Cellular phenotypes of age-associated skeletal muscle mitochondrial abnormalities in rhesus monkeys

Rhesus monkey vastus lateralis muscle was examined histologically for age-associated electron transport system (ETS) abnormalities: fibers lacking cytochrome c oxidase activity (COX(-)) and/or exhibiting succinate dehydrogenase hyperreactivity (SDH(++)). Two hundred serial cross-sections (spanning 1600 microm) were obtained and analyzed for ETS abnormalities at regular intervals. The abundance and length of ETS abnormal regions increased with age. Extrapolating the data to the entire length of the fiber, up to 60% of the fibers were estimated to display ETS abnormalities in the oldest animal studied (34 years) compared to 4% in a young adult animal (11 years). ETS abnormal phenotypes varied with age and fiber type. Middle-aged animals primarily exhibited the COX(-) phenotype, while COX(-)/SDH(++) abnormalities were more common in old animals. Transition region phenotype was affected by fiber type with type 2 fibers first displaying COX(-) and then COX(-)/SDH(++) while type 1 fibers progressed from normal to SDH(++) and then to COX(-)/SDH(++). In situ hybridizations studies revealed an association of ETS abnormalities with deletions of the mitochondrial genome. By measuring cross-sectional area along the length of ETS abnormal fibers, we demonstrated that some of these fibers exhibit atrophy. Our data suggest mitochondrial (mtDNA) deletions and associated ETS abnormalities are contributors to age-associated fiber atrophy.     

Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibers

Skeletal muscle-mass loss with age has severe health consequences, yet the molecular basis of the loss remains obscure. Although mitochondrial DNA (mtDNA)-deletion mutations have been shown to accumulate with age, for these aberrant genomes to be physiologically relevant, they must accumulate to high levels intracellularly and be present in a significant number of cells. We examined mtDNA-deletion mutations in vastus lateralis (VL) muscle of human subjects aged 49-93 years, using both histologic and polymerase-chain-reaction (PCR) analyses, to determine the physiological and genomic integrity of mitochondria in aging human muscle. The number of VL muscle fibers exhibiting mitochondrial electron-transport-system (ETS) abnormalities increased from an estimated 6% at age 49 years to 31% at age 92 years. We analyzed the mitochondrial genotype of 48 single ETS-abnormal, cytochrome c oxidase-negative/succinate dehydrogenase-hyperreactive (COX-/SDH++) fibers from normal aging human subjects and identified mtDNA-deletion mutations in all abnormal fibers. Deletion mutations were clonal within a fiber and concomitant to the COX-/SDH++ region. Quantitative PCR analysis of wild-type and deletion-containing mtDNA genomes within ETS-abnormal regions of single fibers demonstrated that these deletion mutations accumulate to detrimental levels (>90% of the total mtDNA).     

Apoptosis and necrosis mediate skeletal muscle fiber loss in age‐induced mitochondrial enzymatic abnormalities

Sarcopenia, the age‐induced loss of skeletal muscle mass and function, results from the contributions of both fiber atrophy and loss of myofibers. We have previously characterized sarcopenia in FBN rats, documenting age‐dependent declines in muscle mass and fiber number along with increased fiber atrophy and fibrosis in vastus lateralis and rectus femoris muscles. Concomitant with these sarcopenic changes is an increased abundance of mitochondrial DNA deletion mutations and electron transport chain (ETC) abnormalities. In this study, we used immunohistological and histochemical approaches to define cell death pathways involved in sarcopenia. Activation of muscle cell death pathways was age‐dependent with most apoptotic and necrotic muscle fibers exhibiting ETC abnormalities. Although activation of apoptosis was a prominent feature of electron transport abnormal muscle fibers, necrosis was predominant in atrophic and broken ETC‐abnormal fibers. These data suggest that mitochondrial dysfunction is a major contributor to the activation of cell death processes in aged muscle fibers. The link between ETC abnormalities, apoptosis, fiber atrophy, and necrosis supports the hypothesis that mitochondrial DNA deletion mutations are causal in myofiber loss. These studies suggest a progression of events beginning with the generation and accumulation of a mtDNA deletion mutation, the concomitant development of ETC abnormalities, a subsequent triggering of apoptotic and, ultimately, necrotic events resulting in muscle fiber atrophy, breakage, and fiber loss.     

