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

Creatine kinase (CK) isoenzymes are present in all vertebrates. An important property of the creatine kinase system is that its total activity, its isoform distribution, and the concentration of guanidino substrates are highly variable among species and tissues. In the highly organized structure of adult muscles, it has been shown that specific CK isoenzymes are bound to intracellular compartments, and are functionally coupled to enzymes and transport systems involved in energy production and utilization. It is however, not established whether functional coupling and intracellular compartmentation are present in all vertebrates. Furthermore, these characteristics seem to be different among different muscle types within a given species. This study will review some of these aspects. It has been observed that: (1) In heart ventricle, CK compartmentation and coupling characterize adult mammalian cells. It is almost absent in frogs, and is weakly present in birds. (2) Efficient coupling of MM-CK to myosin ATPase is seen in adult mammalian striated muscles but not in frog and bird heart where B-CK is expressed instead of M-CK. Thus, the functional efficacy of bound MM-CK to regulate adenine nucleotide turnover within the myofibrillar compartment seems to be specific for muscles expressing M-CK as an integral part of the sarcomere. (3) Mi-CK expression and/or functional coupling are highly tissue and species specific; moreover, they are subject to short term and long term adaptations, and are present late in development. The mitochondrial form of CK (mi-CK) can function in two modes depending on the tissue: (i) in an <> and (ii) in an <>. The mode of action of mi-CK seems to be related to its precise localization within the mitochondrial intermembrane space, whereas its amount might control the quantitative aspects of the coupling. Mi-CK is highly plastic, making it a strong candidate for fine regulation of excitation-contraction coupling in muscles and for energy transfer in cells with large and fluctuating energy demands in general. (4) Although CK isoforms show a binding specificity, the presence of a given isoform within a tissue or a species only, does not predict its functional role. For example, M-CK is expressed before it is functionally compartmentalized within myofibrils during development. Similarly, the presence of ubiquitous or sarcomeric mi-CK isoforms, is not an index of functional coupling of mi-CK to oxidative phosphorylation. (5) Amongst species or muscles, it appears that a large buffering action of the CK system is associated with rapid contraction and high glycolytic activity. On the other hand, an oxidative metabolism is associated with isoform diversity, increased compartmentation, a subsequent low buffering action and efficient phosphotransfer between mitochondria and energy utilization sites. It can be concluded that, in addition to a high variation of total activity and isoform expression, the role of the CK system also critically depends on its intracellular organization and interaction with energy producing and utilizing pathways. This compartmentation will determine the high cellular efficiency and fine specialization of highly organized and differentiated muscle cells.

Energy balance refers to the dynamic homeostasis of ATP and related forms of chemical potential within cells. This regulation is accomplished mainly by oxidative metabolism in most mammals. This homeostasis matches dynamically the energy demands of cellular ATPases (net decrease in chemical potential energy) with the energy supply by mitochondrial oxidative phosphorylation (net increase in chemical potential energy). Muscle cells are distinguished from most other cell types in their ability to attain energy balance with more than a 10-fold range of ATPase demand. Creatine kinase maintains a near to equilibrium flux: PCr + ADP<-->ATP + Cr. One important function of creatine kinase is to buffer ATP and ADP concentrations. A system of differential equations describe the coupled operation of cellular ATPase, creatine kinase and oxidative phosphorylation. These equations used experimentally measured concentrations of relevant metabolites and enzyme activities to simulate energy balance in muscle cells. The principle of energy balance is adequately illustrated by simulations with only a three component system.

Human carcinoembryonic antigen (CEA), a widely used tumor marker, is a member of a family of cell surface glycoproteins that are overexpressed in many carcinomas. CEA has been shown to function in vitro as a homotypic intercellular adhesion molecule. This correlation of overproduction of an adhesion molecule with neoplastic transformation provoked a test of the effect of CEA on cell differentiation. Using stable CEA transfectants of the rat L6 myoblast cell line as a model system of differentiation, we show that fusion into myotubes and, in fact, the entire molecular program of differentiation, including creatine phosphokinase upregulation, myogenin upregulation, and beta-actin downregulation are completely abrogated by the ectopic expression of CEA. The blocking of the upregulation of myogenin, a transcriptional regulator responsible for the execution of the entire myogenic differentiation program, indicates that CEA expression intercepts the process at a very early stage. The adhesion function of CEA is essential for this effect since an adhesion-defective N domain deletion mutant of CEA was ineffective in blocking fusion and CEA transfectants treated with adhesion-blocking peptides fused normally. Furthermore, CEA transfectants maintain their high division potential, whereas control transfectants lose division potential with differentiation similarly to the parental cell line. Thus the expression of functional CEA on the surface of cells can block terminal differentiation and maintain proliferative potential.

