Saponin-permeabilized Muscle Fibers Research Articles

Effects of aging and exercise on Mitochondria-ER contacts (MERCs) in skeletal muscle

Mitochondrial dysfunction is a prominent factor contributing to age-associated loss of muscle mass and force (a.k.a., sarcopenia). Yet, events preceding age-related mitochondrial dysfunction, which may allow the development of new therapies, remain poorly understood. Physical coupling of the outer mitochondrial membrane (OMM) to the endoplasmic or sarcoplasmic reticulum (ER), termed mitochondria ER contact sites (MERCs), modulate several mitochondrial processes influencing cellular function. In an energetically demanding and primarily post-mitotic tissue, we hypothesized that alterations in MERCs correlate with the development of sarcopenia. We also hypothesized that MERCs respond to exercise training and may contribute to beneficial muscular remodeling in older adults. To address these, we studied three age groups of male C57BL6N mice: young adults (Y, 5 mo. of age), old (O, 20 mo. of age), and very old (VO, 31 mo. of age). An additional group of old mice underwent 8-9 weeks of treadmill training (OET). Muscle mass and contractile function decreased with aging; VO animals displayed ~25% decline in maximum tetanic force vs. Y mice (p<0.0001). Fatigability (assessed via repetitive sub-maximal contractions) increased by ~10% with aging but was attenuated in the OET group (p<0.001). In saponin-permeabilized muscle fibers, aging did not affect mitochondrial oxidative capacity. However, H2O2 emission was nearly doubled in the O group (p<0.01) and this trend was partially alleviated by exercise training. Assessed via TEM, aging decreased the size of MERCs (i.e., length of SR and OMM apposition) by around 20% (p<0.01). Interestingly, OET animals displayed ~50% increase in MERC coverage (i.e., proportion of OMM covered by SR; p<0.0001). Immunoblots of cellular MERC fractions revealed that MERC proteins associated with calcium exchange were altered with age and exercise. The OMM channel, VDAC1, was reduced by 20% in O muscles (p=0.02), while Grp75, a tether protein, was ~25% higher in OET (p=0.02). Quantitative proteomic analyses revealed various MERC proteins being modified by aging and exercise. Our findings indicate that changes in MERC structure and protein composition may underlie early events preceding the development of sarcopenia and that exercise-mediated adaptations at MERCs may contribute to the beneficial adaptations in aging skeletal muscle. Supported by the University of Iowa Fraternal Order of Eagles Diabetes Research Center (UIowa FOEDRC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondrial respiration and H2O2 emission in saponin-permeabilized murine diaphragm fibers: optimization of fiber separation and comparison to limb muscle.

Diaphragm abnormalities in aging or chronic diseases include impaired mitochondrial respiration and H2O2 emission, which can be measured using saponin-permeabilized muscle fibers. Mouse diaphragm presents a challenge for isolation of fibers due to relatively high abundance of connective tissue in healthy muscle that is exacerbated in disease states. We tested a new approach to process mouse diaphragm for assessment of intact mitochondria respiration and ROS emission in saponin-permeabilized fibers. We used the red gastrocnemius (RG) as "standard" limb muscle. Markers of mitochondrial content were two- to fourfold higher in diaphragm (Dia) than in RG (P < 0.05). Maximal O2 consumption (JO2: pmol·s-1·mg-1) in Dia was higher with glutamate, malate, and succinate (Dia 399 ± 127, RG 148 ± 60; P < 0.05) and palmitoyl-CoA + carnitine (Dia 15 ± 5, RG 7 ± 1; P < 0.05) than in RG, but not different between muscles when JO2 was normalized to citrate synthase activity. Absolute JO2 for Dia was two- to fourfold higher than reported in previous studies. Mitochondrial JH2O2 was higher in Dia than in RG (P < 0.05), but lower in Dia than in RG when JH2O2 was normalized to citrate synthase activity. Our findings are consistent with an optimized diaphragm preparation for assessment of intact mitochondria in permeabilized fiber bundles. The data also suggest that higher mitochondrial content potentially makes the diaphragm more susceptible to "mitochondrial onset" myopathy. Overall, the new approach will facilitate testing and understanding of diaphragm mitochondrial function in mouse models that are used to advance biomedical research and human health.

