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Phosphocreatine (PCr) depletion during isometric twitch stimulation at 5 Hz was measured by (31)P-NMR spectroscopy in gastrocnemius muscles of pentobarbital-anesthetized MM creatine kinase knockout (MMKO) vs. wild-type C57B (WT) mice. PCr depletion after 2 s of stimulation, estimated from the difference between spectra gated to times 200 ms and 140 s after 2-s bursts of contractions, was 2.2 +/- 0.6% of initial PCr in MMKO muscle vs. 9.7 +/- 1.6% in WT muscles (mean +/- SE, n = 7, P < 0.001). Initial PCr/ATP ratio and intracellular pH were not significantly different between groups, and there was no detectable change in intracellular pH or ATP in either group after 2 s. The initial difference in net PCr depletion was maintained during the first minute of continuous 5-Hz stimulation. However, there was no significant difference in the quasi-steady-state PCr level approached after 80 s (MMKO 36.1 +/- 3.5 vs. WT 35.5 +/- 4.4% of initial PCr; n = 5-6). A kinetic model of ATPase, creatine kinase, and adenylate kinase fluxes during stimulation was consistent with the observed PCr depletion in MMKO muscle after 2 s only if ADP-stimulated oxidative phosphorylation was included in the model. Taken together, the results suggest that cytoplasmic ADP more rapidly increases and oxidative phosphorylation is more rapidly activated at the onset of contractions in MMKO compared with WT muscles.

The individual functional significance of the various creatine kinase (CK) isoenzymes for myocardial energy homeostasis is poorly understood. Whereas transgenic hearts lacking the M subunit of CK (M-CK) show unaltered cardiac energetics and left ventricular (LV) performance, deletion of M-CK in combination with loss of sarcomeric mitochondrial CK (ScCKmit) leads to significant alterations in myocardial high-energy phosphate metabolites. To address the question as to whether this alteration is due to a decrease in total CK activity below a critical threshold or due to the specific loss of ScCKmit, we studied isolated perfused hearts with selective loss of ScCKmit (ScCKmit(-/-), remaining total CK activity approximately 70%) using (31)P NMR spectroscopy at two different workloads. LV performance in ScCKmit(-/-) hearts (n = 11) was similar compared with wild-type hearts (n = 9). Phosphocreatine/ATP, however, was significantly reduced in ScCKmit(-/-) compared with wild-type hearts (1.02 +/- 0.05 vs. 1.54 +/- 0.07, P < 0.05). In parallel, free [ADP] was higher (144 +/- 11 vs. 67 +/- 7 microM, P < 0.01) and free energy release for ATP hydrolysis (DeltaG(ATP)) was lower (-55.8 +/- 0.5 vs. -58.5 +/- 0.5 kJ/mol, P < 0.01) in ScCKmit(-/-) compared with wild-type hearts. These results demonstrate that M- and B-CK containing isoenzymes are unable to fully substitute for the loss of ScCKmit. We conclude that ScCKmit, in contrast to M-CK, is critically necessary to maintain normal high-energy phosphate metabolite levels in the heart.

Our goal was to determine whether mice genetically altered to lack either creatine kinase (M/MtCK(-/-)) or adenylate kinase (AK(-/-)) show altered properties in the dynamic regulation of myocardial oxygen consumption (MVO(2)). We measured contractile function, oxygen consumption, and the mean response time of oxygen consumption to a step increase in heart rate [i.e., mitochondrial response time (t(mito))] in isolated Langendorff-perfused hearts from wild-type (n = 6), M/MtCK(-/-) (n = 6), and AK(-/-) (n = 4) mice. Left ventricular developed pressure was higher in M/MtCK(-/-) hearts (88.2 +/- 6.8 mmHg) and lower in AK(-/-) hearts (46.7 +/- 9.4 mmHg) compared with wild-type hearts (60.7 +/- 10.1 mmHg) at the basal pacing rate. Developed pressure fell slightly when heart rate was increased in all three groups. Basal MVO(2) at 300 beats/min was 19.1 +/- 2.4, 19.4 +/- 1.5, and 16.3 +/- 1.9 micromol x min(-1) x g dry wt(-1) for M/MtCK(-/-), AK(-/-), and wild type, respectively, which increased to 25.5 +/- 3.7, 25.4 +/- 2.6, and 22.0 +/- 2.6 micromol. min(-1) x g(-1), when heart rate was increased to 400 beats/min. The t(mito) was significantly faster in M/MtCK(-/-) hearts: 3.0 +/- 0.3 versus 7.3 +/- 0.6 and 8.0 +/- 0.4 s for M/MtCK(-/-), AK(-/-), and wild-type hearts, respectively. Our results demonstrate that MVO(2) of M/MtCK(-/-) hearts adapts more quickly to an increase in heart rate and thereby support the hypothesis that creatine kinase acts as an energy buffer in the cytosol, which delays the energy-related signal between sites of ATP hydrolysis and mitochondria.

