Studies of human skeletal muscle mitochondria

CUOCHEMICAL

MEDICINE

Studies

2,

179-189 (1968)

of Human

Skeletal

Muscle

Mitochondria

J. B. PETER Department

of Medicine,

UCLA

School

of Medicine,

Los

Angeles,

California

900%

Received May 1. 1968

Although skeletal muscle mitochondria contribute over 30% of the basal metabolic rate of the intact animal (1)) very little is known of the biochemistry of these organelles isolated from human skeletal muscle. This lack of knowledge stems from several factors, including the relatively small concentration of mitochondria in skeletal muscle compared to liver, kidney, or heart (2) and the dil?lculty in homogenizing skeletal muscle. Previous studies have shown that mitochondria can be isolated from human skeletal muscle (36), and at least one human disease is due to an alteration of respiratory control in skeletal muscle mitochondria (7, 8). However, deficiencies of techniques previously available for isolation of skeletal muscle mitochondria have hampered progress in this important area of human muscle biochemistry. In this paper the characteristics of human skeletal muscle mitochondria isolated from small biopsy specimens by a new improved technique will be described. These characteristics are readily reproducible and provide a solid basis for studies of human skeletal muscle mitochondria in a variety of diseases. MATERIALS

AND METHODS

Muscle was obtained from the quadriceps, gastrocnemius, or deltoid muscle of patients undergoing orthopedic surgery. The type or duration of general anesthesia had no obvious effect on the characteristics of the isolated mitochondria. Generally the muscle sample was obtained as soon after induction of anesthesia as possible. In many instances the tissue studied was from diagnostic biopsy specimens of patients whose complete evaluation, including light microscopy, histochemistry, biochemistry, and electromyography, ultimately proved the muscle to be normal. In these cases, the muscle biopsy was done under local anesthesia with lo/O lidocaine. Care was taken to avoid direct infiltration of the muscle to be removed. Muscle samples as small as 1.0 gm yielded adequate mitochondria for the biochemical studies to be described. No complications 179

180

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I?. PETER

resulted from any biopsy autl the patients were ambulatory immediatel! after operation with pressure dressings in place for 24 hours. Two media are employed in the isolation of human skeletal muscle mitochondria. The first, hereafter referred to ws low-speed medium, contains 50 mM TES,1 100 mM KCI, 50 mM Pi, 5 mM MgSO,, 1 mM ATP, 1 mM EDTA, and 5 mg bovine serum albumin (Pentex) per ml, pH 7.4. The high-speed medium is identical to the low-speed except that ATP and bovine serum albumin are omitted. The low-speed medium is a modification of that first used by Chappell and Perry (9) to isolate mitochondria from pigeon breast muscle. After removal, the muscle was immediately chilled to about 2°C in low-speed medium for transport to the laboratory where gross adipose and fibrous tissue was removed. After rapid weighing in a tared beaker oontaining a small amount of cold low-speed medium the muscle was finely minced with scissors and diluted to 18% (gram wet-weight muscle/ 100 ml cold, low-speed media) and poured into a prechilled 75ml bottle of the Bronwill mechanical homogenizer (Braun, model MSK, Bronwill Scientific, Box 277, Rochester, New York 14601). Chilled glass beads of 1.0 mm diameter obtained from Bronwill were added to the bottle in ratio of 7 gm of beads per gram of wet-weight muscle. Homogenization was then carried out in the C&-cooled homogenizer for 45 seconds at 4000 cycle/min. Cooling during homogenization in this instrument is controlled by a valve regulating the flow of liquid COZ from a storage tank through a stainless-steel capillary to a spout from which the coolant circulates around the bottle. It was impossible to measure the temperature during homogenization but the temperature of the suspension immediately after homogenization was consistently between 3 and 7°C. After the initial homogenate was decanted into precooled 50-ml centrifuge tubes, about 15 ml of low-speed medium was added to the bottle to wash the beads and this was combined with the initial homogenate. After centrifugation at 2-4°C for 10 minutes at 70 g, the resultant supernatant fluid was decanted through two layers of cheesecloth into prechilled centrifuge tubes and spun again at 70 g for 10 minutes. The combined supernatant fluids, all of which had been spun twice at 70g for 10 min*Abbreviations used in this paper include : ADP (adenosineJ’-diphosphatc j, AR (acceptor ratio), ATP (adenosine-5’-triphosphate), DPNH (reduced diphosphopyridine nucleotide or reduced nicotinamide adeninc dinucleotide). EDTA Pi (inorganic orthophosphate), QO, (pl oxygen (ethylenediaminetetraacetate), consumed/mg mitochondrial protein/hour), RCR (respiratory control ratio), TER (N-t& (hydroxymethyl) methyl-Samino-ethane sulfonic acid), TMPD (tetramethylpara-phenylenediamine) _

