Skeletal Muscle Of Patients Research Articles

Skeletal muscle PI3K/Akt signaling and ubiquitin‐related enzyme mRNA expression in lung cancer cachexia

The progressive loss of muscle mass is common in lung cancer. Objective to assess possible changes in signal transduction events that regulate protein synthesis and degradation in skeletal muscle of patients with early lung cancer. Muscle biopsies and blood samples were collected from non-small cell lung cancer (NSCLC) subjects, 8 with and 8 without cachexia, and 8 non-cancer controls undergoing thoracic surgery. The abundance and phosphorylation of proteins of the PI3K/Akt signaling pathway were measured by immunoblotting and expression of ubiquitin ligases and deubiquitinating enzyme USP19 mRNA, by RT-qPCR. Cachectic patients had lost 11.5 ± 1.9% body weight, had lower lean and fat mass and higher serum C-reactive protein, IL-6 and IL-8. Phospho-PRAS40Thr246 and p-FoxO1/3aThr24/32 were higher in cachectic than in other groups, despite similar p-AktSer473, but Akt, PRAS40 and FoxO3a abundance was lower. The abundance of the translational inhibitor eukaryotic initiation factor (eIF) 4E binding protein (4E-BP1) was 65% higher than in controls (P =0.009); eIF4E abundance did not differ. MuRF-1, MAFbx and USP19 mRNA expression was not different among groups. Data show lower expression of components of the PI3K/Akt signaling pathway and greater abundance of 4E-BP1 in early NSCLC cachexia that may indicate decreased mRNA translation in skeletal muscle, which in turn, could lead to muscle loss. (Funded by CIHR)

Increased mitochondrial substrate sensitivity in skeletal muscle of patients with type 2 diabetes

Mitochondrial respiration has been linked to insulin resistance. We studied mitochondrial respiratory capacity and substrate sensitivity in patients with type 2 diabetes (patients), and obese and lean control participants. Mitochondrial respiration was measured in permeabilised muscle fibres by respirometry. Protocols for respirometry included titration of substrates for complex I (glutamate), complex II (succinate) and both (octanoyl-carnitine). Myosin heavy chain (MHC) composition, antioxidant capacity (manganese superoxide dismutase [MnSOD]), citrate synthase activity and maximal oxygen uptake (VO2) were also determined. Insulin sensitivity was determined with the isoglycaemic-hyperinsulinaemic clamp technique. Insulin sensitivity was different (p < 0.05) between the groups (patients<obese controls<lean controls). MnSOD was lower in patients than in lean controls. MHC I content was lowest in patients (37 ± 11% [mean ± SE] vs 53 ± 6% and 56 ± 4%) vs obese controls and lean controls, respectively. VO2 was highest in lean controls (40 ± 3 ml min(-1) kg(-1) [mean ± SE]) compared with patients (25 ± 2) and obese controls (27 ± 2). Mitochondrial content (citrate synthase) was higher (p < 0.05) in lean controls than in patients and obese controls. When normalised for mitochondrial content by citrate synthase, mitochondrial respiratory capacity was similar in all groups. However, the half maximal substrate concentration (C(50)) for complex I was significantly lower (p = 0.03) in patients (1.1 ± 0.2 mmol/l [mean ± SE]) than in obese (2.0 ± 0.3) and lean (1.8 ± 0.3) controls. Likewise, C(50) for complex II was lower (p = 0.02) in patients (3.5 ± 0.2 mmol/l [mean ± SE]) than in obese controls (4.1 ± 0.2), but did not differ from that in lean controls (3.8 ± 0.4). Substrate sensitivity for octanoyl-carnitine did not differ between groups. Increased mitochondrial substrate sensitivity is seen in skeletal muscle from type 2 diabetic patients and is confined to non-lipid substrates. Respiratory capacity per mitochondrion is not decreased with insulin resistance.

