Expression Of Creatine Kinase Research Articles

Effect of selenium status on the response of unfit horses to exercise

Exercise is known to increase reactive oxygen species and alter glutathione peroxidase activity (GPx), a selenoenzyme responsible for neutralising hydrogen peroxide. This study evaluated the effect of selenium (Se) status on the response of unfit horses to mild exercise. 25 mature horses received one of four dietary treatments for 29 weeks: low Se (LS, n=6), adequate Se (AS, sodium selenite, n=6), high organic Se (SP; Sel-Plex®, n=7) or high inorganic Se (SS, sodium selenite, n=6). Total dietary Se concentration for LS, AS, SP and SS was 0.06, 0.12, 0.3 and 0.3 mg/kg respectively. Blood samples were collected before and at 0, 4 and 24 h after a 36 min exercise test covering 4.41 km. Blood samples were evaluated for blood GPx, pro-inflammatory cytokine expression, serum malondialdehyde (MDA), creatine kinase (CK), aspartate aminotransferase (AST) and blood pro-inflammatory cytokine expression. Data were analysed as ANOVA with repeated measures. Prior to the exercise test blood Se was higher (P<0.01) for SP and SS than AS and LS. An effect of treatment and treatment×time (P<0.05) existed for GPx. Post-exercise GPx decreased in LS and did not recover by 24 h while remaining unchanged in AS. GPx increased in SP and decreased in SS post-exercise, but both returned to pre-values at 24 h. Serum MDA was elevated at 24 h (P<0.05), but unaffected by treatment. Serum CK and AST were unaffected by Se status (P≯0.05). Pro-inflammatory cytokine expression was unaffected by Se status, although tumour necrosis factor alpha decreased immediately after exercise followed by an increase post-exercise while granzyme B was elevated immediately post-exercise (time, P<0.05). Overall Se status had minimal effects on exercise response in unfit horses. The differences in post-exercise GPx response may warrant further investigation.

Role of heme oxygenase-1 in process of sevoflurane preconditioning attenuating hypoxla-reoxygenation iniury in cultured neonatal rat cardiomyocytes

Objective To investigate the role of heme oxygenase-1 (HO-1 ) in process of sevoflurane preconditioning attenuating hypoxia-reoxygenation (H/R) injury in cultured neonatal rat cardiomyocytes.Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into four groups ( n ＝ 25 each):control group (group C),group H/R,sevoflurane preconditioning group(group S + H/R),and HO-1 inhibitor zinc protoporphyria (ZnPP) and sevoflurane preconditioning group(group ZnPP + S + H/R).In group H/R,the cardiomyocytes were exposed to 2 hours of hypoxia,followed by 1 hour of reoxygenation.Group S+ H/R received 2.5％ sevoflurane preconditioning for 20 minutes followed by 10 minutes of wash-out before H/R.ZnPP was added to the culture medium with final concentrations of 3 μMol/L 1 hour before sevoflurane preconditioning and H/R in group ZnPP +S + H/R.HO- 1 expression,apoptosis rate,concentration of free calcium ( [ Ca2 + ] i),mitochondrial membrane permeability transition pore (PTP),cytoohrome C ( Cyto C) expression and activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in culture supernatant were detected after reoxygenation.Results Compared with group C,the expression of HO-1 and cytoplasmic Cyto C of cardiomyocytes were up-regulated,mitochondrial Cyto C was down-regulated,while the [Ca2+ ]i,opening degree of PIP,apoptosis rate and activities of LDH and CK in culture supernatant were increased in group H/R.Compared with group H/R,the expression of HO-1 and mitochondrial Cyto C of cardiomyocytes were up-regulated,cytoplasmic Cyto C was down-regulated,while the [Ca2+ ] i,opening degree of PTP,apoptosis rate and activities of LDH and CK in culture supernatant were decreased in group S + H/R.Compared with group S + H/R,the expression of HO-1 and mitochondrial Cyto C of cardiomyocytes were down-regulated,cytoplasmic Cyto C was up-regulated,while the [Ca2+ ]i,opening degree of PTP,apoptosis rate and activities of LDH and CK in culture supematant were increased in group ZnPP + S + H/R.Conclusion The up-regulation of HO-1 is involved in the process of sevoflurane preconditioning attenuating hypoxia-reoxygenation injury in cultured neonatal rat cardiomyocytes. Key words: Hene oxygenase-1 ; Cell hypoxia; Oxygen; Myocytes,cardiac; Sevoflurane