HUMAN

SKELETAL

MUSCLE

MITOCHONDRIA

181

utes were then spun at 3500 g for 10 minutes. This yielded the 3500-g mitochondrial pellet that was diluted to 25% in high-speed medium (grams of wet-weight original muscle per 100 ml of high-speed medium) and spun for 10 minutes at 70 g. The 3500-g pellet was again obtained from this supernate and was finally suspended uniformly by hand in a small Potter-Elvehj em homogenizer. Mitochondrial fractions sedimenting in 10 minutes between 3500 and 7000 g and in 20 minutes between 7000 and 15,000 g were obtained from the initial 3500 g supernatant fluid. These fractions were resuspended in high-speed medium, respun for 10 minutes each at 7000 and 15,000 g respectively and finally suspended uniformly in small volumes of highspeed medium by gentle Potter-Elvehjem homogenization. The protein concentration of the 70-3500 g, 3500-7000 g, and 700015,000 g fractions was determined by the biuret method (10) and highspeed medium was added to give a final mitochondrial protein concentration of 10 mg/ml of high-speed medium. For protein determination deoxycholate was added in final concentration of 0.1 gm per 100 ml to ensure complete solubilization of mitochondrial protein. Crystallized human albumin was used as the standard. The QO, (~1 0, consumed/mg mitochondrial protein/hour), was assayed with a vibrating, platinum electrode polarized at - 0.6V in a GME Oxygraph (Gilson Medical Electronics, Middleton, Wisconsin) at 26”, pH 7.4, in a final volume of 2 ml containing substrates at concentrations stated plus 30 mM Pi, 25 mM TES, 8.0 mM MgSO,, 0.5 mM EDTA, 50 mM KCI, and about 1.0 mg of mitochondrial protein. The ADP/O ratio was calculated from the increment of oxygen consumption induced by the addition of 150 PM ADP. Respiratory control ratios (RCR) were calculated from the oxygraph tracings and are defined as the ratio of “respiratory rate (QOJ in the presence of added ADP (state 3) to respiratory rate after its (ADP) expenditure (state 4)” (11). State 4 respiration as used herein refers to the QO, of mitochondria in the presence of excess substrate, Pi, and oxygen after added ADP has been converted to ATP by oxidative phosphorylation. State 3 is defined as the QO, of mitochondria in the presence of oxygen and added substrate, P,, and ADP (12). As discussed later a less critical estimation of respiratory control may be obtained from the ratio of the QO, in state 3 to the QO, in state 4 before any ADP is added. In this paper the latter estimation of respiratory control will be called the acceptor ratio (AR), using the terminology of Gregg et al. (27). All additions during oxygraph tracings were made in volumes less than 0.6% of the total volume of 2 ml so that corrections for changes in oxygen concentrations are not necessary.

182

J. B. PETER

RESULTS

Figure 1 shows the importance of controlling the amount of mitochondria in the oxygraph chamber if rates of oxygen consumption by different preparations of mitochondria are to be compared. These data refer to mitochondria from rat skeletal muscle, and less extensive studies show a similar change in QO, of human muscle mitochondria depending on the 2 \ .-c 2007 Q) ‘j 180t;

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FIG. 1. Relationship of QO, and concentration conditions were employed with pyruvate-malate obtained from rat skeletal muscle.

