The reliability of histochemical fibre typing of human necropsy muscles

Summary The reliability of muscle fibre typing of post mortem specimens was investigated with special reference to the influence of time and temperature. In specimens stored at +4° C, muscle fibre typing could be reliably performed up to at least ten and fifteen days post mortem for the masseter and biceps brachii muscles respectively. The corresponding figures for storage at room temperature were three and six days. The difference in the preservation of enzyme activity between masticatory and limb muscles might be related to the demonstrated difference in the fibre type composition and thus the enzyme content and energy sources.     

31P NMR study of phosphorus metabolites in fast and slow muscles

1. 31P NMR was used to characterize phosphate pools in perchloric acid extracts of muscles with various composition of muscle fibre types. 2. The white m. pectoralis major (MPM) of chickens 15 min post mortem is characterized by 1.6-times higher relative content of phosphocreatine (PCr) in comparison with mixed leg muscle (LM) of this species. The glycerophosphorylcholine (GPC) does not occur in MPM at NMR detectable level in contrast to the leg muscles. Relative amounts of other phosphates are similar in both muscles. 3. The intermediate MPM of pigeons as well as mixed LM of this species contain 15 min post mortem a very small amount of PCr and ATP but a large amount of inorganic phosphate. Relative content of GPC is higher in leg muscles than in intermediate MPM. 4. Muscles with higher occurrence of white fibres contain relatively more PCr than muscles with lower occurrence of white fibres. 5. The occurrence of GPC seems to be connected with metabolism of red muscle fibres.     

The use of33P-labelled riboprobes forin situ hybridizations: localization of myosin alkali light-chain mRNAs in adult human skeletal muscle

Summary ³³P-labelled probes were used to localize the mRNAs coding for the myosin alkali light-chain isoforms MLC 1f/3f and MLC 1sb in adult human muscles, which are distributed in characteristic fibre type specific patterns. In situ hybridizations of ³³P-labelled probes were compared with probes carrying ³⁵S or digoxigenin labels. Signals of equal strength were obtained with each of the three labels. The preferentially peripheral localization of these mRNAs in the muscle fibres could be clearly seen with all three probes, with digoxigenin probes providing the best resolution. ³³P can serve as a viable alternative in this type of experiment.These experiments with adult human muscles also showed that the post mortem stability of RNA in human muscle is better than generally assumed. We could detect no signs of degradation in RNA prepared from heart ventricle as well as skeletal muscle up to 24 hours post mortem. In situ hybridizations worked equally well in biopsy material as in post mortem samples.     

Re-Evaluation of Sarcolemma Injury and Muscle Swelling in Human Skeletal Muscles after Eccentric Exercise

The results regarding the effects of unaccustomed eccentric exercise on muscle tissue are often conflicting and the aetiology of delayed onset muscle soreness (DOMS) induced by eccentric exercise is still unclear. This study aimed to re-evaluate the paradigm of muscular alterations with regard to muscle sarcolemma integrity and fibre swelling in human muscles after voluntary eccentric exercise leading to DOMS. Ten young males performed eccentric exercise by downstairs running. Biopsies from the soleus muscle were obtained from 6 non-exercising controls, 4 exercised subjects within 1 hour and 6 exercised subjects at 2–3 days and 7–8 days after the exercise. Muscle fibre sarcolemma integrity, infiltration of inflammatory cells and changes in fibre size and fibre phenotype composition as well as capillary supply were examined with specific antibodies using enzyme histochemistry and immunohistochemistry. Although all exercised subjects experienced DOMS which peaked between 1.5 to 2.5 days post exercise, no significant sarcolemma injury or inflammation was detected in any post exercise group. The results do not support the prevailing hypothesis that eccentric exercise causes an initial sarcolemma injury which leads to subsequent inflammation after eccentric exercise. The fibre size was 24% larger at 7–8 days than at 2–3 days post exercise (p<0.05). In contrast, the value of capillary number per fibre area tended to decrease from 2–3 days to 7–8 days post exercise (lower in 5 of the 6 subjects at 7–8 days than at 2–3 days; p<0.05). Thus, the increased fibre size at 7–8 days post exercise was interpreted to reflect fibre swelling. Because the fibre swelling did not appear at the time that DOMS peaked (between 1.5 to 2.5 days post exercise), we concluded that fibre swelling in the soleus muscle is not directly associated with the symptom of DOMS.     

