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Deficiency of S6 kinase (S6K) extends the lifespan of multiple species, but the underlying mechanisms are unclear. To discover potential effectors of S6K-mediated longevity, we performed a proteomics analysis of long-lived rsks-1/S6K C. elegans mutants compared to wild-type animals. We identified the arginine kinase ARGK-1 as the most significantly enriched protein in rsks-1/S6K mutants. ARGK-1 is an ortholog of mammalian creatine kinase, which maintains cellular ATP levels. We found that argk-1 is possibly a selective effector of rsks-1/S6K-mediated longevity and that overexpression of ARGK-1 extends C. elegans lifespan, in part by activating the energy sensor AAK-2/AMPK. argk-1 is also required for the reduced body size and increased stress resistance observed in rsks-1/S6K mutants. Finally, creatine kinase levels are increased in the brains of S6K1 knockout mice. Our study identifies ARGK-1 as a longevity effector in C. elegans with reduced RSKS-1/S6K levels., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin-related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca(2+) ] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction. Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca(2+) ] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca(2+) handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca(2+) ] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca(2+) in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca(2+) both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca(2+) ] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca(2+) overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca(2+) ]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca(2+) , and thereby increase myocardial stiffness., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Statin treatment in association with physical exercise practice can substantially reduce cardiovascular mortality risk of dyslipidemic individuals, but this practice is associated with myopathic event exacerbation. This study aimed to present the most recent results of specific literature about the effects of statins and its association with physical exercise on skeletal musculature. Thus, a literature review was performed using PubMed and SciELO databases, through the combination of the keywords "statin" AND "exercise" AND "muscle", restricting the selection to original studies published between January 1990 and November 2013. Sixteen studies evaluating the effects of statins in association with acute or chronic exercises on skeletal muscle were analyzed. Study results indicate that athletes using statins can experience deleterious effects on skeletal muscle, as the exacerbation of skeletal muscle injuries are more frequent with intense training or acute eccentric and strenuous exercises. Moderate physical training, in turn, when associated to statins does not increase creatine kinase levels or pain reports, but improves muscle and metabolic functions as a consequence of training. Therefore, it is suggested that dyslipidemic patients undergoing statin treatment should be exposed to moderate aerobic training in combination to resistance exercises three times a week, and the provision of physical training prior to drug administration is desirable, whenever possible.

The permeability transition pore (PT-pore) mediates cell death through the dissipation of the mitochondrial membrane potential (ΔΨm). Because the exact composition of the PT-pore is controversial, it is crucial to investigate the actual molecular constituents and regulators of this complex. We found that mitochondrial creatine kinase-1 (CKMT1) is a universal and functionally necessary gatekeeper of the PT-pore, as its depletion induces mitochondrial depolarization and apoptotic cell death. This can be inhibited efficiently by bongkrekic acid, a compound that is widely used to inhibit the PT-pore. However, when the 'classical' PT-pore subunits cyclophilin D and VDAC1 are pharmacologically inhibited or their expression levels reduced, mitochondrial depolarization by CKMT1 depletion remains unaffected. At later stages of drug-induced apoptosis, CKMT1 levels are reduced, suggesting that CKMT1 downregulation acts to reinforce the commitment of cells to apoptosis. A novel high-molecular-mass CKMT1 complex that is distinct from the known CKMT1 octamer disintegrates upon treatment with cytotoxic drugs, concomitant with mitochondrial depolarization. Our study provides evidence that CKMT1 is a key regulator of the PT-pore through a complex that is distinct from the classical PT-pore.

The purpose of this study was to analyse the impact of an intermittent test reproducing the soccer running activity profile on physical performance, subjective ratings and biochemical parameters throughout 72 h recovery. 8 professional soccer players performed the intermittent test on a non-motorised treadmill and data was collected before, immediately after, 24, 48 and 72 h after the test. Squat jump (SJ), countermovement jump (CMJ), peak isometric force (IFpeak), 6-s sprint, repeated sprints test (RS), perceptual ratings (fatigue, muscle soreness, stress), creatine kinase ([CK]) and uric acid ([UA]) were analyzed. After the test, a mean reduction in countermovement jump performance of -8.2% (CI: -12.9 to -3.4, p<0.01) was observed, while perceived fatigue (+2.1±1.7 a.u.; p<0.05), perceived muscle soreness (+1.8±1.5 a.u.; p<0.05), perceived stress (+1.6±1.5 a.u.; p<0.05), creatine kinase (+171±77 IU x l(-1); p<0.01) and uric acid (+168±89 Umol x l(-1); p<0.01) concentrations were significantly increased relative to baseline. No significant effect was found for SJ, IFpeak, 6-s sprint, RS immediately after and throughout the 72 h following the test. In conclusion, soccer running performance does not appear to be the main cause of post soccer match-induced fatigue. Physical data provided by video match analysis systems is insufficient to accurately estimate the level of match fatigue., (© Georg Thieme Verlag KG Stuttgart · New York.)

Purpose: This study aimed to determine whether competitive intermittent exercise in the heat affects recovery, aggravates markers of muscle fiber damage, and delays the recovery of performance and muscle glycogen stores., Methods: Plasma creatine kinase, serum myoglobin, muscle glycogen, and performance parameters (sprint, endurance, and neuromuscular testing) were evaluated in 17 semiprofessional soccer players before, immediately after, and during 48 h of recovery from a match played in 43°C (HOT) and compared with a control match (21°C with similar turf and setup)., Results: Muscle temperature was ∼1°C higher (P < 0.001) after the game in HOT compared with control and reached individual values between 39.9°C and 41.1°C. Serum myoglobin levels increased by more than threefold after the matches (P < 0.01), but values were not different in HOT compared with control, and they were similar to baseline values after 24 h of recovery. Creatine kinase was significantly elevated both immediately and 24 h after the matches, but the response after HOT was reduced compared with control. Muscle glycogen responses were similar across trials and remained depressed for more than 48 h after both matches. Sprint performance and voluntary muscle activation were impaired to a similar extent after the matches (sprint by ∼2% and voluntary activation by ∼1.5%; P < 0.05). Both of these performance parameters as well as intermittent endurance capacity (estimated by a Yo-Yo IR1 test) were fully recovered 48 h after both matches., Conclusion: Environmental heat stress does not aggravate the recovery response from competitive intermittent exercise associated with elevated muscle temperatures and markers of muscle damage, delayed resynthesis of muscle glycogen, and impaired postmatch performance.

