Mitochondrial Creatine Kinase Activity Research Articles

Inhibition of creatine kinase activity from rat cerebral cortex by d-2-hydroxyglutaric acid in vitro

d-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as d-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11–34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.

Inhibition of the Mitochondrial Permeability Transition by Creatine Kinase Substrates: REQUIREMENT FOR MICROCOMPARTMENTATION

Mitochondria from transgenic mice, expressing enzymatically active mitochondrial creatine kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by beta-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in liver, no protective effect on pore opening by creatine and its analog was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.

L-2-Hydroxyglutaric acid inhibits mitochondrial creatine kinase activity from cerebellum of developing rats

l-2-Hydroxyglutaric acid (LGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as l-2-hydroxyglutaric aciduria (LHGA). Although this disorder is predominantly characterized by severe neurological findings and pronounced cerebellum atrophy, the neurotoxic mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of LGA, at 0.25–5mM concentrations, on total creatine kinase (tCK) activity from cerebellum, cerebral cortex, cardiac muscle and skeletal muscle homogenates of 30-day-old Wistar rats. CK activity was measured also in the cytosolic (Cy-CK) and mitochondrial (Mi-CK) fractions from cerebellum. We verified that tCK activity was significantly inhibited by LGA in the cerebellum, but not in cerebral cortex, cardiac muscle and skeletal muscle. Furthermore, CK activity from the mitochondrial fraction was inhibited by LGA, whereas that from the cytosolic fraction of cerebellum was not affected by the acid. Kinetic studies revealed that the inhibitory effect of LGA on Mi-CK was non-competitive in relation to phosphocreatine. Finally, we verified that the inhibitory effect of LGA on tCK was fully prevented by pre-incubation of the homogenates with reduced glutathione (GSH), suggesting that this inhibition is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of creatine kinase activity for energy homeostasis, our results suggest that the selective inhibition of this enzyme activity by increased levels of LGA could be possibly related to the cerebellar degeneration characteristically found in patients affected by l-2-hydroxyglutaric aciduria.

Alterations in the myocardial creatine kinase system precede the development of contractile dysfunction in β 1-adrenergic receptor transgenic mice

The β-adrenergic receptor system not only plays a central role in modulating heart rate and left-ventricular (LV) contractility, but is also involved in the development of heart failure. We have, recently, shown that heart-specific overexpression of the β 1-adrenergic receptor in transgenic mice (TG) initially leads to increased contractility, followed by LV hypertrophy and heart failure. Since one feature for all forms of heart failure are characteristic changes in myocardial energy metabolism, we asked whether alterations in energetics are detectable in these mice before signs of LV impairment are present. Myocardial energetics ( 31P NMR spectroscopy) and LV performance were measured simultaneously in isolated perfused hearts at different workloads. LV performance as well as contractile reserve was identical for hearts of 4-month-old TG and wild-type mice. The ratio of phosphocreatine to ATP (1.16 ± 0.05 vs. 1.46 ± 0.10) and total creatine content (17.6 ± 1.2 vs. 22.6 ± 0.9 mmol/l) were significantly reduced in TG. Furthermore, there was a significant decrease in creatine transporter content (–43%), mitochondrial (–44%) and total creatine kinase (CK) activity (–21%) as well as citrate synthase activity (–25%), indicating impaired oxidative energy generation in TG. In conclusion, these findings of alterations in the CK system, creatine metabolism and mitochondrial proteins in TG hearts prior to the development of LV dysfunction provide further evidence that changes in myocardial energetics play a central role in the deterioration of cardiac function after chronic β-adrenergic stimulation.

Ethylmalonic acid inhibits mitochondrial creatine kinase activity from cerebral cortex of young rats in vitro.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an inherited metabolic disorder biochemically characterized by tissue accumulation of predominantly ethylmalonic acid (EMA) and clinically by neurological dysfunction. In the present study we investigated the in vitro effects of EMA on the activity of the mitochondrial (Mi-CK) and cytosolic (Cy-CK) creatine kinase isoforms from cerebral cortex, skeletal muscle, and cardiac muscle of young rats. CK activities were measured in the mitochondrial and cytosolic fractions prepared from whole-tissue homogenates of 30-day-old Wistar rats. The acid was added to the incubation medium at concentrations ranging from 0.5 to 2.5 mM. EMA had no effect on Cy-CK activity, but significantly inhibited the activity of Mi-CK at 1.0 mM and higher concentrations in the brain. In contrast, both Mi-CK and Cy-CK from skeletal muscle and cardiac muscle were not affected by the metabolite. We also evaluated the effect of the antioxidants glutathione (GSH), ascorbic acid, and alpha-tocopherol and the nitric oxide synthase inhibitor L-NAME on the inhibitory action of EMA on cerebral cortex Mi-CK activity. We observed that the drugs did not modify Mi-CK activity per se, but GSH and ascorbic acid prevented the inhibitory effect of EMA when co-incubated with the acid. In contrast, L-NAME and alpha-tocopherol could not revert the inhibition provoked by EMA on Mi-CK activity. Considering the importance of CK for brain energy homeostasis, it is proposed that the inhibition of Mi-CK activity may be associated to the neurological symptoms characteristic of SCAD deficiency.

