Intermyofibrillar Research Articles

Dietary nitrate preserves mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term immobilization in mice.

Skeletal muscle disuse reduces muscle protein synthesis rates and induces atrophy, events associated with decreased mitochondrial respiration and increased reactive oxygen species. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation attenuates disuse-induced impairments in mitochondrial function and muscle protein synthesis rates. Female C57Bl/6N mice were subjected to single-limb casting (3 or 7days) and consumed drinking water with or without 1mM sodium nitrate. Compared with the contralateral control limb, 3days of immobilization lowered myofibrillar fractional synthesis rates (FSR, P< 0.0001), resulting in muscle atrophy. Although FSR and mitophagy-related proteins were higher in subsarcolemmal (SS) compared with intermyofibrillar (IMF) mitochondria, immobilization for 3days decreased FSR in both SS (P=0.009) and IMF (P=0.031) mitochondria. Additionally, 3days of immobilization reduced maximal mitochondrial respiration, decreased mitochondrial protein content, and increased maximal mitochondrial reactive oxygen species emission, without altering mitophagy-related proteins in muscle homogenate or isolated mitochondria (SS and IMF). Although nitrate consumption did not attenuate the decline in muscle mass or myofibrillar FSR, intriguingly, nitrate completely prevented immobilization-induced reductions in SS and IMF mitochondrial FSR. In addition, nitrate prevented alterations in mitochondrial content and bioenergetics after both 3 and 7days of immobilization. However, in contrast to 3days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR after 7days of immobilization. Therefore, although nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to maintain mitochondrial bioenergetics and transiently preserve mitochondrial protein synthesis rates during short-term muscle disuse. KEY POINTS: Alterations in mitochondrial bioenergetics (decreased respiration and increased reactive oxygen species) are thought to contribute to muscle atrophy and reduced protein synthesis rates during muscle disuse. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation could attenuate immobilization-induced skeletal muscle impairments in female mice. Dietary nitrate prevented short-term (3day) immobilization-induced declines in mitochondrial protein synthesis rates, reductions in markers of mitochondrial content, and alterations in mitochondrial bioenergetics. Despite these benefits and the preservation of mitochondrial content and bioenergetics during more prolonged (7 day) immobilization, nitrate consumption did not preserve skeletal muscle mass or myofibrillar protein synthesis rates. Overall, although dietary nitrate did not prevent atrophy, nitrate supplementation represents a promising nutritional approach to preserve mitochondrial function during muscle disuse.

What triggers eNOS translocation to the mitochondria in the heart: Adrenergic or mechanical stress?

The translocation of eNOS from the cytosol to subsarcolemmal (SSM) and intermyofibrillar (IMF) mitochondrial subfractions has been recently reported as a key trigger of exercise training-induced cardioprotection. The cardiac response to exercise is determined by both intrinsic mechanical and extrinsic adrenergic-dependent signaling pathways. Whether eNOS translocation in response to a single bout of exercise is due to mechanical stretch of the cardiac muscle or adrenergic stress remains to be studied. The aim of this work was to evaluate the impact of cardiac adrenergic or mechanical stress on the translocation of eNOS from the cytosol to the mitochondria. Mechanical stress was obtained on isolated perfused hearts by adjusting the diastolic pressure at 5 (low-stretch) or 15 (high-stretch) mmHg. The β-adrenergic stress was induced in low or high-stretch isolated hearts perfused with 10 nM Isoproterenol for 5 min. The hearts were quick-frozen. The cytosolic, SSM, and IMF + Myofilaments subfractions were obtained by differential centrifugations. The level of eNOS was analyzed by Western blot. High-stretch tended to increase the level of eNOS in both IMF and SSM subfractions without reaching statistical significance. In contrast, Isoproterenol stimulation increased markedly the level of eNOS both in SSM and IMF in low-stretch hearts, with no additional impact of high-stretch. These results suggest that β-adrenergic receptors stimulation rather than mechanical stress of the cardiac muscle can trigger the translocation of eNOS. These results obtained ex-vivo were confirmed in vivo by treating the rats with a single injection of Isoproterenol (1 mg/kg, 30 min) that increased the level of eNOS in mitochondrial subfractions. In this preliminary work, we reported that myocardial acute adrenergic stress, rather than acute stretch, is associated with eNOS translocation from cytosol to mitochondrial subfractions. The role of the eNOS-NO pathway in the normal response of the heart to adrenergic stress remain however to be studied and will be the aim of further studies.

Exercise and Doxorubicin Modify Markers of Iron Overload and Cardiolipin Deficiency in Cardiac Mitochondria.

