In vivo assessment of human skeletal muscle mitochondria respiration in health and disease

One of many problems to be faced when assessing in vivo human muscle mitochondria respiration by phosphorus magnetic resonance spectroscopy (31P-MRS) is the definition of the correct reference population and the values of reference range. To take into account most factors that influence muscle activity as age, sex, physical activity; nutritional state etc., an exceedingly high number of different reference groups are needed. To overcome this problem we developed specific tests to assess separately in vivo the activity and the functionality of muscle mitochondria by 31P-MRS in clinical settings. By activity we refer to muscle whole metabolic activity, i.e. the total oxidative capacity of muscle mitochondria which is influenced by many factors (age, sex, physical activity, nutritional state etc.). By functionality we refer to the qualitative aspects of mitochondrial respiration which depends on the integrity of mitochondrial multienzyme systems and on substrate availability. Our tests have been experienced on some 1200 patients and are currently used to detect deficits of mitochondrial respiration and ion transport in patients with suspected primary or secondary muscle mitochondrial malfunctioning.     

Creatine supplementation improves intracellular Ca2+ handling and survival in mdx skeletal muscle cells

Rapid review, digital recording, on-line quantification, and three-dimensional reconstruction are all essential in the evaluation of intracoronary ultrasound images during coronary interventions. We describe a low-cost method that offers all these necessary features. The proposed method uses the QuickTime compatible video digitizers of standard multimedia Apple Macintosh or PowerPC desktop computers and the freeware software Object Image 1.60.     

Myosin isoforms in skeletal muscle in patients with chronic heart decompensation: distribution and correlation with with exercise tolerance

Chronic heart failure (CHF) is accompanied by a reduced exercise capacity, and the symptoms can be at least in part explained by qualitative and quantitative changes in the skeletal muscle composition and metabolism. We have correlated the myosin heavy chain (MHC) composition of the gastrocnemius in 20 patients with different degrees of CHF to expiratory gases measured during maximal cardiopulmonary exercise testing, NYHA functional class and echocardiographic parameters. MHC composition was determined electrophoretically in skeletal muscle needle microbiopsies and the percent distribution calculated by laser densitometry. There was no correlation between ejection fraction, left ventricular end-diastolic and end-systolic diameters and MHC composition. The percentage of MHC 1 (slow aerobic isoform) was positively correlated with peak VO2 (r2 = 0.5, p = 0.0004), ventilatory threshold (VT, r2 = 0.33, p = 0.008), and O2 pulse (peak VO2/HR, r2 = 0.40, p = 0.003). There was a negative correlation between MHC 2a and 2b (fast isoforms) and peak VO2 (r2 = 0.38 and 0.37, p = 0.004, respectively), VT (r2 = 0.2, p = 0.05; r2 = 0.34, p = 0.007, respectively) and O2 pulse (r2 = 0.39, p = 0.003; r2 = 0.23, p = 0.03, respectively). NYHA functional class was also negatively correlated with the same parameters (r2 = 0.2, p = 0.01; r2 = 0.4, p = 0.001; r2 = 0.34, p = 0.006, respectively) as well as with MHC 1 (r2 = 0.62, p = 0.0001). A positive correlation was found between NYHA functional class and MHC 2a and 2b (r2 = 0.46, p = 0.001; r2 = 0.41, p = 0.002, respectively). The severity of heart failure is paralleled by a shift of the MHC pattern toward the fast MHC 2b. The correlation between the magnitude of the MHCs shift, from the slow aerobic to the fast type, with both clinical parameters (NYHA functional class) and functional measurements (peak VO2, VT, O2 pulse) of exercise capacity seem to suggest that changes in skeletal muscle composition may play a key role in exercise tolerance in patients with CHF.     

