Adaptation of striated muscles to Wolframin deficiency in mice: Alterations in cellular bioenergetics.

BACKGROUND: Wolfram syndrome (WS), caused by mutations in WFS1 gene, is a multi-targeting disease affecting multiple organ systems. Wolframin is localized in the membrane of the endoplasmic reticulum (ER), influencing Ca2+ metabolism and ER interaction with mitochondria, but the exact role of the protein remains unclear. In this study we aimed to characterize alterations in energy metabolism in the cardiac and in the oxidative and glycolytic skeletal muscles in Wfs1-deficiency. METHODS: Alterations in the bioenergetic profiles in the cardiac and skeletal muscles of Wfs1-knock-out (KO) male mice and their wild type male littermates were determined using high resolution respirometry, quantitative RT-PCR, NMR spectroscopy, and immunofluorescence confocal microscopy. RESULTS: Oxygen consumption without ATP synthase activation (leak) was significantly higher in the glycolytic muscles of Wfs1 KO mice compared to wild types. ADP-stimulated respiration with glutamate and malate was reduced in the Wfs1-deficient cardiac as well as oxidative and glycolytic skeletal muscles. CONCLUSIONS: Wfs1-deficiency in both cardiac and skeletal muscles results in functional alterations of energy transport from mitochondria to ATP-ases. There was a substrate-dependent decrease in the maximal Complex I -linked respiratory capacity of the electron transport system in muscles of Wfs1 KO mice. Moreover, in cardiac and gastrocnemius white muscles a decrease in the function of one pathway were balanced by the increase in the activity of the parallel pathway. GENERAL SIGNIFICANCE: This work provides new insights to the muscle involvement at early stages of metabolic syndrome like WS as well as developing glucose intolerance.

Increased Mitochondrial Protein Levels and Bioenergetics in the Musculus Rectus Femoris of Wfs1-Deficient Mice.

Wfs1 deficiency leads to a progressive loss of plasma insulin concentration, which should reduce the consumption of glucose in insulin-dependent tissues, causing a variety of changes in intracellular energy metabolism. Our objective here was to assess the changes in the amount and function of mitochondrial proteins in different muscles of Wfs1-deficient mice. Mitochondrial functions were assayed by high-resolution oxygraphy of permeabilized muscle fibers; the protein amount was evaluated by liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis and mRNA levels of the uncoupler proteins UCP2 and UCP3 by real-time PCR; and citrate synthase (CS) activity was determined spectrophotometrically in muscle homogenates. Compared to controls, there were no changes in proton leak and citrate synthase activity in the heart and m. soleus tissues of Wfs1-deficient mice, but significantly higher levels of both of these factors were observed in the m. rectus femoris; mitochondrial proteins and mRNA of UCP2 were also higher in the m. rectus femoris. ADP-stimulated state 3 respiration was lower in the m. soleus, remained unchanged in the heart, and was higher in the m. rectus femoris. The mitochondrial protein amount and activity are higher in Wfs1-deficient mice, as are mitochondrial proton leak and oxygen consumption in m. rectus femoris. These changes in muscle metabolism may be important for identifying the mechanisms responsible for Wolfram syndrome and diabetes.

2102Ep embryonal carcinoma cells have compromised respiration and shifted bioenergetic profile distinct from H9 human embryonic stem cells.

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells, it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line). We examined three parameters related to cellular bioenergetics: phosphotransfer system, aerobic glycolysis, and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells. 2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85±5.73 vs 64.39±2.55mU/mg of protein) and the expression of AK2 was significantly higher in these cells, while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass, increased proton leak, and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference, and highlight the importance of further research concerning energy metabolism of stem cells.

Proliferation of Human Primary Myoblasts Is Associated with Altered Energy Metabolism in Dependence on Ageing In Vivo and In Vitro.

BACKGROUND. Ageing is associated with suppressed regenerative potential of muscle precursor cells due to decrease of satellite cells and suppressive intramuscular milieu on their activation, associated with ageing-related low-grade inflammation. The aim of the study was to characterize the function of oxidative phosphorylation (OXPHOS), glycolysis, adenylate kinase (AK), and creatine kinase (CK) mediated systems in young and older individuals. MATERIALS AND METHODS. Myoblasts were cultivated from biopsies taken by transcutaneous conchotomy from vastus lateralis muscle in young (20-29 yrs, n = 7) and older (70-79 yrs, n = 7) subjects. Energy metabolism was assessed in passages 2 to 6 by oxygraphy and enzyme analysis. RESULTS. In myoblasts of young and older subjects the rate of OXPHOS decreased during proliferation from passages 2 to 6. The total activities of CK and AK decreased. Myoblasts of passage 2 cultivated from young muscle showed higher rate of OXPHOS and activities of CK and AK compared to myoblasts from older subjects while hexokinase and pyruvate kinase were not affected by ageing. CONCLUSIONS. Proliferation of myoblasts in vitro is associated with downregulation of OXPHOS and energy storage and transfer systems. Ageing in vivo exerts an impact on satellite cells which results in altered metabolic profile in favour of the prevalence of glycolytic pathways over mitochondrial OXPHOS of myoblasts.