Doxorubicin (DOX) is a chemotherapeutic agent highly effective at limiting cancer progression. Despite the efficacy of this anticancer drug, the clinical use of DOX is limited due to cardiotoxicity. The cardiac mitochondria are implicated as the primary target of DOX, resulting in inactivation of electron transport system complexes, oxidative stress, and iron overload. However, it is established that the cardiac mitochondrial subpopulations reveal differential responses to DOX exposure, with subsarcolemmal (SS) mitochondria demonstrating redox imbalance and the intermyofibrillar (IMF) mitochondria showing reduced respiration. In this regard, exercise training is an effective intervention to prevent DOX-induced cardiac dysfunction. Although it is clear that exercise confers mitochondrial protection, it is currently unknown if exercise training mitigates DOX cardiac mitochondrial toxicity by promoting beneficial adaptations to both the SS and IMF mitochondria. To test this, SS and IMF mitochondria were isolated from sedentary and exercise-preconditioned female Sprague Dawley rats exposed to acute DOX treatment. Our findings reveal a greater effect of exercise preconditioning on redox balance and iron handling in the SS mitochondria of DOX-treated rats compared to IMF, with rescue of cardiolipin synthase 1 expression in both subpopulations. These results demonstrate that exercise preconditioning improves mitochondrial homeostasis when combined with DOX treatment, and that the SS mitochondria display greater protection compared to the IMF mitochondria. These data provide important insights into the molecular mechanisms that are in part responsible for exercise-induced protection against DOX toxicity.

Response of Subsarcolemmal and Intermyofibrillar Mitochondria to Exercise and Doxorubicin‐Induced Cardiotoxicity

The chemotherapeutic agent doxorubicin (DOX) is highly effective at limiting cancer progression. However, systemic DOX treatment results in off‐target accumulation within cardiac mitochondria, which can cause aberrant signaling leading to dose‐dependent heart failure in cancer patients and survivors. Exercise has demonstrated preclinical and clinical efficacy in limiting the cardiotoxic effects of DOX by putatively improving mitochondrial quality and function, although this mechanism has not been fully explicated. Therefore, to further understand the molecular determinants of exercise‐mediated protection against DOX‐induced mitochondriopathy and cardiotoxicity we independently examined the cardiac subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subpopulations. Female Sprague Dawley rats were divided (n=10/group) into 1) Sedentary‐Saline; 2) Sedentary‐DOX; 3) Exercise‐Saline; and 4) Exercise‐DOX groups. Exercise groups underwent 10 days of aerobic treadmill running (~70% VO2max, 1hr/day) with 2 days of rest after day 5. Twenty‐four hours following the last exercise bout animals received saline or DOX (20mg/kg IP) treatment. Dependent measures were evaluated 48 hours post treatment. To demonstrate that the exercise preconditioning protocol was sufficient to elicit cardiorespiratory adaptations, all groups performed an exercise tolerance test (ETT). The ETT revealed a significant increase in cardiorespiratory fitness in both exercise‐trained groups, regardless of saline or DOX treatment. In addition, evaluation of the myocardial performance index supported the premise that exercise preconditioning induces a cardioprotective phenotype against DOX toxicity. Further, analysis of the cardiac SS and IMF mitochondrial subfractions indicate that exercise preconditioning protects against DOX‐induced reactive oxygen species (ROS) emission in the SS fraction. Interestingly, IMF mitochondrial ROS emission was not affected by exercise or DOX treatment. In contrast, the IMF mitochondria respiratory control ratio (RCR) was significantly elevated in Exercise‐Saline animals compared to both sedentary groups, while no differences existed in the SS mitochondria. These findings indicate that 1) exercise preconditioning is a beneficial non‐pharmacological approach to improve cardiac outcomes following DOX treatment and that 2) cardiac mitochondrial subfractions are differentially affected by exercise and DOX treatment.

Time-dependent changes in autophagy, mitophagy and lysosomes in skeletal muscle during denervation-induced disuse.