Calcium Homeostasis Is Modified in Skeletal Muscle Fibers of Small Ankyrin1 Knockout Mice.

Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca2+ handling, cytosolic Ca2+ levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca2+ content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca2+ transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca2+ release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of sparks was significantly lower in fibers from 4-month-old KO mice compared to WT. Furthermore, both the amplitude and spatial spread of sparks were significantly smaller in muscle fibers from both 4- and 10-month-old KO mice compared to WT. These data suggest that the absence of sAnk1 results in an impairment of SR Ca2+ release, likely as a consequence of a decreased Ca2+ store due to the reduction of the SR volume in sAnk1 KO muscle fibers.

28 Mitochondrial adaptations to submaximal exercise training in the gluteus medius and triceps brachii of young equine athletes

s / Journal of Equine Veterinary Science 35 (2015) 392e397 395 but the use of electrolyte pastes in races was less common (31%). Analyzing the data set using logistic regression, there was a trend (P 1⁄4 0.10) for metabolic failure to increase 0.66 for each increasing level of alfalfa feeding (0 1⁄4 none, 1 1⁄4 occasional, 2 1⁄4 regular, 3 1⁄4 daily). OR for metabolic failure reduced by 0.10 for each additional ride > 90 km the rider had entered in the last 2 years (P < 0.05). Highest current level of competition and total distance completed over 90 km also approached significance for reducing the risk of metabolic failure. Electrolyte use is common among endurance riders competing at all levels in the UK. In this small survey, experience of horse and rider appeared to have a greater influence on the risk of metabolic failure than electrolyte use or nutrition per se. 28 Mitochondrial adaptations to submaximal exercise training in the gluteus medius and triceps brachii of young equine athletes S.H. White*, L.K. Warren, C. Li, and S. Wohlgemuth University of Florida, Gainesville, FL, USA Exercise training has been shown to enhance mitochondrial biogenesis and efficiency in human and rodent models. We hypothesized that lowto moderate-intensity exercise training would result in similar mitochondrial improvements in skeletal muscle of young horses, and that responses would be muscle dependent. Quarter Horse long-yearlings (mean ± SE, 21 ± 0.2 mo, 410 ± 9 kg) were randomly assigned to either: submaximal exercise training (TR; n 1⁄4 18) comprised of walk-trot-canter exercise in a round-pen or while being ridden 30e45min/d, 3 d/wk; or no controlled exercise (UT; n 1⁄4 6). UT horses were grouphoused on 16-ha pastures, whereas housing for TR horses was split between pasture and stall (~6 h/d). Biopsies were taken from the gluteus medius of all horses (n 1⁄4 24) and from the triceps brachii of a randomly selected subset of TR (n 1⁄4 6) and all UT (n 1⁄4 6) before (wk 0) and after 9 wk of training. Muscle was assessed for activities of cytochrome c oxidase (CCO) and citrate synthase (CS) as indicators of mitochondrial function and number, respectively. Additionally, saponin-permeabilized muscle fibers were analyzed for mitochondrial function via high-resolution respirometry, and data were normalized to mitochondrial number in the sample (CS activity). Data were analyzed using a mixed-model ANOVA. The triceps had greater CS activity (P < 0.0001) and lower CCO activity (P < 0.0001) than the gluteus in all horses; however, enzyme activities were unaffected by time or training. An overall effect of time across both muscle groups and training levels was observed, whereby electron transport system capacity (ETSC) increased (P 1⁄4 0.02), and state 3 respiration (P 1⁄4 0.06) and the respiratory control ratio (P 1⁄4 0.06) tended to increase during the 9-wk observation period. Leak respiration (P 1⁄4 0.01) and state 3 with both complex I and II substrates (GMS; P1⁄4 0.007) were higher across bothmuscle groups in TR compared with UT horses. The gluteus in TR horses appeared to be most greatly affected, as demonstrated by an increase in state 3 with complex I substrates (P 1⁄4 0.04), state 3 GMS (P 1⁄4 0.005), ETSC (P 1⁄4 0.04), and the phosphorylation system control ratio with complex I substrates (P 1⁄4 0.01) from wk 0 to 9. As both young untrained and trained horses exhibited improvements in mitochondrial adaptations over time, these changes could reflect alterations in mitochondria due to growth. Nevertheless, submaximal exercise training appeared to further enhance mitochondrial efficiency in the gluteus medius of young horses.

