Succinate Dehydrogenase Staining Research Articles

Changes in Skeletal Muscle Atrophy over Time in a Rat Model of Adenine-Induced Chronic Kidney Disease

This study evaluated changes over time in skeletal muscle atrophy, expressions of skeletal muscle anabolic and catabolic genes, and mitochondrial activity by skeletal muscle type in an adenine-induced chronic kidney disease (CKD) model. A CKD model was successfully established by feeding male Wistar rats a 0.75% adenine diet for 4 weeks starting at 8 weeks of age. Control and CKD groups were sacrificed at 12 and 20 weeks of age. The back muscles were analyzed histologically, and succinate dehydrogenase (SDH) staining was performed to evaluate mitochondrial activity. Gene expressions of myogenic determination gene number 1 and myogenin as indicators of muscle anabolism, atrogin-1 and muscle RING-finger protein-1 (MuRF1) as indicators of muscle catabolism, and peroxisome proliferator-activated receptor-γ coactivator-1-α as a marker of mitochondrial biogenesis were assessed. Type I and type II muscle cross-sectional areas (CSAs) were decreased at 12 weeks, but type I muscle CSA was recovered at 20 weeks. SDH staining was lower in CKD than in control rats at 12 weeks, but no significant difference was observed at 20 weeks. Increased expressions of myogenin, atrogin-1, and MuRF-1 were observed only at 12 weeks, but no differences were observed at 20 weeks. The adenine-induced CKD rat model appears to show changes in muscle atrophy over time.

Betaine delays age-related muscle loss by mitigating Mss51-induced impairment in mitochondrial respiration via Yin Yang1.

Mitochondrial dysfunction is one of the hallmarks of aging and a leading contributor to sarcopenia. Nutrients are essential for improving mitochondrial function and skeletal muscle health during the aging process. Betaine is a nutrient with potential muscle-preserving properties. However, whether and how betaine could regulate the mitochondria function in aging muscle are poorly understood. We aimed to explore the molecular target and underlying mechanism of betaine in attenuating the age-related mitochondrial dysfunction in skeletal muscle. Young mice (YOU, 2months), old mice (OLD, 15months), and old mice with betaine treatment (BET, 15months) were fed for 12weeks. The effects of betaine on muscle mass, strength, function, and subcellular structure of muscle fibres were assessed. RNA sequencing (RNA-seq) was conducted to identify the molecular target of betaine. The impacts of betaine on mitochondrial-related molecules, superoxide accumulation, and oxidative respiration were examined using western blotting (WB), immunofluorescence (IF) and seahorse assay. The underlying mechanism of betaine regulation on the molecular target to maintain mitochondrial function was investigated by luciferase reporter assay, chromatin immunoprecipitation and electrophoretic mobility shift assay. Adenoassociated virus transfection, succinate dehydrogenase staining (SDH), and energy expenditure assessment were performed on 20-month-old mice for validating the mechanism in vivo. Betaine intervention demonstrated anti-aging effects on the muscle mass (P=0.017), strength (P=0.010), and running distance (P=0.013). Mitochondrial-related markers (ATP5a, Sdha, and Uqcrc2) were 1.1- to 1.5-fold higher in BET than OLD (all P≤0.036) with less wasted mitochondrial vacuoles accumulating in sarcomere. Bioinformatic analysis from RNA-seq displayed pathways related to mitochondrial respiration activity was higher enriched in BET group (NES=-0.87, FDR=0.10). The quantitative real time PCR (qRT-PCR) revealed betaine significantly reduced the expression of a novel mitochondrial regulator, Mss51 (-24.9%, P=0.002). In C2C12 cells, betaine restored the Mss51-mediated suppression in mitochondrial respiration proteins (all P≤0.041), attenuated oxygen consumption impairment, and superoxide accumulation (by 20.7%, P=0.001). Mechanically, betaine attenuated aging-induced repression in Yy1 mRNA expression (BET vs. OLD: 2.06 vs. 1.02, P=0.009). Yy1 transcriptionally suppressed Mss51 mRNA expression both in vitro and in vivo. This contributed to the preservation of mitochondrial respiration, improvement for energy expenditure (P=0.008), and delay of muscle loss during aging process. Altogether, betaine transcriptionally represses Mss51 via Yy1, improving age-related mitochondrial respiration in skeletal muscle. These findings suggest betaine holds promise as a dietary supplement to delay skeletal muscle degeneration and improve age-related mitochondrial diseases.

