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PubMed2004

Expression of muscle creatine kinase gene of mice and interaction of nuclear proteins with MEF-2, E boxes and A/T-rich elements during aging.

The changes in the expression of muscle creatine kinase (MCK) gene in the heart and skeletal muscle of mice during aging were studied. Its expression declines as a function of age in the heart, however, no age-related change is observed in the skeletal muscle. The cis-acting elements, MEF-2, E boxes and A/T rich elements present in the enhancer region of the mouse MCK gene are known to regulate the expression of the gene. Hence, these elements were subcloned and electrophoretic mobility shift assay was carried out to investigate the changes in the binding of the nuclear trans-acting protein factors of the heart with these elements as a function of age. These factors showed specificity for the respective cis-acting elements. Furthermore, the binding of these factors was found to decrease during aging which may contribute to the age-related decline in the expression of the MCK gene and activity of the heart.

AT Rich Sequence↗
PubMed2005

In vitro measurement of enzymatic markers as a tool to detect mouse cardiomyocytes injury.

Primary cultures of cardiomyocytes represent a useful model for analyzing cardiac cell biology as well as pathogenesis of several cardiovascular disorders. Our aim was to standardize protocols for determining the damage of cardiac cells cultured in vitro by measuring the creatine kinase and its cardiac isotype and lactate dehydrogenase activities in the supernatants of mice cardiomyocytes submitted to different protocols of cell lysis. Our data showed that due to its higher specificity, the cardiac isotype creatine kinase was the most sensitive as compared to the others studied enzymatic markers, and can be used to monitor and evaluate cardiac damage in in vitro assays.

Animals↗
PubMed1986

The isoelectric focusing of creatine kinase variants: II. The heterogeneity of creatine kinase in human serum with normal and elevated catalytic concentrations.

An effective and reliable method for the quantitative estimation of creatine kinase-MB, creatine kinase-MM variants and mitochondrial forms of creatine kinase in serum is presented. The high resolving power of isoelectric focusing allows the use of tetrazolium salts and meldola blue for the quantitative measurement without interfering non-specific reduction. The addition of thiol compounds to the agarose medium increases the sensitivity of the method, due to the inhibition of sulfhydryl group oxidation, and prevents enzyme degradation, which is a possible cause of an artificial heterogeneity. Depending upon the type of muscle and the degree of cell damage, we found 3-4 creatine kinase-MM sub-bands in sera with activities below 80 U/l. At elevated creatine kinase activities 3-11 creatine kinase-MM sub-bands were found. The appearance of creatine kinase-MB in serum indicates that damage has occurred to certain organs, especially the cardiac muscle. An organ with moderate or massive cell damage could release, in addition to the sarcoplasmatic creatine kinase variants, other forms with more alkaline isoelectric points (mitochondrial creatine kinase). The presence of such bands in serum of patients correlates with poor prognosis. Besides the separation of creatine kinase-MM sub-bands, creatine kinase-MB, creatine kinase-BB and of macroforms 1 and 2, the advantage of this method is the detection of mitochondrial creatine kinase forms, which in cellulose acetate electrophoresis migrate with creatine kinase-MM.

Catalysis↗
PubMed1997

Sarcoplasmic reticulum function in determining atrioventricular contractile differences in rat heart.

The relationships between the contractile characteristics and the sarcoplasmic reticulum (SR) function of rat atrial and ventricular trabeculae were compared. The isometric developed tension (DT) and the rates of contraction (+ dT/dt) and relaxation (-dT/dt) normalized to cross-sectional area were 3.7, 2.2, and 1.8 times lower, respectively, in intact atrial strips compared with ventricular strips, whereas + dT/dt and -dT/dt (normalized to DT) were 2.3 and 2.8 times higher, respectively, in atria. Atria exhibited a maximal potentiation of DT after shorter rest periods than ventricles and a lower reversal for prolonged rest periods. Caffeine-induced tension transients in saponin-permeabilized fibers suggested that the Ca2+ concentration released in atrial myofibrils reached a lower maximum and decayed more slowly than in ventricular preparations. However, the tension-time integrals indicated an equivalent capacity of sequestrable Ca2+ in SR from both tissues. In atrial, as in ventricular myocardium, the SR Ca2+ uptake was more efficiently supported by ATP produced by the SR-bound MM form of creatine kinase (CK; MM-CK) than by externally added ATP, suggesting a tight functional coupling between the SR Ca2+ adenosinetriphosphatase (ATPase) and MM-CK. The maximal rate of oxalate-supported Ca2+ uptake was two times higher in atrial than in ventricular tissue homogenates. The SR Ca(2+)-ATPase 2a mRNA content normalized to 18S RNA was 38% higher in atria than in ventricles, whereas the amount of mRNA encoding the alpha-myosin heavy chain, calsequestrin, and the ryanodine receptor was similar in both tissues. Thus a lower amount of readily releasable Ca2+ together with a faster uptake rate may partly account for the shorter time course and lower tension development in intact atrial myocardium compared with ventricular myocardium.