I I 1.0 1.2

I 1.4

I I.6

I I 1.8 2.0

of mitochondrial as substrate. The

protein. Standard mitochondria were

protein concentration. The concentration of mitochondrial protein does not, however, significantly affect the respiratory control ratios or ADP/O ratios. A final concentration of 0.5 mg mitochondrial protein per 2 ml gives QO, values that are high enough to be measured accurately, but in this study 1.0 mg of mitochondrial protein was used routinely. Figure 2 shows a typical oxygraph tracing of normal human skeletal muscle mitochondria isolated between 3500 and 7000 g. With various substrates the QO, in state 3 and state 4 is quite reproducible even when the oxygen concentration falls to less than 50%. This reproducibility is also reflected in the ADP/O ratios and the respiratory control ratios. With succinate as substrate and with rotenone present to block oxidation of DPN-linked substrates the average ADP/O ratio of 1.8 closely approximates the value of 2.0 that is generally accepted as normal for carefully prepared mitochondria from liver, heart, or kidney. In Fig. 3 respiratory control in t’he cytochrome oxidase region of the

HUMAN

-

SKELETAL

MUSCLE

0,+=25

15.0 mM PYRUVATE 150 mM MALATE

183

MITOCHONDBIA

AVERAGE: PYRUVATE

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-MALATE

ADP*29 0

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45.0 mM SUCCINATE 5OpLM ROTENONE-’

----

1.0 1179 PROTEIN STANDARD MEDIUM TOTAL VOLUME. 2 T=26O

SUCCINATE

RCR=

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- ROTENONE

3.8

\ ml

01.1’7.4

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20pt4

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FIG. 2. Typical oxygraph tracings with mitochondria were isolated between 3500 pyruvate-malate despite the fact that the ratios approach the “theoretical” of 3.0 rotenone.

0 67 mq PROTEIN STANDARD MEDIUM TOTAL VOLUME= T : 2E.O p” : 7.4

human skeletal muscle mitochondria. The and 7000 g. The average RCR is higher for AR is higher for succinate-rotenone. ADP/O for pyruvate-malate and 2.0 for succinat+

2 ml

T

1.5rnM

ASCORBATE

t ‘ISOpd TMPD

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IOO/LMLO.I5mM TMPD

ADP

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ISpM CYTOCHROYE

c

Fm. 3. Respiratory control at cytochrome oxidase of human skeletal muscle mitorhondria isolated at 3500-7000 g. After addition of ADP the QO, increased from 99 to 125 giving an acceptor ratio of 1.3. an RCR of 1.4, and an ADP/O ratio of OS.

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82

QO,

3500-7000

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fraction

NIT~CHONDRIA

Mitochondrial

~IUSCLE

159

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1

107

3 QO,

1.6

3.1

68

1.3

‘2 3

.4DP/O

2. 9

RCRb

g

SKELETAL

TABLE

48

QOP

70-3500

OF HUMAN

before any ADP is added’ The averages for each substrate are derived from the state ADP/O ratio, and RCR obtained from four or five additions .-\DP to each of ten seppnrxte preparations of mitochondria.

ADP

3

(~1 Oz/mg

in state

4 after

QO,

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l’yruvate-malate (15 mM) (15 mM I Succinate (rotenone) (45n.d (5/d Iscorbate-TMPD-ADP c1.5 mM) (250 PM) (150 &co&ate-TMPD-ADP-cyto ( 1.5 mM) (250 PM‘I (300 DPNH - cyto c 11.5 mM) (15/.&Ml DPNH - eyto c - TMPD (1.5md) (15~~) (250 DPNH - cyto c - TMPD (1.5 mr+d) (15 PM) (250