Introduction: Despite the prevalence, associated comorbidities, and functional consequences of bipolar depression (BPD), underlying disease mechanisms remain unclear. Published studies of individuals with bipolar disorder implicate abnormalities in cellular energy metabolism. This study tests the hypotheses that the forward rate constant (k(for)) of creatine kinase (CK) is altered in older adults with BPD and that CoEnzyme Q10 (CoQ10), known to have properties that enhance mitochondrial function, increases k(for) in elderly individuals with BPD treated with CoQ10 compared with untreated age- and sex-matched controls., Methods: Ten older adults (ages 55 and above) with Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition [DSM IV]) bipolar disorder, current episode depressed and 8 older controls underwent two 4 Tesla (31)Phosphorus magnetic resonance spectroscopy ((31)PMRS) scans 8 weeks apart using a magnetization transfer (MT) acquisition scheme to calculate k(for). The BPD group was treated with open-label CoEnzyme Q10 400 mg/d titrated up by 400 mg/d every 2 weeks to a maximum of 1200 mg/d. The Montgomery Asberg Depression Rating Scale (MADRS) was used to measure depression symptom severity. Baseline k(for) and changes in k(for) were compared between individuals with BPD and controls, not receiving CoQ. Clinical ratings were compared across time and associated with k(for) changes using repeated measures linear regression., Results: The k(for) of CK was nonsignificantly lower for BPD than healthy controls at baseline (BPD mean (standard deviation [SD]) = 0.19 (0.02), control mean (SD) = 0.20 (0.02), Wilcoxon rank sum exact P = .40). The k(for) for both CoQ10-treated BPD and controls increased after 8 weeks (mean increase (SD) = 0.03 (0.04), Wilcoxon signed rank exact P = .01), with no significant difference in 8-week changes between groups (BPD mean change (SD) = 0.03 (0.03), control mean change (SD) = 0.03 (0.05), Wilcoxon rank sum exact P = .91). In an exploratory analysis, depression severity decreased with CoQ10 treatment in the group with BPD (F (3,7) = 4.87, P = .04) with significant reductions in the MADRS at weeks 2 (t (9) = -2.40, P = .04) and 4 (t (9) = -3.80, P = .004)., Conclusions: This study employing the novel MRS technique of MT did not demonstrate significance between group differences in the k(for) of CK but did observe a trend that would require confirmation in a larger study. An exploratory analysis suggested a reduction in depression symptom severity during treatment with high-dose CoEnzyme Q10 for older adults with BPD. Further studies exploring alterations of high-energy phosphate metabolites in geriatric BPD and efficacy studies of CoQ10 in a randomized controlled trial are both warranted.

The aim was to compare the effect of a simulated team sport activity circuit (reflective of the activity demands of Australian football) either with or without body 'contact' on muscle soreness, damage, and performance when the circuit was repeated 48 h later. Eleven male, team-sport athletes completed a 'non-contact' (NCON) and a 'contact' (CON) version of the team sport activity circuit in a crossover design with at least 1 week between trials. The effect of CON and NCON on repeated 15m sprint and vertical jump performance was assessed by completing the same version of the circuit 48 h after the initial trial. The effect on perceived soreness and blood markers of muscle damage and inflammation was also determined. Subsequent performance was affected to a greater extent by CON, with both best and mean sprint times significantly slower 48h following CON (p<0.05), while performance was maintained after NCON. Best and mean vertical jump performance was significantly impaired following CON (p<0.05), while only best vertical jump was affected by NCON (p<0.05). Perceived soreness and pressure sensitivity were elevated following both NCON and CON (p<0.001); however, the increase in soreness was greater with CON (p=0.012). Both CON and NCON resulted in elevated serum creatine kinase, myoglobin and lactate dehydrogenase, while c-reactive protein increased following CON but not NCON. In conclusion, Greater perceived soreness and decrements in performance of the simulated team sport activity circuit when repeated 48 h later were observed following CON., (Copyright © 2011 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.)

The purpose of the present study was to (a) examine player-movement patterns to determine total distance covered during competitive Rugby League match play using global positioning systems (GPSs) and (b) examine pre, during, and postmatch creatine kinase (CK) and endocrine responses to competitive Rugby League match play. Seventeen elite rugby league players were monitored for a single game. Player movement patterns were recorded using portable GPS units (SPI-Pro, GPSports, Canberra, Australia). Saliva and blood samples were collected 24 hours prematch, 30 minutes prematch, 30 minutes postmatch, and then at 24-hour intervals for a period of 5 days postmatch to determine plasma CK and salivary testosterone, cortisol, and testosterone:cortisol ratio (T:C). The change in the dependent variables at each sample collection time was compared to 24-hour prematch measures. Backs and forwards traveled distances 5,747 ± 1,095 and 4,774 ± 1,186 m, respectively, throughout the match. Cortisol and CK increased significantly (p < 0.05) from 30 minutes prematch to 30 minutes postmatch. Creatine kinase increased significantly (p < 0.05) postmatch, with peak CK concentration measured 24 hours postmatch (889.25 ± 238.27 U·L). Cortisol displayed a clear pattern of response with significant (p < 0.05) elevations up to 24 hours postmatch, compared with 24 hours prematch. The GPS was able to successfully provide data on player-movement patterns during competitive rugby league match play. The CK and endocrine profile identified acute muscle damage and a catabolic state associated with Rugby League match play. A return to normal T:C within 48 hours postmatch indicates that a minimum period of 48 hours is required for endocrine homeostasis postcompetition. Creatine kinase remained elevated despite 120 hours of recovery postmatch identifying that a prolonged period of at least 5 days modified activity is required to achieve full recovery after muscle damage during competitive Rugby League match play.

The aim was to investigate the changes in lipid peroxidation, antioxidant enzyme activities, and muscle damage in the same and different exercise intensities during walking and running. Fourteen healthy males participated in this study. The subjects' individual preferred walk-to-run transition speeds (WRTS) were determined. Each subject covered a 1.5-mile distance for 4 exercise tests; walking (WRTS-W) and running (WRTS-R) tests at WRTS, 2 kmxh-1 slower walking than WRTS (WRTS-2) and 2 kmxh-1 faster running than WRTS (WRTS+2). Blood samples were taken pre, immediately, and 30 minutes post each test. The changes in (MDA) and glutathione (GSH) levels and superoxide dismutase (SOD), catalase (CAT), and creatine kinase activities were measured. Oxygen uptake, carbon dioxide output, oxygen uptake per kilogram of body weight, and heart rate during exercises were significantly higher in both the WRTS-W and the WRTS+2 exercises compared with the WRTS-2 and WRTS-R. Oxygen consumption and energy expenditure were higher in walking than in the running exercise at the preferred WRTS and only WRTS-W exercise significantly increased MDA levels. Catalase activities were increased by WRTS-W, WRTS-R, and WRTS+2 exercises. Changes in SOD and CAT activities were not different between walking and running exercises at the preferred WRTS. Total plasma GSH increased in response to WRTS-W exercise, which could be associated with an increase in MDA. Also, total GSH levels 30 minutes postexercise were significantly lower than postexercise in WRTS-2, WRTS-W, and WRTS+2 exercises. Our results indicate that walking and running exercises at the preferred WRTS have different oxidative stress and antioxidant responses.

Physical activity of significant intensity and duration may cause varying degrees of skeletal muscle damage, but it is unclear whether mode of rehydration will attenuate muscle tissue disruption caused by exercise in the heat. To examine the effects of the mode of rehydration on markers of muscle damage (myoglobin and creatine kinase [CK]), 11 healthy active men (age = 23 +/- 4 years, body mass = 80.9 +/- 3.9 kg, height = 180.5 +/- 5.4 cm) completed 4 experimental trials consisting of an exercise dehydration protocol (to -4% of baseline body mass), followed by a rehydration period (oral, intravenous [IV], oral and IV combined, and ad libitum), and finishing with an intense exercise challenge that included treadmill running and sprinting and a box lifting protocol. During rehydration, subjects returned to -2% of baseline body mass unless completing the ad libitum trial during which they consumed fluids as thirst dictated. Myoglobin (Mb) and CK were measured during euhydrated rest. Post-exercise blood was drawn at 1 and 24 hours post exercise challenge for Mb and CK, respectively. Urine was collected during euhydrated rest and 1-hour post exercise challenge for measurement of Mb clearance. Mb concentrations increased significantly from pre (1.06 +/- 0.20, 0.88 +/- 0.07, 1.15 +/- 0.25 and 0.92 +/- 0.06 nmol.L) to post (1.52 +/- 0.28, 1.44 +/- 0.11, 1.71 +/- 0.45 and 1.58 +/- 0.39) for IV, oral, oral and IV combined, and ad libitum, respectively, but were not significantly different among trials. Serum CK concentrations remained within the normal physiological range for all trials. Thus, despite previous research that clearly indicates the benefit of ingesting fluids during exercise to attenuate muscle damage, there were no significant differences between the modes of rehydration on circulating Mb and CK.

