Assessing dystrophies and other muscle diseases at the nanometer scale by atomic force microscopy.

AIM: Atomic force microscopy nanoindentation of myofibers was used to assess and quantitatively diagnose muscular dystrophies from human patients. MATERIALS & METHODS: Myofibers were probed from fresh or frozen muscle biopsies from human dystrophic patients and healthy volunteers, as well as mice models, and Young's modulus stiffness values were determined. RESULTS: Fibers displaying abnormally low mechanical stability were detected in biopsies from patients affected by 11 distinct muscle diseases, and Young's modulus values were commensurate to the severity of the disease. Abnormal myofiber resistance was also observed from consulting patients whose muscle condition could not be detected or unambiguously diagnosed otherwise. DISCUSSION & CONCLUSION: This study provides a proof-of-concept that atomic force microscopy yields a quantitative read-out of human muscle function from clinical biopsies, and that it may thereby complement current muscular dystrophy diagnosis.

Vitamin D: a review on its effects on muscle strength, the risk of fall, and frailty.

Vitamin D is the main hormone of bone metabolism. However, the ubiquitary nature of vitamin D receptor (VDR) suggests potential for widespread effects, which has led to new research exploring the effects of vitamin D on a variety of tissues, especially in the skeletal muscle. In vitro studies have shown that the active form of vitamin D, calcitriol, acts in myocytes through genomic effects involving VDR activation in the cell nucleus to drive cellular differentiation and proliferation. A putative transmembrane receptor may be responsible for nongenomic effects leading to rapid influx of calcium within muscle cells. Hypovitaminosis D is consistently associated with decrease in muscle function and performance and increase in disability. On the contrary, vitamin D supplementation has been shown to improve muscle strength and gait in different settings, especially in elderly patients. Despite some controversies in the interpretation of meta-analysis, a reduced risk of falls has been attributed to vitamin D supplementation due to direct effects on muscle cells. Finally, a low vitamin D status is consistently associated with the frail phenotype. This is why many authorities recommend vitamin D supplementation in the frail patient.

Enhanced muscle strength with carbohydrate supplement two hours before open cholecystectomy: a randomized, double-blind study

Objective: to investigate the effects of preoperative fasting abbreviation with oral supplementation with carbohydrate in the evolution of grip strength in patients undergoing cholecystectomy by laparotomy. Methods : we conducted a clinical, randomizeddouble blind study with adult female patients, aged 18-60 years. Patients were divided into two groups: Control Group, with fasting prescription 6-8h until the time of operation; and Intervention Group, which received prescription of fasting for solids 6-8h before surgery, but ingested an oral supplement containing 12.5% carbohydrate, six (400ml) and two (200ml) hours before theprocedure. The handgrip strength was measured in both hands in both groups, at patient's admission (6h before surgery), the immediate pre-operative time (1h before surgery) and 12-18h postoperatively. Results : we analyzed 27 patients, 14 in the intervention group and 13 in the control group. There was no mortality. The handgrip strength (mean [standard deviation]) was significantly higher in the intervention group in the three periods studied, in at least one hand: preoperatively in the dominant hand (27.8 [2.6] vs 24.1 [3.7] kg; p=0.04), in the immediate preoperative in both hands, and postoperatively in the non-dominant hand (28.5 [3.0] vs 21.3 [5.9] kg; p=0.01). Conclusion : the abbreviation of preoperative fasting to two hours with drink containing carbohydrate improves muscle function in the perioperative period.

Sarcolemmal lipid analysis from mechanically skinned rat muscle fibres.