Deficits in skeletal muscle mitochondrial content and quality are observed following denervation-atrophy. This is due to alterations in the biogenesis of new mitochondria as well as their degradation via mitophagy. The regulation of autophagy and mitophagy over the course of denervation (Den) remains unknown. Further, the time-dependent changes in lysosome content, the end-stage organelle for mitophagy, remain unexplored. Here, we studied autophagic as well as mitophagic pre-lysosomal flux in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria from rat muscle subjected to Den for 1, 3 or 7days. We also assessed flux at 1day post-denervation in transgenic mt-keima mice. Markers of mitochondrial content were reduced at 7days following Den, and Den further resulted in rapid decrements in mitochondrial respiration, along with increased ROS emission. Pre-lysosomal autophagy flux was upregulated at 1 and 3days post-Den but was reduced compared to time-matched sham-operated controls at 7days post-Den. Similarly, pre-lysosomal mitophagy flux was enhanced in SS mitochondria as early as 1 and 3days of Den but decreased in both SS and IMF subfractions following 7days of Den. Lysosome protein content and transcriptional regulators TFEB and TFE3 were progressively enhanced with Den, an adaptation designed to enhance autophagic capacity. However, evidence for lysosome dysfunction was apparent by 7days, which may limit degradation capacity. This may contribute to an inability to clear dysfunctional mitochondria and increased ROS signalling, thereby accelerating muscle atrophy. Thus, therapeutic targeting of lysosome function may help to maintain autophagy and muscle health during conditions of muscle disuse or denervation. KEY POINTS: Denervation is an experimental model of peripheral neuropathies as well as muscle disuse, and it helps us understand some aspects of the sarcopenia of ageing. Muscle disuse is associated with reduced mitochondrial content and function, leading to metabolic impairments within the tissue. Although the processes that regulate mitochondrial biogenesis are understood, those that govern mitochondrial breakdown (i.e. mitophagy) are not well characterized in this context. Autophagy and mitophagy flux, measured up to the point of the lysosome (pre-lysosomal flux rates), were increased in the early stages of denervation, along with mitochondrial dysfunction, but were reduced at later time points when the degree of muscle atrophy was highest. Denervation led to progressive increases in lysosomal proteins to accommodate mitophagy flux, yet evidence for lysosomal impairment at later stages may limit the removal of dysfunctional mitochondria, stimulate reactive oxygen species signalling, and reduce muscle health as denervation time progresses.

Effects of Aging and Caloric Restriction on Fiber Type Composition, Mitochondrial Morphology and Dynamics in Rat Oxidative and Glycolytic Muscles.

Aging is associated with a progressive decline in muscle mass and strength, a process known as sarcopenia. Evidence indicates that mitochondrial dysfunction plays a causal role in sarcopenia and suggests that alterations in mitochondrial dynamics/morphology may represent an underlying mechanism. Caloric restriction (CR) is among the most efficient nonpharmacological interventions to attenuate sarcopenia in rodents and is thought to exert its beneficial effects by improving mitochondrial function. However, CR effects on mitochondrial morphology and dynamics, especially in aging muscle, remain unknown. To address this issue, we investigated mitochondrial morphology and dynamics in the oxidative soleus (SOL) and glycolytic white gastrocnemius (WG) muscles of adult (9-month-old) ad libitum-fed (AL; A-AL), old (22-month-old) AL-fed (O-AL), and old CR (O-CR) rats. We show that CR attenuates the aging-related decline in the muscle-to-body-weight ratio, a sarcopenic index. CR also prevented the effects of aging on muscle fiber type composition in both muscles. With aging, the SOL displayed fragmented SubSarcolemmal (SS) and InterMyoFibrillar (IMF) mitochondria, an effect attenuated by CR. Aged WG displayed enlarged SS and more complex/branched IMF mitochondria. CR had marginal anti-aging effects on WG mitochondrial morphology. In the SOL, DRP1 (pro-fission protein) content was higher in O-AL vs YA-AL, and Mfn2 (pro-fusion) content was higher in O-CR vs A-AL. In the gastrocnemius, Mfn2, Drp1, and Fis1 (pro-fission) contents were higher in O-AL vs A-AL. CR reduced this aging-related increase in Mfn2 and Fis1 content. Overall, these results reveal for the first time that aging differentially impacts mitochondrial morphology and dynamics in different muscle fiber types, by increasing fission/fragmentation in oxidative fibers while enhancing mitochondrial size and branching in glycolytic fibers. Our results also indicate that although CR partially attenuates aging-related changes in mitochondrial dynamics in glycolytic fibers, its anti-aging effect on mitochondrial morphology is restricted to oxidative fibers.

Adenosine Triphosphate Production of Muscle Mitochondria after Acute Exercise in Lean and Obese Humans.