Myogenic differentiation is obligatorily coupled to withdrawal of myoblasts from the cell cycle and is inhibited by specific polypeptide growth factors. To investigate the potential involvement of c-myc in the control of myogenesis, the BC3H1 muscle cell line was stably transfected with a simian virus 40 promoter:c-myc chimeric gene. In quiescent cells in 0.5% serum, the exogenous c-myc gene was expressed at a level more than threefold greater than the level of endogenous c-myc in undifferentiated, proliferating cells of the parental line in 20% serum. The transfected myc gene partially inhibited the expression of both muscle creatine kinase and the nicotinic acetylcholine receptor, but was not sufficient to prevent the induction of these muscle differentiation products upon mitogen withdrawal.

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.

The effect of a diabetic state in the diabetic KK-CAy mouse on calcium activated neutral proteinase (CANP) of hind-limb skeletal muscles was investigated. In the diabetic state, there was an increased sensitivity to activation of CANP by calcium (Ca). In addition, there was an enhancement of maximal activity of the enzyme. The effect was induced by secondary modification of the diabetic state, but not genetical factors. Several lines of evidence suggest that the CANP is responsible for 92 K dalton protein in diabetic skeletal muscles. Among the evidence are the following: a) The 92 K band in the diabetic muscles was lower than in the prediabetic mouse and restored by the addition of 2 mM EDTA and 2 mM EGTA. b) The band was reduced by increasing the Ca content and neutral pH in the non-diabetic normal muscles. c) E-64-C, a CANP inhibitor, restored the 92 K component reduced by the diabetic state. Since the band in denervated muscles was not changed by the Ca chelating agents, the reduction of the band in the diabetic muscles is related with musculotrophic factors, not diabetic neuropathy. These results suggest that diabetic amyotrophy may be regarded as a phenomenon linked to an increase in intracellular Ca ions and an increase in CANP activity.

The fields of muscle physiology and biochemistry have already identified some of the key components of ATPase hydrolysis products that are involved in muscle fatigue. The concentration of the relevant chemical species can be readily measured by nuclear magnetic resonance techniques in muscle. Now the question is: is alteration of cellular energy balance and the normal balance between supply and demand disturbed in fibromyalgia? Since these chemical events account for a very large amount of muscle reduced performance as well as reduction in both velocity and force, at the very least one ought to identify how large these changes are in any patient in whom we are trying to assess the degree to which these chemical changes might be associated with muscle fatigue. An objective chemical criteria for muscle performance is possible with modern noninvasive phosphorus magnetic resonance spectroscopy.

This paper summarizes the data concerning the role of the creatine phosphokinase system in muscle cells with main attention to the cardiac muscle. Creatine phosphokinase isoenzymes play a key role in the intracellular energy transport from mitochondria to myofibrils and other sites of energy utilization. Due to the existence of the creatine phosphate pathway for energy transport, intracellular creatine phosphate concentration is apparently an important regulatory factor for muscle contraction which influences the contractile force by determining the rate of regeneration of ATP directly available for myosin ATPase, and at the same time controls the activator calcium entry into the myoplasm across the surface membrane of the cells.

Several recent studies have demonstrated the presence of creatine kinase and of phosphorylcreatine in a variety of cells besides striated muscle and brain cells. The total creatine kinase and phosphagen levels in these cells encompass a wide range of values. The available data are collected in this article to demonstrate that the variation of the enzyme and phosphagen concentrations is not random but that the two are interrelated. With both the major isoenzymes of creatine kinase, namely the muscle type and the brain type, the basal levels of phosphorylcreatine follow closely the cellular creatine kinase levels. A hypothesis is presented in which the enzyme itself is the major determinant of phosphorylcreatine content by virtue of its ability to act as an intracellular binding protein for creatine derived from extracellular fluid, and also for cellular ADP. The proposed mechanism further predicts that in cells that contain high levels of actin and thus sequester the cytoplasmic free ADP (e.g. most muscle cells), a high level of creatine kinase can effectively regulate the myokinase reaction by its ability to bind ADP. The net effect of such regulation is to conserve the adenine nucleotide pools in the cell. The evolutionary advantage of these two regulatory functions of creatine kinase in terms of energy conservation is discussed.

