Hypoxia-induced gene activity in disused oxidative muscle

Hypoxia is an important modulator of the skeletal muscle's oxidative phenotype. However, little is known regarding the molecular circuitry underlying the muscular hypoxia response and the interaction of hypoxia with other stimuli of muscle oxidative capacity. We hypothesized that exposure of mice to severe hypoxia would promote the expression of genes involved in capillary morphogenesis and glucose over fatty acid metabolism in active or disused soleus muscle of mice. Specifically, we tested whether the hypoxic response depends on oxygen sensing via the alpha-subunit of hypoxia-inducible factor-1 (HIF-1 alpha). Spontaneously active wildtype and HIF-1 alpha heterozygous deficient adult female C57B1/6 mice were subjected to hypoxia (PiO2 70 mmHg). In addition, animals were subjected to hypoxia after 7 days of muscle disuse provoked by hindlimb suspension. Soleus muscles were rapidly isolated and analyzed for transcript level alterations with custom-designed AtlasTM cDNA expression arrays (BD Biosciences) and cluster analysis of differentially expressed mRNAs. Multiple mRNA elevations of factors involved in dissolution and stabilization of blood vessels, glycolysis, and mitochondrial respiration were evident after 24 hours of hypoxia in soleus muscle. In parallel transcripts of fat metabolism were reduced. A comparable hypoxia-induced expression pattern involving complex alterations of the IGF-I axis was observed in reloaded muscle after disuse. This hypoxia response in spontaneously active animals was blunted in the HIF-1 alpha heterozygous deficient mice demonstrating 35% lower HIF-1 alpha mRNA levels. Our molecular observations support the concept that severe hypoxia provides HIF-1-dependent signals for remodeling of existing blood vessels, a shift towards glycolytic metabolism and altered myogenic regulation in oxidative mouse muscle and which is amplified by enhanced muscle use. These findings further imply differential mitochondrial turnover and a negative role of HIF-1 alpha for control of fatty acid oxidation in skeletal muscle exposed to one day of severe hypoxia.

Control of muscle relaxation during anesthesia: a novel approach for clinical routine

During general anesthesia drugs are administered to provide hypnosis, ensure analgesia, and skeletal muscle relaxation. In this paper, the main components of a newly developed controller for skeletal muscle relaxation are described. Muscle relaxation is controlled by administration of neuromuscular blocking agents. The degree of relaxation is assessed by supramaximal train-of-four stimulation of the ulnar nerve and measuring the electromyogram response of the adductor pollicis muscle. For closed-loop control purposes, a physiologically based pharmacokinetic and pharmacodynamic model of the neuromuscular blocking agent mivacurium is derived. The model is used to design an observer-based state feedback controller. Contrary to similar automatic systems described in the literature this controller makes use of two different measures obtained in the train-of-four measurement to maintain the desired level of relaxation. The controller is validated in a clinical study comparing the performance of the controller to the performance of the anesthesiologist. As presented, the controller was able to maintain a preselected degree of muscle relaxation with excellent precision while minimizing drug administration. The controller performed at least equally well as the anesthesiologist.

Role of proton MR for the study of muscle lipid metabolism

1H-MR spectroscopy (MRS) of intramyocellular lipids (IMCL) became particularly important when it was recognized that IMCL levels are related to insulin sensitivity. While this relation is rather complex and depends on the training status of the subjects, various other influences such as exercise and diet also influence IMCL concentrations. This may open insight into many metabolic interactions; however, it also requires careful planning of studies in order to control all these confounding influences. This review summarizes various historical, methodological, and practical aspects of 1H-MR spectroscopy (MRS) of muscular lipids. That includes a differentiation of bulk magnetic susceptibility effects and residual dipolar coupling that can both be observed in MRS of skeletal muscle, yet affecting different metabolites in a specific way. Fitting of the intra- (IMCL) and extramyocellular (EMCL) signals with complex line shapes and the transformation into absolute concentrations is discussed. Since the determination of IMCL in muscle groups with oblique fiber orientation or in obese subjects is still difficult, potential improvement with high-resolution spectroscopic imaging or at higher field strength is considered. Fat selective imaging is presented as a possible alternative to MRS and the potential of multinuclear MRS is discussed. 1H-MRS of muscle lipids allows non-invasive and repeated studies of muscle metabolism that lead to highly relevant findings in clinics and patho-physiology.

