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PubMed2001

Effects of acrylamide on creatine kinase from rabbit muscle.

The mechanism of inhibition of creatine kinase (CK) by acrylamide (Acr) has been examined (in vitro). Within the concentration range of 0 to 1 M, Acr markedly inhibited CK and depleted the protein thiols. Both inactivation and thiol depletion were time- and Acr concentration-dependent. Addition of dithiothreitol (DTT) did not reactivate CK inactivated by Acr. However, CK with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) pre-blocked thiols can be reactivated by DTT after incubation with Acr. The transition-state analogue also had a significant protective effect on CK against Acr inhibition. We conclude that thiol alkylation is a critical event in inactivation of CK by Acr. Furthermore, Acr binding to CK changed its surface charge, which may be the same effect for the toxicity of Acr towards other proteins.

Acrylamide↗
PubMed2002

Effects of aspartate on rabbit muscle creatine kinase and the salt induced molten globule state.

The aspartate (Asp)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, circular dichroism (CD) spectra, native polyacrylamide gel electrophoresis and ultraviolet difference spectra. The results showed that Asp caused inactivation and unfolding of CK, with no aggregation during CK denaturation. The kinetics of CK unfolding followed a one phase process. At higher concentrations of Asp (>2.5mM), the CK dimers were partially dissociated. Inactivation occurred before noticeable conformational change during CK denaturation. Asp denatured CK was mostly reactivated and refolded by dilution. KCl induced the molten globule state with compact structure after CK was denatured with 10mM Asp. These results suggest that the effect of Asp differed from that of other denaturants such as guanidine, HCl or urea during CK unfolding. Asp is a reversible protein denaturant and the molten globule state indicates that intermediates exist during CK folding.

Animals↗
PubMed2006

Myocardial protection by nicorandil during open-heart surgery under cardiopulmonary bypass.

BACKGROUND: To evaluate the myocardial protective effect of nicorandil when used as an adjuvant to cold hyperkalaemic cardioplegia in open-heart surgery. METHODS: Patients who underwent surgery under cardiopulmonary bypass (CPB) for mitral valve replacement (MVR, 23 patients) or coronary artery bypass grafting (CABG, 24 patients) were entered in a double-blind study. The patients were randomized to a nicorandil Group (N) or placebo Group (P). Nicorandil 0.1 mg kg-1 (Group N), or normal saline (Group P), were administered at three time points: (1) after aortic cannulation, but prior to going on CPB, (2) 5 min before aortic cross-clamping and (3) 5 min before reperfusion. The following variables were studied: (a) time until electromechanical arrest after cardioplegia administration (Tarrest), (b) time until return of electromechanical activity after aortic cross-clamp removal (Trecovery), (c) incidence of postoperative myocardial infarction or low output syndromes (d) dysrhythmias requiring intervention after aortic cross-clamp removal and (e) haemodynamic changes after nicorandil administration. RESULTS: The Tarrest after cardioplegia administration was significantly faster in nicorandil group in both MVR and CABG patients (P 75 IU L-1 in MVR patients was significantly lower in the Group N than in placebo patients (P < 0.05). However, in CABG patients there was no such significant difference. The incidence of dysrhythmias requiring intervention after aortic cross-clamp removal was also less in Group N. Administration of 0.1 mg kg-1 boluses of nicorandil did not cause significant haemodynamic changes or precipitate dysrhythmias in any patient. CONCLUSION: Nicorandil enhances the myocardial protective effect of cold hyperkalaemic cardioplegia in cardiac surgery patients.

Adult↗
PubMed2001

Mutagenesis of two acidic active site residues in human muscle creatine kinase: implications for the catalytic mechanism.