125 Effects of Saccharmyces yeast postbiotics on nursey pig muscle fiber cross-sectional area and oxidative capacity

Abstract This study aimed to determine effects of dietary supplementation of Saccharomyces yeast postbiotics (celluTEIN, Puretein Bioscience, LLC, Minneapolis, MN) on nursery pig muscle fiber cross-sectional area (CSA), isoform, and succinate dehydrogenase (SDH) staining intensity. Newly weaned pigs [n = 32; 16 barrows and 16 gilts; 21 d of age, body weight (BW) 6.05 ± 0.24 kg] were stratified by BW within sex, placed in individual pens, and randomly assigned to one of two dietary treatments within each two-pig strata. Dietary treatments included pigs supplemented 0 (CON; n = 16) or 175 g/ton (CT; n = 16) of celluTEIN to their standard three-phase nursery ration. Pigs were harvested at d 35 of feeding and the longissimus lumborum muscle was collected for mean SDH staining intensity and immunohistochemistry analyses. Data were analyzed as a completely randomized design with pig as the experimental unit and treatment as the fixed effect. Statistical significance was determined at P ≤ 0.05. There were no treatment effects on type I and type IIB muscle fiber CSA (P > 0.14), but CT pigs had greater type IIA and type IIX muscle fiber CSA compared with CON (P < 0.05). When not analyzed by fiber isoform, pigs fed CT had a greater (P = 0.02) average muscle fiber CSA than CON pigs. There were no treatment effects on muscle fiber type percentage or SDH intensity of all fiber types (P ≥ 0.23); however, when calculated as a muscle fiber CSA ratio, CT type IIA fibers had a smaller ratio (darker staining/µm2) than CON type IIA fibers (P = 0.05). There were no treatment effects for type I, IIX, and IIB ratios (P > 0.21). In conclusion, supplementing Saccharomyces yeast postbiotics during the nursery period increased muscle fiber hypertrophy and type IIA fiber oxidative capacity.

Resistance exercise following short term muscle disuse leads to enhanced mitochondrial activity and dynamics

Introduction: Short-term muscle unloading (~7-10 days) can be usually experienced following injury or hospitalisation, leading to muscle disuse atrophy, muscle weakness and changes in motor control. In such contexts, exercise countermeasures can be employed to overcome these neuromuscular impairments. Surprisingly, the effects of exercise countermeasures following the exposure to muscle unloading has been scarcely investigated. Hence, this study aimed to investigate the functional and molecular adaptations to a 21-day resistance training (RT) program following short term muscle disuse. We hypothesised that a period of resistance exercise following short-term unloading would speed up the recovery on muscle mass and function mostly by an effcient counteraction of the molecular regulators of muscle disuse atrophy consistent with the exercise modality employed. Methods: A 10-day unilateral lower limb suspension (ULLS) model was applied to eleven healthy young males (22.09 ± 2.91 y), followed by a 21-day RT program for the knee extensors performed at 70% of the 1RM (3 times/wk, 9 sessions in total). Data acquisition was performed before the ULLS period, after 10 days of unloading and after 21 days of resistance exercise. Quadriceps femoris isometric maximum voluntary contraction (MVC) was evaluated by dynamometry and quadriceps CSA was assessed by ultrasonography. Succinate dehydrogenase (SDH, a key component of the citric acid cycle and of the respiratory electron transfer chain) staining was performed on frozen cryosections obtained from VL biopsies, whereas western blot analyses were carried out for several mitochondrial-related proteins and transcriptomics data were obtained by RNA-seq. Results: Following ULLS, QF MVC and CSA decreased (-29.3%, p<0.001, and -4.5%, p<0.05, respectively). After the 21-d of RT, MVC was fully brought back (+42% compared to post-ULLS) whereas QF CSA showed a very marked hypertrophy (+18.6%, p<0.001, compared to post-ULLS). SDH immunoreactivity was not affected by unloading but revealed a significant increase after exercise compared to post ULLS values (~+20%, p<0.001). In addition, mitochondrial dynamics were highly influenced by resistance exercise, as mitochondrial pro-fusion (MFN1, MFN2, OPA1) and fission (DRP1, pDRP1616) proteins were markedly increased in response to RT compared to post-ULLS values (p<0.05). Further, PGC-1a levels showed a trend to decrease after ULLS but even a stronger trend to increase after resistance exercise compared to post-ULLS (p=0.056). Transcriptomics analyses showed that the most differentially expressed genes were identified in the pathways related to oxidative phosphorylation, in which the top regulated genes after resistance training were mitochondrial related genes (CKMT2, UQCRH, ACSS1, MDH1, COX7A2, CYCS, SLC16A1), presenting a marked reduction after ULLS and being significantly enhanced after RT. When compared to baseline, the genes which showed a positive “rebound effect” after resistance exercise were mostly related to mitochondrial function (MT-ATP6, MT-CO2, MT-ND3). Noteworthy, among these latter genes, the top upregulated one was APLNR, coding for apelin receptor protein, a positive regulator of inter-myofibrillar mitochondrial content in sarcopenia and ageing. Conclusions: Resistance exercise was effective in recovering muscle mass and function after 10 days of disuse, but it enhanced mitochondrial activity, biogenesis, and dynamics far beyond the expected effects for such exercise modality. In fact, mitochondrial-related genes expression was the most affected by exercise. This suggests that mitochondrial dynamics could play an essential role in the recovery of neuromuscular alterations induced by short term disuse. We put forward the hypothesis that the prior level of physical activity and a prior exposure to short-term muscle disuse could be driving the nature of adaptations to subsequent resistance training. Funding: The present work was funded by the Italian Space Agency (ASI), MARS-PRE, Project, n. DC-VUM-2017-006. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Label-free analysis of the β-hydroxybutyricacid drug on mitochondrial redox states repairment in type 2 diabetic mice by resonance raman scattering