Creatine kinase catalyzes the reversible transfer of the N-phosphoryl group from phosphocreatine to ADP to generate ATP and plays a key role in highly energy-demanding processes such as muscle contraction and flagellar motility; however, its role in signal transduction (which frequently involves ATP-consuming phosphorylation) and consequent cell-fate decisions remains largely unknown. Here we report that creatine kinase B was significantly up-regulated during the differentiation of double-positive thymocytes into single-positive thymocytes. Ectopic expression of creatine kinase B led to increased ATP level and enhanced phosphorylation of the TCR signaling proteins. Consequentially, transgenic expression of creatine kinase B promoted the expression of Nur77 and Bim proteins and the cell death of TCR signaled thymocyte. In addition, the activation, proliferation and cytokine secretion of T cells were also enhanced by the expression of creatine kinase B transgene. In contrast, treatment of T cells with specific creatine kinase inhibitor or creatine kinase B shRNA resulted in severely impaired T cell activation. Taken together, our results indicate that creatine kinase B plays an unexpected role in modulating TCR-mediated signaling and critically regulates thymocyte selection and T cell activation.

Creatine kinase (CK) is a phosphotransfer kinase that catalyzes the reversible transfer of a phosphate moiety between ADP and creatine and that is highly expressed in skeletal muscle. In fast glycolytic skeletal muscle, deletion of the cytosolic M isoform of CK in mice (M-CK-/-) leads to a massive increase in the oxidative capacity and of mitochondrial volume. This study was aimed at investigating the transcriptional pathways leading to mitochondrial biogenesis in response to CK deficiency. Wild type and M-CK-/- mice of eleven months of age were used for this study. Gastrocnemius muscles of M-CK-/- mice exhibited a dramatic increase in citrate synthase (+120%) and cytochrome oxidase (COX, +250%) activity, and in mitochondrial DNA (+60%), showing a clear activation of mitochondrial biogenesis. Similarly, mRNA expression of the COXI (mitochondria-encoded) and COXIV (nuclear-encoded) subunits were increased by +103 and +94% respectively. This was accompanied by an increase in the expression of the nuclear respiratory factor (NRF2alpha) and the mitochondrial transcription factor (mtTFA). Expression of the co-activator PGC-1alpha, a master gene in mitochondrial biogenesis was not significantly increased while that of PGC-1beta and PRC, two members of the same family, was moderately increased (+45% and +55% respectively). While the expression of the modulatory calcineurin-interacting protein 1 (MCIP1) was dramatically decreased (-68%) suggesting inactivation of the calcineurin pathway, the metabolic sensor AMPK was activated (+86%) in M-CK-/- mice. These results evidence that mitochondrial biogenesis in response to a metabolic challenge exhibits a unique pattern of regulation, involving activation of the AMPK pathway.

Elevated creatine kinase (CK) in the circulation was generally regarded to be a passive release from muscle damage. We utilized proteomic methodologies to characterize amphioxus humoral fluid APPs in response to caudal trauma, and found several spots of CK alterations with up-regulation and pI shift. Its amount and enzyme activity showed a dynamic pattern of APP in humoral fluid accompanied with a reduction in enzyme activity of muscle, whereas there was no significant difference in CK amount of muscle and the other tissues and in CK enzyme activity of the other tissues between different time points of sample collection following caudal trauma. In addition, CK phosphorylation regulation during injury was not achieved by monoclonal antibodies separately against phosphothreonine, phosphotyrosine, and phosphoserine. These results suggested that the CK elevation of humoral fluid might be from muscle, being an active response to caudal trauma rather than a passive release from muscle damage. Therefore, CK ability in response to caudal trauma should be highly concerned.

Objective: To investigate the association between muscle-specific creatine kinase (CKMM) gene polymorphism and the effects of endurance training on running economy., Methods: 102 biologically unrelated male volunteers from northern China performed a 5000-m running programme, with an intensity of 95-105% ventilatory threshold. The protocol was undertaken three times per week and lasted for 18 weeks. Running economy indexes were determined by making the participants run on a treadmill before and after the protocol, and the A/G polymorphism in the 3' untranslated region of CKMM was detected by polymerase chain reaction-restricted fragment length polymorphism (NcoI restriction enzyme)., Results: Three expected genotypes for CKMM-NcoI (AA, AG and GG) were observed in the participants. After training, all running economy indexes declined markedly. Change in steady-state consumption of oxygen, change in steady-state consumption of oxygen by mean body weight, change in steady-state consumption of oxygen by mean lean body weight and change in ventilatory volume in AG groups were larger than those in AA and GG groups., Conclusions: The findings indicate that the CKMM gene polymorphism may contribute to individual running economy responses to endurance training.

The production of ATP in mitochondria depends on the magnesium nuclear spin and magnetic moment of a Mg2+ ion in creatine kinase and ATPase. This suggests that enzymatic synthesis of ATP is an ion-radical process and thus depends on the external magnetic field (magnetobiology originates from this fact) and microwave fields, which control the spin states of ion-radical pairs and affect the ATP synthesis. The chemical mechanism of ATP synthesis and the origin of biological effects of electromagnetic (microwave) fields are discussed.

The fundamental principle of cardiac behaviour is described by the Frank-Starling law relating force of contraction during systole with end-diastolic volume. While both work and respiration rates increase linearly with imposed load, the basis of mechano-energetic coupling in heart muscle has remained a long-standing enigma. Here, we highlight advances made in understanding of complex cellular and molecular mechanisms that orchestrate coupling of mitochondrial oxidative phosphorylation with ATP utilization for muscle contraction. Cardiac system bioenergetics critically depends on an interrelated metabolic infrastructure regulating mitochondrial respiration and energy fluxes throughout cellular compartments. The data reviewed indicate the significance of two interrelated systems regulating mitochondrial respiration and energy fluxes in cells: (1) the creatine kinase, adenylate kinase and glycolytic pathways that communicate flux changes generated by cellular ATPases within structurally organized enzymatic modules and networks; and (2) a secondary system based on mitochondrial participation in cellular calcium cycle, which adjusts substrate oxidation and energy-transducing processes to meet increasing cellular energy demands. By conveying energetic signals to metabolic sensors, coupled phosphotransfer reactions provide a high-fidelity regulation of the excitation-contraction cycle. Such integration of energetics with calcium signalling systems provides the basis for 'metabolic pacing', synchronizing the cellular electrical and mechanical activities with energy supply processes.

A discovery of the huge magnesium isotope effect in enzymatic ATP synthesis provides a new insight into mechanochemistry of enzymes as the molecular machines. It has been found that the catalytic activity values of ATPase, creatine kinase and phosphoglycerate kinase are 2 to 4-fold higher once their active sites contain magnetic (25Mg) not spinless, non-magnetic (24Mg, 26Mg), magnesium cation isotopes. This clearly proves that the ATP synthesis is a spin-selective process involving Mg2+ as the electron accepting reagent. The formation of ATP takes place in an ion-radical pair resulted by two partners, ATP oxyradical and Mg+. The magnesium bivalent cation is a key player in this process, this ion transforms the protein molecule mechanics into a mere chemistry. This ion is a most critical detail of structure of the magnesium dependent phosphorylation enzymes as the mechanochemical molecular machines.