Deep Quantitative Proteomics Reveals Extensive Metabolic Reprogramming and Cancer-Like Changes of Ectopic Endometriotic Stromal Cells.

Endometriosis is a prevalent health condition in women of reproductive age characterized by ectopic growth of endometrial-like tissue in the extrauterine environment. Thorough understanding of the molecular mechanisms underlying the disease is still incomplete. We dissected eutopic and ectopic endometrial primary stromal cell proteomes to a depth of nearly 6900 proteins using quantitative mass spectrometry with a spike-in SILAC standard. Acquired data revealed metabolic reprogramming of ectopic stromal cells with extensive upregulation of glycolysis and downregulation of oxidative respiration, a widespread metabolic phenotype known as the Warburg effect and previously described in many cancers. These changes in metabolism are additionally accompanied by attenuated aerobic respiration of ectopic endometrial stromal cells as measured by live-cell oximetry and by altered mRNA levels of respective enzyme complexes. Our results additionally highlight other molecular changes of ectopic endometriotic stromal cells indicating reduced apoptotic potential, increased cellular invasiveness and adhesiveness, and altered immune function. Altogether, these comprehensive proteomics data refine the current understanding of endometriosis pathogenesis and present new avenues for therapies.

Dilation of human atria: increased diffusion restrictions for ADP, overexpression of hexokinase 2 and its coupling to oxidative phosphorylation in cardiomyocytes.

Cardiac energy metabolism with emphasis on mitochondria was addressed in atrial tissue from patients with overload-induced atrial dilation. Structural remodeling of dilated (D) atria manifested as intracellular accumulation of fibrillar aggregates, lipofuscin, signs of myolysis and autophagy. Despite impaired complex I dependent respiration and increased diffusion restriction for ADP, no changes regarding adenylate and creatine kinase occurred. We observed 7-fold overexpression of HK2 gene in D atria with concomitant 2-fold greater activation of mitochondrial oxygen consumption by glucose, which might represent an adaption to increased energy requirements and impaired mitochondrial function by effectively joining glycolysis and oxidative phosphorylation.

Impaired oxidative phosphorylation in overtrained rat myocardium.

The present study was undertaken to characterize and review the changes in energy metabolism in rat myocardium in response to chronic exhaustive exercise. It was shown that a treadmill exercise program applied for six weeks led the rats into a state characterized by decreased performance, loss of body weight and enhanced muscle catabolism, indicating development of overtraining syndrome. Electron microscopy revealed disintegration of the cardiomyocyte structure, cellular swelling and appearance of peroxisomes. Respirometric assessment of mitochondria in saponin-permeabilized cells in situ revealed a decreased rate of oxidative phosphorylation (OXPHOS) due to diminished control over it by ADP and impaired functional coupling of adenylate kinase to OXPHOS. In parallel, reduced tissue content of cytochrome c was observed, which could limit the maximal rate of OXPHOS. The results are discussed with respect to relationships between the volume of work and corresponding energy metabolism. It is concluded that overtraining syndrome is not restricted to skeletal muscle but can affect cardiac muscle as well.

Distinct organization of energy metabolism in HL-1 cardiac cell line and cardiomyocytes.

Expression and function of creatine kinase (CK), adenylate kinase (AK) and hexokinase (HK) isoforms in relation to their roles in regulation of oxidative phosphorylation (OXPHOS) and intracellular energy transfer were assessed in beating (B) and non-beating (NB) cardiac HL-l cell lines and adult rat cardiomyocytes or myocardium. In both types of HL-1 cells, the AK2, CKB, HK1 and HK2 genes were expressed at higher levels than the CKM, CKMT2 and AK1 genes. Contrary to the saponin-permeabilized cardiomyocytes the OXPHOS was coupled to mitochondrial AK and HK but not to mitochondrial CK, and neither direct transfer of adenine nucleotides between CaMgATPases and mitochondria nor functional coupling between CK-MM and CaMgATPases was observed in permeabilized HL-1 cells. The HL-1 cells also exhibited deficient complex I of the respiratory chain. In conclusion, contrary to cardiomyocytes where mitochondria and CaMgATPases are organized into tight complexes which ensure effective energy transfer and feedback signaling between these structures via specialized pathways mediated by CK and AK isoforms and direct adenine nucleotide channeling, these complexes do not exist in HL-1 cells due to less organized energy metabolism.

Different kinetics of the regulation of respiration in permeabilized cardiomyocytes and in HL-1 cardiac cells. Importance of cell structure/organization for respiration regulation.

