Ultrastructural changes in the red muscle fibers of the quadriceps muscle of the rat femur during increased motor activity

Experiments on rats were made to study the effect of physical exercise running in a treadbahn on ultrastructure of skeletal muscle fibers. The biphasic mechanism of muscle contractile activity was shown. The processes of destruction occurring in red muscle fibers of the intensely working quadriceps femoris were manifested by enlargement of T system and sarcoplasmic reticulum elements, mitochondrial matrix swelling, cryst destruction, vacuolar degeneration of part of the mitochondria, and destruction of individual myofibrils. In addition to destructive changes, the muscle exhibited the recovery processes--physiological regeneration. Those processes included large accumulations of the mitochondria beneath the plasma membrane of muscle fibers, the presence of small mitochondria, division of the mitochondria, transformation of myosatellitocytes to myoblasts, the presence of centrioles in endotheliocytes, and so forth.     

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.     

Mitochondria with Morphology Characteristic for Alzheimer’s Disease Patients Are Found in the Brain of OXYS Rats

Growing evidence suggests that mitochondrial dysfunction is closely linked to the pathogenesis of sporadic Alzheimer’s disease (AD). One of the key contributors to various aspects of AD pathogenesis, along with metabolic dysfunction, is mitochondrial dynamics, involving balance between fusion and fission, which regulates mitochondrial number and morphology in response to changes in cellular energy demand. Recently, Zhang et al. ((2016) Sci. Rep., 6, 18725) described a previously unknown mitochondrial phenotype manifesting as elongated chain-linked mitochondria termed “mitochondria-on-a-string” (MOAS) in brain tissue from AD patients and mouse models of AD. The authors associated this phenotype with fission arrest, but implications of MOAS formation in AD pathogenesis remain to be understood. Here we analyze the presence and number of MOAS in the brain of OXYS rats simulating key signs of sporadic AD. Using electron microscopy, we found MOAS in OXYS prefrontal cortex neuropil in all stages of AD-like pathology, including mani-festation (5-month-old rats) and progression (12–18-month-old rats). The most pronounced elevation of MOAS content (8–fold) in OXYS rats compared to Wistar controls was found at the preclinical stage (20 days) on the background of decreased numbers of non-MOAS elongated mitochondria. From the age of 20 days through 18 months, the percentage of MOAS-containing neuronal processes increased from 1.7 to 8.3% in Wistar and from 13.9 to 16% in OXYS rats. Our results support the importance of the disruption of mitochondrial dynamics in AD pathogenesis and corroborate the existence of a causal link between impaired mitochondrial dynamics and formation of the distinctive phenotype of “mitochondria-on-a-sting”.     

Aging enhances indirect flight muscle fiber performance yet decreases flight ability in Drosophila

We investigated the effects of aging on Drosophila melanogaster indirect flight muscle from the whole organism to the actomyosin cross-bridge. Median-aged (49-day-old) flies were flight impaired, had normal myofilament number and packing, barely longer sarcomeres, and slight mitochondrial deterioration compared with young (3-day-old) flies. Old (56-day-old) flies were unable to beat their wings, had deteriorated ultrastructure with severe mitochondrial damage, and their skinned fibers failed to activate with calcium. Small-amplitude sinusoidal length perturbation analysis showed median-aged indirect flight muscle fibers developed greater than twice the isometric force and power output of young fibers, yet cross-bridge kinetics were similar. Large increases in elastic and viscous moduli amplitude under active, passive, and rigor conditions suggest that median-aged fibers become stiffer longitudinally. Small-angle x-ray diffraction indicates that myosin heads move increasingly toward the thin filament with age, accounting for the increased transverse stiffness via cross-bridge formation. We propose that the observed protein composition changes in the connecting filaments, which anchor the thick filaments to the Z-disk, produce compensatory increases in longitudinal stiffness, isometric tension, power and actomyosin interaction in aging indirect flight muscle. We also speculate that a lack of MgATP due to damaged mitochondria accounts for the decreased flight performance.     