The function of creatine kinase (CK) and its effect on phosphorus metabolites was studied in livers of transgenic mice expressing human ubiquitous mitochondrial CK (CK-Mit) and rat brain CK (CK-B) isoenzymes and their combination. (31)P NMR spectroscopy and saturation transfer were recorded in livers of anesthetized mice to measure high-energy phosphates and hepatic CK activity. CK reaction velocity was related to total enzyme activity irrespective of the isoenzyme expressed, and it increased with increasing concentrations of creatine (Cr). The fluxes mediated by both isoenzymes in both directions (phosphocreatine or ATP synthesis) were equal. Over a 20-fold increase in CK-Mit activity (28-560 micromol. g wet wt(-1). min(-1)), the fraction of phosphorylated Cr increased 1.6-fold. Hepatic free ADP concentrations calculated by assuming equilibrium of the CK-catalyzed reaction in vivo decreased from 84 +/- 9 to 38 +/- 4 nmol/g wet wt. Calculated free ADP levels in mice expressing high levels of CK-B (920-1,635 micromol. g wet wt(-1). min(-1)) were 52 +/- 6 nmol/g wet wt. Mice expressing both isoenzymes had calculated free ADP levels of 36 +/- 4 nmol/g wet wt. These findings indicate that CK-Mit catalyzes its reaction equally well in both directions and can lower hepatic apparent free ADP concentrations.

Creatine kinase (CK) is an abundant enzyme, important for maintenance of high-energy phosphate homeostasis in many tissues including heart. Double-knockout CK (DbKO-CK) mice missing both the muscle (MM) and sarcomeric mitochondrial (ScMit) isoforms of CK have recently been studied. Despite a large change in skeletal muscle function in DbKO-CK mice, there is little functional change in the heart. To investigate whether there are specific changes in cardiac mitochondrial proteins associated with the loss of MM- and ScMit-CK isoforms, we have used difference gel electrophoresis (DIGE) to compare mitochondrial proteins from wild-type and DbKO-CK mice. Mass spectrometry fingerprinting was used to identify 40 spots as known mitochondrial proteins. We have discovered that the loss of MM- and ScMit-CK isoforms did not cause large scale changes in heart mitochondrial proteins. The loss of ScMit-CK was readily detected in the DbKO-CK samples. We have also detected a large decrease in the precursor form of aconitase. Furthermore, two mitochondrial protein differences have been found in the parent mouse strains of the DbKO-CK mice.

Creatine kinase (CK) has been implicated in affecting cell growth, and the CK substrates creatine (Cr) and cyclocreatine (CyCr) have been shown to have anti-tumor activity. The influence of Cr and CyCr on liver regeneration following major hepatectomy was evaluated in normal and transgenic mice expressing the human ubiquitous mitochondrial isoform of CK (CK-mit) or the brain isoform of CK (CK-B) or livers expressing both CK-mit and CK-B (CK-comb). Expression of CK isoenzymes had little effect on liver regeneration in the absence of dietary supplementation with Cr or CyCr as assayed by the increase in liver mass. Dietary supplementation with Cr and CyCr significantly reduced liver growth in normal mice. Liver regeneration was almost completely inhibited in mice expressing CK-mit in the presence of Cr. Livers expressing CK-mit regenerated better than normal livers in the presence of CyCr. In mice expressing CK-B, Cr and CyCr had opposite effects from those found in CK-mit mice. In the presence of CyCr, regeneration was inhibited in livers expressing CK-B, and, in the presence of Cr, CK-B-expressing livers regenerated better than normal livers. The amount of DNA synthesized 2 days after hepatectomy confirmed the results obtained from measurements of liver mass for all groups. Growth and DNA synthesis were completely abolished by Cr in CK-mit mice, whereas CyCr mainly affected growth 2 days after hepatectomy in CK-B-expressing mice. Coexpression of the CK isoforms in CK-comb mice ameliorated the effects detected with either isoform alone. Inhibition of growth by Cr and CyCr was not correlated to water accumulation. These results clearly demonstrate isoenzyme and substrate-specific effects of CK on cell growth.

