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Lactate dehydrogenase isoenzymes: Distribution in fast-twitch red, fast-twitch white, and slow-twitch intermediate fibers of guinea pig skeletal muscle
ARCHIVES
OF
lactate
BIOCHEMISTRY
AND
Dehydrogenase
Red, Fast-Twitch Fibers J. B. PETER,
of Medicine
White,
and
of Guinea
S. SAWAKI,
and Physical Received
(1971)
Isoenzymes:
AND
Departments
144, 304307
BIOPHYSICS
‘Slow-Twitch
University
23, 1970; accepted
in Fast-Twitch Intermediate
Pig Skeletal
R. J. BARNARD, c. A. GILLESPIE
Education,
November
Distribution Muscle
V. R. EDGERTON,
of California, February
Los Angeles,
California
90024
12, 1971
Lactate dehydrogenase activity and LDH isoenzyme distribution were determined in supernatants of skeletal muscles consisting predominately of fast-twitch red, fasttwitch white, or slow-twitch intermediate fibers. The LDH activity was highest in the fast-twitch white. The activity in the fast-twitch red was significantly lower than in the fast-twitch white but significantly greater than in slow-twitch intermediate muscle or the heart. LDHs was found to be the predominant isoenzyme in both the fasttwitch white and fast-twitch red fibers whereas LDHl predominated in both the slowtwitch intermediate fibers and the heart.
Recent histochemical, biochemical, and physiological studies have demonstrated the presence of three distinct fiber types in guinea pig skeletal muscle (1). These three fiber types have been classified as fasttwitch red, fast-twitch white, and slowtwitch intermediate. Each type possesses distinctive combinations of metabolic and mechanical characteristics. The high biochemical and histochemical activities of myosin and actomyosin ATPase in fasttwitch fibers correlate with their fast contraction time (1). These fibers are further subclassified into red or white according to their mitochondrial enzyme activities. Thus, a fast-twitch red fiber has a fast contraction time and high mitochondrial enzyme activity, whereas the fast-twitch white has low activities of mitochondrial enzymes. The lower specific activities of myosin and actomyosin ATPase of slow-twitch intermediate fibers correlate with their slow twitch time. Slow-twitch intermediate fibers have mitochondrial enzyme activities which are intermediate between the fast-twitch red and fast-twitch white fibers (unpublished data). Recent reports (2, 3) demonstrated that 304
the glycogen concentration is high in both fast-twitch red and fast-twitch white fibers whereas it is low in slow-twitch intermediate fibers and heart (cf. Table I). In view of these distinct characteristics among the three fiber types we have investigated the lactate dehydrogenase (LDH) activity and the LDH isoenzyme pattern in muscles which are composed predominately or solely of one of the three fiber types. MATERIALS
AND
METHODS
The soleus (1007, slow-twitch intermediate fibers), the red portion of the vastus lateralis (787, fast-twitch red fibers), the white portion of the vastus lateralis (71% fast-tm-itch white fibers), and heart muscle were studied in seven adult, male guinea pigs. The animals were sacrificed with a sharp blow to the head. The muscles were quickly removed, trimmed, weighed, scissorminced, and homogenized in distilled water (1:3, w/v). The homogenates were then centrifuged for 30 min at 40,OOOg (24’). Samples of the supernatants were placed on 3 X G-cm strips of Cellogel (Chemetron Ltd., Milano, Italy) for separation of the isoenzymes. Electrophoresis was carried out in a SpincoDurrum type electrophoretic cell in which the support stand had been removed. The cell was
LACTATE
DEHYDROGENASE TABLE
COMPSRISON OF LDH
ISOENZYMES
305
I
ACTIVITY OFSKELET~L AND CARDIAC MUSCLE CYTOCHROME a CONCENTRATIOKS
WITH GLYCOGEN AND
12luscle:
White vastus
Red vastus
S&US
Heart
Fiber types:
Fast-twitch white (71%)
Fast-twitch red (78%)
Slow-twitch intermediate (10070)
Slow-twitch
LDH (pmolesjminjg) Glycogen (mg/g wet) tissue wt) Cytochrome Q (nmoles/g) a Dat,a reported
543 I!Z 42
350 i
24
108 f
20
163 zk 27
7.4 zk 0.6
9.7 f
0.8
3.3 f
0.4
5.2 f
0.4”
5.7 + 0.7
32.8 f
2.8
2.3 f
by Lamb
0.3
13.3 3~ 0.6
et al. (3).
