In VivoMicroscopy Reveals Extensive Embedding of Capillaries within the Sarcolemma of Skeletal Muscle Fibers (original) (raw)
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Journal of Experimental Biology, 2012
SUMMARY Diffusion plays a prominent role in governing both rates of aerobic metabolic fluxes and mitochondrial organization in muscle fibers. However, there is no mechanism to explain how the non-homogeneous mitochondrial distributions that are prevalent in skeletal muscle arise. We propose that spatially variable degradation with dependence on O2 concentration, and spatially uniform signals for biogenesis, can account for observed distributions of mitochondria in a diversity of skeletal muscle. We used light and transmission electron microscopy and stereology to examine fiber size, capillarity and mitochondrial distribution in fish red and white muscle, fish white muscle that undergoes extreme hypertrophic growth, and four fiber types in mouse muscle. The observed distributions were compared with those generated using a coupled reaction-diffusion/cellular automata (CA) mathematical model of mitochondrial function. Reaction-diffusion analysis of metabolites such as oxygen, ATP, ADP ...
Journal of Applied Physiology, 2013
Picard M, White K, Turnbull DM. Mitochondrial morphology, topology, and membrane interactions in skeletal muscle: a quantitative threedimensional electron microscopy study. Dynamic remodeling of mitochondrial morphology through membrane dynamics are linked to changes in mitochondrial and cellular function. Although mitochondrial membrane fusion/fission events are frequent in cell culture models, whether mitochondrial membranes dynamically interact in postmitotic muscle fibers in vivo remains unclear. Furthermore, a quantitative assessment of mitochondrial morphology in intact muscle is lacking. Here, using electron microscopy (EM), we provide evidence of interacting membranes from adjacent mitochondria in intact mouse skeletal muscle. Electron-dense mitochondrial contact sites consistent with events of outer mitochondrial membrane tethering are also described. These data suggest that mitochondrial membranes interact in vivo among mitochondria, possibly to induce morphology transitions, for kiss-and-run behavior, or other processes involving contact between mitochondrial membranes. Furthermore, a combination of freeze-fracture scanning EM and transmission EM in orthogonal planes was used to characterize and quantify mitochondrial morphology. Two subpopulations of mitochondria were studied: subsarcolemmal (SS) and intermyofibrillar (IMF), which exhibited significant differences in morphological descriptors, including form factor (means Ϯ SD for SS: 1.41 Ϯ 0.45 vs. IMF: 2.89 Ϯ 1.76, P Ͻ 0.01) and aspect ratio (1.97 Ϯ 0.83 vs. 3.63 Ϯ 2.13, P Ͻ 0.01) and circularity (0.75 Ϯ 0.16 vs. 0.45 Ϯ 0.22, P Ͻ 0.01) but not size (0.28 Ϯ 0.31 vs. 0.27 Ϯ 0.20 m 2 ). Frequency distributions for mitochondrial size and morphological parameters were highly skewed, suggesting the presence of mechanisms to influence mitochondrial size and shape. In addition, physical continuities between SS and IMF mitochondria indicated mixing of both subpopulations. These data provide evidence that mitochondrial membranes interact in vivo in mouse skeletal muscle and that factors may be involved in regulating skeletal muscle mitochondrial morphology. skeletal muscle mitochondria; myofiber; outer mitochondrial membrane; scanning and transmission electron microscopy; mitochondrial reticulum SKELETAL MUSCLE FIBERS PERFORM energetically demanding functions (contraction, rapid cycles of ion transport, gene expression, and protein synthesis) requiring large amounts of ATP Address for reprint requests and other correspondence: M. PicardFig. 4. Physical interactions between SS and IMF mitochondria. Transmission electron micrograph of myofibers in the transverse plane. A: SS and IMF mitochondria are distinct organelles. B and C: some SS and IMF mitochondria form continuous organelles (arrowheads) that coexist in both subcellular compartments. SS, subsarcolemmal; IMF, intermyofibrillar; PM, plasma membrane (sarcolemma); Myofibr, Myofibrils; Cap, capillary. 166 Skeletal Muscle Mitochondrial Morphology • Picard M et al. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: M.P. conception and design of research; M.P. and K.W. performed experiments; M.P. analyzed data; M.P. and D.M.T. interpreted results of experiments; M.P. prepared figures; M.P. drafted manuscript; M.P., K.W., and D.M.T. edited and revised manuscript; M.P., K.W., and D.M.T. approved final version of manuscript. 170 Skeletal Muscle Mitochondrial Morphology • Picard M et al.
