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.

Gene expression in working skeletal muscle

A number of molecular tools enable us to study the mechanisms of muscle plasticity. Ideally, this research is conducted in view of the structural and functional consequences of the exercise-induced changes in gene expression. Muscle cells are able to detect mechanical, metabolic, neuronal and hormonal signals which are transduced over multiple pathways to the muscle genome. Exercise activates many signaling cascades--the individual characteristic of the stress leading to a specific response of a network of signaling pathways. Signaling typically results in the transcription of multiple early genes among those of the well known for and jun family, as well as many other transcription factors. These bind to the promoter regions of downstream genes initiating the structural response of muscle tissue. While signaling is a matter of minutes, early genes are activated over hours leading to a second wave of transcript adjustments of structure genes that can then be effective over days. Repeated exercise sessions thus lead to a concerted accretion of mRNAs which upon translation results in a corresponding protein accretion. On the structural level, the protein accretion manifests itself for instance as an increase in mitochondrial volume upon endurance training or an increase in myofibrillar proteins upon strength training. A single exercise stimulus carries a molecular signature which is typical both for the type of stimulus (i.e. endurance vs. strength) as well as the actual condition of muscle tissue (i.e. untrained vs. trained). Likewise, it is clearly possible to distinguish a molecular signature of an expressional adaptation when hypoxic stress is added to a regular endurance exercise protocol in well-trained endurance athletes. It therefore seems feasible to use molecular tools to judge the properties of an exercise stimulus much earlier and at a finer level than is possible with conventional functional or structural techniques.

Quantitative (1)H magnetic resonance spectroscopy of myoglobin de- and reoxygenation in skeletal muscle: reproducibility and effects of location and disease

1H-magnetic resonance spectroscopy ((1)H-MRS) of deoxymyoglobin (DMb) provides a means to noninvasively monitor the oxygenation state of human skeletal muscle in work and disease. As shown in this work, it also offers the opportunity to measure the absolute tissue content of DMb, the basic oxygen consumption of resting muscle, and the reperfusion characteristics after release of a pressure cuff. The methodology to determine these tissue properties simultaneously at two positions along the calf is presented. The obtained values are in agreement with invasive determinations. The reproducibility of the (1)H-MRS measurements is established for healthy controls and patients with peripheral arterial disease (PAD). A location dependence in axial direction, as well as differences between controls and patients are demonstrated for all parameters. The reoxygenation time in particular is expected to provide a means to quantitatively monitor therapies aimed at improving muscular perfusion in these patients.

Hydrogel-based engineered skeletal muscle grafts normalize heart function early after myocardial infarction

Tissue engineering represents an attractive approach for the treatment of congestive heart failure. The influence of the differentiation of myogenic graft for functional recovery is not defined. We engineered a biodegradable skeletal muscle graft (ESMG) tissue and investigated its functional effect after implantation on the epicardium of an infarcted heart segment. ESMGs were synthesized by mixing collagen (2 mg/mL), Matrigel (2 mg/mL), and rat skeletal muscle cells (10(6)). Qualitative and quantitative aspects of ESMGs were optimized. Two weeks following coronary ligation, the animals were randomized in three groups: ESMG glued to the epicardial surface with fibrin (ESMG, n = 7), fibrin alone (fibrin, n = 5), or sham operation (sham, n = 4). Echocardiography, histology, and immunostaining were performed 4 weeks later. A cohesive three-dimensional tissular structure formed in vitro within 1 week. Myoblasts differentiated into randomly oriented myotubes. Four weeks postimplantation, ESMGs were vascularized and invaded by granulation tissue. Mean fractional shortening (FS) was, however, significantly increased in the ESMG group as compared with preimplantation values (42 +/- 6 vs. 33 +/- 5%, P < 0.05) and reached the values of controlled noninfarcted animals (control, n = 5; 45 +/- 3%; not significant). Pre- and postimplantation FS did not change over these 4 weeks in the sham group and the fibrin-treated animals. This study showed that it is possible to improve systolic heart function following myocardial infarction through implantation of differentiated muscle fibers seeded on a gel-type scaffold despite a low rate of survival.