Current evidence indicates mitochondrial dysfunction in humans with obesity. Acute exercise appears to enhance mitochondrial function in the muscle of nonobese humans, but its effects on mitochondrial function in muscle of humans with obesity are not known. We sought to determine whether acute aerobic exercise stimulates mitochondrial function in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in humans with obesity. We assessed maximal adenosine triphosphate production rate (MAPR) and citrate synthase (CS) activity in isolated SS and IMF mitochondria from subjects with body mass index < 27 kg·m (median age, 25 yr; interquartile range, 22-39 yr) and subjects with body mass index > 32 kg·m (median age, 29 yr; interquartile range, 20-39 yr) before and 3 h after a 45-min cycling exercise at an intensity corresponding to 65% HR reserve. The SS and IMF mitochondria were isolated from muscle biopsies using differential centrifugation. Maximal adenosine triphosphate production rate and CS activities were determined using luciferase-based and spectrophotometric enzyme-based assays, respectively. Exercise increased MAPR in IMF mitochondria in both nonobese subjects and subjects with obesity (P < 0.05), but CS-specific activity did not change in either group (P > 0.05). Exercise increased MAPR supported by complex II in SS mitochondria, in both groups (P < 0.05), but MAPR supported by complex I or palmitate did not increase by exercise in the subjects with obesity (P > 0.05). Citrate synthase-specific activity increased in SS mitochondria in response to exercise only in nonobese subjects (P < 0.05). In nonobese humans, acute aerobic exercise increases MAPR in both SS and IMF mitochondria. In humans with obesity, the exercise increases MAPR in IMF mitochondria, but this response is less evident in SS mitochondria.

Obesity modifies the stoichiometry of mitochondrial proteins in a way that is distinct to the subcellular localization of the mitochondria in skeletal muscle

BackgroundSkeletal muscle mitochondrial content and function appear to be altered in obesity. Mitochondria in muscle are found in well-defined regions within cells, and they are arranged in a way that form distinct subpopulations of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. We sought to investigate differences in the proteomes of SS and IMF mitochondria between lean subjects and subjects with obesity. MethodsWe performed comparative proteomic analyses on SS and IMF mitochondria isolated from muscle samples obtained from lean subjects and subjects with obesity. Mitochondria were isolated using differential centrifugation, and proteins were subjected to label-free quantitative tandem mass spectrometry analyses. Collected data were evaluated for abundance of mitochondrial proteins using spectral counting. The Reactome pathway database was used to determine metabolic pathways that are altered in obesity. ResultsAmong proteins, 73 and 41 proteins showed different (mostly lower) expression in subjects with obesity in the SS and IMF mitochondria, respectively (false discovery rate-adjusted P ≤ 0.05). We specifically found an increase in proteins forming the tricarboxylic acid cycle and electron transport chain (ETC) complex II, but a decrease in proteins forming protein complexes I and III of the ETC and adenosine triphosphate (ATP) synthase in subjects with obesity in the IMF, but not SS, mitochondria. Obesity was associated with differential effects on metabolic pathways linked to protein translation in the SS mitochondria and ATP formation in the IMF mitochondria. ConclusionsObesity alters the expression of mitochondrial proteins regulating key metabolic processes in skeletal muscle, and these effects are distinct to mitochondrial subpopulations located in different regions of the muscle fibers. Trial RegistrationClinicalTrials.gov (NCT01824173).

The effect of aging and calorie restriction on mitochondrial morphology and dynamic in oxidative and glycolytic skeletal muscles

