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 intoleranc

Energia ülekande ja tagasiside regulatsioon oksüdatiivsetes lihasrakkudes.

Recently, it has been hypothesized that in oxidative muscle cells the mitochondria and ATPases form tight complexes, termed as the intracellular energetic units (ICEUs). At present the structure and molecular basis of the ICEUs are unclear and therefore an object of active research. Aims of the study: 1) To assess influence of alteration of the intracellular structure on mentioned units by hypercontraction in normal cardiomyocytes and in HL-1 cells, 2) to confirm existence of ICEUs by mathematical modeling of real experimental data, 3) to investigate whether ICEUs exist also in human heart atrium and in musculus gluteus medius (MGM), 4) to investigate whether these units may in MGM disintegrate during osteoarthritis. Results of the study: Differently from cardiomyocytes, where mitochondria and CaMgATPases are organized into ICEUs 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, the ICEUs do not exist in HL-1 cells due to less organized energy metabolism. Ca2+-induced hypercontraction leads in normal cardiomyocytes to the decreased direct transfer of ADP from ATPases to the mitochondria, thus to impairment of ICEU function. Mathematical modelling shows that in oxidative muscle cells diffusion restrictions of metabolites are not distributed uniformly within cardiac muscles, but are localized in certain areas, which supports the ICEU hypothesis. Energy metabolism in human atria and MGM cells is organized similarly to that in oxidative muscles − in the form of the ICEUs. Pathogenesis of OA involves disintegration of the ICEUs in association with dysfunction of CK-phosphotransfer system and increased diffusion of exogenous ADP to mitochondria. Hiljuti püstitati hüpotees, mille kohaselt oksüdatiivsetes lihasrakkudes moodustavad mitokondrid ja ATPaasid komplekse, e. rakusiseseid energeetilisi üksusi (RSEÜd). Praeguseks pole veel selge nende komplekside struktuuriline ja molekulaarne olemus, mis on seetõttu aktiivse uurimise objektiks. Töö eesmärgid: 1) Hinnata rakusiseste struktuurimuutuste toimet nimetud üksustele normaalsetes südamelihaskiududes hüperkontraktsiooni toimel ja HL-1 südamelihasrakkudes, 2) kinnitada RSEÜ-de olemasolu ka reaalsete eksperimenditulemuste matemaatilise modeleerimise abil, 3) uurida, kas RSEÜ-d eksisteerivad ka inimese südame kojas ja musculus gluteus medius-es (MGM) ning 4) kas nad võivad MGM-es osteoartriidi korral laguneda. Töö tulemused: Erinevalt südamelihasrakkudest, kus mitokondrid ja ATPaasid on organiseeritud RSEÜ-desse, mis kindlustavad efektiivse energia ülekande ja tagasiside nende struktuuride vahel otsese adeniinnukleotiidide ülekande teel ning kreatiinkinaasi ja adenülaatkinaasi isoensüümide vahendusel, puuduvad need üksüsed HL-1 rakkudes vähem organiseeritud energeetilise metabolismi tõttu. Ca2+ poolt põhjustatud hüperkontraktsioon põhjustab normaalsetes südamelihasrakkudes otsese adeniinukleotiidide ülekande nõrgenemise, seega RSEÜde funktsiooni häirumise. Reaalsete eksperimendiandmete matemaatiline modelleerimine näitab, et metaboliitide difusioonitakistused pole jaotunud oksüdatiivses lihasrakus ühtlaselt vaid on lokaliseerunud, mis toetab seega RSEÜ hüpoteesi. Energeetiline metabolism inimese südame kojas ja MGM-es on organiseeritud sarnaselt oksüdatiivsetele lihastele RSEÜde vormis. Osteoartroosi patogenees hõlmab RSEÜ-de osalist lagunemist, mis peegeldub kretiinkinaasse energiaülekande nõrgenemises ja eksogeense ADP suurenenud difusioonis mitokondrisse

Proteomic dataset of wolframin-deficient mouse heart and skeletal muscles.

The data presented in this article are related to the research article entitled "Increased Mitochondrial Protein Levels and Bioenergetics in the musculus rectus femoris of Wfs1-Deficient mice" (Eimre et al., accepted for publication). This dataset reports the analysis of Wfs1-deficient mouse heart, musculus soleus, and white part of musculus rectus femoris by liquid chromatography/tandem mass spectrometry. Label-free quantitative analysis of the mass spectrometry data identified 4056 proteins, with 114, 212, and 1290 proteins differentially expressed (t-test; p m. soleus, and m. rectus femoris, respectively, between the Wfs1-deficient and wild-type groups. Eight proteins were found to be differentially expressed in all mentioned muscles, with 1 protein differently expressed in oxidative (m. soleus and heart) and 88 in skeletal muscles. This dataset supports the cited study and can be used to extend additional analyses. Data are available via ProteomeXchange with identifier PXD011019

Studies of mitochondrial respiration in muscle cells in situ: use and misuse of experimental evidence in mathematical modelling.

