A histochemical analysis of mammalian oxidative skeletal muscle fibres using the enzymes of energetic metabolism

Summary Some histochemical parameters of the three main fibre types of rat vastus lateralis muscle were studied. Succinic dehydrogenase (SDH), creatine kinase (CK), sarcotubular ATPase (SR-ATPase) and mitochondrial ATPase activities were demonstrated in serial sections. The three fibre types, recognised by the distribution pattern of SDH activity, all show high CK activity. However, red Type I oxidative fibres when examined for ATPase and ATPase dependent CK activity, show distinct heterogeneity revealing sub-populations within the same homogeneous fibre type. Three distinct patterns were recognised in the red Type I fibres depending on the distribution of the final reaction product. The present histochemical evidence confirms the fact that subdivision of mammalian skeletal muscle into three fibre types is only approximate and probably more than three types exist.     

A histochemical and ultrastructural study of heterogeneous red fibres of pigeon pectoralis muscle

The oxidative fibres of pigeon pectoralis muscle are 'type II red' on the basis of high myofibrillar adenosine triphosphatase (ATPase) and succinate dehydrogenase (SDH). The ATPase and SDH reactions do not characterize the heterogeneity of the red fibres in the normal pigeon pectoralis. Therefore, lipid, glycogen, phosphorylase, glycogen synthetase, SDH and ATPase reactions were studied in transverse sections of the pigeon pectoralis red fibre. This study has revealed the existence of an inherent heterogeneity between these red fibres. Three sub-populations distinguished were : (1) 'red1' fibres showing low fat but high glycogen and enzymes of glycogen metabolism (EGM); (2) 'red2' fibres displaying higher fat and higher amount of glycogen and EGM; and (3) 'red3' fibres possessing higher fat but lower content of glycogen and EGM. Ultrastructurally, one category of fibres (presumably red2 and red3) showed a higher concentration of fat; these fibres presumably possess higher synthetic capacity and lower (or higher?) utilization. Other mitochondria-loaded red fibres (presumably red1) documented here for the first time, showed fewer and smaller fat droplets indicating that they are 'predominantly lipid utilizers' and are incapable of storing large quantities of lipid. Moreover, the differing histochemical-ultrastructural attributes are presumably correlated with differences in levels of energetic metabolism, heat production and motor units of the red fibres.     

Eccentric and posteccentric contractile behaviour of skeletal muscle a comparative study in frog single fibres and in humans

Eccentric and posteccentric force behaviour in human skeletal muscle and in isolated frog muscle fibres was studied by imposing stretch-and-hold loading conditions during contractions with maximal voluntary effort or under tetanic stimulation in the isolated preparations. The investigations on human muscle were made on the forearm flexors of a group of kayak racers (n = 16; age: 17-22 years) and of schoolgirls (n = 15; age: 17-18 years) with both groups participating in a strength-training programme over 4 (kayak racers) or 3 (girls) months. Half of the training regime consisted of eccentric elements. In the isolated muscle fibres, it could be shown that in the posteccentric hold phase the enhanced force decayed exponentially to the original isometric value with a mean time-constant of 0.35 s (10 degrees C) and of 0.23 (20 degrees C). In the forearm flexor of human subjects similar results were obtained not only qualitatively but even quantitatively (time constant of posteccentric force decay: 0.25-0.37 s). Strength training in both groups did not lead to an enhancement in maximal isometric force alone [mean increase in force 17 (SD 10)%], a well-known and generally accepted fact, but also to a parallel shift in eccentric [21 (SD 10)%] and posteccentric force level. The close similarity between the findings in isolated muscle fibres and in human muscle in situ suggests that the eccentric and posteccentric behaviour must be primarily ascribed to the contractile properties of the muscle fibres themselves. A three-element muscle model with variable visco-elastic properties would appear to be most suitable for simulating the experimental findings.     

Further histochemical properties of rabbit skeletal muscle fibres

Summary The histochemical activities of succinic dehydrogenase (SDH), creatine kinase (CK), sarcoplasmic reticular ATPase (SR-ATPase) and myosin ATPase were studied in serial sections of rabbit adductor muscle. Three fibre types were distinguished depending upon the distribution of the enzyme activities. The type II white fibres posessing minimal SDH showed high myosin ATPase, SR-ATPase and ATPase dependent CK activities. Red oxidative fibres showing high SDH fell into two distinct groups: One category had mainly a peripheral localization of SDH and showed an enzymatic profile identical to that of type II white fibres. The second category of red fibres displayed both a homogeneous distribution of small diformazan granules throughout the fibre as well as a sub-sarcolemmal collection when tested for SDH activity but possessed very low amounts of reaction product of the various enzymes of the energetic metabolism studied. Since it is well established that the myosin ATPase of a fibre correlates with its contraction time, the present histochemical investigation provides further support for this concept by demonstrating the presence of high SR-ATPase and ATPase dependent CK activities in all white and red fibres rich in myosin ATPase.     

