Mitochondrial creatine sensitivity is lost in the D2.mdx model of Duchenne muscular dystrophy and rescued by the mitochondrial-enhancing compound Olesoxime.

Duchenne muscular dystrophy (DMD) is associated with distinct mitochondrial stress responses. Here, we aimed to determine whether the prospective mitochondrial-enhancing compound Olesoxime, prevents early-stage mitochondrial stress in limb and respiratory muscle from D2.mdx mice using a proof-of-concept short-term regimen spanning 10-28 days of age. As mitochondrial-cytoplasmic energy transfer occurs via ATP- or phosphocreatine-dependent phosphate shuttling, we assessed bioenergetics with or without creatine in vitro. We observed that disruptions in Complex I-supported respiration and mH2O2 emission in D2.mdx quadriceps and diaphragm were amplified by creatine demonstrating mitochondrial creatine insensitivity manifests ubiquitously and early in this model. Olesoxime selectively rescued or maintained creatine sensitivity in both muscles, independent of the abundance of respiration-related mitochondrial proteins or mitochondrial creatine kinase cysteine oxidation in quadriceps. Mitochondrial calcium retention capacity and glutathione were altered in a muscle-specific manner in D2.mdx but were generally unchanged by Olesoxime. Treatment reduced serum creatine kinase (muscle damage) and preserved cage hang-time, microCT-based volumes of lean compartments including whole body, hindlimb and bone, recovery of diaphragm force after fatigue, and cross-sectional area of diaphragm type IIX fiber, but reduced type I fibers in quadriceps. Grip strength, voluntary wheel-running and fibrosis were unaltered by Olesoxime. In summary, locomotor and respiratory muscle mitochondrial creatine sensitivities are lost during early stages in D2.mdx mice but are preserved by short-term treatment with Olesoxime in association with specific indices of muscle quality suggesting early myopathy in this model is at least partially attributed to mitochondrial stress.

Impairments in left ventricular mitochondrial bioenergetics precede overt cardiac dysfunction and remodelling in Duchenne muscular dystrophy.

KEY POINTS: Ninety-eight per cent of patients with Duchenne muscular dystrophy (DMD) develop cardiomyopathy, with 40% developing heart failure. While increased propensity for mitochondrial induction of cell death has been observed in left ventricle, it remains unknown whether this is linked to impaired mitochondrial respiratory control and elevated H2 O2 emission prior to the onset of cardiomyopathy. Classic mouse models of DMD demonstrate hyper-regeneration in skeletal muscle which may mask mitochondrial abnormalities. Using a model with less regenerative capacity that is more akin to DMD patients, we observed elevated left ventricular mitochondrial H2 O2 and impaired oxidative phosphorylation in the absence of cardiac remodelling or overt cardiac dysfunction at 4 weeks. These impairments were associated with dysfunctions at complex I, governance by ADP and creatine-dependent phosphate shuttling, which results in a less efficient response to energy demands. Mitochondria may be a therapeutic target for the treatment of cardiomyopathy in DMD. ABSTRACT: In Duchenne muscular dystrophy (DMD), mitochondrial dysfunction is predicted as a response to numerous cellular stressors, yet the contribution of mitochondria to the onset of cardiomyopathy remains unknown. To resolve this uncertainty, we designed in vitro assessments of mitochondrial bioenergetics to model mitochondrial control parameters that influence cardiac function. Both left ventricular mitochondrial responsiveness to the central bioenergetic controller ADP and the ability of creatine to facilitate mitochondrial-cytoplasmic phosphate shuttling were assessed. These measurements were performed in D2.B10-DMDmdx /2J mice - a model that demonstrates skeletal muscle atrophy and weakness due to limited regenerative capacities and cardiomyopathy more akin to people with DMD than classic models. At 4 weeks of age, there was no evidence of cardiac remodelling or cardiac dysfunction despite impairments in ADP-stimulated respiration and ADP attenuation of H2 O2 emission. These impairments were seen at both submaximal and maximal ADP concentrations despite no reductions in mitochondrial content markers. The ability of creatine to enhance ADP's control of mitochondrial bioenergetics was also impaired, suggesting an impairment in mitochondrial creatine kinase-dependent phosphate shuttling. Susceptibly to permeability transition pore opening and the subsequent activation of cell death pathways remained unchanged. Mitochondrial H2 O2 emission was elevated despite no change in markers of irreversible oxidative damage, suggesting alternative redox signalling mechanisms should be explored. These findings demonstrate that selective mitochondrial dysfunction precedes the onset of overt cardiomyopathy in D2.mdx mice, suggesting that improving mitochondrial bioenergetics by restoring ADP, creatine-dependent phosphate shuttling and complex I should be considered for treating DMD patients.