BackgroundMitochondrial redox imbalance underlies the pathophysiology of type2 diabetes mellitus (T2DM), and is closely related to tissue damage and dysfunction. Studies have shown the beneficial effects of dietary strategies that elevate β-hydroxybutyrate (BHB) levels in alleviating T2DM. Nevertheless, the role of BHB has not been clearly elucidated. MethodsWe performed a spectral study to visualize the preventive effects of BHB on blood and multiorgan mitochondrial redox imbalance in T2DM mice via using label-free resonance Raman spectroscopy (RRS), and further explored the impact of BHB therapy on the pathology of T2DM mice by histological and biochemical analyses. FindingsOur data revealed that RRS-based mitochondrial redox states assay enabled clear and reliable identification of the improvement of mitochondrial redox imbalance by BHB, evidenced by the reduction of Raman peak intensity at 750 cm−1, 1128 cm−1 and 1585 cm−1 in blood, tissue as well as purified mitochondria of db/db mice and the increase of tissue mitochondrial succinic dehydrogenase (SDH) staining after BHB treatment. Exogenous supplementation of BHB was also found to attenuate T2DM pathology related to mitochondrial redox states, involving organ injury, blood glucose control, insulin resistance and systemic inflammation. InterpretationOur findings provide strong evidence for BHB as a potential therapeutic strategy targeting mitochondria for T2DM.

Celecoxib ameliorates diabetic sarcopenia by inhibiting inflammation, stress response, mitochondrial dysfunction, and subsequent activation of the protein degradation systems.

Aim: Diabetic sarcopenia leads to disability and seriously affects the quality of life. Currently, there are no effective therapeutic strategies for diabetic sarcopenia. Our previous studies have shown that inflammation plays a critical role in skeletal muscle atrophy. Interestingly, the connection between chronic inflammation and diabetic complications has been revealed. However, the effects of non-steroidal anti-inflammatory drug celecoxib on diabetic sarcopenia remains unclear. Materials and Methods: The streptozotocin (streptozotocin)-induced diabetic sarcopenia model was established. Rotarod test and grip strength test were used to assess skeletal muscle function. Hematoxylin and eosin and immunofluorescence staining were performed to evaluate inflammatory infiltration and the morphology of motor endplates in skeletal muscles. Succinate dehydrogenase (SDH) staining was used to determine the number of succinate dehydrogenase-positive muscle fibers. Dihydroethidium staining was performed to assess the levels of reactive oxygen species (ROS). Western blot was used to measure the levels of proteins involved in inflammation, oxidative stress, endoplasmic reticulum stress, ubiquitination, and autophagic-lysosomal pathway. Transmission electron microscopy was used to evaluate mitophagy. Results: Celecoxib significantly ameliorated skeletal muscle atrophy, improving skeletal muscle function and preserving motor endplates in diabetic mice. Celecoxib also decreased infiltration of inflammatory cell, reduced the levels of IL-6 and TNF-α, and suppressed the activation of NF-κB, Stat3, and NLRP3 inflammasome pathways in diabetic skeletal muscles. Celecoxib decreased reactive oxygen species levels, downregulated the levels of Nox2 and Nox4, upregulated the levels of GPX1 and Nrf2, and further suppressed endoplasmic reticulum stress by inhibiting the activation of the Perk-EIF-2α-ATF4-Chop in diabetic skeletal muscles. Celecoxib also inhibited the levels of Foxo3a, Fbx32 and MuRF1 in the ubiquitin-proteasome system, as well as the levels of BNIP3, Beclin1, ATG7, and LC3Ⅱ in the autophagic-lysosomal system, and celecoxib protected mitochondria and promoted mitochondrial biogenesis by elevating the levels of SIRT1 and PGC1-α, increased the number of SDH-positive fibers in diabetic skeletal muscles. Conclusion: Celecoxib improved diabetic sarcopenia by inhibiting inflammation, oxidative stress, endoplasmic reticulum stress, and protecting mitochondria, and subsequently suppressing proteolytic systems. Our study provides evidences for the molecular mechanism and treatment of diabetic sarcopenia, and broaden the way for the new use of celecoxib in diabetic sarcopenia.