ECG-triggered skeletal muscle stimulation improves hemodynamics and physical performance of heart failure patients

BACKGROUND: Muscular counterpulsation (MCP) was developed for circulatory assistance by stimulation of peripheral skeletal muscles. We report on a clinical MCP study in patients with and without chronic heart failure (CHF). METHODS AND RESULTS: MCP treatment was applied (30 patients treated, 25 controls, all under optimal therapy) for 30 minutes during eight days by an ECG-triggered, battery-powered, portable pulse generator with skin electrodes inducing light contractions of calf and thigh muscles, sequentially stimulated at early diastole. Hemodynamic parameters (ECG, blood pressure and echocardiography) were measured one day before and one day after the treatment period in two groups: Group 1 (9 MCP, 11 no MCP) with ejection fraction (EF) above 40% and Group 2 (21 MCP, 14 no MCP) below 40%. In Group 2 (all patients suffering from CHF) mean EF increased by 21% (p<0.001) and stroke volume by 13% (p<0.001), while end systolic volume decreased by 23% (p<0.001). In Group 1, the increase in EF (6%) and stroke volume (8%) was also significant (p<0.05) but less pronounced than in Group 2. Physical exercise duration and walking distance increased in Group 2 by 56% and 72%, respectively. CONCLUSIONS: Noninvasive MCP treatment for eight days substantially improves cardiac function and physical performance in patients with CHF.

Upregulation of alpha-skeletal muscle actin and myosin heavy polypeptide gene products in degenerating rotator cuff muscles

Impaired function of shoulder muscles, resulting from rotator cuff tears, is associated with abnormal deposition of fat in muscle tissue, but corresponding cellular and molecular mechanisms, likely reflected by altered gene expression profiles, are largely unknown. Here, an analysis of muscle gene expression was carried out by semiquantitative RT-PCR in total RNA extracts of supraspinatus biopsies collected from 60 patients prior to shoulder surgery. A significant increase of alpha-skeletal muscle actin (p = 0.0115) and of myosin heavy polypeptide 1 (p = 0.0147) gene transcripts was observed in parallel with progressive fat deposition in the muscle, assessed on parasagittal T1-weighted turbo-spin-echo magnetic resonance images according to Goutallier. Upregulation of alpha-skeletal muscle actin and of myosin heavy polypeptide-1 has been reported to be associated with increased muscle tissue metabolism and oxidative stress. The findings of the present study, therefore, challenge the hypothesis that increased fat deposition in rotator cuff muscle after injury reflects muscle degeneration.

Training in hypoxia and its effects on skeletal muscle tissue

It is well established that local muscle tissue hypoxia is an important consequence and possibly a relevant adaptive signal of endurance exercise training in humans. It has been reasoned that it might be advantageous to increase this exercise stimulus by working in hypoxia. However, as long-term exposure to severe hypoxia has been shown to be detrimental to muscle tissue, experimental protocols were developed that expose subjects to hypoxia only for the duration of the exercise session and allow recovery in normoxia (live low-train high or hypoxic training). This overview reports data from 27 controlled studies using some implementation of hypoxic training paradigms. Hypoxia exposure varied between 2300 and 5700 m and training duration ranged from 10 days to 8 weeks. A similar number of studies was carried out on untrained and on trained subjects. Muscle structural, biochemical and molecular findings point to a specific role of hypoxia in endurance training. However, based on the available data on global estimates of performance capacity such as maximal oxygen uptake (VO2max) and maximal power output (Pmax), hypoxia as a supplement to training is not consistently found to be of advantage for performance at sea level. There is some evidence mainly from studies on untrained subjects for an advantage of hypoxic training for performance at altitude. Live low-train high may be considered when altitude acclimatization is not an option.

Effects of four-week high-fructose diet on gene expression in skeletal muscle of healthy men

AIMS: A high-fructose diet (HFrD) may play a role in the obesity and metabolic disorders epidemic. In rodents, HFrD leads to insulin resistance and ectopic lipid deposition. In healthy humans, a four-week HFrD alters lipid homoeostasis, but does not affect insulin sensitivity or intramyocellular lipids (IMCL). The aim of this study was to investigate whether fructose may induce early molecular changes in skeletal muscle prior to the development of whole-body insulin resistance. METHODS: Muscle biopsies were taken from five healthy men who had participated in a previous four-week HFrD study, during which insulin sensitivity (hyperinsulinaemic euglycaemic clamp), and intrahepatocellular lipids and IMCL were assessed before and after HFrD. The mRNA concentrations of 16 genes involved in lipid and carbohydrate metabolism were quantified before and after HFrD by real-time quantitative PCR. RESULTS: HFrD significantly (P<0.05) increased stearoyl-CoA desaturase-1 (SCD-1) (+50%). Glucose transporter-4 (GLUT-4) decreased by 27% and acetyl-CoA carboxylase-2 decreased by 48%. A trend toward decreased peroxisomal proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was observed (-26%, P=0.06). All other genes showed no significant changes. CONCLUSION: HFrD led to alterations of SCD-1, GLUT-4 and PGC-1alpha, which may be early markers of insulin resistance.