Mitofusin 2 mutations affect mitochondrial function by mitochondrial DNA depletion

Charcot-Marie-Tooth neuropathy type 2A (CMT2A) is associated with heterozygous mutations in the mitochondrial protein mitofusin 2 (Mfn2) that is intimately involved with the outer mitochondrial membrane fusion machinery. The precise consequences of these mutations on oxidative phosphorylation are still a matter of dispute. Here, we investigate the functional effects of MFN2 mutations in skeletal muscle and cultured fibroblasts of four CMT2A patients applying high-resolution respirometry. While maximal activities of respiration of saponin-permeabilized muscle fibers and digitonin-permeabilized fibroblasts were only slightly affected by the MFN2 mutations, the sensitivity of active state oxygen consumption to azide, a cytochrome c oxidase (COX) inhibitor, was increased. The observed dysfunction of the mitochondrial respiratory chain can be explained by a twofold decrease in mitochondrial DNA (mtDNA) copy numbers. The only patient without detectable alterations of respiratory chain in skeletal muscle also had a normal mtDNA copy number. We detected higher levels of mtDNA deletions in CMT2A patients, which were more pronounced in the patient without mtDNA depletion. Detailed analysis of mtDNA deletion breakpoints showed that many deleted molecules were lacking essential parts of mtDNA required for replication. This is in line with the lack of clonal expansion for the majority of observed mtDNA deletions. In contrast to the copy number reduction, deletions are unlikely to contribute to the detected respiratory impairment because of their minor overall amounts in the patients. Taken together, our findings corroborate the hypothesis that MFN2 mutations alter mitochondrial oxidative phosphorylation by affecting mtDNA replication.

Erythropoietin Treatment Enhances Muscle Mitochondrial Capacity in Humans

Erythropoietin (Epo) treatment has been shown to induce mitochondrial biogenesis in cardiac muscle along with enhanced mitochondrial capacity in mice. We hypothesized that recombinant human Epo (rhEpo) treatment enhances skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity in humans. In six healthy volunteers rhEpo was administered by sub-cutaneous injection over 8 weeks with oral iron (100 mg) supplementation taken daily. Mitochondrial OXPHOS was quantified by high-resolution respirometry in saponin-permeabilized muscle fibers obtained from biopsies of the vastus lateralis before and after rhEpo treatment. OXPHOS was determined with the mitochondrial complex I substrates malate, glutamate, pyruvate, and complex II substrate succinate in the presence of saturating ADP concentrations, while maximal electron transport capacity (ETS) was assessed by addition of an uncoupler. rhEpo treatment increased OXPHOS (from 92 ± 5 to 113 ± 7 pmol·s−1·mg−1) and ETS (107 ± 4 to 143 ± 14 pmol·s−1·mg−1, p < 0.05), demonstrating that Epo treatment induces an upregulation of OXPHOS and ETS in human skeletal muscle.

