Reduced creatine-stimulated respiration in doxorubicin challenged mitochondria: particular sensitivity of the heart

Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its use is limited by a risk of cardiotoxicity, which is not completely understood. Recently, we have shown that DXR impairs essential properties of purified mitochondrial creatine kinase (MtCK), with cardiac isoenzyme (sMtCK) being particularly sensitive. In this study we assessed the effects of DXR on respiration of isolated structurally and functionally intact heart mitochondria, containing sMtCK, in the presence and absence of externally added creatine (Cr), and compared these effects with the response of brain mitochondria expressing uMtCK, the ubiquitous, non-muscle MtCK isoenzyme. DXR impaired respiration of isolated heart mitochondria already after short-term exposure (minutes), affecting both ADP- and Cr-stimulated respiration. During a first short time span (minutes to 1 h), detachment of MtCK from membranes occurred, while a decrease of MtCK activity related to oxidative damage was only observed after longer exposure (several hours). The early inhibition of Cr-stimulated respiration, in addition to impairment of components of the respiratory chain involves a partial disturbance of functional coupling between MtCK and ANT, likely due to interaction of DXR with cardiolipin leading to competitive inhibition of MtCK/membrane binding. The relevance of these findings for the regulation of mitochondrial energy production in the heart, as well as the obvious differences of DXR action in the heart as compared to brain tissue, is discussed.     

Expression of the mitochondrial creatine kinase genes

Mitochondrial Creatine Kinase (MtCK) is responsible for the transfer of high energy phosphate from mitochondria to the cytosolic carrier, creatine, and exists in mammals as two isoenzymes encoded by separate genes. In rats and humans, sarcomere-specific MtCK (sMtCK) is expressed only in skeletal and heart muscle, and has 87% nucleotide identity across the 1257 bp coding region. The ubiquitous isoenzyme of MtCK (uMtCK) is expressed in many tissues with highest levels in brain, gut, and kidney, and has 92% nucleotide identity between the 1254 bp coding regions of rat and human. Both genes are highly regulated developmentally in a tissue-specific manner. There is virtually no expression of sMtCK mRNA prior to birth. Unlike cytosolic muscle CK (MCK) and brain CK (BCK), there is no developmental isoenzyme switch between the MtCKs. Cell culture models representing the tissue-specific expression of either sMtCK or uMtCK are available, but there are no adequate developmental models to examine their regulation. Several animal models are available to examine the coordinate regulation of the CK gene family and include 1) Cardiac Stress by coarctation (sMtCK, BCK, and MCK), 2) Uterus and placenta during pregnancy (uMtCK and BCK), and 3) Diabetes and mitochondrial myopathy (sMtCK, BCK, and MCK). We report the details of these findings, and discuss the coordinate regulation of the genes necessary for high-energy transduction.     

Differential effects of peroxynitrite on human mitochondrial creatine kinase isoenzymes. Inactivation,octamer destabilization,and identification of involved residues

Creatine kinase isoenzymes are very susceptible to free radical damage and are inactivated by superoxide radicals and peroxynitrite. In this study, we have analyzed the effects of peroxynitrite on enzymatic activity and octamer stability of the two human mitochondrial isoenzymes (ubiquitous mitochondrial creatine kinase (uMtCK) and sarcomeric mitochondrial creatine kinase (sMtCK)), as well as of chicken sMtCK, and identified the involved residues. Inactivation by peroxynitrite was concentration-dependent and similar for both types of MtCK isoenzymes. Because peroxynitrite did not lower the residual activity of a sMtCK mutant missing the active site cysteine (C278G), oxidation of this residue is sufficient to explain MtCK inactivation. Mass spectrometric analysis confirmed oxidation of Cys-278 and further revealed oxidation of the C-terminal Cys-358, possibly involved in MtCK/membrane interaction. Peroxynitrite also led to concentration-dependent dissociation of MtCK octamers into dimers. In this study, ubiquitous uMtCK was much more stable than sarcomeric sMtCK. Mass spectrometric analysis revealed chemical modifications in peptide Gly-263-Arg-271 located at the dimer/dimer interface, including oxidation of Met-267 and nitration of Trp-268 and/or Trp-264, the latter being a very critical residue for octamer stability. These data demonstrate that peroxynitrite affects the octameric state of MtCK and confirms human sMtCK as the generally more susceptible isoenzyme. The results provide a molecular explanation of how oxidative damage can lead to inactivation and decreased octamer/dimer ratio of MtCK, as seen in neurodegenerative diseases and heart pathology, respectively.     

Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase

The role of ubiquitous mitochondrial creatine kinase (uMtCK) reaction in regulation of mitochondrial respiration was studied in purified preparations of rat brain synaptosomes and mitochondria. In permeabilized synaptosomes, apparent Km for exogenous ADP, Km (ADP), in regulation of respiration in situ was rather high (110 +/- 11 microM) in comparison with isolated brain mitochondria (9 +/- 1 microM). This apparent Km for ADP observed in isolated mitochondria in vitro dramatically increased to 169 +/- 52 microM after their incubation with 1 muM of dimeric tubulin showing that in rat brain, particularly in synaptosomes, mitochondrial outer membrane permeability for ADP, and ATP may be restricted by tubulin binding to voltage dependent anion channel (VDAC). On the other hand, in synaptosomes apparent Km (ADP) decreased to 25 +/- 1 microM in the presence of 20 mM creatine. To fully understand this effect of creatine on kinetics of respiration regulation, complete kinetic analysis of uMtCK reaction in isolated brain mitochondria was carried out. This showed that oxidative phosphorylation specifically altered only the dissociation constants for MgATP, by decreasing that from ternary complex MtCK.Cr.MgATP (K (a)) from 0.13 +/- 0.02 to 0.018 +/- 0.007 mM and that from binary complex MtCK.MgATP (K (ia)) from 1.1 +/- 0.29 mM to 0.17 +/- 0.07 mM. Apparent decrease of dissociation constants for MgATP reflects effective cycling of ATP and ADP between uMtCK and adenine nucleotide translocase (ANT). These results emphasize important role and various pathophysiological implications of the phosphocreatine-creatine kinase system in energy transfer in brain cells, including synaptosomes.     

Divergent enzyme kinetics and structural properties of the two human mitochondrial creatine kinase isoenzymes

The mitochondrial isoenzymes of creatine kinase (MtCK), ubiquitous uMtCK and sarcomeric sMtCK, are key enzymes of oxidative cellular energy metabolism and play an important role in human health and disease. Very little is known about uMtCK in general, or about sMtCK of human origin. Here we have heterologously expressed and purified both human MtCK isoenzymes to perform a biochemical, kinetic and structural characterization. Both isoenzymes occurred as octamers, which can dissociate into dimers. Distinct Stokes' radii of uMtCK and sMtCK in solution were indicative for conformational differences between these equally sized proteins. Both human MtCKs formed 2D-crystals on cardiolipin layers, which revealed further subtle differences in octamer structure and stability. Octameric human sMtCK displayed p4 symmetry with lattice parameters of 145 A, indicating a 'flattening' of the octamer on the phospholipid layer. pH optima and enzyme kinetic constants of the two human isoenzymes were significantly different. A pronounced substrate binding synergism (Kd > Km) was observed for all substrates, but was most pronounced in the forward reaction (PCr production) of uMtCK and led to a significantly lower Km for creatine (1.01 mM) and ATP (0.11 mM) as compared to sMtCK (creatine, 7.31 mM; ATP, 0.68 mM).     

Diaphragm and cardiac mitochondrial creatine kinases are impaired in sepsis.