filled to the fluid line with 50 mM barbital buffer, pH 8.6. The separation was carried out for 60 min at 4 mA, 100 V. Thereafter the Cellogel strips were incubated for 10 min in a medium cont,aining 50 rnM phosphate buffer (pH 7.4), 3.5 mM NAD, 1.4 rn~ nitroblue tetrazolium (NBT), 8 mM phenazine methosulfate (PMS), and 26 mM Na lactate. After incubation the Cellogel strips were rinsed and stored in 276 acetic acid. The isoenzyme
distribution
(relative
percentage)
was measured
in a Beckman Model R-110 microzone densitometer. For quantitative analysis of LDH activity appropriate dilutions of the remaining supernatant were made in 50 mu phosphate buffer (pH 7.4). The reaction was initiated by adding 0.1 ml snpernat,ant to 2.9 ml of a medium containing 50 rnM phosphate buffer (pH 7.4), 2.0 mM NADH, and 1.0 rn>f Sa pyruvate. The initial rates of oxidation of NADH were measured at 37” in a Gilford 2000 recording spectrophotometer at 340 rnp. Cytochrome concentration of whole homogenates was determined by the method of Schollmeyer and Klingenberg (4) on a Cary recording spectrophotometer. (+lycogeu was determined by t,he method of ?;elsol~ (5) as described previously (2). RESULTS
Quantitative assays for LDH activity (Table I) in the different muscles demonst,rate t,hnt the activity is highest in the white vastus lateralis. The LDH activity in the red vnst,us is significantly lower than t,hat of the white vast,us but is significantly higher than that of the heart or soleus. The electrophoretic distribut,ion of the various LDH isoenzymes also shows significant differences among the different muscles (Fig. 1; Table II). The red and white vasks, which are both fast-twitch, have a predomi-
FIG. 1. LDH
isoenzyme patterns muscles,
of guinea pig
nance of LDH, or muscle-type LDH. Conversely the soleus and heart muscles which are both slow-t’wi-itch muscles have a predominance of LDHl or heart-t)ype LDH. I>ISCUSSION
Since each of the muscles analyzed in this st.udy, was composed predominately or exclusively of a single fiber type (Table I), the data should reflect individual biochemical differences among the different types of fibers. The data show distinct qualitative as well as quantitative differences in LDH of the three fiber types. Although other investi-
PETER ET AL.