Normal mitochondrial function and increased fat oxidation capacity in leg and arm muscles in obese humans

The aim of this study was to investigate mitochondrial function, fibre-type distribution and substrate oxidation during exercise in arm and leg muscles in male postobese (PO), obese (O) and age- and body mass index (BMI)-matched control (C) subjects. The hypothesis of the study was that fat oxidation during exercise might be differentially preserved in leg and arm muscles after weight loss. Indirect calorimetry was used to calculate fat and carbohydrate oxidation during both progressive arm-cranking and leg-cycling exercises. Muscle biopsy samples were obtained from musculus deltoideus (m. deltoideus) and m. vastus lateralis muscles. Fibre-type composition, enzyme activity and O(2) flux capacity of saponin-permeabilized muscle fibres were measured, the latter by high-resolution respirometry. During the graded exercise tests, peak fat oxidation during leg cycling and the relative workload at which it occurred (FatMax) were higher in PO and O than in C. During arm cranking, peak fat oxidation was higher in O than in C, and FatMax was higher in O than in PO and C. Similar fibre-type composition was found between groups. Plasma adiponectin was higher in PO than in C and O, and plasma leptin was higher in O than in PO and C. In O subjects, maximal fat oxidation during exercise and the eliciting relative exercise intensity are increased. This is associated with higher intramuscular triglyceride levels and higher resting non esterified fatty acid (NEFA) concentrations, but not with differences in fibre-type composition, mitochondrial function or muscle enzyme levels compared with Cs. In PO subjects, the changes in fat oxidation are preserved during leg, but not during arm, exercise.

In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography.

A method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin-permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed-polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast-twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest.

Relationships between maximal muscle oxidative capacity and blood lactate removal after supramaximal exercise and fatigue indexes in humans.

The present study investigated whether blood lactate removal after supramaximal exercise and fatigue indexes measured during continuous and intermittent supramaximal exercises are related to the maximal muscle oxidative capacity in humans with different training status. Lactate recovery curves were obtained after a 1-min all-out exercise. A biexponential time function was then used to determine the velocity constant of the slow phase (gamma(2)), which denoted the blood lactate removal ability. Fatigue indexes were calculated during all-out (FI(AO)) and repeated 10-s cycling sprints (FI(Sprint)). Biopsies were taken from the vastus lateralis muscle, and maximal ADP-stimulated mitochondrial respiration (V(max)) was evaluated in an oxygraph cell on saponin-permeabilized muscle fibers with pyruvate + malate and glutamate + malate as substrates. Significant relationships were found between gamma(2) and pyruvate + malate V(max) (r = 0.60, P < 0.05), gamma(2) and glutamate + malate V(max) (r = 0.66, P < 0.01), and gamma(2) and citrate synthase activity (r = 0.76, P < 0.01). In addition, gamma(2), glutamate + malate V(max), and pyruvate + malate V(max) were related to FI(AO) (gamma(2) - FI(AO): r = 0.85; P < 0.01; glutamate + malate V(max) - FI(AO): r = 0.70, P < 0.01; and pyruvate + malate V(max) - FI(AO): r = 0.63, P < 0.01) and FI(Sprint) (gamma(2) - FI(Sprint): r = 0.74, P < 0.01; glutamate + malate V(max) - FI(Sprint): r = 0.64, P < 0.01; and pyruvate + malate V(max) - FI(Sprint): r = 0.46, P < 0.01). In conclusion, these results suggested that the maximal muscle oxidative capacity was related to blood lactate removal ability after a 1-min all-out test. Moreover, maximal muscle oxidative capacity and blood lactate removal ability were associated with the delay in the fatigue observed during continuous and intermittent supramaximal exercises in well-trained subjects.

Preserved response of mitochondrial function to short-term endurance training in skeletal muscle of heart transplant recipients