Statins are highly effective at reducing coronary disease risk. The main side effects of these medications are a variety of skeletal muscle complaints ranging from mild myalgia to frank rhabdomyolysis. To search for physiologic factors possibly influencing statin muscle toxicity, we screened for genetic associations with serum creatine kinase (CK) levels in 102 patients receiving statin therapy for hypercholesteremia. A total of 19 single nucleotide polymorphism (SNPs) were selected from ten candidate genes involved in vascular homeostasis. Multiple linear regression was used to rank the SNPs according to probability of association, and the most significant associations were analyzed in greater detail. SNPs in the angiotensin II Type 1 receptor (AGTR1) and nitric oxide synthase 3 (NOS3) genes were significantly associated with CK activity. These results demonstrate a strong association between CK activity during statin treatment and variability in genes related to vascular function, and suggest that vascular smooth muscle function may contribute to the muscle side effects of statins.

Background: The role of the creatine kinase (CK)/phosphocreatine (PCr) energy buffer and transport system in heart remains unclear. Guanidinoacetate-N-methyltransferase-knockout (GAMT-/-) mice represent a new model of profoundly altered cardiac energetics, showing undetectable levels of PCr and creatine and accumulation of the precursor (phospho-)guanidinoacetate (P-GA). To characterize the role of a substantially impaired CK/PCr system in heart, we studied the cardiac phenotype of wild-type (WT) and GAMT-/- mice., Methods and Results: GAMT-/- mice did not show cardiac hypertrophy (myocyte cross-sectional areas, hypertrophy markers atrial natriuretic factor and beta-myosin heavy chain). Systolic and diastolic function, measured invasively (left ventricular conductance catheter) and noninvasively (MRI), were similar for WT and GAMT-/- mice. However, during inotropic stimulation with dobutamine, preload-recruitable stroke work failed to reach maximal levels of performance in GAMT-/- hearts (101+/-8 mm Hg in WT versus 59+/-7 mm Hg in GAMT-/-; P<0.05). (31)P-MR spectroscopy experiments showed that during inotropic stimulation, isolated WT hearts utilized PCr, whereas isolated GAMT-/- hearts utilized P-GA. During ischemia/reperfusion, GAMT-/- hearts showed markedly impaired recovery of systolic (24% versus 53% rate pressure product recovery; P<0.05) and diastolic function (eg, left ventricular end-diastolic pressure 23+/-9 in WT and 51+/-5 mm Hg in GAMT-/- during reperfusion; P<0.05) and incomplete resynthesis of P-GA., Conclusions: GAMT-/- mice do not develop hypertrophy and show normal cardiac function at low workload, suggesting that a fully functional CK/PCr system is not essential under resting conditions. However, when acutely stressed by inotropic stimulation or ischemia/reperfusion, GAMT-/- mice exhibit a markedly abnormal phenotype, demonstrating that an intact, high-capacity CK/PCr system is required for situations of increased cardiac work or acute stress.

We have investigated the role of the protein ubiquitous mitochondrial creatine kinase (uMtCK) in the formation and stabilization of inner and outer membrane contact sites. Using liver mitochondria isolated from transgenic mice, which, unlike control animals, express uMtCK in the liver, we found that the enzyme was associated with the mitochondrial membranes and, in addition, was located in membrane-coated matrix inclusions. In mitochondria isolated from uMtCK transgenic mice, the number of contact sites increased 3-fold compared with that observed in control mitochondria. Furthermore, uMtCK-containing mitochondria were more resistant to detergent-induced lysis than wild-type mitochondria. We conclude that octameric uMtCK induces the formation of mitochondrial contact sites, leading to membrane cross-linking and to an increased stability of the mitochondrial membrane architecture.

The aims of this prospective study were to determine serum levels of creatine kinase (CK) in overt and subclinical hypothyroidism; to investigate the change in CK levels with treatment; and to evaluate the relationship between free triiodsothyronine (FT3), free thyroxine (FT4), and thyrotropin (TSH) levels and the degree of skeletal muscle involvement, as determined by serum CK levels. Patients with hypothyroidism presenting to our endocrinology clinic were eligible for inclusion in this study. Patients with other causes of CK elevation were excluded. We included 28 patients (25 women and 3 men, ages 41.75 +/- 13.65 years) with overt hypothyroidism, 38 patients (37 women, 1 man, ages 40.55 +/- 10.48 years) with subclinical hypothyroidism, and 30 age- and gender-matched controls (27 women, 3 men, ages 40.81 +/- 11.20 years) in the study. Serum levels of TSH, FT4, FT3, and CK were measured in all subjects. CK elevation was found in 16 patients (57%) with overt hypothyroidism and in 4 patients (10%) with subclinical hypothyroidism. Although a statistically significant elevation of CK levels was found in patients with overt hypothyroidism when compared with patients with subclinical hypothyroidism and controls (p = 0. 0001, p = 0. 01, respectively), no difference was found between the subclinical hypothyroidism and control groups (p = 0.14). In hypothyroid (overt and subclinical) patients, a positive correlation was found between CK and TSH (r = 0.432; p = 0.04), and a negative correlation between CK and FT3 (r = - 0.556; p = 0.002) and between CK and FT4 (r =0.448; p = 0.04). CK levels decreased to normal levels after thyroid function normalized with treatment. In conclusion, skeletal muscle is affected by hypothyroidism more profoundly in cases of overt hypothyroidism, less so when subclinical hypothyroidism is present.

The aim of the present study was to characterize the pharmacological profile of the P2Y(12) receptor for several adenine triphosphate nucleotides in view of their possible roles as partial agonists or true antagonists. Two distinct cellular systems were used: P2Y(1) receptor deficient mouse platelets ( platelets) previously shown to express a native and functional P2Y(12) receptor and 1321 N1 astrocytoma cells stably expressing the human P2Y(12) receptor (1321 N1 P2Y(12)). ADP and its structural analogues inhibited cAMP accumulation in a dose-dependent manner in both platelets and 1321 N1 P2Y(12) cells with a similar rank order of potency, 2 methylthio-ADP (2MeSADP) >>ADP - Adenosine 5'-(betathio) diphosphate (AlphaDPbetaS). Commercial ATP, 2 chloro; ATP (2ClATP) and 2 methylthio-ATP (2MeSATP) also inhibited cAMP accumulation in both cell systems. In contrast, after creatine phosphate (CP)/creatine phosphokinase (CPK) regeneration, adenine triphosphate nucleotides lost their agonistic effect on platelets and behaved as antagonists of ADP (0.5 microm)-induced adenylyl cyclase inhibition with IC(50) of 13.5 +/- 4.8, 838 +/- 610, 1280 +/- 1246 microm for 2MeSATP, ATP and 2ClATP, respectively. In 1321 N1 P2Y(12) cells, CP/CPK regenerated ATP and 2ClATP lost their agonistic effect only when CP/CPK was maintained during the cAMP assay. The stable ATP analogue ATPgammaS antagonized ADPbetaS-induced inhibition of cAMP accumulation in both platelets and 1321 N1 P2Y(12) cells. Thus, ATP and its triphosphate analogues are not agonists but rather antagonists at the P2Y(12) receptor expressed in platelets or transfected cells, provided care is taken to remove diphosphate contaminants and to prevent the generation of diphosphate nucleotide derivatives by cell ectonucleotidases.