Animals↗
PubMed1997

Denaturation by guanidinium chloride of dimeric MM-creatine kinase and its proteinase K-nicked form: evidence for a multiple-step process.

Cytosolic MM-creatine kinase is a homodimeric protein. Each monomer can be cleaved by proteinase K at an exposed surface loop, into two fragments K1 and K2, which remain associated. The nicked protein is thus a heterotetrameric protein, named (K1K2)2, made up of two heterodimers K1K2 linked together by their K1 subunit. In non-denaturing conditions, the cleaved protein does not present any measurable difference compared with uncleaved MM-creatine kinase, except for the loss of enzymatic activity. Comparative equilibrium denaturation of the two oligomeric proteins by guanidinium chloride indicates a multistep process with formation of either compact monomer or compact K1K2 dimer, a molten globule and a pre-molten globule state. In the case of the nicked-enzyme, the molten globule is composed of the two peptides K1 and K2, whereas in the pre-molten globule the interactions between K1 and K2 are too weak to maintain their cohesion. At low guanidinium chloride concentration, the proteinase K-nicked protein exhibits a higher accessibility of one of its tryptophan accompanied by a small decrease in its molar ellipticity suggesting a secondary structure loosening of the K1 peptide. Our results suggest that K1 and K2 are not strictly autonomous unfolding units and thus cannot be considered as independent domains.

Animals↗
PubMed1987

Creatine kinase isoform analysis in the detection and assessment of thrombolysis in man.

Recent demonstrations of the efficacy of intravenous thrombolytic therapy in acute myocardial infarction have emphasized the need for a noninvasive index of successful reperfusion. The tissue form of MM creatine kinase (MM3) is known to undergo posttranslational conversion to modified forms MM2 and MM1 after release into the plasma following acute infarction. Since this conversion is rapid, sustained elevation of plasma MM3 may be a marker of the prolonged creatine kinase release characteristic of nonreperfused infarction. Therefore, we investigated the rate of decline of plasma MM3 in a consecutive series of patients undergoing thrombolytic therapy of acute myocardial infarction, all of whom underwent acute angiography to assess treatment success, as well as in 30 conventionally treated patients. Among 55 patients with angiographically documented successful reperfusion (group IA), the rate of decline of MM3 was 4.18 +/- 1.25%/hr (mean +/- SD); in contrast, the rate of decline was 2.37 +/- 1.11%/hr in 39 patients with angiographically documented unsuccessful reperfusion (group IB) and 1.77 +/- 1.46%/hr among the 30 patients receiving conventional treatment (group II) (p less than .001 for groups IB and II vs group IA). A cutoff value of 3.1%/hr minimized the overlap between the groups; 48/55 (87%) patients with successful reperfusion had a rate of decline of MM3 of 3.1%/hr or more, while 29 of 39 (74%) patients in whom thrombolysis was unsuccessful and 27 of 30 (90%) patients receiving conventional treatment had a rate of decline less than 3.1%/hr (p less than .001 for groups IB and II vs group IA).(ABSTRACT TRUNCATED AT 250 WORDS)

Angioplasty, Balloon↗
PubMed1989

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles.

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.

Animals↗
PubMed2006

The evolution from asparagine or threonine to cysteine in position 146 contributes to generation of a more efficient and stable form of muscle creatine kinase in higher vertebrates.

Creatine kinase, a key enzyme in vertebrate excitable tissues that require large energy fluxes, catalyzes the reversible transfer of phosphate between adenosine triphosphate and creatine. Sequence alignment indicated that the 146th amino acid is cysteine in the muscle creatine kinase of higher vertebrates including Amphibia, Reptilia, Aves and Mammalia. In fishes, it is cysteine in Agnatha and Chondrichthyes, and asparagine or threonine in Osteichthyes, which is the ancestor of Amphibia, Reptilia, Aves and Mammalia. To explore the structural and functional role of this special residue, a series of site-directed mutants of rabbit muscle creatine kinase were constructed, including C146S, C146N, C146T, C146G, C146A, C146D and C146R. A detailed comparison was made between wild-type creatine kinase and the mutants in catalytic activity, physico-chemical properties and structural stability against thermal inactivation and guanidine hydrochloride denaturation. It was found that except for C146S, the mutants had relatively lower catalytic activity and structural stability than Wt-CK. Wt-CK and C146S were the most stable ones, followed by C146N and C146T, and then C146G and C146A, and C146D and C146R were the least stable mutants. These results suggested that the 146th residue plays a crucial role in maintaining the structural stability of creatine kinase, and that the evolution in this amino acid from asparagine or threonine to cysteine contributes to the generation of a more efficient and more stable form of creatine kinase in higher vertebrates.

