be repeated several times. In addition, the ADP-induced respiratory stirnulation can he abolished upon the addition of uncoupler, FCCP. Intact mitochondria from myodystrophic and control muscle do not respire with externally added NADH as substrate (data not shown). As shown in figure 2C, the oligoniycin-inhibited succinate oxidation can be transiently released by the additon of 200300 p M Ca2+.The decrease in the rate of Ca2+stimulated respiration occurs after the rnitochondria have taken up the added Ca2+. The Ca2+/0 ratios were estimated to be 5.4 k 0.4 and 5.7 k 0.2, respectively, for the skeletal muscle mitochondria1 preparations from mydimyd and their normal littermate controls, in close agreement with those reported in the l i t e r a t ~ r e ~for ~'~ other types of mitochondrial preparations. The respiratory and phosphorylating activities of isolated skeletal muscle mitochondria from myd/ myd and their normal littermate controls with various substrates are summarized in table 1. In the case of myodystrophic mitochondria, with pyruvate + malate as substrates, the state 3 respiratory rate is depressed by approximately 28% compared to controls. Similar results were also seen with long- and short-chain fatty acyl esters as substrates. With succinate as substrate, the state 3 respiratory rate exhibited by mydimyd is virtually identical to
MUSCLE & NERVE
Sep/Oct 1979
343
that exhibited by controls. An elevation in the state 4 respiratory rate of myodystrophic mitochondria is seen with both pyruvate + malate and succinate as substrates. The cause of the elevation is not known at the present time. The phosphorylating capacities-expressed as ADP/O ratios-accompanying the oxidation of all substrates tested appear to be normal (table 1). thus indicating that the energy-coupling system of the skeletal muscle mitochondria of mydimyd is operating properly. For comparison, a systematic investigation was also made of the respiratory and phosphorylating activities of heart muscle and liver mitochondrial preparations isolated from the same animals as were the skeletal muscle mitochondria. Data shown in tables 2 and 3 indicate that in liver and heart mitochondria derived from cont.rols and myd/myd, there is no significant difference in either the respiratory or the phosphorylating activities with the substrates tested (except that an elevated state 4 rate is seen with succinate as substrate in liver mitochondria). The reduced mirzus oxidized difference spectrum of rnyodystrophic skeletal muscle mitochondria is shown in figure 3. Pyruvate + rnalate and succinate were used as substrates. A typical mitochondrial cytochrome spectrum was obtained, with clearly distinguishable a and y bands of cytochromes a , a 3 , b, and c. The content of the respiratory-chain pigments of mitochondrial prep-
Cytochrome Content.
arations from both licart and skelctal miiscle is summarized in table 4. In myodystrophic skeletal muscle mitochondria1 preparations, a slight increase in coenzyme Q is noted, whereas in the case of heart mitochondrial preparations, the respiratory chain pigment content of mydlmyd is comparable to that of controls. The ATPase activities of mitochondrial preparations derived from skeletal muscle, heart, and liver of the controls and mydimyd assayed under various conditions are shown in table 5. A stimulation of twofold to threefold by Mg" or DNP was seen. A slightly higher basal ATPase activity was seen in both liver and skeletal muscle mitochondria derived from mydimyd compared to controls. Oligomyciri abolished virtually completely the ATPase activity in heart and liver mitochondria, whereas in the case of skeletal muscle mitochondria, a substantial level of oligomycininsensitive ATPase remained in the mitochondria of both myd/myd and controls. The basal ATPase activity of myodystrophic skeletal muscle mitochondria can be reduced substantially by disrupting the mitochondria with brief sonication (dat.a not shown). This suggests that elevated basal ATPase may be due to some contaminant(s) loosely bound to the mitochondrial membranes.
ATPase Activity.