Trimetazidine improves exercise performance in patients with peripheral arterial disease

Trimetazidine, an inhibitor of free fatty acids (FFA) oxidation, shifts cardiac and muscle metabolism from FFA to glucose utilization. This effect results in a greater production of high energy phosphates and ultimately into an anti-ischemic effect. Whether the anti-ischemic cardiac effects of trimetazidine (TMZ) can be translated to skeletal muscle in patients with claudication is unknown. We investigated the effectiveness of TMZ on functional performance in patients with peripheral arterial disease (PAD) and claudication. One hundred patients with claudication were enrolled in a parallel, double-blind, 3 months study. Patients were randomized to receive TMZ or matching placebo and were included in a domiciliary exercise program, consisting in daily sessions of aerobic and isotonic exercise for at least five days a week. All patients underwent a treadmill test, evaluating maximal walking distance (MWD), and ankle–brachial index (ABI) at baseline and after 3 months. ABI was similar in the two groups at baseline and did not significantly change at the end of the study in either groups (0.83 + 0.04 vs 0.85 + 0.03, TMZ vs placebo, respectively). MWD improved in all patients with exercise training; however, a greater improvement in MWD was observed with TMZ compared to placebo (23% vs 14%, p < 0.0001). Physical training ameliorates functional performance in PAD. The adjunct of TMZ to exercise induces a greater improvement in MWD, suggesting that the inhibition of FFA oxidation improves functional capacity in patients with PAD and claudication.

Aldosterone increases Na+-K+-ATPase activity in skeletal muscle of patients with Conn’s syndrome

In Conn's syndrome, hypokalaemia normally results from renal potassium loss because of the effect of excess aldosterone on Na(+) -K(+) -ATPase in principal cells. Little is known about the effect of aldosterone on cellular potassium redistribution in skeletal muscle. Our study determined the effect of aldosterone on muscle Na(+) -K(+) -ATPase. Muscle biopsies were taken from six patients immediately before and 1 month after adrenalectomy. Ten age-matched subjects with normal levels of circulating aldosterone served as controls. Average plasma aldosterone was significantly higher in presurgery (235·0 ± 51·1 pg/ml) than postsurgery (64·5 ± 25·1 pg/ml) patients. Similarly, Na(+) -K(+) -ATPase activity, relative mRNA expression of α(2) (not α(1) or α(3) ) and β(1) (not β(2) or β(3) ), and protein abundance of α(2) and β(1) subunits were greater in pre- than postsurgery samples (128·7 ± 12·3 vs 79·4 ± 13·3 nmol·mg/protein/h, 2·45 ± 0·31 vs 1·04 ± 0·17, 1·92 ± 0·22 vs1·02 ± 0·14, 2·17 ± 0·33 vs 0·98 ± 0·09 and 1·70 ± 0·17 vs 0·90 ± 0·17, respectively, all P<0·05). The activity and mRNA expression of the α(2) and β(1) subunits correlated well with plasma aldosterone levels (r = 0·71, r = 0·75 and r = 0·78, respectively, all P < 0·01). Our study provides the first evidence in human skeletal muscle that increased plasma aldosterone leads to increased Na(+) -K(+) -ATPase activity via increases in α(2) and β(1) subunit mRNAs and their protein expressions. The increased activity may contribute in part to the induction of hypokalaemia in patients with Conn's syndrome.

The Overexpression of S105N Mutant Rad Leads Intracellular Ca2+ Overload Via Up-Regulation of Cardiac Ryanodine Receptor Activity

Background: The ras-related small G-protein Rad was originally identified from skeletal muscle of patients with type 2 diabetes mellitus. We have recently reported that Rad plays a critical role in generating arrhythmias. The study was aimed to elucidate the role of Rad in intracellular calcium homeostasis. Methods and Results: We developed the transgenic mice that overexpress S105N mutant Rad driven by α-myosin heavy chain promoter. We measure intracellular Ca concentration ([Ca2+]i) from isolated cardiomyocytes by confocal microscopy. The amplitude of [Ca2+]i transient was significantly increased in S105N-Rad-TG cardiomyocytes, compared with littermate. Furthermore, the Ca2+ sparks and the spontaneous Ca2+ waves occurred with greater frequency in S105N-Rad-TG cells, implicating the enhanced activity of SERCA. We recorded L-type calcium currents (ICa-L) and action potentials (APs) from isolated cardiomyocytes using whole cell patch-clamp technique. The peak ICa-L was dramatically larger in the S105N-Rad-TG cells than in littermate. Early afterdepolarization (EAD) and delayed afterdepolarization (DAD) were frequently observed in S105N-Rad-TG. Then, the phosphorylation of RYR2 at Ser2809 and PKA was significantly enhanced in S105N-Rad-TG by Western blot. Conclusions: Our results provided the first evidence that Rad might regulate RYR2 activity possibly via downstream of PKA signaling pathway.