International audienceApplications of permeabilized cell and skinned fiber techniques in combination with methods of mathematical modelling for studies of mitochondrial function in the cell are critically evaluated. Mathematical models may be useful tools for explaining biological phenomena, but only if they are selected by fitting the computing results with real experimental data. Confocal microscopy has been used in experiments with permeabilized cardiomyocytes and myocardial fibers to determine the maximal diffusion distance from medium to the core of cells, which is shown not to exceed 8-10 microm. This is a principal index for correctly explaining high apparent Km for exogenous ADP (200-300 microM) in regulation of mitochondrial respiration in oxidative muscle cells in situ. The best fitting of the results of in silico studies may be achieved by using of the compartmentalized energy transfer model. From these results, it may be concluded that in cardiac muscle cells the mitochondria and ATPases are organized into intracellular energetic units (ICEUs) separated from the bulk phase of cytoplasm by some barriers which limit the diffusion of adenine nucleotides. In contrast, alternative models based on the concept of the cell as homogenous system do not explain the observed experimental phenomena and have led to misleading conclusions. The various sources of experimental and conceptual errors are analyzed

Intracellular diffusion of adenosine phosphates is locally restricted in cardiac muscle.

International audienceRecent studies have revealed the structural and functional interactions between mitochondria, myofibrils and sarcoplasmic reticulum in cardiac cells. Direct channeling of adenosine phosphates between organelles identified in the experiments indicates that diffusion of adenosine phosphates is limited in cardiac cells due to very specific intracellular structural organization. However, the mode of diffusion restrictions and nature of the intracellular structures in creating the diffusion barriers is still unclear, and, therefore, a subject of active research. The aim of this work is to analyze the possible role of two principally different modes of restriction distribution for adenosine phosphates (a) the uniform diffusion restriction and (b) the localized diffusion limitation in the vicinity of mitochondria, by fitting the experimental data with the mathematical model. The reaction-diffusion model of compartmentalized energy transfer was used to analyze the data obtained from the experiments with the skinned muscle fibers, which described the following processes: mitochondrial respiration rate dependency on exogenous ADP and ATP concentrations; inhibition of endogenous ADP-stimulated respiration by pyruvate kinase (PK) and phosphoenolpyruvate (PEP) system; kinetics of oxygen consumption stabilization after addition of 2 mM MgATP or MgADP; ATPase activity with inhibited mitochondrial respiration; and buildup of MgADP concentration in the medium after addition of MgATP. The analysis revealed that only the second mechanism considered--localization of diffusion restrictions--is able to account for the experimental data. In the case of uniform diffusion restrictions, the model solution was in agreement only with two measurements: the respiration rate as a function of ADP or ATP concentrations and inhibition of respiration by PK + PEP. It was concluded that intracellular diffusion restrictions for adenosine phosphates are not distributed uniformly, but rather are localized in certain compartments of the cardiac cells

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

Calcium-induced contraction of sarcomeres changes the regulation of mitochondrial respiration in permeabilized cardiac cells.

International audienceThe relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyocytes and myocardial fibers, and in their 'ghost' preparations (after selective myosin extraction). Up to 3 microm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and a significant decrease in the apparent K(m) for exogenous ADP and ATP in the kinetics of mitochondrial respiration. The V(max) of respiration showed a moderate (50%) increase, with an optimum at 0.4 microm free calcium and a decrease at higher calcium concentrations. At high free-calcium concentrations, the direct flux of ADP from ATPases to mitochondria was diminished compared to that at low calcium levels. All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in 'ghost' preparations after the selective extraction of myosin. Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition to the metabolic signalling via the creatine kinase system

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

Altered mitochondrial apparent affinity for ADP and impaired function of mitochondrial creatine kinase in gluteus medius of patients with hip osteoarthritis.

International audienceThe cellular energy metabolism in human musculus gluteus medius (MGM) under normal conditions and hip osteoarthritis (OA) was explored. The functions of oxidative phosphorylation and energy transport systems were analyzed in permeabilized (skinned) muscle fibers by oxygraphy, in relation to myosin heavy chain (MHC) isoform distribution profile analyzed by SDS-PAGE, and to creatine kinase (CK) and adenylate kinase (AK) activities measured spectrophotometrically in the intact muscle. The results revealed high apparent Km for ADP in regulation of respiration that decreased after addition of creatine in MGM of traumatic patients (controls). OA was associated with increased sensitivity of mitochondrial respiration to ADP, decreased total activities of AK and CK with major reduction in mi-CK fraction, and attenuated effect of creatine on apparent Km for ADP compared with control group. It also included a complete loss of type II fibers in a subgroup of patients with the severest disease grade. It is concluded that energy metabolism in MGM cells is organized into functional complexes of mitochondria and ATPases. It is suggested that because of degenerative remodeling occurring during development of OA, these complexes become structurally and functionally impaired, which results in increased access of exogenous ADP to mitochondria and dysfunction of CK-phosphotransfer system