Sarcomeric Mitochondrial Creatine Kinase Research Articles

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.

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.

Mitochondrial creatine kinase is critically necessary for normal myocardial high-energy phosphate metabolism.

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.

Creatine Kinase and Creatine Transporter in Normal, Wounded, and Diseased Skin

Skin comprises many cell types that are characterized by high biosynthetic activity and increased energy turnover. The creatine kinase system, consisting of creatine kinase isoenzymes and creatine transporter, is known to be important to support the high energy demands in such cells. We analyzed the presence and the localization of these proteins in murine and human skin under healthy and pathologic conditions, using immunoblotting and confocal immunohistochemistry with our recently developed specific antibodies. In murine skin, we found high amounts of brain-type cytosolic creatine kinase coexpressed with lower amounts of ubiquitous mitochondrial creatine kinase, both mainly localized in suprabasal layers of the epidermis, different cell types of hair follicles, sebaceous glands, and the subcutaneous panniculus carnosus muscle. With exception of sebaceous glands, these cells were also expressing creatine transporter. Muscle-type cytosolic creatine kinase and sarcomeric mitochondrial creatine kinase were restricted to panniculus carnosus. Immediately after wounding of murine skin, brain-type cytosolic creatine kinase and a creatine transporter-subspecies were transiently upregulated about 3-fold as seen in immunoblots, whereas the amount of ubiquitous mitochondrial creatine kinase increased during days 10-15 after wounding. Healthy and psoriatic human skin showed a similar coexpression pattern of brain-type cytosolic creatine kinase, ubiquitous mitochondrial creatine kinase, and creatine transporter in this pilot study, with creatine transporter species being upregulated in psoriasis.

Mitochondrial Creatine Kinase and Mitochondrial Outer Membrane Porin Show a Direct Interaction That Is Modulated by Calcium

Mitochondrial creatine kinase (MtCK) co-localizes with mitochondrial porin (voltage-dependent anion channel) and adenine nucleotide translocator in mitochondrial contact sites. A specific, direct protein-protein interaction between MtCK and mitochondrial porin was demonstrated using surface plasmon resonance spectroscopy. This interaction was independent of the immobilized binding partner (porin reconstituted in liposomes or MtCK) or the analyzed isoform (chicken sarcomeric MtCK or human ubiquitous MtCK, human recombinant porin, or purified bovine porin). Increased ionic strength reduced the binding of MtCK to porin, suggesting predominantly ionic interactions. By contrast, micromolar concentrations of Ca(2+) increased the amount of bound MtCK, indicating a physiological regulation of complex formation. No interaction of MtCK with reconstituted adenine nucleotide translocator was detectable in our experimental setup. The relevance of these findings for structure and function of mitochondrial contact sites is discussed.

Changes in mRNA expression profile underlie phenotypic adaptations in creatine kinase-deficient muscles

We have studied the mechanisms that regulate the remodeling of the glycolytic, mitochondrial and structural network of muscles of creatine kinase M (M-CK)/sarcomeric mitochondrial creatine kinase (ScCKmit) knockout mice by comparison of wild-type and mutant mRNA profiles on cDNA arrays. The magnitudes of changes in mRNA levels were most prominent in M-CK/ScCKmit (CK −/−) double mutants but did never exceed those of previously observed changes in protein level for any protein examined. In gastrocnemius of CK −/− mice we measured a 2.5-fold increase in mRNA level for mitochondrial encoded cytochrome c oxidase (COX)-III which corresponds to the increase in protein content. The level of the nuclear encoded mRNAs for COX-IV, H +-ATP synthase-C, adenine nucleotide translocator-1 and insulin-regulatable glucose transporter-4 showed a 1.5-fold increase, also in agreement with protein data. In contrast, no concomitant up-regulation in mRNA and protein content was detected for the mitochondrial inorganic phosphate-carrier, voltage-dependent anion channel and certain glycolytic enzymes. Our results reveal that regulation of transcript level plays an important role, but it is not the only principle involved in the remodeling of mitochondrial and cytosolic design of CK −/− muscles.

A Conserved Negatively Charged Cluster in the Active Site of Creatine Kinase Is Critical for Enzymatic Activity

A Conserved Negatively Charged Cluster in the Active Site of Creatine Kinase Is Critical for Enzymatic Activity

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.