Substratr

CHARAC'TERISTICS

02 RCR

7000-15,000g ADP/O

HUMAN

SKELETAL

MUSCLE

MWOCHONDRIA

l&5

electron transport system is demonstrated by an increase in QO, brought about by adding ADP to mitochondria oxidizing ascorbate with TMPD present to catalyze reduction of cytochrome c (13,14). The ADP/O ratio for ascorbate-TMPD approaches 1. Similar respiratory control exists for DPNH oxidation in the presence of TMPD and cytochrome c (Table 1). Table 1 shows the average QO, values, RCR and the ADP/O ratios of human skeletal muscle mitochondria for several important substrates. Significant reproducible differences are noted in the rates of oxidation of these substrates. The QO, with ascorbate-TMPD exceeds that of succinate or pyruvate-malate and the QO, with the latter does not approach that of ascorbate-TMPD even when cytochrome c is added. Respiratory control ratios of the magnitude shown in Table 1 have not been recorded with human skeletal muscle mitochondria prepared by techniques previously available. In general, the fraction of mitochondria isolated between 3500 and 7000 g exhibits the best biochemical characteristics but the amount of mitochondria in this fraction is low compared with the 70-3500 g fraction. The fewer mitochondria in the 7OtKL15,OOO g fraction show lower respiratory control ratios as might be expected with damaged mitochondria that are probably contaminated with sarcotubular vesicles and fragmented mitochondria containing active ATPases. DISCUSSIOK

Previous studies of human skeletal muscle mitochondria have required biopsies ranging from 10-20 gm of muscle (4). With the techniques described herein biopsy specimens of less than 1.5 gm routinely yield mitochondria sufficient for a variety of metabolic studies. More than 3 mg of mitochondrial protein are obtained from 1.0 gm of muscle and this yield permits assay of the QO, value, and the respiratory control and ADP/O ratios with a variety of substrates. With a mechanical shaker as employed herein the degree of homogenization of muscle minces can be closely controlled. This plus careful regulation of the temperature accounts for the vastly improved biochemical characteristics of the isolated mitochondria. Previous attempts in this laboratory at homogenization using the same solutions but different techniques of homogenization have never yielded mitochondria of the same quantity or quality. Human skeletal muscle mitochondria prepared by previously available techniques do not consistently exhibit a state 3 to state 4 transition. Some investigators (15) attributed this to “contamination of the mitochondria with a Mg2+-stimulated ATPase originating from myofibrils, sarcotubular membranes or submitochondrial particles.” The absence of

186

0.

H. PETEH

:((%ivc ;1TPases in the mitochondri;l prepared by the present technique is illustrated hy the data of Table I and Fig. 2, which show respiratory control ratios averaging over 3 with pyruvate and malatc as suhstrates. The presence of an active ATPase in these preparations would make very unlikely the repeated bursts of respiration with subsequent return to the much slower respiration of state 4 even after multiple additions of ADP. The respiratory control ratio as defined herein (WC XlhTERIALS AND METHODS) is a more critical test of the integrity and purit,y of skeletal muscle mitochondria than is the acceptor ratio (AR), which is defined as the ratio of QO, in state 3 to QO, in st.ate 4 before addition of any nucleotide. Occasionally preparations of mitochondria show high acceptor ratios but rather low respiratory control ratios. This might bc explained by a very low endogenous ADP content of such preparations that limits substrate oxidation despite the presence of an active ATPase. In addition the acceptor ratio is difficult to measure accurately because the ratrs of respiration are less in the absence of any added nucleotide :md hence a somewhat inaccurate estimation of QO, in state 4 i hefore addition of ADP) or a difference in the amount of endogenous ADP can greatly change t,he acceptor ratio. For these reasons respiratory control in skeletal muscle mitochondria as a manifestation of mitochondrial integrity and purity is most critically assayed as the ratio of QO, in stat,c 3 to QO, in state 4 after added ADP has heen utilized hy oxidativc phosphorylation. In addition to high respiratory control ratios and ADP/O ratios with pyruvate-malate, mitochondria isolated by this technique oxidize succinate very rapidly with high respiratory control in contrast to previous preparations of human skeletal muscle mitochondria (4, 7’1. Previous preparations of rat or human skeletal muscle mitochondria have consistently shown low acceptor ratios for the state 4 (no added nucleotidesi t,o 3 transition with succinate, whereas the present preparations shon average rat.ios of 8.0 for this transition and more importantly show ratios averaging over 3 for the state 3 to state 4 (after expenditure of added .4DP) transition. Only recently has significant respiratory control been demonstrated in the terminal cytochrome region of the electron transport system by an increase of respiration when ADP is added to rabbit or rat heart mitochondria (14). Human skeletal muscle mitochondria show a 1.4-fold increase in QO, after addition of ADP to a system oxidizing ascorbateTMPD. That this increase is not due to ADP-stimulated oxidation of endogenous substrates is shown by a similar increase in QO, in the presence of rotenone, which blocks oxidation of DPN-linked substrates (18), or in the presence of antimycin A, which blocks electron transport