ObjectivesWe sought to determine whether intrinsic mitochondrial function and regulation were altered in heart transplant recipients (HTRs) and to investigate the response of mitochondrial function to six-week endurance training in these patients. BackgroundDespite the normalization of central oxygen transport during exercise, HTRs are still characterized by limited exercise capacity, which is thought to result from skeletal muscle metabolic abnormalities. MethodsTwenty HTRs agreed to have vastus lateralis biopsies and exercise testing: before and after training for 12 of them and before and after the same control period for eight subjects unwilling to train. Mitochondrial respiration was evaluated on saponin-permeabilized muscle fibers in the absence or presence (maximum respiration rate [Vmax]) of saturating adenosine diphosphate. ResultsMitochondrial function was preserved at the level of sedentary subjects in untrained HTRs, although they showed 28 ± 5% functional aerobic impairment (FAI). After training, Vmax, citrate synthase, cytochrome coxidase, and mitochondrial creatine kinase (CK) activities were significantly increased by 48%, 40%, 67%, and 53%, respectively (p < 0.05), whereas FAI decreased to 12 ± 5% (p < 0.01). The control of mitochondrial respiration by creatine and mitochondrial CK was also improved (p < 0.01), suggesting that phosphocreatine synthesis and transfer by the mitochondrial CK become coupled to oxidative phosphorylation, as shown in trained, healthy subjects. ConclusionsIn HTRs, the mitochondrial properties of skeletal muscle were preserved and responded well to training, reaching values of physically active, healthy subjects. This suggests that, in HTRs, immunosuppressive drugs do not alter the intrinsic muscle oxidative capacities and that the patients’ physical handicap results from nonmitochondrial mechanisms.

12] Functional imaging of mitochondrial redox state

The use of intrinsic fluorophores allows evaluation of the function of mitochondrial oxidative phosphorylation without having the phototoxic side effects of the various fluorescent mitochondrial dyes. To detect the changes of the mitochondrial redox state in response to alterations of oxidative phosphorylation (OxPhos) at the single cell level, the fluorescence changes of the α-lipoamide dehydrogenase flavin moiety by confocal microscopy is investigated. Functional imaging of the mitochondrial redox state can be performed by microscopic detection of the flavoprotein fluorescence of the mitochondrially localized α-lipoamide dehydrogenase, which is in tight redox equilibrium with the mitochondrial NAD system. Direct detection of the NAD(P)H fluorescence signal for mitochondrial NAD redox state determinations can be performed in digitonin-permeabilized cells or saponin-permeabilized muscle fibers at considerably higher sensitivity. The procedure is applicable for cultured cells having low mitochondrial content. For the determination of putative heterogeneities of the mitochondrial redox state in permeabilized muscle fibers, the application of Fp/NAD(P)H ratio imaging is advantageous, because it offers an increased sensitivity with respect to metabolic alterations of the fluorescence signals. In sum, the monitoring of intrinsic fluorophores allows one to obtain insights into the mitochondrial function of single living cells, avoiding the problems of phototoxicity and photobleaching.

Flux Control of Cytochrome c Oxidase in Human Skeletal Muscle

In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the flux control coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 +/- 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 +/- 0.2-fold excess capacity of COX was determined. In agreement with these findings, the flux control of COX increased, in the presence of the three substrates, from 0.27 +/- 0.03 to 0.36 +/- 0.08. These results indicate a tight in vivo control of respiration by COX in human skeletal muscle. This tight control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased flux control coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme.

Mitochondrial DNA abnormalities in skeletal muscle of patients with sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a neurodegenerative disease affecting the anterior horn cells of the spinal cord and cortical motor neurons. Previous findings have suggested a specific impairment of mitochondrial function in skeletal muscle of at least a limited number of patients. Applying flavoprotein/NAD(P)H autofluorescence imaging of mitochondrial function in saponin-permeabilized muscle fibres, we detected a heterogeneous distribution of the respiratory chain defect among individual fibres in muscle biopsies of patients (11 out of 17) with sporadic amyotrophic lateral sclerosis (SALS). These findings correlate with the presence of cytochrome c oxidase (COX)-negative muscle fibres detected histologically. We established the molecular basis for the decreased activities of NADH:CoQ oxidoreductase and COX in SALS muscle. In the skeletal muscle of the investigated patients, diminished levels (13 out of 17) or multiple deletions (one out of 17) of mitochondrial DNA (mtDNA) were observed. These alterations of mtDNA seem to be related to decreased levels of membrane-associated mitochondrial Mn-superoxide dismutase. Our results support the viewpoint that an oxygen radical-induced impairment of mtDNA is of pathophysiological significance in the aetiology of at least a subgroup of patients with SALS.