Effects of creatine monohydrate ingestion in sedentary and weight-trained older adults

Differential mortality exists in the United States both between racial/ethnic groups and along gradients of socioeconomic status. The specification of statistical models for processes underlying these observed disparities has been hindered by the fact that social and economic quantities are distributed in a highly nonrandom manner throughout the population. We sought to provide a substantive foundation for model development by representing the shape of the income-mortality relation by racial/ethnic group. We used data on black and white men and women from the longitudinal component of the National Health Interview Survey (NHIS), 1986-1990, which provided 1,191,824 person-years of follow-up and 12,165 mortal events. To account for family size when considering income, we used the ratio of annual family income to the federal poverty line for a family of similar composition. To avoid unnecessary categorizations and prior assumptions about model form, we employed kernel smoothing techniques and calculated the continuous mortality surface across dimensions of adjusted income and age for each of the gender and racial/ethnic groups. Representing regions of equal mortality density with contour plots, we observed interactions that need to be accommodated by any subsequent statistical models. We propose two general theories that provide a foundation for more elaborate and testable hypotheses in the future.     

Interactions of mitochondrial ATP synthesis and the creatine kinase equilibrium in skeletal muscle

This study aimed to evaluate the effect of FR128998, (1s,6s)-1-benzyl-10-(3-pyridyl-methyl)-7-thia-10-azaspiro [5,6]-dodecan-11-one 7,7-dioxide hydrochloride, a novel platelet activating factor (PAF) receptor antagonist, on endotoxin lipopolysaccharide-induced disseminated intravascular coagulation in rats. Experimental disseminated intravascular coagulation was induced by an infusion of lipopolysaccharide at 0.25 mg/kg/h for 4 h. Simultaneous infusion of FR128998 (0.25 and 1.0 mg/kg/h) with lipopolysaccharide dose dependently inhibited thrombocytopenia, but not leukopenia. The changes in coagulation parameters of disseminated intravascular coagulation, i.e., prolongation of activated partial thromboplastin time and elevated levels of fibrinogen/fibrin degradation products, were also prevented by the treatment with FR128998. In addition, FR128998 attenuated the increase in serum tumor necrosis factor (TNF) which appeared during the initial stage of disseminated intravascular coagulation. FR128998 (10 microM) also inhibited the TNF production by peripheral blood leukocytes or alveolar macrophages stimulated by lipopolysaccharide in vitro. Furthermore, TNF production induced by PAF itself in vitro was also inhibited in the presence of FR128998. These data indicate that PAF plays a pivotal role in the development of disseminated intravascular coagulation via TNF production.     

A Na(+)-dependent creatine transporter in rabbit brain, muscle, heart, and kidney. cDNA cloning and functional expression

A cDNA has been cloned from rabbit brain that is a new member of the emerging family of Na(+)-dependent plasma membrane transporters for several neurotransmitters and structurally related compounds. Functional expression of this cDNA in COS-7 cells identifies its product as a Na(+)- and Cl(-)-dependent creatine transporter with a Km of approximately 35 microM. Its creatine transporter activity is efficiently antagonized by 3-guanidinopropionate, a well characterized alternative substrate of creatine transport in several tissues, and by 4-guanidinobutyrate. More distant structural analogues of creatine are much less efficient or inactive as antagonists, indicating a high substrate specificity of the transporter. Northern blot hybridization detects the expression of its mRNA in most tissues tested, most prominently in kidney, heart, and muscle, but not in liver and intestine. A full-length cDNA clone was also isolated from a muscle cDNA library and found to contain the same coding sequence. Substrate affinity and specificity of the cloned transporter are very similar to the endogenous creatine transporter of the COS-7 cells and to the creatine transport activities of several cell types described in the literature. Moreover, its mRNA is most abundant in the tissues known to possess high creatine uptake capacity.     

