Cytoarchitectural and metabolic adaptations in muscles with mitochondrial and cytosolic creatine kinase deficiencies

We have blocked creatine kinase (CK) mediated phosphocreatine (PCr) <==> ATP transphosphorylation in mitochondria and cytosol of skeletal muscle by knocking out the genes for the mitochondrial (ScCKmit) and the cytosolic (M-CK) CK isoforms in mice. Animals which carry single or double mutations, if kept and tested under standard laboratory conditions, have surprisingly mild changes in muscle physiology. Strenuous ex vivo conditions were necessary to reveal that MM-CK absence in single and double mutants leads to a partial loss of tetanic force output. Single ScCKmit deficiency has no noticeable effects but in combination the mutations cause slowing of the relaxation rate. Importantly, our studies revealed that there is metabolic and cytoarchitectural adaptation to CK defects in energy metabolism. The effects involve mutation type-dependent alterations in the levels of AMP, IMP, glycogen and phosphomonoesters, changes in activity of metabolic enzymes like AMP-deaminase, alterations in mitochondrial volume and contractile protein (MHC isoform) profiles, and a hyperproliferation of the terminal cysternae of the SR (in tubular aggregates). This suggests that there is a compensatory resiliency of loss-of-function and redirection of flux distributions in the metabolic network for cellular energy in our mutants.     

Functional aspects of the X-ray structure of mitochondrial creatine kinase: a molecular physiology approach

Mitochondrial creatine kinase (Mi-CK) is a central enzyme in energy metabolism of tissues with high and fluctuating energy requirements. In this review, recent progress in the functional and structural characterization of Mi-CK is summarized with special emphasis on the solved X-ray structure of chicken Mib-CK octamer (Fritz-Wolf et al., Nature 381, 341-345, 1996). The new results are discussed in a historical context and related to the characteristics of CK isoforms as known from a large number of biophysical and biochemical studies. Finally, two hypothetical functional aspects of the Mi-CK structure are proposed: (i) putative membrane binding motifs at the top and bottom faces of the octamer and (ii) a possible functional role of the central 20 A channel.     

Expression of brain-type creatine kinase and ubiquitous mitochondrial creatine kinase in the fetal rat brain: evidence for a nuclear energy shuttle

To test the hypothesis that embryonic brain cells utilize a creatine phosphate energy shuttle, we examined the pattern of creatine kinase (CK) isoform expression and localization in the fetal rat brain. Moderate levels of CK activity are present at embryonic day 14 (7 U/mg protein) and decrease slightly until 3 days postpartum followed by a rapid, fourfold up-regulation to adult levels by 1 month (18 U/mg protein). In parallel with changes in enzyme activity, there is a biphasic and coordinate pattern of expression of brain-type CK (BCK) and ubiquitous mitochondrial CK (uMtCK) determined by nondenaturing electrophoresis and immunoblot analysis. The localization of CK isoforms was examined by immunocytochemistry, and, during the fetal period, BCK and uMtCK immunoreactivity was detected throughout the central and peripheral nervous system, especially in neuroepithelial regions of the cerebral vesicles and spinal cord. In large cells within the olfactory neuroepithelium and ventral spinal cord, differential compartmentation of CK isoforms was evident, with BCK localized primarily in cell nuclei, whereas uMtCK immunoreactivity was present in the cell body (but not within nuclei). In olfactory bulb neuroepithelium, both isoforms were expressed in the middle zone of the germinal layer associated with DNA synthesis. In embryonic skeletal and cardiac muscle, which also express BCK, the same compartmentation of BCK was seen, with BCK localized primarily in the cell nucleus of cardiac and skeletal myoblasts. These results demonstrate a coordinate pattern of expression and compartmentation of BCK and uMtCK isoforms in the fetal brain that, in some cells, provides the anatomic basis for a nuclear energy shuttle.     

Theoretical modelling of some spatial and temporal aspects of the mitochondrion/creatine kinase/myofibril system in muscle

After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional 'coupling' between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters phi and psi that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible 'coupling' are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when phi or psi is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when psi or psi is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless phi = psi = 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the 'adenine nucleotide flux' at the ends of the model into 'creatine flux' in the middle; the overall net flux is small, but only zero if phi = psi. A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model.     