Experimental analysis of error sources in fibre type counts of biopsies in horses

The contribution to total variance of different error sources in fibre type counts of equine gluteus medius muscle biopsies was determined to quantify and possibly improve the resolution of the method. Fibre types were defined on the basis of myosin heavy chain immunostaining. Errors were determined at levels: (1) positioning the insertion channel, (2) positioning the needle tip (3) biopsy heterogeneity (4) observer interpretation. Errors at levels 1 and 2 were considerable. Confidence intervals for individual observations were +/- 10-15%. In longitudinal studies a group size of 4 animals is necessary to resolve fibre composition changes of 10%. Comparison with multiple counts on post mortem specimens showed that local muscle fibre heterogeneity is responsible to a considerable extent for the error variance. Variance is effectively reduced by processing 3-4 shavings from the same insertion channel.     

Function and cytochemical characteristics of postural limb muscles of the rhesus monkey: A telemetered EMG and immunofluorescence study

Using telemetered electromyography and immunocytochemical fibre typing (of both fresh frozen and preserved specimens), the present paper demonstrates clearly that at the elbow, knee, and ankle joints, the rhesus monkey (Macaca mulatta) is endowed with one extensor-muscle head specialized for posture. These postural heads are distinguished by (a) recruitment at low levels to maintain joint position against the effect of gravity, (b) recruitment near maximum levels during walking, and (c) high content and relatively large size of slow, fatigue-resistant (type I) muscle fibres. The nonpostural heads of the investigated muscles were recruited at levels correlated to the strenuousness of the effort and are notable by the small percentage and size of slow muscle fibres. The postmortem stability of the structural properties of myosin makes immunocytochemical fibre typing suitable for the study of preserved cadavers.     

Proteolytic and physicochemical mechanisms involved in meat texture development.

Development in meat texture is a complex process originating very likely from a softening of the structural elements, especially myofibrils. This process probably involves two sets of mechanisms: 1) an enzymatic mechanism involving at least two of the three proteolytic systems so far identified and present in this tissue, namely lysosomal (cathepsins) and calcium dependent (calpains) proteinases; 2) a physicochemical mechanism based on the important post mortem rise in muscle osmotic pressure which could be twice as high as in live animals. Despite the large progress in muscle enzymology, the nature of the proteinases responsible for the post mortem proteolysis associated with the development in meat texture is still not clearly established. In the present review, data obtained from two different approaches attempting to answer this question were analysed. The first one was based on the identification of a set of structural and biochemical changes associated with meat texture development and to examine which proteolytic system or proteinase would be able to reproduce them when incubated with either myofibrils or muscle fibres as substrate. The second tentatively relates the rate and the extent of the changes in meat texture to the proteolytic equipment of the tissue. The first approach led to the conclusion that changes in muscle proteins and structure can be only explained by considering a synergistic action of both lysosomal and calcium-dependent proteinases. From the second, it was concluded that the process of meat texture development did not depend on the proteinase levels but was related to their initial potential efficiency assessed by measurement of the enzyme/inhibitor ratio. With respect to the physicochemical mechanisms, the post mortem rise in muscle osmotic pressure was shown to be responsible for some biochemical changes occurring in myofibrils. This was further substantiated by the fact that the greatest osmotic pressure values were observed in muscles exhibiting highest tenderising rate. On the other hand we provide evidence suggesting that the substrate, namely myofibrils, might constitute an important limiting step of the efficiency of both types of mechanism. Taken together, the findings presented emphasize that improvement of our knowledge in this field will greatly depend on the development of basic research on these different topics notably: 1) the mechanisms by which proteinases activities are regulated in living and post mortem muscles; and 2) the myofibrillar structure, especially in slow-twitch or type I muscles.     

Skeletal muscle biopsy studies of cardiac patients

Eleven patients diagnosed and treated for congestive cardiomyopathy (COCM) of unknown aetiology, and another 10 patients, with congestive alcoholic heart muscle disease (ACOCM) were studied. Muscle biopsy samples were obtained from the vastus lateralis (VL) and the gastrocnemius (G) muscles. In part of the sample muscle the fibre pattern was classified by means of ATPase activity staining, a technique based on the pH lability of the fibres concerned. Fibre typing and area measurements were carried out by light microscope. The other part of the sample was used as muscle homogenate of which the Ca2+-activated ATPase activity as well as citrate synthetase (CS) and aldolase activities were measured. No significant difference was found in these enzyme activities between the two groups of patients. The proportion of the slow twitch (ST) fibres in the VL, mainly in the patients with ACOCM, was lower as compared to data for healthy subjects. A similar tendency was revealed for G. In both muscles tested, the area of ST fibres was smaller in the ACOCM group. The fast twitch (FT) fibre area proved to be slightly different in the two groups of subjects tested. Occurrence of degenerative signs in the histological tests was higher in the ACOCM than in the COCM group. It was concluded that differences in the skeletal muscles of patients with ACOCM and COCM may primarily account for the alcoholism. The disease of the heart muscle has little effect on the function of skeletal muscle. Even so, a low amount or lack of physical activity may have an unfavourable influence on the skeletal muscles of patients with heart muscle disease.     