Inhibition of mitochondrial creatine kinase activity by D-2-hydroxyglutaric acid in cerebellum of young rats.

D-2-Hydroxyglutaric aciduria (DHGA) is a neurometabolic disorder biochemically characterized by tissue accumulation and excretion of high amounts of D-2-hydroxyglutaric acid (DGA). Although the affected patients have predominantly severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In previous studies we have demonstrated that DGA, at concentrations as low as 0.25 mM, significantly decreased creatine kinase activity and other parameters of energy metabolism in cerebral cortex of young rats. In the present study, we investigated the effect of DGA (0.25-5 mM) on total creatine kinase (tCK) activity, as well as on CK activity in cytosolic (Cy-CK) and mitochondrial (Mi-CK) preparations from cerebellum of 30-day-old Wistar rats in order to test whether the inhibitory effect of DGA on CK was tissue specific. We verified that tCK (22% inhibition) and Mi-CK (40% inhibition) activities were moderately inhibited by DGA at concentrations of 2.5 mM and higher, in contrast to Cy-CK, which was not affected by the acid. Kinetic studies revealed that the inhibitory effect of DGA was noncompetitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by preincubation of the homogenates with reduced glutathione, suggesting that the inhibition of CK activity by DGA is possibly mediated by modification of essential thiol groups of the enzyme. Our present results therefore demonstrate a relatively weak inhibitory effect of DGA on cerebellum Mi-CK activity, as compared to that provoked in cerebral cortex, and may possibly be related to the neuropathology of DHGA, characterized by cerebral cortex abnormalities.

Reduced creatine kinase activity in transgenic amyotrophic lateral sclerosis mice

Creatine (Cr), the substrate of the creatine kinase (CK) isoenzymes, was shown to be neuroprotective in several models of neurodegeneration, including amyotrophic lateral sclerosis (ALS). In order to investigate the mechanism of this beneficial effect, we determined CK activities and mitochondrial respiration rates in tissues from G93A transgenic mice, which overexpress a mutant form of human superoxide dismutase associated with familial ALS (FALS). While respiration rates of mitochondria from G93A transgenic or wild-type control mice isolated from spinal cord showed no difference, a significant and dramatic loss of CK activity could be detected in these tissues. In homogenates from spinal cord of G93A transgenic mice, CK activity decreased to 49% and in mitochondrial fractions to 67% compared to CK activities in wild-type control mice. Feeding the G93A transgenic mice with 2% Cr, the same tissues showed no statistically significant increase of CK activity compared to regular fed G93A transgenic mice. Experiments with isolated mitochondria, however, showed that Cr and adenosine triphosphate (ATP) protected mitochondrial CK activity against peroxynitrite-induced inactivation, which may play a role in tissue damage in neurodegeneration. Our data provide evidence for oxidative damage to the CK system in ALS, which may contribute to impaired energy metabolism and neurodegeneration.

Response of mitochondrial function to hypothyroidism in normal and regenerated rat skeletal muscle.

