BB creatine kinase and myogenic differentiation

Abstract. Antisera specific for the B monomer of creatine kinase (B-CK), the M monomer of creatine kinase (M-CK), and muscle-specific myosin heavy chain (MHC) were used to investigate the biochemical characteristics of individual cells in primary myogenic cultures. Through the use of immunocytochemical techniques, in conjunction with 3H-thy-midine autoradiography, it was determined that (1) all of the terminally differentiated myoblasts contained B-CK in addition to M-CK and MHC, (2) none of the cycling cells contained M-CK or MHC, (3) a fraction (7.5%) of the cycling cells contained B-CK, and (4) the cycling, B-CK positive cells divided once, and only once, and produced two terminally differentiated myoblasts. These results indicate that myogenic precursors in vitro are a phenotypically heterogeneous cell population and that the appearance of B-CK in cycling myogenic cells is a biochemical manifestation of a distinct precursor compartment in the chicken skeletal myogenic lineage.     

Fixation and immunofluorescent analysis of creatine kinase isozymes in embryonic skeletal muscle

Pectoral muscles from chicken embryos of various ages were examined with immunofluorescent and radiolabeled probes for the presence of brain-type creatine kinase (B-CK), muscle-specific creatine kinase (M-CK), muscle-specific myosin heavy chain (MHC), and cycling cells. The diffusible creatine kinase isozymes were not detectable by indirect immunofluorescence after standard histological fixation of embryonic muscle. However, a fixation procedure was devised that permitted immunodetection of the creatine kinase isozymes (particularly B-CK) in embryonic tissue from all stages of development studied. B-CK, M-CK, and MHC were all detected in post-mitotic muscle cells, but only B-CK was detected in cycling cells. Correlations between these findings and in vitro observations of a deterministic muscle lineage are discussed.     

Specific proteolytic modification of creatine kinase isoenzymes. Implication of C-terminal involvement in enzymic activity but not in subunit-subunit recognition.

We are using the isoenzymes of creatine kinase (CK) to investigate the effect of specific proteolytic modification on the abilities of enzyme subunits to establish precise subunit-subunit recognition in vitro. Previous work by others has shown that treatment of the MM isoenzyme of rabbit CK with Proteinase K results in a specific proteolytic modification and inactivation of the enzyme. In the present work, we show that both the MM and BB isoenzymes of chicken CK are also specifically modified by Proteinase K, resulting in over 98% loss of catalytic activity and approx. 10% decreases in subunit molecular masses of the enzymes. Similar reactions appear to occur when the isoenzymes are treated with Pronase E. Limited amino acid sequence analysis of intact and Proteinase K-modified MM-CK suggests that the proteolytic modification results from a single peptide-bond cleavage occurring between alanine residues 328 and 329, about 50 amino acid residues from the C-terminal end; the active-site cysteine residue was recovered in the large protein fragment of modified M-CK subunits. Proteolytically modified M-CK and B-CK subunits were able to refold and reassociate into dimeric structures after treatment with high concentrations of LiCl and at low pH. Thus the proteolytically modified CK subunits retain their ability to refold and to establish precise subunit-subunit recognition in vitro.     

Regulation of creatine kinase induction in differentiating mouse myoblasts.

The regulation of creatine kinase (CK) induction during muscle differentiation was analyzed with MM14 mouse myoblasts. These cells withdraw from the cell cycle and commit to terminal differentiation when fed with mitogen-depleted medium. Myoblasts contained trace amounts of an isozyme of brain CK (designated BB-CK), but differentiation was accompanied by the induction of two other isozymes of muscle and brain CKs (designated MM-CK and MB-CK). Increased CK activity was detectable within 6 h of mitogen removal, 3 h after the first cells committed to differentiation and 6 h before fusion began. By 48 h, MM-CK activity increased more than 400-fold, MB-CK activity increased more than 150-fold, and BB-CK activity increased more than 10-fold. Antibodies prepared against purified mouse MM-CK cross-reacted with muscle and brain CKs (designated M-CK and B-CK, respectively) from a variety of species and were used to demonstrate that the increase in enzymatic activity was paralleled by an increase in the protein itself. CK antibodies were also used to aid in identifying cDNA clones to M-CK. cDNA sequences which corresponded to protein-coding regions cross-hybridized with B-CK mRNA; however, a subclone containing the 3'-nontranslated region was unique and was used to quantitate M-CK mRNA levels during myoblast differentiation. M-CK mRNA was not detectable in myoblasts, but within 5 to 6 h of mitogen withdrawal (6 to 7 h before fusion begins) it accumulated to about 30 molecules per cell. By 24 h, myotubes contained approximately 1,100 molecules per nucleus of M-CK mRNA.     