A conserved negatively charged cluster in the active site of creatine kinase is critical for enzymatic activity.

Creatine kinase catalyzes the reversible transphosphorylation of creatine by MgATP. From the sequence homology and the molecular structure of creatine kinase isoenzymes, we have identified several highly conserved residues with a potential function in the active site: a negatively charged cluster (Glu(226), Glu(227), Asp(228)) and a serine (Ser(280)). Mutant proteins E226Q, E226L, E227Q, E227L, D228N, and S280A/S280D of human sarcomeric mitochondrial creatine kinase were generated by in vitro mutagenesis, expressed in Escherichia coli, and purified to homogeneity. Their overall structural integrity was confirmed by CD spectroscopy and gel filtration chromatography. The enzymatic activity of all proteins mutated in the negatively charged cluster was extremely low (0.002-0.4% of wild type) and showed apparent Michaelis constants (K(m)) similar to wild type, suggesting that most of the residual activity may be attributed to wild-type revertants. Mutations of Ser(280) led to higher residual activities and altered K(m) values; S280A showed an increase of K(m) for phosphocreatine (65-fold), creatine (6-fold), and ATP (6-fold); S280D showed a decrease of K(m) for creatine (6-fold). These results, together with the transition state structure of the homologous arginine kinase (Zhou, G., Somasundaram, T., Blanc, E., Parthasarathy G., Ellington, W. R., and Chapman, M. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8449-8454), strongly suggest a critical role of Glu(226), Glu(227), and Asp(228) in substrate binding and catalysis and point to Glu(227) as a catalytic base.

Crystal structure of human ubiquitous mitochondrial creatine kinase

Creatine kinase (CK), catalyzing the reversible trans-phosphorylation between ATP and creatine, plays a key role in the energy metabolism of cells with high and fluctuating energy requirements. We have solved the X-ray structure of octameric human ubiquitous mitochondrial CK (uMtCK) at 2.7 A resolution, representing the first human CK structure. The structure is very similar to the previously determined structure of sarcomeric mitochondrial CK (sMtCK). The cuboidal octamer has 422 point group symmetry with four dimers arranged along the fourfold axis and a central channel of approximately 20 A diameter, which extends through the whole octamer. Structural differences with respect to sMtCK are found in isoform-specific regions important for octamer formation and membrane binding. Octameric uMtCK is stabilized by numerous additional polar interactions between the N-termini of neighboring dimers, which extend into the central channel and form clamp-like structures, and by a pair of salt bridges in the hydrophobic interaction patch. The five C-terminal residues of uMtCK, carrying positive charges likely to be involved in phospholipid-binding, are poorly defined by electron density, indicating a more flexible region than the corresponding one in sMtCK. The structural differences between uMtCK and sMtCK are consistent with biochemical studies on octamer stability and membrane binding of the two isoforms.

Crystal structure of human ubiquitous mitochondrial creatine kinase.

Creatine kinase (CK), catalyzing the reversible trans-phosphorylation between ATP and creatine, plays a key role in the energy metabolism of cells with high and fluctuating energy requirements. We have solved the X-ray structure of octameric human ubiquitous mitochondrial CK (uMtCK) at 2.7 A resolution, representing the first human CK structure. The structure is very similar to the previously determined structure of sarcomeric mitochondrial CK (sMtCK). The cuboidal octamer has 422 point group symmetry with four dimers arranged along the fourfold axis and a central channel of approximately 20 A diameter, which extends through the whole octamer. Structural differences with respect to sMtCK are found in isoform-specific regions important for octamer formation and membrane binding. Octameric uMtCK is stabilized by numerous additional polar interactions between the N-termini of neighboring dimers, which extend into the central channel and form clamp-like structures, and by a pair of salt bridges in the hydrophobic interaction patch. The five C-terminal residues of uMtCK, carrying positive charges likely to be involved in phospholipid-binding, are poorly defined by electron density, indicating a more flexible region than the corresponding one in sMtCK. The structural differences between uMtCK and sMtCK are consistent with biochemical studies on octamer stability and membrane binding of the two isoforms.

Myofibrillar or mitochondrial creatine kinase deficiency alone does not impair mouse diaphragm isotonic function.