Proficiency testing of creatine kinase and creatine kinase-2: the experience of the Ontario Laboratory Proficiency Testing Program

The Ontario Laboratory Proficiency Testing Program has regularly monitored the analytical performance of total creatine kinase (CK) (approximately 230 participants) and CK isoenzyme-2 (CK-MB) (approximately 160 participants) throughout the entire province. Consistently, a wide dispersion of results has been observed not only between different analyzer systems but also among identical analyzers. Accordingly, the results of the last three proficiency surveys for these analytes were examined statistically to establish both the extent of these variations and the range of values reported for the male upper reference ranges. The results of many of the analyzer systems were significantly different from each other, as were many of the reference ranges. This unsatisfactory situation may only be remedied by the use of reference materials as shown by others. The consequences of these findings also effect the reliability of epidemiological surveys such as the WHO MONICA Project (Circulation 1994;90:583-612), which monitors deaths due to heart disease and includes cardiac enzyme results in its criteria.     

Equilibrium intermediates in the unfolding pathway of creatine kinase

The unfolding of creatine kinase in various concentrations of guanidine hydrochloride of increasing concentrations has been investigated by combination of size-exclusion chromatography (SEC) with other methods. There are two peaks in the profiles of SEC in GuHCl at moderate concentrations, showing that unfolding of creatine kinase goes through dimeric and monomeric intermediates with increasing guanidine hydrochloride concentrations. Both intermediates have relatively compact structure and retain considerable ordered structure.     

The utilisation of creatine and its analogues by cytosolic and mitochondrial creatine kinase

We have investigated the utilisation of four analogues of creatine by cytosolic Creatine Kinase (CK), using 31P-NMR in the porcine carotid artery, and by mitochondrial CK (Mt-CK), using oxygen consumption studies in isolated heart mitochondria and skinned fibers. Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22 degrees C, containing 11 mM glucose as substrate, and supplemented with either 20 mM beta-guanidinopropionic acid (beta-GPA), methyl-guanidinopropionic acid (m-GPA), guanidinoacetic acid (GA) or cyclocreatine (cCr). All four analogues entered the tissue and became phosphorylated by CK as seen by 31 P-NMR, Inhibition of oxidative metabolism by 1 mM cyanide after accumulation of the phosphorylated analogue resulted in the utilisation of PCr, beta-GPA-P, GA-P and GA-P over a similar time course (approximately 2 h), despite very different kinetic properties of these analogues in vitro. cCr-P was utilised at a significantly slower rate, but was rapidly dephosphorylated in the presence of both 1 mM iodoacetate and cyanide (to inhibit both glycolysis and oxidative metabolism respectively). The technique of creatine stimulated respiration was used to investigate the phosphorylation of the analogues by Mt-CK, Isolated mitochondria were subjected to increasing [ATP], whereas skinned fibres received a similar protocol with increasing [ADP]. There was a significant stimulation of respiration by creatine and cCr in isolated mitochondria (decreased K(m) and increased Vmax vs control), but none by GA, mGPA or beta-GPA (also in skinned fibres), indicating that these latter analogues were not utilised by Mt-CK. These results demonstrate differences in the phosphorylation and dephosphorylation of creatine and its analogues by cytosolic CK and Mt-CK in vivo and in vitro.     

Cardiac markers in the early hours of acute myocardial infarction: clinical performance of creatine kinase, creatine kinase MB isoenzyme (activity and mass concentration), creatine kinase MM and MB subform ratios, myoglobin and cardiac troponin T

We compared early markers of acute myocardial infarction (AMI) in the first 6 h from the onset of symptoms in 133 non-traumatized patients arriving at the emergency department with chest pain suggestive of AMI. Clinical performance parameters were calculated on the basis of 45 patients with AMI and 88 patients with a non-AMI diagnosis. At admission and in the first 0-3 h after the onset of chest pain the creatine kinase-MB (CK-MB) subform ratio was the most sensitive test at a comparable specificity level of 0.95. In the time interval of 3-5 h, myoglobin, the CK-MB mass concentration and the CK-MB subform ratio were associated with the greatest areas under receiver operating characteristic (ROC) curves, but differences between these tests were small and non-significant. At 6 h from the onset of pain, differences in clinical performance between the same three tests were even smaller whether or not samples drawn after the start of thrombolytic treatment were included in the test comparison. For confirmation of AMI at 6 h after onset of pain, CK-MB (activity and mass concentration) demonstrated the highest positive likelihood ratio, and for exclusion of AMI at 6 h the CK-MB subform ratio was associated with the highest negative likelihood ratio. However, differences between the CK-MB subform ratio, CK-MB mass concentration and myoglobin were not significant as estimated by the substantial overlap between the confidence intervals of the likelihood ratios and the ROC areas at 6 h. Cardiac troponin T (cTnT) demonstrated an ROC area equal to the CK-MB isoform ratio and myoglobin at 6 h. However, the likelihood ratio for ruling out AMI was lower, mostly due to the elevated cTnT in unstable coronary disease not defined as AMI. We conclude that the CK-MB subform ratio, CK-MB mass concentration and myoglobin do not demonstrate any significant differences in clinical performance for ruling in or ruling out acute myocardial infarction at 6 h after the onset of chest pain.     