Membrane lipid composition, which includes phospholipid (PL) headgroup, and fatty acid (FA) saturation, has been shown to affect cellular function. The sarcolemma (SL) membrane is integral to skeletal muscle function and health. Previous studies assessing SL lipid composition are limited as they have 1) restricted analysis to a PL level and neglected FA composition and 2) relied on aggressive membrane isolation procedures resulting in t-tubule and sarcoplasmic reticulum contamination and unknown levels of nuclear and mitochondrial contamination. Thus, to overcome these limitations, this study assessed a method of individually skinned skeletal muscle fibres as an alternative to analyze complete sarcolemmal membrane lipid composition. The major findings of this study were 1) complete SL lipid composition can be obtained 2) the SL had higher sphingomyelin content than previous studies and 3) the SL membrane had minimal nuclear and mitochondrial contamination and was void of contamination from sarcoplasmic reticulum and t-tubules.

Force potentiation as a modulator of contractile performance: Implications for control of skeletal muscle force and energetics of work

This thesis investigated the modulation of dynamic contractile function and energetics of work by posttetanic potentiation (PTP). Mechanical experiments were conducted in vitro using software-controlled protocols to stimulate/determine contractile function during ramp shortening, and muscles were frozen during parallel incubations for biochemical analysis. The central feature of this research was the comparison of fast hindlimb muscles from wildtype and skeletal myosin light chain kinase knockout (skMLCK-/-) mice that does not express the primary mechanism for PTP: myosin regulatory light chain (RLC) phosphorylation. In contrast to smooth/cardiac muscles where RLC phosphorylation is indispensable, its precise physiological role in skeletal muscle is unclear. It was initially determined that tetanic potentiation was shortening speed dependent, and this sensitivity of the PTP mechanism to muscle shortening extended the stimulation frequency domain over which PTP was manifest. Thus, the physiological utility of RLC phosphorylation to augment contractile function in vivo may be more extensive than previously considered. Subsequent experiments studied the contraction-type dependence for PTP and demonstrated that the enhancement of contractile function was dependent on force level. Surprisingly, in the absence of RLC phosphorylation, skMLCK-/- muscles exhibited significant concentric PTP; consequently, up to ~50% of the dynamic PTP response in wildtype muscle may be attributed to an alternate mechanism. When the interaction of PTP and the catchlike property (CLP) was examined, we determined that unlike the acute augmentation of peak force by the CLP, RLC phosphorylation produced a longer-lasting enhancement of force and work in the potentiated state. Nevertheless, despite the apparent interference between these mechanisms, both offer physiological utility and may be complementary in achieving optimal contractile function in vivo. Finally, when the energetic implications of PTP were explored, we determined that during a brief period of repetitive concentric activation, total work performed was ~60% greater in wildtype vs. skMLCK-/- muscles but there was no genotype difference in High-Energy Phosphate Consumption or Economy (i.e. HEPC: work). In summary, this thesis provides novel insight into the modulatory effects of PTP and RLC phosphorylation, and through the observation of alternative mechanisms for PTP we further develop our understanding of the history-dependence of fast skeletal muscle function.

Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of α1-syntrophin and α-dystrobrevin in skeletal muscles of mdx mice. Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence–optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signalling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain–extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction–induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

Myostatin is a key mediator between energy metabolism and endurance capacity of skeletal muscle

Myostatin (Mstn) participates in the regulation of skeletal muscle size and has emerged as a regulator of muscle metabolism. Here, we hypothesized that lack of myostatin profoundly depresses oxidative phosphorylation-dependent muscle function. Toward this end, we explored Mstn/ mice as a model for the constitutive absence of myostatin and AAV-mediated overexpression of myostatin propeptide as a model of myostatin blockade in adult wild-type mice. We show that muscles from Mstn/ mice, although larger and stronger, fatigue extremely rapidly. Myostatin deficiency shifts muscle from aerobic toward anaerobic energy metabolism, as evidenced by decreased mitochondrial respiration, reduced expression of PPAR transcriptional regulators, increased enolase activity, and exercise-induced lactic acidosis. As a consequence, constitutively reduced myostatin signaling diminishes exercise capacity, while the hypermuscular state of Mstn/ mice increases oxygen consumption and the energy cost of running. We wondered whether these results are the mere consequence of the congenital fiber-type switch toward a glycolytic phenotype of constitutive Mstn/ mice. Hence, we overexpressed myostatin propeptide in adult mice, which did not affect fiber-type distribution, while nonetheless causing increased muscle fatigability, diminished exercise capacity, and decreased Pparb/d and Pgc1a expression. In conclusion, our results suggest that myostatin endows skeletal muscle with high oxidative capacity and low fatigability, thus regulating the delicate balance between muscle mass, muscle force, energy metabolism, and endurance capacity.

Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy

Myostatin regulates skeletal muscle size via the activin receptor IIB (ActRIIB). However, its effect on muscle energy metabolism and energy dependent muscle function remains largely unexplored. This question needs to be solved urgently since various therapies for neuromuscular diseases based on blockade of ActRIIB signaling are being developed. Here we show in mice that four months of pharmacological abrogation of ActRIIB signaling by treatment with soluble ActRIIB-Fc triggers extreme muscle fatigability. This is associated with elevated serum lactate levels and a severe metabolic myopathy in the mdx mouse, an animal model of Duchenne muscular dystrophy. Blockade of ActRIIB signaling down-regulates Porin, a crucial ADP/ATP shuttle between cytosol and mitochondrial matrix leading to a consecutive deficiency of oxidative phosphorylation as measured by in vivo Phophorus Magnetic Resonance Spectroscopy (31P-MRS). Further, ActRIIB blockade reduces muscle capillarization, which further compounds the metabolic stress. We show that ActRIIB regulates key determinants of muscle metabolism, such as Pparβ, Pgc1α, and Pdk4 thereby optimizing different components of muscle energy metabolism. In conclusion, ActRIIB signaling endows skeletal muscle with high oxidative capacity and low fatigability. The severe metabolic side effects following ActRIIB blockade caution against deploying this strategy, at least in isolation, for treatment of neuromuscular disorders.

Targeting myocardial remodelling to develop novel therapies for heart failure: a position paper from the Working Group on Myocardial Function of the European Society of Cardiology

The failing heart is characterized by complex tissue remodelling involving increased cardiomyocyte death, and impairment of sarcomere function, metabolic activity, endothelial and vascular function, together with increased inflammation and interstitial fibrosis. For years, therapeutic approaches for heart failure (HF) relied on vasodilators and diuretics which relieve cardiac workload and HF symptoms. The introduction in the clinic of drugs interfering with beta-adrenergic and angiotensin signalling have ameliorated survival by interfering with the intimate mechanism of cardiac compensation. Current therapy, though, still has a limited capacity to restore muscle function fully, and the development of novel therapeutic targets is still an important medical need. Recent progress in understanding the molecular basis of myocardial dysfunction in HF is paving the way for development of new treatments capable of restoring muscle function and targeting specific pathological subsets of LV dysfunction. These include potentiating cardiomyocyte contractility, increasing cardiomyocyte survival and adaptive hypertrophy, increasing oxygen and nutrition supply by sustaining vessel formation, and reducing ventricular stiffness by favourable extracellular matrix remodelling. Here, we consider drugs such as omecamtiv mecarbil, nitroxyl donors, cyclosporin A, SERCA2a (sarcoplasmic/endoplasmic Ca(2 +) ATPase 2a), neuregulin, and bromocriptine, all of which are currently in clinical trials as potential HF therapies, and discuss novel molecular targets with potential therapeutic impact that are in the pre-clinical phases of investigation. Finally, we consider conceptual changes in basic science approaches to improve their translation into successful clinical applications.