306
TABLE II L~CT~TE Muscle
White vastus Red vastus Soleus
Heart,
LDHI
LDHz
o.oa 5.5 f 68.4 i 57.0 f
DEHYDROGENASE
o.oa 2.3 2.9 3.7
9.5 f 25.4 f 27.7 f
ISOENZYME DISTRIBUTION LDH3
1.1 f: 0.2 1.6 1.3 1.8
15.1 f 3.2 f 9.0 f
1.5 0.9 1.9
LDH4
LDHs
8.8 f 1.1
90.2 f 1.0
21.2 f: 1.1 2.0 f 0.6 2.8 f 0.5
48.3 f 4.7 0.8 f 0.3 2.7 zt 0.8
a LDHl and LDHz bands were observed in the white vastus but. were characteristically too light to be quantitated on the densitometer. gators (6-11) have reported LDH activity and isoenzyme patterns for visibly red and white muscles, it is difficult to correlate their data with that presented here because recent work has demonstrated that mammalian muscles which are red in appearance can be composed of high percentages of either slow-twitch intermediate or fasttwit’ch red fibers (1). The percentages of LDHI, LDH2, and LDH3 in the red vastus are small, but these are most likely a true reflection of t’he actual LDH isoenzyme composition of fast-twitch red fibers and not due to contamination of the muscle sample with slow-twitch intermediate fibers because this portion of the vastus lateralis contains less than 4% slow-twitch intermediate fibers. Although t,he physiological significance of the different LDH isoenzymes has not been established, Kaplan and his associates (8,12) have suggested that LDHI, which is inhibited in vitro by high pyruvate concentrations, is found in tissues with a high aerobic capacity and thus may function to funnel pyruvate into the Krebs’ cycle. Conversely LDH5, which is not inhibited by high pyruvate concentrations, is found in tissues with a high anaerobic capacity and thus may be important in the reduction of pyruvate to lactate. The high percentage of LDHl in the soleus, which has moderate aerobic (cytochrome a) and low anaerobic (glycogen and LDH) capacities, as well as t,he high percentage of LDHB in the white vastus, which is characterized by low aerobic and high anaerobic capacities, support the concepts expressed by Kaplan et al. (8, 12). These findings, along with the reported increase in LDH5 in human heart muscle exposed to
decreased oxygen supply (13) suggest that LDH6 is an important enzyme for glycolysis. In addition the red vastus which has high LDHS as well as high cytochrome a and glycogen may have a high capacity for glycolysis in addition to the well-known capacity for oxidative phosphorylation. Examination of the red vastus, white vastus, and soleus muscles of the guinea pig reveals that the fast-twitch red, fast-twitch white, and slow-twitch intermediate fibers can be readily distinguished by t’heir LDH activity and isoenzyme pattern. Since the isoenzyme composition of skeletal muscles from the same species is so variable, the or “heart-type” are terms “muscle-type” inadequate for describing isoenzymes of LDH. ACKNOWLEDGMENTS Supported by NIH Grants NS 07587, HD 02584, and NS 08590. R. J. Barnard is a postdoctoral fellow of the Muscular Dystrophy Associations of America. REFERENCES 1. BARNARD, R. J., EDGERTON, V. R., FURUKAWA, T., AND PETER, J. B., Amer. J. Physiol., 220, 410 (1971). 2. GILLESPIE, C. A., SIMPSON, D. R., AND EDGERTON, V. R., J. Histochem. Cystochem. 18, 552 (1970). 3. L.4MB, D. R., PETER, J. B., JEFFRESS, R. N., AND WALLACE, H. A., Amer. J. Physiol. 217, 1628 (1969). M., 4. SCHOLLMEYER, P., AND KLINGENBERG, Biochem. 2. 336, 426 (1962). 5. NELSON, N., J. Biol. Chem. 163, 375 (1944). 6. GARCIA-BURUEL, L., GARCIA-BURUEL, V. M., GREEN, L., AND SUBIN, D. K., Neurology 16, 491 (1966). 7. BLANCHAER, M. C., BND V.4N WIJHE, M., Amer. J. Physiol. 202, 827 (1962).
LACTATE
DEHYDROGENASE
8. DAWSON, D. M., KAPLAN,N. O., AND GOODFRIEND, T. L., Science 143, 929 (1964). 9. BLANCHAER, M. C., VAN WIJHE, M., AND MOZERSKY, D., J. Histochem. Cytochem. 11, 500 (1963). 10. TaKASU, T., AND HUGHES, B. P., .I. Neural. Neurosurg. Psychiat. 32, 175 (1969).
ISOENZYMES
307
11. VAN WIJHE, M., BLANCHAER, M. C., AND ST. GEORGE-STUBBS, S., J. Histochem. Cytothem. 12, 6008 (1964). 12. CAHN, R. D., KAPLAN, N. O., LEVINE, L. AND ZWILLING, E., Science 136, 962 (1962). 13. BALLO, J. M., AND MESSER, J. V., Biochem. Biophys. Res. Commun. 33, 487 (1968).