Alterations in nitric oxide synthase isoforms in acute lower limb ischemia and reperfusion

Alterations in nitric oxide synthase (NOS) are implicated in ischemia and ischemia-reperfusion injury. Changes in the 3 NOS isoforms in human skeletal muscle subjected to acute ischemia and reperfusion were studied. Muscle biopsies were taken from patients undergoing total knee replacement. Distribution of the specific NOS isoforms within muscle sections was studied using immunohistochemistry. NOS mRNA levels were measured using real-time reverse transcription-polymerase chain reaction and protein levels studied using Western blotting. NOS activity was also assessed using the citrulline assay. All 3 NOS isoforms were found in muscle sections associated with muscle fibers and microvessels. In muscle subjected to acute ischemia and reperfusion, NOS I/neuronal NOS mRNA and protein were elevated during reperfusion. NOS III/endothelial NOS was also upregulated at the protein level during reperfusion. No changes in NOS II/inducible NOS expression or NOS activity occurred. In conclusion, alterations in NOS I and III (neuronal NOS and endothelial NOS) at different levels occurred after acute ischemia and reperfusion in human skeletal muscle; however, this did not result in increased NOS activity. In the development of therapeutic agents based on manipulation of the NO pathway, targeting the appropriate NOS isoenzymes may be important.

Expression of atrophy mRNA relates to tendon tear size in supraspinatus muscle

Skeletal muscle atrophy and fatty infiltration develop after tendon tearing. The extent of atrophy serves as one prognostic factor for the outcome of surgical repair of rotator cuff tendon tears. We asked whether mRNA of genes involved in regulation of degradative processes leading to muscle atrophy, ie, FOXOs, MSTN, calpains, cathepsins, and transcripts of the ubiquitin-proteasome pathway, are overexpressed in the supraspinatus muscle in patients with and without rotator cuff tears. We evaluated biopsy specimens collected during surgery of 53 consecutive patients with different sizes of rotator cuff tendon tears and six without tears. The levels of corresponding gene transcripts in total RNA extracts were assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Supraspinatus muscle atrophy was assessed by MRI. The area of muscle tissue (or atrophy), decreased (increased) with increasing tendon tear size. The transcripts of CAPN1, UBE2B, and UBE3A were upregulated more than twofold in massive rotator cuff tears as opposed to smaller tears or patients without tears. These atrophy gene products may be involved in cellular processes that impair functional recovery of affected muscles after surgical rotator cuff repair. However, the damaging effects of gene products in their respective proteolytic processes on muscle structures and proteins remains to be investigated.

Proton diffusion tensor spectroscopy of metabolites in human muscle in vivo

PURPOSE To study the apparent diffusivity and its directionality for metabolites of skeletal muscle in humans in vivo by (1) H magnetic resonance spectroscopy. METHODS The diffusion tensors were determined on a 3 Tesla MR system using optimized acquisition and processing methods including an adapted STEAM sequence with orientation-dependent diffusion weighting, pulse-triggering with individually adapted delays, eddy-current correction schemes, median filtering, and simultaneous prior-knowledge fitting of all related spectra. RESULTS The average apparent diffusivities, as well as the fractional anisotropies of taurine (ADCav  = 0.74 × 10(-3) s/mm(2) , FA = 0.46), creatine (ADCav  = 0.41 × 10(-3)  s/mm(2) , FA = 0.33), trimethylammonium compounds (ADCav  = 0.48 × 10(-3)  s/mm(2) , FA = 0.34), carnosine (ADCav  = 0.46 × 10(-3)  s/mm(2) , FA = 0.47), and water (ADCav  = 1.5 × 10(-3)  s/mm(2) , FA = 0.36) were estimated. The diffusivities of most metabolites and water were significantly different from each other. Diffusion was found to be anisotropic and the diffusion tensors showed tensor correlation coefficients close to 1 and were hence found to be essentially coaligned. The magnitudes of apparent metabolite diffusivities were largely ordered according to molecular weight, with taurine as the smallest molecule diffusing fastest, both along and across the fiber direction. CONCLUSION Diffusivities, directional dependence of diffusion and fractional anisotropies of (1) H MRS-visible muscle metabolites were presented. It was shown that metabolites share diffusion directionality with water and have similar fractional anisotropies, hinting at similar diffusion barriers. Magn Reson Med, 2014. © 2014 Wiley Periodicals, Inc.