Muscle fibres: applications for the study of the metabolic consequences of enzyme deficiencies in skeletal muscle

Mitochondrial function in saponin-permeabilized muscle fibres can be studied by high-resolution respirometry, laser-excited fluorescence spectroscopy and fluorescence microscopy. We applied these techniques to study metabolic effects of changes in the pattern of mitochondrial enzymes in skeletal muscle of patients with chronic progressive external ophthalmoplegia or Kearns-Sayre syndrome harbouring large-scale deletions of mitchondrial DNA (mtDNA). In all patients combined deficiencies of respiratory chain enzymes containing mitochondrially encoded subunits were observed. The citrate synthase-normalized activity ratios of these enzymes decreased linearly with increasing mtDNA heteroplasmy. This indicates the absence of any well-defined mutation thresholds for mitochondrial enzyme activities in the entire skeletal muscle. We applied metabolic control analysis to perform a quantitative estimation of the metabolic influence of the observed enzyme deficiencies. For patients with degrees of mtDNA heteroplasmy below about 60% we observed at almost normal maximal rates of respiration an increase in flux control coefficients of complexes I and IV. Permeabilized skeletal-muscle fibres of patients with higher degrees of mtDNA heteroplasmy and severe enzyme deficiencies exhibited additionally decreased maximal rates of respiration. This finding indicates the presence of a 'metabolic threshold' which can be assessed by functional studies of muscle fibres providing the link to the phenotypic expression of the mtDNA mutation in skeletal muscle.

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres.

The effects of the sarcoplasmic reticulum (SR) Ca2+ pump inhibitor cyclopiazonic acid (CPA) were studied in saponin-permeabilized frog skeletal muscle fibres. Release of Ca2+ from the SR was triggered by brief (2 s) applications of 40 mM caffeine at 2-min intervals. Changes in [Ca2+] within the fibre were monitored continuously using Fura-2 fluorescence. At a bathing [Ca2+] of 100 nM, introduction of 20 microM CPA induced a slow release of Ca2+ from the SR. The following one to two caffeine-induced Ca2+ transients were markedly increased in amplitude and duration. Thereafter, the caffeine-induced Ca2+ transients decreased progressively and were barely detectable 6-7 min after introduction of CPA. However, increasing the bathing [Ca2+] or increasing the Ca2+ loading period resulted in a partial recovery of the caffeine-induced Ca2+ transients, suggesting that pump inhibition is incomplete, even in the presence of 100 microM CPA. The slow Ca2+ efflux induced by CPA was insensitive to ryanodine, but absent following abolition of SR Ca2+ pump activity by ATP withdrawal. These results suggest that the caffeine-induced Ca2+ transient reflects a balance between efflux via the SR Ca2+ channel and reuptake by the Ca pump. Ca2+ release upon addition of CPA may result from inhibition of SR Ca2+ uptake, which reveals a tonic Ca2+ efflux that is independent of the Ca2+ release channels.

Impairment of mitochondrial function in skeletal muscle of patients with amyotrophic lateral sclerosis

In skeletal muscle homogenates of 14 patients with sporadic amyotrophic lateral sclerosis, an approximately twofold lower specific activity of NADH:CoQ oxidoreductase in comparison to an age matched control group ( n=28) was detected. This finding was confirmed by a detailed analysis of mitochondrial oxidative phosphorylation in skeletal muscle using saponin-permeabilized muscle fibers. (i) A significantly lowered maximal glutamate+malate and pyruvate+malate supported respiration of saponin-permeabilized fibers was detected in the patients group. (ii) Titrations with the specific inhibitor of NADH:CoQ oxidoreductase amytal revealed a higher sensitivity of respiration to this inhibitor indicating an elevated flux control coefficient of this enzyme. (iii) Applying functional imaging of mitochondria using ratios of NAD(P)H and flavoprotein autofluorescence images of saponin-permeabilized fibers we detected the presence of partially respiratory chain inhibited mitochondria on the single fiber level. A secondary defect of mitochondrial function due to the neurogenic changes in muscle seems to be unlikely since no mitochondrial abnormalities were detectable in biopsies of patients with spinal muscular atrophy. These results support the viewpoint that an impairment of mitochondria may be of pathophysiological significance in the etiology of amyotrophic lateral sclerosis.