OF
lactate
BIOCHEMISTRY
AND
Dehydrogenase
Red, Fast-Twitch Fibers J. B. PETER,
of Medicine
White,
and
of Guinea
S. SAWAKI,
and Physical Received
(1971)
Isoenzymes:
AND
Departments
144, 304307
BIOPHYSICS
‘Slow-Twitch
University
23, 1970; accepted
in Fast-Twitch Intermediate
Pig Skeletal
R. J. BARNARD, c. A. GILLESPIE
Education,
November
Distribution Muscle
V. R. EDGERTON,
of California, February
Los Angeles,
California
90024
12, 1971
Lactate dehydrogenase activity and LDH isoenzyme distribution were determined in supernatants of skeletal muscles consisting predominately of fast-twitch red, fasttwitch white, or slow-twitch intermediate fibers. The LDH activity was highest in the fast-twitch white. The activity in the fast-twitch red was significantly lower than in the fast-twitch white but significantly greater than in slow-twitch intermediate muscle or the heart. LDHs was found to be the predominant isoenzyme in both the fasttwitch white and fast-twitch red fibers whereas LDHl predominated in both the slowtwitch intermediate fibers and the heart.
Recent histochemical, biochemical, and physiological studies have demonstrated the presence of three distinct fiber types in guinea pig skeletal muscle (1). These three fiber types have been classified as fasttwitch red, fast-twitch white, and slowtwitch intermediate. Each type possesses distinctive combinations of metabolic and mechanical characteristics. The high biochemical and histochemical activities of myosin and actomyosin ATPase in fasttwitch fibers correlate with their fast contraction time (1). These fibers are further subclassified into red or white according to their mitochondrial enzyme activities. Thus, a fast-twitch red fiber has a fast contraction time and high mitochondrial enzyme activity, whereas the fast-twitch white has low activities of mitochondrial enzymes. The lower specific activities of myosin and actomyosin ATPase of slow-twitch intermediate fibers correlate with their slow twitch time. Slow-twitch intermediate fibers have mitochondrial enzyme activities which are intermediate between the fast-twitch red and fast-twitch white fibers (unpublished data). Recent reports (2, 3) demonstrated that 304
the glycogen concentration is high in both fast-twitch red and fast-twitch white fibers whereas it is low in slow-twitch intermediate fibers and heart (cf. Table I). In view of these distinct characteristics among the three fiber types we have investigated the lactate dehydrogenase (LDH) activity and the LDH isoenzyme pattern in muscles which are composed predominately or solely of one of the three fiber types. MATERIALS
AND
METHODS
The soleus (1007, slow-twitch intermediate fibers), the red portion of the vastus lateralis (787, fast-twitch red fibers), the white portion of the vastus lateralis (71% fast-tm-itch white fibers), and heart muscle were studied in seven adult, male guinea pigs. The animals were sacrificed with a sharp blow to the head. The muscles were quickly removed, trimmed, weighed, scissorminced, and homogenized in distilled water (1:3, w/v). The homogenates were then centrifuged for 30 min at 40,OOOg (24’). Samples of the supernatants were placed on 3 X G-cm strips of Cellogel (Chemetron Ltd., Milano, Italy) for separation of the isoenzymes. Electrophoresis was carried out in a SpincoDurrum type electrophoretic cell in which the support stand had been removed. The cell was
LACTATE
DEHYDROGENASE TABLE
COMPSRISON OF LDH
ISOENZYMES
305
I
ACTIVITY OFSKELET~L AND CARDIAC MUSCLE CYTOCHROME a CONCENTRATIOKS
WITH GLYCOGEN AND
12luscle:
White vastus
Red vastus
S&US
Heart
Fiber types:
Fast-twitch white (71%)
Fast-twitch red (78%)
Slow-twitch intermediate (10070)
Slow-twitch
LDH (pmolesjminjg) Glycogen (mg/g wet) tissue wt) Cytochrome Q (nmoles/g) a Dat,a reported
543 I!Z 42
350 i
24
108 f
20
163 zk 27
7.4 zk 0.6
9.7 f
0.8
3.3 f
0.4
5.2 f
0.4”
5.7 + 0.7
32.8 f
2.8
2.3 f
by Lamb
0.3
13.3 3~ 0.6
et al. (3).