The effect of chronic heart failure and type 2 diabetes on insulin-stimulated endothelial function is similar and additive

AimChronic heart failure is associated with endothelial dysfunction and insulin resistance. The aim of this investigation was to study insulin-stimulated endothelial function and glucose uptake in skeletal muscles in patients with heart failure in comparison to patients with type 2 diabetes.MethodsTwenty-three patients with systolic heart failure and no history of diabetes, seven patients with both systolic heart failure and type 2 diabetes, 19 patients with type 2 diabetes, and ten healthy controls were included in the study. Endothelial function was studied by venous occlusion plethysmography. Insulin-stimulated endothelial function was assessed after intra-arterial infusion of insulin followed by co-infusion with serotonin in three different dosages. Forearm glucose uptake was measured during the insulin infusion.ResultsPatients with systolic heart failure had impaired insulin-stimulated endothelial function. The percentage increase in blood flow during co-infusion with insulin and serotonin dose response study was 24.74% ± 6.16%, 23.50% ± 8.32%, and 22.29% ± 10.77% at the three doses respectively, compared to the healthy control group 45.96% ± 11.56%, 67.40% ± 18.11% and 84.57% ± 25.73% (P = 0.01). Insulin-stimulated endothelial function was similar in heart failure patients and patients with type 2 diabetes, while it was further deteriorated in patients suffering from both heart failure and diabetes with a percentage increase in blood flow of 19.15% ± 7.81%, −2.35% ± 11.76%, and 5.82% ± 17.70% at the three doses of serotonin, respectively. Forearm glucose uptake was impaired in patients with heart failure compared to healthy controls (P = 0.03) and tended to be further impaired by co-existence of diabetes (P = 0.08).ConclusionSystolic heart failure and type 2 diabetes result in similar vascular insulin resistance and reduced muscular insulin-stimulated glucose uptake. The effects of systolic heart failure and type 2 diabetes appear to be additive.

Ischemia- Reperfusion Challenge in Human Skeletal Muscle: Study in Knee Arthroplasty Surgery

Background: There are many studies that analyze ischemia-reperfusion injury in different organs (brain, heart...) but they are very scarce in skeletal muscle. The few studies performed in humans show partial results, and none include morphological, inflammatory, biochemical, metabolic and hemodynamic alterations. The objective of our study was to typify ischemic and ischemia-reperfusion injury in skeletal muscle in patients undergoing total knee arthroplasty from several points of view: morphological, biochemical, metabolic and hemodynamical, as well as to evaluate the protective effect of a single-dose of corticoids on the above-mentioned parameters. Methods: Forty-one patients participated in the study. They were randomly assigned to either group A: no corticoid administered; or to group B: a single dose of methylprednisolone was administered before performing ischemia. Blood samples were drawn at different intervals for determination of inflammatory biomarkers and necrosis enzymes, and three muscle biopsies were performed for histological studies. Results: The ischemia period ranged from 50 to 84 minutes in both groups. There were no histological differences either between the groups. We found significant differences in inflammatory markers: there was a higher increase in C-reactive protein, erythrocyte sedimentation rate and Interleukin-6 in group A, while the leukocyte count was higher in group B. \ Conclusions: Human skeletal muscle can bear periods of ischemia followed by reperfusion as far as 1.5 h without showing any sign of structural, ultrastructural or inmunoshistochemical damage. We have described the biochemical changes that take place in patients submitted to total knee arthroplasty with tourniquet under spinal anesthesia during the first 48 h post-procedure. Adding a single dose of methylprednisolone 7 mg/kg IV immediately prior to surgery significantly reduced the levels of some markers of morbidity.