With aging, skeletal muscles progressively loose mass and function, a biological process known as sarcopenia. Recent data indicate that accumulation of mitochondrial dysfunction plays a major role in the development of sarcopenia and also suggested that alterations in mitophagy and mitochondrial dynamics/morphology may represent mechanisms causing this accumulation of dysfunctional mitochondria in the aged muscles. One most efficient lifestyle and non‐pharmacological intervention to attenuate sarcopenia in rodents is calorie restriction (CR). The hypothesis currently put forward to explain the protective and “anti‐aging” effects of caloric restriction is that CR would attenuate the effects of aging on mitochondrial function. However, the specific effects of CR on many aspects of mitochondrial biology, especially in the aged muscle, remain unclear or unknown. In particular, the effects of aging and CR on skeletal muscle mitochondrial morphology and dynamics, which are known to impact mitochondrial function, remain largely under investigated. The objectives of the present study were (i) to investigate the effects of aging on mitochondrial morphology and dynamic in glycolytic and oxidative skeletal muscles, define whether CR can attenuate the effects of aging on mitochondrial morphology and (iii) determine the effects of CR and aging on the muscular phenotype.Three groups of male Sprague Dawley rats were studied: 1‐adult (9‐month‐old) ad‐libitum fed (AL) (A‐AL); 2‐old (21‐month‐old) AL fed (O‐AL) and; 3‐old (21‐month‐old) calorie restricted (CR; 40% calorie restricted for 13 months; O‐CR) rats. The morphology of SubSarcolemmal (SS) and InterMyoFibrillar (IMF) mitochondria was assessed using a 2‐dimensional transmission electron microscopy approach (TEM) in the oxidative soleus (SOL) and glycolytic white gastrocnemius (WG) muscles. Westernblot analyses were performed to assess the contents of proteins regulating mitochondrial dynamics (fusion protein: Mfn2; Fission proteins: Drp1 and Fis1) in the SOL and WG.CR attenuated the impact of aging on the muscle mass to body weight ratio (a marker of sarcopenia). The SOL of O‐AL rats displayed fragmented SS and IMF mitochondria vs A‐AL rats. CR attenuated this aging‐related fragmentation of SS and IMF mitochondria in the SOL. The WG of O‐AL rats displayed enlarged SS mitochondria and more complex and branched IMF mitochondria vs their younger counterparts. In the WG, CR did not attenuate the effects of aging on mitochondrial morphology. In the SOL, the contents of Drp1 and Fis1 were higher in O‐AL vs A‐AL rats. When compared to the SOL of Y‐AL rats, O‐CR rats displayed higher contents of both Drp1 and Mfn2. In the WG, the content of Mfn2 and Drp1 was higher in O‐AL vs A‐AL rats. No difference between O‐CR and A‐AL rats were observed for proteins regulating mitochondrial dynamics.Taken altogether, our results indicate that the effects of aging and calorie restriction on mitochondrial morphology and dynamic are complex and muscle‐specific.Support or Funding InformationThis work was funded by grants from the Natural Sciences and Engineering Council of Canada (NSERC; #RGPIN‐2014‐04668), the Quebec Research Network on Aging and the FQRS (FRQS‐35184) awarded to Gilles Gouspillou. Jean‐Philippe Leduc‐Gaudet is supported by a CIHR Vanier Doctoral Scholarship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Plasma Amino Acids Stimulate Uncoupled Respiration of Muscle Subsarcolemmal Mitochondria in Lean but Not Obese Humans.

Obesity is associated with mitochondrial dysfunction in skeletal muscle. Increasing the plasma amino acid (AA) concentrations stimulates mitochondrial adenosine triphosphate (ATP) production in lean individuals. To determine whether acute elevation in plasma AAs enhances muscle mitochondrial respiration and ATP production in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in obese adults. Assessment of SS and IMF mitochondrial function during saline (i.e., control) and AA infusions. Eligible participants were healthy lean (body mass index, <25 kg/m2; age, 37 ± 3 years; n = 10) and obese (body mass index >30 kg/m2; age 35 ± 3 years; n = 11) subjects. Single trial of saline infusion followed by AA infusion. SS and IMF mitochondria were isolated from muscle biopsies collected at the end of the saline and AA infusions. Mitochondrial respiration and ATP production. AA infusion increased adenosine 5'-diphosphate (ADP)-stimulated respiration and ATP production rates of SS mitochondria in the lean (P < 0.05), but not obese, subjects. Furthermore, AA infusion increased the uncoupled (i.e., non-ADP-stimulated) respiration of SS mitochondria in the lean subjects only (P < 0.05). AA infusion had no effect on any of these parameters in IMF mitochondria in either lean or obese subjects (P > 0.05). Increasing the plasma AA concentrations enhances the capacity for respiration and ATP production of muscle SS, but not IMF, mitochondria in lean individuals, in parallel with increases in uncoupled respiration. However, neither of these parameters increases in muscle SS or IMF mitochondria in obese individuals.

Photothermal imaging of skeletal muscle mitochondria.

The morphology and topology of mitochondria provide useful information about the physiological function of skeletal muscle. Previous studies of skeletal muscle mitochondria are based on observation with transmission, scanning electron microscopy or fluorescence microscopy. In contrast, photothermal (PT) microscopy has advantages over the above commonly used microscopic techniques because of no requirement for complex sample preparation by fixation or fluorescent-dye staining. Here, we employed the PT technique using a simple diode laser to visualize skeletal muscle mitochondria in unstained and stained tissues. The fine mitochondrial network structures in muscle fibers could be imaged with the PT imaging system, even in unstained tissues. PT imaging of tissues stained with toluidine blue revealed the structures of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria and the swelling behavior of mitochondria in damaged muscle fibers with sufficient image quality. PT image analyses based on fast Fourier transform (FFT) and Grey-level co-occurrence matrix (GLCM) were performed to derive the characteristic size of mitochondria and to discriminate the image patterns of normal and damaged fibers.

Unique morphological characteristics of mitochondrial subtypes in the heart: the effect of ischemia and ischemic preconditioning.