The impact of acute and chronic exercise on Nrf2 expression in relation to markers of mitochondrial biogenesis in human skeletal muscle.

PURPOSE: To examine the relationship between changes in nuclear factor erythroid 2-related factor 2 (Nrf2) expression and markers of mitochondrial biogenesis in acutely and chronically exercised human skeletal muscle. METHODS: The impact of acute submaximal endurance (END) and supramaximal interval (Tabata) cycling on the upregulation of Nrf2 (and its downstream targets), nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (TFAM) mRNA expression was examined in healthy young males (n = 10). The relationship between changes in citrate synthase (CS) maximal activity and the protein content of Nrf2, heme oxygenase 1 (HO-1), NRF-1, and TFAM was also investigated following 4 weeks of Tabata in a separate group of males (n = 21). RESULTS: Nrf2, NRF-1, and HO-1 mRNA expression increased after acute exercise (p < 0.05), whereas the increase in superoxide dismutase 2 (SOD2) mRNA expression approached significance (p = 0.08). Four weeks of Tabata increased CS activity and Nrf2, NRF-1, and TFAM protein content (p < 0.05), but decreased HO-1 protein content (p < 0.05). Training-induced changes in Nrf2 protein were strongly correlated with NRF-1 (r = 0.63, p < 0.01). When comparing protein content changes between individuals with the largest (HI: + 23%) and smallest (LO: - 1%) observed changes in CS activity (n = 8 each), increases in Nrf2 and TFAM protein content were apparent in the HI group only (p < 0.02) with medium-to-large effect sizes for between-group differences in changes in Nrf2 (ηp2=0.15) and TFAM (ηp2 = 0.12) protein content. CONCLUSION: Altogether, our findings support a potential role for Nrf2 in exercise-induced mitochondrial biogenesis in human skeletal muscle.

Synergistic activation of mitochondrial metabolism and the glutathione redox couple protects HepG2 hepatocarcinoma cells from palmitoylcarnitine-induced stress.

Fatty acid stress can have divergent effects in various cancers. We explored how metabolic and redox flexibility in HepG2 hepatocarcinoma cells mediates protection from palmitoylcarnitine. HepG2 cells, along with HCT 116 and HT29 colorectal cancer cells were incubated with 100 µM palmitoylcarnitine for up to 48 h. Mitochondrial H2O2 emission, glutathione, and cell survival were assessed in HT29 and HepG2 cells. 100 µM palmitoylcarnitine promoted early growth in HepG2 cells by ~8% after 48 h versus decreased cell survival observed in HT29 and HCT 116 cells. Palmitoylcarnitine increased mitochondrial respiration at physiological and maximal concentrations of ADP, while lowering cellular lactate content in HepG2 cells, suggesting a switch to mitochondrial metabolism. HepG2 cell growth was associated with an early increase in H2O2 emission by 10 min, followed by a decrease in H2O2 at 24 h that corresponded with increased glutathione content, suggesting a redox-based compensatory mechanism. In contrast, abrogation of HT29 cell proliferation was related to decreased mitochondrial respiration (likely due to cell death) and decreased glutathione. Concurrent glutathione depletion with BSO prevented palmitoylcarnitine-induced growth in HepG2 cells, indicating that glutathione was critical for promoting growth following palmitoylcarnitine. Inhibiting UCP2 with genipin sensitized HepG2 cells to palmitoylcarnitine, suggesting that activation of UCP2 may be a 2nd redox-based mechanism conferring protection. These findings suggest that HepG2 cells possess inherent metabolic and redox flexibility relative to HT29 cells that confers protection from palmitoylcarnitine-induced stress via adaptive increases in mitochondrial respiratory control, glutathione buffering, and induction of UCP2.

