Abnormal mitochondria and sarcoplasmic changes in rabbit skeletal muscle induced by immobilization

Immobilization of the rabbit knee in extended position results in damage to the vastus intermedius profundus (VIP) muscle. To examine the mechanisms involved in initiation of the injury, we studied the light and electron microscopic morphology of the VIP muscle, as well as the activity and distribution of NADH tetrazolium reductase (NADH-TR) in the affected muscle, and determined serum total creatine kinase (CK) activity in immobilized rabbits. The VIP muscle of the immobilized right hindlimb was removed at various time points (10 h, 24 h, 36 h and 48-72 h, n=5 for each time point). The nonimmobilized left hindlimb and five nonimmobilized animals served as controls. No morphological changes were observed by light microscopy within 48-72 h in routine stainings. Transient ultrastructural abnormalities, including abnormal cristae, matrix lucencies and mild swelling of mitochondria, were observed between 10 h and 36 h of immobilization, subsiding by 48-72 h. On the other hand, progressive disorganization of myofibrils with breaking-up of Z-bands and an increase in the number and size of sarcoplasmic lipid vacuoles was seen with increasing duration of immobilization. NADH-TR activity at subsarcolemmal locations had decreased by 10 h and disappeared by 24 h of immobilization, while the intermyofibrillar mitochondria remained unaltered. Serum total CK activity began to increase by 2 h of immobilization and reached a peak by 24 h. The results indicate that already a few hours of immobilization of the rabbit knee in extension leads to signs of metabolic disturbance of the VIP muscle and sarcolemmal leakage. The simultaneous occurrence of transient mitochondrial abnormalities, transient CK efflux and progressive myofibrillar damage suggests the operation of multiple adverse mechanisms already at the onset of disuse muscle atrophy.     

In vitro evidence of baicalein's inhibition of the metabolism of zidovudine (AZT)

Background

Herb-drug interaction (HDI) has been regarded as a key factor limiting the clinical application of herbs and drugs.

Aims

Potential baicalein-zidovudine (AZT) interaction was predicted in the present study.

Methods

In vitro evaluation of baicalein''s inhibition towards human liver microsomes (HLMs)-catalyzed metabolism of zidovudine (AZT) was performed. Dixon and Lineweaver-Burk plots were used to determine the inhibition kinetic type, and second plot with the slopes from Lineweaver-Burk plot versus the concentrations of baicalein was employed to calculate the inhibition parameter (Ki). In combination with the in vivo concentration of baicalein, in vitro-in vivo extrapolation (IVIVE) was carried out to predict in vivo baicalein-AZT interaction.

Results

Competitive inhibition of baicalein towards AZT metabolism was demonstrated, and the K_i value was calculated to be 101.2 µM. The value of AUCi/AUC was calculated to be 2.

Conclusion

Potential baicalein-AZT interaction was indicated in the present study, indicating the need for monitoring when AZT is co-administrated with baicalein or baicalein-containing herbs.     

Enhancement of HIV-specific cytotoxic T lymphocyte responses by zidovudine (AZT) treatment.

Zidovudine or 3'-azido-2'-3'-dideoxy-thymidine (AZT) is an antiviral drug widely used to treat HIV-infected patients. Because cytotoxic T lymphocytes (CTL) are thought to contribute actively to resistance against HIV-induced disease, we studied sequentially 10 HIV-infected individuals under zidovudine treatment for a period of 6-12 months. For a given patient all lymphocyte suspensions corresponding to the complete zidovudine therapy period were tested on the same day and on the same target cells. Patients were selected for expression of HLA-A2 and/or HLA-A3 class I transplantation antigen. HLA-restricted cytotoxicity specific for env, gag and nef HIV proteins was quantified for each patient at 6 week intervals. The data clearly indicated that zidovudine has a beneficial effect on the CTL response during the first 6-12 weeks of treatment, inducing cytotoxicity levels up to 100-fold stronger than base line. This effect was usually short lived. However, patients who maintained strong levels of cytotoxicity had better clinical and survival outlook than patients who had lost all detectable cytotoxic lymphocytes. It is proposed that AZT, among other effects, delays the onset of disease in HIV-infected patients by contributing to the stimulation of the HIV-specific CTL response.     