Creatine kinase (CK)-catalysed ATP-phosphocreatine (PCr) exchange is considered to play a key role in energy homeostasis of the brain. This study assessed the metabolic and anatomical consequences of partial or complete depletion of this system in transgenic mice without cytosolic B-CK (B-CK-/-), mitochondrial ubiquitous CK (UbCKmit-/-), or both isoenzymes (CK -/-), using non-invasive quantitative magnetic resonance (MR) imaging and spectroscopy. MR imaging revealed an increase in ventricle size in a subset of B-CK-/- mice, but not in animals with UbCKmit or compound CK mutations. Mice lacking single CK isoenzymes had normal levels of high-energy metabolites and tissue pH. In the brains of CK double knockouts pH and ATP and Pi levels were also normal, even though PCr had become completely undetectable. Moreover, a 20-30% decrease was observed in the level of total creatine and a similar increase in the level of neuronal N-acetyl-aspartate compounds. Although CKs themselves are not evenly distributed throughout the CNS, these alterations were uniform and concordant across different brain regions. Changes in myo-inositol and glutamate peaks did appear to be mutation type and brain area specific. Our results challenge current models for the biological significance of the PCr-CK energy system and suggest a multifaceted role for creatine in the brain.

Creatine kinase (CK) has a central role in skeletal muscle, acting as a fast energy buffer and shuttle between sites of energy production (mitochondria) and consumption (cross-bridges and ion pumps). Unexpectedly, isolated fast-twitch skeletal muscle cells of mice deficient in both cytosolic and mitochondrial CK (CK-/-) are highly fatigue resistant during stimulation protocols that stress aerobic metabolism. We have now studied different aspects of mitochondrial function in CK-/- skeletal muscle. Intact, single fibres of flexor digitorum brevis (FDB) muscles were fatigued by repeated tetanic stimulation (70 Hz, 350 ms duration, duty cycle 0.14). Under control conditions, CK-/- FDB fibres were more fatigue resistant than wild-type fibres. However, after mitochondrial inhibition with cyanide, force declined markedly faster in CK-/- fibres than in wild-type fibres. The rapid force decline in CK-/- fibres was not due to decreased myoplasmic [Ca2+] during tetani (measured with indo-1), which in these fibres remained virtually constant during fatigue in the presence of cyanide. Intact, single fibres of highly oxidative soleus muscles were fatigued by repeated tetani (50 Hz, 500 ms duration, duty cycle 0.5). All CK-/- soleus fibres tested (n = 9) produced > 40 % force at the end of the fatiguing stimulation period (500 tetani), whereas force fell to < 40 % before 500 tetani in two of three wild-type fibres. Mitochondrial [Ca2+] (measured with rhod-2 and confocal microscopy) increased during repeated tetanic stimulation in CK-/- but not in wild-type FDB fibres. In conclusion, mitochondria and energy shuttling operate effectively in CK-/- fibres and this is associated with an increase in mitochondrial [Ca2+].

Background: Little is known about the effects of weight loss on the relation between skeletal muscle enzymes and energy metabolism., Objective: This study was performed retrospectively to investigate the relation between skeletal muscle enzymes and 24-h energy metabolism in obese persons before and after weight loss., Design: Ten women and 9 men [with body mass indexes (in kg/m(2)) > 30] underwent a 15-wk weight-loss program (-700 kcal/d). Body weight and composition, 24-h energy metabolism (whole-body indirect calorimetry), and maximal activities of phosphofructokinase (EC 2.7.1.11), creatine kinase (CK; EC 2.7.3.2), citrate synthase (CS; EC 4.1.3.7), 3-hydroxyacyl-CoA dehydrogenase (HADH; EC 1.1.1.35), and cytochrome-c oxidase (COX; EC 1.9.3.1) were determined from biopsy samples of the vastus lateralis taken before and after weight loss., Results: Before weight loss, fat-free mass (FFM) was the only predictor of 24-h energy expenditure (R(2) = 0.70, P < 0.001), whereas the cumulative variance in sleeping metabolic rate explained by FFM and fat mass (FM) was 83% (P < 0.001). After weight loss, CS (r = 0.45, P = 0.05) and COX (r = 0.65, P < 0.01) were significantly associated with 24-h energy expenditure, whereas CK (r = 0.53, P < 0.05), CS (r = 0.45, P < 0.05), COX (r = 0.64, P < 0.01), and HADH (r = 0.45, P = 0.05) were all significant correlates of sleeping metabolic rate. After weight loss, FFM, FM, and COX explained 84% (P < 0.01) of the variance in 24-h energy expenditure, whereas FFM, FM, and CK all contributed to the cumulative variance in sleeping metabolic rate explained by this model (R(2) = 0.82, P < 0.05)., Conclusion: Maximal activities of key skeletal muscle enzymes contribute to the variability in 24-h energy metabolism in reduced-obese persons.

To define the relation between the phosphoryl transfer via creatine kinase and the ability to recover from an ischemia-reperfusion challenge, the authors chemically inhibited creatine kinase activity with iodoacetamide (IAm) and then measured myocardial recovery after 2, 10, or 30 min of global ischemia followed by 30 min of reperfusion in the isolated, arterially perfused interventricular septa of the rabbit heart. During normoxia, IAm (0.5 M perfused for 15 min) did not by itself modify developed tension, maximal rate of tension development, or resting tension. In ischemia, IAm pretreatment increased the rate of developed tension loss and highly diminished developed tension recovery after reperfusion for all the ischemia periods tested. Moreover, IAm significantly enhanced the maximal increase in the resting tension induced by 10 or 30 min of ischemia plus reperfusion. Lactate dehydrogenase activity in reperfusion was also significantly increased over untreated septa. On the basis of the present results, the authors suggest that the aggravating effects exhibited by IAm on the ischemic myocardium are compatible with its creatine kinase inhibition properties and that creatine kinase activity is essential for full recovery from an ischemia-reperfusion challenge.

Phosphocreatine (PCr) depletion during isometric twitch stimulation at 5 Hz was measured by (31)P-NMR spectroscopy in gastrocnemius muscles of pentobarbital-anesthetized MM creatine kinase knockout (MMKO) vs. wild-type C57B (WT) mice. PCr depletion after 2 s of stimulation, estimated from the difference between spectra gated to times 200 ms and 140 s after 2-s bursts of contractions, was 2.2 +/- 0.6% of initial PCr in MMKO muscle vs. 9.7 +/- 1.6% in WT muscles (mean +/- SE, n = 7, P < 0.001). Initial PCr/ATP ratio and intracellular pH were not significantly different between groups, and there was no detectable change in intracellular pH or ATP in either group after 2 s. The initial difference in net PCr depletion was maintained during the first minute of continuous 5-Hz stimulation. However, there was no significant difference in the quasi-steady-state PCr level approached after 80 s (MMKO 36.1 +/- 3.5 vs. WT 35.5 +/- 4.4% of initial PCr; n = 5-6). A kinetic model of ATPase, creatine kinase, and adenylate kinase fluxes during stimulation was consistent with the observed PCr depletion in MMKO muscle after 2 s only if ADP-stimulated oxidative phosphorylation was included in the model. Taken together, the results suggest that cytoplasmic ADP more rapidly increases and oxidative phosphorylation is more rapidly activated at the onset of contractions in MMKO compared with WT muscles.