Creatine kinase (CK) catalyzes the reversible phosphorylation of the guanidine substrate, creatine, by MgATP. Although several X-ray crystal structures of various isoforms of creatine kinase have been published, the detailed catalytic mechanism remains unresolved. A crystal structure of the CK homologue, arginine kinase (AK), complexed with the transition-state analogue (arginine-nitrate-ADP), has revealed two carboxylate amino acid residues (Glu225 and Glu314) within 2.8 A of the proposed transphosphorylation site. These two residues are the putative catalytic groups that may promote nucleophilic attack by the guanidine amino group on the gamma-phosphate of ATP. From primary sequence alignments of arginine kinases and creatine kinases, we have identified two homologous creatine kinase acidic amino acid residues (Glu232 and Asp326), and these were targeted for examination of their potential roles in the CK mechanism. Using site-directed mutagenesis, we have made several substitutions at these two positions. The results indicate that of these two residues the Glu232 is the likely catalytic residue while Asp326 likely performs a role in properly aligning substrates for catalysis.

Amino Acid Sequence↗
PubMed2002

Rapid time-resolved immunofluorometric assay for the measurement of creatine kinase in serum and whole blood samples.

A rapid and simple immunochemical method was developed for the assessment of the creatine kinase (MM) isoenzyme [CK(MM)], a protein marker linked with animal welfare and meat quality. The one-step time-resolved immunofluorometric assay produced quantitative results from serum or whole blood samples in 20 min. The analytical limit of detection (mean + 2s) for the immunoassay was 17 ng/mL (n = 6), and the functional limit of detection for the analysis of porcine whole blood samples was 426 ng/mL (n = 24). The working range of the method was linear up to 50 micro g/mL, and the within-assay precision varied between 2.1 and 10.9%. The analysis of porcine serum samples showed that the results from the immunoassay method and colorimetric CK enzyme activity determination were highly correlated (r(2) = 0.965, n = 17, p < 0.001). The practicability of the assay was demonstrated by the analysis of 300 porcine whole blood samples in a slaughterhouse environment.

Animals↗
PubMed2003

Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice.

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

Animals↗
PubMed2003

Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types.

The muscle creatine kinase (MCK) gene is expressed at high levels only in differentiated skeletal and cardiac muscle. The activity of the cloned enhancer-promoter has previously been shown to be dependent on the Trex element which is specifically bound by a yet unidentified nuclear factor, TrexBF. We have further characterized the function of the Trex site by comparing wild-type and Trex-mutated MCK transgenes in five mouse skeletal muscles: quadriceps, extensor digitorum longus (EDL), soleus, diaphragm, and distal tongue, as well as in heart ventricular muscle. Several types of statistical analysis including analysis of variance (ANOVA) and rank sum tests were used to compare expression between muscle types and between constructs. Upon mutation of the Trex site, median transgene expression levels decreased 3- to 120-fold in the muscles examined, with statistically significant differences in all muscles except the EDL. Expression in the largely slow soleus muscle was more affected than in the EDL, and expression in the distal tongue and diaphragm muscles was affected more than in soleus. Median expression of the transgene in ventricle decreased about 18-fold upon Trex mutation. Transfections into neonatal rat myocardiocytes confirmed the importance of the Trex site for MCK enhancer activity in heart muscle, but the effect is larger in transgenic mice than in cultured cells.

Animals↗
PubMed2001

A cellular model system of differentiated human myotubes.

The aim of this study was to select an effective and stable protocol for the differentiation of human satellite cells (Sc) and to identify the optimal time period for the experimental use of differentiated human Sc-cultures. In order to identify the differentiation conditions which give a good survival of myotubes and a high grade of differentiation, Sc-cultures were induced to differentiate in media supplemented with either 2% fetal calf serum (FCS) 2% horse serum (HS) or 10% HS. Based on higher CK-activities in cultures differentiating in FCS-supplemented media compared to horse sera, fetal calf serum was chosen to induce differentiation. The ATP, DNA and protein content increased during the first 4 days after induction of differentiation and was followed by a period with minor changes. The maximal differences of ATP, DNA and protein between days 4-10 were evaluated and the differences in the three components were found to be less than 20% of the average value with a certainity of more than 0.9. Day 8-myotubes were investigated morphologically and were found immunoreactive for fast myosin, and expressed areas with clear cross striation. We recommend the use of differentiated Sc-cultures in the period from day 4 to 8 after induction of differentiation as only minor differentation-related changes will take place in the cells during this period of time.

