Intermyofibrillar Mitochondria Research Articles

A single bout of resistance exercise triggersmitophagy, potentially involving the ejection of mitochondria in human skeletal muscle.

The present study aimed to investigate the effects of a single bout of resistance exercise on mitophagy in human skeletal muscle (SkM). Eight healthy men were recruited to complete an acute bout of one-leg resistance exercise. SkM biopsies were obtained one hour after exercise in the resting leg (Rest-leg) and the contracting leg (Ex-leg). Mitophagy was assessed using protein-related abundance, transmission electron microscopy (TEM), and fluorescence microscopy. Our results show that acute resistance exercise increased pro-fission protein phosphorylation (DRP1Ser616) and decreased mitophagy markers such as PARKIN and BNIP3L/NIX protein abundance in the Ex-leg. Additionally, mitochondrial complex IV decreased in the Ex-leg when compared to the Rest-leg. In the Ex-leg, TEM and immunofluorescence images showed mitochondrial cristae abnormalities, a mitochondrial fission phenotype, and increased mitophagosome-like structures in both subsarcolemmal and intermyofibrillar mitochondria. We also observed increased mitophagosome-like structures on the subsarcolemmal cleft and mitochondria in the extracellular space of SkM in the Ex-leg. We stimulated human primary myotubes with CCCP, which mimics mitophagy induction in the Ex-leg, and found that BNIP3L/NIX protein abundance decreased independently of lysosomal degradation. Finally, in another human cohort, we found a negative association between BNIP3L/NIX protein abundance with both mitophagosome-like structures and mitochondrial cristae density in the SkM. The findings suggest that a single bout of resistance exercise can initiate mitophagy, potentially involving mitochondrial ejection, in human skeletal muscle. BNIP3L/NIX is proposed as a sensitive marker for assessing mitophagy flux in SkM.

Functional and Morphological Differences of Muscle Mitochondria in Chronic Fatigue Syndrome and Post-COVID Syndrome.

Patients suffering from chronic fatigue syndrome (CFS) or post-COVID syndrome (PCS) exhibit a reduced physiological performance capability. Impaired mitochondrial function and morphology may play a pivotal role. Thus, we aimed to measure the muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity and assess mitochondrial morphology in CFS and PCS patients in comparison to healthy controls (HCs). Mitochondrial OXPHOS capacity was measured in permeabilized muscle fibers using high-resolution respirometry. Mitochondrial morphology (subsarcolemmal/intermyofibrillar mitochondrial form/cristae/diameter/circumference/area) and content (number and proportion/cell) were assessed via electron microscopy. Analyses included differences in OXPHOS between HC, CFS, and PCS, whereas comparisons in morphology/content were made for CFS vs. PCS. OXPHOS capacity of complex I, which was reduced in PCS compared to HC. While the subsarcolemmal area, volume/cell, diameter, and perimeter were higher in PCS vs. CFS, no difference was observed for these variables in intermyofibrillar mitochondria. Both the intermyofibrillar and subsarcolemmal cristae integrity was higher in PCS compared to CFS. Both CFS and PCS exhibit increased fatigue and impaired mitochondrial function, but the progressed pathological morphological changes in CFS suggest structural changes due to prolonged inactivity or unknown molecular causes. Instead, the significantly lower complex I activity in PCS suggests probably direct virus-induced alterations.

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.

Adaptive increases in respiratory capacity and O2 affinity of subsarcolemmal mitochondria from skeletal muscle of high-altitude deer mice.