Insulin Resistance Is Not Conserved in Myotubes Established from Women with PCOS

BackgroundPolycystic ovary syndrome (PCOS) is the most common endocrine disorder among premenopausal women, who often develop insulin resistance. We tested the hypothesis that insulin resistance in skeletal muscle of patients with polycystic ovary syndrome (PCOS) is an intrinsic defect, by investigating the metabolic characteristics and gene expression of in vitro differentiated myotubes established from well characterized PCOS subjects.MethodsUsing radiotracer techniques, RT-PCR and enzyme kinetic analysis we examined myotubes established from PCOS subjects with or without pioglitazone treatment, versus healthy control subjects who had been extensively metabolically characterized in vivo. Results Myotubes established from PCOS and matched control subjects comprehensively expressed all insulin-sensitive biomarkers; glucose uptake and oxidation, glycogen synthesis and lipid uptake. There were no significant differences between groups either at baseline or during acute insulin stimulation, although in vivo skeletal muscle was insulin resistant. In particular, we found no evidence for defects in insulin-stimulated glycogen synthase activity between groups. Myotubes established from PCOS patients with or without pioglitazone treatment also showed no significant differences between groups, neither at baseline nor during acute insulin stimulation, although in vivo pioglitazone treatment significantly improved insulin sensitivity. Consistently, the myotube cultures failed to show differences in mRNA levels of genes previously demonstrated to differ in PCOS patients with or without pioglitazone treatment (PLEK, SLC22A16, and TTBK).ConclusionThese results suggest that the mechanisms governing insulin resistance in skeletal muscle of PCOS patients in vivo are not primary, but rather adaptive.Trial RegistrationClinicalTrials.gov NCT00145340

FTO Is Increased in Muscle During Type 2 Diabetes, and Its Overexpression in Myotubes Alters Insulin Signaling, Enhances Lipogenesis and ROS Production, and Induces Mitochondrial Dysfunction

OBJECTIVEA strong association between genetic variants and obesity was found for the fat mass and obesity-associated gene (FTO). However, few details are known concerning the expression and function of FTO in skeletal muscle of patients with metabolic diseases.RESEARCH DESIGN AND METHODSWe investigated basal FTO expression in skeletal muscle from obese nondiabetic subjects and type 1 and type 2 diabetic patients, compared with age-matched control subjects, and its regulation in vivo by insulin, glucose, or rosiglitazone. The function of FTO was further studied in myotubes by overexpression experiments.RESULTSWe found a significant increase of FTO mRNA and protein levels in muscle from type 2 diabetic patients, whereas its expression was unchanged in obese or type 1 diabetic patients. Moreover, insulin or glucose infusion during specific clamps did not regulate FTO expression in skeletal muscle from control or type 2 diabetic patients. Interestingly, rosiglitazone treatment improved insulin sensitivity and reduced FTO expression in muscle from type 2 diabetic patients. In myotubes, adenoviral FTO overexpression increased basal protein kinase B phosphorylation, enhanced lipogenesis and oxidative stress, and reduced mitochondrial oxidative function, a cluster of metabolic defects associated with type 2 diabetes.CONCLUSIONSThis study demonstrates increased FTO expression in skeletal muscle from type 2 diabetic patients, which can be normalized by thiazolidinedione treatment. Furthermore, in vitro data support a potential implication of FTO in oxidative metabolism, lipogenesis and oxidative stress in muscle, suggesting that it could be involved in the muscle defects that characterize type 2 diabetes.

Impact of the Mitochondrial Genetic Background in Complex III Deficiency

BackgroundIn recent years clinical evidence has emphasized the importance of the mtDNA genetic background that hosts a primary pathogenic mutation in the clinical expression of mitochondrial disorders, but little experimental confirmation has been provided. We have analyzed the pathogenic role of a novel homoplasmic mutation (m.15533 A>G) in the cytochrome b (MT-CYB) gene in a patient presenting with lactic acidosis, seizures, mild mental delay, and behaviour abnormalities.MethodologySpectrophotometric analyses of the respiratory chain enzyme activities were performed in different tissues, the whole muscle mitochondrial DNA of the patient was sequenced, and the novel mutation was confirmed by PCR-RFLP. Transmitochondrial cybrids were constructed to confirm the pathogenicity of the mutation, and assembly/stability studies were carried out in fibroblasts and cybrids by means of mitochondrial translation inhibition in combination with blue native gel electrophoresis.Principal FindingsBiochemical analyses revealed a decrease in respiratory chain complex III activity in patient's skeletal muscle, and a combined enzyme defect of complexes III and IV in fibroblasts. Mutant transmitochondrial cybrids restored normal enzyme activities and steady-state protein levels, the mutation was mildly conserved along evolution, and the proband's mother and maternal aunt, both clinically unaffected, also harboured the homoplasmic mutation. These data suggested a nuclear genetic origin of the disease. However, by forcing the de novo functioning of the OXPHOS system, a severe delay in the biogenesis of the respiratory chain complexes was observed in the mutants, which demonstrated a direct functional effect of the mitochondrial genetic background.ConclusionsOur results point to possible pitfalls in the detection of pathogenic mitochondrial mutations, and highlight the role of the genetic mtDNA background in the development of mitochondrial disorders.