Seasonal cycles of mitochondrial ADP sensitivity and oxidative capacities in trout oxidative muscle

Mitochondria from red myotomal muscle of rainbow trout, Oncorhynchus mykiss, showed seasonal cycles of their maximal rates of substrate oxidation (nmol · min⁻¹ mg⁻¹ mitochondrial protein) and their apparent ADP affinity (Km_app), as well as in the thermal sensitivity of these properties. Increases in the maximal capacity of pyruvate oxidation were sufficient to compensate for seasonal changes in temperature, except during the winter months when rates at habitat temperature were depressed relative to other periods. The ADP affinity of isolated mitochondria was highest during cold months. Thus, the Km_app for ADP at habitat temperature showed less seasonal variation than the ADP Km_app at a given temperature. A loss in ADP affinity with decreasing temperature occurred through much of the year, and only was definitively suppressed in December and July. Both the ADP affinity and the maximal oxidative capacities of muscle mitochondria seem to be regulated parameters. Accepted: 10 June 1999     

Slow and fast fatigable frog muscle fibres: electrophysiological and histochemical characteristics

Continuous activity of isolated frog gastrocnemius muscle fibres provoked by repetitive stimulation of 5 Hz was used as an experimental model for fatigue development in different fibre types. Parameter changes of the elicited intracellular action potentials and mechanical twitches during the period of uninterrupted activity were used as criteria for fatigue evaluation. Slow fatigable muscle fibre (SMF) and fast fatigable muscle fibre (FMF) types were distinguished depending on the duration of their uninterrupted activity, which was significantly longer in SMFs than in FMFs. The normalized changes of action potential amplitude and duration were significantly smaller in FMFs than in SMFs. The average twitch force and velocity of contraction and relaxation were significantly higher in FMFs than in SMFs. Myosin ATPase (mATPase) and succinate dehydrogenase activity were studied by histochemical assessment in order to validate the fibre type classification based on their electrophysiological characteristics. Based on the relative mATPase reactivity, the fibres of the studied muscle were classified as one of five different types (1-2, 2, 2-3, 3 and tonic). Smaller sized fibres (tonic and type 3) expressed higher succinate dehydrogenase activity than larger sized fibres (type 1-2, 2), which is related to the fatigue resistance. The differences between fatigue development in SMFs and FMFs during continuous activity were associated with fibre-type specific mATPase and succinate dehydrogenase activity.     

Classification of bovine muscle fibres by different histochemical techniques

The classification of bovine muscle fibres is of particular interest for the food industry because meat tenderness depends in part on the proportion of the different types of fibres. It is, therefore, important to define reliable methods for classifying fibre types. There are several classification systems. One is based on contractile type alone, as revealed by myofibrillar ATPase activity or with antibodies against myosin heavy chain. Others take both contractile and metabolic types into account. In this study, the classifications of fibres obtained by these three systems were compared on the semitendinosus and longissimus thoracis muscles of 35 Charolais bulls. Only the use of antibodies allowed the identification of a proportion of hybrid fibres containing two isoforms of fast myosin heavy chain (2a and 2b). In addition, the combination of metabolic types showed that the metabolism of these hybrid fibres differed according to the muscle.     