Increasing mean arterial blood pressure in sepsis: effects on fluid balance, vasopressor load and renal function.

INTRODUCTION: The objective of this study was to evaluate the effects of two different mean arterial blood pressure (MAP) targets on needs for resuscitation, organ dysfunction, mitochondrial respiration and inflammatory response in a long-term model of fecal peritonitis. METHODS: Twenty-four anesthetized and mechanically ventilated pigs were randomly assigned (n = 8/group) to a septic control group (septic-CG) without resuscitation until death or one of two groups with resuscitation performed after 12 hours of untreated sepsis for 48 hours, targeting MAP 50-60 mmHg (low-MAP) or 75-85 mmHg (high-MAP). RESULTS: MAP at the end of resuscitation was 56 ± 13 mmHg (mean ± SD) and 76 ± 17 mmHg respectively, for low-MAP and high-MAP groups. One animal each in high- and low-MAP groups, and all animals in septic-CG died (median survival time: 21.8 hours, inter-quartile range: 16.3-27.5 hours). Norepinephrine was administered to all animals of the high-MAP group (0.38 (0.21-0.56) mcg/kg/min), and to three animals of the low-MAP group (0.00 (0.00-0.25) mcg/kg/min; P = 0.009). The high-MAP group had a more positive fluid balance (3.3 ± 1.0 mL/kg/h vs. 2.3 ± 0.7 mL/kg/h; P = 0.001). Inflammatory markers, skeletal muscle ATP content and hemodynamics other than MAP did not differ between low- and high-MAP groups. The incidence of acute kidney injury (AKI) after 12 hours of untreated sepsis was, respectively for low- and high-MAP groups, 50% (4/8) and 38% (3/8), and in the end of the study 57% (4/7) and 0% (P = 0.026). In septic-CG, maximal isolated skeletal muscle mitochondrial Complex I, State 3 respiration increased from 1357 ± 149 pmol/s/mg to 1822 ± 385 pmol/s/mg, (P = 0.020). In high- and low-MAP groups, permeabilized skeletal muscle fibers Complex IV-state 3 respiration increased during resuscitation (P = 0.003). CONCLUSIONS: The MAP targets during resuscitation did not alter the inflammatory response, nor affected skeletal muscle ATP content and mitochondrial respiration. While targeting a lower MAP was associated with increased incidence of AKI, targeting a higher MAP resulted in increased net positive fluid balance and vasopressor load during resuscitation. The long-term effects of different MAP targets need to be evaluated in further studies.

A novel animal model for external anal sphincter insufficiency

Abstract PURPOSE: Reliable animal models are essential to evaluate future therapeutic options like cell-based therapies for external anal sphincter insufficiency. The goal of our study was to describe the most reliable model for external sphincter muscle insufficiency by comparing three different methods to create sphincter muscle damage. METHODS: In an experimental animal study, female Lewis rats (200-250 g) were randomly assigned to three treatment groups (n = 5, each group). The external sphincter muscle was weakened in the left dorsal quadrant by microsurgical excision, cryosurgery, or electrocoagulation by diathermy. Functional evaluation included in vivo measurements of resting pressure, spontaneous muscle contraction, and contraction in response to electrical stimulation of the afferent nerve at baseline and at 2, 4, and 6 weeks after sphincter injury. Masson's trichrome staining and immunofluorescence for skeletal muscle markers was performed for morphological analysis. RESULTS: Peak contraction after electrical stimulation was significantly decreased after sphincter injury in all groups. Contraction forces recovered partially after cryosurgery and electrocoagulation but not after microsurgical excision. Morphological analysis revealed an incomplete destruction of the external sphincter muscle in the cryosurgery and electrocoagulation groups compared to the microsurgery group. CONCLUSIONS: For the first time, three different models of external sphincter muscle insufficiency were directly compared. The animal model using microsurgical sphincter destruction offers the highest level of consistency regarding tissue damage and sphincter insufficiency, and therefore represents the most reliable model to evaluate future therapeutic options. In addition, this study represents a novel model to specifically test the external sphincter muscle function.