Changes in physicochemical parameters and α-crystallin expression in the lens during cataract development in OXYS rats

The pathogenesis of cataract is associated with oxidative stress and with altered crystallin expression but it is still understood incompletely. In this study, the senescence-accelerated OXYS rats were used as a model. The first biomicro-scopic signs of cataract in OXYS rats were registered at the age of 1.5 months; at 3 months morbidity reached 90%, and at 6 months it reached 100%. Cataract manifestation progresses: at 24 months mature cataract was detected in 90% of eyes of OXYS rats, whereas in 80% of Wistar rat eyes only initial signs of this disease were detected. Analysis of lens redox-parameters has shown that in OXYS rats the intensity of tryptophan fluorescence is higher, the GSH content being higher at 2 months but during formation of mature cataract at 13, 18, and 24 months being lower than in Wistar rats. Decrease in solubility of OXYS rat lens proteins was observed at the age of 13 months. At the age of 3 months gene expression of αA-crystallin and αB-crystallin was 3-fold and 25% lower, respectively, than in Wistar rats. At the age of 14 months there was a 27-fold decrease in expression of αB-crystallin in OXYS rats and it became 21-fold lower than in control. Proteins are synthesized in lens epithelial cells and dystrophic changes in senile cataract result in decrease in structural protein expression. The changes observed in OXYS rats are evidently associated with the dystrophic changes in lens epithelium, which we have described earlier, and are consistent with the model of senile cataract.     

Skeletal muscle fiber, nerve, and blood vessel breakdown in space-flown rats

Histochemical and ultrastructural analyses were performed postflight on hind limb skeletal muscles of rats orbited for 12.5 days aboard the unmanned Cosmos 1887 biosatellite and returned to Earth 2 days before sacrifice. The antigravity adductor longus (AL), soleus, and plantaris muscles atrophied more than the non-weight-bearing extensor digitorum longus, and slow muscle fibers were more atrophic than fast fibers. Muscle fiber segmental necrosis occurred selectively in the AL and soleus muscles; primarily, macrophages and neutrophils infiltrated and phagocytosed cellular debris. Granule-rich mast cells were diminished in flight AL muscles compared with controls, indicating the mast cell secretion contributed to interstitial tissue edema. Increased ubiquitination of disrupted myofibrils implicated ubiquitin in myofilament degradation. Mitochondrial content and succinic dehydrogenase activity were normal, except for subsarcolemmal decreases. Myofibrillar ATPase activity of flight AL muscle fibers shifted toward the fast type. Absence of capillaries and extravasation of red blood cells indicated failed microcirculation. Muscle fiber regeneration from activated satellite cells was detected. About 17% of the flight AL end plates exhibited total or partial denervation. Thus, skeletal muscle weakness associated with spaceflight can result from muscle fiber atrophy and segmental necrosis, partial motor denervation, and disruption of the microcirculation.     

Physical and Functional Association of Lactate Dehydrogenase (LDH) with Skeletal Muscle Mitochondria

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO₂) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO₂. However, subsequent addition of NAD⁺ significantly increased JO₂, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD⁺-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.     

Oxidation of guanine in liver and lung DNA of prematurely aging OXYS rats

Immunofluorescence assay was applied for determination of 8-oxoguanine (8-oxoG) in DNA. The 8-oxoG content in liver and lung DNA of 2- and 18-month-old Wistar rats was compared with that of prematurely aging OXYS rats. It was shown that for rats of both strains, 8-oxoG content in lung DNA compared with liver DNA was 1.7-2.0-fold and 1.3-1.7-fold higher for 2- and 18-month-old rats, respectively. However, the degree of oxidative damage in liver DNA of OXYS rats was 2.4- (p < 0.01) and 1.5-fold (p < 0.05) higher for 2- and 18-month-old animals, respectively, than that in liver DNA of Wistar rats. Oxidation of guanine in lung DNA of OXYS rats was 2- (p < 0.01) and 1.7-fold (p < 0.05) higher for 2- and 18-month-old animals, respectively, than that in lung DNA of Wistar rats. The data indicate that elevated DNA oxidative damage in various organs of OXYS rats may be an important factor of accelerated aging and progression of age-related diseases--cataract, macular dystrophy, hypertension, osteoporosis, cognitive and behavioral dysfunctions, and also lung and liver pathologies.     

