Adipophilin distribution and colocalisation with lipid droplets in skeletal muscle

Intramyocellular lipids (IMCL) are stored as discrete lipid droplets which are associated with a number of proteins. The lipid droplet-associated protein adipophilin (the human orthologue of adipose differentiation-related protein) is ubiquitously expressed and is one of the predominant lipid droplet-proteins in skeletal muscle. The aim of this study was to investigate the subcellular distribution of adipophilin in human muscle fibres and to measure the colocalisation of adipophilin with IMCL. Muscle biopsies from six lean male cyclists (BMI 23.4 ± 0.4, aged 31 ± 2 years, W _max 346 ± 8) were stained for myosin heavy chain type 1, IMCL, adipophilin and mitochondria using immunofluorescence and viewed with widefield and confocal fluorescence microscopy. The present study shows that like IMCL, the adipophilin content is ~twofold greater in type I skeletal muscle fibres and is situated in the areas between the mitochondrial network. Colocalisation analysis demonstrated that 61 ± 2% of IMCL contain adipophilin. Although the majority of adipophilin is contained within IMCL, 36 ± 4% of adipophilin is not associated with IMCL. In conclusion, this study indicates that the IMCL pool is heterogenous, as the majority but not all IMCL contain adipophilin.     

Influence of environmental temperature and energy intake on skeletal muscle respiratory enzymes and morphology

Influence of a cold (10 degrees C) or warm (35 degrees C) environment and a high or low level of energy intake on respiratory enzyme activities has been investigated in porcine skeletal muscle. Scanning microdensitometry was used to measure the reaction products from mitochondrial enzymes in individual slow- and fast-twitch muscle fibres. A cold environment was found to increase the activity of succinate dehydrogenase in both types of muscle fibre (P less than 0.001 for dark fibres, P less than 0.01 for light fibres) from young growing animals. Enzyme activity was also increased in animals on a low compared with a high energy intake (P less than 0.01) when living at 10 degrees C but not at 35 degrees C. Similar findings were obtained for NADH diaphorase and cytochrome oxidase aa3. The numbers of slow-twitch muscle fibres also increased after exposure to cold (P less than 0.01) and as a result of a low energy intake (P less than 0.01). These results are similar to those obtained in other species after exercise or as a result of peripheral arterial insufficiency. The extent to which they could be related to local tissue hypoxia or to changes in metabolic hormones is discussed.     

Volume density and distribution of mitochondria in harbor seal (<Emphasis Type="Italic">Phoca vitulina</Emphasis>) skeletal muscle

Recent studies have shown that harbor seals (Phoca vitulina) have an increased skeletal muscle mitochondrial volume density that may be an adaptation for maintaining aerobic metabolism during diving. However, these studies were based on single samples taken from locomotory muscles. In this study, we took multiple samples from a transverse section of the epaxial (primary locomotory) muscles and single samples from the m. pectoralis (secondary locomotory) muscle of five wild harbor seals. Average mitochondrial volume density of the epaxial muscles was 5.6%, which was 36.6% higher than predicted for a terrestrial mammal of similar mass, and most (82.1%) of the mitochondria were interfibrillar, unlike athletic terrestrial mammals. In the epaxial muscles, the total mitochondrial volume density was significantly greater in samples collected from the deep (6.0%) compared with superficial (5.0%) regions. Volume density of mitochondria in the pectoralis muscle was similar (5.2%) to that of the epaxial muscles. Taken together, these adaptations reduce the intracellular distance between mitochondria and oxymyoglobin and increase the mitochondrial diffusion surface area. This, in combination with elevated myoglobin concentrations, potentially increases the rate of oxygen diffusion into mitochondria and prevents diffusion limitation so that aerobic metabolism can be maintained under low oxygen partial pressure that develops during diving.     