Myosin-ATPase fibre typing of chemically skinned muscle fibres

Summary The efficacy of myosin (M)-ATPase fibre typing to differentiate fibre types in chemically (EGTA) skinned muscle fibres was investigated. Cryosections or single fibres from isolated bundles of chemically skinned rat extensor digitorum longus (EDL) and soleus (SOL) muscles were stained for M-ATPase activity. The results indicate that two major fibre types (type I and II, Brooke & Kaiser, 1970) can be indentified, as well as subgrouping of the type II fibres into types IIa and IIb. Thus, chemically skinning muscle fibres appears to have no detrimental effects on subsequent M-ATPase fibre typing.     

Parallel measurement of brain acetylcholinesterase and the muscarinic cholinergic receptor in the diagnosis of acute, lethal poisoning by anti-cholinesterase pesticides

The activity of acetylcholinesterase (AChE) and the density of muscarinic cholinergic binding receptors (mCBR) were measured in brains from normal Japanese quail (Coturnix coturnix japonica) and from quail after lethal intoxication with diazinon. These were measured in brains from whole heads held at 25 C for 0 to 8 days after death. The maximum relative loss of activity due to post mortem decomposition alone during 8 days was 13% and 10% for AChE and mCBR, respectively. During post mortem decomposition, the ratio of AChE: mCBR activities remained constant at approximately 1.3:1 in normal brains while it was always less than or equal to 0.5:1 after intoxication with diazinon. Normal AChE activity could be estimated from mCBR density. Parallel measurement of AChE and mCBR may assist in the post mortem diagnosis of death due to acute poisoning with anti-cholinesterase pesticides when control specimens are not available.     

Effect of delay in cryofixation on the elemental composition of biopsies and post mortem specimens of the thyroid gland

The time between the excision and cryofixation of a biopsy is most important regarding its elemental composition as demostrated by an investigation of the thyroid glands of rats and pigs. Biopsies taken and cryofixed immediately served as control specimens. Biopsies that were allowed to stand at room temperature for 20 min before cryofixation and specimens cryofixed at 1 h post mortem were also investigated. Significant changes in the ion concentration of the cells and colloid were apparent in biopsies in which cryofixation was delayed for 20 min and in thyroids cryofixed 1 h post mortem. It was demonstrated that redistribution of electrolytes occurs within 1h post mortem and that similar changes occur in biopsies allowed to stand for 20 min at room temperature before cryofixation. The results stress the importance of immediate cryofixation after surgical excision of a biopsy. This is especially important since numerous elemental changes due to delayed cryofixation resemble those which occur in pathological processes.     

Phosphorus-31 nuclear magnetic resonance study of post mortem catabolism and intracellular pH in intact excised rabbit muscle

Phosphorus-31 nuclear magnetic resonance has been used to study the post mortem catabolism of high-energy phosphate compounds and the associated intracellular pH variation in pure fast- and slow-twitch rabbit muscles and in rabbit muscle with mixed fiber types. Comparative results from pure fiber types are reported for the first time. Large amounts of glycerophosphorylcholine (14.1 mumol/g fresh tissue) are found in the internal conoidal bundle (ICB), a pure oxidative slow twitch muscle, whereas the m. psoas major (PM), a pure glycolytic fast twitch muscle and the m. gastrocnemius caput medialis (GCM), with mixed fiber types, are devoid of the same metabolite. The total content of phosphorylated metabolites is constant among the three muscle types. The time-dependent post mortem changes in phosphorylated metabolites display the expected rapid drop in phosphocreatine and a simultaneous increase in intracellular inorganic phosphate. However, the ATP level remains constant during more than 2 h. Rate constants for metabolite breakdown and apparent ATPase activity have been determined. The comparative kinetics of intracellular acidosis at 25 degrees C yield rates of 3.3 X 10(-3) pH unit/min for PM, 2.7 X 10(-3) pH unit/min for GCM and 3.0 X 10(-3) pH unit/min for ICB. Initial intracellular pH values are 7.07, 7.20 and 7.02, respectively. Upon aging, the heterogeneity of the Pi signal reflects the existence of cellular compartments with different internal pH. The results suggest that the more intense low-pH Pi signal arises from the sarcoplasmic reticulum while the less intense resonance would reflect the sarcoplasmic higher pH. The temperature effect on post mortem catabolism in the 15-25 degrees C range has been documented. As expected, phosphocreatine and ATP breakdown increase with temperature but at a higher rate for slow-twitch ICB than for fast-twitch PM.     