Aerobic energy demands have led to the evolution of complex mitochondrial reticula in highly oxidative muscles, but the extent to which metabolic challenges can be met with adaptive changes in physiology of specific mitochondrial fractions remains unresolved. We examined mitochondrial mechanisms supporting adaptive increases in aerobic performance in deer mice (Peromyscus maniculatus) adapted to the hypoxic environment at high altitude. High-altitude and low-altitude mice were born and raised in captivity, and exposed as adults to normoxia or hypobaric hypoxia (12kPa O2 for 6-8weeks). Subsarcolemmal and intermyofibrillar mitochondria were isolated from the gastrocnemius, and a comprehensive substrate titration protocol was used to examine mitochondrial physiology and O2 kinetics by high-resolution respirometry and fluorometry. High-altitude mice had greater yield, respiratory capacity for oxidative phosphorylation, and O2 affinity (lower P50 ) of subsarcolemmal mitochondria compared to low-altitude mice across environments, but there were no species difference in these traits in intermyofibrillar mitochondria. High-altitude mice also had greater capacities of complex II relative to complexes I + II and higher succinate dehydrogenase activities in both mitochondrial fractions. Exposure to chronic hypoxia reduced reactive oxygen species (ROS) emission in high-altitude mice but not in low-altitude mice. Our findings suggest that functional changes in subsarcolemmal mitochondria contribute to improving aerobic performance in hypoxia in high-altitude deer mice. Therefore, physiological variation in specific mitochondrial fractions can help overcome the metabolic challenges of life at high altitude.

CYB5R3 overexpression preserves skeletal muscle mitochondria and autophagic signaling in aged transgenic mice

Cytochrome b5 reductase 3 (CYB5R3) overexpression activates respiratory metabolism and exerts prolongevity effects in transgenic mice, mimicking some of the salutary effects of calorie restriction. The aim of our study was to understand how CYB5R3 overexpression targets key pathways that modulate the rate of aging in skeletal muscle, a postmitotic tissue with a greater contribution to resting energy expenditure. Mitochondrial function, autophagy and mitophagy markers were evaluated in mouse hind limb skeletal muscles from young-adult (7 months old) and old (24 months old) males of wild-type and CYB5R3-overexpressing genotypes. Ultrastructure of subsarcolemmal and intermyofibrillar mitochondria was studied by electron microscopy in red gastrocnemius. CYB5R3, which was efficiently overexpressed and targeted to skeletal muscle mitochondria regardless of age, increased the abundance of complexes I, II, and IV in old mice and prevented the age-related decrease of complexes I, III, IV, and V and the mitofusin MFN-2. ATP was significantly decreased by aging, which was prevented by CYB5R3 overexpression. Coenzyme Q and the mitochondrial biogenesis markers TFAM and NRF-1 were also significantly diminished by aging, but CYB5R3 overexpression did not protect against these declines. Both aging and CYB5R3 overexpression upregulated SIRT3 and the mitochondrial fission markers FIS1 and DRP-1, although with different outcomes on mitochondrial ultrastructure: old wild-type mice exhibited mitochondrial fragmentation whereas CYB5R3 overexpression increased mitochondrial size in old transgenic mice concomitant with an improvement of autophagic recycling. Interventions aimed at stimulating CYB5R3 could represent a valuable strategy to counteract the deleterious effects of aging in skeletal muscle.Graphical abstract

Intrinsic Mitochondrial Function Impacts the Outcomes of Metformin Treatment on Skeletal Muscle Mitochondrial Morphology in Aged Rat