Skeletal muscle cells from insulin-resistant (non-diabetic) individuals are susceptible to insulin desensitization by palmitate

We recently demonstrated that in vivo insulin resistance is not retained in cultured skeletal muscle cells. In the present study, we tested the hypothesis that treating cultured skeletal muscle cells with fatty acids has an effect on insulin action which differs between insulin-sensitive and insulin-resistant subjects. Insulin effects were examined in myotubes from 8 normoglycemic non-obese insulin-resistant and 8 carefully matched insulin-sensitive subjects after preincubation with or without palmitate, linoleate, and 2-bromo-palmitate. Insulin-stimulated glycogen synthesis decreased by 27 +/- 5 % after palmitate treatment in myotubes from insulin-resistant, but not from insulin-sensitive subjects (1.50 +/- 0.08-fold over basal vs. 1.81 +/- 0.09-fold, p = 0.042). Despite this observation, we did not find any impairment in the PI 3-kinase/PKB/GSK-3 pathway. Furthermore, insulin action was not affected by linoleate and 2-bromo-palmitate. In conclusion, our data provide preliminary evidence that insulin resistance of skeletal muscle does not necessarily involve primary defects in insulin action, but could represent susceptibility to the desensitizing effect of fatty acids and possibly other environmental or adipose tissue-derived factors.

Effects of troglitazone on cellular differentiation, insulin signaling, and glucose metabolism in cultured human skeletal muscle cells

To determine the immediate effect of thiazolidinediones on human skeletal muscle, differentiated human myotubes were acutely (1 day) and myoblasts chronically (during the differentiation process) treated with troglitazone (TGZ). Chronic TGZ treatment resulted in loss of the typical multinucleated phenotype. The increase of muscle markers typically observed during differentiation was suppressed, while adipocyte markers increased markedly. Chronic TGZ treatment increased insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and membranous protein kinase B/Akt (PKB/Akt) Ser-473 phosphorylation more than 4-fold. Phosphorylation of p42/44 mitogen-activated protein kinase (42/44 MAPK/ERK) was unaltered. Basal glucose uptake as well as both basal and insulin-stimulated glycogen synthesis increased approximately 1.6- and approximately 2.5-fold after chronic TGZ treatment, respectively. A 2-fold stimulation of PI 3-kinase but no other significant TGZ effect was found after acute TGZ treatment. In conclusion, chronic TGZ treatment inhibited myogenic differentiation of that human muscle while inducing adipocyte-specific gene expression. The effects of chronic TGZ treatment on basal glucose transport may in part be secondary to this transdifferentiation. The enhancing effect on PI 3-kinase and PKB/Akt involved in both differentiation and glycogen synthesis appears to be pivotal in the cellular action of TGZ.

Ramipril increases the protein level of skeletal muscle IRS-1 and alters protein tyrosine phosphatase activity in spontaneously hypertensive rats

To investigate mechanisms by which angiotensin converting enzyme (ACE)-inhibition increases insulin sensitivity, spontaneously hypertensive (SH) rats were treated with or without ramipril (1 mg/kg per day) for 12 weeks. Insulin binding and protein levels of insulin receptor substrate-1 (IRS-1), p85-subunit of phosphatidylinositol 3'-kinase (p85) and Src homology 2 domain-containing phosphatase-2 (SHP2) were then determined in hindlimb muscle and liver. Additionally, protein tyrosine phosphatase (PTPase) activities towards immobilized phosphorylated insulin receptor or phosphorylated IRS-1 of membrane (MF) and cytosolic fractions (CF) of these tissues were measured. Ramipril treatment increased IRS-1-protein content in muscle by 31+/-9% (P<0.05). No effects were observed on IRS-1 content in liver or on insulin binding or protein expression of p85 or SHP2 in both tissues. Ramipril treatment also increased dephosphorylation of insulin receptor by muscle CF (22.0+/-1.0%/60 min compared to 16.8+/-1.5%/60 min; P<0.05), and of IRS-1 by liver MF (37.2+/-1.7%/7.5 min compared to 33.8+/-1.7%/7.5 min; P<0.05) and CF (36.8+/-1.0%/7.5 min compared to 33.2+/-1.0%/7.5 min; P<0.05). We conclude that the observed effects of ACE-inhibition by ramipril on the protein expression of IRS-1 and on PTPase activity might contribute to its effect on insulin sensitivity.