Senescent tumor cells in colorectal cancer are characterized by elevated enzymatic activity of complexes 1 and 2 in oxidative phosphorylation.

Cellular senescence is defined as an irreversible cell cycle arrest caused by various internal and external insults. While the metabolic dysfunction of senescent cells in normal tissue is relatively well-established, there is a lack of information regarding the metabolic features of senescent tumor cells. Publicly available single-cell RNA-sequencing data from the GSE166555 and GSE178341 datasets were utilized to investigate the metabolic features of senescent tumor cells. To validate the single-cell RNA-sequencing data, we performed senescence-associated β-galactosidase (SA-β-Gal) staining to identify senescent tumor cells in fresh frozen colorectal cancer tissue. We also evaluated nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase (NADH-TR) and succinate dehydrogenase (SDH) activity using enzyme histochemical methods and compared the staining with SA-β-Gal staining. MTT assay was performed to reveal the complex 1 activity of the respiratory chain in in-vitro senescence model. Single-cell RNA-sequencing data revealed an upregulation in the activity of complexes 1 and 2 in oxidative phosphorylation, despite overall mitochondrial dysfunction in senescent tumor cells. Both SA-β-Gal and enzyme histochemical staining using fresh frozen colorectal cancer tissues indicated a high correlation between SA-β-Gal positivity and NADH-TR/SDH staining positivity. MTT assay showed that senescent colorectal cancer cells exhibit higher absorbance in 600 nm wavelength. Senescent tumor cells exhibit distinct metabolic features, characterized by upregulation of complexes 1 and 2 in the oxidative phosphorylation pathway. NADH-TR and SDH staining represent efficient methods for detecting senescent tumor cells in colorectal cancer.

Loss of Farnesoid X receptor (FXR) accelerates dysregulated glucose and renal injury in db/db mice.

End-stage renal disease is primarily caused by diabetic kidney disease (DKD). The Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has anti-inflammatory, lipid-lowering and hypoglycemic properties. It also inhibits renal fibrosis. Although its physiological role is not fully understood, it also plays a role in the control of diabetic nephropathy (DN). In the present study, we examined male FXR & leptin receptor double knockout mice, in which weight, blood glucose, body fat, and other indicators were monitored. After 6 months of rearing, blood and urine samples were collected and biochemical parameters were measured. Fibrosis was assessed by Masson's stain, while the assessment of the resuscitation case's condition was performed using succinate dehydrogenase (SDHA) stain immunohistochemistry, which measures aerobic respiration. Expression of molecules such as connective tissue growth factor (CTGF), SMAD family members 3 (Smad3) and 7 (Smad7), and small heterodimer partner were detected by RT-PCR and Western blotting as part of the application. FXR knockout decreased body weight and body fat in db/db mice, but increased blood glucose, urine output, and renal fibrosis. Primary mesangial cells (P-MCs) from FXR+/+ mice stimulated with transforming growth factor β1 (TGFβ1) showed significantly higher levels of related fibrosis factors, TGFβ1 and Smad3 mRNA and protein, and significantly reduced levels of Smad7. These effects were reversed by the action of FXR agonist chenodeoxycholic acid (CDCA). P-MCs from FXR-/- mice stimulated with TGFβ1 resulted in an increase in the expression and protein levels of collagen I and TGFβ1, and the addition of CDCA had no significant effect on TGFβ1 stimulation. However, compared with FXR+/+db/db mice, the rate of oxygen consumption, the rate of carbon dioxide production, and the rate of energy conversion were increased in FXR-/-db/db mice, whereas the SDHA succinate dehydrogenase, a marker enzyme for aerobic respiration, was significantly decreased. These results provide evidence that FXR plays a critical role in the regulation of mesangial cells in DN. The likely mechanism is that aberrant FXR expression activates TGFβ1, which induces extracellular matrix accumulation through the classical Smad signaling pathway, leading to mitochondrial dysfunction.

Capillary rarefaction during bed rest is proportionally less than fibre atrophy and loss of oxidative capacity.