The fatty acid derivative palmitoylcarnitine abrogates colorectal cancer cell survival by depleting glutathione.

Previous evidence suggests that palmitoylcarnitine incubations trigger mitochondrial-mediated apoptosis in HT29 colorectal adenocarcinoma cells, yet nontransformed cells appear insensitive. The mechanism by which palmitoylcarnitine induces cancer cell death is unclear. The purpose of this investigation was to examine the relationship between mitochondrial kinetics and glutathione buffering in determining the effect of palmitoylcarnitine on cell survival. HT29 and HCT 116 colorectal adenocarcinoma cells, CCD 841 nontransformed colon cells, and MCF7 breast adenocarcinoma cells were exposed to 0 µM, 50 µM, and 100 µM palmitoylcarnitine for 24-48 h. HCT 116 and HT29 cells showed decreased cell survival following palmitoylcarnitine compared with CCD 841 cells. Palmitoylcarnitine stimulated H2O2 emission in HT29 and CCD 841 cells but increased it to a greater level in HT29 cells due largely to a higher basal H2O2 emission. This greater H2O2 emission was associated with lower glutathione buffering capacity and caspase-3 activation in HT29 cells. The glutathione-depleting agent buthionine sulfoximine sensitized CCD 841 cells and further sensitized HT29 cells to palmitoylcarnitine-induced decreases in cell survival. MCF7 cells did not produce H2O2 when exposed to palmitoylcarnitine and were able to maintain glutathione levels. Furthermore, HT29 cells demonstrated the lowest mitochondrial oxidative kinetics vs. CCD 841 and MCF7 cells. The results demonstrate that colorectal cancer is sensitive to palmitoylcarnitine due in part to an inability to prevent oxidative stress through glutathione-redox coupling, thereby rendering the cells sensitive to elevations in H2O2. These findings suggest that the relationship between inherent metabolic capacities and redox regulation is altered early in response to palmitoylcarnitine.

Phosphodiesterase type 3A (PDE3A), but not type 3B (PDE3B), contributes to the adverse cardiac remodeling induced by pressure overload.

Phosphodiesterase type 3 (PDE3) inhibitors block the cAMP hydrolyzing activity of both PDE3 isoforms, PDE3A and PDE3B, which have distinct roles in the heart. Although PDE3 inhibitors improve cardiac function in heart disease patients, they also increase mortality. Nevertheless, PDE3 inhibitors can provide benefit to non-ischemic heart disease patients and are used extensively to treat heart failure in dogs. Since the isoform-dependence of the complex cardiac actions of PDE3 inhibition in diseased hearts remains unknown, we assessed the effects of PDE3 inhibitors as well as gene ablation of PDE3A or PDEB in mice following the induction of non-ischemic heart disease by pressure-overload with transverse-aortic constriction (TAC). As expected, after 6 weeks of TAC, mice exhibited left ventricular contractile dysfunction, dilation, hypertrophy and interstitial fibrosis, in association with increased macrophage numbers, activation of p38 MAPK and elevated PDE3 activity. Chronic PDE3 inhibition with milrinone (MIL), at doses that did not affect either cardiac contractility or arterial blood pressure, profoundly attenuated the adverse ventricular remodeling, reduced macrophage number and diminished p38-MAPK activation induced by TAC. Surprisingly, whole-body ablation of PDE3A, but not PDE3B, provided similar protection against TAC-induced adverse ventricular remodeling, and the addition of MIL to mice lacking PDE3A provided no further protection. Our results support the conclusion that PDE3A plays an important role in adverse cardiac remodeling induced by chronic pressure overload in mice, although the underlying biochemical mechanisms remain to be fully elucidated. The implications of this conclusion on the clinical use of PDE3 inhibitors are discussed.

Early myopathy in Duchenne muscular dystrophy is associated with elevated mitochondrial H2 O2 emission during impaired oxidative phosphorylation.