The aim of this study was to investigate the mechanism of cellular regulation of mitochondrial respiration in permeabilized cardiac cells with clearly different structural organization: (i) in isolated rat cardiomyocytes with very regular mitochondrial arrangement, (ii) in HL-1 cells from mouse heart, and (iii) in non-beating (NB HL-1 cells) without sarcomeres with irregular and dynamic filamentous mitochondrial network. We found striking differences in the kinetics of respiration regulation by exogenous ADP between these cells: the apparent Km for exogenous ADP was by more than order of magnitude (14 times) lower in the permeabilized non-beating NB HL-1 cells without sarcomeres (25+/-4 microM) and seven times lower in normally cultured HL-1 cells (47+/-15 microM) than in permeabilized primary cardiomyocytes (360+/-51 microM). In the latter cells, treatment with trypsin resulted in dramatic changes in intracellular structure that were associated with 3-fold decrease in apparent Km for ADP in regulation of respiration. In contrast to permeabilized cardiomyocytes, in NB HL-1 cells creatine kinase activity was low and the endogenous ADP fluxes from MgATPases recorded spectrophotometrically by the coupled enzyme assay were not reduced after activation of mitochondrial oxidative phosphorylation by the addition of mitochondrial substrates, showing the absence of ADP channelling in the NB HL-1 cells. While in the permeabilized cardiomyocytes creatine strongly activated mitochondrial respiration even in the presence of powerful competing pyruvate kinase-phosphoenolpyruvate system, in the NB HL-1 cells the stimulatory effect of creatine was not significant. The results of this study show that in normal adult cardiomyocytes and HL-1 cells intracellular local restrictions of diffusion of adenine nucleotides and metabolic feedback regulation of respiration via phosphotransfer networks are different, most probably related to differences in structural organization of these cells.

Oxidative phosphorylation and its coupling to mitochondrial creatine and adenylate kinases in human gastric mucosa.

Energy metabolism in gastrobiopsy specimens of the antral and corpus mucosa, treated with saponin to permeabilize the cells, was studied in patients with gastric diseases. The results show twice lower oxidative capacity in the antral mucosa than in the corpus mucosa and the relative deficiency of antral mitochondria in complex I. The mucosal cells expressed mitochondrial and cytosolic isoforms of creatine kinase and adenylate kinase (AK). Creatine (20 mM) and AMP (2 mM) markedly stimulated mitochondrial respiration in the presence of submaximal ADP or ATP concentrations, and creatine reduced apparent Km for ADP in stimulation of respiration, which indicates the functional coupling of mitochondrial kinases to oxidative phosphorylation. Addition of exogenous cytochrome c increased ADP-dependent respiration, and the large-scale cytochrome c effect (>or=20%) was associated with suppressed stimulation of respiration by creatine and AMP in the mucosal preparations. These results point to the impaired mitochondrial outer membrane, probably attributed to the pathogenic effects of Helicobacter pylori. Compared with the corpus mucosa, the antral mucosa exhibited greater sensitivity to such type of injury as the prevalence of the large-scale cytochrome c effect was twice higher among the latter specimens. Active chronic gastritis was associated with decreased respiratory capacity of the corpus mucosa but with its increase in the antral mucosa. In conclusion, human gastric mucosal cells express the mitochondrial and cytosolic isoforms of CK and AK participating in intracellular energy transfer systems. Gastric mucosa disease is associated with the altered functions of these systems and oxidative phosphorylation.

Structure-function relationships in the regulation of energy transfer between mitochondria and ATPases in cardiac cells.