Previous studies indicate that ATP formation by the electron transport chain is impaired in sepsis. However, it is not known whether sepsis affects the mitochondrial ATP transport system. We hypothesized that sepsis inactivates the mitochondrial creatine kinase (MtCK)-high energy phosphate transport system. To examine this issue, we assessed the effects of endotoxin administration on mitochondrial membrane-bound creatine kinase, an important trans-mitochondrial ATP transport system. Diaphragms and hearts were isolated from control (n = 12) and endotoxin-treated (8 mg.kg(-1).day(-1); n = 13) rats after pentobarbital anesthesia. We isolated mitochondria using techniques that allow evaluation of the functional coupling of mitochondrial creatine kinase MtCK activity to oxidative phosphorylation. MtCK functional activity was established by 1) determining ATP/creatine-stimulated oxygen consumption and 2) assessing total creatine kinase activity in mitochondria using an enzyme-linked assay. We examined MtCK protein content using Western blots. Endotoxin markedly reduced diaphragm and cardiac MtCK activity, as determined both by ATP/creatine-stimulated oxygen consumption and by the enzyme-linked assay (e.g., ATP/creatine-stimulated mitochondrial respiration was 173.8 +/- 7.3, 60.5 +/- 9.3, 210.7 +/- 18.9, was 67.9 +/- 7.3 natoms O.min(-1).mg(-1) in diaphragm control, diaphragm septic, cardiac control, and cardiac septic samples, respectively; P < 0.001 for each tissue comparison). Endotoxin also reduced diaphragm and cardiac MtCK protein levels (e.g., protein levels declined by 39.5% in diaphragm mitochondria and by 44.2% in cardiac mitochondria; P < 0.001 and P = 0.009, respectively, comparing sepsis to control conditions). Our data indicate that endotoxin markedly impairs the MtCK-ATP transporter system; this phenomenon may have significant effects on diaphragm and cardiac function.     

C-terminal lysines determine phospholipid interaction of sarcomeric mitochondrial creatine kinase

High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.     

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.     

Acute toxicity of doxorubicin on isolated perfused heart: response of kinases regulating energy supply

Doxorubicin (DXR) is a widely used and efficient anticancer drug. However, its application is limited by the risk of severe cardiotoxicity. Impairment of cardiac high-energy phosphate homeostasis is an important manifestation of both acute and chronic DXR cardiotoxic action. Using the Langendorff model of the perfused rat heart, we characterized the acute effects of 1-h perfusion with 2 or 20 microM DXR on two key kinases in cardiac energy metabolism, creatine kinase (CK) and AMP-activated protein kinase (AMPK), and related them to functional responses of the perfused heart and structural integrity of the contractile apparatus as well as drug accumulation in cardiomyocytes. DXR-induced changes in CK were dependent on the isoenzyme, with a shift in protein levels of cytosolic isoenzymes from muscle-type CK to brain-type CK, and a destabilization of octamers of the mitochondrial isoenzyme (sarcometric mitochondrial CK) accompanied by drug accumulation in mitochondria. Interestingly, DXR rapidly reduced the protein level and phosphorylation of AMPK as well as phosphorylation of its target, acetyl-CoA-carboxylase. AMPK was strongly affected already at 2 microM DXR, even before substantial cardiac dysfunction occurred. Impairment of CK isoenzymes was mostly moderate but became significant at 20 microM DXR. Only at 2 microM DXR did upregulation of brain-type CK compensate for inactivation of other isoenzymes. These results suggest that an impairment of kinase systems regulating cellular energy homeostasis is involved in the development of DXR cardiotoxicity.     

Metabolic control analysis of integrated energy metabolism in permeabilized cardiomyocytes - experimental study

The main focus of this research was to apply Metabolic Control Analysis to quantitative investigation of the regulation of respiration by components of the Mitochondrial Interactosome (MI, a supercomplex consisting of ATP Synthasome, mitochondrial creatine kinase (MtCK), voltage dependent anion channel (VDAC), and tubulin) in permeabilized cardiomyocytes. Flux control coefficients (FCC) were measured using two protocols: 1) with direct ADP activation, and 2) with MtCK activation by creatine (Cr) in the presence of ATP and pyruvate kinase-phosphoenolpyruvate system. The results show that the metabolic control is much stronger in the latter case: the sum of the measured FCC is 2.7 versus 0.74 (ADP activation). This is consistent with previous data showing recycling of ADP and ATP inside the MI due to the functional coupling between MtCK and ANT and limited permeability of VDAC for these compounds, PCr being the major energy carrier between the mitochondria and ATPases. In physiological conditions, when the MI is activated, the key sites of regulation of respiration in mitochondria are MtCK (FCC = 0.93), adenine nucleotide translocase ANT (FCC = 0.95) and CoQ cytochrome c oxidoreductase (FCC = 0.4). These results show clearly that under the physiological conditions the energy transfer from mitochondria to the cytoplasm is regulated by the MI supercomplex and is very sensitive to metabolic signals.     