Cell metabolism, 2015
Skeletal muscle fibers differentiate into specific fiber types with distinct metabolic properties determined by their reliance on oxidative phosphorylation (OXPHOS). Using in vivo approaches, we find that OXPHOS-dependent fibers, compared to glycolytic fibers, contain elongated mitochondrial networks with higher fusion rates that are dependent on the mitofusins Mfn1 and Mfn2. Switching of a glycolytic fiber to an oxidative IIA type is associated with elongation of mitochondria, suggesting that mitochondrial fusion is linked to metabolic state. Furthermore, we reveal that mitochondrial proteins are compartmentalized to discrete domains centered around their nuclei of origin. The domain dimensions are dependent on fiber type and are regulated by the mitochondrial dynamics proteins Mfn1, Mfn2, and Mff. Our results indicate that mitochondrial dynamics is tailored to fiber type physiology and provides a rationale for the segmental defects characteristic of aged and diseased muscle fibers.
Interaction between myoglobin and mitochondria in rat skeletal muscle
Journal of Applied Physiology, 2012
The mechanisms underlying subcellular oxygen transport mediated by myoglobin (Mb) remain unclear. Recent evidence suggests that, in the myocardium, transverse diffusion of Mb is too slow to effectively supply oxygen to meet the immediate mitochondrial oxygen demands at the onset of muscle contractions. The cell may accommodate the demand by maintaining the distribution of Mb to ensure a sufficient O2 supply in the immediate vicinity of the mitochondria. The present study has verified the co-localization of Mb with mitochondria by using biochemical histological and electron microscopy analyses. Immunohistochemical and electron microscopy analysis indicates a co-localization of Mb with mitochondria. Western blotting confirms the presence of Mb colocalizes with the mitochondrial fraction and appears more prominently in slow-twitch oxidative than in fast-twitch glycolytic muscle. In particular, Mb interacts with cytochrome c oxidase-subunit IV. These results suggest that a direct Mb-med...
Maximal diffusion-distance within skeletal muscle can be estimated from mitochondrial distributions
Respiration Physiology, 1990
Mitochondrial volume density (V,[mit]) distributions were measured with a test pattern of concentric rings centered upon randomly chosen capillaries in oxidative skeletal muscle ceils of two Antarctic fishes, Trematomus newnesi and Notothenia gibberifrons. Vv(mit) in both species was highest in the ring closest to the capillary, minimal further from the capillary (at a distance that was characteristic for each species), and rose in the annuli furthest from the capillary. Plots of Vv(mit) against total area between each ring and the central capillary fit the form of a second-order polynomial (r2> 0.9). If Po2 or blood-borne metabolite concentration predicates the pattern of Vv(mit) distribution, minimal Vv(mit) is at the same position as the minimum in concentration or gaseous partial pressure of capillary-supplied commodities. This minimum is the boundary between cylinders of tissue being supplied by adjacent capillaries, and thus delineates the maximal diffusion-distance for capillary-supplied commodities. Maximal diffusion-distance (#m) for T. newnesi-26.23 + 1.64; N. gibberifrons = 21.45 + 0.51. For 02, maximal diffusion-distance conventionally is referred to as Krogh's radius, R. With an easily obtained estimate of numerical capillary density, these R values can be used to calculate a capillary tortuosity constant (c[k,0]) and capillary length density (Jr [c,f]). c(k,0) values were also determined using an established method, and R and Jv(c,f) values calculated from these values did not significantly differ from values determined from mitochondrial distributions. Mitochondrial distribution analysis may more accurately reflect changes in capillary blood flow and heterogeneity of diffusion and solubility constants within muscle than currently existing techniques. Similar distributions of V,,(mit) reported for several species of vertebrates suggest wide applicability of the method. Animal, antarctic fish; Capillaries, density in skeletal muscle, tortuosity; Diffusion, of 02 in tissue; Mitochondria, distribution of-in skeletal muscle; Skeletal muscle, diffusion distance, capillary supply Several methods exists to estimate maximal diffusion-distances for capillary-supplied commodities within skeletal muscle, including the capillary-domains method (Hoofd et al., 1985), the closest individual method (Kayar et al., 1982), and the concentric circle method (Kayar and Banchero, 1982). All of these techniques are based upon August Krogh's pioneering model of oxygen delivery to striated muscle cells (Krogh, 1919).
Journal of Experimental Biology, 1996
This paper integrates the results of a series of studies on the supply of O2 and substrates for oxidative muscle metabolism and draws conclusions on the role of structural design in partitioning and limiting substrate supply. The studies compared dogs and goats exercising at different intensities and combined physiological, biochemical and morphometric investigations. In both species, the rate of fatty acid oxidation reached an upper limit at low exercise intensities, and only glucose consumption was increased at higher exercise intensities. The supply of both glucose and fatty acids from the capillaries reached maximal rates at low exercise intensities; this limitation is related to the design of the sarcolemma as calculations suggest that the endothelium introduces only a small resistance to substrate flux. From these findings, it appears that the capillaries are designed to satisfy O2 supply up to maximal O2 demand. The increase in substrate supply to the mitochondria at higher e...