Muscle disuse from bed rest or spaceflight results in losses in muscle mass, strength and oxidative capacity. Capillary rarefaction may contribute to muscle atrophy and the reduction in oxidative capacity during bed rest. Artificial gravity may attenuate the negative effects of long-term space missions or bed rest. The aim of the present study was to assess (1) the effects of bed rest on muscle fibre size, fibre type composition, capillarization and oxidative capacity in the vastus lateralis and soleus muscles after 6 and 55days of bed rest and (2) the effectiveness of artificial gravity in mitigating bed-rest-induced detriments to these parameters. Nineteen participants were assigned to a control group (control, n=6) or an intervention group undergoing 30min of centrifugation (n=13). All underwent 55days of head-down tilt bed rest. Vastus lateralis and soleus biopsies were taken at baseline and after 6 and 55days of bed rest. Fibre type composition, fibre cross-sectional area, capillarization indices and oxidative capacity were determined. After just 6days of bed rest, fibre atrophy (-23.2±12.4%, P<0.001) and reductions in capillary-to-fibre ratio (C:F; 1.97±0.57 vs. 1.56±0.41, P<0.001) were proportional in both muscles as reflected by a maintained capillary density. Fibre atrophy proceeded at a much slower rate between 6 and 55days of bed rest (-11.6±12.1% of 6days, P=0.032) and was accompanied by a 19.1% reduction in succinate dehydrogenase stain optical density (P<0.001), without any further significant decrements in C:F (1.56±0.41 vs. 1.49±0.37, P=0.459). Consequently, after 55days of bed rest, the capillary supply-oxidative capacity ratio of a fibre had increased by 41.9% (P<0.001), indicating a capillarization in relative excess of oxidative capacity. Even though the heterogeneity of capillary spacing (LogR SD) was increased after 55days by 12.7% (P=0.004), tissue oxygenation at maximal oxygen consumption of the fibres was improved after 55days bed rest. Daily centrifugation failed to blunt the bed-rest-induced reductions in fibre size and oxidative capacity and capillary rarefaction. The relationship between fibre size and oxidative capacity with the capillary supply of a fibre is uncoupled during prolonged bed rest as reflected by a rapid loss of muscle mass and capillaries, followed at later stages by a more than proportional loss of mitochondria without further capillary loss. The resulting excessive capillary supply of the muscle after prolonged bed rest is advantageous for the delivery of substrates needed for subsequent muscle recovery.

Dysregulation of Skeletal Muscle Mitochondrial Turnover and Dynamics Occurs During Late Symptomatic Stages of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disorder caused by a mutation in the survival motor neuron 1 (SMN1) gene and it is the leading genetic cause of infant mortality. Recently approved genetic therapies designed to augment full length SMN protein in the central nervous system fail to ameliorate abnormal skeletal muscle features, which strongly suggests an important role for SMN protein in skeletal muscle homeostasis. This study aimed to characterize key modifiers of skeletal muscle mitochondrial turnover and dynamics during disease progression in a pre‐clinical murine model of SMA in vivo. Additionally, we investigated the effects of a single dose of exercise on these mitochondrial biology‐regulating pathways in the skeletal muscle of SMA mice. Muscle samples were collected from wild‐type (WT) and Smn2B/‐ (SMA) mice at postnatal day 9 (P9), P13, and P21 to examine skeletal muscle‐specific disease progression. Mitochondrial content did not differ between genotypes at all timepoints as evident by similar levels of mitochondrial oxidative phosphorylation proteins, succinate dehydrogenase staining, and citrate synthase content. However, mRNA content of key genes responsible for regulating mitochondrial quality such as nuclear respiratory factor 2, mitochondrial transcription factor A, and p53 were significantly higher in SMA mice versus WT at P21. Additionally, we observed elevated mitochondrial fission activity as indicated by a 2‐fold increase (p &lt; 0.05) in dynamin related protein 1 (DRP1) activation. Concomitantly, the expression of several mitophagy proteins including BLC2 interacting protein 3, parkin, and PTEN‐induced kinase 1 were significantly increased by 2.9‐, 2.7‐, and 2‐fold, respectively, compared to WT animals at P21. Interestingly, we observed blunted (p &lt; 0.05) inclusion of optic atrophy 1 exon 4b, a key exon in mitochondrial biogenesis, between WT and SMA mice at P21. Acute exercise significantly increased the inhibition of DRP1‐mediated mitochondrial fission activity in the muscles of SMA mice relative to sedentary SMA mice. However, a single exercise dose failed to alter the expression of mitophagy proteins up to three hours after running. Our data demonstrate that skeletal muscle mitochondrial health is compromised in SMA mice due to elevated fission and mitophagy processes. Furthermore, SMA mice display aberrant alternative splicing of Opa1 transcripts that may in turn hinder mitochondrial biogenesis in later disease stages. We also highlight that an acute bout of treadmill running elicits pro‐fusion signaling to potentially improve the mitochondrial reticulum. Collectively, this study is the first to reveal mitochondrial dysfunction in SMA skeletal muscle and outlines signalling associated with mitochondrial plasticity following a single dose of exercise.