Presence of embryonic myosin in normal postural muscles of the adult rat

Indirect immunofluorescence was used to localize embryonic myosin heavy chains in soleus, adductor longus, tibialis anterior, plantaris, and extensor digitorum longus muscles of 6-month-old rats. A monoclonal antibody (2B6), specifically recognizing rat embryonic myosin, was applied to unfixed, transverse, frozen sections. The number of embryonic myosin-positive (EMP) extrafusal fibers was expressed as a percentage of the total number of fibers. EMP extrafusal fibers were only seen in the soleus and adductor longus muscles, both postural muscles. Approximately 1% of the soleus muscle fibers appeared positively stained for embryonic myosin. The majority of such fibers had a small diameter (<500 ), appeared intensely fluorescent, and typically contained central nuclei. Re-expression of embryonic myosin due to spontaneous fiber denervation is not a likely factor in this study, since alpha-bungarotoxin and N-CAM localization were restricted to the motor end-plate region of EMP fibers. Since embryonic myosin was shown to disappear in all normal-sized myofibers by 2 to 3 months of age, the results suggest that the EMP extrafusal fibers seen in postural muscles of 6 to 12-month-old animals are regenerating myofibers. We speculate that a small number of muscle fibers may be regenerating in normal, adult postural muscles, in response to fiber damage possibly caused by excessive recruitment or overloading.     

EMG-normalised kinase activation during exercise is higher in human gastrocnemius compared to soleus muscle

In mice, certain proteins show a highly confined expression in specific muscle groups. Also, resting and exercise/contraction-induced phosphorylation responses are higher in rat skeletal muscle with low mitochondrial content compared to muscles with high mitochondrial content, possibly related to differential reactive oxygen species (ROS)-scavenging ability or resting glycogen content. To evaluate these parameters in humans, biopsies from soleus, gastrocnemius and vastus lateralis muscles were taken before and after a 45 min inclined (15%) walking exercise bout at 69% VO2(max) aimed at simultaneously activating soleus and gastrocnemius in a comparable dynamic work-pattern. Hexokinase II and GLUT4 were 46-59% and 26-38% higher (p<0.05) in soleus compared to the two other muscles. The type I muscle fiber percentage was highest in soleus and lowest in vastus lateralis. No differences were found in protein expression of signalling proteins (AMPK subunits, eEF2, ERK1/2, TBC1D1 and 4), mitochondrial markers (F1 ATPase and COX1) or ROS-handling enzymes (SOD2 and catalase). Gastrocnemius was less active than soleus measured as EMG signal and glycogen use yet gastrocnemius displayed larger increases than soleus in phosphorylation of AMPK Thr172, eEF2 Thr56 and ERK 1/2 Thr202/Tyr204 when normalised to the mean relative EMG-signal. In conclusion, proteins with muscle-group restricted expression in mice do not show this pattern in human lower extremity muscle groups. Nonetheless the phosphorylation-response is greater for a number of kinase signalling pathways in human gastrocnemius than soleus at a given activation-intensity. This may be due to the combined subtle effects of a higher type I muscle fiber content and higher training status in soleus compared to gastrocnemius muscle.     

Aging affects different human muscles in various ways. An image analysis of the histomorphometric characteristics of fiber types in human masseter and vastus lateralis muscles from young adults and the very old