Selective Decrease of Components of the Creatine Kinase System and ATP Synthase Complex in Chronic Chagas Disease Cardiomyopathy

BackgroundChronic Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC) and ischemic (IC) cardiomyopathies.Methodology/Principal FindingsMyocardium homogenates from CCC (N = 5), IC (N = 5) and IDC (N = 5) patients, as well as from heart donors (N = 5) were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit) and muscular creatine kinase (CKM) and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels.Conclusions/SignificanceThe selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.

Besides Huntington's disease, does brain-type creatine kinase play a role in other forms of hearing impairment resulting from a common pathological cause?

Hearing impairment following cochlear damage due to noise trauma, ototoxicity caused by aminoglycoside antibiotics, or age-related cochlear degeneration was linked to a common pathogenesis involving the formation of reactive oxygen species (ROS). Cochleae are more vulnerable to oxidative stress than other organs because of the high metabolic demands of their mechanosensory hair cells in response to sound stimulation. We recently showed that patients and mice with Huntington's disease (HD) have hearing impairment and that the dysregulated phosphocreatine (PCr)-creatine kinase (CK) system may account for this auditory dysfunction. Given the importance of noninvasive biomarkers and the easy access of hearing tests, the symptom of hearing loss in HD patients may serve as a useful clinical indicator of disease onset and progression of HD. We also showed that dietary creatine supplementation rescued the impaired PCr-CK system and improved the expression of cochlear brain-type creatine kinase (CKB) in HD mice, thereby restoring their hearing. Because creatine is an antioxidant, we postulated that creatine might enhance expression of CKB by reducing oxidative stress. In addition to HD-related hearing impairment, inferior CKB expression and/or an impaired PCr-CK system may also play an important role in other hearing impairments caused by elevated levels of ROS. Most importantly, dietary supplements may be beneficial to patients with these hearing deficiencies.

Dysregulated brain creatine kinase is associated with hearing impairment in mouse models of Huntington disease

Huntington disease (HD) is a degenerative disorder caused by expanded CAG repeats in exon 1 of the huntingtin gene (HTT). Patients with late-stage HD are known to have abnormal auditory processing, but the peripheral auditory functions of HD patients have yet to be thoroughly assessed. In this study, 19 HD patients (aged 40-59 years) were assessed for hearing impairment using pure-tone audiometry and assessment of auditory brainstem responses (ABRs). PTA thresholds were markedly elevated in HD patients. Consistent with this, elevated ABR thresholds were also detected in two mouse models of HD. Hearing loss thus appears to be an authentic symptom of HD. Immunohistochemical analyses demonstrated the presence of mutant huntingtin that formed intranuclear inclusions in the organ of Corti of HD mice, which might interfere with normal auditory function. Quantitative RT-PCR and Western blot analyses further revealed reduced expression of brain creatine kinase (CKB), a major enzyme responsible for ATP regeneration via the phosphocreatine-creatine kinase (PCr-CK) system, in the cochlea of HD mice. Treatment with creatine supplements ameliorated the hearing impairment of HD mice, suggesting that the impaired PCr-CK system in the cochlea of HD mice may contribute to their hearing impairment. These data also suggest that creatine may be useful for treating the hearing abnormalities of patients with HD.