Upregulation of thioredoxin contributes to inhibiting diabetic hearing impairment

AimsHair cell reduction was related to diabetes-induced hearing loss. Oxidative stress, endoplasmic reticulum stress, and autophagy participate in this process. Thioredoxin (Trx) is a protein with many biological functions which can regulate them. In this study, aiming to clarify protective effect of Trx on diabetic hearing loss and to identify an early potential therapeutic target for diabetic hearing impairment in the future. MethodsTrx transgenic (Tg) mice were used to establish a diabetic model by intraperitoneally injecting streptozotocin (STZ) and with/without SF or PX12 treatment. Succinate dehydrogenase (SDH) staining was used to evaluate the loss of hair cells. The relative expression of related proteins and genes was detected using western blotting and qRT-PCR. ResultsIn vivo, loss of outer hair cells was observed. However, it can be delayed Trx overexpression. Moreover, the expression of PGC-1α, bcl-2 and LC3 was increased in Tg(+)-DM mice compared with Tg(-)-DM mice. The expression of ASK1, Txnip, GRP78, CHOP and p62 was decreased in Tg(+)-DM mice compared with Tg(-)-DM mice. ConclusionsUpregulation of Trx protects diabetes-induced cochlear hair cells reduction. The underlying mechanisms were related to the regulation of ER stress through ASK1 and the mitochondrial pathway or autophagy via Txnip.

MiR-183 and miR-96 orchestrate both glucose and fat utilization in skeletal muscle.

Our knowledge of the coordination of fuel usage in skeletal muscle is incomplete. Whether and how microRNAs are involved in the substrate selection for oxidation is largely unknown. Here we show that mice lacking miR-183 and miR-96 have enhanced muscle oxidative phenotype and altered glucose/lipid homeostasis. Moreover, loss of miR-183 and miR-96 results in a shift in substrate utilization toward fat relative to carbohydrates in mice. Mechanistically, loss of miR-183 and miR-96 suppresses glucose utilization in skeletal muscle by increasing PDHA1 phosphorylation via targeting FoxO1 and PDK4. On the other hand, loss of miR-183 and miR-96 promotes fat usage in skeletal muscle by enhancing intramuscular lipolysis via targeting FoxO1 and ATGL. Thus, our study establishes miR-183 and miR-96 as master coordinators of fuel selection and metabolic homeostasis owing to their capability of modulating both glucose utilization and fat catabolism. Lastly, weshow that loss of miR-183 and miR-96 can alleviate obesity andimprove glucose metabolism in high-fat diet-induced mice, suggesting that miR-183 and miR-96 may serve as therapeutic targets for metabolic diseases.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

Antioxidant Apigenin Relieves Age-Related Muscle Atrophy by Inhibiting Oxidative Stress and Hyperactive Mitophagy and Apoptosis in Skeletal Muscle of Mice.

Skeletal muscle atrophy in the aged causes loss in muscle mass and functions. Naturally occurring antioxidant flavonoid apigenin is able to ameliorate obesity- and denervation-induced muscle atrophies, but its effects on age-related muscle atrophy remain unknown. We hypothesized that apigenin can relieve muscle atrophy in aged mice, probably through special effects on reactive oxygen species and enzymes with antioxidant functions. For the male mice of the study, apigenin showed significant dose-dependent effects in relieving aging-related muscle atrophy according to results of frailty index as indicator of frailty associated with aging, grip strength, and running distance. Apigenin also improved myofiber size and morphological features and increased mitochondria number and volume, as manifested by succinate dehydrogenase staining and transmission electron microscopy. Our tests also suggested that apigenin promoted activities of enzymes such as superoxide dismutase and glutathione peroxidase for antioxidation and those for aerobic respiration such as mitochondrial respiratory enzyme complexes I, II, and IV, increased ATP, and enhanced expression of genes such as peroxisome proliferator-activated receptor-γ coactivator 1α, mitochondrial transcription factor A, nuclear respiratory factor-1, and ATP5B involved in mitochondrial biogenesis. The data also suggested that apigenin inhibited Bcl-2/adenovirus E1B 19kD-interacting protein 3 and DNA fragmentation as indicators of mitophagy and apoptosis in aged mice with skeletal muscle atrophy. Together, the results suggest that apigenin relieves age-related skeletal muscle atrophy through reducing oxidative stress and inhibiting hyperactive autophagy and apoptosis.