Accumulation of creatine kinase mRNA during myogenesis: molecular cloning of a B-creatine kinase cDNA

Cytosolic creatine kinase isoenzymes MM, MB, and BB are assembled from M or B subunits which occur in different relative amounts in specific tissues. The accumulation of mRNAs encoding the M and B subunits was measured during myogenesis in culture. The relative concentration of the two mRNAs was determined by hybridization with a M-CK cDNA probe isolated previously and a B-CK cDNA probe, the cloning and characterization of which is reported here. The B-CK cDNA hybridizes specifically to a 1.6-kb mRNA found in brain and gizzard but not in adult skeletal muscle tissue. The M-CK cDNA hybridizes to a smaller mRNA 1.4-kb long which is specific to skeletal muscle. In culture, the B-CK mRNA is transiently induced and then declines to a low but detectable level.     

Approaching the multifaceted nature of energy metabolism: inactivation of the cytosolic creatine kinases via homologous recombination in mouse embryonic stem cells

To study the physiological role of the creatine kinase/phosphocreatine (CK/PCr) system in cells and tissues with a high and fluctuating energy demand we have concentrated on the site-directed inactivation of the B- and M-CK genes encoding the cytosolic CK protein subunits. In our approach we used homologous recombination in mouse embryonic stem (ES) cells from strain 129/Sv. Using targeting constructs based on strain 129/Sv isogenic DNA we managed to ablate the essential exons of the B-CK and M-CK genes at reasonably high frequencies. ES clones with fully disrupted B-CK and two types of M-CK gene mutations, a null (M-CK^–) and leaky (M-CK¹) mutation, were used to generate chimaeric mutant mice via injection in strain C57BL/6 derived blastocysts. Chimaeras with the B-CK null mutation have no overt abnormalities but failed to transmit the mutation to their offspring. For the M-CK^– and M-CK¹ mutations successful transmission was achieved and heterozygous and homozygous mutant mice were bred. Animals deficient in MM-CK are phenotypically normal but lack muscular burst activity. Fluxes through the CK reaction in skeletal muscle are highly impaired and fast fibres show adaptation in cellular architecture and storage of glycogen. Mice homozygous for the leaky M-CK allele, which have 3-fold reduced MM-CK activity, show normal fast fibres but CK fluxes and burst activity are still not restored to wildtype levels.     

Intracellular targeting of isoproteins in muscle cytoarchitecture

Part of the muscle creatine kinase (MM-CK) in skeletal muscle of chicken is localized in the M-band of myofibrils, while chicken heart cells containing myofibrils and BB-CK, but not expressing MM-CK, do not show this association. The specificity of the MM-CK interaction was tested using cultured chicken heart cells as "living test tubes" by microinjection of in vitro generated MM-CK and hybrid M-CK/B-CK mRNA with SP6 RNA polymerase. The resulting translation products were detected in injected cells with isoprotein-specific antibodies. M-CK molecules and translation products of chimeric cDNA molecules containing the head half of the B-CK and the tail half of the M-CK coding regions were localized in the M-band of the myofibrils. The tail, but not the head portion of M-CK is essential for the association of M-CK with the M-band of myofibrils. We conclude that gross biochemical properties do not always coincide with a molecule's specific functions like the participation in cell cytoarchitecture which may depend on molecular targeting even within the same cellular compartment.     