Oxidative alterations of proteins by reactive oxygen species (ROS) have been implicated in the progression of aging and age-related neurodegenerative disorders such as Alzheimer's disease (AD). Protein carbonyls, a marker of protein oxidation, are increased in AD brain, indicating that oxidative modification of proteins is relevant in AD. Oxidative damage can lead to several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, to neuronal death. Identification of specific targets of protein oxidation represents a crucial step in establishing a relationship between oxidative modification and neuronal death in AD, and was partially achieved previously in our laboratory through immunochemical detection of creatine kinase BB and beta-actin as specifically oxidized proteins in AD brain versus control brain. However, this process is laborious, requires the availability of specific antibodies, and, most importantly, requires a reasonable guess as to the identity of the protein in the first place. In this study, we present the first proteomics approach to identify specifically oxidized proteins in AD, by coupling 2D fingerprinting with immunological detection of carbonyls and identification of proteins by mass spectrometry. The powerful techniques, emerging from application of proteomics to neurodegenerative disease, reveal the presence of specific targets of protein oxidation in Alzheimer's disease (AD) brain: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain. Proteomics offers a rapid means of identifying oxidatively modified proteins in aging and age-related neurodegenerative disorders without the limitations of the immunochemical detection method.

The individual functional significance of the various creatine kinase (CK) isoenzymes for myocardial energy homeostasis is poorly understood. Whereas transgenic hearts lacking the M subunit of CK (M-CK) show unaltered cardiac energetics and left ventricular (LV) performance, deletion of M-CK in combination with loss of sarcomeric mitochondrial CK (ScCKmit) leads to significant alterations in myocardial high-energy phosphate metabolites. To address the question as to whether this alteration is due to a decrease in total CK activity below a critical threshold or due to the specific loss of ScCKmit, we studied isolated perfused hearts with selective loss of ScCKmit (ScCKmit(-/-), remaining total CK activity approximately 70%) using (31)P NMR spectroscopy at two different workloads. LV performance in ScCKmit(-/-) hearts (n = 11) was similar compared with wild-type hearts (n = 9). Phosphocreatine/ATP, however, was significantly reduced in ScCKmit(-/-) compared with wild-type hearts (1.02 +/- 0.05 vs. 1.54 +/- 0.07, P < 0.05). In parallel, free [ADP] was higher (144 +/- 11 vs. 67 +/- 7 microM, P < 0.01) and free energy release for ATP hydrolysis (DeltaG(ATP)) was lower (-55.8 +/- 0.5 vs. -58.5 +/- 0.5 kJ/mol, P < 0.01) in ScCKmit(-/-) compared with wild-type hearts. These results demonstrate that M- and B-CK containing isoenzymes are unable to fully substitute for the loss of ScCKmit. We conclude that ScCKmit, in contrast to M-CK, is critically necessary to maintain normal high-energy phosphate metabolite levels in the heart.

ATP-sensitive K(+) (K(ATP)) channels in the heart are normally closed by high intracellular ATP, but are activated during ischemia to promote cellular survival. These channels are heteromultimers composed of Kir6.2 subunit, an inwardly rectifying K(+) channel core, and SUR2A, a regulatory subunit implicated in ligand-dependent regulation of channel gating. Here, we have shown that the muscle form (M-LDH), but not heart form (H-LDH), of lactate dehydrogenase is directly physically associated with the sarcolemmal K(ATP) channel by interacting with the Kir6.2 subunit via its N-terminus and with the SUR2A subunit via its C-terminus. The species of LDH bound to the channel regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of M-LDH in the channel protein complex was required for opening of K(ATP) channels during ischemia and ischemia-resistant cellular phenotype. We conclude that M-LDH is an integral part of the sarcolemmal K(ATP) channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the K(ATP) channels activity that is essential for cell protection against ischemia.

Our goal was to determine whether mice genetically altered to lack either creatine kinase (M/MtCK(-/-)) or adenylate kinase (AK(-/-)) show altered properties in the dynamic regulation of myocardial oxygen consumption (MVO(2)). We measured contractile function, oxygen consumption, and the mean response time of oxygen consumption to a step increase in heart rate [i.e., mitochondrial response time (t(mito))] in isolated Langendorff-perfused hearts from wild-type (n = 6), M/MtCK(-/-) (n = 6), and AK(-/-) (n = 4) mice. Left ventricular developed pressure was higher in M/MtCK(-/-) hearts (88.2 +/- 6.8 mmHg) and lower in AK(-/-) hearts (46.7 +/- 9.4 mmHg) compared with wild-type hearts (60.7 +/- 10.1 mmHg) at the basal pacing rate. Developed pressure fell slightly when heart rate was increased in all three groups. Basal MVO(2) at 300 beats/min was 19.1 +/- 2.4, 19.4 +/- 1.5, and 16.3 +/- 1.9 micromol x min(-1) x g dry wt(-1) for M/MtCK(-/-), AK(-/-), and wild type, respectively, which increased to 25.5 +/- 3.7, 25.4 +/- 2.6, and 22.0 +/- 2.6 micromol. min(-1) x g(-1), when heart rate was increased to 400 beats/min. The t(mito) was significantly faster in M/MtCK(-/-) hearts: 3.0 +/- 0.3 versus 7.3 +/- 0.6 and 8.0 +/- 0.4 s for M/MtCK(-/-), AK(-/-), and wild-type hearts, respectively. Our results demonstrate that MVO(2) of M/MtCK(-/-) hearts adapts more quickly to an increase in heart rate and thereby support the hypothesis that creatine kinase acts as an energy buffer in the cytosol, which delays the energy-related signal between sites of ATP hydrolysis and mitochondria.

Creatine kinases are important in maintaining cellular-energy homeostasis, and neuroprotective effects have been attributed to the administration of creatine and creatine-like compounds. Herein we examine whether ablation of the cytosolic brain-type creatine kinase (B-CK) in mice has detrimental effects on brain development, physiological integrity or task performance. Mice deficient in B-CK (B-CK-/-) showed no gross abnormalities in brain anatomy or mitochondrial ultrastructure, but had a larger intra- and infrapyramidal mossy fibre area. Nuclear magnetic resonance spectroscopy revealed that adenosine triphosphate (ATP) and phosphocreatine (PCr) levels were unaffected, but demonstrated an apparent reduction of the PCr left arrow over right arrow ATP phosphorus exchange capacity in these mice. When assessing behavioural characteristics B-CK-/- animals showed diminished open-field habituation. In the water maze, adult B-CK-/- mice were slower to learn, but acquired the spatial task. This task performance deficit persisted in 24-month-old, aged B-CK-/- mice, on top of the age-related memory decline normally seen in old animals. Finally, a delayed development of pentylenetetrazole-induced seizures (creating a high-energy demand) was observed in B-CK-/- mice. It is suggested that the persistent expression of the mitochondrial isoform ubiquitous mitochondrial CK (UbCKmit) in the creatine/phospho-creatine shuttle provides compensation for the loss of B-CK in the brain. Our studies indicate a role for the creatine-phosphocreatine/CK circuit in the formation or maintenance of hippocampal mossy fibre connections, and processes that involve habituation, spatial learning and seizure susceptibility. However, for fuelling of basic physiological activities the role of B-CK can be compensated for by other systems in the versatile and robust metabolic-energy network of the brain.

To understand the role of creatine kinase (CK) in cardiac excitation-contraction coupling, CK-deficient mice (CK-/-) were studied in vitro and in vivo. In skinned fibers, the kinetics of caffeine-induced release of Ca2+ was markedly slowed in CK-/- mice with a partial restoration when glycolytic substrates were added. These abnormalities were almost compensated for at the cellular level: the responses of Ca2+ transient and cell shortening to an increased pacing rate from 1 Hz to 4 Hz were normal with a normal post-rest potentiation of shortening. However, the post-rest potentiation of the Ca2+ transient was absent and the cellular contractile response to isoprenaline was decreased in CK-/- mice. In vivo, echocardiographically determined cardiac function was normal at rest but the response to isoprenaline was blunted in CK-/- mice. Previously described compensatory pathways (glycolytic pathway and closer sarcoplasmic reticulum-mitochondria interactions) allow a quasi-normal SR function in isolated cells and a normal basal in vivo ventricular function, but are not sufficient to cope with a large and rapid increase in energy demand produced by beta-adrenergic stimulation. This shows the specific role of CK in excitation-contraction coupling in cardiac muscle that cannot be compensated for by other pathways.