OXIDATIVE METABOLISM DURING THE TIME-COURSE OF DISUSE ATROPHY IN MALE AND FEMALE MICE

Muscle loss is an important predictor of morbidity and mortality across a variety of diseases. Males and females appear to differ on clinical outcomes in relation to disuse-induced muscle atrophy, however reasons for these different responses have not been investigated. PURPOSE: To investigate measures of muscle oxidative metabolism during the time-course of disuse atrophy in male and female mice. METHODS: Disuse atrophy was induced using hindlimb unloading in 50 male and 50 female mice for 0 (CON), 1, 2, 3, or 7 days (n=~10/group). Muscle sections of the tibialis anterior were stained for succinate dehydrogenase (SDH). Cross sectional area (CSA) by SDH staining was used to assess the effect of disuse on different muscle fiber phenotypes. mRNA content of Pparα was measured in the gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. Data were analyzed within each sex by one way ANOVA and trend analysis. A p<0.05 indicated statistical significance. RESULTS: CSA of SDH positive fibers progressively decreased in both male and female mice. CON animals (male and female) had SDH positive fiber CSA of ~400 μm2 and 7 day unloaded animals had CSAs of ~300 μm2. Both male and female mice had an SDH negative CSA of ~650 μm2, with no significant differences in fiber CSA noted across groups. In the gastrocnemius muscle, Pparα content was ~50-60% lower at 1 day of unloading in males and females and remained depressed in all experimental groups. In soleus muscles of females, Pparα was ~60% lower at days 1, 2, and 3 compared to CON, but then recovered back to CON levels. Whereas in males, Pparα was ~60% lower with 1 day of unloading and remained depressed in 1, 2, 3, and 7 day groups. In females, there were no differences in Pparα content in EDL across all groups. In males, there was ~50-75% lower Pparα in EDL content that reached statistical significance at 2 days and remained depressed throughout intervention groups. CONCLUSION: Disuse results in muscle loss in males and females and appears to result in similar alterations to oxidative metabolism across multiple tissues. Future studies should investigate if improving oxidative metabolism is protective against disuse atrophy in males and females. This study was funded by the National Institutes of Health, Award number: R15 AR069913/AR/NIAMS

Novel role of Tieg1 in muscle metabolism and mitochondrial oxidative capacities.

Tieg1 is involved in multiple signalling pathways, human diseases, and is highly expressed in muscle where its functions are poorly understood. We have utilized Tieg1 knockout (KO) mice to identify novel and important roles for this transcription factor in regulating muscle ultrastructure, metabolism and mitochondrial functions in the soleus and extensor digitorum longus (EDL) muscles. RNA sequencing, immunoblotting, transmission electron microscopy, MRI, NMR, histochemical and mitochondrial function assays were performed. Loss of Tieg1 expression resulted in altered sarcomere organization and a significant decrease in mitochondrial number. Histochemical analyses demonstrated an absence of succinate dehydrogenase staining and a decrease in cytochrome c oxidase (COX) enzyme activity in KO soleus with similar, but diminished, effects in the EDL. Decreased complex I, COX and citrate synthase (CS) activities were detected in the soleus muscle of KO mice indicating altered mitochondrial function. Complex I activity was also diminished in KO EDL. Significant decreases in CS and respiratory chain complex activities were identified in KO soleus. 1 H-NMR spectra revealed no significant metabolic difference between wild-type and KO muscles. However, 31 P spectra revealed a significant decrease in phosphocreatine and ATPγ. Altered expression of 279 genes, many of which play roles in mitochondrial and muscle function, were identified in KO soleus muscle. Ultimately, all of these changes resulted in an exercise intolerance phenotype in Tieg1 KO mice. Our findings have implicated novel roles for Tieg1 in muscle including regulation of gene expression, metabolic activity and organization of tissue ultrastructure. This muscle phenotype resembles diseases associated with exercise intolerance and myopathies of unknown consequence.

Ontogenetic change in the amount and position of slow-oxidative myotomal muscle in relationship to regional endothermy in juvenile yellowfin tuna Thunnus albacares.

Myotomal slow-oxidative muscle (SM) powers continuous swimming and generates heat needed to maintain elevated locomotor muscle temperatures (regional endothermy) in tunas. This study describes how the amount and distribution of myotomal SM increases with fish size and age in juvenile yellowfin tuna Thunnus albacares in relationship to the development of regional endothermy. In T. albacares juveniles 40-74 mm fork length (LF ; n = 23) raised from fertilised eggs at the Inter-American Tropical Tuna Commission Achotines Laboratory in Panama and larger juveniles (118-344 mm LF ; n = 5) collected by hook and line off of Oahu, Hawaii, USA, SM was identified by histochemical staining for the mitochondrial enzyme succinic dehydrogenase or by colour (in the two largest individuals). The cross-sectional area of myotomal SM at 60% LF , a position with maximal percentage of SM in larger T. albacares, increased exponentially with LF . The percentage of total cross-sectional area composed of SM at 60% LF increased significantly with both LF and age, suggesting that SM growth occurs throughout the size range of T. albacares juveniles studied. In addition, the percentage of SM at 60% LF that is medial increased asymptotically with LF . The increases in amount of SM and medial SM, along with the development of the counter-current heat-exchanger blood vessels that retain heat, allow larger tuna juveniles to maintain elevated and relatively stable SM temperatures, facilitating range expansion into cooler waters.