NADH Oxidase Activity. 'The reduced state 3 respiratory rates of mydfmyd skeletal muscle mitochon-
Table 1. Respiratory and phosphorylative activities in isolated skeletal muscle mitochondria from control and myodystrophic (mydimyd) mice a Respiratory rates (nAtoms Oirninimg protein) Substrate Pyruvate
+
Souiceb
State 3
State 4
Respiratory control index
ADPIO
5.3 2 0.2 3.1 rt 0.2c
2.9 + 0.1 2.8 % 0.1
control rnydimyd
228 f 10 164 f 7c
47* 57
control mydimyd
176 f 21 184 f 21
69f 9 99 f 12e
2.6 ? 0.2 2.0 ? 0.2e
2.0 f 0.1 2.0 2 0.2
control rnydimyd
1082 6 74 f 4'
53% 3 502 3
2.1 + 0.1 1.5 % 0.1"
2.7 % 0.1 2.5 % 0.1
41+ 2 46+ 4
20201 1 . 4 ? 0.1'
2.6 5 0.1 2.6 f 0.1
*
3 34
malate Succinate
+
rotenone Palmityl carnitine
+
malate Acetyl carnitine
+
control mydimyd
82f 5 64 f 7d
malate aExperimenta/ conditions are as described in Materials and Methods. Values are expressed as mean f SE bThe number of experiments is given in parentheses. cStaOstica//ydifferent from controls ( p
2:340-348
1979
DEFECTIVE OXIDATIVE METABOLISM OF MYODYSTROPHIC SKELETAL MUSCLE MITOCHONDRIA C. P. LEE, PhD, M. E. MARTENS, BA, L. JANKULOVSKA, BS, and M. A. NEYMARK, PhD
T h e pioneering study of I.uft et a]'' on isolated skeletal muscle mitochondria] preparations of a patient with severe hypermetabolism of nonthyroid origin constituted the first report of' altered mitochondrial structure and function associated with a neuromuscular disease. This and subsequent studies revealed that in Luft disease the skeletal muscle mitochondria exhibited loosely coilpled respiration with virtually no respiratory c o n t r ~ l ~ and - . ' ~possessed ~~~ a relatively high basal A T P ~ S ~and ~ * an ~ " abnormal energy-lin ked Ca2+ transport s y ~ t e mSince . ~ the original report of Lmft disease, reports of altered mitochondrial structUre2,15,16,19,31.34.39,42 and func~~on~,8-14.1fi.17.24.3"'"6.3R.40.4" associated with human neuromuscular disease have appeared. These studies have shown that in addition to altered rates of- respiration, loose coupling, and high basal ATPase activity, muscle mitochon-
From the Department of Biochemistry Wayne State University School of Medicine, Detroit, MI. Acknowledgments. This work has been supported by grants from the Muscular Dystrophy Association and the National Institutes of Health. Address reprint requests to Dr. Lee at the Department of Biochemistry, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, Received for publication March 2, 1979; revised manuscript accepted for publication May 7, 1979. 0148-639)(/0205/0340$OO.OO/O 1979 Houghton Mifflin Professional Publishers
340
Mitochondria1 Disorder
dria in various neuromuscular diseases have also exhibited decreases in phosphorylation efficiencies, expressed as P/O ratio^,^*'^.'^ cytochrome content14.40 and ATPase activity"" as well as in fatty acid metabolism.'~R~'0~'2.*4~~' Such abnormalities in mitochondrial structure and function have also been reported in certain animal models of muscular dystrophy, such as the strain 129 dystrophic mouse (dy/dy)'X,23,50,41 and the dystrophic hamSter.26,32.43,47-49 Mouse myodystrophy (myd/myd), a new animal model for muscular dystrophy resulting from a spontaneous recessive mutation on chromosome 8, was first identified clinically and histopathologically by Lane et alZ1in 1976. Mydimyd is characterized by the variation in fiber size, loss of striation, and central migration of nuclei of skeletal muscle seen in dy/dy, an animal model of muscular dystrophy since 1955.33In contrast to dy/dy, no abnormal accumulation of fat and connective tissue is found with mydlmyd. A systematic investigation of the cellular energy metabolism of myd/ myd and their normal littermate controls has recently been initiated in our laboratory. In this article, we report results concerning the electron transfer and energy transfer capacities of mitochondria] preparations isolated from liver, heart, and skeletal muscle of myd/myd and their normal littermate controls. Our data indicate an impairment in the NADH CoQ region of the respiratory
MUSCLE & NERVE
Sep/Oct 1979
chain of skeletal muscle of mydimyd. Part of this investigation has recently been briefly reportcd.2’ MATERIALS AND METHODS