BACKGROUND: Muscle wasting and weakness in Duchenne muscular dystrophy (DMD) causes severe locomotor limitations and early death due in part to respiratory muscle failure. Given that current clinical practice focuses on treating secondary complications in this genetic disease, there is a clear need to identify additional contributions in the aetiology of this myopathy for knowledge-guided therapy development. Here, we address the unresolved question of whether the complex impairments observed in DMD are linked to elevated mitochondrial H2 O2 emission in conjunction with impaired oxidative phosphorylation. This study performed a systematic evaluation of the nature and degree of mitochondrial-derived H2 O2 emission and mitochondrial oxidative dysfunction in a mouse model of DMD by designing in vitro bioenergetic assessments that attempt to mimic in vivo conditions known to be critical for the regulation of mitochondrial bioenergetics. METHODS: Mitochondrial bioenergetics were compared with functional and histopathological indices of myopathy early in DMD (4 weeks) in D2.B10-DMDmdx /2J mice (D2.mdx)-a model that demonstrates severe muscle weakness. Adenosine diphosphate's (ADP's) central effect of attenuating H2 O2 emission while stimulating respiration was compared under two models of mitochondrial-cytoplasmic phosphate exchange (creatine independent and dependent) in muscles that stained positive for membrane damage (diaphragm, quadriceps, and white gastrocnemius). RESULTS: Pathway-specific analyses revealed that Complex I-supported maximal H2 O2 emission was elevated concurrent with a reduced ability of ADP to attenuate emission during respiration in all three muscles (mH2 O2 : +17 to +197% in D2.mdx vs. wild type). This was associated with an impaired ability of ADP to stimulate respiration at sub-maximal and maximal kinetics (-17 to -72% in D2.mdx vs. wild type), as well as a loss of creatine-dependent mitochondrial phosphate shuttling in diaphragm and quadriceps. These changes largely occurred independent of mitochondrial density or abundance of respiratory chain complexes, except for quadriceps. This muscle was also the only one exhibiting decreased calcium retention capacity, which indicates increased sensitivity to calcium-induced permeability transition pore opening. Increased H2 O2 emission was accompanied by a compensatory increase in total glutathione, while oxidative stress markers were unchanged. Mitochondrial bioenergetic dysfunctions were associated with induction of mitochondrial-linked caspase 9, necrosis, and markers of atrophy in some muscles as well as reduced hindlimb torque and reduced respiratory muscle function. CONCLUSIONS: These results provide evidence that Complex I dysfunction and loss of central respiratory control by ADP and creatine cause elevated oxidant generation during impaired oxidative phosphorylation. These dysfunctions may contribute to early stage disease pathophysiology and support the growing notion that mitochondria are a potential therapeutic target in this disease.

Holo-lipocalin-2-derived siderophores increase mitochondrial ROS and impair oxidative phosphorylation in rat cardiomyocytes.

Lipocalin-2 (Lcn2), a critical component of the innate immune response which binds siderophores and limits bacterial iron acquisition, can elicit spillover adverse proinflammatory effects. Here we show that holo-Lcn2 (Lcn2-siderophore-iron, 1:3:1) increases mitochondrial reactive oxygen species (ROS) generation and attenuates mitochondrial oxidative phosphorylation in adult rat primary cardiomyocytes in a manner blocked by N-acetyl-cysteine or the mitochondria-specific antioxidant SkQ1. We further demonstrate using siderophores 2,3-DHBA (2,3-dihydroxybenzoic acid) and 2,5-DHBA that increased ROS and reduction in oxidative phosphorylation are direct effects of the siderophore component of holo-Lcn2 and not due to apo-Lcn2 alone. Extracellular apo-Lcn2 enhanced the potency of 2,3-DHBA and 2,5-DHBA to increase ROS production and decrease mitochondrial respiratory capacity, whereas intracellular apo-Lcn2 attenuated these effects. These actions of holo-Lcn2 required an intact plasma membrane and were decreased by inhibition of endocytosis. The hearts, but not serum, of Lcn2 knockout (LKO) mice contained lower levels of 2,5-DHBA compared with wild-type hearts. Furthermore, LKO mice were protected from ischemia/reperfusion-induced cardiac mitochondrial dysfunction. Our study identifies the siderophore moiety of holo-Lcn2 as a regulator of cardiomyocyte mitochondrial bioenergetics.

The superoxide dismutase mimetic tempol does not alleviate glucocorticoid-mediated rarefaction of rat skeletal muscle capillaries.