Three subsets of mitochondria have been described in adult cardiomyocytes - intermyofibrillar (IMF), subsarcolemmal (SSM), and perinuclear (PN). They have been shown to differ in physiology, but whether they also vary in morphological characteristics is unknown. Ischemic preconditioning (IPC) is known to prevent mitochondrial dysfunction induced by acute myocardial ischemia/reperfusion injury (IRI), but whether IPC can also modulate mitochondrial morphology is not known. Morphological characteristics of three different subsets of adult cardiac mitochondria along with the effect of ischemia and IPC on mitochondrial morphology will be investigated. Mouse hearts were subjected to the following treatments (N=6 for each group): stabilization only, IPC (3x5 min cycles of global ischemia and reperfusion), ischemia only (20 min global ischemia); and IPC and ischemia. Hearts were then processed for electron microscopy and mitochondrial morphology was assessed subsequently. In adult cardiomyocytes, IMF mitochondria were found to be more elongated and less spherical than PN and SSM mitochondria. PN mitochondria were smaller in size when compared to the other two subsets. SSM mitochondria had similar area to IMF mitochondria but their sphericity measures were similar to PN mitochondria. Ischemia was shown to increase the sphericity parameters of all 3 subsets of mitochondria; reduce the length of IMF mitochondria, and increase the size of PN mitochondria. IPC had no effect on mitochondrial morphology either at baseline or after ischemia. The three subsets of mitochondria in the adult heart are morphologically different. IPC does not appear to modulate mitochondrial morphology in adult cardiomyocytes.

Effects of doxorubicin on cardiac muscle subsarcolemmal and intermyofibrillar mitochondria

Doxorubicin (DOX) is a highly effective chemotherapeutic used in the treatment of a broad spectrum of malignancies. However, clinical use of DOX is highly limited by cumulative and irreversible cardiomyopathy that occurs following DOX treatment. The pathogenesis of DOX-induced cardiac muscle dysfunction is complex. However, it has been proposed that the etiology of this myopathy is related to mitochondrial dysfunction, as a result of the dose-dependent increase in the mitochondrial accumulation of DOX. In this regard, cardiac muscle possesses two morphologically distinct populations of mitochondria. Subsarcolemmal (SS) mitochondria are localized just below the sarcolemma, whereas intermyofibrillar (IMF) mitochondria are found between myofibrils. Mitochondria in both regions exhibit subtle differences in biochemical properties, giving rise to differences in respiration, lipid composition, enzyme activities and protein synthesis rates. Based on the heterogeneity of SS and IMF mitochondria, we hypothesized that acute DOX administration would have distinct effects on each cardiac mitochondrial subfraction. Therefore, we isolated SS and IMF mitochondria from the hearts of female Sprague-Dawley rats 48h after administration of DOX. Our results demonstrate that while SS mitochondria appear to accumulate greater amounts of DOX, IMF mitochondria demonstrate a greater apoptotic and autophagic response to DOX exposure. Thus, the divergent protein composition and function of the SS and IMF cardiac mitochondria result in differential responses to DOX, with IMF mitochondria appearing more susceptible to damage after DOX treatment.

Twenty‐eight days of exposure to 3454 m increases mitochondrial volume density in human skeletal muscle

The role of hypoxia on skeletal muscle mitochondria is controversial. Studies superimposing exercise training on hypoxic exposure demonstrate an increase in skeletal muscle mitochondrial volume density (Mito(VD)) over equivalent normoxic training. In contrast, reductions in both skeletal muscle mass and Mito(VD) have been reported following mountaineering expeditions. These observations may, however, be confounded by negative energy balance, which may obscure the results. Accordingly we sought to examine the effects of high altitude hypoxic exposure on mitochondrial characteristics, with emphasis on Mito(VD), while minimizing changes in energy balance. For this purpose, skeletal muscle biopsies were obtained from nine lowlanders at sea level (Pre) and following 7 and 28 days of exposure to 3454 m. Maximal ergometer power output, whole body weight and composition, leg lean mass and skeletal muscle fibre area all remained unchanged following the altitude exposure. Transmission electron microscopy determined that intermyofibrillar (IMF) Mito(VD) was augmented (P = 0.028) by 11.5 ± 9.2% from Pre (5.05 ± 0.9%) to 28 Days (5.61 ± 0.04%). In contrast, there was no change in subsarcolemmal (SS) Mito(VD). As a result, total Mito(VD) (IMF + SS) was increased (P = 0.031) from 6.20 ± 1.5% at Pre to 6.62 ± 1.4% at 28 Days (7.8 ± 9.3%). At the same time no changes in mass-specific respiratory capacities, mitochondrial protein or antioxidant content were found. This study demonstrates that skeletal muscle Mito(VD) may increase with 28 days acclimation to 3454 m.