Sequence homology and structure predictions of the creatine kinase isoenzymes

Comparisons of the protein sequences and gene structures of the known creatine kinase isoenzymes and other guanidino kinases revealed high homology and were used to determine the evolutionary relationships of the various guamidino kinases. A CK framework is defined, consisting of the most conserved sequence blocks, and diagnostic boxes are identified which are characteristic for anyone creatine kinase isoenzyme (e.g. for vertebrate B-CK) and which may serve to distinguish this isoenzyme from all others (e.g. from M-CKs and Mi-CKs). Comparison of the guanidino kinases by near-UV and far-UV circular dichroism further indicates pronounced conservation of secondary structure as well as of aromatic amino acids that are involved in catalysis.Abbreviations GuaK guanidino kinase - CK creatine kinase - B-and M-CK brain and muscle cytosolic CK isoenzyme - Mi-CK mitochondrial CK isoenzyme - ArgK arginine kinase - Cr creatine - PCr phosphorylcreatine - PArg phosphorylarginine     

Expression of creatine kinase isoenzymes in myogenic cell lines.

Investigation of creatine kinase isoenzyme activity in several cloned myogenic cell lines showed differences in B-type subunit expression. In cultures of myoblasts isolated from rat skeletal muscle by selective cell plating and in the cell lines M58 and M41, the activity of the mononucleated cells was of the BB isoenzyme. After cell fusion, MM, MB, and BB isoenzymes were present; the main activity was of the MM isoenzyme. In the myogenic lines L8 and L84, in cultures of mononucleated cells, creatine kinase activity was absent or barely detectable. The high creatine kinase activity after cell fusion was of the MM type. No BB and MB activity was detected in these lines at any stage of differentiation. The difference in expression of creatine kinase isoenzymes seems not to affect the expression of other parameters of differentiation.     

Immunoassay of muscle-specific creatine kinase with a monoclonal antibody and application to myogenesis and muscular dystrophy.

A competition e.l.i.s.a. (enzyme-linked immunosorbent assay) is described that enables direct measurement of the muscle-specific polypeptide of chick creatine kinase (M-CK) in extracts of differentiating muscle-cell cultures and in blood plasma samples, even in the presence of embryonic, or brain-type, creatine kinase. The characteristics of the assay can be considerably improved by the use of a monoclonal antibody, CK-ART, instead of rabbit antisera, and we offer an explanation for this in terms of heterogeneity of antibody affinities in polyclonal antisera. In addition to native enzyme, the assay will measure creatine kinase unfolded and inactivated by 8 M-urea treatment. During chick muscle differentiation in vitro, M-CK increased from 7.5% of the total creatine kinase at 24h to 76.0% at 143h, in good agreement with isoenzyme separation data. As a percentage of the total cell protein, M-CK increased by 156-340-fold over the same period and constituted 0.38-0.56% of the total protein in late cultures. E.l.i.s.a. measurements on 17-20-day embryonic thigh-muscle extracts, which contain almost exclusively M-CK, agree well with enzyme activity and radioimmunoassay. M-CK constituted 0.7-1.6% of the total protein in 17-19-day embryonic thigh muscle. Plasma M-CK concentrations in normal 2-8-week-old chickens were found to be in the range 0.5-0.9 micrograms/ml. Plasma concentrations of 32-56 micrograms/ml were found in 8-week-old dystrophic chickens by both e.l.i.s.a. and enzyme-activity measurements. The results suggest that inactive or unfolded forms of M-CK do not normally exist, in any significant amounts, in cell and tissue extracts or in freshly prepared samples of plasma.     

Differentiation in cultures derived from embryonic chicken muscle: II. Phosphorylase histochemistry and fluorescent antibody staining for creatine kinase and aldolase

The presence or absence of five proteins (glycogen phosphorylase, aldolase A, aldolase C, creatine kinase M, creatine kinase B) in the various classes of cells found in primary cultures derived from embryonic chick breast muscle was investigated using cytological staining methods. Histochemical staining for phosphorylase and indirect fluorescent antibody staining for aldolase A and C as well as for creatine kinases M and B showed the following: All five proteins were found in the many myotubes present in standard medium cultures and in the very few myotubes found in cultures containing 5-bromodeoxyuridine (10^?5M). The elongated bipolar cells prevented from fusing in medium containing EGTA also contain all five proteins. The flattened myogenic cells that predominate in the 5-bromodeoxyuridine-treated cultures contain no phosphorylase or creatine kinase M, though many of them contain creatine kinase B and aldolases A and C. These results are interpreted as indicating that: (1) phosphorylase and creatine kinase M, but not aldolase A, are suitable all-or-none markers for terminal muscle differentiation; (2) the small amounts of creatine kinase M detected in electrophoreses of 5-bromodeoxyruridine-treated cultures can be accounted for by the few myotubes present and are not due to “protodifferentiation” of large numbers of cells; (3) proteins typical of differentiated muscle are produced only in cells that have passed through the last step in myogenesis that is susceptible to 5-bromodeoxyuridine inhibition, and (4) if fusion is blocked by reducing the concentration of calcium ions, accumulation of characteristic muscle proteins can continue in those cells that have initiated terminal differentiation.     