Sustained elevations in circulating glucocorticoids elicit reductions in skeletal muscle microvascular content, but little is known of the underlying mechanisms. We hypothesized that glucocorticoid-induced oxidative stress contributes to this phenomenon. In rats that were implanted with corticosterone (CORT) or control pellets, CORT caused a significant decrease in muscle glutathione levels and a corresponding increase in protein carbonylation, an irreversible oxidative modification of proteins. Decreased endothelial nitric oxide synthase and increased endothelin-1 mRNA levels were detected after 9 days of CORT, and blood flow to glycolytic muscles was diminished. Control and CORT rats were treated concurrently with drinking water containing the superoxide dismutase mimetic tempol (172 mg/L) or the α-1 adrenergic receptor antagonist prazosin (50 mg/L) for 6 or 16 days. Both tempol and prazosin alleviated skeletal muscle protein carbonylation. Tempol failed to prevent CORT-mediated capillary rarefaction and was ineffective in restoring skeletal muscle blood flow. In contrast, prazosin blocked capillary rarefaction and restored skeletal muscle blood flow to control levels. The failure of tempol to prevent CORT-induced skeletal muscle microvascular rarefaction does not support a dominant role of superoxide-induced oxidative stress in this process. Although a decrease in protein carbonylation was observed with prazosin treatment, our data suggest that the maintenance of skeletal muscle microvascular content is related more closely with counteracting the CORT-mediated influence on skeletal muscle vascular tone.

Adipose tissue depot specific differences of PLIN protein content in endurance trained rats.

Adipose tissue is classified as either white (WAT) or brown (BAT) and differs not only by anatomical location but also in function. WAT is the main source of stored energy and releases fatty acids in times of energy demand, whereas BAT plays a role in regulating non-shivering thermogenesis and oxidizes fatty acids released from the lipid droplet. The PLIN family of proteins has recently emerged as being integral in the regulation of fatty acid storage and release in adipose tissue. Previous work has demonstrated that PLIN protein content varies among adipose tissue depots, however an examination of endurance training-induced depot specific changes in PLIN protein expression has yet to be done. Male Sprague-dawley rats (n = 10) underwent 8-weeks of progressive treadmill training (18-25 m/min for 30-60 min at 10% incline) or remained sedentary as control. Following training, under isoflurane induced anesthesia epidydmal (eWAT), inguinal subcutaneous (iWAT) and intrascapular brown adipose tissue (BAT) was excised, and plasma was collected. Endurance training resulted in an increase in BAT PLIN5 and iWAT PLIN3 content, while there was no difference in PLIN protein content in endurance trained eWAT. Interestingly, endurance training resulted in a robust increase in ATGL and CGI-58 in eWAT alone. Together these results suggest the potential of a depot specific function of PLIN3 and PLIN5 in adipose tissue in response to endurance training.

Skeletal site-specific effects of endurance running on structure and strength of tibia, lumbar vertebrae, and mandible in male Sprague-Dawley rats.

Bone microarchitecture, bone mineral density (BMD), and bone strength are affected positively by impact activities such as running; however, there are discrepancies in the magnitude of these effects. These inconsistencies are mainly a result of varying training protocols, analysis techniques, and whether or not the skeletal sites measured are weight bearing. This study's purpose was to determine the effects of endurance running on sites that experience different weight bearing and load. Eight-week-old male Sprague-Dawley rats (n = 20) were randomly assigned to either a group with a progressive treadmill running protocol (25 m/min for 1 h, incline of 10%) or a nontrained control group for 8 weeks. The trabecular structure of the tibia, lumbar vertebra (L3), and mandible and the cortical structure at the tibia midpoint were measured using microcomputed tomography to quantify bone volume fraction (i.e., bone volume divided by total volume (BV/TV)), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and cortical thickness. BMD at the proximal tibia, lumbar vertebrae (L1-L3), and mandible was measured using dual energy X-ray absorptiometry. The tibia midpoint strength was measured by 3-point bending using a materials testing system. Endurance running resulted in superior bone structure at the proximal tibia (12% greater BV/TV (p = 0.03), 14% greater Tb.N (p = 0.01), and 19% lower Tb.Sp (p = 0.05)) but not at other sites. Contrary to our hypothesis, mandible bone structure was altered after endurance training (8% lower BV/TV (p < 0.01) and 15% lower Tb.Th (p < 0.01)), which may be explained by a lower food intake, resulting in less mechanical loading from chewing. These results highlight the site-specific effects of loading on the skeleton.