European Journal of Applied Physiology and Occupational Physiology, 1991
Biopsies from the medial gastrocnemius muscle of three experienced endurance runners who had completed an ultramarathon run (160 km) the previous day were assessed for their oxidative characteristics (fibre types, capillarization and mitochondria content). Also, a regional comparison was made for fibres located centrally (completely surrounded by other fibres) versus fibres located peripherally (next to the interfascicular space) and the capillarization of these peripheral fibres was determined. Subsarcolemmal mitochondria were abundant and 'compartmentalized' close to the capillaries. The number of capillaries around fibres ranged from 5.8 to 8.5 and 5.7 to 8.5, and the number of capillaries.mm-2 ranged from 665 to 810 and 727 to 762, for type I (slow twitch) and type II (fast twitch) fibres, respectively. Central fibres contained a greater number of capillaries and more capillaries.mm-2 than their peripheral counterparts. Peripheral fibres contained more capillaries, ktm-1 between fibres than at the interfascicular space. Type I fibres were more distributed (63%-78%) and larger than type II fibres. An abundance of subsarcolemmal mitochondria located close to the capillaries, efficient capillary proliferation between fibres where sharing can occur and greater relative distribution and size of type I fibres are, collectively, efficient characteristics of extreme endurance training.
Oxygen control of intracellular distribution of mitochondria in muscle fibers
Biotechnology and Bioengineering, 2013
Mitochondrial density in skeletal muscle fibers is governed by the demand for aerobic ATP production, but the heterogeneous distribution of these mitochondria appears to be governed by constraints associated with oxygen diffusion. We propose that each muscle fiber has an optimal mitochondrial distribution at which it attains a near maximal rate of ATP consumption (R ATPase) while mitochondria are exposed to a minimal oxygen concentration, thus minimizing reactive oxygen species (ROS) production. We developed a coupled reaction-diffusion/cellular automata (CA) mathematical model of mitochondrial function and considered 4 fiber types in mouse extensor digitorum longus (EDL) and soleus (SOL) muscle. The developed mathematical model uses a reaction-diffusion analysis of metabolites including oxygen, ATP, ADP, phosphate and phosphocreatine (PCr) involved in energy metabolism and mitochondrial function. A CA approach governing mitochondrial life cycles in response to the metabolic state of the fiber was superimposed and coupled to the reaction-diffusion approach. The model results show the sensitivity of important model outputs such as the R ATPase , effectiveness factor (η) and average oxygen concentration available at each mitochondrion to local oxygen concentration in the fibers through variation in the CA model parameter θ det , which defines the sensitivity of mitochondrial death to the oxygen concentration. The predicted optimal mitochondrial distributions matched previous experimental findings. Deviations from this optimal distribution corresponding to higher CA model parameter values (a more uniform mitochondrial distribution) lead to lower aerobic rates. In contrast, distributions corresponding to lower CA model parameter values (a more asymmetric distribution) lead to an increased exposure of mitochondria to oxygen, usually without substantial increases in aerobic rates, which would presumably result in increased ROS production and thus increased risks of cytotoxicity.
Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells
Nature Protocols, 2008
Analysis of mitochondrial function is central to the study of intracellular energy metabolism, mechanisms of cell death and pathophysiology of a variety of human diseases, including myopathies, neurodegenerative diseases and cancer. However, important properties of mitochondria differ in vivo and in vitro. Here, we describe a protocol for the analysis of functional mitochondria in situ, without the isolation of organelles, in selectively permeabilized cells or muscle fibers using digitonin or saponin. A specially designed substrate/inhibitor titration approach allows the step-by-step analysis of several mitochondrial complexes. This protocol allows the detailed characterization of functional mitochondria in their normal intracellular position and assembly, preserving essential interactions with other organelles. As only a small amount of tissue is required for analysis, the protocol can be used in diagnostic settings in clinical studies. The permeabilization procedure and specific titration analysis can be completed in 2 h.
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 2006
Capillary electrophoresis (CE) with postcolumn laser-induced fluorescence detection (LIF) was used to analyze single skeletal muscle fibers from young and old rats. Due to selective labeling of mitochondria with 10-N-nonyl acridine orange, the zeptomole (10 À21 mole) sensitivity, and the high separation power, three properties of individual mitochondrial particles were revealed: the number, the distributions of cardiolipin, and their electrophoretic mobilities. Type I fibers had more mitochondrial particles and cardiolipin per particle than did type IIb fibers from rats of similar age. Individual fibers of the same fiber type from young rats contained more mitochondrial particles and cardiolipin per particle than did fibers from old rats. There were fiber typespecific and age-specific differences in the electrophoretic mobility of individual mitochondrial particles. The CE-LIF results of individual mitochondrial particles are the first of their kind in that they reveal fiber type-specific and age-specific differences that are not obviously noticed in bulk measurements of heterogeneous tissues.