Cyto-inactivation instantly induced by microwave ablation on thyroid nodules

Objective: To explore the effect of microwave ablation on thyroid nodules cell activity by the reaction of key enzyme of cell activation. Methods: From November 2017 to February 2018, 104 patients with 120 thyroid nodules underwent ultrasound-guided microwave ablation at Super-minimally Invasive Medicals, Shanghai International Medical Center, aged 14-55 years, 42 males and 62 females.Twice core needle biopsy were performed before and after thermal ablation.The specimen were using hematoxylin and eosin (HE) staining and enzyme histochemical staining with include succinate dehydrogenase (SDH) and nicotinamide adeninedinucleotide phosphate diaphorase (NADPH-d), respectively, and observe under microscope. Results: Enzyme histochemical staining showed that the positive rate of SDH and NADPH-d in the marginal region and transitional region were 100% before ablation, and were 0% immediately after ablation.The positive rate of SDH and NADPH-d histochemical staining in the same area before and immediately after ablation was statistically significant (P<0.05). Shortly after microwave ablation, the tissue structure and cell morphology showed no obvious alteration in HE stained sections, but in sections with enzyme histochemical staining, the activity of SDH and NADPH-d in ablated tissue disappeared.The accuracy rate of pathologic diagnosis was 100% after ablation. Conclusions: SDH and NADPH-d enzyme activity may be better in evaluating the short-term efficacy of microwave ablation of thyroid nodules than HE staining.

Deletion Of Muscle Acsl1 Caused Myopathy And Fiber Switch

Skeletal muscle exhibits plasticity and complexity in the choice of fuel substrate. During starvation, muscle initiates protein degradation to provide amino acids for liver gluconeogenesis, during acute exercise, muscle uses glucose as the primary fuel, while during rest or prolonged exercise, muscle switches to use primarily fatty acids. Defective fatty acid metabolism is one of the leading causes of metabolic myopathy. Long chain acyl‐CoA synthetase isoform‐1 (ACSL1) converts long chain fatty acids to long chain acyl‐CoAs, before acyl‐CoAs enter mitochondria for β‐oxidation or are esterified to form complex lipids. Although 5 Acsl isoforms exist, deletion of Acsl1 reduces 90% of ACS activity and fatty acid oxidation (FAO) in gastrocnemius, indicating that ACSL1 is the predominant ACSL isoform in the muscle. The objective of this study was to examine how reduced FAO through deletion of Acsl1 affects muscle physiology. We hypothesized that ACSL1‐directed fatty acid metabolism is essential for energy balance in muscle, and that lack of ACSL1 causes myopathy. To test this hypothesis, we used a mouse model in which ACSL1 is specifically deleted in skeletal muscle (Acsl1M−/−). Compared to control mice (Acsl1flox/flox), Acsl1M−/− mice demonstrated 3.4‐ and 1.5‐fold increases in plasma creatine kinase and aspartate aminotransferase activities, indicating that deletion of Acsl1 led to muscle damage. Gastrocnemius from Acsl1M−/− mice also showed the presence of central nuclei, a marker of muscle regeneration. In addition, we detected expression of caspase 3 protein only in Acsl1M−/− muscle, suggesting that the apoptosis pathway was involved. Different muscle fiber types prefer to metabolize different substrates. To investigate the consequence of Acsl1 deletion on muscle fiber type, succinate dehydrogenase staining was performed and it showed a significantly increased percentage of oxidative fibers in Acsl1M−/− gastrocnemius compared to controls. In addition, gene expression of myosin heavy chain I, an oxidative muscle fiber marker, was 6 fold higher in Acsl1M−/− glycolytic fibers than in controls, suggesting a switch from glycolytic to oxidative fibers. We further hypothesized that the fiber switch in Acsl1M−/− gastrocnemius muscle was caused by increased damage to glycolytic fibers compared to oxidative fibers. In summary, deletion of Acsl1 caused muscle damage and regeneration and promoted a glycolytic to oxidative fiber switch. Ongoing studies are examining protein turnover to determine its contribution to muscle damage.Support or Funding InformationSupported by DK56598.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.