Relationship of upper-limb and thoracic muscle strength to 6-min walk distance in COPD patients

Study objectives: This study was developed to investigate the influence of thoracic and upperlimb muscle function on 6-min walk distance (6MWD) in patients with COPD.Design: A prospective, cross-sectional study.Setting: the pulmonary rehabilitation center of a university hospital.Patients: Thirty-eight patients with mild to very severe COPD were evaluated.Measurements and results: Pulmonary function and baseline dyspnea index (BDI) were assessed, handgrip strength, maximal inspiratory pressure (Pimax), and 6MWD were measured, and the one-repetition maximum (1RM) was determined for each of four exercises (bench press, lat pull down, leg extension, and leg press) performed on gymnasium equipment. Quality of life was assessed using the St. George Respiratory Questionnaire (SGRQ). We found statistically significant positive correlations between 6MWD and body weight (r = 0.32; p < 0.05), BDI (r = 0.50; p < 0.01), FEV, (r = 0.33; p < 0.05), PImax (r = 0.53; p < 0.01), and all values of 1RM. A statistically significant negative correlation was observed between 6MWD and dyspnea at the end of the 6-min walk test (r = -0.29; p < 0.05), as well as between 6MWD and the SGRQ activity domain (r = -0.45; p < 0.01) and impact domain (r = -0.34; p < 0.05) and total score (r = -0.40; p < 0.01). Multiple regression analysis selected body weight, BDI, Pimax, and lat pull down IRM as predictive factors for 6MWD (R-2 = 0.589).Conclusions: the results of this study showed the importance of the skeletal musculature of the thorax and upper limbs in submaximal exercise tolerance and could open new perspectives for training programs designed to improve functional activity in COPD patients.

Direct effects of colchicine on myocardial function - Studies in hypertrophied and failing spontaneously hypertensive rats

The aging spontaneously hypertensive rat (SHR) is a model in which the transition from chronic stable left ventricular hypertrophy to overt heart failure can be observed. Although the mechanisms for impaired function in hypertrophied and failing cardiac muscle from the SHR have been studied, none accounts fully for the myocardial contractile abnormalities. The cardiac cytoskeleton has been implicated as a possible cause for myocardial dysfunction. If an increase in microtubules contributes to dysfunction, then myocardial microtubule disruption by colchicine should promote an improvement in cardiac performance. We studied the active and passive properties of isolated left ventricular papillary muscles from 18- to 24-month-old SHR with evidence of heart failure (SHR-F, n=6), age-matched SHR without heart failure (SHR-NF, n=6), and age-matched normotensive Wistar-Kyoto rats (WKY, n=5). Mechanical parameters were analyzed before and up to 90 minutes after the addition of colchicine (10(-5), 10(-4), and 10(-3) mol/L). In the baseline state, active tension (AT) developed by papillary muscles from the WKY group was greater than for SHR-NF and SHR-F groups (WKY 5.69+/-1.47 g/mm(2) [mean+/-SD], SHR-NF 3.41+/-1.05, SHR-F 2.87+/-0.26; SHR-NF and SHR-F P<0.05 versus WKY rats). The passive stiffness was greater in SHR-F than in the WKY and SHR-NF groups (central segment exponential stiffness constant, K-cs: SHR-F 70+/-25, SHR-NF 44+/-17, WKY 41+/-13 [mean+/-SD]; SHR-F P<0.05 versus; SHR-NF and WKY rats). AT did not improve after 10, 20, and 30 minutes of exposure to colchicine (10(-5), 10(-4), and 10(-3) mol/L) in any group. In the SHR-F group, AT and passive stiffness did not change after 30 to 90 minutes of colchicine exposure (10(-4) mol/L). In summary, the data in this study fail to demonstrate improvement of intrinsic muscle function in SHR with heart failure after colchicine. Thus, in the SHR there is no evidence that colchicine-induced cardiac microtubular depolymerization affects the active or passive properties of hypertrophied or failing left ventricular myocardium.