Oxygen dependence of flux control of cytochrome c oxidase – implications for mitochondrial diseases

The oxygen dependence of cytochrome c oxidase control on succinate oxidation was investigated in saponin-permeabilized muscle fibers and isolated mitochondria from mouse quadriceps muscle applying metabolic control analysis. For this cyanide titrations of the oxygen consumption in the presence of succinate+rotenone were performed at different oxygen concentrations in the medium. While with isolated mitochondria high flux control coefficients were detected only at oxygen concentrations close to the K M value of cytochrome c oxidase, with saponin-permeabilized fibers a significant increase of cytochrome c oxidase flux control was already observed below 130 μM oxygen. The result is in line with the high oxygen sensitivity of maximal respiration of saponin-permeabilized muscle fibers (P 50=33 μM) caused most probably by oxygen diffusion gradients through the fiber lattice. The oxygen dependence of cytochrome c oxidase flux control in muscle fibers can explain the pathological phenotype of mild cytochrome c oxidase deficiencies in mitochondrial myopathies.

Use of saponin-permeabilized muscle fibers for the diagnosis of mitochondrial diseases.

Use of saponin-permeabilized muscle fibers for the diagnosis of mitochondrial diseases.

The effects of essential oil of Mentha x villosa on skeletal muscle of the toad

The effects of the essential oil of Mentha x villosa (EOMV) was investigated in saponin-permeabilized fibres and in intact sartorius muscle of the toad. In intact muscle EOMV at concentrations from 1 to 100 μg/mL blocked the contraction induced by high (80 mM) potassium and induced potentiation of caffeine-induced muscle contraction. From 1 to 6 mg/mL EOMV elicited contraction in both depolarized (by 110 mM K+) and non-depolarized (4 mM K+) muscle fibres. This contraction was blocked by procaine. At 3 mg/mL EOMV depolarized the normal resting potential from −80.77 to −71.21 mV, but did not alter the transmembrane potential in the presence of 60 mM K+. EOMV (1 mg/mL) induced contraction of saponin-permeabilized muscle fibres with an intact sarcoplasmic reticulum (SR). These data suggest that EOMV: (i) induces muscle contraction by releasing Ca2+ from SR; (ii) impairs the excitation–contraction coupling at a step before Ca2+ release from the SR. © 1997 John Wiley & Sons, Ltd.

The effects of essential oil ofMentha x villosa on skeletal muscle of the toad

The effects of the essential oil of Mentha x villosa (EOMV) was investigated in saponin-permeabilized fibres and in intact sartorius muscle of the toad. In intact muscle EOMV at concentrations from 1 to 100 μg/mL blocked the contraction induced by high (80 mM) potassium and induced potentiation of caffeine-induced muscle contraction. From 1 to 6 mg/mL EOMV elicited contraction in both depolarized (by 110 mM K+) and non-depolarized (4 mM K+) muscle fibres. This contraction was blocked by procaine. At 3 mg/mL EOMV depolarized the normal resting potential from −80.77 to −71.21 mV, but did not alter the transmembrane potential in the presence of 60 mM K+. EOMV (1 mg/mL) induced contraction of saponin-permeabilized muscle fibres with an intact sarcoplasmic reticulum (SR). These data suggest that EOMV: (i) induces muscle contraction by releasing Ca2+ from SR; (ii) impairs the excitation–contraction coupling at a step before Ca2+ release from the SR. © 1997 John Wiley & Sons, Ltd.