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.     

Analysis of myogenesis by somatic cell hybridization: III. Myogenic competence of hybrids derived from rat L6 myoblasts and mouse primary fibroblasts and myoblasts

Hybrid cells derived from rat L6 myoblasts and mouse primary fibroblasts (M x F hybrids), as well as those derived from rat L6 myoblasts and mouse primary myoblasts (M x M hybrids), were examined for their ability to engage in myogenesis as judged by muscle fiber formation plus the expression of skeletal muscle myosin and creatine kinase (CK). Of 172 primary hybrid colonies scored, 59% were myogenic in the M x F fusion and 97% exhibited muscle fiber formation in the M x M fusion. Individual hybrid clones from each cross were isolated, expanded and analyzed for myogenic capabilities as well. All three M x M and all ten M x F isolated clones exhibited preferential elimination of mouse chromosomes. Nonetheless, all were capable of fusing spontaneously and of elaborating skeletal muscle myosin and CK. The three M x M hybrids expressed only MM-CK whereas nine out of ten M x F hybrids produced all three CK isoenzymes (MM, MB, BB). These results suggest that M X M hybrids express CK patterns reminiscent of the rat L6 parental cells while M X F hybrids apparently mimic mouse muscle fiber CK patterns. Various models are discussed which address these phenomena.     

Hemin enhances differentiation and maturation of cultured regenerated skeletal myotubes

Satellite cells, isolated from marcaine-damaged rat skeletal muscle, differentiate in culture to form contracting, cross-striated myotubes. Addition of 20 microM hemin (ferriprotoporphyrin IX chloride) to the culture medium resulted in increases in the number, size, and alignment of myotubes; in the number of myotubes that exhibited cross-striations; and in the strength and frequency of myotube contractions. Hemin increased satellite cell fusion by 27%, but decreased cell proliferative rate by 30%. Hemin increased the specific activity of creatine kinase (CK), a sensitive indicator of muscle differentiation, by 157%. Separation of CK isoenzymes by agarose gel electrophoresis showed that hemin increased only the muscle-specific CK isoenzymes (MM-CK and MB-CK). Thus, hemin seems to duplicate some of the effects of innervation on cultured myotubes by increasing contraction frequency and strength, appearance of cross-striations, and muscle-specific isoenzymes. In contrast, 3-amino-1,2,4-triazole, an inhibitor of heme biosynthesis, decreased the number of cross-striated myotubes, the strength and frequency of myotube contractions, and CK activity. These inhibitory effects were reversed by hemin. Collectively, these results demonstrate a physiologically significant role for heme in myotube maturation.     

Isoenzyme-specific interaction of muscle-type creatine kinase with the sarcomeric M-line is mediated by NH(2)-terminal lysine charge-clamps

Creatine kinase (CK) is located in an isoenzyme-specific manner at subcellular sites of energy production and consumption. In muscle cells, the muscle-type CK isoform (MM-CK) specifically interacts with the sarcomeric M-line, while the highly homologous brain-type CK isoform (BB-CK) does not share this property. Sequence comparison revealed two pairs of lysine residues that are highly conserved in M-CK but are not present in B-CK. The role of these lysines in mediating M-line interaction was tested with a set of M-CK and B-CK point mutants and chimeras. We found that all four lysine residues are involved in the isoenzyme-specific M-line interaction, acting pair-wise as strong (K104/K115) and weak interaction sites (K8/K24). An exchange of these lysines in MM-CK led to a loss of M-line binding, whereas the introduction of the very same lysines into BB-CK led to a gain of function by transforming BB-CK into a fully competent M-line-binding protein. The role of the four lysines in MM-CK is discussed within the context of the recently solved x-ray structures of MM-CK and BB-CK.     

Kinetic, thermodynamic, and developmental consequences of deleting creatine kinase isoenzymes from the heart. Reaction kinetics of the creatine kinase isoenzymes in the intact heart

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.     