Creatine kinase (CK) provides ATP buffering in skeletal muscle and is expressed as 1) cytosolic myofibrillar CK (M-CK) and 2) sarcomeric mitochondrial CK (ScCKmit) isoforms that differ in their subcellular localization. The diaphragm (Dia) expresses both M-CK and ScCKmit in abundance. We compared the power and work output of 1) control CK-sufficient (Ctl), 2) M-CK-deficient [M-CK(-/-)], 3) ScCKmit-deficient [ScCKmit(-/-)], and 4) combined M-CK/ScCKmit-deficient null mutant [CK(-/-)] Dia during repetitive isotonic activations to determine the effect of CK phenotype on Dia function. Maximum power was obtained at approximately 0.4 tetanic force in all groups. M-CK(-/-) and ScCKmit(-/-) Dia were able to sustain power and work output at Ctl levels during repetitive isotonic activation (75 Hz, 330-ms duration repeated each second at 0.4 tetanic force load), and the duration of sustained Dia shortening was 67 +/- 4 s in M-CK(-/-), 60 +/- 4 s in ScCKmit(-/-), and 62 +/- 5 s in Ctl Dia. In contrast, CK(-/-) Dia power and work declined acutely and failed to sustain shortening altogether by 40 +/- 6 s. We conclude that Dia power and work output are not absolutely dependent on the presence of either M-CK or ScCKmit, whereas the complete absence of CK acutely impairs Dia shortening capacity during repetitive activation.

Alterations in the expression and activity of creatine kinase-M and mitochondrial creatine kinase subunits in skeletal muscle following prolonged intense exercise in rats.

Creatine kinase (CK) isoenzymes are important structural and energy metabolism components in skeletal muscle. In this study, CK isoenzyme alterations were examined in male rats, with an 8% body mass weight attached to their tail. The rats were either forced to swim for 5 h (5S, n = 51), or were pre-trained for 8 days and then forced to swim for 5 h (T5S, n = 48). Rats were sacrificed either immediately (0 h PS), 3 h (3 h PS), or 48 h post-swimming (48 h PS). Serum CK was increased significantly (P < 0.01) 6.2- and 2.0-fold at 0 h PS following the 5S and T5S protocols, respectively. However, training (T5S protocol) significantly (P < 0.01) decreased CK release. Soleus and white gastrocnemius (WG) CK activity was significantly decreased following the 5S protocol (P < 0.05), but not following the T5S protocol. The CK-M activity of the soleus muscle was significantly (P < 0.05) decreased at 0 h PS following both the 5S and T5S protocols, and returned to control values at 3 h PS. The CK-M activity of the WG was significantly (P < 0.05) decreased at 0 h PS following the 5S protocol. Sarcomeric mitochondrial CK (sCK-Mit) was decreased significantly (P < 0.01) at 0 h PS (20%), 3 h PS (14%), 24 h PS (22%), and 48 h PS (15%) following the 5S protocol. However, sCK-Mit was decreased significantly (P < 0.01) only at 0 h PS (7%) following the T5S. The results of this study demonstrate that prolonged intense exercise causes a loss of skeletal muscle CK-M and sCK-Mit activity and that training prior to the prolonged intense exercise attenuates the exercise-induced CK-M and sCK-Mit loss in both red and white skeletal muscles.

Structural and functional implications of the amino acid sequences of dimeric, cytoplasmic and octameric mitochondrial creatine kinases from a protostome invertebrate.