Amino Acid Sequence↗
PubMed2001

Structure of human muscle creatine kinase.

The crystal structure of human muscle creatine kinase has been determined by the molecular-replacement method and refined at 3.5A resolution. The structures of both the monomer and the dimer closely resemble those of the other known structures in the creatine kinase family. Two types of dimers, one with a non-crystallographic twofold symmetry axis and the other with a crystallographic twofold symmetry axis, were found to exist simultaneously in the crystal. These dimers form an infinite "double-helix"-like structure along an unusual long crystallographic 3(1) axis.

Creatine Kinase↗
PubMed2003

Asparagine 285 plays a key role in transition state stabilization in rabbit muscle creatine kinase.

To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site-directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with the major cause of activity loss being a reduction in k(cat) in comparison to the recombinant native CK. The data for N285Q still fit a random-order, rapid-equilibrium mechanism, with either MgATP or creatine binding first with affinities very nearly equal to those for native CK. However, the affinity for the binding of the second substrate is reduced approximately 10-fold, suggesting that addition of a single methylene group at position 285 disrupts the symphony of substrate binding. The data for the N285A mutant only fit an ordered binding mechanism, with MgATP binding first. Isosteric replacement to form the N285D mutant has almost no effect on the K(M) values for either creatine or MgATP, thus the decrease in activity is due almost entirely to a 5000-fold reduction in k(cat). Using the quenching of the intrinsic CK tryptophan fluorescence by added MgADP (Borders et al. 2002), it was found that, unlike native CK, none of the mutants have the ability to form a quaternary TSAC. We use these data to propose that asparagine 285 indeed plays a key role in transition state stabilization in the reaction catalyzed by creatine kinase and other phosphagen kinases.

Adenosine Diphosphate↗
PubMedDate not supplied

Mutations in the dystrophin gene are associated with sporadic dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is the major indication for heart transplantation. Approximately 30% of all DCM is thought to be inherited, while 70% is sporadic. Mutations in the dystrophin gene have been associated with the uncommon X-linked form of DCM. We hypothesized that missense mutations and other less severe mutations of the dystrophin gene might predispose to the common form of sporadic DCM. To test this hypothesis, 22kb of genomic dystrophin DNA was scanned with DOVAM-S in each of the 22 patients with sporadic DCM, including all 79 coding sequences and splice junctions, as well as six alternative exon 1 dystrophin isoforms (484kb, total). Three putative new mutations (IVS5+1 G>T, K18N, and F3228L) and seven polymorphisms were identified. The splice site mutation IVS5+1 is predicted to cause skipping of exon 5, which is within a region containing an actin binding site. The missense mutations occur at amino acids that display substantial evolutionary conservation. Screening of 236 control individuals failed to identify these three mutations. The three patients with putative mutations had CK-MM (creatine kinase, skeletal muscle) levels greater than 250 units while the 14 patients without mutations for which CK-MM were available had values ranging from 20 to 200. The first comprehensive mutation scanning of the exons and splice junctions of the dystrophin gene in patients with sporadic DCM presents the evidence that point mutations are associated with sporadic DCM without clinical evidence of skeletal myopathy. It may be prudent to measure CK-MM in all patients with dilated cardiomyopathy to identify candidates at high risk for dystrophin mutations.

Adolescent↗
PubMed1984

Carboxypeptidase-catalyzed hydrolysis of C-terminal lysine: mechanism for in vivo production of multiple forms of creatine kinase in plasma.

Human myocardial creatine kinase isoenzyme MM is present as a single form in tissue, but upon its release into plasma two additional forms, with faster anodal migration, are apparent on polyacrylamide electrophoresis. We designate the three forms as MM3, MM2, and MM1 in increasing order of anodal mobility. When tissue creatine kinase isoenzyme MM (MM3) is incubated with either carboxypeptidase N or carboxypeptidase B it is converted into the two additional forms, MM2 and MM1. The carboxy terminal amino acid of human, canine, and rabbit tissue MM3 was determined to be lysine, a specific substrate for carboxypeptidases N and B. Evidently the mechanism for the production of multiple forms of creatine MM in human plasma is the hydrolysis of a positively charged C-terminal lysine residue from one M subunit (MM2), followed by hydrolysis of the C-terminal lysine from the other subunit (MM1).