Immunological detection of m- and μ-calpains in the skeletal muscle of Marchigiana cattle

Calpains are Ca²⁺-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15^th day post mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.Key words: m-calpain, µ-calpain, skeletal muscle, Marchigiana cattle, immunohistochemistry, Electron Microscopy.     

Different rates of glycolysis affect glycolytic activities and protein properties in turkey breast muscle

Protein alterations of turkey breast muscles (Pectoralis major) were investigated at 20 min and 24 h post mortem. Specific activities, quantities and kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase A were also determined at 20 min post mortem. Based on the pH values at 20 min post mortem, two groups of samples were classified as rapid glycolysis group (RG; pH20 min = 5.80 ± 0.07, n = 20) and normal glycolysis group (NG; pH20 min = 6.21 ± 0.01, n = 20). RG had lower specific activities of GAPDH and aldolase A than NG while Vm and Km values of both enzymes were not different between groups. RG showed lower high ionic strength (HIS) and pellet protein extractabilities at 20 min post mortem. It also had lower low ionic strength (LIS) and HIS protein extractabilities at 24 h post mortem. Besides pellet protein, muscular protein extractabilities at 24 h post mortem were higher than at 20 min post mortem. From SDS-PAGE of samples at 24 h post mortem, RG exhibited lower band intensities at 45 and 200 kDa, which were further identified as actin and myosin heavy chain (MHC), respectively. Western blots revealed that relative amounts of actin and MHC at 20 min post mortem were not different between groups. However, RG muscles had less relative amount of actin at 24 h post mortem. It also indicated that amounts of actin and MHC increased with regard to post mortem time.     

Comparison of Magnetic Resonance Imaging in Live vs. Post Mortem Rat Brains

Magnetic Resonance Imaging (MRI) is an increasingly popular technique for examining neurobiology in rodents because it is both noninvasive and nondestructive. MRI scans can be acquired from either live or post mortem specimens. In vivo scans have a key advantage in that subjects can be scanned at multiple time-points in longitudinal studies. However, repeated exposure to anesthesia and stress may confound studies. In contrast, post mortem scans offer improved image quality and increased signal-to-noise ratio (SNR) due to several key advantages: First, the images are not disrupted by motion and pulsation artifacts. Second, they allow the brain tissue to be perfused with contrast agents, enhancing tissue contrast. Third, they allow longer image acquisition times, yielding higher resolution and/or improved SNR. Fourth, they allow assessment of groups of animals at the same age without scheduling complications. Despite these advantages, researchers are often skeptical of post mortem MRI scans because of uncertainty about whether the fixation process alters the MRI measurements. To address these concerns, we present a thorough comparative study of in vivo and post mortem MRI scans in healthy male Wistar rats at three age points throughout adolescence (postnatal days 28 through 80). For each subject, an in vivo scan was acquired, followed by perfusion and two post mortem scans at two different MRI facilities. The goal was to assess robustness of measurements, to detect any changes in volumetric measurements after fixation, and to investigate any differential bias that may exist between image acquisition techniques. We present this volumetric analysis for comparison of 22 anatomical structures between in vivo and post mortem scans. No significant changes in volumetric measurements were detected; however, as hypothesized, the image quality is dramatically improved in post mortem scans. These findings illustrate the validity and utility of using post mortem scans in volumetric neurobiological studies.     

Non-enzymatic weakening of myofibrillar structures during conditioning of meat: calcium ions at 0.1 mM and their effect on meat tenderization.

The tenderness of meat is set by the properties of connective tissue and myofibrils. Skeletal muscle connective tissues become firm with chronological aging concomitantly with the increase in intermolecular non-reducing cross-links of collagen, and this process toughens meat, however, connective tissues hardly change during conditioning of meat. Therefore, the tenderization of meat during post mortem aging, or to put it more precisely, during post rigor aging, stems for the most part from changes in myofibril structures. My research derives its origin on findings of two kinds of post mortem changes in myofibril structures; i) fragmentation of myofibrils; and ii) restoration of rigor-shortened sarcomeres. These results were published in 1967 [1], and were, thereafter proved by many workers to be closely related to meat tenderization. I report in this paper the essential molecular mechanisms of these phenomena, and of structural changes in connectin or titin filaments. All of them are non-enzymatically induced by 0.1 mM calcium ion, which is the ultimate concentration of sarcoplasmic calcium ion in post mortem muscles.     