Serum creatine kinase in fasciocutaneous and musculocutaneous flap surgery

BACKGROUND: Stage III and IV squamous cell cancers of the head and neck are often unresectable at presentation and are associated with poor disease-free and overall survival rates. A phase II study using concurrent cisplatin and radiotherapy in advanced head and neck cancer indicated impressive local-regional control and survival with organ preservation. METHODS: A multicentered phase II study was undertaken consisting of 1.8 Gy fraction radiotherapy for 2 weeks followed by 1.2 Gy BID hyperfractionation to 46.8 Gy. Continuous infusion cisplatin 20 mg/m2 was given on days 1 through 4 and 22 through 25. Biopsy of the primary tumor was done at this point, and patients with clinical and pathologic complete response continued with hyperfractionated radiotherapy to 75.6 Gy plus simultaneous carboplatin 25 mg/m2 BID for 12 consecutive days. Residual disease at 46.8 Gy required curative surgery. RESULTS: Seventy-four patients entered the study, and 73 completed their treatment. Twenty were stage III and 54 were stage IV. Fifty patients had involved regional lymph nodes. Treatment was well tolerated with only one grade IV hematologic toxicity. At 46.8 Gy, biopsy revealed a complete response in 75% of the primary sites and 47% of the nodes. Only 12 patients required resection of the primary lesion. At 4 years (median follow-up is 26 months), 29 patients have recurred. CONCLUSIONS: Accelerated hyperfractionated radiotherapy with concurrent chemotherapy in stage III and IV head and neck cancer yields excellent local-regional control with organ preservation. This protocol is intensive, and some patients have distant failures.     

Synthesis and differential properties of creatine analogues as inhibitors for human creatine kinase isoenzymes

Fourteen new creatine analogues, all with a guanidine function and either a polar or an apolar group instead of the creatine carboxylic function, were tested as potential inhibitors for human creatine kinase by kinetic analysis of their effects on the reaction rate. Only compounds bearing an apolar aromatic moiety, which was spaced from the guanidine function by at least two bonds, proved to have a significant inhibitory activity and showed a mixed-type inhibition similar to that of creatine. Among these compounds 2,6-dichlorobenzylguanidine (Ki = 5.6 mM and 39.8 mM for muscle-type and brain-type creatine kinases, respectively) and 3-(2,6-dichlorophenyl)propylguanidine (Ki = 15 mM and 4.5 mM) were the more potent inhibitors and showed a significant isoenzyme selectivity between muscle- and brain-type creatine kinases. Our results are in agreement with recent data that suggest the location of a hydrophobic pocket near the guanidine-binding domain of the enzyme. The observed selectivity in isoenzyme inhibition may be useful to study structural differences in catalytic centers.     

Pathogenetic aspects of the L-arginine-NO metabolic pathway in arteriosclerosis and possible therapeutic aspects

A yearly pattern in the occurrence of uterine cervical cancer (UCC), obtained from cytological examinations reported as type V (cases concluding a malignant alteration), has been previously shown for data obtained in the Monterrey Metropolitan Area (state of Nuevo Leon, Mexico) for a span of 10 years (1978-1987), with a peak of relative incidence in the month of February being high stable for consecutive years. With the aim of extending and validating those results, we analyzed the monthly totals of positive detected cases of UCC in the states of Nuevo Leon, Chihuahua, Coahuila, and Tamaulipas (covering most of Northern Mexico) during the same period. To eliminate bias due to the seasonal variation in the number of screening smears, data were first expressed in relation to the number of cytological examinations done the same month. The least-squares fit of a 1-year cosine curve to the data of relative incidence in the four states reveals a statistically significant yearly pattern (p = .008), with a maximum of relative incidence in February almost double that during the rest of the year. Results indicate that the relative incidence of UCC is higher than the yearly average during the winter, with secondary peaks in May and October. In view of the nonsinusoidal waveform in the incidence of UCC, we undertook a multiple-component analysis, allowing several cosine functions to be simultaneously fitted to the data. Results indicate that the yearly pattern in the relative incidence of UCC can be represented by a model that includes two components with periods of 12 and 4 months (p = .004). The same model can be documented as statistically significant independently for each of the four states. These results, summarizing over 2200 positive cases of UCC detected in more than 1,100,000 screening smears, are in full agreement with those found previously for part of the state of Nuevo Leon and reveal a highly stable and predictable yearly pattern of variation in the relative incidence of UCC in Northern Mexico.     