Thyroid hormone receptors are down-regulated in skeletal muscle of patients with non-thyroidal illness syndrome secondary to non-septic shock

Non-thyroidal illness syndrome (NTIS) is related to changes in thyroid hormone (TH) physiology. Skeletal muscle (SM) plays a major role in metabolism, and TH regulates SM phenotype and metabolism. We aimed to characterize the SM of non-septic shock NTIS patients in terms of: i) expression of genes and proteins involved in TH metabolism and actions; and ii) NFKB's pathway activation, a responsible factor for some of the phenotypic changes in NTIS. We also investigated whether the patient's serum can induce in vitro the effects observed in vivo. Serum samples and SM biopsies from 14 patients with non-septic shock NTIS and 11 controls. Gene and protein expression and NFKB1 activation were analyzed by quantitative PCR and immunoblotting. Human SM cell (HSkMC) cultures to investigate the effects of patient's serum on TH action mediators. Patients with non-septic shock NTIS showed higher levels of pro-inflammatory cytokines than controls. Expression of TRβ (THRB), TRα1 (THRA), and retinoid X receptor γ (RXRG) was decreased in NTIS patients. RXRA gene expression was higher, but its protein was lower in NTIS than controls, suggesting the existence of a post-transcriptional mechanism that down-regulates protein levels. NFKB1 pathway activation was not different between NTIS and control patients. HSkMC incubated with patient's serum increased TH receptor and RXRG gene expression after 48 h. Patients with non-septic shock NTIS showed decreased expression of TH receptors and RXRs, which were not related to increased activation of the NFKB1 pathway. These findings could not be replicated in cultures of HSkMCs incubated in the patient's serum.

Opposite effects of pioglitazone and rosiglitazone on mitochondrial respiration in skeletal muscle of patients with type 2 diabetes

Skeletal muscle insulin resistance has been linked to mitochondrial dysfunction. We examined how improvements in muscular insulin sensitivity following rosiglitazone (ROSI) or pioglitazone (PIO) treatment would affect muscle mitochondrial function in patients with type 2 diabetes mellitus (T2DM). Muscle biopsies were obtained from 21 patients with T2DM before and after 12 weeks on either ROSI (4 mg once daily) [n = 12; age, 59.2 +/- 2.2 years; body mass index (BMI), 29.6 +/- 0.7 kg/m(2)] or PIO (30 mg once daily) (n = 9; age, 56.3 +/- 2.4 years; BMI, 29.5 +/- 1.5 kg/m(2)). An age- and BMI-matched control group was also included (n = 8; age, 61.8 +/- 2.3 years; BMI, 28.4 +/- 0.6 kg/m(2)). Insulin sensitivity, citrate synthase- and beta-hydroxyacyl-CoA-dehydrogenase (HAD) activity, intramuscular triglyceride (IMTG) and protein content of complexes I-IV were measured, while mitochondrial respiration per milligram muscle was measured in saponin-treated skinned muscle fibres using high-resolution respirometry. Mitochondrial respiration per milligram muscle was lower in T2DM compared to controls at baseline and decreased during ROSI treatment but increased during PIO treatment. Citrate synthase activity and average protein content of complexes I-IV were unchanged in the ROSI group, but protein content of complexes II and III increased during PIO treatment. Insulin sensitivity improved in all patients, but IMTG levels were unchanged. We show opposite effects of ROSI and PIO on mitochondrial respiration, and also show that insulin sensitivity can be improved independently of changes in mitochondrial respiration. We confirm that mitochondrial respiration is reduced in T2DM compared to age- and BMI-matched control subjects.