Myodystrophic mice and their normal littermate controls were purchased from Jackson Laboratory, Bar Harbor, Maine. All of the mice were from 10 to 16 weeks old at the time of the experiments. Each assay was made with pooled specimens from two t o four mice. Preparation of Mitochondria. Skeletal muscle mitochondria were isolated from hind leg and back muscle of mydimyd o r control mice by the procedure of Makinen and Lee,29 with slight modification. We have previously shown that the conditions of incubation of skeletal muscle from canine, human, and rat with proteinase are critical in order to achieve good-quality mitochondrial preparations with optimal yield.‘g We therefore conducted a systematic investigation to determine the optimal conditions for the isolation of intact skeletal muscle mitochondria from mydimyd. Thr-ee parameters-the mitochondrial protein recovery, thc state-3 respiratory rate with pyruvate + malate as substrates, and the accompanying respiratory control-were analyzed as a function of t h e pr-oteinase concentration in the isolation process. Muscle tissue derived from two to four mice (approximately 1.8-2.5 g of muscle per mouse) was freed of all connective and fatty tissue, minced finely with scissors, and rinsed thoroughly with isotonic KCI followed by several rinsings with Chappell-Perry7 medium. This medium consisted of 100 mM KC1, 50 m M Tris-HC1, pH 7.5, 1 mM ATP, 5 mM MgCl, and 1 mM EDTA. All procedures were carried out on ice. The minced tissue was suspended in the Chappell-Perry medium (10 mlig tissue), to which was added varying. amounts of Nagarse. l’he mixture was stirred frequently, and the pH was kept constant at 7.2. After 5 miri of’ incubation, additional Chappell-Perry medium (10 mlig tissue) was added to the mixture which was homogenized with a Tekmar Tissuemizer (Tekmar Company, Cincinnati, OH) for 15 sec. The pH was readjusted to 7.3 with 0.1 N HC1 and the homogenate was centrifuged at 600 x g for 10 min. The supernatant was decanted and filtered through one layer of cheesecloth and centrifuged at 14,000 x g for 10 min. The resulting supernatant was discarded and the pellet was resuspended in the following medium (volume equal to the volumc of the original homogenate): 100 m M KCI, 50 m M Tris-HCI, pH 7.5, 0.2 mM ATP,
Mitochondria1 Disorder
1 mM MgCL 0.2 mM EDTA, and 1% defatted bovine serum albumin (BSA). T h e suspension was centrifuged at 7,000 x g for 10 min; then the resulting pellet was resuspended in one-half the volume of the above-modified Chappell-Perry medium but without BSA. This was centrifuged at 3,500 x g for I0 min. Occasionally, a pale “fluffy” layer appeared which could be easily removed by gentle shaking of the centrifuge tube. The pellets were rinsed lightly with 0.25 AMsucrose and resuspended in 0.25 M sucrose to give an approximate content of 10-25 mg mitochondrial protein per milliliter. It is apparent from the data shown in figure 1 that 3 mg proteinase per gram of tissue with an incubation period of 5 min was the optimal condition. The mitochondrial yield per gram of muscle of mydimyd was cornparablc to that of controls. All subsequent experiments were performed with mitochondrial preparations isolated under these conditions. Heart muscle mitochondria were prepared by the method of Tyler and G ~ n z using e ~ ~5 mg proteinase per gram of heart tissue. Two to four hearts ranging in weight from 80 to 100 mg each were used for each assay. Liver mitochondria were isolated essentially according to the method of Johnson and Lardy.” We found that the quality of the mitochondrial preparations could be improved considerably if a medium consisting of 0.25 M sucrose plus 0.5 mM EDTA was used for the initial homogenization step. The homogenate was centrifuged at 600 x g for 10 min; the resulting supernatant was then centrifuged at 15,000 x g for 5 min. T h e mitochondria] pellet was washed twice with the sucroseEDTA medium and then once with 0.25 M sucrose. The pellet was finally resuspended in 0.25 M sucrose to give approximately 20-30 mg mitochondrial protein per milliliter. Two to four livers ranging in weight from 0.8- 1.2 g each were pooled for mitochondrial isolation. There was no significant difference in muscle or organ weight between myd/myd and their normal littermate controls. Statistical differences were assessed using the unpaired Student’s t test.
Substratc oxidation rates and phosphorylating capacities (ADP/O ratios) were determined p~larographically~ by means of a Clark oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH) fitted into a thermostatted plexiglass chamber with a capacity of 1.0 ml. T h e reaction mixture consisted of 150 mM sucrose, 25 m M Tris-
Assays.
MUSCLE & NERVE
SepIOct 1979
341
.---A -
-
Protein Recovery
4 .O
250
c
=
Control
=
Dystrophic
Respiratory Control Index -
Respiratory Rate State 3 (Pyr.tMal.1 0.0
$ 3.0
6.0
4-
b \
4.0
q i
n
100
I .o
2 .o
1
0
2.0
I
4.0
0
2.0
4.0
I 2.0
I
I 4.0
Proteinose (mg/g tissue)
I
Figure 7. Hfect of proteinase on skeletal muscle miiochondrfal preparations isolated from myodystrophic mice. Experimental conditions are as described in Materials and Methods. Respiratory control index is the ratio of the state 3 to state 4 respiratory rates. State 3 = presence of ADP; state 4 = absence of ADP.