Aging is the main risk factor for many costly, irreversible, and comorbid chronic diseases. The anti‐diabetic drug Metformin is being considered as an anti‐aging treatment although little is known about its effects in healthy individuals. Previous data from our lab suggest that metformin is beneficial to individuals that are relatively insulin resistant and may be detrimental to those who are insulin sensitive. We aimed to understand how intrinsic function of mitochondria affects metformin treatment outcomes in skeletal muscle. We hypothesized that the effects of metformin on skeletal muscle mitochondrial morphology and remodeling will differ based on intrinsic mitochondrial function. To test our hypothesis, we used the High‐Capacity Runner/Low‐Capacity Runner (HCR/LCR) rat model system which comprises two selectively bred lines of genetically heterogeneous rats that diverge for exercise capacity, cardiovascular risk factors, lifespan, and healthspan. In drinking water, we treated six HCR and four LCR male rats (age 18 months) with a dose of 100 mg/kg/day metformin for a week, followed by three weeks of 200 mg/kg/day. We included HCR and LCR control groups (n=5) for a total of four groups. A week prior to sacrifice, we initiated deuterium oxide (D2O) labeling to assess protein turnover and electroporated DNA constructs that encode for mitochondrial matrix‐targeted YFP and tdTomato‐tagged TOM20, a protein located on the mitochondrial outer membrane. We analyzed protein turnover in hindlimb muscles using GC‐MS, and prepared histological sections of the Tibialis Anterior (TA) muscle for further analysis of mitochondrial morphology. Normalized to tibial length, the mass of the gastrocnemius (‐16.6%) and plantaris (‐20.8%) muscles were lower in metformin treated HCR rats compared to untreated, while the normalized mass of the TA muscle was lower in both HCR (‐15%) and LCR (‐20.1%) metformin treated rats compared to control. There were no significant differences in mean fiber cross sectional area (CSA) of the TA muscle. We detected no significant differences in bulk protein synthesis of both the myofibrillar and mitochondrial fractions of the TA, gastrocnemius, and soleus muscles. The number of mitochondrial branches observed in a cross section of TA fibers from metformin treated LCR rats were 30.5% lower compared to control LCR rats. However, mean CSA of each mitochondrial branch in metformin treated LCR rats increased by 82.8% compared to control LCR rats, which, together with the decrease in branch number, resulted in maintenance of the cumulative mitochondrial CSA in each fiber. Analysis based on mitochondrial sub‐populations showed that the subsarcolemmal mitochondrial population was more impacted than intermyofibrillar mitochondria. There were no significant differences in mitochondrial morphology in control vs metformin treated HCR rats. Our data supports our hypothesis that metformin has differential effects in skeletal muscle based on intrinsic mitochondrial function prior to treatment. Additional studies are needed to determine what impact these disparities have on the aging process.

PERM1 interacts with the MICOS-MIB complex to connect the mitochondria and sarcolemma via ankyrin B

Skeletal muscle subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria subpopulations have distinct metabolic activity and sensitivity, though the mechanisms that localize SSM to peripheral areas of muscle fibers are poorly understood. A protein interaction study and complexome profiling identifies PERM1 interacts with the MICOS-MIB complex. Ablation of Perm1 in mice reduces muscle force, decreases mitochondrial membrane potential and complex I activity, and reduces the numbers of SSM in skeletal muscle. We demonstrate PERM1 interacts with the intracellular adaptor protein ankyrin B (ANKB) that connects the cytoskeleton to the plasma membrane. Moreover, we identify a C-terminal transmembrane helix that anchors PERM1 into the outer mitochondrial membrane. We conclude PERM1 functions in the MICOS-MIB complex and acts as an adapter to connect the mitochondria with the sarcolemma via ANKB.

The influence of microwave electromagnetic radiation on rat heart morphogenesis during thyroidectomy