This study is an attempt to objectively evaluate age-related changes in human muscles by use of histomorphometric methods. Aging in humans induces dramatic transformations in the skeletal muscles but little is known as to whether or not the aging processes per se may affect all muscles equally. In this study aging of two human muscles with different functions, origin and nerve supply is compared. Sections were cut from masseter and vastus lateralis muscles obtained from young adults aged 18-24 years and from the very old aged 90-102 years. Muscle fiber types were classified with the traditional myofibrillar ATPase staining. Various histomorphometric parameters of the different fiber types in human masseter and vastus lateralis muscle sections were obtained by image analyses to evaluate the age-related changes in the muscle fibers. The following variables were calculated: the number of each fiber type per photographed area; the area of each fiber and two indicators for the shape of the muscle fibers. In the aging muscles there was no relative preferential loss of a fiber type. High numbers of intermediate ATPase-stained fibers (IM fibers) were found in some old vastus muscles but were only sporadic in young vastus muscles. However, there was no change in the percentage distribution of intermediate ATPase-stained fibers when young and very old human masseter muscles were compared. Incubation of the sections with antimyosin antibodies showed that the IM fibers in old masseter and old vastus contained different myosin heavy chains. Thus ATPase activity and anti-myosin staining displayed a somewhat different pattern of fiber type distribution. The main changes in the shape and area indicated that type I fibers in the masseter became more circular while in the vastus they decreased significantly in size. The type II fibers in the vastus became very small and deviated significantly from circularity whereas the type II fibers in the masseter only exhibited a decrease in the size of the fibers. Histomorphometric measurements show that aging affects different human muscles in various ways.     

Seven skeletal muscles rich in slow muscle fibers may function to sustain neutral position in the rodent hindlimb

Skeletal muscles consist of slow-twitch and fast-twitch muscle fibers, which have distinct physiological and biochemical properties. The muscle fiber composition determines the contractile velocity and fatigability of a particular skeletal muscle. We analyzed the systemic distribution of slow muscle fibers in all rodent skeletal muscles by myosin ATPase staining and found that only seven hindlimb skeletal muscles were extremely rich in slow muscle fibers. These included the mouse piriformis (56.5%), gluteus minimus (35.7%), vastus intermedius (24.7%), quadratus femoris (69.9%), adductor brevis (44.3%), gracilis (24.6%), and soleus muscles (35.1%). In mice, the relative proportion of slow muscle fibers did not exceed 15% in skeletal muscles in other regions. The distribution of slow muscle fibers was well conserved in rats and rabbits. The soleus muscle is an important antigravity muscle in both rodents and humans; therefore, these skeletal muscles rich in slow muscle fibers might play an important role in sustaining neutral alignment of the lower extremity.     

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.     

Effect of growth on muscle capillarity and fiber type composition in rat diaphragm

The effect of growth on the capillarity and fiber type composition of the diaphragm, soleus and extensor digitorum longus (EDL) muscles of rats weighing between 55 and 330 g have been studied. Muscle samples obtained from the anesthetized rat were rapidly frozen and sliced transversely in a cryostat. The sections were stained histochemically by the SDH method and the myosin ATPase method after preincubation at pH 4.3 to typify fibers (FG, FOG and SO fibers). To visualize capillaries, the myosin ATPase method after preincubation at pH 4.0 was used. The percentage of FOG fibers decreased in all muscles with growth. While the FG and SO fibers increased in the diaphragm, SO fibers increased in the soleus, and FG fibers increased in the EDL. The capillary density showed a hyperbolic decrease with growth in all muscles, while the number of capillaries around each fiber increased in all muscles with growth. It is concluded that growth causes the changing properties of the motoneurons and the new capillary formation in the diaphragm muscle, as well as the soleus and EDL muscles.     