Study of age-dependent structural and functional changes of mitochondria in skeletal muscles and heart of naked mole rats (<Emphasis Type="Italic">Heterocephalus glaber</Emphasis>)

Morphometric analysis of mitochondria in skeletal muscles and heart of 6- and 60-month-old naked mole rats (Heterocephalus glaber) revealed a significant age-dependent increase in the total area of mitochondrial cross-sections in studied muscle fibers. For 6- and 60-month-old animals, these values were 4.8 ± 0.4 and 12.7 ± 1.8%, respectively. This effect is mainly based on an increase in the number of mitochondria. In 6-month-old naked mole rats, there were 0.23 ± 0.02 mitochondrial cross-sections per μm² of muscle fiber, while in 60-month-old animals this value was 0.47 ± 0.03. The average area of a single mitochondrial cross-section also increased with age in skeletal muscles–from 0.21 ± 0.01 to 0.29 ± 0.03 μm². Thus, naked mole rats show a drastic enlargement of the mitochondrial apparatus in skeletal muscles with age due to an increase in the number of mitochondria and their size. They possess a neotenic type of chondriome accompanied by specific features of mitochondrial functioning in the state of oxidative phosphorylation and a significant decrease in the level of matrix adenine nucleotides.     

Fine structure of fast-twitch and slow-twitch guinea pig muscle fibers

The guinea pig soleus muscle is a convenient model for the study of slow-twitch intermediate (STI) fiber ultrastructure because it is composed entirely of fibers of this class. Such fibers were compared with fast-twitch red (FTR) and fast-twitch white (FTW) fibers from the vastus lateralis muscle. FTW fibers are characterized by small, sparse mitochondria, a narrow Z line and, an extensive sarcoplasmic reticulum arranged primarily in longitudinal profiles at the A band and with numerous expansions at the I band. Abundant mitochondria with a dense matrix and subsarcolemmal and perinuclear aggregations are typical of FTR fibers. These fibers contain a plexus of sarcoplasmic reticulum at the A band and a less extensive network at the I band. The Z lines are wider (890 ± 74 Å) than those of FTW fibers (582 ± 62 Å). STI intermediate fibers are distinguished from other types by wide Z lines (1205 ± 58 Å), a faint M band, and a less extensive sarcoplasmic reticulum. Compared to FTR fibers, STI fiber mitochondria are usually smaller with less notable subsarcolemmal accumulations. FTW fibers have a more limited capillary supply, rarely contain lipid inclusions, and thus may be restricted to phasic activity. Extensive capillarity, mitochondrial and lipid context, and fast contraction times indicate possible phasic and tonic roles for FTR fibers. STI fibers, characterized by numerous lipid inclusions, extensive capillarity, relatively numerous mitochondria, but slow contraction-relaxation cycles, are morphologically suited for tonic muscle activity.     

Age-dependent increase of etheno-DNA-adducts in liver and brain of ROS overproducing OXYS rats

Reactive oxygen species (ROS) and lipid peroxidation (LPO) play a role in aging and degenerative diseases. To correlate oxidative stress and LPO-derived DNA damage, we determined etheno-DNA-adducts in liver and brain from ROS overproducing OXYS rats in comparison with age-matched Wistar rats. Liver DNA samples from 3- and 15-month-old OXYS and Wistar rats were analyzed for 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) by immunoaffinity/32P-postlabelling. While epsilondA and epsilondC levels were not different in young rats, adduct levels were significantly higher in old OXYS rats when compared to old Wistar or young OXYS rats. Frozen rat brain sections were analyzed for epsilondA by immunostaining of nuclei. Brains from old OXYS rats accumulated epsilondA more frequently than age-matched Wistar rats. Our results demonstrate increased LPO-induced DNA damage in organs of OXYS rats which correlates with their known shorter life-span and elevated frequency of chronic degenerative diseases.     