Statin Therapy and the Expression of Genes that Regulate Calcium Homeostasis and Membrane Repair in Skeletal Muscle

In skeletal muscle of patients with clinically diagnosed statin-associated myopathy, discrete signs of structural damage predominantly localize to the T-tubular region and are suggestive of a calcium leak. The impact of statins on skeletal muscle of non-myopathic patients is not known. We analyzed the expression of selected genes implicated in the molecular regulation of calcium and membrane repair, in lipid homeostasis, myocyte remodeling and mitochondrial function. Microscopic and gene expression analyses were performed using validated TaqMan custom arrays on skeletal muscle biopsies of 72 age-matched subjects who were receiving statin therapy (n = 38), who had discontinued therapy due to statin-associated myopathy (n = 14), and who had never undergone statin treatment (n = 20). In skeletal muscle, obtained from statin-treated, non-myopathic patients, statins caused extensive changes in the expression of genes of the calcium regulatory and the membrane repair machinery, whereas the expression of genes responsible for mitochondrial function or myocyte remodeling was unaffected. Discontinuation of treatment due to myopathic symptoms led to a normalization of gene expression levels, the genes encoding the ryanodine receptor 3, calpain 3, and dystrophin being the most notable exceptions. Hence, even in clinically asymptomatic (non-myopathic) patients, statin therapy leads to an upregulation in the expression of genes that are concerned with skeletal muscle regulation and membrane repair.

Effect of physical training on mitochondrial respiration and reactive oxygen species release in skeletal muscle in patients with obesity and type 2 diabetes

Studies have suggested a link between insulin resistance and mitochondrial dysfunction in skeletal muscles. Our primary aim was to investigate the effect of aerobic training on mitochondrial respiration and mitochondrial reactive oxygen species (ROS) release in skeletal muscle of obese participants with and without type 2 diabetes. Type 2 diabetic men (n = 13) and control (n = 14) participants matched for age, BMI and physical activity completed 10 weeks of aerobic training. Pre- and post-training muscle biopsies were obtained before a euglycaemic-hyperinsulinaemic clamp and used for measurement of respiratory function and ROS release in isolated mitochondria. Training significantly increased insulin sensitivity, maximal oxygen consumption and muscle mitochondrial respiration with no difference between groups. When expressed in relation to a marker of mitochondrial density (intrinsic mitochondrial respiration), training resulted in increased mitochondrial ADP-stimulated respiration (with NADH-generating substrates) and decreased respiration without ADP. Intrinsic mitochondrial respiration was not different between groups despite lower insulin sensitivity in type 2 diabetic participants. Mitochondrial ROS release tended to be higher in participants with type 2 diabetes. Aerobic training improves muscle respiration and intrinsic mitochondrial respiration in untrained obese participants with and without type 2 diabetes. These adaptations demonstrate an increased metabolic fitness, but do not seem to be directly related to training-induced changes in insulin sensitivity.

Therapeutic Potential of Proteasome Inhibition in Duchenne and Becker Muscular Dystrophies

Duchenne muscular dystrophy (DMD) and its milder allelic variant, Becker muscular dystrophy (BMD), result from mutations of the dystrophin gene and lead to progressive muscle deterioration. Enhanced activation of proteasomal degradation underlies critical steps in the pathogenesis of the DMD/BMD dystrophic process. Previously, we demonstrated that treatment with the proteasome inhibitor MG-132 rescues the cell membrane localization of dystrophin and the dystrophin glycoprotein complex in mdx mice, a natural genetic mouse model of DMD. The current work aims to thoroughly define the therapeutic potential in dystrophinopathies of Velcade, a drug that selectively blocks the ubiquitin-proteasome pathway. Velcade is particularly intriguing since it has been approved for the treatment of multiple myeloma. Therefore, its side effects in humans have been explored. Velcade effects were analyzed through two independent methodological approaches. First, we administered the drug systemically in mdx mice over a 2-week period. In this system, Velcade restores the membrane expression of dystrophin and dystrophin glycoprotein complex members and improves the dystrophic phenotype. In a second approach, we treated with the compound explants from muscle biopsies of DMD or BMD patients. We show that the inhibition of the proteasome pathway up-regulates dystrophin, alpha-sarcoglycan, and beta-dystroglycan protein levels in explants from BMD patients, whereas it increases the proteins of the dystrophin glycoprotein complex in DMD cases.