Evaluation of serum creatine kinase levels in ectopic pregnancy

OBJECTIVE: To analyze whether increased serum creatine kinase (CK) levels are useful in early detection of ectopic pregnancy (EP). DESIGN: Prospective cohort study. SETTING: Patients in a university-based reproductive endocrinology and infertility practice. PATIENTS: Infertile women who achieved clinical pregnancy. INTERVENTIONS: Serum CK with isoenzymes levels were drawn after sonographic evaluation in patients achieving clinical pregnancy. MAIN OUTCOME MEASURES: Comparison of serum CK levels in patients with EP versus those with normal and abnormal intrauterine pregnancy (IUP), both separately and together as one group. RESULTS: There was no significant difference in mean total CK levels for patients with EP (82.25 +/- 81.39 IU/L; conversion factor to SI unit, 1.00) versus normal IUP (62.54 +/- 44.79 IU/L), abnormal IUP (55.15 +/- 3.46 IU/L), or all IUP (60.87 +/- 40.72 IU/L). The mean gestational ages were similar in all three groups: EP, 46.78 +/- 6.65 days; normal IUP, 47.56 +/- 8.29 days; and abnormal IUP, 47.86 +/- 13.30 days. CONCLUSION: Serum CK levels do not help to predict EP for infertility patients achieving conception. To assist in preventing maternal morbidity, a more discriminative test is needed to identify this condition early in gestation.     

Maintained coupling of oxidative phosphorylation to creatine kinase activity in sarcomeric mitochondrial creatine kinase-deficient mice

The importance of mitochondrial creatine kinase (mi-CK) in oxidative muscle was tested by studying the functional properties of in situ mitochondria in saponin-skinned muscle fibres from sarcomeric mi-CK-deficient (mutant) mice. Biochemical analyses showed that the lack of mi-CK in mutant muscle was associated with a decrease in specific activity of MM-CK in mutant ventricle, and increase in mutant soleus (oxidative) muscle. Lactate dehydrogenase activity and isoenzyme analysis showed an increased glycolytic metabolism in mutant soleus. No change was observed in ventricular muscle. In control animals, the apparent K(m) of mitochondrial respiration for ADP in ventricle and soleus (232 +/- 36 and 381 +/- 63 microM, respectively) was significantly reduced in the presence of creatine (52 +/- 8 and 45 +/- 12 microM, respectively). There was no change in the K(m) in oxidative fibres from mutant mice (258 +/- 27 and 399 +/- 66 microM, respectively) compared with control, though surprisingly, it was also significantly decreased in the presence of creatine (144 +/- 8 and 150 +/- 27 microM, respectively) despite the absence of mi-CK. It is proposed that in mutant (and perhaps normal) oxidative tissue, cytosolic MM-CK can relocate to the outer mitochondrial membrane, where it is coupled to oxidative phosphorylation by close proximity to porin, and the adenine nucleotide translocase. Such an effect can preserve the functioning of the CK shuttle and the energetic properties of mi-CK deficient tissue.     

Purification and characterization of human sarcomeric mitochondrial creatine kinase

PURPOSE: A dose-escalation study of irinotecan hydrochloride (CPT-11) combined with fixed-dose cisplatin was conducted to determine the maximum-tolerated dose (MTD), dose-limiting toxicities, and objective response rate in patients with advanced gastric cancer. PATIENTS AND METHODS: Twenty-four patients with or without prior chemotherapy were enrolled. All patients were assessable for toxicities and response. On day 1, CPT-11 was administered as a 90-minute intravenous (I.V.) infusion, which was followed 2 hours later by a 120-minute I.V. infusion of cisplatin 80 mg/m2. CPT-11 alone at the same dose was administered again on day 15. The treatment was repeated every 4 weeks until disease progression was observed. The initial dose of CPT-11 was 60 mg/m2, and was escalated in increments of 10 mg/m2 until severe or life-threatening toxicity was observed. RESULTS: The MTD of this combination was CPT-11 80 mg/m2. At this dose level, 16.7% of patients (two of 12) had leukopenia of less than 1,000/microL, 66.7% (eight of 12) had neutropenia of less than 500/microL, and 16.7% (two of 12) had severe diarrhea of grade 4 during the first course. The dose-limiting toxicity was neutropenia. Ten patients achieved a partial response (PR), and the overall response rate was 41.7% among 24 patients (95% confidence interval, 21.9% to 61.4%). CONCLUSION: The recommended dose and schedule is CPT-11 70 mg/m2 on days 1 and 15 and cisplatin 80 mg/m2 on day 1 every 4 weeks. This combination of CPT-11 and cisplatin, considered to be active against advanced gastric cancer with acceptable toxicity, should be further assessed in a phase II study.     