Adenosine Triphosphate↗
PubMed2002

Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres.

The biochemical basis for the regulation of fibre-type determination in skeletal muscle is not well understood. In addition to the expression of particular myofibrillar proteins, type I (slow-twitch) fibres are much higher in mitochondrial content and are more dependent on oxidative metabolism than type II (fast-twitch) fibres. We have previously identified a transcriptional co-activator, peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1 alpha), which is expressed in several tissues including brown fat and skeletal muscle, and that activates mitochondrial biogenesis and oxidative metabolism. We show here that PGC-1 alpha is expressed preferentially in muscle enriched in type I fibres. When PGC-1 alpha is expressed at physiological levels in transgenic mice driven by a muscle creatine kinase (MCK) promoter, a fibre type conversion is observed: muscles normally rich in type II fibres are redder and activate genes of mitochondrial oxidative metabolism. Notably, putative type II muscles from PGC-1 alpha transgenic mice also express proteins characteristic of type I fibres, such as troponin I (slow) and myoglobin, and show a much greater resistance to electrically stimulated fatigue. Using fibre-type-specific promoters, we show in cultured muscle cells that PGC-1 alpha activates transcription in cooperation with Mef2 proteins and serves as a target for calcineurin signalling, which has been implicated in slow fibre gene expression. These data indicate that PGC-1 alpha is a principal factor regulating muscle fibre type determination.

Animals↗
PubMed2004

Suv39h histone methyltransferases interact with Smads and cooperate in BMP-induced repression.

Smad proteins transduce signals from transforming growth factor-beta (TGF-beta) superfamily ligands to regulate the expression of target genes. In order to identify novel partners of Smad proteins in transcriptional regulation, we performed a two-hybrid screen using Smad5, a protein that is activated predominantly by bone morphogenetic protein (BMP) signaling. We identified an interaction between Smad5 and suppressor of variegation 3-9 homolog 2 (Suv39h2), a chromatin modifier enzyme. Suv39h proteins are histone methyltransferases that methylate histone H3 on lysine 9, resulting in transcriptional repression or silencing of target genes. Biochemical studies in mammalian cells demonstrated that Smad5 binds to both known mammalian isoforms of Suv39h proteins, and that Smad proteins activated by the TGF-beta signaling pathway, Smad2 and Smad3, do not bind with significant affinity. Functional studies using the muscle creatine kinase (MCK) promoter, which is suppressed by BMP signaling, demonstrate that Suv39h proteins and Smads cooperate to repress promoter activity. These data suggest a model where association of Smad proteins with Suv39h methyltransferases can repress or silence genes involved in developmental processes, and argues that inefficient gene repression may result in the alteration of the differentiated phenotype. Thus, examination of the Smad-Suv interaction may provide insight into the mechanism of phenotypic determination mediated by BMP signaling.

Animals↗
PubMed2002

PKC-regulated myogenesis is associated with increased tyrosine phosphorylation of FAK, Cas, and paxillin, formation of Cas-CRK complex, and JNK activation.

Previous reports suggest that PKC plays an important role in regulating myogenesis. However, the regulatory signaling pathways are not fully understood. We examined the effects of PKC downregulation on signaling events during skeletal muscle differentiation. We found that downregulation of PKC results in increased myogenesis in C2C12 cells as measured by creatine kinase activity and myogenin expression. We showed that, during differentiation, downregulation of PKC expression results in increased tyrosine phosphorylation of FAK, Cas, and paxillin, concomitant with enhanced Cas-CrkII complex formation, which leads to activation of JNK2. But in proliferated muscle cells, PKC inhibition results in FAK and Cas tyrosine dephosphorylation. Further, disruption of actin cytoskeleton by cytochalasin D prevents the activation of FAK and Cas as well as the formation of Cas-CrkII complex stimulated by PKC downregulation during muscle cell differentiation. Finally, we observed that PKC downregulation increases the tyrosine phosphorylation of focal adhesion associated proteins. Based on the above data, we propose that PKC downregulation results in enhanced tyrosine phosphorylation of FAK, Cas, and paxillin, thus promoting the establishment of Cas-CrkII complex, leading to activation of JNK and that these interactions are dependent upon the integrity of actin cytoskeleton during muscle cell differentiation. Data presented here significantly contribute to elucidating the regulatory role of PKC in myogenesis possibly through integrin signaling pathway.