Influence of spaceflight on rat skeletal muscle

The size, succinate dehydrogenase (SDH) and alpha-glycerolphosphate dehydrogenase (GPD) activities, and alkaline myofibrillar adenosinetriphosphatase (ATPase) staining properties were determined from quantitative histochemical analyses of single fibers from five hindlimb muscles of six male rats exposed to a 7-day National Aeronautics and Space Administration spaceflight mission (SL-3). These same properties were determined in a group of ground-based control rats housed under simulated environmental conditions. The wet weight of each of the flight muscles was significantly reduced relative to control. However, the loss of mass varied from 36% in the soleus to 15% in the extensor digitorum longus. The cross-sectional areas of fibers in the flight muscles also were reduced, except for the dark ATPase fibers in the medial gastrocnemius. The greatest relative fiber atrophy occurred in the muscles with the highest proportion of light ATPase fibers. An increase in the percentage of dark ATPase fibers also was observed in flight muscles with a predominance of light ATPase fibers. Also, there was an increase in the biochemically determined myofibrillar ATPase activity of tissue sections of the flight soleus. No changes in histochemical or biochemical measures of ATPase activity were observed in the flight extensor digitorum longus. In general, the SDH activity of flight muscles was maintained, whereas GPD activity either was maintained or increased. Based on a metabolic profile of ATPase, SDH, and GPD, there was an increase in the proportion of fast oxidative-glycolytic fibers in some muscles.     

Enzyme profiles of single muscle fibers never exposed to normal neuromuscular activity.

Do muscle fiber properties commonly associated with fiber types in adult animals and the population distribution of these properties require normal activation patterns to develop? To address this issue, the activity of an oxidative [succinic dehydrogenase (SDH)] and a glycolytic [alpha-glycerophosphate dehydrogenase (GPD)] marker enzyme, the characteristics of myosin adenosinetriphosphatase (myosin ATPase, alkaline preincubation), and the cross-sectional area of single fibers were studied. The soleus and medial gastrocnemius of normal adult cats were compared with cats that 6 mo earlier had been spinally transected at T12-T13 at 2 wk of age. In control cats, SDH activity was higher in dark than light ATPase fibers in the soleus and higher in light than dark ATPase fibers in the medial gastrocnemius. After transection, SDH activity was similar to control in both muscles. GPD activity appeared to be elevated in some fibers in each fiber type in both muscles after transection. The cross-sectional areas most affected by spinal transection were light ATPase fibers of the soleus and dark ATPase fibers of the medial gastrocnemius, the predominant fiber type in each muscle. These data demonstrate that although the muscle fibers of cats spinalized at 2 wk of age presumably were never exposed to normal levels of activation, the activity of an oxidative marker enzyme was maintained or elevated 6 mo after spinal transection. Furthermore, although the absolute enzyme activities in some fibers were elevated by transection, three functional protein systems commonly associated with fiber types, i.e., hydrolysis of ATP by myosin ATPase and glycolytic (GPD) and oxidative (SHD) metabolism, developed in a coordinated manner typical of normal adult muscles.     

Rat skeletal muscle triacylglycerol utilization during exhaustive swimming

The utilization of triacylglycerol in slow oxidative, fast oxidative-glycolytic, and fast glycolytic skeletal muscle fiber types was examined in rats subjected to a prolonged exhaustive swim. Significant reductions of intramuscular triacylglycerol occurred following 2 h and 40 min of swimming in all muscles containing a predominance of slow oxidative and fast oxidative-glycolytic fibers, which possess a high capacity for free fatty acid oxidation. Triacylglycerol content in the soleus decreased by 48%, and reductions of 41, 29, and 27% were measured in the red vastus lateralis, red gastrocnemius, and plantaris muscles, respectively. In the white vastus lateralis and white gastrocnemius muscles (fast glycolytic fibers) triacylglycerol concentrations were unaffected. In all muscles the variability of intramuscular triacylglycerol measurements between animals was 20-50% and the within animal variance (right vs. left hindlimb) was similar. Analytical repeatability was approximately 10% in all muscles and significantly less than the between- and within-animal variances. It was concluded that a real biological variation exists in the triacylglycerol content of all rat skeletal muscles and that intramuscular triacylglycerol is an important energy source during prolonged exercise of moderate intensity.     

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.     

Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia.