Flight duration and flight muscle ultrastructure of unfed hawk moths

Flight muscle breakdown has been reported for many orders of insects, but the basis of this breakdown in insects with lifelong dependence on flight is less clear. Lepidopterans show such muscle changes across their lifespans, yet how this change affects the ability of these insects to complete their life cycles is not well documented. We investigated the changes in muscle function and ultrastructure of unfed aging adult hawk moths (Manduca sexta). Flight duration was examined in young, middle-aged, and advanced-aged unfed moths. After measurement of flight duration, the main flight muscle (dorsolongitudinal muscle) was collected and histologically prepared for transmission electron microscopy to compare several measurements of muscle ultrastructure among moths of different ages. Muscle function assays revealed significant positive correlations between muscle ultrastructure and flight distance that were greatest in middle-aged moths and least in young moths. In addition, changes in flight muscle ultrastructure were detected across treatment groups. The number of mitochondria in muscle cells peaked in middle-aged moths. Many wild M. sexta do not feed as adults; thus, understanding the changes in flight capacity and muscle ultrastructure in unfed moths provides a more complete understanding of the ecophysiology and resource allocation strategies of this species.     

Lipofuscin granule dynamics during development of age-related macular degeneration

The pigment epithelium cell structure and therapeutic effect of antioxidant SkQ1, selectively penetrating into mitochondria from eye drops, were studied upon development in OXYS rats of age-related retinopathy as a model of macular degeneration. The characteristic dynamics and ultrastructural peculiarities of the layer of electron-dense cytoplasmic structures of the pigment epithelium apex part and incorporated lipofuscin granules were revealed. The therapy of OXYS animals for 68 days using 250 nM SkQ1 drops decreased the extent of development of age-related macular degeneration. Electron-microscopic investigation showed that SkQ1 prevented development of ultrastructural changes in the pigment epithelium characteristic of macular degeneration, the condition of which after therapy with SkQ1 drops corresponded to ultrastructure of pigment epithelium in Wistar rats of the same age having no symptoms of retinal damage. It is supposed that ultrastructural changes in the electron-dense layer upon development of age-related macular degeneration are indicative of disturbances in the optical cycle functioning, especially of disturbances in functioning of photoreceptor membranes.     

Altered distribution of mitochondria in rat soleus muscle fibers after spaceflight.

The influence of spaceflight on the distribution of succinate dehydrogenase (SDH) activity throughout the cross section of fibers in the soleus was studied in five male rats and in five rats maintained under ground-based simulated flight conditions (control). The flight (COSMOS 1887) was 12.5 days in duration, and the animals were killed approximately 2 days after return to 1 G. Fibers were classified as slow-twitch oxidative or fast-twitch oxidative-glycolytic in histochemically prepared tissue sections. The distribution of SDH activity throughout the cross section of 20-30 fibers (each type) was determined using quantitative histochemical and computer-assisted image analysis techniques. In all the fibers, the distribution of SDH activity was significantly higher in the subsarcolemmal than in intermyofibrillar region. After spaceflight the entire regional distribution of SDH activity was significantly altered in the slow-twitch oxidative fibers. The fast-twitch oxidative-glycolytic fibers of the spaceflight muscles exhibited a significantly lower SDH activity only in their subsarcolemmal region. These data suggest that when determining the influence of spaceflight on muscle fiber oxidative metabolism enzymes, it is important to consider the location of the enzyme throughout the cross section of a fiber. Furthermore the functional properties of the soleus that depend on the metabolic support of mitochondria in the subsarcolemmal region may be primarily affected by exposure to microgravity.     

