Activity patterns of phosphofructokinase,glyceraldehydephosphate dehydrogenase,lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle

Summary Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.     

Activity patterns of phosphofructokinase, glyceraldehydephosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle.

Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.     

Metabolic and physiologic characteristics of skeletal muscle determine its response to clenbuterol treatment

beta-Adrenoceptor agonists are reported to induce skeletal muscle hypertrophy and hence serve as valuable adjunct to the treatment of wasting disorders. In the present study, we attempted to find out whether metabolic and physiologic characteristics of fibres are important in determining skeletal muscle response to clenbuterol (an adrenergic receptor agonist) therapy, as proposed in the treatment of wasting disorders. The treatment of mice with clenbuterol (2 mg/kg body wt for 30 days) resulted in skeletal muscle hypertrophy, more common amongst fast-twitch glycolytic fibres/muscle, with increase in body mass and a parallel rise in muscle mass to body mass ratio. Measurement of fibre diameters in soleus (rich in slow-twitch oxidative fibres), ALD or anterior latissimus dorsi (with a predominance of fast-twitch glycolytic fibres) and gastrocnemius (a mixed-type of muscle) from clenbuterol-treated mice for 30 days revealed noticeable increase in the per cent population of narrow slow-twitch fibre and a corresponding decline in white-type or fast-twitch glycolytic fibres in gastrocnemius and ALD. As revealed by counting of muscle cells in soleus, narrow red fibres declined with corresponding increase in white-type glycolytic fibres population. A significant decline in the succinic dehydrogenase activity was observed, thereby suggesting abnormality in oxidative activity of skeletal muscles in response to clenbuterol therapy.     

Myosin light chain patterns of individual fast and slow-twitch fibres of rabbit muscles

Summary Single muscle fibres were isolated by microdissection from freeze-dried samples of rabbit psoas and soleus muscles. The individual fibres were typed according to qualitative histochemical reactions for succinate dehydrogenase or NADH-tetrazolium reductase and for alkaline Ca²⁺-activated myofibrillar myosin ATPase after acid or alkaline preincubation. Methods are described for electrophoretic analysis by means of polyacrylamide disc electrophoresis in the presence of SDS of total myofibrillar proteins in single fibres after pre-extraction of soluble proteins. Fast-twitch white fibres revealed a myosin light chain pattern characteristic of fast-type myosin with three light chains of apparent molecular weights of 22,300 (LC₁), 18,400 (LC₂) and 16,000 (LC₃). Fast-twitch red fibres were indistinguishable in this respect from fast-twitch white fibres and showed an identical pattern of myosin light chains. Slow-twitch fibres could be characterized by a myosin light chain pattern typical of myosin of slow-twitch muscles with peptides of the apparent molecular weights of 23,500 (LC_1Sa), 23,000 (LC_1Sb) and 18,500 (LS_2S). Slow-twitch fibres isolated from soleus as well as from psoas muscle were indistinguishable with regard to their myosin light chain patterns, thus suggesting that fibres of the same histochemical type correspond in their myosin light chain patterns irrespective of their origin from different muscles.Dedicated to the memory of Ernest Gutmann who has contributed so much to our knowledge on differentiation of muscle and who died on August 6, 1977     

Effect of exercise on muscle fibre composition and enzyme activities of skeletal muscles in young rats

Young Wistar rats underwent dynamic (D) or static (S) exercise from the 5th to 35th day after birth. Histochemical and biochemical analysis were performed in the extensor digitorum longus (EDL) and the soleus muscle (SOL). Lactate dehydrogenase (LDH) (regulating anaerobic metabolism) and citrate synthase (CS) and hydroxyacyl-CoA dehydrogenase (HAD) (both regulating aerobic metabolism) activities were determined spectrophotometrically. An increase of the fast oxidative-glycolytic (FOG) muscle fibres was found in the slow SOL muscle in both trained groups, i.e. by 10% in group D and by 7% in group S in comparison with the C group. The EDL muscle fibre distribution did not differ from those of control animals in respect to the slow oxidative (SO) fibre type. A higher percentage of FOG fibres by 19% was found in group D contrary to a decreased number of the fast glycolytic (FG) muscle fibres in this trained group. The greatest increase of CS (EDL 185%, SOL 176%) and HAD (EDL 83%, SOL 178%) activities were found in group D as compared with control group (C). Only small differences were observed in LDH activity. The values of characteristic enzyme activity ratios show that dynamic training resulted in an elevation of oxidative capacity of skeletal muscle, while the static load led preferentially along the glycolytic pathway. It may be concluded that an adaptive response to the training load during early postnatal development is different due to the type of exercise (dynamic or static) and/or the type of skeletal muscle (fast or slow).     