C1 (pH 7.5), and 10 mM phosphate (pH 7 . 5 ) . Additions were as indicated in figure 2. In the case of succinate oxidation, 2.5 p M rotenone was also included. Acetyl carnitine, when added, was present at a concentration of 5 mM. The final volume was 1.0 ml, and the assay temperature was 30°C. NADH oxidase activity was determined spectrophotometrically at 340 nm. Skeletal muscle mitochondria (1 50-300 pg protein) were suspended in 3.0 ml of medium consisting of 150 mM sucrose, 25 m M Tris-C1, and 10 mM phosphate buffer (pH 7.5), and were sonicated for 50 sec at 70 watts with a Branson Sonifer, Model 185 (Branson Sonic Power Co., Danbury, CT). T h e sonicates were assayed for NADH oxidase activity directly without further fractionation. The assays were performed at 25°C. NADH (200 p M ) was used to initiate the reaction. ATPase activity was determined by the methods of Lindberg and E r n ~ t e r . 'The ~ reaction mixture consisted of 150 mM sucrose, 50 mM TrisHC1 (pH 7.5), and 0.1-0.4 mg mitochondrial protein. The reaction was initiated by the addition of 5 mM ATP. When indicated, 4 mM MgS04,0.6 mM
342
Mitochondria1 Disorder
dinitrophenol (DNP), and/or 2 p g oligoniycin were also added. Final volume was 1.0 ml; assay ternperature, 37"C, and incubation time, 10 min. Protein content was determined by the method of Lowry et aIz7using crystalline BSA as standard. The respiratory chain pigment content of mitochondrial preparations was calculated from reduced rninws oxidized difference spectra recorded at room temperature by means of an Aminco DW-2 spectrophotometer (American instrument Company, Silver Spring. MD). Mitochondria were suspended to a protein concentration of 1.5 mg/ml in a medium that consisted of 150 mM sucrose, 20 mM Tris-C1, 10 mM potassium phosphate (pH 7.5): and 1.7 mM ADP. The mitochondrial suspension was placed in reference arid sample cuvettes, and the oxidized minus oxidized spectrum was recorded to obtain the baseline. After the addition of 5 mM succinate to the sample cuvette, the reaction mixture was incubated at room temperature until cytochrome reduction was complete (approximately 2-3 rnin). The reduced minus oxidized spectrum was then recorded. Concentrations of respiratory chain components were calculated
MUSCLE & NERVE
SepiOct 1979
Figure 2 Poiarographrc tracings of myodystrophrc skeietai muscle mitochondria oxidizmg various substrates Experimental conditions are as described in Materials and Methods
using the fdlowing millimolar extinction coefficients: cytochrome n (605-630 nm), 24.0;45cytochrome u 3 (445-455 nm), 80.0;4" cytochrome 6 (562-575 rim), 20.0:6 cytochrome c + c , (551-540 nm), l Y . l : 3and flavoprotein (465-510 nm), 1 1.0.3 Coenzyme Q was determined as described by Redusing paired mitochondria1 preparations derived from mydirnyd and their normal littermate controls in each set of determinations. Crystalline R nhtilis proteinase (Nagarse) was obtained from Teikoku Chemical Co., Osaka, Japan. Carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) was a gift from Dr. Peter Hytler, E. I . DuPont de Neniours and Co., Wilmington, DE. Other chemicals of the purest grades available were obtained commercially. Glassredistilled water was used throughout the investigation.
Reagents.
RESULTS AND DISCUSSION
Figure 2 shows typical polarographic tracings exhibited by skeletal muscle mitochondria1 preparations derived from mydimyd with pyruvate + malate (A), palniityl carnitine malate (B), and succinate rotenone (C) as substrates. These results show that the rate of respiration of all substrates tested is dependent on the presence (state 3) and absence Respiratory and Phosphorylating Activities.