Alterations in skeletal muscle gene expression in the rat with chronic congestive heart failure

Congestive heart failure is often associated with skeletal muscle abnormalities that contribute to early fatigue and acidosis. Up to the present time, however, the mechanisms responsible for these changes are unclear. Myocardial infarctions were produced by coronary ligation in adult Sprague-Dawley rats. At 20 weeks, 10 control rats, and 15 animals with heart failure [defined by elevated LVEDP (26.1 +/- 3.1 v 2.5 +/- 0.5 mmHg) and RV hypertrophy (300 +/- 21 g v 158 +/- 9 mg)] underwent in vivo measurements of total body, and soleus total protein and myosin heavy chain (MHC) synthesis by [3H]leucine constant infusion. Soleus muscle was also analysed for protein content, and MHC isoenzyme content by SDS-PAGE. Northern blotting also was used to determine levels of the mRNA's encoding type I, IIa, IIb, and IIx MHC, alpha-skeletal actin, COX III, SDH and GAPDH. Soleus muscles in heart failure rats were smaller than controls (112 +/- 6 v 126 +/- 5 mg) and the degree of atrophy was significant when corrected for body mass (0.38 +/- 0.02 v 0.46 +/- 0.02 mg/g. P = 0.007). Although there was no significant difference in plasma leucine flux (an index of whole-body protein synthesis), soleus muscle total and MHC synthesis was reduced in heart failure animals. Whereas the Type I MHC isoenzyme (beta MHC) was the only MHC detected in the soleus of control animals, type II MHC isoenzyme comprised 11.8 +/- 3.1% of the MHC in the heart failure group. Furthermore, steady-state mRNA levels encoding beta MHC were significantly depressed in the heart failure rats, where those encoding Types IIb and IIx MHC were increased. Steady-state mRNA levels of alpha-skeletal actin, cytochrome C oxidase (COX III) and succinate dehydrogenase (SDH) were also significantly depressed. This animal model of chronic heart failure is associated with quantitative and qualitative alterations in skeletal muscle gene expression that are similar to those reported in skeletal muscle of patients with chronic heart failure. The altered phenotype and impaired metabolic capacity may contribute to exercise intolerance in CHF.     

Differential membrane localization and intermolecular associations of alpha-dystrobrevin isoforms in skeletal muscle

alpha-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length alpha-dystrobrevin-1 (84 kD), and COOH-terminal truncated alpha-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that alpha-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. alpha-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, alpha-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro-translated proteins, alpha-dystrobrevin-2 binds dystrophin, whereas alpha-dystrobrevin-1 binds both dystrophin and utrophin. alpha-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, alpha-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of alpha-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the alpha-dystrobrevins.     

Altered expression of myosin heavy chain in human skeletal muscle in chronic heart failure

To explore further alterations in skeletal muscle in chronic heart failure (CHF), we examined myosin heavy chain (MHC) isoforms from biopsies of the vastus lateralis in nine male patients with class II-III (CHF) (left ventricular ejection fraction (LVEF) 26 +/- 11%, peak oxygen consumption (peak VO2) 12.6 +/- 2 mL.kg-1.min-1) and nine age-matched sedentary normal males (NL). The relative content of MHC isoforms I, IIa, and IIx was determined by gel electrophoresis as follows: The normal sedentary group (NL) had a higher percent of MHC type I when compared with the patients (NL 48.4 +/- 7% vs CHF patients 24 +/- 21.6%, P < 0.05, no difference between MCH IIa (NL 45.1 +/- 10.5% vs CHF 56.0 +/- 12.5%), and CHF patients had a higher relative content of MHC type IIx than did the normal group (NL 6.5 +/- 9.6% vs CHF 20.0 +/- 12.9%, P < 0.05. Three of nine patients had no detectable MHC type I. In patients relative expression of MHC type I (%) was related to peak VO2 (r = 0.70, P < 0.05). Our results indicate that major alterations in MHC isoform expression are present in skeletal muscle in CHF. These alterations parallel previously reported changes in fiber typing that may affect contractile function i skeletal muscle and possibly exercise performance. The absence of MHC type I in some CHF patients suggests that skeletal muscle changes in this disorder are not solely a result of deconditioning, buy may reflect a specific skeletal muscle myopathy in this disorder.     