It has been shown that the contents of mitochondria and mitochondrial isoenzyme of creatine phosphokinase are almost identical in skeletal and heart muscles. In mitochondria from both types of muscle creatine phosphokinase is functionally coupled to ATP-ADP translocase. This kind of coupling ensures complete conversion of mitochondrial ATP energy into the energy of creatine phosphate and effective control of oxidative phosphorylation by the creatine phosphokinase reaction. It has also been shown that all isoenzymes of creatine phosphokinase from heart and skeletal muscle have very similar kinetic properties. Significant differences have been found to exist between isoenzyme patterns of these muscles and also in distribution of different isoenzymes in the cells. In skeletal muscle cells creatine phosphokinase is present mainly as cytosolic MM isoenzyme; about 6% of total cellular activity is localised also in mitochondria. Due to high activity of cytosolic isoenzyme the total activity of creatine phosphokinase is about three times higher in skeletal muscle than in cardiac muscle. It has been also shown that phosphoenolpyruvate and glucose-6-phosphate do not have any inhibitory effect on creatine phosphokinase.

Glycerinated rabbit psoas muscle fibers containing native CPK, ATPase, and myokinase activities were used and isometric contraction and relaxation responses to either ADP or ATP + CP or to ATP alone in the presence and absence of P1, P5-di(adenosine-5'-pentaphosphate), a myokinase inhibitor, were compared. In previous (14) work it was shown that CP generated more efficient and faster contraction and relaxation of glycerinated muscle fibers than ATP. The present work deals with the role of myokinase in the differential response of fibers to CP and ATP. Inhibition of the myokinase activity of these fibers caused slight diminution of the rate of contraction at physiological concentrations of ATP. Uninhibited fibers were not able to reach maximum contraction, because the tension began to drop gradually even in the presence of Ca2+. Addition of Ap5A permitted maximum contraction and the ability to stay at the contracted state. In the case of CP + adenosine nucleotides (ATP or ADP), myokinase activity decreased the rate of tension development which was statistically significant after 5-7 sec of contraction. Thus, a higher tension was obtainable when myokinase was inhibited. At high concentration of adenine nucleotides (greater than 2 mM) and in the absence of Ap5A, not only the maximum tension never was reached, but a spontaneous drop in tension was observed before addition of EGTA, as was seen with ATP alone. Relaxation was faster and more complete in the presence of uninhibited myokinase activity except that the ADP was low (125 mM). These observations provide further evidence for a close functional interaction of these three enzymes in the mechanism of contraction and relaxation, giving further support to the notion of the creatine-phosphocreatine energy shuttle.

Development proceeds by way of a discrete yet overlapping series of biosynthetic and restructuring events that result in the continued molding of tissues and organs into highly restricted and specialized states required for adult function. Individual molecules and cells are replaced by molecular and cellular variants, called isoforms; these arise and function during embryonic development or later life. Isoforms, whether molecular or cellular, have been identified by their structural differences, which allow separation and characterization of each variant. These isoforms play a central and controlling role in the continued and dynamic remodeling that takes place during development. Descriptions of the individual phases of the orderly replacement of one isoform for another provides an experimental context in which the process of development can be better understood.

BC3H1 is a cell line that undergoes a musclelike pattern of differentiation under the appropriate conditions. We have examined the control of the synthesis of proteins characteristic of differentiated muscle in these cells as a function of their position in the cell cycle. We define two positions in the cell cycle where BC3H1 cells can remain stably quiescent. G1d is a restriction point early in the G1 portion of the cell cycle that permits the synthesis of muscle-specific proteins and is probably identical to G0. The second restriction point, G1q, occurs approximately 4 hr later in the G1 portion of the cell cycle and does not permit the synthesis of muscle-specific proteins. Movement of the cells from G1d to G1q occurs when fibroblast growth factor is added to the cells and is reversed when this growth factor is removed. Repression of the synthesis of muscle-specific proteins occurs when fibroblast growth factor is added to cells in G1d. In the case of the muscle form of creatine phosphokinase (M-CPK), the decline in the rate synthesis of this protein is a consequence of a decreased level of its mRNA. By contrast, the repression of alpha-actin synthesis, a protein synthesized only in differentiated cells, appears to be controlled at the translational level. The effect of fibroblast growth factor and other mitogens in these cells require activation of tyrosine kinase(s), but the intracellular targets of these kinases are not known. Studies by others suggest that activation of the ras oncogene can mimic the action of mitogenic polypeptides on these and other muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)