The in vivo cellular impact of age-associated mitochondrial DNA mutations is unknown. We hypothesized that mitochondrial DNA deletion mutations contribute to the fiber atrophy and loss that cause sarcopenia, the age-related decline of muscle mass and function. We examined 82,713 rectus femoris muscle fibers from Fischer 344 x Brown Norway F1 hybrid rats of ages 5, 18, and 38 months through 1000 microns by serial cryosectioning and histochemical staining for cytochrome c oxidase and succinate dehydrogenase. Between 5 and 38 months of age, the rectus femoris muscle in the hybrid rat demonstrated a 33% decrease in mass concomitant with a 30% decrease in total fibers at the muscle mid-belly. We observed significant increases in the number of mitochondrial abnormalities with age from 289 +/- 8 ETS abnormal fibers in the entire 5-month-old rectus femoris to 1094 +/- 126 in the 38-month-old as calculated from the volume density of these abnormalities. Segmental mitochondrial abnormalities contained mitochondrial DNA deletion mutations as revealed by laser capture microdissection and whole mitochondrial genome amplification. Muscle fibers harboring mitochondrial deletions often displayed atrophy, splitting and increased steady-state levels of oxidative nucleic damage. These data suggest a causal role for age-associated mitochondrial DNA deletion mutations in sarcopenia.     

Histological and histochemical comparisons of muscle spindles in three hind limb muscles of the guinea pig.

Guinea pig soleus, medial gastrocnemius and vastus lateralis muscles were compared for spindle density and distribution, number of intrafusal fibers per spindle and histochemical appearance of the axial bundle. A total of 326 spindles was used in the comparisons. Spindle density was over four times greater in the soleus than in either the medial gastrocnemius or vastus lateralis. In the soleus the spindles were distributed at random, but in the other two muscles no spindles were found in those fascicles in which fast-twitch glycolytic extrafusal fibers predominated. The average number of intrafusal fibers per spindle varied by less than 5% between the three kinds of muscles. About 80% of all spindles located had four intrafusal fibers, two of the nuclear bag type and two of the nuclear chain type. The histochemical appearance of the axial bundle was the same in each kind of muscle. Based on intensities of the myofibrillar adenosine triphosphatase reaction product at polar regions nuclear bag fibers were separable into two histochemical groups; nuclear chain fibers were of only one histochemical type.     

Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis

Skeletal muscle is a heterogeneous tissue comprised of fibers with different morphological, functional, and metabolic properties. Different muscles contain varying proportions of fiber types; therefore, accurate identification is important. A number of histochemical methods are used to determine muscle fiber type; however, these techniques have several disadvantages. Immunofluorescence analysis is a sensitive method that allows for simultaneous evaluation of multiple MHC isoforms on a large number of fibers on a single cross-section, and offers a more precise means of identifying fiber types. In this investigation we characterized pure and hybrid fiber type distribution in 10 rat and 10 mouse skeletal muscles, as well as human vastus lateralis (VL) using multicolor immunofluorescence analysis. In addition, we determined fiber type-specific cross-sectional area (CSA), succinate dehydrogenase (SDH) activity, and α-glycerophosphate dehydrogenase (GPD) activity. Using this procedure we were able to easily identify pure and hybrid fiber populations in rat, mouse, and human muscle. Hybrid fibers were identified in all species and made up a significant portion of the total population in some rat and mouse muscles. For example, rat mixed gastrocnemius (MG) contained 12.2% hybrid fibers whereas mouse white tibialis anterior (WTA) contained 12.1% hybrid fibers. Collectively, we outline a simple and time-efficient method for determining MHC expression in skeletal muscle of multiple species. In addition, we provide a useful resource of the pure and hybrid fiber type distribution, fiber CSA, and relative fiber type-specific SDH and GPD activity in a number of rat and mouse muscles.     

Skeletal muscle histology and biochemistry of an elite sprinter, the African cheetah