Mitochondrial Interactosome in Energy Metabolism in Healthy and Cancer Cells

In this study we investigated intracellular distribution of β-tubulin isotypes, expression of mitochondrial creatine kinase (MtCK) and their roles in Mitochondrial Interactosome (supercomplex formed by ATP synthasom, ANT, MtCK, VDAC-tubulin and other VDAC-linker proteins) in adult rat cardiomyocytes, in cancerous HL-1 cells of cardiac phenotype and several other cancer cells lines. Immunofluorescent and western blot analysis of adult cardiac muscle cells revealed the presence of all β-tubulin isotypes with the very regular localization of tubulin II(β2) corresponding to mitochondrial “crystal like” pattern. Antibodies against tubulin IV(β4) revealed characteristic staining of microtubular network. Polymerized transversal lines of tubulin III(β3) matched with Z-lines (alpha-actinin) fixing, probably, microtubular network to sarcomer structure. Tubulin I(β5) distribution was diffusely spotted and fragmentary polymerized. In cancer cardiac HL-1 and other cancer cell types MI remodelling is characterized by the absence of II(β2). Additionally, these cells do not contain tubulin IV(β4) isotype creating micro-tubular network in adult cardiomyocytes and have diffusely distributed tubulin I(β5) and III(β3) isotypes. Other characteristic of cancer cells was the absence of both sarcomeric and ubiquitous mitochondrial isoform of creatine kinase (MtCK). In cardiac cells respiration is controlled by creatine via activation of MtCK within MI. Knowing that the extremely low apparent Km for free ADP in cancer cells as well as in isolated mitochondria is associated with the lack of tubulin II(β2) isotype, we can assume that tubulin II(β2) can specifically control the voltage dependent anion channel (VDAC) selective permeability resulting in adenine nucleotides micro-compartmentation within the MI. The absence of II(β2) tubulin isotype and MtCK isoforms induce significant alterations of phenotype and regulatory mechanisms of cancer cells energy metabolism. By these mechanisms, all mitochondrial ATP can be captured to maintain activated glycolysis and lactate production, known as Warburg effect.

Proteomic and transcriptomic analysis of heart failure due to volume overload in a rat aorto-caval fistula model provides support for new potential therapeutic targets - monoamine oxidase A and transglutaminase 2

BackgroundChronic hemodynamic overloading leads to heart failure (HF) due to incompletely understood mechanisms. To gain deeper insight into the molecular pathophysiology of volume overload-induced HF and to identify potential markers and targets for novel therapies, we performed proteomic and mRNA expression analysis comparing myocardium from Wistar rats with HF induced by a chronic aorto-caval fistula (ACF) and sham-operated rats harvested at the advanced, decompensated stage of HF.MethodsWe analyzed control and failing myocardium employing iTRAQ labeling, two-dimensional peptide separation combining peptide IEF and nano-HPLC with MALDI-MS/MS. For the transcriptomic analysis we employed Illumina RatRef-12v1 Expression BeadChip.ResultsIn the proteomic analysis we identified 2030 myocardial proteins, of which 66 proteins were differentially expressed. The mRNA expression analysis identified 851 differentially expressed mRNAs.ConclusionsThe differentially expressed proteins confirm a switch in the substrate preference from fatty acids to other sources in the failing heart. Failing hearts showed downregulation of the major calcium transporters SERCA2 and ryanodine receptor 2 and altered expression of creatine kinases. Decreased expression of two NADPH producing proteins suggests a decreased redox reserve. Overexpression of annexins supports their possible potential as HF biomarkers. Most importantly, among the most up-regulated proteins in ACF hearts were monoamine oxidase A and transglutaminase 2 that are both potential attractive targets of low molecular weight inhibitors in future HF therapy.

Increased Cardiac Alpha-Myosin Heavy Chain in Left Atria and Decreased Myocardial Insulin-Like Growth Factor (IGF-I) Expression Accompany Low Heart Rate in Hibernating Grizzly Bears