Octamers of mitochondrial creatine kinase isoenzymes differ in stability and membrane binding

Octamer stability and membrane binding of mitochondrial creatine kinase (MtCK) are important for proper functioning of the enzyme and were suggested as targets for regulatory mechanisms. A quantitative analysis of these properties, using fluorescence spectroscopy, gel filtration, and surface plasmon resonance, revealed substantial differences between the two types of MtCK isoenzymes, sarcomeric (sMtCK) and ubiquitous (uMtCK). As compared with human and chicken sMtCK, human uMtCK showed a 23-34 times slower octamer dissociation rate, a reduced reoctamerization rate and a superior octamer stability as deduced from the octamer/dimer ratios at thermodynamic equilibrium. Octamer stability of sMtCK increased with temperature up to 30 degrees C, indicating a substantial contribution of hydrophobic interactions, while it decreased in the case of uMtCK, indicating the presence of additional polar dimer/dimer interactions. These conclusions are consistent with the recently solved x-ray structure of the human uMtCK (Eder, M., Fritz-Wolf, K., Kabsch, W., Wallimann, T., and Schlattner, U. (2000) Proteins 39, 216-225). When binding to 16% cardiolipin membranes, sMtCK showed slightly faster on-rates and higher affinities than uMtCK. However, human uMtCK was able to recruit the highest number of binding sites on the vesicle surface. The observed divergence of ubiquitous and sarcomeric MtCK is discussed with respect to their molecular structures and the possible physiological implications.     

High efficiency of energy flux controls within mitochondrial interactosome in cardiac intracellular energetic units

The aim of our study was to analyze a distribution of metabolic flux controls of all mitochondrial complexes of ATP-Synthasome and mitochondrial creatine kinase (MtCK) in situ in permeabilized cardiac cells. For this we used their specific inhibitors to measure flux control coefficients (C(vi)(JATP)) in two different systems: A) direct stimulation of respiration by ADP and B) activation of respiration by coupled MtCK reaction in the presence of MgATP and creatine. In isolated mitochondria the C(vi)(JATP) were for system A: Complex I - 0.19, Complex III - 0.06, Complex IV 0.18, adenine nucleotide translocase (ANT) - 0.11, ATP synthase - 0.01, Pi carrier - 0.20, and the sum of C(vi)(JATP) was 0.75. In the presence of 10mM creatine (system B) the C(vi)(JATP) were 0.38 for ANT and 0.80 for MtCK. In the permeabilized cardiomyocytes inhibitors had to be added in much higher final concentration, and the following values of C(vi)(JATP) were determined for condition A and B, respectively: Complex I - 0.20 and 0.64, Complex III - 0.41 and 0.40, Complex IV - 0.40 and 0.49, ANT - 0.20 and 0.92, ATP synthase - 0.065 and 0.38, Pi carrier - 0.06 and 0.06, MtCK 0.95. The sum of C(vi)(JATP) was 1.33 and 3.84, respectively. Thus, C(vi)(JATP) were specifically increased under conditions B only for steps involved in ADP turnover and for Complex I in permeabilized cardiomyocytes within Mitochondrial Interactosome, a supercomplex consisting of MtCK, ATP-Synthasome, voltage dependent anion channel associated with tubulin βII which restricts permeability of the mitochondrial outer membrane.     

Mitochondrial creatine kinase in human health and disease

Mitochondrial creatine kinase (MtCK), together with cytosolic creatine kinase isoenzymes and the highly diffusible CK reaction product, phosphocreatine, provide a temporal and spatial energy buffer to maintain cellular energy homeostasis. Mitochondrial proteolipid complexes containing MtCK form microcompartments that are involved in channeling energy in form of phosphocreatine rather than ATP into the cytosol. Under situations of compromised cellular energy state, which are often linked to ischemia, oxidative stress and calcium overload, two characteristics of mitochondrial creatine kinase are particularly relevant: its exquisite susceptibility to oxidative modifications and the compensatory up-regulation of its gene expression, in some cases leading to accumulation of crystalline MtCK inclusion bodies in mitochondria that are the clinical hallmarks for mitochondrial cytopathies. Both of these events may either impair or reinforce, respectively, the functions of mitochondrial MtCK complexes in cellular energy supply and protection of mitochondria form the so-called permeability transition leading to apoptosis or necrosis.     