Calcineurin signaling and PGC-1α expression are suppressed during muscle atrophy due to diabetes

PGC-1α is a transcriptional coactivator that controls energy homeostasis through regulation of glucose and oxidative metabolism. Both PGC-1α expression and oxidative capacity are decreased in skeletal muscle of patients and animals undergoing atrophy, suggesting that PGC-1α participates in the regulation of muscle mass. PGC-1α gene expression is controlled by calcium- and cAMP-sensitive pathways. However, the mechanism regulating PGC-1α in skeletal muscle during atrophy remains unclear. Therefore, we examined the mechanism responsible for decreased PGC-1α expression using a rodent streptozotocin (STZ) model of chronic diabetes and atrophy. After 21 days, the levels of PGC-1α protein and mRNA were decreased. We examined the activation state of CREB, a potent activator of PGC-1α transcription, and found that phospho-CREB was paradoxically high in muscle of STZ-rats, suggesting that the cAMP pathway was not involved in PGC-1α regulation. In contrast, expression of calcineurin (Cn), a calcium-dependent phosphatase, was suppressed in the same muscles. PGC-1α expression is regulated by two Cn substrates, MEF2 and NFATc. Therefore, we examined MEF2 and NFATc activity in muscles from STZ-rats. Target genes MRF4 and MCIP1.4 mRNAs were both significantly reduced, consistent with reduced Cn signaling. Moreover, levels of MRF4, MCIP1.4, and PGC-1α were also decreased in muscles of CnAα −/− and CnAβ −/− mice without diabetes indicating that decreased Cn signaling, rather than changes in other calcium- or cAMP-sensitive pathways, were responsible for decreased PGC-1α expression. These findings demonstrate that Cn activity is a major determinant of PGC-1α expression in skeletal muscle during diabetes and possibly other conditions associated with loss of muscle mass.

Cardiovascular disease in patients with inflammatory rheumatic disease is associated with up‐regulation of markers of inflammation in cardiac microvessels and cardiomyocytes

Various inflammatory rheumatic diseases (IRDs) are associated with increased mortality due to cardiovascular disease. The aim of this study was to investigate heart biopsy specimens obtained from patients undergoing coronary artery bypass grafting and compare markers of inflammation and endothelial cell activation in the cardiac and skeletal muscle of patients with and those without IRD. Paired biopsy specimens of cardiac and skeletal muscle were obtained from 22 consecutive patients with IRD and 8 patients without IRD, all of whom were undergoing coronary artery bypass grafting. The biopsy specimens were evaluated in a blinded manner by conventional microscopy and digital image analysis for cell markers (CD3, CD4, CD8, CD68, CD163, and CD31), HLA (HLA-ABC, HLA-DR, and HLA-DQ), adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1), and proinflammatory cytokines (interleukin-1alpha, interleukin-1beta, and tumor necrosis factor). Patients with IRD had significantly higher expression of adhesion molecules, proinflammatory cytokines, and all classes of HLA on cardiomyocytes and endothelial cells but no increase on mononuclear cells in the myocardium compared with patients without IRD. Furthermore, cardiac muscle from patients with IRD displayed significantly higher local expression of inflammation and activation of cardiac microvessels compared with skeletal muscle from the same patients. Patients with cardiovascular disease had increased expression of adhesion molecules, HLA, and proinflammatory cytokines in heart tissue, indicating local inflammation involving microvessels and cardiomyocytes that could play a role in the pathogenesis of cardiovascular disease. The more pronounced changes in patients with IRD compared with patients without IRD might contribute to the increased risk of cardiovascular disease and premature death in patients with IRD.

Resistance Training Prevents Deterioration in Quadriceps Muscle Function During Acute Exacerbations of Chronic Obstructive Pulmonary Disease

Exacerbations of chronic obstructive pulmonary disease (COPD) acutely reduce skeletal muscle strength and result in long-term loss of functional capacity. To investigate whether resistance training is feasible and safe and can prevent deteriorating muscle function during exacerbations of COPD. Forty patients (FEV(1) 49 +/- 17% predicted) hospitalized with a severe COPD exacerbation were randomized to receive usual care or an additional resistance training program during the hospital admission. Patients were followed up for 1 month after discharge. Primary outcomes were quadriceps force and systemic inflammation. A muscle biopsy was taken in a subgroup of patients to assess anabolic and catabolic pathways. Resistance training did not yield higher systemic inflammation as indicated by C-reactive protein levels and could be completed uneventfully. Enhanced quadriceps force was seen at discharge (+9.7 +/- 16% in the training group; -1 +/- 13% in control subjects; P = 0.05) and at 1 month follow-up in the patients who trained. The 6-minute walking distance improved after discharge only in the group who received resistance training (median 34; interquartile range, 14-61 m; P = 0.002). In a subgroup of patients a muscle biopsy showed a more anabolic status of skeletal muscle in patients who followed training. Myostatin was lower (P = 0.03) and the myogenin/MyoD ratio tended to be higher (P = 0.08) in the training group compared with control subjects. Resistance training is safe, successfully counteracts skeletal muscle dysfunction during acute exacerbations of COPD, and may up-regulate the anabolic milieu in the skeletal muscle. Clinical trial registered with www.clinicaltrials.gov (NCT00877084).