The function of creatine kinase (CK) and its effect on phosphorus metabolites was studied in livers of transgenic mice expressing human ubiquitous mitochondrial CK (CK-Mit) and rat brain CK (CK-B) isoenzymes and their combination. (31)P NMR spectroscopy and saturation transfer were recorded in livers of anesthetized mice to measure high-energy phosphates and hepatic CK activity. CK reaction velocity was related to total enzyme activity irrespective of the isoenzyme expressed, and it increased with increasing concentrations of creatine (Cr). The fluxes mediated by both isoenzymes in both directions (phosphocreatine or ATP synthesis) were equal. Over a 20-fold increase in CK-Mit activity (28-560 micromol. g wet wt(-1). min(-1)), the fraction of phosphorylated Cr increased 1.6-fold. Hepatic free ADP concentrations calculated by assuming equilibrium of the CK-catalyzed reaction in vivo decreased from 84 +/- 9 to 38 +/- 4 nmol/g wet wt. Calculated free ADP levels in mice expressing high levels of CK-B (920-1,635 micromol. g wet wt(-1). min(-1)) were 52 +/- 6 nmol/g wet wt. Mice expressing both isoenzymes had calculated free ADP levels of 36 +/- 4 nmol/g wet wt. These findings indicate that CK-Mit catalyzes its reaction equally well in both directions and can lower hepatic apparent free ADP concentrations.

Exercise-induced muscle damage is a well documented phenomenon that often follows unaccustomed and sustained metabolically demanding activities. This is a well researched, but poorly understood area, including the actual mechanisms involved in the muscle damage and repair cycle. An integrated model of muscle damage has been proposed by Armstrong and is generally accepted. A more recent aspect of exercise-induced muscle damage to be investigated is the potential of estrogen to have a protective effect against skeletal muscle damage. Estrogen has been demonstrated to have a potent antioxidant capacity that plays a protective role in cardiac muscle, but whether this antioxidant capacity has the ability to protect skeletal muscle is not fully understood. In both human and rat studies, females have been shown to have lower creatine kinase (CK) activity following both eccentric and sustained exercise compared with males. As CK is often used as an indirect marker of muscle damage, it has been suggested that female muscle may sustain less damage. However, these findings may be more indicative of the membrane stabilising effect of estrogen as some studies have shown no histological differences in male and female muscle following a damaging protocol. More recently, investigations into the potential effect of estrogen on muscle damage have explored the possible role that estrogen may play in the inflammatory response following muscle damage. In light of these studies, it may be suggested that if estrogen inhibits the vital inflammatory response process associated with the muscle damage and repair cycle, it has a negative role in restoring normal muscle function after muscle damage has occurred. This review is presented in two sections: firstly, the processes involved in the muscle damage and repair cycle are reviewed; and secondly, the possible effects that estrogen has upon these processes and muscle damage in general is discussed. The muscle damage and repair cycle is presented within a model, with particular emphasis on areas that are important to understanding the potential effect that estrogen has upon these processes.

Cardiac sarcolemmal ATP-sensitive K+ (KATP) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of cells with the membrane excitability. Based on previous functional studies, we have hypothesized that creatine kinase (CK) may be a part of the sarcolemmal KATP channel protein complex. The inside-out and whole cell patch clamp electrophysiology applied on guinea pig cardiomyocytes showed that substrates of CK regulate KATP channels activity. Following immunoprecipitation of guinea-pig cardiac membrane fraction with the anti-SUR2 antibody, Coomassie blue staining revealed, besides Kir6.2 and SUR2A, a polypeptide at approximately 48 kDa. Western blotting analysis confirmed the nature of putative Kir6.2 and SUR2A, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified p48 kDa as a muscle form of CK. In addition, the CK activity was found in the anti-SUR2A immunoprecipitate and the cross reactivity between an anti-CK antibody and the anti-SUR2A immunoprecipitate was observed as well as vice verse. Further results obtained at the level of recombinant channel subunits demonstrated that CK is directly physically associated with the SUR2A, but not the Kir6.2, subunit. All together, these results suggest that the CK is associated with SUR2A subunit in vivo, which is an integral part of the sarcolemmal KATP channel protein complex.

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.

Creatine kinase (CK) is an abundant enzyme, important for maintenance of high-energy phosphate homeostasis in many tissues including heart. Double-knockout CK (DbKO-CK) mice missing both the muscle (MM) and sarcomeric mitochondrial (ScMit) isoforms of CK have recently been studied. Despite a large change in skeletal muscle function in DbKO-CK mice, there is little functional change in the heart. To investigate whether there are specific changes in cardiac mitochondrial proteins associated with the loss of MM- and ScMit-CK isoforms, we have used difference gel electrophoresis (DIGE) to compare mitochondrial proteins from wild-type and DbKO-CK mice. Mass spectrometry fingerprinting was used to identify 40 spots as known mitochondrial proteins. We have discovered that the loss of MM- and ScMit-CK isoforms did not cause large scale changes in heart mitochondrial proteins. The loss of ScMit-CK was readily detected in the DbKO-CK samples. We have also detected a large decrease in the precursor form of aconitase. Furthermore, two mitochondrial protein differences have been found in the parent mouse strains of the DbKO-CK mice.

The development of ideas concerning the buffer and transport functions of the creatine kinase system is described. The concept of ATP compartmentation at sites of its production and utilization is critically analyzed. Kinetic, thermodynamic, and structural data used as a basis for a hypothesis on the structural and functional coupling of mitochondrial creatine kinase and adenine nucleotide translocase are comprehensively analyzed, and experimental evidence inconsistent with this hypothesis is presented. It seems that the mitochondrial creatine kinase may serve to equilibrate ADP concentration in the intermembrane space with fluctuating ADP concentrations in the cytoplasm. It is suggested that creatine kinase molecules bound to other intracellular structures (e.g., to myofibrils) may equilibrate local ADP concentrations with those present in the cytoplasm.

The subcellular mechanisms responsible for myocardial depression during sepsis remain unclear. Recent data suggest a role for impaired energy generation and utilization, resulting in altered contractile function. Here, we studied the energetic and mechanical properties of skinned fibers isolated from rabbit ventricle in a nonlethal but hypotensive model of endotoxemia. Thirty-six hours after lipopolysaccharide (LPS) injection (in the presence of altered myocardial contractility), mitochondrial respiration, coupling between oxidation and phosphorylation, and creatine kinase function were similar in preparations from endotoxemic (LPS) and control animals. The maximal Ca2+-activated force was similar in LPS and control preparations. However, the Ca2+ concentration corresponding to half-maximal force (pCa50, where pCa = -log10[Ca2+]) was 5.55 +/- 0.01 (n = 11) in LPS fibers versus 5.61 +/- 0.01 (n = 10) in control fibers (p < 0.01). Both protein kinase A (PKA) and alkaline phosphatase treatment led to the disappearance in the difference between control and LPS pCa50 values. Incubation of control fibers with the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) did not change the Ca2+ sensitivity after subsequent skinning, whereas isoproterenol decreased pCa50 from 5.62 +/- 0.01 to 5.55 +/- 0.01 (p < 0.01). These data suggest that during sepsis, cardiac mitochondrial and creatine kinase systems remain unaltered, whereas protein phosphorylation decreases myofibrillar Ca2+ sensitivity and may contribute to the depression of cardiac contractility.