Nitric oxide inhibits cardiac energy production via inhibition of mitochondrial creatine kinase

Nitric oxide biosynthesis in cardiac muscle leads to a decreased oxygen consumption and lower ATP synthesis. It is suggested that this effect of nitric oxide is mainly due to the inhibition of the mitochondrial respiratory chain enzyme, cytochrome c oxidase. However, this work demonstrates that nitric oxide is able to inhibit soluble mitochondrial creatine kinase (CK), mitochondrial CK bound in purified mitochondria, CK in situ in skinned fibres as well as the functional activity of mitochondrial CK in situ in skinned fibres. Since mitochondrial isoenzyme is functionally coupled to oxidative phosphorylation, its inhibition also leads to decreased sensitivity of mitochondrial respiration to ADP and thus decreases ATP synthesis and oxygen consumption under physiological ADP concentrations.     

Mitochondrial creatine kinase in contact sites: interaction with porin and adenine nucleotide translocase, role in permeability transition and sensitivity to oxidative damage

The creatine/phosphocreatine circuit provides an efficient energy buffering and transport system in a variety of cells with high and fluctuating energy requirements. It connects sites of energy production (mitochondria, glycolysis) with sites of energy consumption (various cellular ATPases). The cellular creatine/phosphocreatine pool is linked to the ATP/ADP pool by the action of different isoforms of creatine kinase located at distinct subcellular compartments. Octameric mitochondrial creatine kinase (MtCK), together with porin and adenine nucleotide translocase, forms a microcompartment at contact sites between inner and outer mitochondrial membranes and facilitates the production and export into the cytosol of phosphocreatine. MtCK is probably in direct protein-protein contact with outer membrane porin, whereas interaction with inner membrane adenine nucleotide translocase is rather mediated by acidic phopholipids (like cardiolipin) present in significant amounts in the inner membrane. Octamer-dimer transitions of MtCK as well as different creatine kinase substrates have a profound influence on controlling mitochondrial permeability transition (MPT). Inactivation by reactive oxygen species of MtCK and destabilization of its octameric structure are factors that contribute to impairment of energy homeostasis and facilitated opening of the MPT pore, which eventually lead to tissue damage during periods of ischemia/reperfusion.     

Influence of mitochondrial creatine kinase on the mitochondrial/extramitochondrial distribution of high energy phosphates in muscle tissue: evidence for a leak in the creatine shuttle

The influence of mitochondrial creatine kinase on subcellular high energy systems has been investigated using isolated rat heart mitochondria, mitoplasts and intact heart and skeletal muscle tissue.In isolated mitochondria, the creatine kinase is functionally coupled to oxidative phosphorylation at active respiratory chain, so that it catalyses the formation of creatine phosphate against its thermodynamic equilibrium. Therefore the mass action ratio is shifted from the equilibrium ratio to lower values. At inhibited respiration, it is close to the equilibrium value, irrespective of the mechanism of the inhibition. The same results were obtained for mitoplasts under conditions where the mitochondrial creatine kinase is still associated with the inner membrane.In intact tissue increasing amounts of creatine phosphate are found in the mitochondrial compartment when respiration and/or muscle work are increased. It is suggested that at high rates of oxidative phosphorylation creatine phosphate is accumulated in the intermembrane space due to the high activity of mitochondrial creatine kinase and the restricted permeability of reactants into the extramitochondrial space. A certain amount of this creatine phosphate leaks into the mitochondrial matrix.This leak is confirmed in isolated rat heart mitochondria where creatine phosphate is taken up when it is generated by the mitochondrial creatine kinase reaction. At inhibited creatine kinase, external creatine phosphate is not taken up. Likewise, mitoplasts only take up creatine phosphate when creatine kinase is still associated with the inner membrane. Both findings indicate that uptake is dependent on the functional active creatine kinase coupled to oxidative phosphorylation.Creatine phosphate uptake into mitochondria is inhibited with carboxyatractyloside. This suggests a possible role of the mitochondrial adenine nucleotide translocase in creatine phosphate uptake.Taken together, our findings are in agreement with the proposal that creatine kinase operates in the intermembrane space as a functional unit with the adenine nucleotide translocase in the inner membrane for optimal transfer of energy from the electron transport chain to extramitochondrial ATP-consuming reactions.     