Localization of creatine kinase isoenzymes in myofibrils. I. Chicken skeletal muscle

Purified, repeatedly washed, skeletal muscle myofibrils contain approx. 0.2 U of creatine kinase (CK) activity (equivalent to 2.5 micrograms CK) per milligram dry weight; this firmly bound CK activity is estimated to represent 3-5% of the total cellular CK. It had been shown previously that the myofibrillar CK, which can be quantitatively extracted at low ionic strength and purified to homogeneity, is very similar, if not identical, to the bulk MM-CK. It is shown that the two protein preparations also have the same peptide pattern after cyanogen bromide fractionation and very similar specific activities, confirming their identity. The earlier demonstration that the bound CK is specifically located at the M-lines of isolated myofibrils has been confirmed by immunofluorescence. Antibodies directed against purified MM- and BB-CK were used in the indirect fluorescent antibody technique to study the specificity of myofibril binding sites for different forms of CK. With myofibrils from adult muscle, which has only MM-CK, as well as from early developmental stages in which BB-CK is the predominant isoenzyme, M-type CK was localized exclusively at the M-line, while greater or lesser amounts of B-type CK were found at the Z-line. The data provide strong evidence that the MM-CK at the M-lines in skeletal myofibrils is not adventitiously bound but is rather an integral element in the M-line structure. The amount of CK bound is reasonably consistent with the earlier proposal that the CK molecules might be the transverse M-bridges and appears to be sufficient to regenerate all of the ATP hydrolyzed during muscle contraction.     

Isoenzyme-directed selection and characterization of anti-creatine kinase single chain Fv antibodies from a human phage display library

Epitopes differing among isoenzymes of creatine kinase (CK) are apparently limited in number and poorly immunogenic in vivo. Especially for the BB-CK isoenzyme, very few monoclonal antibodies (mAb) are available. Here, we use in vitro selection with a synthetic human phage display antibody library and develop isoenzyme competition and peptide panning strategies to obtain human single chain Fv (scFv) antibodies against specific CK isoenzymes. We isolated and characterized seven scFv clones that recognize native as well as denatured cytosolic BB-CK in ELISA, immunoblot, immunofluorescence histochemistry and surface plasmon resonance (SPR) spectroscopy. To a variable but minor degree, they also react with cytosolic MM-CK, but not with mitochondrial CK isoenzymes. Epitope mapping revealed that the scFv antibodies recognize different BB-CK epitopes, including the N-terminus and the isoenzyme-specific box, a highly conserved sequence of unknown function for which no mAb were available so far. With a K(D) of 3.5-9.6 x 10(-7) M, the isolated scFv compare favorably with mouse mAb and may overcome certain of their limitations. Our results demonstrate the advantages of in vitro antibody selection for the generation of isoenzyme-specific antibodies.     

Why is creatine kinase a dimer? Evidence for cooperativity between the two subunits

The dimeric chicken brain type isoenzyme of creatine kinase (BB-CK) was mutated by a C283S amino acid exchange in the catalytic site to produce a basically inactive dimer (B*B*-CK). The mutated enzyme showed a residual activity of about 4% compared to the wild-type, whereas substrate binding parameters were not altered. The inactivated dimer was hybridized with native dimeric muscle enzyme (MM-CK) to produce a partially inactivated MB*-CK heterodimeric hybrid and also to a his-tagged BB-CK (hBhB-CK) resulting in a partially inactive hBB*-CK homodimer. The generated hybrids were purified by chromatography. The V(max) and substrate binding parameters K(m) and K(d) were determined for both directions of the CK reaction and compared to the parameters of the wild-type enzymes (MM-, BB-, hBhB-, MB-CK). In the direction of ATP synthesis (reverse reaction), the MB*- and hBB*-CK hybrids showed a decrease of V(max) to 34% and 32%, respectively, compared to the unmodified wild-type isoform. The inactivation of a single subunit in MB*-CK led to an increase in the K(d) value resulting in an significant substrate synergism, not seen with the MB-CK wild-type enzyme. In the direction of phosphocreatine synthesis (forward reaction), the modified hybrids showed a decrease of V(max) to 50% of the wild-type enzymes and no significant alterations of the K(m) and K(d) parameters. These results strongly suggest an enzymatic cooperativity of the two subunits in the reverse reaction but independent catalytic function in the forward reaction.     