Alteration in skeletal muscle mitochondria of cold-acclimated rats: Association with enhanced metabolic response to noradrenaline

This paper reports a search for structural changes in skeletal muscle mitochondria of cold-acclimated rats. Histochemical studies (succinic dehydrogenase) show that there appears to be a higher proportion of red fibers in the semitendinosus muscle of the cold-acclimated rat and that the white region of this muscle contains fibers which resemble intermediate fibers. Electron micrographs show an apparently larger number of small mitochondria in both red and white fibers. Counts of mitochondria isolated from skeletal muscle show that there are more mitochondria per gram of both red and white muscle in the cold-acclimated rat than in the non-acclimated control rat. Each mitochondrion contains less protein and less cytochrome oxidase. Thus the mitochondrial mass per gram of red and white muscle is not altered, as indicated by the unchanged content of mitochondrial protein and of cytochrome oxidase per gram of muscle. Thus there appears to be a repackaging of mitochondrial material into smaller units in the skeletal muscle of the cold-acclimated rat. The alteration is shown to be associated with the adaptive state of the rat. No change occurs in muscle mitochondria of cold-acclimated rats in which the development of the enhanced metabolic response to noradrenaline, a measure of the extent of adaptation, is inhibited by treatment with oxytetracycline. The change in skeletal muscle mitochondria disappears when the enhanced metabolic response to noradrenaline in rats which are already cold-climated is reversed by treating the rats with oxytetracycline while they continue to live in the cold. The change in muscle mitochondria also disappears when the cold-acclimated rat undergoes deacclimation after return to room temperature. The alteration in muscle mitochondria is thus not associated either with shivering or with a high metabolic rate. Skeletal muscle of the cold-acclimated rat is known to be an important site of heat production in the course of nonshivering thermogenesis; that is, it can undergo a considerable increase in metabolic rate in the absence of shivering on exposure of the cold-acclimated rat to cold. The metabolic basis of nonshivering thermogenesis is in an enhanced capacity of the tissues of the cold-acclimated rat, principally skeletal muscle, to respond by an increase in metabolic rate to the large amounts of noradrenaline secreted by the nerve endings of the sympathetic nervous system as a consequence of cold-exposure. The mechanism by which the metabolic response to noradrenaline in the cold-acclimated rat can be enhanced is unknown. The structural alteration observed in the skeletal muscle mitochondria of the cold-acclimated rat may indicate a functional alteration responsible for the enhanced capacity of the muscle to respond to noradrenaline by an increase in metabolic rate.     

Cathepsin K and matrix metalloprotease activities in bone tissue of the OXYS rats during the development of osteoporosis

The activity of osteoclast-specific cysteine protease, cathepsin K, and matrix metalloproteases (MMPs) has been investigated in bone tissue of senescence-accelerated OXYS rats and in Wistar rats. At the age of 3 month (the period preceding manifestation of osteoporosis in OXYS rats) cathepsin K activity was higher whereas MMP activity was lower in Wistar rats. At the age of 14 months Wistar rats cathepsin K activity increased and MMP activity decreased. The age-related changes in bone cathepsin K and MMP activity of OXYS rats had opposite direction. Thus, despite of marked manifestations of osteoporosis previously found by us in OXYS rats (the decrease in mineralization density of the bone tissue and its resorption) no interstrain differences in cathepsin K and MMPs were found between Wistar and OXYS rats. Activity of a universal protease inhibitor, α₂-macroglobulin, was higher in serum of 14-month old OXYS rats than in Wistar rats of the same age. The role of cathepsin K activation in resorption of bone tissue in the development of osteoporosis in senescence-accelerated OXYS rats is discussed.     