HUMAN

SHELETAL

MUSCLE

MITOCHONDRIA

187

at the level of cytochrome b. Also the amount of endogenous substrate in human or rat skeletal muscle mitochondria is very low as has been repeatedly demonstrated by failure of QO, of these mitochondria to rise after addition of ADP in the absence of exogenous substrate. In human skeletal muscle mitochondria oxidizing ascorbate-TMPD the ADP/O ratio approaches the expected value of 1. Oxidation of added DPNH is negligible in the absence of added cytochrome c or TMPD, as might be expected for tightly coupled, nonswollen mitochondria (16). Using somewhat different conditions Hedman et al. (5) found a QO, value of about 19 &mg mitochondrial protein/ hour for skeletal muscle mitochondria oxidizing DPNH in the presence of added cytochrome c and ADP. The QO, value with DPNH-cytochrome c of our mitochondria averaged 23 ~1 O,/mg protein/hour and some respiratory control was manifest by a small increase in QO, after addition of ADP. In confirmation of previous work (5) the oxidation of DPNH in the presence of cytochrome c by human skeletal muscle mitochondria is not consistently inhibited by rotenone, whereas this reaction in rat skeletal muscle mitochondria has an .average QO? of about 150 and is inhibited by very low concentrations of rotenone (5, 17). Possibly the reduced exogenous cytochrome c interacts only slowly with cytochrome oxidase of human skeletal muscle mitochondria. Furt.her studies of these differences are needed. This new technique for isolation of mitochondria from small biopsy samples of human skeletal muscle should greatly facilitate the study of mitochondrial biochemistry in a variety of diseases. Several muscle diseases are proposed to manifest defects of mitochondrial metabolism and can readily be investigated with the methods described herein. Luft et al. (7)) using less satisfactory techniques, have already clearly defined n human muscle disease due to deficient respiratory control of skeletal muscle mitochondria. Studies in this laboratory have shown that in Duchenne dystrophy, dystrophia myotonica, thyrotoxic myopathy, clenervation atrophy, and at least one type of paroxysmal rhabdomyolysis with normal glycolpsis, the oxidative phosphorylation of skeletal muscle mitochondria is normal (19-21). Other studies have defined a myopathy characterized by hist,ochemical evidence of mitochondrial hyperactivity (22, 23). With the techniques described herein an inordinate increase in amount of mitochondria was demonstrated (19). This represents the first biochemical demonstrat,ion of increased amounts of mitochondria with normal respiratory control in human myopathy. Preliminary studies have defined another new myopathy probably due to a block of electron transport at the level of cytochrome b (20). Jlitochondria prepared hp this technique have been examined electron

188

J. B.

PETER

microscopically by the method of Baudhuin (24) that allows sampling of small aliquots of mitochondrial suspensions. A high degree of purity of the preparations and an extraordinary preservation of the structure of human skeletal muscle mitochondria so isolated has been demonstrated (25). The supernatants fluids from the 15,000 g mitochondrial pellet may be used for isolation of sarcotubular vesicles. The ATPase and calciumaccumulating capacities of such vesicles from human skeletal muscle have been studied in a few human diseases (26). Such studies have been furthered in this laboratory and in one patient with severe hypothyroidism the capacity for calcium accumulation by isolated vesicles was greatly decreased (21). Possibly this accounts for the muscle spasms and very prolonged relaxation phase of the deep tendon reflexes that the patient manifested. It is obvious that in studies of human muscle disease the techniques of choice should yield reproducible results with the smallest biopsy specimens possible, and the methods should be so designed that a variety of systems can be studied. The methods of muscle homogenization described herein fulfill this important. criterion. SUMMARY