Effect of cold environment on skeletal muscle mitochondria in growing rats

Summary Growing rats (4 weeks old) were kept for 3 weeks at 11° C and 24° C respectively. The cold-adapted animals showed a significantly higher oxygen consumption (64%). Volume density of subsarcolemmal and interfibrillar mitochondria as well as volume density of fat droplets were estimated in M. soleus and the diaphragm of both groups. In cold-adapted animals, the total volume of mitochondria was significantly increased by 24% in diaphragm and 37% in M. soleus. The volume of subsarcolemmal mitochondria was almost doubled in each muscle, but the volume of interfibrillar mitochondria did not change significantly. The surface of the inner mitochondrial membranes per unit volume of mitochondrion in M. soleus was significantly increased both in interfibrillar and subsarcolemmal mitochondria, whereas the surface of the outer mitochondrial membranes per unit volume of mitochondrion was increased only in the subsarcolemmal mitochondria. The volume of fat droplets in the diaphragm and M. soleus of cold adapted animals increased significantly by 62% and 150% respectively.     

A Novel MitoTimer Reporter Gene for Mitochondrial Content,Structure, Stress,and Damage in Vivo

Mitochondrial dysfunction plays important roles in many diseases, but there is no satisfactory method to assess mitochondrial health in vivo. Here, we engineered a MitoTimer reporter gene from the existing Timer reporter gene. MitoTimer encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized. Confocal microscopy confirmed targeting of the MitoTimer protein to mitochondria in cultured cells, Caenorhabditis elegans touch receptor neurons, Drosophila melanogaster heart and indirect flight muscle, and mouse skeletal muscle. A ratiometric algorithm revealed that conditions that cause mitochondrial stress led to a significant shift toward red fluorescence as well as accumulation of pure red fluorescent puncta of damaged mitochondria targeted for mitophagy. Long term voluntary exercise resulted in a significant fluorescence shift toward green, in mice and D. melanogaster, as well as significantly improved structure and increased content in mouse FDB muscle. In contrast, high-fat feeding in mice resulted in a significant shift toward red fluorescence and accumulation of pure red puncta in skeletal muscle, which were completely ameliorated by voluntary wheel running. Hence, MitoTimer allows for robust analysis of multiple parameters of mitochondrial health under both physiological and pathological conditions and will be highly useful for future research of mitochondrial health in multiple disciplines in vivo.     

Mitochondrial function, content and ROS production in rat skeletal muscle: effect of high-fat feeding

A high intake of dietary fat has been suggested to diminish mitochondrial functioning in skeletal muscle, possibly attributing to muscular fat accumulation. Here we show however, that an 8-week high-fat dietary intervention did not affect intrinsic functioning of rat skeletal muscle mitochondria assessed by respirometry, neither on a carbohydrate- nor on a lipid-substrate. Interestingly, PPARGC1A protein increased by approximately 2-fold upon high-fat feeding and we observed inconsistent results on different markers of mitochondrial density. Mitochondrial ROS production, assessed by electron spin resonance spectroscopy remained unaffected. Intramyocellular lipid levels increased significantly illustrating that a reduced innate mitochondrial function is not a prerequisite for intra-muscular fat accumulation.     

脂滴与线粒体相互作用的研究进展

肥胖和多种代谢类疾病的发生有着密切的关系,而导致肥胖的脂肪多以中性脂的形式储存于细胞的一种细胞器——脂滴中。越来越多的研究表明,脂滴能够和其它细胞器发生相互作用,而它和线粒体的相互作用可能与Ⅱ型糖尿病的形成密切相关:非正常的脂滴和线粒体的相互作用有可能是导致细胞胰岛素抵抗的重要原因。我们通过对脂滴表面蛋白质组学、脂滴与线粒体的空间位置,以及相关蛋白等研究的总结,结合本实验室的研究结果,对脂滴与线粒体相互作用的物质基础及可能方式、受骨骼肌有氧运动的影响,及其与骨骼肌胰岛素抵抗发生的关系等,进行了讨论。     