Although thyroid hormones induce a well known decrease in muscle oxidative capacity, nothing is known concerning their effects on mitochondrial function and regulation in situ. Similarly, the influence of regeneration process is not completely understood. We investigated the effects of hypothyroidism on mitochondrial function in fast gastrocnemius (GS) and slow soleus (SOL) muscles either intact or having undergone a cycle of degeneration/regeneration (Rg SOL) following a local injection of myotoxin. Thyroid hormone deficiency was induced by thyroidectomy and propylthiouracyl via drinking water. Respiration was measured in muscle fibres permeabilised by saponin in order to assess the oxidative capacity of the muscles and the regulation of mitochondria in situ. Oxidative capacities were 8.9 in SOL, 8.5 in Rg SOL and 5.9 micromol O2/min/g dry weight in GS and decreased by 52, 42 and 39% respectively (P < 0.001) in hypothyroid rats. Moreover, the Km of mitochondrial respiration for the phosphate acceptor ADP exhibited a two-fold decrease in Rg SOL and intact SOL by hypothyroidism (P < 0.01), while mitochondrial creatine kinase activity and sensitivity of mitochondrial respiration to creatine were not altered. The results of this study demonstrate that hypothyroidism markedly altered the sensitivity of mitochondrial respiration to ADP but not to creatine in SOL muscles, suggesting that mitochondrial regulation could be partially controlled by thyroid hormones. On the other hand, mitochondrial function completely recovered following regeneration/degeneration, suggesting that thyroid hormones are not involved in the regeneration process per se.

Heart failure affects mitochondrial but not myofibrillar intrinsic properties of skeletal muscle.

Congestive heart failure (CHF) induces abnormalities in skeletal muscle that are thought to in part explain exercise intolerance. The aim of the present study was to determine whether these changes actually result in contractile or metabolic functional alterations and whether they are muscle type specific. With a rat model of CHF (induced by aortic banding), we studied mitochondrial function, mechanical properties, and creatine kinase (CK) compartmentation in situ in permeabilized fibers from soleus (SOL), an oxidative slow-twitch muscle, and white gastrocnemius (GAS), a glycolytic fast-twitch muscle. Animals were studied 7 months after surgery, and CHF was documented on the basis of anatomic data. Alterations in skeletal muscle phenotype were documented with an increased proportion of fast-type fiber and fast myosin heavy chain, decreased capillary-to-fiber ratio, and decreased citrate synthase activity. Despite a slow-to-fast phenotype transition in SOL, no change was observed in contractile capacity or calcium sensitivity. However, muscles from CHF rats exhibited a dramatic decrease in oxidative capacities (oxygen consumption per gram of fiber dry weight) of 35% for SOL and 45% for GAS (P:<0.001). Moreover, the regulation of respiration with ADP and mitochondrial CK and adenylate kinase was impaired in CHF SOL. Mitochondrial CK activity and content (Western blots) were dramatically decreased in both muscles. CHF results in alterations in both mitochondrial function and phosphotransfer systems but unchanged myofibrillar function in skeletal muscles, which suggests a myopathy of metabolic origin in CHF.

Calcineurin Co-regulates Contractile and Metabolic Components of Slow Muscle Phenotype

Activation of the transcription factor nuclear factor of activated T cells by the calcium-sensitive serine/threonine phosphatase calcineurin has been proposed as one of the molecular mechanisms by which motor nerve activity establishes the slow muscle phenotype. To investigate whether the calcineurin pathway can regulate the large spectrum of slow muscle characteristics in vivo, we treated rats for three weeks with cyclosporin A (an inhibitor of calcineurin). In soleus (slow muscle), but not in plantaris (fast muscle), the proportion of slow myosin heavy chain (MHC-1) and slow sarcoplasmic reticulum ATPase (SERCA2a) was decreased, whereas that of fast MHC (MHC-2A) and fast SERCA1 increased, indicating a slow to fast contractile phenotype transition. Cytosolic isoforms of creatine kinase and lactate dehydrogenase (most abundant in fast fibers), as well as mitochondrial creatine kinase and citrate synthase activities (elevated in fast/oxidative fibers) were dose dependently increased by cyclosporin A treatment in soleus muscle, with no change in plantaris. Calcineurin catalytic subunit was more abundant in soleus muscle fibers compared with plantaris. Taken together these results suggest that the calcineurin pathway co-regulates a set of multigenic protein families involved in the transition between slow oxidative (type I) to fast oxidative (type IIa) phenotype in soleus muscle.