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

Summary This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.     

Quantitative histochemistry of three mouse hind-limb muscles: the relationship between calcium-stimulated myofibrillar ATPase and succinate dehydrogenase activities

Summary A quantitative modification of Meijer's calcium-lead capture method, for the demonstration of calcium-stimulated myofibrillar ATPase activity at physiological pH, is described. A range of myofibrillar ATPase activities has been found among fast muscle fibres in two mouse hind-limb muscles. The myofibrillar ATPase activity of fast muscle fibres is 1.5–3 times higher than the myofibrillar ATPase activity of slow muscle fibres.Myofibrillar ATPase activities and succinate dehydrogenase activities of individual muscle fibres have been determined in serial sections. Activities of the two enzymes are correlated positively in soleus (fast and slow fibres), and negatively in plantaris (almost all fast) and extensor digitorum longus muscle (all fast). However, this correlation is not significant among the oxidative fibres in the extensor digitorum longus. The fibres of the latter muscle cannot be classified satisfactorily into two sub-types.     

Skeletal muscle fiber type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression in fed and food-deprived rats

Fiber type specificity of pyruvate dehydrogenase (PDH) phosphatase (PDP) was determined in fed (CON) and 48-h food-deprived (FD) rats. PDP activity and isoform protein content were determined in soleus (slow-twitch oxidative), red gastrocnemius (RG; fast-twitch oxidative glycolytic), and white gastrocnemius (WG; fast-twitch glycolytic) muscles. When normalized for mitochondrial volume, there was no difference in PDP activity between muscle types or CON and FD. When expressed per gram wet tissue weight, PDP activity was higher in RG compared with soleus and WG in both CON and FD rats. PDP activities from CON muscles were 1.48 +/- 0.19, 2.68 +/- 0.65, and 1.20 +/- 0.33 nmol x min(-1) x g wet tissue wt(-1) in soleus, RG, and WG, respectively, and decreased in FD muscles (1.22 +/- 0.22, 2.00 +/- 0.57, and 0.84 +/- 0.18 nmol x min(-1) x g wet tissue wt(-1)). This correlated with increased PDP2 protein, however, only in RG, as PDP2 was not detectable in soleus or WG. PDP1 protein was not responsive to food deprivation in all fiber types. In conclusion, PDP activity and protein content were higher in fast-twitch oxidative glycolytic muscles from CON and FD rats, identifying a unique inter- and intramuscular distribution. FD induced a small but significant decrease in PDP activity that was partially due to decreases in PDP2 protein. As a result, coordinate changes to PDP activity opposite to those of the other regulatory enzyme, PDH kinase, during food deprivation would maximize the inactivation of skeletal muscle PDH and enhance carbohydrate conservation during periods of limited carbohydrate supply.     

Effects of beta 1- vs. beta 1- beta 2-blockade on training adaptations in rat skeletal muscle

The effect of selective vs. nonselective beta-blockade on fast-twitch [extensor digitorum longus (EDL)] and slow-twitch [soleus (SOL)] muscle enzyme activities following endurance training were characterized. Citrate synthase (CS), lactate dehydrogenase (LDH), and beta-hydroxyacyl-CoA dehydrogenase (HAD) activities were compared in SOL and EDL muscles of trained (T), metoprolol-trained (MT), propranolol-trained (PT), and sedentary (C) rats. Following 8 wk of treadmill running (1 h/day, 5 days/wk at approximately 30 m/min), LDH activity was depressed approximately 20% (P less than 0.05) in both SOL and EDL in only the PT rats, indicating inhibition of beta 2-mediated anaerobic glycolysis. EDL CS activity was similarly elevated in all three trained groups compared with sedentary controls. In SOL muscle, however, a drug attenuation effect was observed so that CS activity was increased only in the T (P less than 0.01) and MT (P less than 0.05) groups. HAD enzyme activity was increased somewhat (P less than 0.10) in SOL muscle in only the T group, but more so (P less than 0.05) in EDL in all three trained groups. The above findings suggest a training-induced selectivity effect not only with respect to beta 1-vs. beta 1-beta 2-blockers, but also with respect to muscle fiber type.     

The effect of torbafylline on enzyme activities in fast and slow muscles with limited blood supply.