Animals↗
PubMed2005

Transition state stabilization by six arginines clustered in the active site of creatine kinase.

Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.

Adenosine Diphosphate↗
PubMed2004

Gene transfer of connexin43 into skeletal muscle.

Cellular cardiomyoplasty using skeletal myoblasts may be beneficial for infarct repair. One drawback to skeletal muscle cells is their lack of gap junction expression after differentiation, thus preventing electrical coupling to host cardiomyocytes. We sought to overexpress the gap junction protein connexin43 (Cx43) in differentiated skeletal myotubes, using retroviral, adenoviral, and plasmid-mediated gene transfer. All strategies resulted in overexpression of Cx43 in cultured myotubes, but expression of Cx43 from constitutive viral promoters caused significant death upon differentiation. Dye transfer studies showed that surviving myotubes contained functional gap junctions, however. Retrovirally transfected myoblasts did not express Cx43 after grafting into the heart, possibly due to promoter silencing. Adenovirally transfected myoblasts expressed abundant Cx43 after forming myotubes in cardiac grafts, but grafts showed signs of injury at 1 week and had died by 2 weeks. Interestingly, transfection of already differentiated myotubes with adenoviral Cx43 was nontoxic, implying a window of vulnerability during differentiation. To test this hypothesis, Cx43 was expressed from the muscle creatine kinase (MCK) promoter, which is active only after myocyte differentiation. The MCK promoter resulted in high levels of Cx43 expression in differentiated myotubes but did not cause cell death during differentiation. MCK-Cx43-transfected myoblasts formed viable cardiac grafts and, in some cases, Cx43-expressing myotubes were in close apposition to host cardiomyocytes, possibly allowing electrical coupling. Thus, high levels of Cx43 during skeletal muscle differentiation cause cell death. When, however, expression of Cx43 is delayed until after differentiation, using the MCK promoter, myotubes are viable and express gap junction proteins after grafting in the heart. This strategy may permit electrical coupling of skeletal and cardiac muscle for cardiac repair.

Adenoviridae↗
PubMed2005

Dimerisation of myomesin: implications for the structure of the sarcomeric M-band.

The sarcomeric M-band is thought to provide a link between the thick and the elastic filament systems. So far, relatively little is known about its structural components and their three-dimensional organisation. Myomesin seems to be an essential component of the M-band, since it is expressed in all types of vertebrate striated muscle fibres investigated and can be found in its mature localisation pattern as soon as the first myofibrils are assembled. Previous work has shown that the N-terminal and central part of myomesin harbour binding sites for myosin, titin and muscle creatine kinase. Intrigued by the highly conserved domain layout of the C-terminal half, we screened for new interaction partners by yeast two-hybrid analysis. This revealed a strong interaction of myomesin with itself. This finding was confirmed by several biochemical assays. Our data suggest that myomesin can form antiparallel dimers via a binding site residing in its C-terminal domain 13. We suggest that, similar to alpha-actinin in the Z-disc, the myomesin dimers cross-link the contractile filaments in the M-band. The new and the already previously identified myomesin interaction sites are integrated into the first three-dimensional model of the sarcomeric M-band on a molecular basis.

Animals↗
PubMed2002

M-LDH serves as a sarcolemmal K(ATP) channel subunit essential for cell protection against ischemia.

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.

Adenocarcinoma↗
PubMed2002

Overexpression of chemokine-like factor 2 promotes the proliferation and survival of C2C12 skeletal muscle cells.

Chemokine-like factor 1 (CKLF1) is a novel cytokine first cloned from U937 cells. It contains different splicing forms and has chemotactic effects on a wide spectrum of cells both in vitro and in vivo; it can also stimulate the regeneration of skeletal muscle cells in vivo, but the mechanism remains unclear. To probe the myogenesis function of CKLF2, which is the largest isoform of CKLFs, C2C12 murine myoblasts were stably transfected with human CKLF2 eukaryotic expression vector. Compared with control vector transfected C2C12 cells, CKLF2 overexpression causes accelerated myoblast proliferation as determined by cell counting and [(3)H]TdR incorporation assays. In addition, CKLF2 overexpression also promotes cell differentiation, which was determined by higher expression levels of myogenin, creatine kinase, myosin and the accelerated myoblast fusion. Further analysis also indicates that CKLF2 could activate the transcription activity of the bHLH/MyoD and MEF2 families. Finally, DNA synthesis and myotube formation could also be promoted by growing C2C12 cells in conditioned media from CKLF2-transfected cells. These findings strongly suggest a role for human CKLF2 in regulation of skeletal muscle myogenesis.

Animals↗