+
Mitochondria1 Disorder
+
(state 4) of added ADP. The state 3 to state 4 cycle can be repeated several times. In addition, the ADP-induced respiratory stirnulation can he abolished upon the addition of uncoupler, FCCP. Intact mitochondria from myodystrophic and control muscle do not respire with externally added NADH as substrate (data not shown). As shown in figure 2C, the oligoniycin-inhibited succinate oxidation can be transiently released by the additon of 200300 p M Ca2+.The decrease in the rate of Ca2+stimulated respiration occurs after the rnitochondria have taken up the added Ca2+. The Ca2+/0 ratios were estimated to be 5.4 k 0.4 and 5.7 k 0.2, respectively, for the skeletal muscle mitochondria1 preparations from mydimyd and their normal littermate controls, in close agreement with those reported in the l i t e r a t ~ r e ~for ~'~ other types of mitochondrial preparations. The respiratory and phosphorylating activities of isolated skeletal muscle mitochondria from myd/ myd and their normal littermate controls with various substrates are summarized in table 1. In the case of myodystrophic mitochondria, with pyruvate + malate as substrates, the state 3 respiratory rate is depressed by approximately 28% compared to controls. Similar results were also seen with long- and short-chain fatty acyl esters as substrates. With succinate as substrate, the state 3 respiratory rate exhibited by mydimyd is virtually identical to
MUSCLE & NERVE
Sep/Oct 1979
343
that exhibited by controls. An elevation in the state 4 respiratory rate of myodystrophic mitochondria is seen with both pyruvate + malate and succinate as substrates. The cause of the elevation is not known at the present time. The phosphorylating capacities-expressed as ADP/O ratios-accompanying the oxidation of all substrates tested appear to be normal (table 1). thus indicating that the energy-coupling system of the skeletal muscle mitochondria of mydimyd is operating properly. For comparison, a systematic investigation was also made of the respiratory and phosphorylating activities of heart muscle and liver mitochondrial preparations isolated from the same animals as were the skeletal muscle mitochondria. Data shown in tables 2 and 3 indicate that in liver and heart mitochondria derived from cont.rols and myd/myd, there is no significant difference in either the respiratory or the phosphorylating activities with the substrates tested (except that an elevated state 4 rate is seen with succinate as substrate in liver mitochondria). The reduced mirzus oxidized difference spectrum of rnyodystrophic skeletal muscle mitochondria is shown in figure 3. Pyruvate + rnalate and succinate were used as substrates. A typical mitochondrial cytochrome spectrum was obtained, with clearly distinguishable a and y bands of cytochromes a , a 3 , b, and c. The content of the respiratory-chain pigments of mitochondrial prep-
Cytochrome Content.
arations from both licart and skelctal miiscle is summarized in table 4. In myodystrophic skeletal muscle mitochondria1 preparations, a slight increase in coenzyme Q is noted, whereas in the case of heart mitochondrial preparations, the respiratory chain pigment content of mydlmyd is comparable to that of controls. The ATPase activities of mitochondrial preparations derived from skeletal muscle, heart, and liver of the controls and mydimyd assayed under various conditions are shown in table 5. A stimulation of twofold to threefold by Mg" or DNP was seen. A slightly higher basal ATPase activity was seen in both liver and skeletal muscle mitochondria derived from mydimyd compared to controls. Oligomyciri abolished virtually completely the ATPase activity in heart and liver mitochondria, whereas in the case of skeletal muscle mitochondria, a substantial level of oligomycininsensitive ATPase remained in the mitochondria of both myd/myd and controls. The basal ATPase activity of myodystrophic skeletal muscle mitochondria can be reduced substantially by disrupting the mitochondria with brief sonication (dat.a not shown). This suggests that elevated basal ATPase may be due to some contaminant(s) loosely bound to the mitochondrial membranes.
ATPase Activity.
NADH Oxidase Activity. 'The reduced state 3 respiratory rates of mydfmyd skeletal muscle mitochon-
Table 1. Respiratory and phosphorylative activities in isolated skeletal muscle mitochondria from control and myodystrophic (mydimyd) mice a Respiratory rates (nAtoms Oirninimg protein) Substrate Pyruvate
+
Souiceb
State 3
State 4
Respiratory control index
ADPIO
5.3 2 0.2 3.1 rt 0.2c
2.9 + 0.1 2.8 % 0.1
control rnydimyd
228 f 10 164 f 7c
47* 57
control mydimyd
176 f 21 184 f 21
69f 9 99 f 12e
2.6 ? 0.2 2.0 ? 0.2e
2.0 f 0.1 2.0 2 0.2
control rnydimyd
1082 6 74 f 4'
53% 3 502 3
2.1 + 0.1 1.5 % 0.1"
2.7 % 0.1 2.5 % 0.1
41+ 2 46+ 4
20201 1 . 4 ? 0.1'
2.6 5 0.1 2.6 f 0.1
*
3 34
malate Succinate
+
rotenone Palmityl carnitine
+
malate Acetyl carnitine
+
control mydimyd
82f 5 64 f 7d
malate aExperimenta/ conditions are as described in Materials and Methods. Values are expressed as mean f SE bThe number of experiments is given in parentheses. cStaOstica//ydifferent from controls ( p