Effects of ACE inhibition and β-receptor blockade on energy metabolism in rats postmyocardial infarction

Chronic treatment with beta-receptor blockers or angiotensin-converting enzyme (ACE) inhibitors in heart failure can reduce mortality and improve left ventricular function, but the mechanisms involved in their beneficial action remain to be fully defined. Our hypothesis was that these agents prevent the derangement of cardiac energy metabolism. Rats were subjected to myocardial infarction (MI) or sham operation. Thereafter, animals were treated with bisoprolol, captopril, or remained untreated. Two months later, cardiac function was measured in the isolated heart by a left ventricular balloon (pressure-volume curves), and energy metabolism of residual intact myocardium was analyzed in terms of total and isoenzyme creatine kinase (CK) activity, steady-state levels (ATP, phosphocreatine), and turnover rates (CK reaction velocity) of high-energy phosphates (31P nuclear magnetic resonance) and total creatine content (HPLC). Bisoprolol and partially captopril prevented post-MI hypertrophy and partially prevented left ventricular contractile dysfunction. Residual intact failing myocardium in untreated, infarcted hearts showed a 25% decrease of the total, a 26% decrease of MM-, and a 37% decrease of the mitochondrial CK activity. Total creatine was reduced by 15%, phosphocreatine by 21%, and CK reaction velocity by 41%. Treatment with bisoprolol or captopril largely prevented all of these changes in infarcted hearts. Thus the favorable functional effects of beta-receptor blockers and ACE inhibitors post-MI are accompanied by substantial beneficial effects on cardiac energy metabolism.

Free radical-induced inactivation of creatine kinase: influence on the octameric and dimeric states of the mitochondrial enzyme (Mib-CK)

Free radicals of X-ray-induced water radiolysis, either directly or indirectly via their reaction products, reduce the activity of both dimeric cytoplasmic muscle-type creatine kinase (MM-CK) and octameric mitochondrial creatine kinase (Mi-CK) to virtually zero. Similarly values of the characteristic D(37)-dose of enzyme inactivation (dose required to reduce enzyme activity to 37%) were found for the two isoenzymes of CK under identical conditions. Octamer stability was not significantly affected within the dose range considered. However, both the dissociation of octamers into dimers by a transition-state analogue complex (TSAC), and the reassociation of the dimers into octamers, showed dose-dependent reduction. Binding of the TSAC to the active centre was found to protect the enzyme against inactivation by free radicals. No protection was observed for the radiation-induced decrease of the endogenous tryptophan fluorescence. The experimental results are in line with the following interpretation: (i) the reduction of Mi(b)-CK dimer association is due to free radical-induced modification of Trp-264, situated at the dimer/dimer interface; (ii) the active-site Trp-223 is not a prime target for free radicals and is not involved in the inactivation of the enzyme; (iii) the inhibition of TSAC-induced dissociation of Mi(b)-CK, like enzyme inactivation, is primarily due to a modification of the active-site Cys-278.

Subcellular creatine kinase alterations. Implications in heart failure.

We have tested the hypothesis that decreased functioning of creatine kinase (CK) at sites of energy production and utilization may contribute to alterations in energy fluxes and calcium homeostasis in congestive heart failure (CHF). Heart failure was induced by aortic banding in 3-week-old rats. Myofilaments, sarcoplasmic reticulum (SR), mitochondrial functions, and CK compartmentation were studied in situ using selective membrane permeabilization of left ventricular fibers with detergents (saponin for mitochondria and SR and Triton X-100 for myofibrils). Seven months after surgery, animals were in CHF. A decrease in total CK activity could be accounted for by a 4-fold decrease in activity and content (Western blots) of mitochondrial CK and a 30% decrease in M isoform of CK (MM-CK) activity. In myofibrils, maximal force, crossbridge kinetics, and alpha-myosin heavy-chain expression decreased, whereas calcium sensitivity of tension development remained unaltered. Myofibrillar CK efficacy was unchanged. Calcium uptake capacities of SR were estimated from the surface of caffeine-induced tension transient (SCa) after loading with different substrates. In CHF, SCa decreased by 23%, and phosphocreatine was 2 times less efficient in enhancing calcium uptake. Oxidative capacities of the failing myocardium measured as oxygen consumption per gram of fiber dry weight decreased by 28%. Moreover, the control of respiration by creatine, ADP, and AMP was severely impaired. Our observations provide evidence that alterations in CK compartmentation may contribute to alterations of energy fluxes and calcium homeostasis in CHF.

Octamer-dimer transitions of mitochondrial creatine kinase in heart disease.