Functional Efficacy of Dystrophin Expression from Plasmids Delivered to mdx Mice by Hydrodynamic Limb Vein Injection

In these studies we delivered by hydrodynamic limb vein (HLV) injection plasmid DNA (pDNA) expressing the full-length mouse dystrophin gene to skeletal muscles throughout the hind limbs of the mdx mouse model for Duchenne muscular dystrophy (DMD). We evaluated the levels and stability of dystrophin expression and measured the resulting muscle protection, using Evans blue dye (EBD) to mark the damaged myofibers. Plasmid delivery was as efficient in the dystrophic mice as in wild-type mice and equally efficient in young adult and old mice, as long as the dose of pDNA was adjusted for the target muscle weight. The HLV gene delivery procedure was tolerated well by the dystrophic mice and repeat injections could be performed over an extended period of time. Multiple gene deliveries additively increased the amount of dystrophin protein and also increased the percentages of dystrophin-expressing myofibers. Plasmids expressing dystrophin from a cytomegalovirus (CMV) promoter construct containing the HMG1 intron provided stable dystrophin expression for the life of the mouse and provided significant benefit to the limbs. EBD staining showed that dystrophin gene delivery preserved myofibers in the CMV-HMGi-mDys-injected leg by 2.5- to 5-fold in large groups of muscles and by 2.5-fold throughout the injected legs, compared with the contralateral control legs injected with a nonexpressing plasmid. A similar degree of protection was measured in young adult mice evaluated soon after the last gene delivery and in aged mice injected over an extended period of time. This degree of protection resulted from 18 to 20% of the normal level of dystrophin protein, with 11-16% dystrophin-expressing myofibers. These studies show promise for the use of HLV injections to deliver therapeutic doses of full-length dystrophin-expressing plasmids for long-lasting protection of skeletal muscles in patients with DMD.

Expression and Functional Significance of Nicotinamide N-methyl Transferase in Skeletal Muscles of Patients with Chronic Obstructive Pulmonary Disease

Nicotinamide N-methyl transferase (NNMT) is highly expressed in quadriceps muscles of patients with chronic obstructive pulmonary disease (COPD). However, its expression in the diaphragm of these patients has not been assessed. The functional significance of NNMT induction in skeletal muscles of patients with COPD is also unknown. (1) To compare NNMT expressions in the diaphragm and quadriceps muscles of patients with COPD. (2) To identify the influence of proinflammatory cytokines on NNMT expression. (3) To assess the influence of NNMT on indices of myogenesis (satellite cell migration and proliferation) and the defense against oxidative stress. Costal diaphragm muscle biopsies were acquired from 13 patients with moderate and severe COPD and 8 control subjects. Quadriceps muscle biopsies were obtained from 12 patients with COPD and 14 control subjects. NNMT expressions were significantly elevated in the diaphragm and quadriceps muscles of patients with COPD; however, the relative induction of NNMT expression was greater in the quadriceps muscle (10-fold) than it was in the diaphragm (2-fold). NNMT expressions correlated negatively with the severity of COPD and limb muscle wasting. In skeletal myoblasts, NNMT expression was significantly induced by IL-6, transforming growth factor beta, and tumor necrosis factor-alpha. Overexpression of NNMT in myoblasts triggered a significant increase in proliferation and migration, but had no influence on cell death. Carbonyl formation, induced by exposing myoblasts to H(2)O(2), was significantly attenuated when NNMT was overexpressed. Up-regulation of NNMT expression in the skeletal muscles of patients with COPD may represent an adaptive response designed to improve myogenesis and defend against oxidative stress.