It is known that electromagnetic radiation affects the functional systems of living beings, in particular, the endocrine and cardiovascular systems and directly the heart cells and their functioning. In this case, the influence of electromagnetic radiation significantly affects the properties of the cardiovascular system and changes physiological processes in it. Moreover, sensitivity to signals is higher for biosystems with a higher level of organization. The method of simulating hypothyroidism for rats makes it possible to develop an experimental model for analyzing morphogenetic changes in the rat heart. Under conditions of hypothyroidism modeling, destructive changes in muscle fibers are observed in the myocardium after thyroidectomy, but after microwave irradiation with an exposure of 45 minutes, a regenerative-compensatory reconstruction of the structural components of the myocardium of the heart wall occurs. After thyroidectomy and microwave irradiation with an exposure of 120 minutes, an increase in degenerative and destructive processes in the heart myocardium was observed. For the first time, a comparative ultramicroscopic study using electron microscopy was carried out. It showed that after electromagnetic irradiation with an exposure of 45 minutes, the general structure of the mitochondrial apparatus does not change. Irradiation with an exposure of 120 minutes causes the destruction of subsarcolemal and paranuclear organelles, edema and degradation of intermyofibrillar mitochondria. After exposure to electromagnetic radiation with the exposure of 45 minutes in a hypothyroid state, a compensatory restructuring of the energy apparatus of the contractile cardiomyocytes of the left ventricle occurs due to the formation of mitochondria and an increase in their contact interaction. After exposure to radiation for 120 minutes, the development of destructive-degenerative processes in the mitochondrial apparatus of left ventricular cardiomyocytes, deepening of damage to intermitochondrial contacts was observed. The most significant increase in the relative volume of the endothelium in the areas of the rat heart myocardium was observed for the case of microwave irradiation only (without experimental hypothyroidism) with exposure duration of 45 minutes. Immunohistochemical study both for the case of microwave irradiation only and for the case of microwave irradiation under conditions of experimental hypothyroidism allows us to conclude that morphogenetic transformations are highly active in the myocardium of the heart when the duration of exposure is 45 minutes. This study will make it possible to develop recommendations for persons with thyroid diseases on the duration of daily exposure of staying in electromagnetic field emitted by devices with characteristics similar to the case under study.

Non-uniform Distribution of Inner Mitochondrial Membrane Calcium Transport Mechanisms in the Cardiac Muscle

Energy homeostasis in the cardiac muscle is critical to assure uninterrupted circulation. The primary energy source of the adult cardiomyocyte is the mitochondrial oxidative ATP production. Metabolic feedback loops and the ATP buffering creatine kinase-phosphocreatine system serve to replenish the ATP levels consumed at each heartbeat; however, they may not sufficiently support the abrupt performance increase needed during fight-or-flight or similar stress responses. To that end, a feed-forward loop has evolved that stimulates the electron donor generators for the respiratory chain, the calcium sensitive mitochondrial matrix dehydrogenases, via mitochondrial calcium uptake during the excitation-contraction coupling. This feed-forward loop, also referred as ‘excitation-bioenergetics coupling’, involves local calcium transfer at the membrane contacts formed between the sarcoplasmic reticulum calcium release units and intermyofibrillar mitochondria. These contacts are secured by protein tethers and our recent studies suggest that they are also areas with strong distribution anomalies of the primary mitochondrial calcium uptake and extrusion mechanisms of the inner mitochondrial membrane. Namely, while the contact area is a hotspot for the mitochondrial calcium uniporter complex (MCUC), the Na+/Ca2+ exchanger NCLX is largely excluded from there, even though it is abundant outside the contact area. In this talk, I will present our ideas why such a biased distribution is a favorable arrangement for effective mitochondrial matrix calcium signal generation. Furthermore, potential molecular mechanisms that are responsible for the non-uniform distribution of the MCUC components will be discussed.

Ultrastructure of mitochondrial apparatus of left ventricle cardiomyocyte of rats after different exposures of electromagnetic radiation under hypothyroidism.