Mitochondrial creatine kinase (Mi-CK) occurs in dimeric and octameric forms, both in vitro and in vivo. The Mi-CK octamer, however, is the predominant form in vivo and is important for various functions of the protein. In the present study we show for the first time a significant decrease of the octamer/dimer ratio in vivo, related to ischemia-induced damage, and a similar decrease of octamer stability in vitro, induced by peroxynitrite (PN) radicals. We used animal models to induce ischemia in two different ways: acute ischemia in intact heart (Langendorff perfusion) and chronic ischemia in vivo (LAD-infarction). In both models, impairment of heart function and mitochondrial energy metabolism was associated with a significant decrease of Mi-CK octamer/dimer ratios and of Mi-CK activities. These findings, together with recent data showing that the formation of PN is induced in ischemia and that Mi-CK is a prime target of peroxynitrite (PN)-induced damage, suggest that oxygen radicals generated during ischemia and reoxygenation could be an important factor for the decreased octamer stability. To test this hypothesis, we studied the effect of PN on pure Mi-CK in vitro, both on dissociation of octamers and reassociation of dimers. At 1 m m PN 66% of Mi-CK octamers dissociated into dimers, whereas octamerization of PN-modified dimers was already completely inhibited at 100 microm PN. Our data indicate that PN-induced damage could be responsible for the octamer-dimer transition of Mi-CK in ischemia. A loss of Mi-CK octamers would impair the channeling of high energy phosphate out of mitochondria and hence heart function in general.

Nitric oxide inhibits cardiac energy production via inhibition of mitochondrial creatine kinase

Nitric oxide biosynthesis in cardiac muscle leads to a decreased oxygen consumption and lower ATP synthesis. It is suggested that this effect of nitric oxide is mainly due to the inhibition of the mitochondrial respiratory chain enzyme, cytochrome c oxidase. However, this work demonstrates that nitric oxide is able to inhibit soluble mitochondrial creatine kinase (CK), mitochondrial CK bound in purified mitochondria, CK in situ in skinned fibres as well as the functional activity of mitochondrial CK in situ in skinned fibres. Since mitochondrial isoenzyme is functionally coupled to oxidative phosphorylation, its inhibition also leads to decreased sensitivity of mitochondrial respiration to ADP and thus decreases ATP synthesis and oxygen consumption under physiological ADP concentrations.

Characterization of isoforms of human mitochondrial creatine kinase by isoelectric focusing

High enzyme activity of mitochondrial creatine kinase (creatine- N-phosphotransferase, mCK, EC 2.7.3.2) was detected in serum from a patient with advanced carcinoma of the rectum and its isoforms were characterized by means of isoelectric focusing (IEF). Three forms of mCK, membrane-bound (p I 6.9–7.0), octameric (p I 7.0–7.9) and dimeric (p I 6.7, 6.8, 6.9 and 7.0), were detected in the fresh serum. These three forms of mCK were converted to five dimeric isoforms, and these were characterized as one reduced form (p I 7.0) and four oxidized (p I 6.6, 6.7, 6.8 and 6.9) forms upon treatment with urea, hydrogen peroxide or 2-mercaptoethanol (2-ME). The C-terminal of the mCKs was concluded to be a lysine residue because the mCKs treated with carboxypeptidase B migrated to positions closer to the anode than did those not treated with carboxypeptidase B. Therefore, four bands were concluded to represent one reduced-delysined isoform (p I 6.4) and three oxidized-delysined isoforms (p I 6.1, 6.2 and 6.3). The broad octameric mCK band disappeared and a narrow band focused at p I 6.8–6.9 appeared upon probable delysination of the mCKs. Thus, the number of lysine residues at the C-terminal of the octamer was concluded to be variable due to variable catalysis by carboxypeptidase N in the plasma. mCKs seemed to be inactivated during conversion from a membrane-bound form to dimeric oxidized-delysined forms via the octameric, dimeric reduced and oxidized forms.

Mitochondrial creatine kinase functional development in post-natal rat skeletal muscle. A combined polarographic/31P NMR study.

Mitochondrial creatine kinase (Mi-CK) function in viable mitochondria from developing rat skeletal muscle was assessed both by polarographic measurements of creatine-induced respiration and 31P NMR spectroscopy measurements of phosphocreatine (PCr) synthesis. Creatine-induced respiration was observed in very young rats and increased by 50% to 35 days of age. PCr synthesis was present in 7 day old animals and increased by 300% reaching levels measured in 35 day and adult muscle. Unlike reports showing Mi-CK enzymatic activities but no mitochondrial function in several situations, a concomitant progression of enzymatic activity and mitochondrial function was evidenced during the developmental stages of skeletal muscle Mi-CK in altricious animals. These results correlated with the progressive pattern of muscle differentiation during development of motricity in such animals. The observation that Mi-CK is functional in skeletal muscle mitochondria very early after birth, strongly favors the notion that adaptations in skeletal muscle of Mi-CK knock-out mice occur early.