Fucose expression in skeletal muscle: a lectin histochemical study

Summary Binding sites for three fucose specific lectins, Aleuria aurantia agglutinin (AAA), Lotus tetragonolobus agglutinin (LTA) and Ulex europeus I agglutinin (UEA I), were investigated in sections from normal human and rat muscles, in muscle from patients with Duchenne muscular dystrophy (DMD) and in denervated and devascularized rat muscle. In normal human and rat muscle AAA detected fucosylated glycocompounds in the sarcoplasm, sarcolemma, interfibre connective tissue and vascular structures. In normal human muscle addition of fucose to the AAA incubation medium or treatment of the sections with formaldehyde followed by periodic oxidation before lectin incubation strongly inhibited the staining at all sites other than endothelial cells. In normal rat muscle the same staining procedures strongly inhibited the AAA binding at all sites other than the sarcolemma. Incubation with LTA resulted in a diffuse reaction around the vascular structures in rat muscle, while in human muscle a moderate, homogeneous staining was present in all muscle fibres. Treatment of the sections with formaldehyde and periodic acid before incubation with LTA resulted in strongly labelled muscle capillaries in both human and rat muscle. The only elements in the muscle tissues that were stained with UEA I were human endothelial cells. In denervated and devascularized rat muscle incubation with AAA revealed a novel fucose expression that appeared intracellularly in some necrotic fibres. The AAA-positive fucose residues in the sarcolemma of normal muscle fibres that were resistant to periodic acid oxidation could not be shown by AAA in denervated muscle. In DMD muscle a cryptic sarcolemmal fucose expression could be shown with AAA. It is suggested that both the sarcoplasm and sarcolemma of diseased muscle fibres show altered fucose expression.     

X-ray diffraction studies of the contractile mechanism in single muscle fibres

The molecular mechanism of muscle contraction was investigated in intact muscle fibres by X-ray diffraction. Changes in the intensities of the axial X-ray reflections produced by imposing rapid changes in fibre length establish the average conformation of the myosin heads during active isometric contraction, and show that the heads tilt during the elastic response to a change in fibre length and during the elementary force generating process: the working stroke. X-ray interference between the two arrays of myosin heads in each filament allows the axial motions of the heads following a sudden drop in force from the isometric level to be measured in situ with unprecedented precision. At low load, the average working stroke is 12 nm, which is consistent with crystallographic studies. The working stroke is smaller and slower at a higher load. The compliance of the actin and myosin filaments was also determined from the change in the axial spacings of the X-ray reflections following a force step, and shown to be responsible for most of the sarcomere compliance. The mechanical properties of the sarcomere depend on both the motor actions of the myosin heads and the compliance of the myosin and actin filaments.     

Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres

Summary A histochemical technique was developed for the quantitative determination of succinic dehydrogenase (SDH) activity in muscle cross-sections using 1-methoxyphenazine methosulphate (mPMS) as the exogenous electron carrier, and azide as an inhibitor of cytochrome oxidase. The optimal composition of the incubation medium for the SDH reaction was determined. This histochemical procedure was compared to one using phenazine methosulphate (PMS) instead of mPMS and cyanide instead of azide. The substitution of mPMS and azide resulted in a substantial decrease in the non-specific reduction of nitroblue tetrazolium (NBT; the reaction indicator), i.e., nothing dehydrogenase activity. With mPMS and azide in the reaction medium, the production of NBT formazan was linear for at least 9 min during the enzymic reaction. This compared to a non-linear reduction of NBT during the initial stages of the reactions (SDH and nothing dehydrogenase) when using PMS and cyanide. The use of both mPMS and azide also eliminated the production of NBT monoformazan which occurred with PMS and cyanide. This procedure was shown to meet various criteria established for the quantification of histochemical reactions.     

Transitional stages in the histochemical development of muscle fibres during post-natal growth

Synopsis Serial frozen sections of longissimus dorsi muscles from seven pigs at different live weights (13 to 127 kg) were reacted for ATPase by the calcium method at an alkaline pH and for NADH oxidative activity. One hundred muscle fibres from each animal were identified individually in serial sections and their staining intensity was measured with a microscope photometer at 600 nm. For each section, staining intensity of fibres (% tranmission) was measured and converted to the nearest one-tenth unit of the range from the darkest to the lightest staining fibres. Frequency of occurrence of fibre types was plotted on a 10×10 grid using the range co-ordinates for NADH oxidative activity (vertical) and ATPase activity (horizontal). The commonly recognized histochemical fibre types in this muscle appeared as crowded areas in the grid but, in many cases, these areas were part of a continuous L shaped distribution. In fibres having an ATPase staining intensity of 1.0 and 0.9 units of the range, a continuous but skewed distribution with regard to NADH oxidative activity was detected. In fibres with NADH oxidative activity of 0.6 to 1.0 units of the range, a continuous but irregular distribution with regard to ATPase activity was detected. Within this range, there was some evidence of a growth-related shift towards weaker ATPase activity.     