To establish a skeletal muscle profile for elite sprinters, we obtained muscle biopsy samples from the vastus lateralis, gastrocnemius and soleus of African cheetahs (Acinonyx jubatus). Muscle ultrastructure was characterized by the fiber type composition and mitochondrial volume density of each sample. Maximum enzyme activity, myoglobin content and mixed fiber metabolite content were used to assess the major biochemical pathways. The results demonstrate a preponderance of fast-twitch fibers in the locomotor muscles of cheetahs; 83% of the total number of fibers examined in the vastus lateralis and nearly 61% of the gastrocnemius were comprised of fast-twitch fibers. The total mitochondrial volume density of the limb muscles ranged from 2.0 to 3.9% for two wild cheetahs. Enzyme activities reflected the sprinting capability of the cheetah. Maximum activities for pyruvate kinase and lactate dehydrogenase in the vastus lateralis were 1519.00 ± 203.60 and 1929.25±482.35 μmol min⁻¹ · g wet wt⁻¹, respectively, and indicated a high capacity for glycolysis. This study demonstrates that the locomotor muscles of cheetahs are poised for anaerobically based exercise. Fiber type composition, mitochondrial content and glycolytic enzyme capacities in the locomotor muscles of these sprinting cats are at the extreme range of values for other sprinters bred or trained for this activity including greyhounds, thoroughbred horses and elite human athletes. Accepted: 5 June 1997     

Structural and metabolic characteristics of human soleus fibers after long duration spaceflight

The fiber size decline, alterations in fiber metabolic potential and increase of connective tissue component were shown in human m. vastus lateralis after short and long-duration space flights and in m.soleus and m.vastus lateralis after 120 day head down tilt bed rest. It is known from rat and monkey studies that the exposure to weightlessness leads to the most pronounced changes in postural muscles, e.g. m.soleus. It was shown that 17 day space flight induced significant decrease of fiber cross-sectional area and slow-to-fast fiber type transformation in human soleus. But in the cited work the fiber population under study was limited like in most single fiber technique analyses. The present study was purposed to investigate the structural and metabolic properties of soleus muscle in Russian cosmonauts exposed to 129-day space flight on board of the International Space Station.     

Fiber type changes in rat skeletal muscle after intense interval training

Female Sprague-Dawley rats were subjected to a ten week training program to determine the influence of intense interval running on the fiber type composition of selected hindlimb muscles; soleus (S), plantaris (P), deep vastus lateralis (DVL), and superficial vastus lateralis (SVL). The muscles of one hindlimb were used for histochemical ATPase analysis to determine the distribution of fiber types and those of the contralateral hindlimb were assayed biochemically for citrate synthase activity (an aerobic marker). Training induced a significant increase in citrate synthase activity in each muscle section. The largest absolute increase occurred in the DVL and the largest relative increase occurred in the SVL. The distribution of fiber types within the S (85% slow-twitch) and SVL (100% fast-twitch) remained unchanged with training. However, significant increases in the percentage of type I (slow-twitch) fibers in both the P (2-fold) and DVL (3-fold) were observed with concomitant decreases in the type II (fast-twitch) population. In addition, training induced significant changes in the fast-twitch subtype populations of the DVL (IIB----IIA). These data suggest exercise-induced fiber type transformations occurring both within the fast-twitch population and between fast-twitch and slow-twitch fibers in certain hindlimb muscles of the rat following a high intensity interval training program.     

Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection

Laser-capture microdissection was coupled with PCR to define the mitochondrial genotype of aged muscle fibers exhibiting mitochondrial enzymatic abnormalities. These electron transport system (ETS) abnormalities accumulate with age, are localized segmentally along muscle fibers, are associated with fiber atrophy and may contribute to age-related fiber loss. DNA extracted from single, 10 µm thick, ETS abnormal muscle fibers, as well as sections from normal fibers, served as templates for PCR-based deletion analysis. Large mitochondrial (mt) DNA deletion mutations (4.4–9.7 kb) were detected in all 29 ETS abnormal fibers analyzed. Deleted mtDNA genomes were detected only in the regions of the fibers with ETS abnormalities; adjacent phenotypically normal portions of the same fiber contained wild-type mtDNA. In addition, identical mtDNA deletion mutations were found within different sections of the same abnormal region. These findings demonstrate that large deletion mutations are associated with ETS abnormalities in aged rat muscle and that, within a fiber, deletion mutations are clonal. The displacement of wild-type mtDNAs with mutant mtDNAs results in concomitant mitochondrial enzymatic abnormalities, fiber atrophy and fiber breakage.