Expression Of Mitochondrial Creatine Kinase Research Articles

Desmin Knock-Out Cardiomyopathy: A Heart on the Verge of Metabolic Crisis.

Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.

Adaptation to chronic hypoxia is associated with increased expression of mitochondrial creatine kinase and hexokinase in the rat myocardium

Adaptation to chronic hypoxia is associated with increased expression of mitochondrial creatine kinase and hexokinase in the rat myocardium

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.

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.

Developmental changes in regulation of mitochondrial respiration by ADP and creatine in rat heart in vivo.

In saponin-skinned muscle fibers from adult rat heart and m. soleus the apparent affinity of the mitochondrial oxidative phosphorylation system for ADP (Km = 200-400 microM) is much lower than in isolated mitochondria (Km = 10-20 microM). This suggests a limited permeability of the outer mitochondrial membrane (OMM) to adenine nucleotides in slow-twitch muscle cells. We have studied the postnatal changes in the affinity of mitochondrial respiration for ADP, in relation to morphological alterations and expression of mitochondrial creatine kinase (mi-CK) in rat heart in vivo. Analysis of respiration of skinned fibers revealed a gradual decrease in the apparent affinity of mitochondria to ADP throughout 6 weeks post partum that indicates the development of mechanism which increasingly limits the access of ADP to mitochondria. The expression of mi-CK started between the 1st and 2nd weeks and reached the adult levels after 6 weeks. This process was associated with increases in creatine-activated respiration and affinity of oxidative phosphorylation to ADP thus reflecting the progressive coupling of mi-CK to adenine nucleotide translocase. Laser confocal microscopy revealed significant changes in rearrangement of mitochondria in cardiac cells: while the mitochondria of variable shape and size appeared to be random-clustered in the cardiomyocytes of 1 day old rat, they formed a fine network between the myofibrils by the age of 3 weeks. These results allow to conclude that in early period of development, i.e. within 2-3 weeks, the diffusion of ADP to mitochondria becomes progressively restricted, that appears to be related to significant structural rearrangements such as formation of the mitochondrial network. Later (after 3 weeks) the control shifts to mi-CK, which by coupling to adenine nucleotide translocase, allows to maximally activate the processes of oxidative phosphorylation despite limited access of ADP through the OMM.

Inhibition of ubiquitous mitochondrial creatine kinase expression in HeLa cells by an antisense oligodeoxynucleotide

Antisense strategy has been used to inhibit the synthesis of the human ubiquitous mitochondrial creatine kinase (Mi-CK) in HeLa cells. Indeed, elevated levels of Mi-CK in the serum of some cancer patients seem to be an adverse pronostic indicator (for refs see Wallimann T and Hemmer W, Mol Cell Biochem 133/134: 193-220, 1994). A phosphorothioate oligonucleotide, complementary to the second intron-exon splice junction site of the human ubiquitous Mi-CK pre-mRNA was shown to inhibit Mi-CK synthesis by 80% without modifying F1-ATPase beta subunit expression or hampering HeLa cell growth. This inhibition was correlated to a decrease of the Mi-CK mRNA level that could be determined quantitatively after amplification of reverse transcription products (RT) in the presence of varying concentrations of internal standard competitors. This study also demonstrated that the Mi-CK mRNA copy number was much lower in HeLa cells than that of the cytosolic creatine kinase isoform, B-CK. The antisense-induced decrease in Mi-CK mRNA and protein level influenced neither the expression of B-CK which uses up the phosphocreatine produced by Mi-CK during the phosphocreatine shuttle, nor that of another nuclear encoded mitochondrial gene, the F1-ATPase subunit which provides ATP to Mi-CK. In conclusion, an elevated Mi-CK expression is not required for cancer cell growth and therefore, Mi-CK is not a significant limiting factor for the growth of the cancer cells which contain it. In addition, a decrease in Mi-CK synthesis does not induce a change in the expression of mitochondrial F1-ATPase which provides ATP to Mi-CK or in the expression of cytosolic B-CK which is involved together with Mi-CK in the phosphocreatine shuttle. Therefore, the use of the phosphocreatine shuttle as a process mandatory for the active growth of some cancer cells is questioned.