The present study discusses the role of structural organization of cardiac cells in determining the mechanisms of regulation of oxidative phosphorylation and interaction between mitochondria and ATPases. In permeabilized adult cardiomyocytes, the apparent K(m) (Michaelis-Menten constant) for ADP in the regulation of respiration is far higher than in mitochondria isolated from the myocardium. Respiration of mitochondria in permeabilized cardiomyocytes is effectively activated by endogenous ADP produced by ATPases from exogenous ATP, and the activation of respiration is associated with a decrease in the apparent K(m) for ATP in the regulation of ATPase activity compared with this parameter in the absence of oxidative phosphorylation. It has also been shown that a large fraction of the endogenous ADP stimulating respiration remains inaccessible for the exogenous ADP trapping system, consisting of pyruvate kinase and phosphoenolpyruvate, unless the mitochondrial structures are modified by controlled proteolysis. These data point to the endogenous cycling of adenine nucleotides between mitochondria and ATPases. Accordingly, the current hypothesis is that in cardiac cells, mitochondria and ATPases are compartmentalized into functional complexes (ie, intracellular energetic units [ICEUs]), which appear to represent a basic pattern of organization of energy metabolism in these cells. Within the ICEUs, the mitochondria and ATPases interact via different routes: creatine kinase-mediated phosphoryltransfer; adenylate kinase-mediated phosphoryltransfer; and direct ATP and ADP channelling. The function of ICEUs changes not only after selective proteolysis, but also during contraction of cardiomyocytes caused by an increase in cytosolic Ca(2+) concentration up to micromolar levels. In these conditions, the apparent K(m) for exogenous ADP and ATP in the regulation of respiration markedly decreases, and more ADP becomes available for the exogenous pyruvate kinase-phosphoenolpyruvate system, which indicates altered barrier functions of the ICEUs. Thus, structural changes transmitted from the contractile apparatus to mitochondria clearly participate in the regulation of mitochondrial function due to alterations in localized restriction of the diffusion of adenine nucleotides. The importance of strict structural organization in cardiac cells emerged drastically from experiments in which the regulation of mitochondrial respiration was assessed in a novel cardiac cell line, that is, beating and nonbeating HL-1 cells. In these cells, the mitochondrial arrangement is irregular and dynamic, whereas the sarcomeric structures are either absent (in nonbeating HL-1 cells) or only rarely present (in beating HL-1 cells). In parallel, the apparent K(m) for exogenous ADP in the regulation of respiration was much lower than that in permeabilized primary cardiomyocytes, and trypsin treatment exerted no impact on the low K(m) value for ADP, in contrast to adult cardiomyocytes where it caused a marked decrease in this parameter. The HL-1 cells were also characterized by the absence of direct exchange of adenine nucleotides. The results further support the concept that the ICEUs in adult cardiomyocytes are products of complex structural organization developed to create the most optimal conditions for effective energy transfer and feedback between mitochondria and ATPases.

Compartmentation of energy metabolism in atrial myocardium of patients undergoing cardiac surgery.

The parameters of oxidative phosphorylation and its interaction with creatine kinase (CK)- and adenylate kinase (AK)-phosphotransfer networks in situ were studied in skinned atrial fibers from 59 patients undergoing coronary artery bypass surgery, valve replacement/correction and atrial septal defect correction. In atria, the mitochondrial CK and AK are effectively coupled to oxidative phosphorylation, the MM-CK is coupled to ATPases and there exists a direct transfer of adenine nucleotides between mitochondria and ATPases. Elimination of cytoplasmic ADP with exogenous pyruvate kinase was not associated with a blockade of the stimulatory effects of creatine and AMP on respiration, neither could it abolish the coupling of MM-CK to ATPases and direct transfer of adenine nucleotides. Thus, atrial energy metabolism is compartmentalized so that mitochondria form functional complexes with adjacent ATPases. These complexes isolate a part of cellular adenine nucleotides from their cytoplasmic pool for participating in energy transfer via CK- and AK-networks, and/or direct exchange. Compared to atria in sinus rhythm, the fibrillating atria were larger and exhibited increased succinate-dependent respiration relative to glutamate-dependent respiration and augmented proton leak. Thus, alterations in mitochondrial oxidative phosphorylation may contribute to pathogenesis of atrial fibrillation.

Intracellular energetic units in healthy and diseased hearts.

BACKGROUND: The present review examines the role of intra-cellular compartmentation of energy metabolism in vivo. OBJECTIVE: To compare the kinetics of the activation of mitochondrial respiration in skinned cardiac fibres by exogenous and endogenous adenine nucleotides in dependence of the modulation of cellular structure and contraction. METHODS: Saponin-permeabilized cardiac fibres or cells were analyzed using oxygraphy and confocal microscopy. RESULTS: Mitochondria respiration in fibres or cells was upregulated by cumulative additions of ADP to the medium with an apparent K(m) of 200 muM to 300 muM. When respiration was stimulated by endogenous ADP produced by intracellular ATPases, a near maximum respiration rate was achieved at an ADP concentration of less than 20 muM in the medium. A powerful ADP-consuming system, consisting of pyruvate kinase and phosphoenolpyruvate, that totally suppressed the activation of respiration by exogenous ADP, failed to abolish the stimulation of respiration by endogenous ADP, but did inhibit respiration after the cells were treated with trypsin. The addition of up to 4 muM of free Ca(2+) to the actively respiring fibres resulted in reversible hypercontraction associated with a decreased apparent K(m) for exogenous ADP. These changes were fully abolished in fibres after the removal of myosin by KCl treatment. CONCLUSIONS: Mitochondria and ATPases, together with cytoskeletal proteins that establish the structural links between mitochondria and sarcomeres, form complexes - intracellular energetic units (ICEUs) - in cardiac cells. Within the ICEUs, the mitochondria and ATPases interact via specialized energy transfer systems, such as the creatine kinase- and adenylate kinase-phosphotransfer networks, and direct ATP channelling. Disintegration of the structure and function of ICEUs results in dyscompartmentation of adenine nucleotides and may represent a basis for cardiac diseases.