Acid phosphatase activity in soleus and plantaris muscle fibres of normal and dystrophic hamsters. A quantitative histochemical study

The activity of acid phosphatase in skeletal muscle fibres of the plantaris and soleus of normal and dystrophic male hamsters was quantified using a histochemical post-coupling semipermeable membrane technique. Although the absolute levels of activity were found to vary widely from one animal to another, the ratio of the mean activities in the two muscles in each animal was virtually constant. In normal muscles, the ratio was about 0.73 and in dystrophic muscles, about 0.77. The activity in plantaris muscle fibres was always significantly lower than that in the corresponding soleus fibres, and in normal fibres compared to dystrophic ones. Another difference was that in normal fibres the mean activity declined to a constant level in mature animals older than about 3 months. In contrast, the activity in dystrophic muscles appeared to fall exponentially throughout life. The functional significance of these findings is discussed.     

Architectural and histochemical analysis of the biceps brachii muscle of the horse.

The biceps brachii of horses is a complex muscle subdivided into two heads which may subserve distinct functions. The lateral head contains a large percentage of type I myofibers. This region is largely composed of short fibers (5-7 mm long) arranged in a pinnate fashion and heavily invested with connective tissue. The medial head contains fewer type I fibers and is composed of relatively longer myofibers (15-20 mm long), also arranged in a pinnate fashion but less heavily invested with connective tissue. It is hypothesized that the lateral muscle head of biceps brachii contributes to the postural role of the muscle in the forelimb passive stay apparatus. The medial head, with its longer fibers and generally fast fiber population may be most important during dynamic activity such as walking, trotting and running.     

Temperature adaptation and the contractile properties of live muscle fibres from teleost fish

Summary The contractile properties of swimming muscles have been investigated in marine teleosts from Antarctic (Trematomus lepidorhinus, Pseudochaenichthys georgianus), temperate (Pollachius virens, Limanda limanda, Agonis cataphractus, Callionymus lyra), and tropical (Abudefduf abdominalis, Thalassoma duperreyi) latitudes. Small bundles of fast twitch fibres were isolated from anterior myotomes and/or the pectoral fin adductor profundis muscle (m. add. p). Live fibre preparations were viable for several days at in vivo temperatures, but became progressively inexcitable at higher or lower temperatures. The stimulation frequency required to produce fused isometric tetani increased from 50 Hz in Antarctic species at 0°C to around 400 Hz in tropical species at 25°C. Maximum isometric tension (P_o) was produced at the normal body temperature (NBT) of each species (Antarctic, 0–2°C; North Sea and Atlantic, 8–10°C; Indo-West Pacific, 23–25°C). P₀ values at physiological temperatures (200–300 kN·m^–2) were similar for Antarctic, temperate, and tropical species. A temperature induced tension hysteresis was observed in muscle fibres from some species. Exposure to <0°C in Antarctic and <2°C in temperate fish resulted in the temporary depression of tension over the whole experimental range, an effect reversed by incubation at higher temperatures. At normal body temperatures the half-times for activation and relaxation of twitch and tetanic tension increased in the order Antarctic>temperate>tropical species. Relaxation was generally much slower at temperatures <10°C in fibres from tropical than temperate fish. Q₁₀ values for these parameters at NBTs were 1.3 2.1 for tropical species, 1.7–2.6 for temperate species, and 1.6–3.5 for Antarctic species. The forcevelocity (P-V) relationship was studied in selected species using iso-velocity releases and the data below 0.8 P₀ iteratively fitted to Hill's equation. The P-V relation at NBT was found to be significantly less curved in Antarctic than temperate species. The unloaded contraction velocity (V_max) of fibres was positively correlated with NBT increasing from about 1 muscle fibre length·s^–;1 in an Antarctic fish (Trematomus lepidorhinus) at 1°C to around 16 muscle fibre lengths·s^–1 in a tropical species (Thalassoma duperreyi) at 24°C. It is concluded that although muscle contraction in Antarctic fish shows adaptations for low temperature function, the degree of compensation achieved in shortening speed and twitch kinetics is relatively modest.Abbreviations ET environmental temperature - m. add. p major adductor profundis - m. add. s. major adductor superficialis - NBT normal body temperature - P ₀ maximum isometric tension - P-V force velocity - SR sarcoplasmic reticulum - T 1/2 a half activation time - T 1/2 r half relaxation time - V _max unloaded contraction     

Altered glycolytic and oxidative capacities of skeletal muscle contribute to insulin resistance in NIDDM