Grizzly bears (Ursus arctos horribilis) tolerate extended periods of extremely low heart rate during hibernation without developing congestive heart failure or cardiac chamber dilation. Left ventricular atrophy and decreased left ventricular compliance have been reported in this species during hibernation. We evaluated the myocardial response to significantly reduced heart rate during hibernation by measuring relative myosin heavy-chain (MyHC) isoform expression and expression of a set of genes important to muscle plasticity and mass regulation in the left atria and left ventricles of active and hibernating bears. We supplemented these data with measurements of systolic and diastolic function via echocardiography in unanesthetized grizzly bears. Atrial strain imaging revealed decreased atrial contractility, decreased expansion/reservoir function (increased atrial stiffness), and decreased passive-filling function (increased ventricular stiffness) in hibernating bears. Relative MyHC-α protein expression increased significantly in the atrium during hibernation. The left ventricle expressed 100% MyHC-β protein in both groups. Insulin-like growth factor (IGF-I) mRNA expression was reduced by ∼50% in both chambers during hibernation, consistent with the ventricular atrophy observed in these bears. Interestingly, mRNA expression of the atrophy-related ubiquitin ligases Muscle Atrophy F-box (MAFBx) and Muscle Ring Finger 1 did not increase, nor did expression of myostatin or hypoxia-inducible factor 1α (HIF-1α). We report atrium-specific decreases of 40% and 50%, respectively, in MAFBx and creatine kinase mRNA expression during hibernation. Decreased creatine kinase expression is consistent with lowered energy requirements and could relate to reduced atrial emptying function during hibernation. Taken together with our hemodynamic assessment, these data suggest a potential downregulation of atrial chamber function during hibernation to prevent fatigue and dilation due to excessive work against an optimally filled ventricle, a response unpredicted by the Frank-Starling mechanism.

Trans‐10, cis‐12 conjugated linoleic acid inhibits skeletal muscle differentiation and GLUT4 expression independently from NF‐κB activation

The capacity of skeletal muscle to contribute to glucose homeostasis depends on muscular insulin sensitivity. The expression of glucose transporter (GLUT)-4 is increased during myoblast differentiation, a process essential in maintenance of adult muscle. Therefore, processes that affect muscle differentiation may influence insulin dependent glucose homeostasis. Conjugated linoleic acids, and in particular trans-10, cis-12 CLA (t10, c12-CLA), are potent inducers of NF-kB in cultured skeletal myotubes, and NF-kB activation inhibits muscle differentiation. The aims of this study were to evaluate whether CLAs inhibit myogenic differentiation and lower GLUT4 mRNA expression and to address the involvement of NF-kB activation in potential effects of CLA on these processes. Incubation of C2C12 cells with t10, c12-CLA blocked the formation of myotubes, which was accompanied by reduced expression of the muscle specific genes creatine kinase, myogenin, myosin heavy chain perinatal and myosin heavy chain IIB, as well as decreased GLUT4 mRNA levels. However, genetic blockade of NF-kB was not sufficient to restore reduced myosin heavy chain protein expression following t10, c12-CLA treatment. Surprisingly, in contrast to myotubes, t10, c12-CLA was not able to activate NF-kB transcriptional activity in myoblasts. In conclusion, t10, c12-CLA inhibits myogenic differentiation and GLUT4 expression, independently from NF-kB activation.

Life of mice - development of cardiac energetics

Life of mice - development of cardiac energetics

Bisphenol A impacts cardiomyocyte differentiation in vitro by modulating cardiac protein expression

introduction: Bisphenol A (BPA) is an environmental toxin commonly found in plastics and is able to mimic the actions of endogenous steroid hormones. BPA binds and activates intracellular estrogen receptors (eRα and eRβ) and estrogen related receptor γ (eRRγ), all of which are present in cardiomyocytes. however, it is unclear how BPA impacts the heart. We hypothesized that BPA modulates the expression of proteins regulating cardiac structure, energy and calcium homeostasis during cardiomyocyte differentiation in vitro. Methods: We differentiated h9C2 cells into cardiomyocytes in hormone-replete (Rm) or hormone-depleted (hD) media. We co-treated the cells with graded amounts of Bpa and pure anti-estrogen ICI 182,780, which blocks eRα and eRβ activity. Immunoblotting measured the expression of the structural protein β-myosin heavy chain (βmhC), calcium homeostasis protein sarcoendoplasmic reticulum calcium aTpase (seRCa2a), and the cardiac energy-producing protein creatine kinase (CK). results: expression of these proteins was hormone-dependent during cardiomyocyte differentiation, with expression highest in Rm media after 72 or 96 hours of differentiation. adding 10-8 m BPA to hD media increased cardiac structural (βmhC), energy (CK), and calcium homeostasis (seRCa2a) protein expression. Conversely, 10-7 m BPA added to Rm media decreased protein expression. Co-treatment with ICI 182,780 reduced Bpa-mediated induction of seRCa2a and CK expression in hD media. discussion: BPA modulates cardiac structure, calcium and energy homeostasis protein expression during cardiomyocyte differention in vitro. moreover, the data suggest that BPA mediates these changes in protein expression through activation of cardiomyocyte eRα, eRβ, or eRRγ.