1. Activities of a glycolytic enzyme--lactate dehydrogenase, LDH, and two oxidative enzymes--citrate synthase (CS), a marker for TCA cycle entry, and 3-hydroxyacyl-CoA dehydrogenase (HAD), which indicates the capacity for beta-oxidation of endogenous lipids, were measured in fast (tibialis anterior, TA, and extensor digitorum longus, EDL) and slow (soleus, SOL) muscles of Sprague-Dawley rats with intact and limited blood supply, and following treatment with the xanthine derivative torbafylline (Hoechst, Werk Albert, Wiesbaden). 2. Limitation of blood supply by unilateral ligation of the common iliac artery increased activity of LDH in fast muscles, and activity of CS and HAD in soleus. 3. Torbafylline treatment caused an increased LDH activity in intact fast muscles and decreased it in soleus, although the relative capacity for anaerobic and aerobic metabolism (indicated by the ratio of LDH and CS activities) remained unchanged in all cases. 4. Whilst having little effect on oxidative enzyme activity of fast muscles, torbafylline decreased the activity of CS but increased activity of HAD in soleus, suggesting a greater reliance on lipid metabolism. 5. The effect of arterial ligation on enzyme activity was ameliorated by treatment with torbafylline, possibly due to its effect on the microcirculation.     

Fibre-type specificity and effect of thyroid hormone on glucose 6-phosphate dehydrogenase activity in normal and denervated skeletal muscles of the rat

Summary Glucose-6-phosphate dehydrogenase activity increases following denervation of rat skeletal muscle. The specificity of this effect to muscle fibre type was studied. Basal activity of the dehydrogenase was higher in soleus, a muscle composed predominantly of type I fibres, than in extensor digitorum longus, a muscle composed predominantly of type IIa and b fibres. The enzymatic activity of the soleus was also greater than that of the red (RQ) and white (WQ) portions of quadriceps muscle (predominantly type IIa and type IIb fibres, respectively). Following denervation, glucose-6-phosphate dehydrogenase increased in extensor digitorum longus and RQ, but not in WQ or the soleus. Following chronic treatment of rats with 3,3,5-triiodothyronine, which converts type I muscle fibres to type II, the dehydrogenase activity increased in both denervated soleus and extensor digitorum longus. It is concluded that the effect of denervation on glucose-6-phosphate dehydrogenase activity is selective for type IIa (fast oxidative-glycolytic) muscle fibres.     

The immunohistochemical location of cathepsin L in rabbit skeletal muscle

Summary The immunohistochemical location of cathepsin L in rabbit soleus, plantaris and psoas muscles was investigated using the peroxidase-anti-peroxidase (PAP) technique. The amount of enzyme detected varied according to the fibre type, which were identified by histochemical staining of serial sections for succinate dehydrogenase and alkali-stable myosin ATPase. In the three muscles studied labelling was strongest in the highly oxidative fibres and weaker in the other fibre types with least staining in the fast white fibres. Immunoreactive cathepsin L appeared to be most concentrated at the periphery of muscle fibres, especially near to the nuclei, although some staining was seen throughout the fibres.     

The immunohistochemical location of cathepsin L in rabbit skeletal muscle. Evidence for a fibre type dependent distribution

The immunohistochemical location of cathepsin L in rabbit soleus, plantaris and psoas muscles was investigated using the peroxidase-anti-peroxidase (PAP) technique. The amount of enzyme detected varied according to the fibre type, which were identified by histochemical staining of serial sections for succinate dehydrogenase and alkali-stable myosin ATPase. In the three muscles studied labelling was strongest in the highly oxidative fibres and weaker in the other fibre types with least staining in the fast white fibres. Immunoreactive cathepsin L appeared to be most concentrated at the periphery of muscle fibres, especially near to the nuclei, although some staining was seen throughout the fibres.     