Greater Yield of Subsarcolemmal Mitochondria from Skeletal Muscle of Obese Subjects with No Change in Intrinsic Mitochondrial Function

Whether obesity influences content and functional capacity of skeletal muscle mitochondria (MITO) remains unresolved. In this study, biopsies of vastus lateralis from lean and obese human subjects were used to isolate subsarcolemmal (SS) and intermyofibrillar (IMF) MITO. IMF MITO were liberated using a protease. The SS and IMF were pelleted separately, using differential centrifugation. Skeletal muscle from obese subjects gave higher MITO protein yield, per g wet muscle vs. skeletal muscle from lean subjects in the SS but not in the IMF. There was a trend for higher isolated yields of citrate synthase (CS) activity, a marker for MITO content, in the muscle of obese subjects vs. lean subjects in SS, but not in IMF MITO. O2 consumption (JO) and ATP production rates measured with NAD‐linked substrates (malate+pyruvate+glutamate), were not different in obese vs. lean in either SS or IMF MITO expressed either per mg MITO protein or per unit CS activity. The similarities in MITO function observed in SS and IMF from both lean and obese human muscle, suggests comparable functional capacity per unit MITO, regardless of the location within the myocyte, or whether the MITO reside in skeletal muscle from lean or obese subjects. However, higher total yield of both protein and CS activities in the SS MITO from the muscle of obese subjects, point to the possibility for greater content of SS MITO in muscle of obese individuals. This conclusion must be qualified; these results may be due to a greater fractional yield of MITO during the isolation procedure of skeletal muscle from obese subjects.

SIRT1 overexpression in skeletal muscle in vivo induces increased insulin sensitivity and enhanced complex I but not complex II-V functions in individual subsarcolemmal and intermyofibrillar mitochondria.

SIRT1 is known to improve insulin resistance (IR), but whether this effect is direct or not is still unclear, and this question has not been addressed in vivo in the skeletal muscle. Therefore, we sought to test if acute overexpression of SIRT1 in skeletal muscle of high-fat diet (HFD) rats in vivo would affect subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial complexes I-V activities and antioxidant enzymes thereby improving insulin action. In vivo electrotransfer was used to overexpress SIRT1 in the skeletal muscle of rats fed HFD for 12weeks. Skeletal muscle insulin sensitivity and downstream effects of SIRT1 on AMPK, SIRT3, and mitochondrial biogenesis were studied. Citrate synthase (CS), complexes I-V, oxidative stress, and antioxidant levels were assessed in SS and IMF mitochondria. HFD rats showed skeletal muscle IR as well as decreased SIRT1 and SIRT3 expressions, mitochondrial DNA (mtDNA), and mitochondrial biogenesis (p < 0.05). SS and IMF mitochondria displayed lower CS, complexes I-V, and antioxidant enzyme activities (p < 0.05). By contrast, moderate (~2.5 folds) SIRT1 overexpression attenuated HFD-induced skeletal muscle IR. This improvement was associated with increased AMPK, PGC-1α, SIRT3, and mtDNA expressions as well as SS and IMF mitochondrial CS and complexes I-V activities. Importantly, SIRT1 overexpression largely restored antioxidant enzyme activities and enhanced complex I but not complexes II-V functions in individual SS and IMF mitochondria. This study suggests that SIRT1 overexpression improved IR at least partly by targeting complex I functions of SS and IMF mitochondria through the activation of SIRT1 and SIRT3.

Mechanical ventilation triggers abnormal mitochondrial dynamics and morphology in the diaphragm.

The diaphragm is a unique skeletal muscle designed to be rhythmically active throughout life, such that its sustained inactivation by the medical intervention of mechanical ventilation (MV) represents an unanticipated physiological state in evolutionary terms. Within a short period after initiating MV, the diaphragm develops muscle atrophy, damage, and diminished strength, and many of these features appear to arise from mitochondrial dysfunction. Notably, in response to metabolic perturbations, mitochondria fuse, divide, and interact with neighboring organelles to remodel their shape and functional properties-a process collectively known as mitochondrial dynamics. Using a quantitative electron microscopy approach, here we show that diaphragm contractile inactivity induced by 6 h of MV in mice leads to fragmentation of intermyofibrillar (IMF) but not subsarcolemmal (SS) mitochondria. Furthermore, physical interactions between adjacent organellar membranes were less abundant in IMF mitochondria during MV. The profusion proteins Mfn2 and OPA1 were unchanged, whereas abundance and activation status of the profission protein Drp1 were increased in the diaphragm following MV. Overall, our results suggest that mitochondrial morphological abnormalities characterized by excessive fission-fragmentation represent early events during MV, which could potentially contribute to the rapid onset of mitochondrial dysfunction, maladaptive signaling, and associated contractile dysfunction of the diaphragm.