Backgroud. The dynamics of morphogenetic rearrangements of myocardial mitochondriom under the influence of hypothyroidism and electromagnetic radiation remains insufficiently studied. These circumstances determine the relevance and need to study the mitochondrial reactions of cardiomyocytes in these conditions. Objective. Determination of the reactions of the contractile cardiomyocytes mitochondrial apparatus of the left ventricle of rat heart under hypothyroidism on the effect of various exposures to electromagnetic radiation. Methods. We studied samples of the intramural zone of the free wall of left ventricle in sexually mature rats. Experimental animals were divided into 6 groups: 1) control (intact rats) 2) rats with hypothyroidism; 3) rats after general electromagnetic irradiation with an exposure of 45 minutes 4) rats after irradiation with an exposure of 120 minutes 5) rats with hypothyroidism after irradiation with an exposure of 45 minutes 6) rats with hypothyroidism after irradiation with an exposure of 120 minutes. The hypothyroid state was modeled using thyroidectomy. The effect of electromagnetic radiation was carried out daily for 10 days on the whole body. Ultrastructural analysis of the mitochondrial apparatus was performed using transmission electron microscopy. Results. Experimental hypothyroidism in rats was accompanied by an increase in mitochondrial heteromorphism of contractile cardiomyocytes of the left ventricle, cristolysis of myofibrillar mitochondria, and the appearance of giant organelles with severe swelling of the matrix. After irradiation with an exposure of 45 minutes, the overall structure of the mitochondrial apparatus is unchanged. An exposure of 120 minutes caused the destruction of subsarcolemmal and paranuclear organelles, edema and degradation of intermyofibrillar mitochondria. After exposure for 45 minutes under conditions of a hypothyroid state, mitochondria in paranuclear localization were represented by enlarged organelles with a vacuolar type of damage, without signs of destruction of the external membranes, moderate cristolysis, with an electronically clear matrix. Exposure for 120 minutes in animals with hypothyroidism led to edema and destruction of most mitochondria, destruction of cristae, effects of excessive tension on preserved organelles, damage to the structure of intermitochondrial contacts, and inhibition of reproduction of mitochondria. Conclusion. After exposure to electromagnetic radiation for 45 minutes under hypothyroid conditions, there is a compensatory restructuring of the energy apparatus of contractile cardiomyocytes of the left ventricle due to neoplasm of mitochondria and an increase in their contact interaction. After the action of irradiation for 120 minutes, the development of destructive-degenerative processes in the mitochondrial apparatus of left ventricular cardiomyocytes is observed, and damage to intermitochondrial contacts is extended.

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.

Assessing Mitochondrial Bioenergetics in Isolated Mitochondria from Mouse Heart Tissues Using Oroboros 2k-Oxygraph.

In this chapter, we describe detailed protocols for measuring high-resolution respirometry on mitochondria extracted from adult whole mouse heart using the Oroboros 2k-Oxygraph system. The method provides detailed procedures for the preparation of mitochondria and measurement of high-resolution respirometry in response to various respiration inhibitions. The method described in this chapter could discern the different respiration rate on mitochondria extracted from two spatially distinct mitochondrial subpopulations, subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IFM). These approaches can easily be translated to other cells and tissues.

Hypertrophic cardiomyopathy. Cardiomyocyte ultrastructure, the specific or stereotypic signs

To study cardiomyocyte (CMC) ultrastructural changes in the interventricular septum (IVS) of patients with HCM and evaluate their specificity for this pathology. IVS myocardial samples taken from 44 HCM patients aged 18-59 years at IVS myoectomy underwent an electron microscopic study. The diameter of CMCs and their nuclei was measured in semithin sections. A morphometric examination of the IVS myocardium in HCM patients revealed moderate hypertrophy of CMCs and their nuclei, the diameters of which averaged 23.7±4.4 and 5.2±0.9 μm, respectively. The IVS CMCs were characterized by the ultrastructural signs of hypertrophy: the larger size and number of structures ensuring contractile and synthetic functions; the myocytes contained higher amounts of myofibrils, intermyofibrillar mitochondria, granular endoplasmic reticulum cisterns, and free ribosomes. On the contrary, some CMCs had fewer myofibrils in the perinuclear region, which is an adaptive change under hemodynamic overload conditions. In addition, a number of myocytes displayed signs of dystrophic changes: the appearance of lipofuscin granules, myelin figures, phagosomes, lipid droplets, and vacuoles, which can fill all free sarcoplasmic zones. Ultrastructural changes characteristic of hypertrophy were found in IVS CMCs in HCM patients. In addition, there was partial myofibrillar loss and dystrophic changes in a number of myocytes, which are stereotypic compensatory-adaptive changes under hemodynamic overload conditions. All the above-mentioned changes in the CMC ultrastructure are characteristic of myocardial hypertrophy, but not specific for hypertrophic cardiomyopathy.

Managing the power grid: how myoglobin can regulate PO2 and energy distribution in skeletal muscle.