Exercise-induced oxidative stress in humans: cause and consequences

The observation that muscular exercise is associated with oxidative stress in humans was first reported over 30 years ago. Since this initial report, numerous studies have confirmed that prolonged or high-intensity exercise results in oxidative damage to macromolecules in both blood and skeletal muscle. Although the primary tissue(s) responsible for reactive oxygen species (ROS) production during exercise remains a topic of debate, compelling evidence indicates that muscular activity promotes oxidant production in contracting skeletal muscle fibers. Mitochondria, NADPH oxidase, PLA₂-dependent processes, and xanthine oxidase have all been postulated to contribute to contraction-induced ROS production in muscle but the primary site of contraction-induced ROS production in muscle fibers remains unclear. Nonetheless, contraction-induced ROS generation has been shown to play an important physiological function in the regulation of both muscle force production and contraction-induced adaptive responses of muscle fibers to exercise training. Although knowledge in the field of exercise and oxidative stress has grown markedly during the past 30 years, this area continues to expand and there is much more to be learned about the role of ROS as signaling molecules in skeletal muscle.     

The beneficial role of exercise in mitigating doxorubicin-induced Mitochondrionopathy

Doxorubicin (DOX) is a widely used antineoplastic agent for a wide range of cancers, including hematological malignancies, soft tissue sarcomas and solid tumors. However, DOX exhibits a dose-related toxicity that results in life-threatening cardiomyopathy. In addition to the heart, there is evidence that DOX toxicity extends to other organs. This general toxicity seems to be related to mitochondrial network structural, molecular and functional impairments. Several countermeasures for these negative effects have been proposed, being physical exercise, not only one of the most effective non-pharmacologic strategy but also widely recommended as booster against cancer-related fatigue.It is widely accepted that mitochondria are critical sensors of tissue functionality, both modulated by DOX and exercise. Therefore, this review focuses on the current understanding of the mitochondrial-mediated mechanisms underlying the protective effect of exercise against DOX-induced toxicity, not only limited to the cardiac tissue, but also in other tissues such as skeletal muscle, liver and brain. We here analyze recent developments regarding the beneficial effects of exercise targeting mitochondrial responsive phenotypes against redox changes, mitochondrial bioenergetics, apoptotic, dynamics and quality control signalling affected by DOX treatment.     

IP3 receptor blockade restores autophagy and mitochondrial function in skeletal muscle fibers of dystrophic mice

Duchenne muscular dystrophy (DMD) is characterized by a severe and progressive destruction of muscle fibers associated with altered Ca²⁺ homeostasis. We have previously shown that the IP₃ receptor (IP₃R) plays a role in elevating basal cytoplasmic Ca²⁺ and that pharmacological blockade of IP₃R restores muscle function. Moreover, we have shown that the IP₃R pathway negatively regulates autophagy by controlling mitochondrial Ca²⁺ levels. Nevertheless, it remains unclear whether IP₃R is involved in abnormal mitochondrial Ca²⁺ levels, mitochondrial dynamics, or autophagy and mitophagy observed in adult DMD skeletal muscle. Here, we show that the elevated basal autophagy and autophagic flux levels were normalized when IP₃R was downregulated in mdx fibers. Pharmacological blockade of IP₃R in mdx fibers restored both increased mitochondrial Ca²⁺ levels and mitochondrial membrane potential under resting conditions. Interestingly, mdx mitochondria changed from a fission to an elongated state after IP₃R knockdown, and the elevated mitophagy levels in mdx fibers were normalized. To our knowledge, this is the first study associating IP₃R1 activity with changes in autophagy, mitochondrial Ca²⁺ levels, mitochondrial membrane potential, mitochondrial dynamics, and mitophagy in adult mouse skeletal muscle. Moreover, these results suggest that increased IP₃R activity in mdx fibers plays an important role in the pathophysiology of DMD. Overall, these results lead us to propose the use of specific IP₃R blockers as a new pharmacological treatment for DMD, given their ability to restore both autophagy/mitophagy and mitochondrial function.     