Resveratrol improves high-fat diet induced insulin resistance by rebalancing subsarcolemmal mitochondrial oxidation and antioxidantion.

Although resveratrol (RES) is thought to be a key regulator of insulin sensitivity in rodents, the exact mechanism underlying this effect remains unclear. Therefore, we sought to investigate how RES affects skeletal muscle oxidative and antioxidant levels of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial populations in high-fat diet (HFD)-induced insulin resistance (IR) rats. Systemic and skeletal muscle insulin sensitivity together with expressions of several genes related to mitochondrial biogenesis and skeletal muscle SIRT1, SIRT3 protein levels were studied in rats fed a normal diet, a HFD, and a HFD with intervention of RES for 8 weeks. Oxidative stress levels and antioxidant enzyme activities were assessed in SS and IMF mitochondria. HFD fed rats exhibited obvious systemic and skeletal muscle IR as well as decreased SIRT1 and SIRT3 expressions, mitochondrial DNA (mtDNA), and mitochondrial biogenesis (p < 0.05). Both SS and IMF mitochondria demonstrated elevated reactive oxygen species (ROS) and malondialdehyde (MDA) levels. In addition, SS mitochondrial antioxidant enzyme activities were significantly lower, while IMF mitochondrial antioxidant enzyme activities were higher (p < 0.05). By contrast, RES treatment protected rats against diet induced IR, increased SIRT1 and SIRT3 expressions, mtDNA, and mitochondrial biogenesis (p < 0.05). Moreover, the activities of SS and IMF mitochondrial antioxidant enzymes were increased, which reverted the increased SS mitochondrial oxidative stress levels (p < 0.05). This study suggests that RES ameliorates insulin sensitivity consistent with improved SIRT3 expressions and rebalance between SS mitochondrial oxidative stress and antioxidant competence in HFD rats.

Characterization of muscle morphology and satellite cells, and expression of muscle-related genes in skeletal muscle of juvenile and adult Megalobrama amblycephala

Hyperplasia and hypertrophy are two distinct processes of skeletal muscle growth regulated by four myogenic regulatory factors (MRFs, contains MyoD, Myf5, Mrf4 and myogenin (MyoG)) and myostatin (MSTN). In this study, characterization of muscle morphology and satellite cells in juvenile (1-year-old) and adult (2-year-old) Megalobrama amblycephala was described. Compared with 1-year-old M. amblycephala, the diameter scope of epaxial, horizontal septum and hypaxial muscle fibers including red and white muscle in 2-year-old fish exhibited broader with dramatic reduction in frequency distribution of <20μm diameter, nevertheless observable increase in frequency distribution of >50μm diameter. Intermyofibrillar (IM) nuclei were also found except numerous subsarcolemmal (SS) nuclei in 2-year-old fish, whereas only SS nuclei were observed in 1-year-old fish. Immunofluorescence results showed that more satellite cells existed in red muscle than white muscle in 1-year-old fish, rather than 2-year-old fish. Moreover, we observed predominant increase in the mRNA levels of MyoD, Myf5, Mrf4, and MSTN during muscle development of fish in 2-year-old fish except MyoG.

Substrate-specific changes in mitochondrial respiration in skeletal and cardiac muscle of hibernating thirteen-lined ground squirrels

During torpor, the metabolic rate (MR) of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is considerably lower relative to euthermia, resulting in part from temperature-independent mitochondrial metabolic suppression in liver and skeletal muscle, which together account for ~40% of basal MR. Although heart accounts for very little (<0.5%) of basal MR, in the present study, we showed that respiration rates were decreased up to 60% during torpor in both subsarcolemmal (SS) and intermyofibrillar (IM) mitochondria from cardiac muscle. We further demonstrated pronounced seasonal (summer vs. winter [i.e., interbout] euthermia) changes in respiration rates in both mitochondrial subpopulations in this tissue, consistent with a shift in fuel use away from carbohydrates and proteins and towards fatty acids and ketones. By contrast, these seasonal changes in respiration rates were not observed in either SS or IM mitochondria isolated from hind limb skeletal muscle. Both populations of skeletal muscle mitochondria, however, did exhibit metabolic suppression during torpor, and this suppression was 2- to 3-fold greater in IM mitochondria, which provide ATP for Ca(2+)- and myosin ATPases, the activities of which are likely quite low in skeletal muscle during torpor because animals are immobile. Finally, these changes in mitochondrial respiration rates were still evident when standardized to citrate synthase activity rather than to total mitochondrial protein.