Adaptive conditioning of skeletal muscle in a large animal model (Sus domesticus)

Recognition of the adaptive capacity of mammalian skeletal muscle has opened the way to a number of clinical applications. For most of these, the fast, fatigue-susceptible fibres need to be transformed stably to fast, fatigue-resistant fibres that express the 2A myosin heavy chain isoform. The thresholds for activity-induced change are size-dependent, so although the requisite patterns of electrical stimulation are known for the rabbit, in humans these same patterns would produce type 1 fibre characteristics, with an undesirable loss of contractile speed and power. We have used histochemistry, immunohistochemistry and electrophoretic separations to evaluate a possible conditioning regime in a large animal model. Stimulation of the porcine latissimus dorsi muscle with a phasic 30-Hz pattern for up to 41 days converted all type 2X and 2A/2X fibres to 2A with only a small increase in the type 1 population, from 17% to 22%. Stimulation for longer periods increased the proportion of type 1 fibres to 52%. Based on this model, stimulation regimes designed to achieve a stable 2A phenotype in humans should deliver fewer stimulating impulses, possibly by a factor of 2, than the pattern assessed here. Any such pattern needs to be tested for at least 8 weeks.     

Computerized calculation of in vitro generation of ATP and creatine phosphate induced by respiration in human muscle mitochondria

A computer program is presented which calculates the action of adenylate kinase and creatine kinase during the process of adenosine-5'-triphosphate generation by the mitochondria in human skeletal muscle 600 X g supernatants. The results show that the mass action ratios of the two kinase reactions are not influenced by incubation time or decreased rate of oxidative phosphorylation as a result of mitochondrial dysfunction. The program is useful for establishing optimum initial adenosine-5'-diphosphate and creatine concentrations in the assay medium.     

Benefit of oral zinc supplementation as an adjunct to zidovudine (AZT) therapy against opportunistic infections in aids

Zinc is perhaps the most important trace element for immune function. Congenital or acquired zinc deficiencies are associated with immune abnormalities and increased susceptibility to infectious diseases. AIDS subjects suffer from reduced zinc bioavailability, more severe in stage IV than in stage III. Such zinc deficiency causes, among other effects, a profound reduction in the biological activity of one of the thymic hormones, thymulin (zinc-facteur-timique-serique, ZnFTS). With these premises, zinc sulphate was administered orally at a daily dose of 200 mg for 30 days to AZT-treated stage III subjects with generalized lymphadenopathy (17 subjects) and stage IV subgroup C1 (12 subjects) AIDS patients. 18 stage III subjects with generalized lymphoadenopathy and 10 stage IV subgroup C1 subjects treated only with AZT served as controls. Zinc sulphate supplementation of stage III and in stage IV CI patients was followed by an increase or a stabilization in the body weight and an increase of the number of CD4⁺ cells and the plasma level of active zinc-bound thymulin. The frequency of opportunistic infectious episodes in the 24 months following entry into the study was reduced after zinc supplementation in stage IV C1 subjects (11 infections vs 25 in controls) and delayed in stage III zinc-treated subjects (1 infection/24 months vs 13 infections/24 months in controls). The effect of zinc on opportunistic infections is restricted to infections due to Pneumocystis carinii and Candida, whereas no variations have been observed in the frequencies of cytomegalovirus and toxoplasma infections. These data may support the benefit of zinc as an adjunct to AZT therapy in AIDS pathology.     

Neuropsychological outcome of zidovudine (AZT) treatment of patients with AIDS and AIDS-related complex

Two hundred eighty-one patients with the acquired immunodeficiency syndrome (AIDS) or advanced AIDS-related complex were enrolled in a double-blind, placebo-controlled trial of the efficacy and safety of orally administered zidovudine (azidothymidine or AZT). Significant clinical benefits and adverse experiences have been reported from this trial. Because neuropsychiatric dysfunction is often associated with human immunodeficiency virus (HIV) infection, a brief affective and neuropsychological examination was administered over 16 weeks of the trial to evaluate any changes in neuropsychological function that occurred with drug administration. Patients receiving zidovudine, particularly those with AIDS, showed improved cognition as compared with patients receiving placebo. There were no changes in affective symptoms. The zidovudine recipients also had a statistically significant reduction in the intensity of symptomatic distress during the trial that may account in part for the observed cognitive changes. Some improvement in various cognitive measures was also seen in patients with AIDS-related complex. The results of this study suggest HIV-associated cognitive abnormalities may be partially ameliorated after the administration of zidovudine.     

Changes in intracellular ion activities induced by adrenaline in human and rat skeletal muscle

To study the stimulating effect of adrenaline (ADR) on active Na⁺/K⁺ transport we used double-barrelled ion-sensitive micro-electrodes to measure the activities of extracellular K⁺ (aK_e) and intracellular Na⁺ (aNa_i) in isolated preparations of rat soleus muscle, normal human intercostal muscle and one case of hyperkalemic periodic paralysis (h.p.p.). In these preparations bath-application of ADR (10⁻⁶ M) resulted in a membrane hyperpolarization and transient decreasesaK_e andaNa_i which could be blocked by ouabain (3×10⁻⁴ M). In the h.p.p. muslce a continuous rise ofaNa_i induced by elevation ofaK_e to 5.2 mM could be stopped by ADR. In addition, the intracellular K⁺ activity (aK_i), the free intracellular Ca²⁺ concentration (pCa_i) and intracellular pH (pH_i) were monitored in rat soleus muscle. During ADRaK_i increased, pH_i remained constant and intracellular Ca²⁺ apparently decreased. In conclusion, our data show that ADR primarily stimulates the Na⁺/K⁺ pump in mammalian skeletal muscle. This stimulating action is not impaired in the h.p.p. muscle. Parts of the results have been presented to the German Physiological Society (Ballanyi and Grafe 1987)     