Antenatal Glucocorticoid Therapy Accelerates ATP Production With Creatine Kinase Increase in the Growth-Enhanced Fetal Rat Heart

Previous study has demonstrated the increase of several cardiac function-related proteins, including creatine kinase (CK) as an important enzyme in the process of ATP synthesis in the fetal heart of rats administered glucocorticoid (GC) antenatally. In the present study the effect of antenatal GC administration on the CK expression in fetal and neonatal hearts was demonstrated. Dexamethasone was administered to pregnant rats on days 19 and 20 of gestation. The mRNA levels of the CK isoforms, CK-M and Mi-CK, in 21-day-old fetal and 1-day-old neonatal hearts were significantly increased after antenatal GC administration. CK protein levels were also increased in both cultured cardiomyocytes and the mitochondria of the hearts. Uptake of 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethyl-benzimidazolocarbocyanine iodide by mitochondria was significantly increased. An increased ATP level accompanied the CK increase in the neonatal hearts. Furthermore, in vitro these effects were mediated though the GC receptor of cardiomyocytes. Peroxisome proliferator-activated receptor gamma as the upstream transcription factor of CK was significantly increased in fetal hearts. These results suggest that antenatal GC administration accelerates ATP synthesis through increased CK and may contribute to maturation of the premature heart so that it is ready for preterm delivery. (Circ J 2010; 74: 171 - 180).

P53 and Metabolism: The GAMT Connection

In this issue of Molecular Cell, Ide et al. (2009) have identified the enzyme guanidinoacetate methyltransferase (GAMT) that regulates creatine metabolism as a p53 target involved in apoptosis, reactive oxygen species (ROS), and fatty acid metabolism.

Distal-atrophe Paresen, Hohlfüße und CK-Erhöhung – Ein differenzialdiagnostisches Update

Distale Myopathien sind eine heterogene Erkrankungsgruppe, die mit distalen Paresen und Atrophien einhergehen. Ätiologisch liegt teilweise eine Genmutation verschiedener Strukturproteine zugrunde. Die Miyoshi-Myopathie ist eine autosomal-rezessiv vererbte Dysferlinopathie die zudem charakteristisch mit einer deutlichen Wadenatrophie und einer massiven CK-Erhöhung einhergeht. Es wird über einen 24-jährigen Patienten mit Hohlfüßen, Krallenzehen und Atrophie der Wadenmuskulatur sowie distal und beinbetonten Paresen aller Extremitäten, massiv erhöhter Kreatinkinase und fehlender Expression von Dysferlin berichtet und die prinzipiell breite Differenzialdiagnose distal-atropher Paresen diskutiert.

Functional overload in ground squirrel plantaris muscle fails to induce myosin isoform shifts

We performed 2 wk of mechanical overload by synergist ablation on plantaris muscles from a small rodent hibernator, Spermophilus lateralis. While this muscle displays prominent myosin heavy-chain (MyHC) isoform shifts during hibernation, sensitivity to mechanical loading as a stimulus for muscle mass and isoform plasticity has not been demonstrated. Squirrel muscles, whether during hibernation or not, potentially are less sensitive to mechanical unloading, but we hypothesized that increased loading would produce the typical mammalian response of greater plantaris mass and MyHC shifts. Mechanical overload produced a 50% increase in muscle mass but, surprisingly, no changes in MyHC isoform protein or mRNA expression, despite previously observed fast-to-slow MyHC isoform switching during hibernation. Citrate synthase enzyme activity, as well as mRNA expression of creatine kinase and the muscle growth factor myostatin, were all unchanged. The mRNA expression of critical muscle atrophy genes decreased by 50% during hypertrophy, including ubiquitin ligases MuRF1 and MAFbx, and the related transcription factor FOXO-1a. Insulin-like growth factor (IGF-1) and hypoxia-inducible factor (HIF-1alpha) mRNA expression was elevated by 400% and 150%. Fast-to-slow MyHC isoform shifts appear unnecessary to support the increased recruitment of the plantaris muscle, shifts which are seen in other rodent models. Our results are consistent with muscular activity during interbout arousals as a potential mechanism to preserve muscle mass, but illustrate the primary importance of other seasonal factors besides patterns of muscle activation which must act in concert to alter MyHC isoforms and muscle fiber type during hibernation.