Actin Cytoskeleton↗
PubMed2005

First United Kingdom Heart and Renal Protection (UK-HARP-I) study: biochemical efficacy and safety of simvastatin and safety of low-dose aspirin in chronic kidney disease.

BACKGROUND: Patients with chronic kidney disease are at increased risk for cardiovascular disease, but the efficacy and safety of simvastatin and aspirin are unknown in this patient group. METHODS: Patients were randomly assigned in a 2 x 2 factorial design to the administration of: (1) 20 mg of simvastatin daily versus matching placebo, and (2) 100 mg of modified-release aspirin daily versus matching placebo. RESULTS: Overall, 448 patients with chronic kidney disease were randomly assigned (242 predialysis patients with a creatinine level > or = 1.7 mg/dL [> or =150 micromol/L], 73 patients on dialysis therapy, and 133 patients with a functioning transplant). Compliance with study treatments was 80% at 12 months. Allocation to treatment with 100 mg of aspirin daily was not associated with an excess of major bleeds (aspirin, 4 of 225 patients [2%] versus placebo, 6 of 223 patients [3%]; P = not significant [NS]), although there was a 3-fold excess of minor bleeds (34 of 225 [15%] versus 12 of 223 patients [5%]; P = 0.001). Among those with predialysis renal failure or a functioning transplant at baseline, aspirin did not increase the number of patients who progressed to dialysis therapy (7 of 187 [4%] versus 6 of 188 patients [3%]; P = NS) or experienced a greater than 20% increase in creatinine level (63 of 187 patients [34%] versus 56 of 188 patients [30%]; P = NS). After 12 months of follow-up, allocation to 20 mg of simvastatin daily reduced nonfasting total cholesterol levels by 18% (simvastatin, 163 mg/dL [4.22 mmol/L] versus placebo, 196 mg/dL [5.08 mmol/L]; P < 0.0001), directly measured low-density lipoprotein cholesterol levels by 24% (89 mg/dL [2.31 mmol/L] versus 114 mg/dL [2.96 mmol/L]; P < 0.0001), and triglyceride levels by 13% (166 mg/dL [1.87 mmol/L] versus 186 mg/dL [2.10 mmol/L]; P < 0.01), but there was no significant effect on high-density lipoprotein cholesterol levels (2% increase; P = NS). Allocation to simvastatin therapy was not associated with excess risk for abnormal liver function test results or elevated creatine kinase levels. CONCLUSION: During a 1-year treatment period, simvastatin, 20 mg/d, produced a sustained reduction of approximately one quarter in low-density lipoprotein cholesterol levels, with no evidence of toxicity, and aspirin, 100 mg/d, did not substantially increase the risk for a major bleeding episode. Much larger trials are now needed to assess whether these treatments can prevent vascular events.

Adult↗
PubMed2002

Misunderstandings, misperceptions, and mistakes in the management of the inflammatory myopathies.

Many misconceptions persist concerning fundamental issues related to the idiopathic inflammatory myopathies. Such misconceptions can lead to frank mistakes in the diagnosis and management of these disorders. In some cases, these misperceptions have resulted from overreliance on out-of-date information and "classic" articles that are no longer classic! In other instances, misperceptions persist because of the many voids in our understanding of these diseases. This review uses case presentations to highlight important caveats in diagnosing and managing the common idiopathic inflammatory myopathies.

Adrenal Cortex Hormones↗
PubMed2002

Prognostic significance of creatine kinase-MB elevation after percutaneous coronary intervention in patients with chronic renal dysfunction.