Changes in the metabolic profile of the equine gluteus medius as a function of sampling depth

1. Cross sections from the middle of the gluteus medius were removed from 10 adult horses and used to evaluate changes in histochemically determined muscle fiber type and biochemically determined metabolic enzyme activities as a function of sample depth. 2. Muscle fiber types determined using histochemical methods for myosin ATPase (pH 9.4) and succinic dehydrogenase (SDH) activity indicated percent fast-twitch glycolytic (FG) muscle fibers decreased and slow-twitch oxidative (SO) fibers increased as a function of increasing sampling depth. 3. Percent histochemically determined fast-twitch oxidative glycolytic (FOG) fibers decreased slightly only in the deepest region of the gluteus medius. 4. Citrate synthase (CS) enzymatic activity, used as a marker for mitochondrial oxidative potential, increased 2.5-fold in activity per g of muscle protein from 1 to 8 cm sampling depth. 5. 3-hydroxyacyl-CoA dehydrogenase (HAD) enzymatic activity, used as a marker for lipid oxidation potential, increased 3-fold in activity per g of muscle protein when the depth increased from 1 to 8 cm. 6. Phosphorylase (PS) enzymatic activity, used as a marker for potential glycogen utilization, decreased 50% in activity per g of muscle protein when going from 1 to 8 cm. 7. Lactate dehydrogenase (LDH) enzymatic activity, used as a marker for anaerobic glycolytic potential, decreased about 50% in activity as the sampling depth increased from 1 to 8 cm. 8. In summary, the superficial portion of the equine gluteus medius was found to be more glycolytic and less aerobic in its metabolic profile than deeper regions. The muscle became progressively more aerobic and less glycolytic with increasing sampling depth.     

Lactate and malate dehydrogenases in the muscles and male genital tract of the rabbit.

Average lactate dehydrogenase (LDH) isoenzyme patterns the content of H subunits, total LDH activity, total malate dehydrogenase (MDH) activity and the m- MDH/s-MDH ratio were determined in twelve muscles and the male genital tract of the rabbit. LDH(1) was the predominant form in the heart, soleus and masseter muscles, LDH(3) in the lingual muscles and LDH(5) in the other muscles analysed. In the muscles, an increase in the percentual proportion of M subunits was accompanied, by a proportional increase in total LDH activity and a decrease in total MDH activity, especially m-MDH. LDH isoenzyme patterns and LDH and MDH activities are useful for obtaining some idea about the proportion of individual muscle fibres. Activity accounted for by H subunits was roughly the same in all the muscles analysed, indicating that the synthesis of H subunits is independent of the type of muscle fibre and of the oxygen supply of the muscular tissue, and also that isoenzymes composed chiefly of H subunits are not localized preferentially in the mitochondria. Similar relationships between LDH isoenzymes and LDH and MDH activities were found in the testicular and epididymal tissues. The tests and the head of the epididymis mainly contain LDH isoenzymes composed of H subunits. The total LDH activity in these tissues is relatively low and their MDH activity is relatively high compared with the body and tail of the epididymis. The proportion of H subunits in the ampulla, the seminal vesicles, the coagulating glands and the prostate is also high. Cowper's glands have a high LDH(5) and LDH(4) concentration. One of two LDHx isoenzymes were found in the testes and spermatozoa.     

Changes in ATPase and SDH reactions of the rat extrafusal and intrafusal muscle fibres after preincubations at different pH.

The dependence of adenosine-triphosphatase (ATPase) and succinic dehydrogenase (SDH) histochemical reactions on the pH of the preincubation medium was studied in serial cross sections of 1- to 6-month-old rat extensor digitorum longus (EDL) and soleus (SOL) muscles. The use of a wide spectrum of pH values confirmed the previous results showing that: (1) according to their ATPase and SDH reactions 3 types of extrafusal muscle fibres, i.e., fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG) and slow-twitch oxidative (SO) and 3 types of intrafusal muscle fibres, i.e. typical and intermediate nuclear bag fibres and nuclear chain fibres were observed; (2) only acid preincubation (pH 4.35) is necessary to demonstrate the reversal of the ATPase reaction; while (3) alkali preincubation (pH 10.4) does not provide any new important information as compared with ATPase without preincubation. Furthermore, it was shown that: (4) fast-twitch muscle fibres exhibited high ATPase activity on preincubations at pH 4.9 to 10.4, slow-twitch fibres had very high ATPase activity on preincubation at pH 4.3 and 4.5; (5) after preincubation at pH 4.5 two types of FOG fibres were observed, differing in their ATPase activity; (6) in both muscles there were fibres with intermediate ATPase activity both after acid and/or alkali preincubations; (7) the intrafusal muscle fibres exhibited some specific characteristics when compared with extrafusal fibres. In contrast to the ATPase reactions, SDH activity was decreased equally, in both extra- and intrafusal fibres, with increasing acidity and alkality of the preincubation medium.     