Insulin signaling and action in cultured skeletal muscle cells from lean healthy humans with high and low insulin sensitivity

The aim of these studies was to investigate whether insulin resistance is primary to skeletal muscle. Myoblasts were isolated from muscle biopsies of 8 lean insulin-resistant and 8 carefully matched insulin-sensitive subjects (metabolic clearance rates as determined by euglycemic-hyperinsulinemic clamp: 5.8 +/- 0.5 vs. 12.3 +/- 1.7 ml x kg(-1) x min(-1), respectively; P < or = 0.05) and differentiated to myotubes. In these cells, insulin stimulation of glucose uptake, glycogen synthesis, insulin receptor (IR) kinase activity, and insulin receptor substrate 1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity were measured. Furthermore, insulin activation of protein kinase B (PKB) was compared with immunoblotting of serine residues at position 473. Basal glucose uptake (1.05 +/- 0.07 vs. 0.95 +/- 0.07 relative units, respectively; P = 0.49) and basal glycogen synthesis (1.02 +/- 0.11 vs. 0.98 +/- 0.11 relative units, respectively; P = 0.89) were not different in myotubes from insulin-resistant and insulin-sensitive subjects. Maximal insulin responsiveness of glucose uptake (1.35 +/- 0.03-fold vs. 1.41 +/- 0.05-fold over basal for insulin-resistant and insulin-sensitive subjects, respectively; P = 0.43) and glycogen synthesis (2.00 +/- 0.13-fold vs. 2.10 +/- 0.16-fold over basal for insulin-resistant and insulin-sensitive subjects, respectively; P = 0.66) were also not different. Insulin stimulation (1 nmol/l) of IR kinase and PI 3-kinase were maximal within 5 min (approximately 8- and 5-fold over basal, respectively), and insulin activation of PKB was maximal within 15 min (approximately 3.5-fold over basal). These time kinetics were not significantly different between groups. In summary, our data show that insulin action and signaling in cultured skeletal muscle cells from normoglycemic lean insulin-resistant subjects is not different from that in cells from insulin-sensitive subjects. This suggests an important role of environmental factors in the development of insulin resistance in skeletal muscle.

Association between statin-associated myopathy and skeletal muscle damage

BACKGROUND: Many patients taking statins often complain of muscle pain and weakness. The extent to which muscle pain reflects muscle injury is unknown. METHODS: We obtained biopsy samples from the vastus lateralis muscle of 83 patients. Of the 44 patients with clinically diagnosed statin-associated myopathy, 29 were currently taking a statin, and 15 had discontinued statin therapy before the biopsy (minimal duration of discontinuation 3 weeks). We also included 19 patients who were taking statins and had no myopathy, and 20 patients who had never taken statins and had no myopathy. We classified the muscles as injured if 2% or more of the muscle fibres in a biopsy sample showed damage. Using reverse transcriptase polymerase chain reaction, we evaluated the expression levels of candidate genes potentially related to myocyte injury. RESULTS: Muscle injury was observed in 25 (of 44) patients with myopathy and in 1 patient without myopathy. Only 1 patient with structural injury had a circulating level of creatine phosphokinase that was elevated more than 1950 U/L (10x the upper limit of normal). Expression of ryanodine receptor 3 was significantly upregulated in patients with biopsy evidence of structural damage (1.7, standard error of the mean 0.3). INTERPRETATION: Persistent myopathy in patients taking statins reflects structural muscle damage. A lack of elevated levels of circulating creatine phosphokinase does not rule out structural muscle injury. Upregulation of the expression of ryanodine receptor 3 is suggestive of an intracellular calcium leak.