The cDNA and deduced amino-acid sequences for dimeric and octameric isoforms of creatine kinase (CK) from a protostome, the polychaete Chaetopterus variopedatus, were elucidated and then analysed in the context of available vertebrate CK sequences and the recently determined crystal structure of chicken sarcomeric mitochondrial CK (MiCK). As protostomes last shared a common ancestor with vertebrates roughly 700 million years ago, observed conserved residues may serve to confirm or reject contemporary hypotheses about the roles of particular amino acids in functional/structural processes such as dimer/octamer formation and membrane binding. The isolated cDNA from the dimeric CK consisted of 1463 nucleotides with an open reading frame of 1116 nucleotides encoding a 372-amino-acid protein having a calculated molecular mass of 41.85 kDa. The percentage identity of C. variopedatus dimeric CK to vertebrate CK is as high as 69%. The octameric MiCK cDNA is composed of 1703 nucleotides with an open reading frame of 1227 nucleotides. The first 102 nucleotides of the open reading frame encode a 34-amino-acid leader peptide whereas the mature protein is composed of 375 amino acids with a calculated molecular mass of 42.17 kDa. The percentage identity of C. variopedatus MiCK to vertebrate CK is as high as 71%. This similarity is also evident in residues purported to be important in the structure and function of dimeric and octameric CK: (a) presence of seven basic amino acids in the C-terminal end thought to be important in binding of MiCK to membranes; (b) presence of a lysine residue (Lys110 in chicken MiCK) also thought to be involved in membrane binding; and (c) presence of a conserved tryptophan thought to be important in dimer stabilization which is present in all dimeric and octameric guanidino kinases. However, C. variopedatus MiCK lacks the N-terminal heptapeptide present in chicken MiCK, which is thought to mediate octamer stabilization. In contrast with vertebrate MiCK, polychaete octamers are very stable indicating that dimer binding into octamers may be mediated by additional and/or other residues. Phylogenetic analyses showed that both octamer and dimer evolved very early in the CK lineage, well before the divergence of deuterostomes and protostomes. These results indicate that the octamer is a primitive feature of CK rather than being a derived and advanced character.

Oxidative myocytes of heart and skeletal muscle express abundant sarcomeric mitochondrial creatine kinase.

Sarcomeric mitochondrial creatine kinase catalyzes the reversible transfer of a high energy phosphate between ATP and creatine. To study cellular distribution of the kinase, we performed immunocytochemical studies using a peptide antiserum specific for the kinase protein. Our results demonstrated that the sarcomeric mitochondrial creatine kinase gene is abundantly expressed in heart and skeletal muscle, with no protein detected in other tissues examined, including brain, lung, liver, spleen, kidney, bladder, testis, stomach, intestine, and colon. RNA blot study showed that there is no detectable expression of the kinase mRNA in the thymus gland. In heart and skeletal muscle, the kinase protein is expressed in atrial and ventricular cardiomyocytes and a subpopulation of skeletal myofibres. In skeletal muscle, fast myosin heavy chain co-localization studies demonstrated that the sarcomeric mitochondrial creatine kinase is highly expressed in type 1, slow-oxidative and type 2A, fast-oxidative-glycolytic myofibres. We conclude that the kinase gene is abundantly expressed in oxidative myocytes of heart and skeletal muscle and may contribute to oxidative capacity of these cells.

Octamer Formation and Coupling of Cardiac Sarcomeric Mitochondrial Creatine Kinase Are Mediated by Charged N-terminal Residues

Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.

Mitochondrial Creatine Kinase Is a Prime Target of Peroxynitrite-induced Modification and Inactivation

The reaction of peroxynitrite (PN) with sarcomeric mitochondrial creatine kinase (Mib-CK; EC 2.7.3.2) was observed at different stages of complexity (i) with purified Mi-CK, (ii) with enzyme bound on isolated mitoplasts, and (iii) within intact respiring mitochondria. Creatine-stimulated respiration was abolished by PN concentrations likely to be physiological and far before the respiratory chain itself was affected, thus demonstrating that Mi-CK is a prime target for inactivation by PN in intact mitochondria. The inactivation by PN of Mi-CK was reversed by 22% with 2-mercaptoethanol. More remarkable protective effects were noticed with the full set of CK substrates, e.g. 30 and 50% protection with MgATP plus creatine and MgADP plus phosphocreatine, respectively, but not with each substrate alone. These data indicate an involvement of the active-site Cys-278 residue of Mi-CK in this process. Furthermore, changes in endogenous tryptophan fluorescence intensity and spectral changes after reaction of Mi-CK with PN suggest additional modifications of Trp and Tyr residues. PN-inactivated Mi-CK can no longer be efficiently converted into dimers by incubation with reagents inducing a transition state analog complex at the active site. Thus, obviously, upon reaction of octameric Mi-CK with PN, the octamer-dimer equilibrium of Mi-CK is also affected. The consequences for cellular energy homeostasis and calcium handling are discussed.

Absence of myofibrillar creatine kinase and diaphragm isometric function during repetitive activation.