Creatine kinase in epithelium of the inner ear.

Epithelium of the inner ear in the gerbil and mouse was examined immunocytochemically for presence of creatine kinase (CK). Marginal cells of the cochlear stria vascularis and dark cells and transitional cells of the vestibular system were found to contain an abundance of the MM isozyme (MM-CK). CK in these cells concurs with that which is coupled to Na,K-ATPase in other cells and is considered to supply ATP for the Na,K-ATPase that mediates the high KCl of endolymph. Inner hair cells revealed content of the BB isozyme and in this respect resembled the energy-transducing photoreceptor cells in retina. In addition, outer phalangeal (Deiters') cells stained for both MM- and BB-CK whereas inner phalangeal cells evidenced content of only the BB isozyme. Immunolocalization of CK appeared similar in mouse and gerbil inner ear. Specificity of the staining was affirmed by observations in agreement with those reported for CK in various cell types and by staining with antisera from more than one source.     

Localization of creatine kinase isoenzymes in myofibrils. II. Chicken heart muscle

Chicken heart muscle contains almost exclusively the BB isoenzyme of creatine kinase (CK), its myofibrils, moreover, lack an M-line. This tissue thus provides an interesting contrast to skeletal muscle, in which some of the MM-CK present as predominant CK isoenzyme is bound at the myofibrillar M-line. Approx. 2% of the total CK activity in a chicken heart homogenate remains bound to the myofibrillar fraction after repeated washing cycles; both the fraction and the absolute amount of CK bound are about threefold lower than in skeletal muscle. Almost all of the bound enzyme is located within the Z-line region of each sarcomere, as revealed by indirect fluorescent-antibody staining with antiserum against purified chicken BB-CK. After incubation with exogenous purified MM-CK, positive immunofluorescent staining for M- type CK at the H-region of heart myofibrils was observed, along with weaker fluorescence in the Z-line region. Chicken heart myofibrils may thus possess binding sites for both M and B forms of CK.     

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.     

Creatine kinase binding and possible role in chemically skinned guinea-pig taenia coli.

The activity and role of creatine kinase (CK) associated with contractile proteins of smooth muscle have been investigated using skinned guinea-pig taenia coli fibers. Total CK activity was 163 +/- 22 IU/g (ww) and agarose electrophoresis showed BB, MB, and MM isoforms (BB-CK being the predominant isoenzyme). After skinning for 1 h with Triton X-100, BB-CK was specifically associated with the myofibrils, representing 22% of the preskinned CK activity. When relaxed fibers were exposed to pCa 9 in the presence of 250 microM ADP, 0 ATP and 12 mM PCr, tension was not significantly different from resting tension, but changing to pCa 4.5 caused the fibers to generate 59.1 +/- 5.2 percent of maximal tension. When a high-tension rigor state was achieved (250 microM ADP, 0 ATP, 0 PCr, and pCa 9), the addition of 12 mM PCr effected significant relaxation. These observations implicate an endogenous form of BB-CK, associated with the myofilaments and capable of producing enough ATP for submaximal tension generation and significant relaxation from rigor conditions. It was also shown that ADP is bound to the myofibrils and available for rephosphorylation by BB-CK. These results suggest co-localization of ATPase, MLCK and CK on the contractile proteins of the taenia coli. This enzymic association may play a role in the compartmentation of adenine nucleotides in smooth muscle.     

Inhibition of chicken myogenesis invitro by partially purified interferon

Continuous treatment of cultured chicken myoblasts with partially purified chicken interferon (IFN) inhibited myotube formation and the expression of MM-creatine kinase (MM-CK) at day 3, followed by continued MM-CK inhibition and concomitant stimulation of BB-creatine kinase (BB-CK) at day 4. Inhibition of MM-CK was also seen in IFN-treated cells prevented from fusing with EGTA. The degree of inhibition at day 3 depended on IFN dose over a range of 2.5–250 international units (IU)/ml; there was no evidence of cytotoxicity. Thus, IFN appears to inhibit the expression of muscle-specific traits during myogenesis.     