Purification and characterization of cytoplasmic creatine kinase isozymes ofXenopus laevis

The soluble creatine kinase isozymes CK-II, CK-III, and CK-IV fromXenopus laevis have been purified to apparent homogeneity and their subunits characterized by means of molecular weight, peptide pattern, and dissociation-reassociation experiments. CK-III and CK-IV are homodimeric isozymes whose subunits are distinct in both molecular weight (42,000 and 41,000, respectively) andStaphylococcus aureus V₈ peptide pattern. In dissociation-reassociation experiments, those two subunits do form active heterodimeric isozymes with one another or with rabbit M-CK subunits. Hybrid CK-III/IV isozymes occur also during embryonic differentiation and in adult heart muscle, whereas most other adult tissues contain only homodimeric CK-III or CK-IV isozymes. The CK-II isozyme is a heterodimer composed of one CK-III subunit and another subunit specific to CK-II (M _r =41,000). Neitherin vivo norin vitro does this subunit seem able to form homodimers or heterodimers with CK-IV and rabbit M-CK subunits. If we take into account the apparent association of CK-I isozyme with cellular organelles, these results corroborate earlier statements and suggest that the CK isozyme system ofX. laevis is encoded by at least four differentially regulated genomic loci.     

Occurrence of heterogenous forms of the subunits of creatine kinase in various muscle and nonmuscle tissues and their behaviour during myogenesis

Purified, homodimeric creatine kinases from chicken were subjected to two-dimensional gel analysis under dissociating conditions. Each of the subunits M-creatine kinase and B-creatine kinase was resolved into a basic and an acidic subspecies with very similar mobilities in the sodium dodecylsulfate dimension. The M-creatine kinase subspecies were found in myogenic cells, fast muscle, slow muscle and the B-creatine kinase subspecies were present in heart, gizzard and brain. The creatine kinase subunits were identified in these tissues by a variety of methods like immunoreplicas of two-dimensional gels, immunoprecipitations, or coelectrophoresis with purified creatine kinase and all gave the same results. In the course of myogenic development in vitro the subspecies were synthesized coordinately and no indication was found for a differential regulation of any of the subspecies of the creatine kinase subunits. No radioactive phosphorus was incorporated into either one of the subspecies, hence phosphorylation could be ruled out as the source of heterogeneity. Furthermore, peptide mapping analysis of partial proteolytic digests did not reveal differences among the subspecies of the same subunit. Not only chicken but also rat creatine kinase displayed this type of heterogeneity. All subspecies were observed after translation of chicken RNA in a cell-free protein-synthesizing system. The heterogeneity probably might best be explained by the existence of multiple, but closely related genes for the creatine kinase subunits.     

Multiple isoforms and an unusual cathodic isoform of creatine kinase from channel catfish (Ictalurus punctatus)

In vertebrates, the creatine kinase (CK) family consists of two cytosolic and two mitochondrial isoforms. The two cytosolic isoforms are the muscle type (M-CK) and the brain type (B-CK). Here we report multiple CK isoenzymes in the diploid channel catfish (Ictalurus punctatus) with one unusual cathodic isoform that was previously found only in pathological situations in human. The cathodic CK isoform existed only in the channel catfish stomach, ovary, and spleen, but not in any other species analyzed such as tilapia, smallmouth bass, chicken, or rat. Two genes encode the multiple forms of the channel catfish M-CK cDNAs. M-CK1 has three alleles, M-CK1.1, M-CK1.2, and M-CK1.3, while M-CK2 has just one allele as determined by analysis of 17 cDNA clones and by allele-specific PCR. M-CK1 encodes a protein of 381 amino acids and the M-CK2 cDNA encodes a protein of 380 amino acids. The two cDNAs shared an 86% identity and both have the nine diagnostic boxes for cytosolic CKs and thus are of cytosolic origin. The M-CK1 gene was isolated, sequenced, and characterized and its promoter should be useful for transgenic research for muscle-specific expression.     