Simoneau, Jean-Aimé, and David E. Kelley. Alteredglycolytic and oxidative capacities of skeletal muscle contribute toinsulin resistance in NIDDM. J. Appl.Physiol. 83(1): 166-171, 1997.The insulinresistance of skeletal muscle in glucose-tolerant obese individuals isassociated with reduced activity of oxidative enzymes and adisproportionate increase in activity of glycolytic enzymes. Becausenon-insulin-dependent diabetes mellitus (NIDDM) is a disordercharacterized by even more severe insulin resistance of skeletal muscleand because many individuals with NIDDM are obese, the present studywas undertaken to examine whether decreased oxidative and increasedglycolytic enzyme activities are also present in NIDDM. Percutaneousbiopsy of vatus lateralis muscle was obtained in eight lean (L) andeight obese (O) nondiabetic subjects and in eight obese NIDDM subjectsand was assayed for marker enzymes of the glycolytic[phosphofructokinase, glyceraldehyde phosphate dehydrogenase,hexokinase (HK)] and oxidative pathways [citrate synthase(CS), cytochrome-c oxidase], aswell as for a glycogenolytic enzyme (glycogen phosphorylase) and amarker of anaerobic ATP resynthesis (creatine kinase). Insulinsensitivity was measured by using the euglycemic clamp technique.Activity for glycolytic enzymes (phosphofructokinase, glyceraldehyephosphate dehydrogenase, HK) was highest in subjects with subjects with NIDDM, following the order of NIDDM > O > L, whereas maximumvelocity for oxidative enzymes (CS,cytochrome-c oxidase) was lowest in subjects with NIDDM. The ratio between glycolytic andoxidative enzyme activities within skeletal muscle correlatednegatively with insulin sensitivity. The HK/CS ratio had the strongestcorrelation (r = 0.60, P < 0.01) with insulinsensitivity. In summary, an imbalance between glycolytic and oxidativeenzyme capacities is present in NIDDM subjects and is more severe thanin obese or lean glucose-tolerant subjects. The altered ratio betweenglycolytic and oxidative enzyme activities found in skeletal muscle ofindividuals with NIDDM suggests that a dysregulation betweenmitochondrial oxidative capacity and capacity for glycolysis is animportant component of the expression of insulin resistance.

     

Biochemical verification of quantitative histochemical analysis of succinate dehydrogenase activity in skeletal muscle fibres

Summary The purpose of this investigation was to examine critically the validity of a computerized quantitative microphotometric histochemical technique for the determination of succinate dehydrogenase (SDH) activity in skeletal muscle fibres. Sections from the anterior costal diaphragm were removed from Fischer-344 rats (n = 12) and assayed histochemically to determine SDH activity. The SDH activity in individual muscle fibres was computed using a computerized microphotometric histochemical technique which involves measurement of the optical density of deposited diformazan derived from nitroblue tetrazolium within the fibres. To validate the histochemical technique, whole muscle SDH activities were calculated from the histochemical procedure and were compared to SDH activities determined from whole muscle homogenates via a standard quantitative biochemical assay. The mean within-day variability of the computerized microphotometric histochemical technique of determining SDH activity was 6% (range = 0.5–10.9%) for an area containing ~50 fibres and 6.1% (range = 1.05–14.9%) for an individual muscle fibre. Similarly, the mean between-day variability of the microphotometric histochemical technique of determining SDH activity was 5.9% (range = 2.6–13.9%) for an area containing ~50 fibres and 6.6% (range = 2.2–13.9%) for an individual muscle fibre. The inter-class correlation coefficient between biochemically determined SDH activity and histochemically determined SDH activity was r = 0.83 (p < 0.05). Collectively, these data demonstrate that the quantitative histochemical technique of Blanco et al. (1988) is both valid and reliable in the determination of SDH activity in skeletal muscle fibres.     

Lymphatic muscle: a review of contractile function

A recent surge in lymphangiogenesis research has led to a greater understanding of lymphatic endothelial cell biology. However, a general understanding of lymphatic muscle cell biology lags far behind its endothelial counterpart. Lymphatics at the level of the collecting vessels and higher contain muscular walls capable of both tonic and phasic contractions, which both generate and regulate lymph flow. Because lymphatic contraction is crucial to lymphatic function, a solid understanding of lymphatic muscle development and function is necessary to understand lymphatic biology. This review summarizes the current body of lymphatic muscle research and addresses important questions that are currently unanswered.