Changes in ATPase and SDH reactions of the rat extrafusal and intrafusal muscle fibres after preincubations at different pH

Summary The dependence of adenosine-triphosphatase (ATPase) and succinic dehydrogenase (SDH) histochemical reactions on the pH of the preincubation medium was studied in serial cross sections of 1- to 6-month-old rat extensor digitorum longus (EDL) and soleus (SOL) muscles.The use of a wide spectrum of pH values confirmed the previous results showing that: (1) according to their ATPase and SDH reactions 3 types of extrafusal muscle fibres, i.e., fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG) and slow-twitch oxidative (SO) and 3 types of intrafusal muscle fibres, i.e. typical and intermediate nuclear bag fibres and nuclear chain fibres were observed; (2) only acid preincubation (pH 4.35) is necessary to demonstrate the reversal of the ATPase reaction; while (3) alkali preincubation (pH 10.4) does not provide any new important information as compared with ATPase without preincubation. Furthermore, it was shown that: (4) fast-twitch muscle fibres exhibited high ATPase activity on preincubations at pH 4.9 to 10.4, slow-twitch fibres had very high ATPase activity on preincubation at pH 4.3 and 4.5; (5) after preincubation at pH 4.5 two types of FOG fibres were observed, differing in their ATPase activity; (6) in both muscles there were fibres with intermediate ATPase activity both after acid and/or alkali preincubations; (7) the intrafusal muscle fibres exhibited some specific characteristics when compared with extrafusal fibres.In contrast to the ATPase reactions, SDH activity was decreased equally, in both extra- and intrafusal fibres, with increasing acidity and alkality of the preincubation medium.     

Lactate dehydrogenase isoenzyme spectrum of fast and slow muscles with innervation disorders

A relative content of muscle fibers of various types and the spectrum of lactate dehydrogenase (LDH) isozymes were studied in fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles of newborn rats, of those aged 2, 3 weeks and one month and of adult rats after neonatal sciatic denervation and application of 0.5 mM colchicine solution to the sciatic nerve. No muscle fibers of various types were found (from the level of succinate dehydrogenase activity) in one-month-old rats, whereas the control and fast-twitch muscles showed A, B and C types and the slow-twitch one B and C types. The denervation brought about an increase in the content of LDH4 and LDH5 in both the muscles, while colchicine application gave rise to an increase in LDH2 activity, diminution of LDH1 in the fast-twitch muscle and elevation of LDH4 in the slow-twitch one. The data obtained attest to the retardation of muscle differentiation under application of the colchicine-induced blockade of axoplasmic transport.     

Changes in lactate dehydrogenase and malate dehydrogenase activities during hypoxia and after temperature acclimation in the armored fish, Rhinelepis strigosa (Siluriformes, Loricariidae)

Lactate (LDH) and malate dehydrogenase (MDH) of white skeletal muscle of fishes acclimated to 20, 25 and 30 degrees C and thereafter submitted to hypoxia were studied in different substrate concentrations. Significant differences for LDH and MDH of white muscle enzyme activities are described here for the first time in Rhinelepis strigosa of fishes acclimated to 20 degrees C and submitted to hypoxia for six hours. LDH presented a significant decrease in enzyme affinity for pyruvate in acute hypoxia, for fishes acclimated to 20 degrees C and an increase for fishes acclimated to 30 degrees C.     

Developmental Changes in Lactate Dehydrogenase and Aldolase Activity of the A2G-adr Mouse with Abnormal Muscle Function: Further Comparison with the 129Re-dy Mutant

Abstract: Lactate dehydrogenase and aldolase activity were reduced in lateral gastrocnemius muscle from two mouse mutants, A2G- adr and 129Re- dy , with abnormal muscle function. The activities of both of these enzymes were significantly reduced in the lateral gastrocnemius muscle from the A2G- adr mice at ages varying from 2 weeks to 32 weeks, whereas the activities in the soleus, heart, liver, and brain were the same as in the control animals. The lactate dehydrogenase isoenzymes in the lateral gastrocnemius and soleus muscles from the A2G mice were quantified, and although those of the soleus were comparable in mutant and control muscle, the lateral gastrocnemius from the adr mutant had reduced activity of LDH 5 and increased activities of the other four isoenzymes. The findings suggest that the adr mutation is expressed in the white (Type II) muscle fibres and not in the red (Type I) fibres or in any of the organs studied. It is suggested that the initiation of differentiation into Type II fibres from the embryonic form is absent or delayed in the A2G mutant. The reduced activities of lactate dehydrogenase and aldolase in 129Re- dy muscle confirm the findings of other workers.