Creatine kinase (CK) provides ATP buffering in skeletal muscle and is expressed as 1) cytosolic myofibrillar CK (M-CK) and 2) sarcomeric mitochondrial CK (ScCKmit) isoforms that differ in their subcellular localization. We compared the isometric contractile and fatigue properties of 1) control CK-sufficient (Ctl), 2) M-CK-deficient (M-CK[-/-]), and 3) combined M-CK/ScCKmit-deficient null mutant (CK[-/-]) diaphragm (Dia) to determine the effect of the absence of M-CK activity on Dia performance in vitro. Baseline contractile properties were comparable across groups except for specific force, which was approximately 16% lower in CK[-/-] Dia compared with M-CK[-/-] and Ctl Dia. During repetitive activation (40 Hz, (1)/(3) duty cycle), force declined in all three groups. This decline was significantly greater in CK[-/-] Dia compared with Ctl and M-CK[-/-] Dia. The pattern of force decline did not differ between M-CK[-/-] and Ctl Dia. We conclude that Dia isometric muscle function is not absolutely dependent on the presence of M-CK, whereas the complete absence of CK acutely impairs isometric force generation during repetitive activation.

Elements Regulating Cardiomyocyte Expression of the Human Sarcomeric Mitochondrial Creatine Kinase Gene in Transgenic Mice

Sarcomeric mitochondrial creatine kinase (sMtCK) is one component of a multiprotein, high energy channeling complex consisting of porin, mitochondrial creatine kinase, and adenine nucleotide translocase. To study the transcriptional mechanisms specifying sMtCK gene expression to the heart, transgenic mice were created carrying the 5'-flanking sequences of the human sMtCK gene ligated upstream of the human growth hormone (hGH) reporter gene. RNA blot hybridization demonstrated that the human sMtCK sequence, -485 to +6 base pair (bp), did not activate reporter gene expression to a detectable level. However, the human sMtCK sequence, -921 to +6 bp, expressed the hGH reporter gene at a high level in heart and skeletal muscle and at a very low level in esophagus and kidney, and it did not express the hGH gene in other organs tested (brain, lung, liver, spleen, bladder, uterus, and stomach). In situ hybridization revealed that reporter gene transcription was specified to cardiac and skeletal myocytes, recapitulating precisely the expression pattern of the endogenous gene. Sequence analysis identified several consensus binding sites between -921 and -757 bp, including four GATT motifs, one E box, and one MEF2 site. Further analysis of a third transgenic mouse strain demonstrated that the human sMtCK sequence, -757 to +6 bp, did not direct detectable expression of the hGH reporter gene. We conclude that this 160-bp genomic sequence, from -921 to -757 bp, is necessary in specifying expression of the human sMtCK gene to the oxidative and highly metabolically active heart tissue.

Crystalline mitochondrial inclusion bodies isolated from creatine depleted rat soleus muscle.

Rats were fed a 2% guanidino propionic acid diet for up to 18 weeks to induce cellular creatine depletion by inhibition of creatine uptake by this creatine analogue. Ultrastructural analysis of creatine depleted tissues showed that mitochondrial intermembrane inclusion bodies appeared in all skeletal muscles analysed, after 11 weeks of feeding. Heart had relatively few even after 18 weeks of analogue feeding and none were evident in kidney, brain or liver. These structures were strongly immuno-positive for sarcomeric mitochondrial creatine kinase and upon removal from mitochondria, the inclusion bodies were shown to diffract to a resolution of 2.5 nm. Two-dimensional image analysis and three-dimensional reconstruction revealed arrays of creatine kinase octamers with additional components between the octameric structures. The same mitochondria had a 3-fold higher extractable specific creatine kinase activity than controls. Molecular mass gel filtration of inclusion body containing mitochondrial extracts from analogue fed rat solei revealed mitochondrial creatine kinase eluting as an aggregate of an apparent molecular mass > or = 2,000 kDa. Mitochondrial creatine kinase of control soleus mitochondrial extract eluted as an octamer, with a molecular mass of 340 kDa. Respiration measurements of control solei mitochondria displayed creatine mediated stimulation of oxidative phosphorylation that was absent in analogue-fed rat solei mitochondria. The latter also had 19% and 14% slower rates of state 4 and maximal state 3 respiration, respectively, than control mitochondria. These results indicate that mitochondrial creatine kinase co-crystallises with another component within the inter membrane space of select mitochondria in creatine depleted skeletal muscle, and is inactive in situ.