Homogenization of minces of human skeletal muscle by glass beads in a COe-cooled mechanical shaker facilitates control of temperature and degree of homogenizat,ion. This results in larger yields of mitochondria with excellent biochemical characteristics in contrast to previous preparations of such mitochondrin that showed evidence of damage and contamination. Human skeletal muscle mitochondria isolated by this technique exhibit the properties of mitochondria isolated from other, more readily homogenized tissues. This study establishes the characteristics of normal human skeletal muscle mitochondria and these can now be used for comparison with mitochondria isolated in a variety of diseases that affect muscle. .ICKNOWLEDGMENTS The assistance of K. Stempel and J. Armstrong is gratefully acknowledged. SupIwrted by grants from NIH HD 02584. NB 07587, GM 15759 and FR 00238 and II> the American Cancer Society P-430. REFERENCES 1.

FIELD,

J.,

ZND,

BELDING,

H. S.,

AND

MARTIN,

A. W., .I. Cell Comp. Phhl/siol. 14.

143 ( 1939). 2. KLINQENBERQ, 3. ERNSTER,

M., Engeb. Physiol. Biol. Chem. Expptl. Pharrnakol. 55, 131 (1964). L. D., IKKOS, D., AND LUFT, R., Nature 184, 1851 (1959).

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41, 1776 (1962). 8. ERNSTER, L., AND Lulrr, It., Exptl. Cell Res. 32,26 (1963). 9. CHAPPELL, J. B., .~ND PERRY, S. V., Nature 173, 1094 (1954). 10. GORNALL, A. G., BARnAWILL, C. J., ANn DAVID, M. M., J. Biol. Chem. 177, 751 ( 1949). 11. CHANCY, B., in Ciba Foundation Symposium on the Regulation of Cell Metabolism (G. E. W. Wolstenholme and C. M. O’Connor, cds.), p. 91. Little, Brown, Boston, Massaehusetta, 1959. 12. CHANCE, B., ANn WILLIAMS, G. R., J. Biol. Chem. 217, 429 (1955). 13. JACOBS, E. E., Biochem. Biophys. Res. Commun. 3,536 (1960). 14. PACKER, I,., MARCHANT, R. H., ANn ComrnEN, E., Biochim. Bioph.ys. Acta 78, 214

(1963). 15. ERNSTER, L., Axn NORDENBBAND, II., in “Methods in Enzymology” (R. W. Estabrook and M. E. Pullman, eds.), Vol. X, p. 86, Academic Press, New York, 1967. 16. LEHNINGER, A. L., Harvey Lectures 49, 176 (1955). 17. PETER, J. B., 1967, unpublished observations. 18. ERNSTF,R, I,., DALLNEB, G., ANn AZZONE, G. F., J. Biol. Chem. 238, 1124 (1963). 19. PETEB, J. B., Clin. Res. 16,349 (1968). 20. PETER, J. B., Clin. Res. 16,159 (1968). 21. PETER, J. B., J. Clin. Invest. 47, 78a (1968). 22. COLEMAN, H. F., NIENHUIS, A. W., BROWN, W. J., IMUNSAT. T. L., AND PEARSOK, C. M., J. Am. Med. Assoc. 199,624 (1967). 23. PRICE, H. M., GORDON, G. B., MUNSAT, T. I,., AND PEARSON. C. M.. J. Xeuropath. and Exptl. Neural. 26, 475 (1967). 24. BAunHuIN, P., Emn, P., ANn BERTHET, J., J. Cell Biol. 32, 181 (1967). 25. DUNN, R. F., WoRsFoLn, M., ANn PETER, J. B., Proc. Electron Microscop. Sot. Am. (1968) in press. 26. SAMAHA, F. J., SCHROEDER, J. M., REBEIZ, J., AND ADAMS, K. D., Arch. Neural. 17,

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