Nitric oxide synthase II in rat skeletal muscles

Constitutive expression of nitric oxide synthase (NOS) II was found in rat hindlimb muscles by immunohistochemistry and western blotting during development from embryonic day 21 to the adult stage of 75 days. The immunohistochemical NOS II expression pattern was related to the physiological metabolic fibre types SO (slow-oxidative), FOG I, II (fast-oxidative glycolytic; I more glycolytic, II more oxidative) and FG (fast-glycolytic) and to the myosin-based fibre types I and IIA, IIB (IIX not separated) identified in serial sections by enzyme histochemistry and immunohistochemistry. In adult muscles only the small population of FOG II fibres, which is a part of both IIA and IIB fibre population, showed NOS II immunoreactivity. This is the reason that only weak NOS II expression in adult hindlimb muscles has been detected by western blotting. Hindlimb muscles of embryonic, neonatal and young rats of 8 days expressed more NOS II as compared with adult rat hindlimb muscles. This can be explained by the findings that before the age of 21 days fast fibres were metabolically undifferentiated, all of them were NOS II positive and contribute to the NOS II expression of the muscle. In muscles of diabetic rats the NOS II expression was elevated indicating an inhibition of glucose uptake into the muscle fibres of diabetic muscles. Our findings suggest that the NOS II may be designated both as constitutive and inducible.     

Evidence of fibre hyperplasia in human skeletal muscles from healthy young men? A left-right comparison of the fibre number in whole anterior tibialis muscles

Cross-sections (thickness 10 microns) of whole autopsied left and right anterior tibialis muscles of seven young previously healthy right-handed men (mean age 23 years, range 18-32 years) were prepared for light-microscope enzyme histochemistry. Muscle cross-sectional area and total number of fibres, mean fibre size (indirectly determined) and proportion of the different fibre types (type 1 and type 2 on basis of myofibrillar adenosine triphosphatase characteristics), in each muscle cross-section were determined. The analysis showed that the cross-sectional area of the left muscle was significantly larger (P less than 0.05), and the total number of fibres was significantly higher (P less than 0.05), than for the corresponding right muscle. There was no significant difference for the mean fibre size or the proportion of the two fibre types. The results imply that long-term asymmetrical low-level daily demands on muscles of the left and the right lower leg in right-handed individuals provide enough stimuli to induce an enlargement of the muscles on the left side, and that this enlargement is due to an increase in the number of muscle fibres (fibre hyperplasia). Calculations based on the data also explain why the underlying process of hyperplasia is difficult, or even impossible, to detect in standard muscle biopsies.     

Effect of genotype and overfeeding on lipid deposition in myofibres and intramuscular adipocytes of breast and thigh muscles of ducks

We conducted a study to evaluate the effects of genotype (Muscovy, Pekin and their crossbred, hinny and mule) and overfeeding (14 days from 12 weeks of age) on lipid deposition in myofibres and intramuscular adipocytes of breast and thigh muscles of ducks. Birds of the four genotypes were also reared contemporaneously with a growing diet distributed ad libitum. Muscle samples (Pectoralis major and Sartorius) were collected at 14 weeks of age on 8 ducks per treatment. The muscle fibre typing, the total lipid and triglyceride contents in myofibres and the relative surface occupied by adipocytes on the cross-sectional area of the muscles were determined by histological and image analysis. Overfeeding induced a marked increase of body weight but had no significant effect on the muscle weight, the cross-sectional area (CSA) of myofibres and the muscle typology. In muscles, overfeeding induced a large accumulation of lipids, mainly in adipocytes whose relative surface increased 1.5 fold in P. major and 2.1 fold in Sartorius and an increase in triglyceride content of fast twitch oxydo-glycolytic and glycolytic fibres in P. major only (+ 37 and + 16% respectively). Genotype had no significant effect on the muscle typology. By comparison with the other genotypes, Muscovy ducks exhibited the highest body weight, the highest muscle weight which could partly be explained by the highest fibre CSA and the lowest intramuscular fat content in adipocytes and myofibres (only fast twitch oxydo-glycolytic fibres in P. major). We observed the reverse situation for the Pekin ducks. The crossbred ducks always presented intermediate values except for body weight.