Regulation of T Cell Development and Activation by Creatine Kinase B

Creatine kinase catalyzes the reversible transfer of the N-phosphoryl group from phosphocreatine to ADP to generate ATP and plays a key role in highly energy-demanding processes such as muscle contraction and flagellar motility; however, its role in signal transduction (which frequently involves ATP-consuming phosphorylation) and consequent cell-fate decisions remains largely unknown. Here we report that creatine kinase B was significantly up-regulated during the differentiation of double-positive thymocytes into single-positive thymocytes. Ectopic expression of creatine kinase B led to increased ATP level and enhanced phosphorylation of the TCR signaling proteins. Consequentially, transgenic expression of creatine kinase B promoted the expression of Nur77 and Bim proteins and the cell death of TCR signaled thymocyte. In addition, the activation, proliferation and cytokine secretion of T cells were also enhanced by the expression of creatine kinase B transgene. In contrast, treatment of T cells with specific creatine kinase inhibitor or creatine kinase B shRNA resulted in severely impaired T cell activation. Taken together, our results indicate that creatine kinase B plays an unexpected role in modulating TCR-mediated signaling and critically regulates thymocyte selection and T cell activation.

Differential proteome profiling analysis of male and female murine skeletal muscle following a single bout of exercise

Identifying the array of proteins that change expression following a single bout of eccentrically‐biased exercise in skeletal muscle will shed light on the molecular mechanisms underlying adaptation, as well as their gender specificity. Exercise‐naive male and female mice (C57BL/10ScSnJ 000476 +/+) ran downhill (‐15°) on a treadmill at 25 m s−1 for 15 min and 800 ug of total biceps brachii extract was electrophoresed on pH 5‐8 2‐D gels. The resulting spots that changed at least +/‐ 2‐fold and were significantly different (p<0.05) relative to unexercised controls of the appropriate gender using PDQuest 8.0 were analyzed by LC/MS‐MS and identified using BioWorks 3.3.1. This analysis showed significant proteome remodeling subsequent to downhill running. The expression change patterns were followed at 0, 24, 48, 72 and 168 hrs post‐exercise (PX) and significant changes were observed in 4 NCBI KOG groups. Both myofibrillar and cytoskeletal protein remodelings were noted, as well as decreased expression of creatine kinase. Nearly all of the glycolytic enzymes were up‐regulated. The cellular heat shock protein responses were significant and specific. Roughly the same number of protein spots changed in females and males, but the females peaked at 24 hr PX, whereas in males the peak was at 72 hr PX. Supported by NSF 0420971, the Smith College Blakeslee Fund and HHMI.

Impact of myocardial inflammation on cytosolic and mitochondrial creatine kinase activity and expression

The disturbance of myocardial energy metabolism has been discussed as contributing to the progression of heart failure. Little however is known about the cardiac mitochondrial/cytosolic energy transfer in murine and human inflammatory heart disease. We examined the myocardial creatine kinase (CK) system, which connects mitochondrial ATP-producing and cytosolic ATP-consuming processes and is thus of central importance to the cellular energy homeostasis. The time course of expression and enzymatic activity of mitochondrial (mtCK) and cytosolic CK (cytCK) was investigated in Coxsackievirus B3 (CVB3)-infected SWR mice, which are susceptible to the development of chronic myocarditis. In addition, cytCK activity and isoform expression were analyzed in biopsies from patients with chronic inflammatory heart disease (n = 22). Cardiac CVB3 titer in CVB3-infected mice reached its maximum at 4 days post-infection (pi) and became undetectable at 28 days pi; cardiac inflammation cumulated 14 days pi but persisted through the 28-day survey. MtCK enzymatic activity was reduced by 40% without a concurrent decrease in mtCK protein during early and acute MC. Impaired mtCK activity was correlated with virus replication and increased level of interleukine 1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and elevated catalase expression, a marker for intracellular oxidative stress. A reduction in cytCK activity of 48% was observed at day 14 pi and persisted to day 28 pi. This restriction was caused by a decrease in cytCK subunit expression but also by direct inhibition of specific cytCK activity. CytCK activity and expression were also reduced in myocardial biopsies from enterovirus genome-negative patients with inflammatory heart disease. The decrease in cytCK activity correlated with the number of infiltrating macrophages. Thus, viral infection and myocardial inflammation significantly influence the myocardial CK system via restriction of specific CK activity and down-regulation of cytCK protein. These changes may contribute to the progression of chronic inflammatory heart disease and malfunction of the heart.