The voltage dependence of depolarization-induced calcium release in isolated skeletal muscle triads

We demonstrate for the first time in this study that triadic vesicles derived from skeletal muscle display a voltage dependence of depolarization-induced calcium release similar to that found in intact muscle. We confirm previous studies by Dunn (1989) which demonstrated that changes in extravesicular potassium induced membrane potential changes in isolated transverse tubules with the voltage sensitive dye DiSC(3)-5. Depolarization-induced calcium release was studied in isolated triadic vesicles through similar changes in extravesicular [K] while clamping extravesicular Ca⁺⁺ to submicromolar concentrations. The amplitude of fast phase of calcium release, identified as depolarizationinduced calcium release, varied with the percentage of transverse tubules in the preparation (determined through ³ H-PN200-110 specific activity) and different levels of depolarization. Threshold activation of calcium release was obtained with a 40.5 mV potential change; maximal calcium release was obtained with a 75 to 81 mV potential change. Boltzmann fits to the normalized depolarization induced calcium release plotted against the membrane potential change yielded a voltage dependence (k = 4.5 mV per e-fold change) very similar to that found in intact muscle (k = 3–4 mV per e-fold change; Baylor, Chandler & Marshall 1978, 1983; Miledi et al., 1981). Substitution of methanesulfonate for propionate as the impermeant ion or addition of valinomycin in the depolarizing solutions had little effect on the voltage dependence of calcium release.We thank Dr. Judith Heiny for her helpful discussions about voltagesensitive fluorescent dyes. This work was supported by the American Heart Association (Ohio Affiliate) grant MV-90 and the State of Ohio Research Challenge Grant.     

4Pi microscopy of quantum dot-labeled cellular structures

The most prominent restrictions of fluorescence microscopy are the limited resolution and the finite signal. Established conventional, confocal, and multiphoton microscopes resolve at best approximately 200nm in the focal plane and only 500nm in depth. Additionally, organic fluorophores and fluorescent proteins are bleached after 10(4)-10(5) excitation cycles. To overcome these restrictions, we synergistically combine the 3- to 7-fold improved axial resolution of 4Pi microscopy with the greatly enhanced photostability of semiconductor quantum dots. Co-localization studies of immunolabeled microtubules and mitochondria demonstrate the feasibility of this approach for routine biological measurements. In particular, we visualize the three-dimensional entanglement of the two networks with unprecedented detail.     

The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle

Resting skeletal muscle is a major contributor to adaptive thermogenesis, i.e., the thermogenesis that changes in response to exposure to cold or to overfeeding. The identification of the "furnace" that is responsible for increased heat generation in resting muscle has been the subject of a number of investigations. A new state of myosin, the super relaxed state (SRX), with a very slow ATP turnover rate has recently been observed in skeletal muscle (Stewart et al. in Proc Natl Acad Sci USA 107:430-435, 2010). Inhibition of the myosin ATPase activity in the SRX was suggested to be caused by binding of the myosin head to the core of the thick filament in a structural motif identified earlier by electron microscopy. To be compatible with the basal metabolic rate observed in vivo for resting muscle, most myosin heads would have to be in the SRX. Modulation of the population of this state, relative to the normal relaxed state, was proposed to be a major contributor to adaptive thermogenesis in resting muscle. Transfer of only 20% of myosin heads from the SRX into the normal relaxed state would cause muscle thermogenesis to double. Phosphorylation of the myosin regulatory light chain was shown to transfer myosin heads from the SRX into the relaxed state, which would increase thermogenesis. In particular, thermogenesis by myosin has been proposed to play a role in the dissipation of calories during overfeeding. Up-regulation of muscle thermogenesis by pharmaceuticals that target the SRX would provide new approaches to the treatment of obesity or high blood sugar levels.