filled to the fluid line with 50 mM barbital buffer, pH 8.6. The separation was carried out for 60 min at 4 mA, 100 V. Thereafter the Cellogel strips were incubated for 10 min in a medium cont,aining 50 rnM phosphate buffer (pH 7.4), 3.5 mM NAD, 1.4 rn~ nitroblue tetrazolium (NBT), 8 mM phenazine methosulfate (PMS), and 26 mM Na lactate. After incubation the Cellogel strips were rinsed and stored in 276 acetic acid. The isoenzyme
distribution
(relative
percentage)
was measured
in a Beckman Model R-110 microzone densitometer. For quantitative analysis of LDH activity appropriate dilutions of the remaining supernatant were made in 50 mu phosphate buffer (pH 7.4). The reaction was initiated by adding 0.1 ml snpernat,ant to 2.9 ml of a medium containing 50 rnM phosphate buffer (pH 7.4), 2.0 mM NADH, and 1.0 rn>f Sa pyruvate. The initial rates of oxidation of NADH were measured at 37” in a Gilford 2000 recording spectrophotometer at 340 rnp. Cytochrome concentration of whole homogenates was determined by the method of Schollmeyer and Klingenberg (4) on a Cary recording spectrophotometer. (+lycogeu was determined by t,he method of ?;elsol~ (5) as described previously (2). RESULTS
Quantitative assays for LDH activity (Table I) in the different muscles demonst,rate t,hnt the activity is highest in the white vastus lateralis. The LDH activity in the red vnst,us is significantly lower than t,hat of the white vast,us but is significantly higher than that of the heart or soleus. The electrophoretic distribut,ion of the various LDH isoenzymes also shows significant differences among the different muscles (Fig. 1; Table II). The red and white vasks, which are both fast-twitch, have a predomi-
FIG. 1. LDH
isoenzyme patterns muscles,
of guinea pig
nance of LDH, or muscle-type LDH. Conversely the soleus and heart muscles which are both slow-t’wi-itch muscles have a predominance of LDHl or heart-t)ype LDH. I>ISCUSSION
Since each of the muscles analyzed in this st.udy, was composed predominately or exclusively of a single fiber type (Table I), the data should reflect individual biochemical differences among the different types of fibers. The data show distinct qualitative as well as quantitative differences in LDH of the three fiber types. Although other investi-
PETER ET AL.