Effects of acute and chronic ingestion of tolbutamide in the chicken

The effects of acute and chronic ingestion of tolbutamide were studied in the growing chicken. After an oral load of 100 or 25 mg tolbutamide/kg b.w., plasma insulin levels increased in a dose-dependent manner but to relatively low levels for about 10 min, while 10-20 min following tolbutamide, plasma glucose levels were markedly decreased and remained so for 2--5 hr. After 100 mg tolbutamide/kg, the profound hypoglycaemia which developed, was generally accompanied by symptoms resembling an hypoglycaemic coma: panting, muscular flacidity and convulsion. Body temperature and plasma calcium levels were not changed during and after tolbutamide-induced insulin release. In the chicken, tolbutamide response is therefore characterized by a fugitive insulin release and a profound and prolonged hypoglycaemia which suggest that the action of insulin is potentiated by other factors. Chronic ingestion of tolbutamide in the diet transiently (for one week) increased the live body weight of a dose of 400 mg tolbutamide/kg of diet. Long term (5 weeks) fasting plasma glucose levels were unchanged and fasting plasma insulin levels were decreased in the chronic tolbutamide treated chickens.     

Expression of neural cell adhesion molecules and neurofilament protein isoforms in skeletal muscle tumors

In a series of rhabdomyosarcomas, the expression of neural cell adhesion molecules (NCAM) and neurofilament isoforms was probed in frozen sections. It was found that NCAM was widely expressed in rhabdomyosarcomas without relation to subtype or differentiation level. Neurofilament isoforms were found throughout all subtypes but were largely restricted to those neurofilament isoforms that are expressed early in neurogenesis, that is, poorly phosphorylated low- and medium-weight isoforms. It was concluded that the expression of these "neural" markers is widespread and does not signify a neural tumor.     

Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney

This experiment was an attempt to use the soybean residue derived from the production of "soy milk". The residue contains about 18% protein, 70% carbohydrates and 7.5% lipid as fish feed for rearing common carp, Cyprinus carpio. There were 4 types of diets: (1) soybean residue, (2) soybean residue digested with Papain, (3) soybean residue (64%) mixed with beef liver (34%) and (4) same mixture as (3) but digested with Papain. The results indicate that the percentage increase in weight and length of fish feeding with beef liver supplemented diets was higher than those feeding with soybean residue alone. This was possibly due to the fact that beef liver was able to supplement the nutrient deficiency in soybean. The two types of feeds (2 and 4) digested with Papain also yielded significantly better fish growth in terms of weight and length gains, than their counterparts without digestion. Furthermore, the water turbidity of the tanks added with digested feeds was significantly less, as Papain was able to hyrolyse the protein substrates suspended in the water, and thus lowered the turbidity.     

Effects of creatine supplementation on repetitive sprint performance and body composition in competitive swimmers

In a double-blind and randomized manner, 18 male and female junior competitive swimmers supplemented their diets with 21 g.day-1 of creatine monohydrate (Cr) or a maltodextrin placebo (P) for 9 days during training. Prior to and following supplementation, subjects performed three 100-m freestyle sprint swims (long course) with 60 s rest/recovery between heats. In addition, subjects performed three 20-s arm ergometer maximal-effort sprint tests in the prone position with 60 s rest/recovery between sprint tests. Significant differences were observed among swim times, with Cr subjects swimming significantly faster than P subjects following supplementation in Heat 1 and significantly decreasing swim time in the second 100-m sprint. There was also some evidence that cumulative time to perform the three 100-m swims was decreased in the Cr group. Results indicate that 9 days of Cr supplementation during swim training may provide some ergogenic value to competitive junior swimmers during repetitive sprint performance.     

Expression of different isoforms of nitric oxide synthase in experimentally denervated and reinnervated skeletal muscle