Managing the power grid: how myoglobin can regulate PO2 and energy distribution in skeletal muscle.

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).

Skeletal muscle atrophy and dysfunction in breast cancer patients: role for chemotherapy-derived oxidant stress.

How breast cancer and its treatments affect skeletal muscle is not well defined. To address this question, we assessed skeletal muscle structure and protein expression in 13 women who were diagnosed with breast cancer and receiving adjuvant chemotherapy following tumor resection and 12 nondiseased controls. Breast cancer patients showed reduced single-muscle fiber cross-sectional area and fractional content of subsarcolemmal and intermyofibrillar mitochondria. Drugs commonly used in breast cancer patients (doxorubicin and paclitaxel) caused reductions in myosin expression, mitochondrial loss, and increased reactive oxygen species (ROS) production in C2C12 murine myotube cell cultures, supporting a role for chemotherapeutics in the atrophic and mitochondrial phenotypes. Additionally, concurrent treatment of myotubes with the mitochondrial-targeted antioxidant MitoQ prevented chemotherapy-induced myosin depletion, mitochondrial loss, and ROS production. In patients, reduced mitochondrial content and size and increased expression and oxidation of peroxiredoxin 3, a mitochondrial peroxidase, were associated with reduced muscle fiber cross-sectional area. Our results suggest that chemotherapeutics may adversely affect skeletal muscle in patients and that these effects may be driven through effects of these drugs on mitochondrial content and/or ROS production.

Mitochondrial Calcium Increase Induced by RyR1 and IP3R Channel Activation After Membrane Depolarization Regulates Skeletal Muscle Metabolism.

Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP3-receptor (IP3R) calcium channels are necessary for the mitochondrial Ca2+ increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling).Methods: Mitochondria matrix and cytoplasmic Ca2+ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca2+ sensor (CEPIA3mt) or a cytoplasmic Ca2+ sensor (RCaMP). The role of intracellular Ca2+ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP3R and Dantrolene for RyR1) and a genetic approach (shIP3R1-RFP). O2 consumption was detected using Seahorse Extracellular Flux Analyzer.Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca2+ levels. Mitochondrial Ca2+ uptake required functional inositol IP3R and RyR1 channels. Inhibition of either channel decreased basal O2 consumption rate but only RyR1 inhibition decreased ATP-linked O2 consumption. Cell membrane depolarization-induced Ca2+ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca2+ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca2+-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber.Conclusion: Ca2+-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an “excitation-metabolism” coupling process in skeletal muscle fibers.

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.

MitoQ improves mitochondrial dysfunction in heart failure induced by pressure overload

Heart failure remains a major public-health problem with an increase in the number of patients worsening from this disease. Despite current medical therapy, the condition still has a poor prognosis. Heart failure is complex but mitochondrial dysfunction seems to be an important target to improve cardiac function directly.Our goal was to analyze the effects of MitoQ (100 µM in drinking water) on the development and progression of heart failure induced by pressure overload after 14 weeks. The main findings are that pressure overload-induced heart failure in rats decreased cardiac function in vivo that was not altered by MitoQ. However, we observed a reduction in right ventricular hypertrophy and lung congestion in heart failure animals treated with MitoQ. Heart failure also decreased total mitochondrial protein content, mitochondrial membrane potential in the intermyofibrillar mitochondria. MitoQ restored membrane potential in IFM but did not restore mitochondrial protein content. These alterations are associated with the impairment of basal and stimulated mitochondrial respiration in IFM and SSM induced by heart failure. Moreover, MitoQ restored mitochondrial respiration in heart failure induced by pressure overload. We also detected higher levels of hydrogen peroxide production in heart failure and MitoQ restored the increase in ROS production. MitoQ was also able to improve mitochondrial calcium retention capacity, mainly in the SSM whereas in the IFM we observed a small alteration. In summary, MitoQ improves mitochondrial dysfunction in heart failure induced by pressure overload, by decreasing hydrogen peroxide formation, improving mitochondrial respiration and improving mPTP opening.