Dose response of endotoxin on hepatocyte and muscle mitochondrial respiration in vitro.

INTRODUCTION Results on mitochondrial dysfunction in sepsis are controversial. We aimed to assess effects of LPS at wide dose and time ranges on hepatocytes and isolated skeletal muscle mitochondria. METHODS Human hepatocellular carcinoma cells (HepG2) were exposed to placebo or LPS (0.1, 1, and 10 μg/mL) for 4, 8, 16, and 24 hours and primary human hepatocytes to 1 μg/mL LPS or placebo (4, 8, and 16 hours). Mitochondria from porcine skeletal muscle samples were exposed to increasing doses of LPS (0.1-100 μg/mg) for 2 and 4 hours. Respiration rates of intact and permeabilized cells and isolated mitochondria were measured by high-resolution respirometry. RESULTS In HepG2 cells, LPS reduced mitochondrial membrane potential and cellular ATP content but did not modify basal respiration. Stimulated complex II respiration was reduced time-dependently using 1 μg/mL LPS. In primary human hepatocytes, stimulated mitochondrial complex II respiration was reduced time-dependently using 1 μg/mL LPS. In isolated porcine skeletal muscle mitochondria, stimulated respiration decreased at high doses (50 and 100 μg/mL LPS). CONCLUSION LPS reduced cellular ATP content of HepG2 cells, most likely as a result of the induced decrease in membrane potential. LPS decreased cellular and isolated mitochondrial respiration in a time-dependent, dose-dependent and complex-dependent manner.

Detection of muscle wasting in patients with chronic heart failure using C-terminal agrin fragment: results from the Studies Investigating Co-morbidities Aggravating Heart Failure (SICA-HF).

AIMS Skeletal muscle wasting affects 20% of patients with chronic heart failure and has serious implications for their activities of daily living. Assessment of muscle wasting is technically challenging. C-terminal agrin-fragment (CAF), a breakdown product of the synaptically located protein agrin, has shown early promise as biomarker of muscle wasting. We sought to investigate the diagnostic properties of CAF in muscle wasting among patients with heart failure. METHODS AND RESULTS We assessed serum CAF levels in 196 patients who participated in the Studies Investigating Co-morbidities Aggravating Heart Failure (SICA-HF). Muscle wasting was identified using dual-energy X-ray absorptiometry (DEXA) in 38 patients (19.4%). Patients with muscle wasting demonstrated higher CAF values than those without (125.1 ± 59.5 pmol/L vs. 103.8 ± 42.9 pmol/L, P = 0.01). Using receiver operating characteristics (ROC), we calculated the optimal CAF value to identify patients with muscle wasting as >87.5 pmol/L, which had a sensitivity of 78.9% and a specificity of 43.7%. The area under the ROC curve was 0.63 (95% confidence interval 0.56-0.70). Using simple regression, we found that serum CAF was associated with handgrip (R = - 0.17, P = 0.03) and quadriceps strength (R = - 0.31, P < 0.0001), peak oxygen consumption (R = - 0.5, P < 0.0001), 6-min walk distance (R = - 0.32, P < 0.0001), and gait speed (R = - 0.2, P = 0.001), as well as with parameters of kidney and liver function, iron metabolism and storage. CONCLUSION CAF shows good sensitivity for the detection of skeletal muscle wasting in patients with heart failure. Its assessment may be useful to identify patients who should undergo additional testing, such as detailed body composition analysis. As no other biomarker is currently available, further investigation is warranted.