The contribution of ATP-generating systems to Na+ pump (Na+-K+-ATPase) function was studied in Xenopus laevis A6 kidney epithelia apically permeabilized with digitonin. The ouabain-inhibitable Na+ pump current (I(P)) was measured in the presence of otherwise impermeant inhibitors and/or substrates at Na+ and K+ concentrations that allowed near-maximal pump function. Confocal fluorescence microscopy after apical addition of sulfosuccinimidobiotin (molecular weight of 443) showed that all cells were permeabilized. Less than 15% of the endogenous lactate dehydrogenase and creatine kinase (CK) were released into the apical medium. The I(P) was approximately 5 microA/cm2 in the presence of D-glucose. Blocking glycolysis with 2-deoxy-D-glucose or oxidative phosphorylation with antimycin A decreased it by > or = 50%. Exogenously added ATP prevented these decreases fully or partially, respectively. Two CK isoforms were detected, one likely being mitochondrial and the other corresponding to mammalian B isoform of CK. Phosphocreatine partially restored Na+ pump activity during inhibition of either ATP synthesis pathway. In conclusion, the ATP used by Na+ pumps of apically digitonin-permeabilized A6 epithelia is generated to a similar extent by glycolysis and oxidative phosphorylation. The CK system can partially support the ATP supply to the Na+ pumps.

The role of creatine kinase in regulation of myocardial respiration was studied in vivo as a function of maturation. Unidirectional creatine kinase flux (JCK), phosphocreatine to gamma-ATP, was measured in newborn lambs (age 3-9 days, n = 8) and mature sheep (age 30-60 days, n = 6) using 31P saturation transfer techniques, and total creatine kinase activity was measured using standard methods. Myocardial oxygen consumption (MVO2) was measured simultaneously via an extracorporeal shunt from the coronary sinus as cardiac work was increased via epinephrine (1-3 micrograms.kg-1.min-1). Findings were as follows: 1) baseline newborn JCK was markedly lower than in mature sheep despite higher levels of MVO2, and this could be related to a decrease in total creatine kinase activity; 2) JCK was substantially higher than the rate of ATP synthesis in both groups at baseline rates of oxygen consumption; and 3) JCK decreased significantly in newborns during increases in MVO2, whereas there was no change in flux rate in the mature sheep during even larger relative changes in work and oxygen consumption. These data imply that creatine kinase does not limit oxidative phosphorylation. However, this enzyme system probably maintains at least an indirect role in respiratory control that is a function of the myocardial developmental state.

Skinned rat papillary muscles and purified preparations of rat cardiac myofibrils were used to study the nature of the interaction of creatine kinase with cardiac myofibrils. High activity of creatine kinase (2 IU/mg protein in fibers and 0.9 IU/mg in purified myofibrils) was due mostly to reversibly bound enzyme. This activity could be removed and rebound. The process of creatine kinase rebinding was characterized by apparent Km value of 0.14 mg/ml (approximately equal to 2 X 10(6) M). Rebinding of creatine kinase to cardiac myofibrils restored the phenomenon of functional compartmentation of adenine nucleotides in myofibrillar space and restored the ability of phosphocreatine to decrease the rigor tension in the presence of MgADP. The physiological experiments with quick length changes showed that rebinding of creatine kinase to skinned papillary muscle also restored Ca sensitivity, increased maximal tension development, decreased stiffness, and restored the tension recovery after quick length changes in muscle under condition of inhibition of endogenous creatine kinase by 1-fluoro-2,4-dinitrobenzene. It is concluded that creatine kinase reversibly bound to cardiac myofibrils is involved in the energy supply for cardiac contraction.