Expression And Purification Of A Stable, Monomeric Creatine Kinase

Three isoform gene families of creatine kinase (CK) are present in animals. Two of these, mitochondrial and cytoplasmic CKs, are obligate oligomers . There is substantial evidence for functional interaction between subunits. Attempts at generating active, monomeric CKs have failed or in one case produced ephemerally active but unstable monomers. A third CK isoform, the so-called flagellar CK, is monomeric but is composed of three complete, contiguous CK domains. Each of these domains is catalytically competent but there is clear interaction between active sites (Hoffman et al., FEBS J 275: 646-654 [2008]). In the present effort, we have used a flagellar CK expression construct as a platform to engineer, express and purify a single domain, monomeric CK. Boundaries between the three domains (D1, D2 and D3) were identified by comparison of key catalytic residues and predicted secondary structural elements. A cDNA coding for D2 was amplified by PCR and inserted into an expression vector. Subsequent expression and purification yielded a recombinant CK which was stable as evidenced by the retention of activity over several weeks. Size exclusion chromatography showed that this CK was monomeric as expected, with a mass similar to the predicted Mr based on the amino acid composition. The engineering of a monomeric CK in the present effort clearly shows that oligomerization is not required for catalysis. Conventional wisdom supports the view that CKs evolved from a related phosphotransferase, arginine kinase (AK). AKs are typically monomeric. It seems likely that oligomerization occurred later in the evolution of CKs perhaps due to the selective pressure for targeting to and binding in intracellular compartments. (Supported by NSF grant IOB-0542236 to WRE).

The transcriptional cascade associated with creatine kinase down-regulation and mitochondrial biogenesis in mice sarcoma

The tissue-specific expressions of creatine kinase (CK) isoforms are regulated by the coordinated action of various transcription factors. The myogenic differentiation factor D (MyoD) family of proteins and the myocyte-specific enhancer binding factor 2 family of transcription factors are important in regulating the muscle-specific expression of cytosolic muscle-type CK (MCK) and mitochondrial CKs. As reported in some related studies, TNF-α mediated degradation of MyoD and myogenin mRNA may lead to severe muscle wasting and cachexia, which is characterized by a low transcript level of MCK and myosin heavy chain proteins. In our previous study, we reported on a complete loss of total CK activity and expression when sarcoma was induced in mouse skeletal muscle (Patra et al. FEBS J. 275 (2008) 3236–3247). This study aimed at investigating the transcriptional cascade of CK down-regulation in carcinogen-induced sarcoma in mouse muscle. Both CK deficiency and enhanced nitric oxide synthase (NOS) were known to augment mitochondrial biogenesis, so we also explored the activation of the transcriptional cascade of mitochondrial biogenesis in this cancer. We observed the activation of the TNF-α-mediated nitric oxide production pathway with NFκB activation and concomitant degradation of MyoD and myogenin mRNA. Exploration of mitochondrial biogenesis revealed high cytochrome c oxidase activity and mitochondrial DNA content in sarcoma. The PGC-related co-activator seems to have a major role in regulating mitochondrial biogenesis by upregulating nuclear respiratory factors and mitochondrial transcription factor A. From the above findings, it can be concluded that severe muscle degeneration leads to CK down-regulation in sarcoma, and that the stimulation of mitochondrial biogenesis indicated a scenario representing both CK deficiency and NOS overexpression on the one hand, and altered bioenergetic profiling on the other.