Denervated muscle fibers express enhanced levels of stress and apoptosis-associated proteins and undergo apoptosis. In experimentally denervated and reinnervated rat facial muscle, we now evaluate changes in the expression patterns of different isoforms of nitric oxide synthase (NOS)-generating nitric oxide (NO), which mediates oxidative stress and apoptosis. Physiological expression of NOS corresponds to a constant sarcolemmal staining pattern for neuronal NOS (nNOS) and a patchy sarcolemmal and weak sarcoplasmic labeling for the endothelial NOS-isoform, with no expression for inducible NOS (iNOS). Denervated muscle displayed distinct downregulation of nNOS with preserved expression of dystrophin. Also, denervated and immediately reinnervated muscle fibers showed decreased expression of nNOS. However, muscle fibers reinnervated for 10 weeks revealed a restored physiological expression of nNOS. There were no changes in the expression of endothelial and inducible NOS. As NO is known to induce growth arrest and collapse of neuronal growth cones, downregulation of NOS may contribute to promotion of axonal regeneration by aiding formation of new endplates. NO is upregulated in reinnervated muscle fibers and thus prevents polyneural hyperinnervation by extrajunctional synapses. Furthermore, downregulation of NOS during denervation is compatible with the finding that low levels of NO contribute to apoptosis instead of necrosis in disease states of oxidative stress.     

Glycosphingolipids of skeletal muscle: I. Subcellular distribution of neutral glycosphingolipids and gangliosides in rabbit skeletal muscle

The development of polymers with different surface properties and surface modifications of intraocular lenses (IOL) should reduce foreign body reactions after implantation by reducing the surface hydrophobicity of the lenses. It was examined how far such surface variations influenced the adhesiveness of bacteria. The most common organism isolated from cases of postoperative endophthalmitis is Staphylococcus epidermidis. For this reason, three strains of this species, the type strain ATCC 14990 and two clinical isolates (8687, 6579 I), with different hydrophobic surfaces, were studied. IOL made of PMMA, silicone, and a copolymer as well as PMMA lenses with modified surfaces (unpolished, polished, silanized, and heparinized) were used. Bacteria were radiolabelled with 3H-thymidine and the adherent bacteria were calculated per mm2 of lens surface. The three strains adhered better to the unpolished surface of silicone than to PMMA. Treatment of PMMA surface by polishing diminished the differences between the strains. An influence of hydrophobic interactions on the adherence of S. epidermidis ATCC 14990 was demonstrated. The adherence of this hydrophobic type strain was clearly reduced by heparinization of the PMMA surface. In contrast, the hydrophilic catheter isolate 6579 I adhered better to modified surfaces. This strain differed clearly in its PFGE pattern from both hydrophobic strains. Hydrophobic interactions play a role in the bacterial adherence to intraocular lenses in vitro and in vivo. Modifications of polymer surfaces, however, can result in rather different effects depending on the bacterial surface composition and properties.     

A sensitive electrophoretic method for the quantification of myosin heavy chain isoforms in horse skeletal muscle: histochemical and immunocytochemical verifications

In adult horses, three myosin heavy chain (MyHC) isoforms can be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunohistochemistry using specific anti-MyHC monoclonal antibodies. This report studies the suitability of a consistent SDS-PAGE technique for quantifying MyHC profiles in homogenized cryostate sections of equine gluteus medius muscle biopsies (n = 18). The method used (previously described by R. J. Talmadge and R. R. Roy; J. Appl. Physiol. 1993, 75, 2337-2340) resolved MyHCs in three bands: I, IIB or IIX, and IIA from the fastest to the slowest migration band. The success rate of the protocol for yielding three well-differentiated MyHC bands was 100% and a subsequent quantification by densitometry for each MyHC isoform was obtained in all 18 muscle biopsies. The results obtained with this electrophoretic method were compared with routine myofibrillar adenosine triphosphatase histochemistry and immunohistochemistry using specific anti-MyHC monoclonal antibodies. The percent composition of the three electrophoretically separated MyHC isoforms (I, IIA and IIB or IIX) showed strong positive correlation with percentages of the area occupied in the biopsies by the three major fiber types (I, IIA, and IIB) identified histochemically (r = 0.96, P < 0.001) and immunohistochemically (r = 0.94, P < 0.01). It can be concluded that the electrophoretic method used here for measuring MyHC content is a valid alternative for muscle fiber typing in horses. As it is less costly and time-consuming than both qualitative histochemistry and immunohistochemistry, the method offers new prospects for application in equine experimental studies and veterinary medicine.