Brain-type creatine kinase BB-CK interacts with the Golgi Matrix Protein GM130 in early prophase

Creatine kinase (CK) isoenzymes are essential for storing, buffering and intracellular transport of “energy-rich” phosphate compounds in tissues with fluctuating high energy demand such as muscle, brain and other tissues and cells where CK is expressed. In brain and many non-muscle cells, ubiquitous cytosolic “brain-type” BB-CK and ubiquitous mitochondrial CK (uMtCK) act as components of a phosphocreatine shuttle to maintain cellular energy pools and distribute energy flux. To date, still relatively little is known about direct coupling of functional dimeric BB-CK with other partner proteins or enzymes that are important for cell function. Using a global yeast two-hybrid (Y2H) screen with monomeric B-CK as bait and a representative brain cDNA library to search for interaction partners of B-CK with proteins of the brain, we repeatedly identified the cis-Golgi Matrix protein (GM130) as recurrent interacting partner of B-CK. Since HeLa cells also express both BB-CK and GM130, we subsequently used this cellular model system to verify and characterize the BB-CK-GM130 complex by GST-pulldown experiments, as well as by in vivo co-localization studies with confocal microscopy. Using dividing HeLa cells, we report here for the first time that GM130 and BB-CK co-localize specifically in a transient fashion during early prophase of mitosis, when GM130 plays an important role in Golgi fragmentation that starts also at early prophase. These data may shed new light on BB-CK function for energy provision for Golgi-fragmentation that is initiated by cell signalling cascades in the early phases of mitosis.     

Heterogeneous cellular expression of creatine kinase isoenzyme during normal rat heart development

The degree to which developmentally related alterations in cardiac creatine kinase (CK) activity reflect modification of CK isoenzyme gene expression remains uncertain. The present studies addressed this question by assessing multiple aspects of CK in rat heart during the perinatal to adult transition. In addition to whole tissue, isolated and purified muscle and nonmuscle cells were studied, as well as myofibrillar, mitochondrial, and cytosolic subcellular fractions. Whole homogenate CK enzyme specific activity nearly doubled during the weanling to adult developmental period. Muscle cell CK activity increased by a similar magnitude. Nonmuscle cell activity decreased. In the adult heart, both myofibrillar and mitochondrial CK activities were augmented versus the weanling heart. The cytoplasmic fraction activity held constant during development. Electrophoretic isoenzyme analyses of both weanling and adult cardiac muscle cells indicated the presence of mitochondrial CK and MM-CK isoforms. Weanling heart nonmuscle cells contained mitochondrial, MM, MB, and BB isoforms; however, BB isoform was not detected in the adult heart nonmuscle cells. Arrhenius plots provided information regarding heart muscle and nonmuscle cell alterations during development. CK activation energies were also determined for whole tissue, muscle/nonmuscle cells, myofibrils, mitochondria, and cytosol. Results demonstrate that heterogeneous muscle/nonmuscle cellular composition and differential myofibrillar/mitochondrial subcellular composition account for normal, developmentally related changes in heart CK enzyme activity. CK isoenzyme gene expression changes were not detected in cardiac muscle cells, and transition of CK-B to CK-M gene expression is limited to nonmuscle cells during normal, weanling to adult development in the rat heart.     

Separation of mitochondrial creatine kinase (MiMi-CK) from cytosolic creatine kinase isoenzymes by Cibachrome-Blue affinity chromatography

The mitochondrial isoenzyme of creatine kinase (MiMi-CK) was separated by affinity chromatography on Cibachrome-Blue-Sepharose (Sepharose-Blue, Pharmacia). While the soluble CK isoforms (BB-CK and MM-CK) were specifically eluted by raising the pH of the column buffer from pH 6.0 to pH 8.0, MiMi-CK remained bound under these conditions but was specifically eluted by subsequent addition of ADP to the pH 8.0 buffer. This one-step method allows a fast and efficient separation of MiMi-CK from MM-and BB-CK isoenzymes and at the same time an enrichment of MiMi-CK by about 50-fold. Since MiMi-CK can be assayed separately after isolation by affinity chromatography on Sepharose-Blue, this method may be of clinical importance.     