Ultrastructure of skeletal muscle fibers in monkeys after space flight

It is known that exposure of humans and animals to microgravity causes reduction in the cross-sected area of muscle fibers and muscle atrophy. These changes also involve ultrastructural alterations in muscle fibers. Therefore primates, that are physiologically close to humans, are to be examined to help a better understanding of the nature of these ultrastructural changes is muscles and muscle fibers. Although failed to find any relevant published data on the quantitative aspects of ultrastructural changes in muscle fibers of space-flown primates we believe that it is important to examine these aspects. The postflight study of monkey's m. soleus, and m. vastus lateralis did not reveal any significant changes in volume density of the myofibrillar apparatus. Mitochondria of m. soleus showed a distinct reduction in volume density, being more obvious in the subsarcolemmal zone than in the central one. Mitochondria of m. vastus lateralis showed a decrease (P > 0.05) in volume density. Following the flight, m. soleus and m. vastus lateralis of the monkeys showed a significant increase in the mean area of myofibrils, and a trend towards a decrease in the number of myofibrils per 100 micron 2. Besides, m. soleus showed a significant increase in the mean area of mitochondria, and a trend towards a decrease in the number of mitochondria per 100 micron 2. In m. vastus lateralis of the monkeys after space flight the number opf mitochondria tended to decrease and the mean area showed differential changes. It can be postulated that these phenomena may be associated with a reduction in the diffusion surface of mitochondria resulting from the diminished myofibrillar volume.     

Differences in proton leak kinetics, but not in UCP3 protein content, in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from fed and fasted rats

We have investigated the effect of 24-h fasting on basal proton leak and uncoupling protein (UCP) 3 expression at the protein level in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. In fed rats, the two mitochondrial populations displayed different proton leak, but the same protein content of UCP3. In addition, 24-h fasting, both at 24 and 29 degrees C, induced an increase in proton leak only in subsarcolemmal mitochondria, while UCP3 content increased in both the populations. From the present data, it appears that UCP3 does not control the basal proton leak of skeletal muscle mitochondria.     

Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters

Two-photon excitation fluorescence microscopy (TPEFM) permits the investigation of the topology of intercellular events within living animals. TPEFM was used to monitor the distribution of mitochondrial reduced nicotinamide adenine dinucleotide (NAD(P)H) in murine skeletal muscle in vivo. NAD(P)H fluorescence emission was monitored (~460 nm) using 710–720 nm excitation. High-resolution TPEFM images were collected up to a depth of 150 μm from the surface of the tibialis anterior muscle. The NAD(P)H fluorescence images revealed subcellular structures consistent with subsarcolemmal, perivascular, intersarcomeric, and paranuclear mitochondria. In vivo fiber typing between IIB and IIA/D fibers was possible using the distribution and content of mitochondria from the NAD(P)H fluorescence signal. The intersarcomeric mitochondria concentrated at the Z-line in the IIB fiber types resulting in a periodic pattern with a spacing of one sarcomere (2.34 ± 0.17 μm). The primary inner filter effects were nearly equivalent to water, however, the secondary inner filter effects were highly significant and dynamically affected the observed emission frequency and amplitude of the NAD(P)H fluorescence signal. These data demonstrate the feasibility, and highlight the complexity, of using NAD(P)H TPEFM in skeletal muscle to characterize the topology and metabolic function of mitochondria within the living mouse.     

Subsarcolemmal mitochondria and capillarization of soleus muscle fibers in young rats subjected to an endurance training

Summary Rats, 6 weeks old, were subjected to a program of endurance running for 3, 6 and 12 weeks. 0.5 to 0.8 m thick sections of Epon embedded soleus muscles were studied with morphometric methods.In cross-sections the area occupied by subsarcolemmal mitochondria was independent of the age, but was 53% higher after 12 weeks of training. The mean depth of the zones with subsarcolemmal mitochondria increased only 15% to about 0.9 m. Thus, the subsarcolemmal mitochondria showed a pronounced spreading at the muscle fiber surface in trained muscles. — The number of capillaries per fiber decreased slightly in controls and increased not significantly in trained muscles.It is concluded that the subsarcolemmal mitochondria supply the energy for the active transport of metabolites through the sarcolemma in oxidative muscle fibers, and that they are the limiting factor for endurance performance of the soleus muscle fibers because the changes in the capillarization were only small. It is suggested that the subsarcolemmal and the interfibrillar mitochondria have different functions and may therefore represent different types of mitochondria which can be distinguished by their morphology as well as by their biochemical properties.