Decreased mitochondrial creatine kinase activity in dystrophic chicken breast muscle alters creatine-linked respiratory coupling

Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT (p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36-44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 mM creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20-50 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent KD, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered.     

Mitochondria-cytoskeleton interaction: distribution of β-tubulins in cardiomyocytes and HL-1 cells

Mitochondria-cytoskeleton interactions were analyzed in adult rat cardiomyocytes and in cancerous non-beating HL-1 cells of cardiac phenotype. We show that in adult cardiomyocytes βII-tubulin is associated with mitochondrial outer membrane (MOM). βI-tubulin demonstrates diffused intracellular distribution, βIII-tubulin is colocalized with Z-lines and βIV-tubulin forms microtubular network. HL-1 cells are characterized by the absence of βII-tubulin, by the presence of bundles of filamentous βIV-tubulin and diffusely distributed βI- and βIII-tubulins. Mitochondrial isoform of creatine kinase (MtCK), highly expressed in cardiomyocytes, is absent in HL-1 cells. Our results show that high apparent K(m) for exogenous ADP in regulation of respiration and high expression of MtCK both correlate with the expression of βII-tubulin. The absence of βII-tubulin isotype in isolated mitochondria and in HL-1 cells results in increased apparent affinity of oxidative phosphorylation for exogenous ADP. This observation is consistent with the assumption that the binding of βII-tubulin to mitochondria limits ADP/ATP diffusion through voltage-dependent anion channel of MOM and thus shifts energy transfer via the phosphocreatine pathway. On the other hand, absence of both βII-tubulin and MtCK in HL-1 cells can be associated with their more glycolysis-dependent energy metabolism which is typical for cancer cells (Warburg effect).     

Characterization of a brain particulate bound form of creatine kinase

A bound form of creatine kinase associated with brain particulate was characterized by isoelectric focusing, antigenicity and chromatography and compared to muscle (MM), brain (BB), and heart mitochondrial isoenzymes. On partial purification and isoelectric focusing, the solubilized enzyme has a pl of 7.3, similar to the pl of muscle creatine kinase MM, pl 6.8, but different from brain creatine kinase BB, which precipitates on isoelectric focusing in sucrose or glycerol stabilized media at its calculated pl of 5.6. Gel filtration chromatography of deoxycholate solubilized particulate creatine kinase on Sephadex Gl50 reveals an estimated molecular weight of approximately 80,000 daltons. The brain particulate enzyme is antigenically distinct from both muscle and rat heart mitochondrial creatine kinase isoenzymes but has antigenic similarity with soluble cytoplasmic brain BB. The situation may be analogous to that found with rat heart mitochondria and rat heart cytoplasmic isoenzymes which we have shown to exhibit antigenic similarity even though differences in electrophoretic and amino acid composition have been demonstrated; however, the confident determination that the particulate enzyme is a separate isoenzyme will have to await amino acid analysis.     

Isolation and characterization of the gene and cDNA encoding human mitochondrial creatine kinase

Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy metabolism. Nuclear genes encode three known CK subunits: cytoplasmic muscle (MCK), cytoplasmic brain (BCK), and mitochondrial (MtCK). We have isolated the gene and cDNA encoding human placental MtCK. By using a dog heart MCK cDNA-derived probe, the 7.0-kb EcoRI fragment from one cross-hybridizing genomic clone was isolated and its complete nucleotide sequence determined. A region of this clone encoded predicted amino acid sequence identical to residues 15-26 of the human heart MtCK NH2-terminal protein sequence. The human placental MtCK cDNA was isolated by hybridization to a genomic fragment encoding this region. The human placental MtCK gene contains 9 exons encoding 416 amino acids, including a 38-amino acid transit peptide, presumably essential for mitochondrial import. Residues 1-14 of human placental MtCK cDNA-derived NH2-terminal sequence differ from the human heart MtCK protein sequence, suggesting that tissue-specific MtCK mRNAs are derived from multiple MtCK genes. RNA blot analysis demonstrated abundant MtCK mRNA in adult human ventricle and skeletal muscle, low amounts in placenta and small intestine, and a dramatic increase during in vitro differentiation induced by serum-deprivation in the non-fusing mouse smooth muscle cell line, BC3H1. These findings demonstrate coordinate regulation of MtCK and cytosolic CK gene expression and support the phosphocreatine shuttle hypothesis.