Coordinated synthesis and degradation of actin and myosin in a variety of myogenic and non-myogenic cells

The turnover of myosin and actin in both muscle and non-muscle cells in culture was investigated. By the double-label criterion, myosin and actin were coordinately synthesized and degraded in replicating, mononucleated fibroblasts, chondrocytes, BUdR-suppressed myogenic cells, and in post-mitotic, multinucleated myotubes. Myosin and actin were among the most stable proteins in each cell type. In single label ‘pulse-chase’ experiments, the half-lives of myosin and actin in all replicating, mononucleated cells were 2.5–3 days; in myotubes, however, they were approx. 6 days. Myosin and actin labelled in replicating presumptive myoblasts and chased until the cells ceased replicating and fused into multinucleated myotubes retained the degradation rate of 3 days; this differed from Jhe rate of 6 days shown for myosin and actin newly-synthesized in post-mitotic myotubes. The type of myosin synthesized in the mother presumptive myoblast, then, is transmitted to the postmitotic daughters. This myosin, however, is more rapidly degraded than the definitive myosin that is synthesized in the myotube.     

Discrete subcellular localization of a cytoplasmic and a mitochondrial isozyme of creatine kinase in intestinal epithelial cells

Two isozymes of creatine kinase have been purified differentially from mitochondrial and cytoplasmic subfractions of intestinal epithelial cells. These intestinal epithelial cell creatine kinases were indistinguishable from the cytoplasmic (B-CK) and mitochondrial (Mi-CK) creatine kinase isozymes of brain when compared by SDS-PAGE, cellulose polyacetate electrophoresis, and peptide mapping. In intestinal epithelial cells, immunolocalization of the Mi-CK isozyme indicates that it is associated with long, thin mitochondria, which are excluded from the brush border at the apical end of each cell. In contrast, immunolocalization of the B-CK isozyme indicates that it is concentrated distinctly in the brush border terminal web domain. Although absent from the microvilli, B-CK also is distributed diffusely throughout the cytoplasm. Terminal web localization of B-CK was maintained in glycerol-permeabilized cells and in isolated brush borders, indicating that B-CK binds to the brush border structure. The abundance and localization of the mitochondrial and cytoplasmic creatine kinase isozymes suggest that they are part of a system that temporally and/or spatially buffers dynamic energy requirements of intestinal epithelial cells.     

Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly

The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin- treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits accumulate, and ultimately make functional receptors in AChR- expressing cells.     

Acute changes of myocardial creatine kinase gene expression under beta-adrenergic stimulation

Creatine kinase (CK) plays a crucial role in myocardial energy metabolism. Alterations in CK gene expression are found in hypertrophied and failing heart, but the mechanisms behind these changes are unclear. This study tests the hypothesis that increased adrenergic stimulation, which is observed in heart failure, induces changes of myocardial CK-activity, -isoenzyme distribution and -gene expression that are characteristic of the failing and hypertrophied heart. Isolated rat hearts were perfused (constant pressure of 80 mmHg) with red cell suspensions. Following a 20-min warm-up period, perfusion for 3 h with 10(-8) M (iso 3 h) or without (control 3 h) isoproterenol was started or experiments were immediately terminated (control 0 h). Left ventricular tissue was analyzed for total CK-activity, CK-isoenzyme distribution and, by use of quantitative RT-PCR, for B-CK, M-CK, mito-CK and GAPDH- (as internal standard) mRNA. After beta-adrenergic stimulation (iso 3 h) but not after control perfusion (control 3 h) a roughly threefold increase in B-CK mRNA levels and a decrease in M-CK mRNA levels by 18% was found. There were no significant differences among the three groups in total CK-activity and in distribution of CK-MM, CK-BB, CK-MB and mito-CK. Thus, beta-adrenergic stimulation induces a switch in CK gene expression from M-CK to B-CK, which is characteristic for the hypertrophied and failing heart. This may be interpreted as an adaptive mechanism making energy transduction via CK more efficient at times of increased metabolic demand.