Cytosolic localization of Fox proteins in motor neurons of G93A SOD1 mice.

NeuN is a nuclear protein expressed exclusively in mature neurons and has served for many years as a reliable neuronal marker in immunohistochemical labeling studies. In 2009, NeuN was identified as Fox3, one of three closely related RNA binding proteins important in pre-mRNA splicing. During the course of a previous study using G93A SOD1 mice, a model of amyotrophic lateral sclerosis (ALS), we observed that NeuN was significantly redistributed to the cytosol. Since altered splicing may be important in the pathogenesis of ALS, we compared the localization (predominantly nuclear or cytosolic) of all three Fox proteins in the lumbar spinal cord of wild-type and G93A SOD1 mice before and after the development of clinical signs of disease. The Fox proteins regulate their own splicing, and we also examined the major Fox protein isoforms in nuclear and cytosolic fractions of lumbar spinal cord by Western blotting. We report here that Fox3 and Fox2 undergo a major cytosolic relocalization in this ALS model that increases with age and that is associated with progressive alterations in the splicing profiles of all three Fox proteins.

Increases in skeletal muscle ATGL and its inhibitor G0S2 following 8 weeks of endurance training in metabolically different rat skeletal muscles.

Adipose triglyceride lipase (ATGL) catalyzes the rate-limiting removal of the first fatty acid from a triglyceride. ATGL is activated by comparative gene identification-58 and inhibited by G(0)/G(1) switch gene-2 protein (G0S2). Research in other tissues and cell culture indicates that inhibition is dependent on relative G0S2-to-ATGL protein content. G0S2 may also have several roles within mitochondria; however, this has yet to be observed in skeletal muscle. The purpose of this study was to determine if muscle G0S2 relative to ATGL content would decrease to facilitate intramuscular lipolysis following endurance training. Male Sprague-Dawley rats (n = 10; age 51-53 days old) were progressively treadmill trained at a 10% incline for 8 wk ending with 25 m/min for 1 h compared with control. Sciatic nerve stimulation for hind-limb muscle contraction (and lipolysis) was administered for 30 min to one leg, leaving the opposing leg as a resting control. Soleus (SOL), red gastrocnemius (RG), and white gastrocnemius were excised from both legs following stimulation or control. ATGL protein increased in all trained muscles. Unexpectedly, G0S2 protein was greater in the trained SOL and RG. In RG-isolated mitochondria, G0S2 also increased with training, yet mitochondrial G0S2 content was unaltered with acute contraction; therefore, any role of G0S2 in the mitochondria does not appear to be acutely mediated by content alone. In summary, G0S2 increased with training in oxidative muscles and mitochondria but not following acute contraction, suggesting that inhibition is not through relative G0S2-to-ATGL content but through more complicated intracellular mechanisms.

Characterization of lipolytic inhibitor G(0)/G(1) switch gene-2 protein (G0S2) expression in male Sprague-Dawley rat skeletal muscle compared to relative content of adipose triglyceride lipase (ATGL) and comparitive gene identification-58 (CGI-58).

The rate-limiting enzyme in lipolysis, adipose triglyceride lipase (ATGL), is activated by comparative gene identification-58 (CGI-58) and inhibited by the G(0)/G(1) switch gene-2 (G0S2) protein. It is speculated that inhibition of ATGL is through a dose dependent manner of relative G0S2 protein content. There is little work examining G0S2 expression in lipolytic tissues, and the relative expression across oxidative tissues such as skeletal muscle has not yet been described. Three muscles, soleus (SOL), red gastrocnemius (RG), and white gastrocnemius (WG) were excised from 57-day old male Sprague-Dawley rats (n = 9). QRT-PCR was used for mRNA analysis, and western blotting was conducted to determine protein content. ATGL and G0S2 protein content were both greatest in the lipolytic SOL, with the least amount of both ATGL and G0S2 protein content found in the WG. CGI-58 protein content however did not mirror ATGL and G0S2 protein content, since the RG had the greatest CGI-58 protein content when compared to the SOL and WG. When comparing our tissues based on CGI-58-to-ATGL ratio and G0S2-to-ATGL ratio, it was discovered that contrary to oxidative demand, the glycolytic WG had the greatest activator CGI-58-to-ATGL ratio with the oxidative SOL having the least, and no differences in G0S2-to-ATGL across the three muscle types. These data suggest that the content of G0S2 relative to the lipase in skeletal muscle would not predict lipolytic potential.