BACKGROUND: Multiple studies have demonstrated a relationship between creatine kinase-MB (CK-MB) elevation after percutaneous coronary intervention (PCI) and increased late mortality within the general population. Because CK-MB is frequently elevated in renal disease even in the absence of myocardial injury, the clinical significance of CK-MB elevation after PCI in patients with renal insufficiency has been questioned. METHODS: We sought to examine the association between elevated CK-MB after PCI and late mortality in 190 consecutive patients with chronic renal insufficiency (serum creatinine > or =2.0 mg/dL) undergoing PCI at the Cleveland Clinic between January 1997 and March 2000. Of the total group, 20 patients undergoing PCI for acute myocardial infarction, cardiogenic shock, or both were excluded. Follow-up was 99.4% complete at a mean duration of 24.8 +/- 11.2 months (range 5-43 months). RESULTS: CK-MB elevation above the upper limit of normal after intervention was detected in 33 patients (19.4%). Baseline characteristics were not significantly different between the CK-MB elevation group and the normal CK-MB group. Late mortality, however, was significantly higher among patients with postprocedural CK-MB elevation (36.4% vs 17.5%, P =.017). Cox proportional hazard model revealed CK-MB elevation as an independent predictor of late mortality (hazard ratio 2.44, 95% CI 1.14-5.24, P =.02), in addition to New York Heart Association class (hazard ratio 1.35, 95% CI 1.05-1.73, P =.02). CONCLUSIONS: This analysis of patients with chronic renal insufficiency undergoing PCI suggests that postprocedural CK-MB elevation is an independent predictor of late mortality even in the presence of renal dysfunction.

Aged↗
PubMed2001

Overexpression of the LAR (leukocyte antigen-related) protein-tyrosine phosphatase in muscle causes insulin resistance.

Previous reports indicate that the expression and/or activity of the protein-tyrosine phosphatase (PTP) LAR are increased in insulin-responsive tissues of obese, insulin-resistant humans and rodents, but it is not known whether these alterations contribute to the pathogenesis of insulin resistance. To address this question, we generated transgenic mice that overexpress human LAR, specifically in muscle, to levels comparable to those reported in insulin-resistant humans. In LAR-transgenic mice, fasting plasma insulin was increased 2.5-fold compared with wild-type controls, whereas fasting glucose was normal. Whole-body glucose disposal and glucose uptake into muscle in vivo were reduced by 39-50%. Insulin injection resulted in normal tyrosyl phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) in muscle of transgenic mice. However, phosphorylation of IRS-2 was reduced by 62%, PI3' kinase activity associated with phosphotyrosine, IRS-1, or IRS-2 was reduced by 34-57%, and association of p85alpha with both IRS proteins was reduced by 39-52%. Thus, overexpression of LAR in muscle causes whole-body insulin resistance, most likely due to dephosphorylation of specific regulatory phosphotyrosines on IRS proteins. Our data suggest that increased expression and/or activity of LAR or related PTPs in insulin target tissues of obese humans may contribute to the pathogenesis of insulin resistance.

Animals↗
PubMed2002

Coupling of cell energetics with membrane metabolic sensing. Integrative signaling through creatine kinase phosphotransfer disrupted by M-CK gene knock-out.

Transduction of metabolic signals is essential in preserving cellular homeostasis. Yet, principles governing integration and synchronization of membrane metabolic sensors with cell metabolism remain elusive. Here, analysis of cellular nucleotide fluxes and nucleotide-dependent gating of the ATP-sensitive K+ (K(ATP)) channel, a prototypic metabolic sensor, revealed a diffusional barrier within the submembrane space, preventing direct reception of cytosolic signals. Creatine kinase phosphotransfer, captured by 18O-assisted 31P NMR, coordinated tightly with ATP turnover, reflecting the cellular energetic status. The dynamics of high energy phosphoryl transfer through the creatine kinase relay permitted a high fidelity transmission of energetic signals into the submembrane compartment synchronizing K(ATP) channel activity with cell metabolism. Knock-out of the creatine kinase M-CK gene disrupted signal delivery to K(ATP) channels and generated a cellular phenotype with increased electrical vulnerability. Thus, in the compartmentalized cell environment, phosphotransfer systems shunt diffusional barriers and secure regimented signal transduction integrating metabolic sensors with the cellular energetic network.

Adenosine Diphosphate↗