Interactions of mitochondrial ATP synthesis and the creatine kinase equilibrium in skeletal muscle

This study aimed to evaluate the effect of FR128998, (1s,6s)-1-benzyl-10-(3-pyridyl-methyl)-7-thia-10-azaspiro [5,6]-dodecan-11-one 7,7-dioxide hydrochloride, a novel platelet activating factor (PAF) receptor antagonist, on endotoxin lipopolysaccharide-induced disseminated intravascular coagulation in rats. Experimental disseminated intravascular coagulation was induced by an infusion of lipopolysaccharide at 0.25 mg/kg/h for 4 h. Simultaneous infusion of FR128998 (0.25 and 1.0 mg/kg/h) with lipopolysaccharide dose dependently inhibited thrombocytopenia, but not leukopenia. The changes in coagulation parameters of disseminated intravascular coagulation, i.e., prolongation of activated partial thromboplastin time and elevated levels of fibrinogen/fibrin degradation products, were also prevented by the treatment with FR128998. In addition, FR128998 attenuated the increase in serum tumor necrosis factor (TNF) which appeared during the initial stage of disseminated intravascular coagulation. FR128998 (10 microM) also inhibited the TNF production by peripheral blood leukocytes or alveolar macrophages stimulated by lipopolysaccharide in vitro. Furthermore, TNF production induced by PAF itself in vitro was also inhibited in the presence of FR128998. These data indicate that PAF plays a pivotal role in the development of disseminated intravascular coagulation via TNF production.     

Non-identical behaviour of the subunits of rabbit muscule creatine kinase

1. The dimeric enzyme creatine kinase (ATP: creatine N-phospotransferase, EC 2.7.3.2) from rabbit muscle was reacted with three separate reagents, each of which specifically modifies one thiol group per subunit. 2. The reactions of the enzyme with these reagents (4-chloro-7-nitrobenzofurazan, 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetate) all behave as normal second-order processes. This indicates that the thiol groups on the two subunits of the enzyme react at the same rate as each other in all three cases. 3. The effects of various ligands (Mg2+, ADP and creatine, and combinations of these) on the kinetics of the reactions were studied. In all cases the reactions behave as normal second-order processes. 4. In the presence of the ligand combination Mg2+ plus ADP plus creatine plus nitrate, which has been postulated to form a "transition state analogue" complex with the enzyme, the reactions of the thiol group show considerable deviation from second-order kinetics. This indicates that the thiol groups on the two subunits react at different rates from each other. A similar effect is also noted in the presence of the combination ADP plus creatine plus nitrate. 5. The binding of ADP to the enzyme (studied by equilibrium dialysis) is hyperbolic in the absence of other ligands or in the presence of Mg2+ or Mg2+ plus creatine. The dissociation constant is similar in all three cases. 6. In the presence of creatine plus nitrate (with or without Mg2+) the bindings of ADP to the enzyme is tightened considerably and the binding plots indicate the presence of either negative interactions between the subunits or two distinct types of binding sites. 7. Possible causes for the observed non-identical behaviour of the two subunits of the enzyme are discussed.     

Mental capacity and cognitive functioning: Developmental and social class differences.

Hypothesized that social class performance differences of same-age children would be negligible on pure mental (M) capacity measures but would be greater on tasks that confound other variables with M capacity. This hypothesis is anchored in a neo-Piagetian theory of cognitive development (J. Pascual-Leone; see record 1971-07908-001), which gives a central role to an attentional mechanism of limited M capacity that grows monotonically with age. 268 Israeli 8-, 10-, and 12-yr-olds of low or high SES completed 10 cognitive developmental tasks that measured pure M capacity, short-term memory, verbal IQ, and spatial-analytic ability. Results show that M capacity developed similarly in same-age Ss, regardless of SES. Significant differences among the investigated populations were found only in performing tasks that confounded factors of learning and style. Results support the notion that a cognitive development process exists that is universal in stage sequences and in the rate and timing of development. (20 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)