Mitochondrial Bioenergetics and Fiber Type Assessments in Microbiopsy vs. Bergstrom Percutaneous Sampling of Human Skeletal Muscle.

Microbiopsies of human skeletal muscle are increasingly adopted by physiologists for a variety of experimental assays given the reduced invasiveness of this procedure compared to the classic Bergstrom percutaneous biopsy technique. However, a recent report demonstrated lower mitochondrial respiration in saponin-permeabilized muscle fiber bundles (PmFB) prepared from microbiopsies vs. Bergstrom biopsies. We hypothesized that ADP-induced contraction (rigor) of smaller length microbiopsy PmFB causes a greater reduction in maximal respiration vs. Bergstrom, such that respiration could be increased by a myosin II ATPase-inhibitor (Blebbistatin; BLEB). Eleven males and females each received a 2 mm diameter percutaneous microbiopsy and a 5 mm diameter Bergstrom percutaneous biopsy in opposite legs. Glutamate/malate (5/0.5 mM)-supported respiration in microbiopsy PmFB was lower than Bergstrom at submaximal concentrations of ADP. 5 µM BLEB reduced this impairment such that there were no differences relative to Bergstrom ± BLEB. Surprisingly, pyruvate (5 mM)-supported respiration was not different between either biopsy technique ±BLEB, whereas BLEB increased succinate-supported respiration in Bergstrom only. H2O2 emission was lower in microbiopsy PmFB compared to Bergstrom PmFB in the presence of BLEB. Microbiopsies contained fewer type I fibers (37 vs. 47%) and more type IIX fibers (20 vs. 8%) compared to Bergstrom possibly due to sampling site depth and/or longitudinal location. These findings suggest that smaller diameter percutaneous biopsies yield lower glutamate-supported mitochondrial respiratory kinetics which is increased by preventing ADP-induced rigor with myosin inhibition. Microbiopsies of human skeletal muscle can be utilized for assessing mitochondrial respiratory kinetics in PmFB when assay conditions are supplemented with BLEB, but fiber type differences with this method should be considered.

Higher PLIN5 but not PLIN3 content in isolated skeletal muscle mitochondria following acute in vivo contraction in rat hindlimb.

Contraction-mediated lipolysis increases the association of lipid droplets and mitochondria, indicating an important role in the passage of fatty acids from lipid droplets to mitochondria in skeletal muscle. PLIN3 and PLIN5 are of particular interest to the lipid droplet-mitochondria interaction because PLIN3 is able to move about within cells and PLIN5 associates with skeletal muscle mitochondria. This study primarily investigated: 1) if PLIN3 is detected in skeletal muscle mitochondrial fraction; and 2) if mitochondrial protein content of PLIN3 and/or PLIN5 changes following stimulated contraction. A secondary aim was to determine if PLIN3 and PLIN5 associate and whether this changes following contraction. Male Long Evans rats (n = 21; age, 52 days; weight = 317 ± 6 g) underwent 30 min of hindlimb stimulation (10 msec impulses, 100 Hz/3 sec at 10-20 V; train duration 100 msec). Contraction induced a ~50% reduction in intramuscular lipid content measured by oil red-O staining of red gastrocnemius muscle. Mitochondria were isolated from red gastrocnemius muscle by differential centrifugation and proteins were detected by western blotting. Mitochondrial PLIN5 content was ~1.6-fold higher following 30 min of contraction and PLIN3 content was detected in the mitochondrial fraction, and unchanged following contraction. An association between PLIN3 and PLIN5 was observed and remained unaltered following contraction. PLIN5 may play a role in mitochondria during lipolysis, which is consistent with a role in facilitating/regulating mitochondrial fatty acid oxidation. PLIN3 and PLIN5 may be working together on the lipid droplet and mitochondria during contraction-induced lipolysis.