306
TABLE II L~CT~TE Muscle
White vastus Red vastus Soleus
Heart,
LDHI
LDHz
o.oa 5.5 f 68.4 i 57.0 f
DEHYDROGENASE
o.oa 2.3 2.9 3.7
9.5 f 25.4 f 27.7 f
ISOENZYME DISTRIBUTION LDH3
1.1 f: 0.2 1.6 1.3 1.8
15.1 f 3.2 f 9.0 f
1.5 0.9 1.9
LDH4
LDHs
8.8 f 1.1
90.2 f 1.0
21.2 f: 1.1 2.0 f 0.6 2.8 f 0.5
48.3 f 4.7 0.8 f 0.3 2.7 zt 0.8
a LDHl and LDHz bands were observed in the white vastus but. were characteristically too light to be quantitated on the densitometer. gators (6-11) have reported LDH activity and isoenzyme patterns for visibly red and white muscles, it is difficult to correlate their data with that presented here because recent work has demonstrated that mammalian muscles which are red in appearance can be composed of high percentages of either slow-twitch intermediate or fasttwit’ch red fibers (1). The percentages of LDHI, LDH2, and LDH3 in the red vastus are small, but these are most likely a true reflection of t’he actual LDH isoenzyme composition of fast-twitch red fibers and not due to contamination of the muscle sample with slow-twitch intermediate fibers because this portion of the vastus lateralis contains less than 4% slow-twitch intermediate fibers. Although t,he physiological significance of the different LDH isoenzymes has not been established, Kaplan and his associates (8,12) have suggested that LDHI, which is inhibited in vitro by high pyruvate concentrations, is found in tissues with a high aerobic capacity and thus may function to funnel pyruvate into the Krebs’ cycle. Conversely LDH5, which is not inhibited by high pyruvate concentrations, is found in tissues with a high anaerobic capacity and thus may be important in the reduction of pyruvate to lactate. The high percentage of LDHl in the soleus, which has moderate aerobic (cytochrome a) and low anaerobic (glycogen and LDH) capacities, as well as t,he high percentage of LDHB in the white vastus, which is characterized by low aerobic and high anaerobic capacities, support the concepts expressed by Kaplan et al. (8, 12). These findings, along with the reported increase in LDH5 in human heart muscle exposed to
decreased oxygen supply (13) suggest that LDH6 is an important enzyme for glycolysis. In addition the red vastus which has high LDHS as well as high cytochrome a and glycogen may have a high capacity for glycolysis in addition to the well-known capacity for oxidative phosphorylation. Examination of the red vastus, white vastus, and soleus muscles of the guinea pig reveals that the fast-twitch red, fast-twitch white, and slow-twitch intermediate fibers can be readily distinguished by t’heir LDH activity and isoenzyme pattern. Since the isoenzyme composition of skeletal muscles from the same species is so variable, the or “heart-type” are terms “muscle-type” inadequate for describing isoenzymes of LDH. ACKNOWLEDGMENTS Supported by NIH Grants NS 07587, HD 02584, and NS 08590. R. J. Barnard is a postdoctoral fellow of the Muscular Dystrophy Associations of America. REFERENCES 1. BARNARD, R. J., EDGERTON, V. R., FURUKAWA, T., AND PETER, J. B., Amer. J. Physiol., 220, 410 (1971). 2. GILLESPIE, C. A., SIMPSON, D. R., AND EDGERTON, V. R., J. Histochem. Cystochem. 18, 552 (1970). 3. L.4MB, D. R., PETER, J. B., JEFFRESS, R. N., AND WALLACE, H. A., Amer. J. Physiol. 217, 1628 (1969). M., 4. SCHOLLMEYER, P., AND KLINGENBERG, Biochem. 2. 336, 426 (1962). 5. NELSON, N., J. Biol. Chem. 163, 375 (1944). 6. GARCIA-BURUEL, L., GARCIA-BURUEL, V. M., GREEN, L., AND SUBIN, D. K., Neurology 16, 491 (1966). 7. BLANCHAER, M. C., BND V.4N WIJHE, M., Amer. J. Physiol. 202, 827 (1962).
LACTATE
DEHYDROGENASE
8. DAWSON, D. M., KAPLAN,N. O., AND GOODFRIEND, T. L., Science 143, 929 (1964). 9. BLANCHAER, M. C., VAN WIJHE, M., AND MOZERSKY, D., J. Histochem. Cytochem. 11, 500 (1963). 10. TaKASU, T., AND HUGHES, B. P., .I. Neural. Neurosurg. Psychiat. 32, 175 (1969).
ISOENZYMES
307
11. VAN WIJHE, M., BLANCHAER, M. C., AND ST. GEORGE-STUBBS, S., J. Histochem. Cytothem. 12, 6008 (1964). 12. CAHN, R. D., KAPLAN, N. O., LEVINE, L. AND ZWILLING, E., Science 136, 962 (1962). 13. BALLO, J. M., AND MESSER, J. V., Biochem. Biophys. Res. Commun. 33, 487 (1968).