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.     

An embryonic pineal body as a multipotent system in cell differentiation

The differentiating potency of pineal cells from 8-day quail embryos was studied with cell culture. It was found that the differentiation of striated muscle fibres occurred abundantly in the pineal cells cultured in hypertonic culture conditions. Muscle nature of these fibres was confirmed by utilizing the antiserum against the striated muscle type creatine kinase (MM-CK). When CO2, NAHCO3, NaCl, KCl and MgCl2 were added in hypertonic concentrations, extensive myogenesis occurred in cultured pineal cells. Myogenesis in pineal cultures began as early as 2 days and, after 3 days in the medium with 75 mM additional NaCl, reached 100-fold when compared with that in the isotonic medium. Muscle fibres from pineal cells in culture were similar in morphology to the skeletal muscle fibres of mesodermal origin in situ. Myogenesis of pineal cells under hypertonic conditions was accompanied by the synthesis of a unique 56 x 10(3) Mr protein, which was not found in the intrinsic muscle cells. Clonal cell culture revealed that about 80% of clonable pineal cells were myogenic precursors. Pineal cells of 8-day quail embryos were not only myogenic but oculopotent (melanogenic and lentoidogenic) in cultures. This study examined whether multipotential progenitor cells with both potentials are present in the pineal or not. The results showed that at least 16% of all clonable pineal cells were multipotent precursors. The embryonic pineal is considered to be a typical multipotent system in parallel with the pigmented and neural retina, the neural crest and the teratocarcinoma.     

Effect of denervation on the distribution and developmental transition of phosphoglycerate mutase and creatine phosphokinase isozymes in rat muscles of different fiber-type composition

Phosphoglycerate mutase (PGM) and creatine phosphokinase (CK) occur as three isozymes (types MM, MB and BB) in mammals and these exhibit similar transitions during skeletal muscle development. To study the influence of innervation on this transition and on the maintenance of the isozyme phenotype in mature muscle, we have determined the changes produced by sciatic neurectomy in neonatal and adult rat hindlimb muscles. In 40-day-old rats, denervation decreased both PGM and CK activity, the effect being more pronounced in the fast-twitch extensorum digitorum longus (EDL) and gastrocnemius muscles than in the slow-twitch soleus muscle. It also produced a progressive increase in the proportion of MB- and BB-PGM isozymes in EDL and gastrocnemius but not in soleus, and an increase of MB- and BB-CK isozymes in all three muscles. In 5-day-old rats, denervation prevented the developmental increase of PGM and CK activity in all three muscles. Denervation also prevented the normal decrease in the relative amounts of the MB and BB isozymes of both enzymes which occur during postnatal muscle development. These results can be explained by the different effects of denervation upon slow and fast muscles, and by the distinct distribution of PGM and CK isozymes in rat type I and II muscle fibers.     

Turnover of the creatine kinase subunits in chicken myogenic cell cultures and in fibroblasts.

The rates of degradation of creatine kinase subunits, M-CK and B-CK subunits, were measured in cultured myogenic cells and in subcultured fibroblasts. In differentiated myogenic cells, the myotubes, both M-CK and B-CK subunits are synthesized. Their rates of degradation were compared. The M-CK subunits is slightly more stable and is degraded with an average apparent half-life of 75 h, whereas that of the B-CK subunit was shorter with 63 h. The turnover properties of M-CK subunit from soluble and of myofibril-bound MM-CK homodimeric creatine kinase isoenzyme isolated from breast muscle of young chickens were identical. The apparent half-life of the B-CK subunit was also determined in subcultured fibroblasts and 5-bromo-2'-deoxyuridine-treated cells, and found to be shorter than in myotubes (46 h and 37 h respectively). Similar observations were made for myosin heavy chain, actin and total acid-precipitable material. It appears therefore that proteins are in general degraded more slowly in differentiated myogenic cells. The differences in the stability of M-CK and B-CK subunits in myotubes probably do not reflect a major regulatory mechanism of the creatine kinase isoenzyme transition.