Muscle Unloading Induces Slow to Fast Transitions in Myofibrillar but not Mitochondrial Properties. Relevance to Skeletal Muscle Abnormalities in Heart Failure

Muscle deconditioning is a common observation in patients with congestive heart failure (CHF), chronic obstructive pulmonary disease, neuromuscular diseases or prolonged bed rest. To gain further insight into metabolic and mechanical properties of deconditioned slow-twitch (soleus) or fast-twitch (EDL) skeletal muscles, we induced experimental muscle deconditioning by hindlimb suspension (HS) in rats for 3 weeks. Cardiac muscle was also studied. Besides profound muscle atrophy, increased proportion of fast type II fibers as well as fast myosin isoenzymes, we found decreased calcium sensitivity of Triton X-100 skinned fiber bundles of soleus muscle directed towards the fast muscle phenotype. Glycolytic enzymes such as hexokinase and pyruvate kinase were increased, and the LDH isoenzyme pattern was clearly shifted from an oxidative to an anaerobic profile. Creatine kinase (CK) and myokinase activities were increased in HS soleus towards EDL values. Moreover, the M-CK mRNA level was greatly increased in soleus, with no change in EDL. However, oxygen consumption rate assessedin situin saponin skinned fibers (12.5±0.8 in C and 15.1±0.9μmol O2/min/g dw in HS soleus compared to 7.3±1.3μmol O2/min/g dw in control EDL), as well as mitochondrial CK (mi-CK) and citrate synthase activities, were preserved in HS soleus. Following deconditioning no change in Kmfor ADP of mitochondrial respiration, either in the absence (511±92 in C and 511±111μmin HS soleus compared to 9±4μMin control EDL) or presence of creatine (88±10 in C and 95±16μmin HS soleus compared to 32±9μMin control EDL), was found. The results show that muscle deconditioning induces a biochemical and functional slow to fast phenotype transition in myofibrillar and cytosolic compartments of postural muscle, but not in the mitochondrial compartment, suggesting that these compartments are differently regulated under conditions of decreased activity.

Effects of γ-glutamylcysteine ethyl ester on heart mitochondrial creatine kinase activity: involvement of sulfhydryl groups

To study the mechanism of the protective effect of γ-glutamylcysteine ethyl ester, mitochondrial creatine kinase activity of rat heart was measured. γ-Glutamylcysteine ethyl ester had a protective effect against the depression of creatine kinase activity induced by xanthine+xanthine oxidase or hydrogen peroxide. γ-Glutamylcysteine ethyl ester also prevented the depression of creatine kinase activity induced by N-ethylmaleimide. It is suggested that the protective effect of γ-glutamylcysteine ethyl ester is related to oxygen free radicals or to reduction of the sulfhydryl groups of the enzyme which were previously oxidized by oxygen free radicals.

Role of nitric oxide in skeletal muscle: synthesis, distribution and functional importance.

Over the last two decades, nitric oxide (NO) has been established as a novel mediator of biological processes, ranging from vascular control to long-term memory, from tissue inflammation to penile erection. This paper reviews recent research which shows that NO and its derivatives also are synthesized within skeletal muscle and that NO derivatives influence various aspects of muscle function. Individual muscle fibres express one or both of the constitutive NO synthase (NOS) isoforms. Type I (neuronal) NOS is localized to the sarcolemma of fast fibres; type III (endothelial) NOS is associated with mitochondria. Isolated skeletal muscle produces NO at low rates under resting conditions and at higher rates during repetitive contraction. NO appears to mediate cell-cell interactions in muscle, including vasodilation and inhibition of leucocyte adhesion. NO also acts directly on muscle fibres to alter cell function. Muscle metabolism appears to be NO-sensitive at several sites, including glucose uptake, glycolysis, mitochondrial oxygen consumption and creatine kinase activity. NO also modulates muscle contraction, inhibiting force output by altering excitation-contraction coupling. The mechanisms of NO action are likely to include direct effects on redox-sensitive regulatory proteins, interaction with endogenous reactive oxygen species, and activation of second messengers such as cyclic guanosine monophosphate (cGMP). In conclusion, research published over the past few years makes it clear that skeletal muscle produces NO and that endogenous NO modulates muscle function. Much remains to be learned, however, about the physiological importance of NO actions and about their underlying mechanisms.