Creatine kinase (CK) exists as a family of isoenzymes in excitable tissue. We studied isolated perfused hearts from mice lacking genes for either the main muscle isoform of CK (M-CK) or both M-CK and the main mitochondrial isoform (Mt-CK) to determine 1) the biological significance of CK isoenzyme shifts, 2) the necessity of maintaining a high CK reaction rate, and 3) the role of CK isoenzymes in establishing the thermodynamics of ATP hydrolysis. (31)P NMR was used to measure [ATP], [PCr], [P(i)], [ADP], pH, as well as the unidirectional reaction rate of PCr--> [gamma-P]ATP. Developmental changes in the main fetal isoform of CK (BB-CK) were unaffected by loss of other CK isoenzymes. In hearts lacking both M- and Mt-CK, the rate of ATP synthesis from PCr was only 9% of the rate of ATP synthesis from oxidative phosphorylation demonstrating a lack of any high energy phosphate shuttle. We also found that the intrinsic activities of the BB-CK and the MM-CK isoenzymes were equivalent. Finally, combined loss of M- and Mt-CK (but not loss of only M-CK) prevented the amount of free energy released from ATP hydrolysis from increasing when pyruvate was provided as a substrate for oxidative phosphorylation.

The perpetual and vigorous nature of heart muscle work requires efficient myocardial energetics. This depends not only on adequate ATP production, but also on efficient delivery of ATP to muscle ATPases and rapid removal of ADP and other by-products of ATP hydrolysis. Indeed, recent evidence indicates that defects in communication between ATP-producing and ATP-consuming cellular sites are a major factor contributing to energetic deficiency in heart failure. In particular, the failing myocardium is characterized by reduced catalytic activity of creatine kinase, adenylate kinase, carbonic anhydrase, and glycolytic enzymes, which collectively facilitate ATP delivery and promote removal of ADP, Pi, and H+ from cellular ATPases. Although energy transfer through adenylate kinase and glycolytic enzymes has been recognized as an adaptive mechanism supporting compromised muscle energetics, in the failing myocardium the total compensatory potential of these systems is diminished. A gradual accumulation of defects at various steps in myocardial energetic signaling, along with compromised compensatory mechanisms, precipitates failure of the whole cardiac energetic system, ultimately contributing to myocardial dysfunction. These advances in our understanding of the molecular bioenergetics in heart failure provide a new perspective toward improving the energetic balance of the failing myocardium.

Chronic ingestion of ethanol (EtOH) produces physiological and morphological alterations in skeletal muscle. The effects of EtOH on skeletal muscle have been studied in experimental animals or on biopsies from alcoholic patients. However, alterations in skeletal muscle from alcoholic patients could be secondary to the effects of EtOH on the nervous system. In this study, by assaying the action of EtOH on primary skeletal muscle cell cultures, we provide evidence of its direct effect on skeletal muscle proliferation and differentiation. The results indicate that EtOH: (1) significantly inhibits skeletal muscle cell proliferation at the beginning of the proliferation phase; (2) delays skeletal muscle differentiation, shown by the significant changes in the evolution of the percentage of the creatine kinase isozymes; (3) has no significant effect on skeletal muscle DNA or protein content during the proliferation phase.

The efficiency of stimulation of mitochondrial respiration in permeabilized muscle cells by ADP produced at different intracellular sites, e.g. cytosolic or mitochondrial intermembrane space, was evaluated in wild-type and creatine kinase (CK)-deficient mice. To activate respiration by endogenous production of ADP in permeabilized cells, ATP was added either alone or together with creatine. In cardiac fibers, while ATP alone activated respiration to half of the maximal rate, creatine plus ATP increased the respiratory rate up to its maximum. To find out whether the stimulation by creatine is a consequence of extramitochondrial [ADP] increase, or whether it directly correlates with ADP generation by mitochondrial CK in the mitochondrial intermembrane space, an exogenous ADP-trap system was added to rephosphorylate all cytosolic ADP. Under these conditions, creatine plus ATP still increased the respiration rate by 2.5 times, compared with ATP alone, for the same extramitochondrial [ADP] of 14 microM. Moreover, this stimulatory effect of creatine, observed in wild-type cardiac fibers disappeared in mitochondrial CK deficient, but not in cytosolic CK-deficient muscle. It is concluded that respiration rates can be dissociated from cytosolic [ADP], and ADP generated by mitochondrial CK is an important regulator of oxidative phosphorylation.

Objective: The aim of this study was to test the utility of serum creatine kinase (CK) isoenzyme determinations as a marker of tissue injury in preterm newborns with respiratory distress syndrome (RDS)., Patients and Methods: Two groups of neonates were studied, 26 suffering from RDS who required mechanical ventilation and 20 healthy newborns with gestational ages, hours of life and birth weights similar to the first group. The activity of CK and its isoenzymes was determined in the bronchial aspirate and serum samples that were obtained before and 24 hours after exogenous surfactant therapy. The isoenzymes were separated by electrophoresis on agarose gel and their activity expressed as a percentage of the total CK. Total proteins were quantified in the bronchial aspirate and CK enzymatic activity expressed in U/mg of protein x 10-3., Results: The CK-BB isoenzyme was significantly increased (p < 0.001) in the serum of infants with RDS compared with the control group. In the bronchial aspirate, the isoenzymatic study showed that the CK-BB isoenzyme represented 98-100% of the total enzymatic CK activity., Conclusions: The study shows significant differences in the CK isoenzyme patterns of neonates with RDS compared to controls. An increase in serum levels of the CK-BB isoenzyme could be an effective marker of tissue injury in lung disease in the newborn.

Early estimation of the efficacy of thrombolysis in acute myocardial infarction is of great clinical importance because the appearance of coronary reperfusion changes therapeutic and diagnostic procedures and decreases the mortality rate. Previous studies showed that the analysis of activity of creatinine kinase (CPK) measured in regular, short periods of time after thrombolysis night be useful in the diagnosis of reperfusion equally to coronary angiography. The aim of the study was to estimate the usefulness of the analysis of creatine kinase (CPK) and its isoenzyme (CK-MB) in the diagnosis of coronary reperfusion in patients with acute myocardial infarction after thrombolytic therapy. The study was performed in 50 patients with acute myocardial infarction admitted to our Cardiology Department, of these 42 were men aged from 34 to 68 and 8 were women aged from 43 to 70. 28 patients had acute inferior myocardial infarction, 22 patients--acute anterior myocardial infarction. All patients were administered 300 mg of aspirin after admission and then 150 mg of aspirin daily and 1,500,000 IU of streptokinase i.v. within 1 hour. Venous blood samples for determination of CPK and CK-MB were obtained every 3 hours during the first 48 h and once a day at 8 a.m from 3rd to 11th day. All patients underwent coronary angiography 2-4 weeks after thrombolysis. The study showed that in patients with reperfusion, activities of CPK and CK-MB three hours after thrombolysis were higher than 30% of later peak. These findings show the usefulness of this criterion in early, non-invasive estimation of efficacy of thrombolysis. Determination of activity of isoenzyme CK-MB during thrombolytic therapy is not necessary, because it evaluates similarly to CPK. We showed that electrocardiographic and enzymatic criteria are comparable in estimation of efficacy of thrombolytic therapy.

After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional 'coupling' between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters phi and psi that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible 'coupling' are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when phi or psi is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when psi or psi is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless phi = psi = 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the 'adenine nucleotide flux' at the ends of the model into 'creatine flux' in the middle; the overall net flux is small, but only zero if phi = psi. A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model.