Extreme endurance training evidence of capillary and mitochondria compartmentalization in human skeletal muscle

Summary Biopsies from the medial gastrocnemius muscle of three experienced endurance runners who had completed an ultramarathon run (160 km) the previous day were assessed for their oxidative characteristics (fibre types, capillarization and mitochondria content). Also, a regional comparison was made for fibres located centrally (completely surrounded by other fibres) versus fibres located peripherally (next to the interfascicular space) and the capillarization of these peripheral fibres was determined. Subsarcolemmal mitochondria were abundant and compartmentalized close to the capillaries. The number of capillaries around fibres ranged from 5.8 to 8.5 and 5.7 to 8.5, and the number of capillaries·mm^–2 ranged from 665 to 810 and 727 to 762, for type I (slow twitch) and type 11 (fast twitch) fibres, respectively. Central fibres contained a greater number of capillaries and more capillaries·mm^–2 than their peripheral counterparts. Peripheral fibres contained more capillaries · m^–1 between fibres than at the interfascicular space. Type I fibres were more distributed (63%–78%) and larger than type II fibres. An abundance of subsarcolemmal mitochondria located close to the capillaries, efficient capillary proliferation between fibres where sharing can occur and greater relative distribution and size of type I fibres are, collectively, efficient characteristics of extreme endurance training.     

Sarcoglycans in human skeletal muscle and human cardiac muscle: a confocal laser scanning microscope study

Sarcoglycans are a subcomplex of transmembrane proteins which are part of the dystrophin-glycoprotein complex. They are expressed in the skeletal, cardiac and smooth muscle. Although numerous studies have been conducted on the sarcoglycan subcomplex in skeletal and cardiac muscle, the manner of the distribution and localization of these proteins along the nonjunctional sarcolemma is not clear. We therefore carried out an indirect immunofluorescence study on surgical biopsies of normal human skeletal muscle and of healthy human atrial myocardium biopsies of patients affected by valvulopathy. Our results indicate that, in skeletal muscle, sarcoglycans have a costameric distribution and all colocalize with each other. Only in a few cases did the alpha-sarcoglycan not colocalize with other sarcoglycans. In addition, these glycoproteins can be localized in different fibers either in the regions of the sarcolemma over band I or band A. In cardiac muscle, our results show a costameric distribution of all proteins examined and, unlike in skeletal muscle, they show a constant colocalization of all sarcoglycans with each other, along with a consistent localization of these proteins in the region of the sarcolemma over band I. In our opinion, this situation seems to confirm the hypothesis of a correlation between the region of the sarcolemma occupied by costameric proteins and the metabolic type, fast or slow, of the muscular fibers. These data, besides opening a new line of research in understanding interactions between the sarcoglycans and other transmembrane proteins, could also be extended to skeletal and cardiac muscles affected by neuromuscular and cardiovascular pathologies to understand possible structural alterations.     

Biomimetic model of skeletal muscle isometric contraction: I. an energetic-viscoelastic model for the skeletal muscle isometric force twitch

This paper describes a revision of the Hill-type muscle model so that it will describe the chemo-mechanical energy conversion process (energetic) and the internal-element stiffness variation (viscoelastic) during a skeletal muscle isometric force twitch contraction. The derivation of this energetic-viscoelastic model is described by a first-order linear ordinary differential equation with constant energetic and viscoelastic coefficients. The model has been implemented as part of a biomimetic model, which describes the excitation-contraction coupling necessary to drive the energetic-viscoelastic model. Finally, the energetic-viscoelastic model is validated by comparing its isometric force-time profile with that of various muscles reported in the literature.     

Changes in postnatal skeletal muscle development induced by alternative immobilization model in female rat

The objective of this study was to adapt a model of hind limb immobilization to newly weaned female rats and to determine the morphology of shortened soleus and plantaris muscles. Female Wistar rats were divided into three groups: control zero (n = 3) and control and free (n = 8), animals aged 21 and 31 days, respectively, submitted to no intervention, and immobilized (n = 25), animals aged 21 days submitted to immobilization for 10 days and sacrificed at 31 days of age. The device used for immobilization had advantages such as easy connection, good fit, and low cost. The immobilized rats showed a reduction in muscle fiber area and in connective tissue. The adaptation of this immobilization model originally used for adult rats was an excellent alternative for newly weaned rats and was also efficient in inducing significant hind limb disuse.