Correlation between morphology and function of the upper lip: a longitudinal evaluation

In order to evaluate the relationship between the morphology of the upper lip and muscle activity in a sample of 38 subjects (17 males and 21 females) with Angle Class II division 1 malocclusions, cephalometric and electromyographic analyses were conducted. The sample was subdivided into either predominantly nose or mouth breathers. The individuals were evaluated at two different periods, with a 2 year interval. At the first observation, the subjects were 11 years to 14 years 11 months of age and at the second observation, 13 years 4 months to 16 years 6 months of age. Height and thickness of the upper lip were measured on lateral cephalograms with the aid of a digital pachymeter. For each individual, electromyographic records were obtained of the orbicularis oris superior muscle at rest and in a series of 12 movements. The electromyographic data were normalized as a function of amplitude, for achievement of the percentage value of each movement. Pearson and Spearman correlation tests were applied.The results showed some correlation between morphology and muscle function (at a confidence level of 95 per cent). However, as the values of the correlation coefficient (r) were too low to establish associations between variables, it was concluded that the dimensions of the upper lip are not correlated with muscle activity.

Comparative mechanical study of isolated papillary muscle from Wistar-Kyoto and Wistar rats

Isolated papillary muscles have often been used in myocardial mechanical function studies. The objective of the present study was to compare the mechanical function of papillary muscle isolated from left ventricle between Wistar (W) and Wistar-Kyoto (WKY) rats of different ages (1, 3, 6 and 12 months), in order to examine whether there is a difference in intrinsic mechanical properties of muscle between the two rat strains. Muscles were perfused with Krebs-Henseleit solution at 28°C and studied isometrically and isotonically at a stimulation rate of 0.2 Hz. The W and WKY showed statistically significant differences during both isometric and isotonic contractions. During isometric contraction? (l) the peak developed tension (DT) and + dT/dt were lower in WKY rats in the 1 mo groups, (2) the resting tension (RT) was greater in WKY at 3, 6 and 12 mo. (3) time to peak tension (TPT) was greater in WKY at 3 and 12 mo, (4) time for tension to fall from peak to 50% of peak tension (RT 1/2) was greater in WKY at 3 mo and (5) - dT/dt was lower in WKY at 1 and 3 mo. During isotonic contraction, (1) the peak shortening (PS) and -dL/dt were lower in WKY at 12 mo, (2) the time to peak shortening (TPS) was greater in WKY at 3 and 12 mo; (3) + dL/dt was lower in WKY at 3, 6, and 12 mo and (4) the relative variation of length (Lmax-PS)/Lmax was greater in WKY at 6 and 12 mo. These data showed a difference in mechanical behaviour of the papillary muscle between Wistar and Wistar-Kyoto rats of different age.

Cellular and molecular mechanisms of apoptosis in skeletal muscle atrophy

Apoptosis is necessary for maintaining the integrity of proliferative tissues, such as epithelial cells of the gastrointestinal and integumentary systems. The role of apoptosis in post-mitotic tissues, such as skeletal muscle, is less well defined, but several lines of evidence suggest that it occurs in both myofiber and other interstitial muscle cell types. Apoptosis of myonuclei likely contributes to the loss of muscle mass, but the mechanisms underlying this process are largely unknown. Caspase-dependent as well as caspase-independent pathways have been implicated, and the mode by which atrophy is induced likely determines the apoptotic mechanisms that are utilized. It remains to be determined whether a decrease in apoptosis will alleviate atrophy and distinct research strategies may be required to clarify the different causes of skeletal muscle mass loss. In this review, it was also speculated that apoptosis is a normal regulatory process that the myofiber can use to reduce the number of nuclear domains, thus ensuring optimal cell functions according to the mechanical load imposed on the muscle. ©FUNPEC-RP.

Hypertrophied myocardium is more dependent on extracellular calcium than the normal cardiac muscle

Background: The aim of this study was to analyze stable hypertrophied myocardial function and its response to inotropic maneuvers in rats submitted to renovascular hypertension for a 10-week period (RHT group, n=10). Material/Methods: Myocardial performance was studied in isolated left ventricle papillary muscles in isometric contraction under the following conditions: at postrest contraction of 30 seconds (PRC), at extracellular calcium (ECa 2+) chloride concentration of 1.25 and 5.20 mM, and after beta-adrenergic stimulation with 10 -6 M isoproterenol (ISOP). Results: The results were compared with normotensive Wistar controls rats (C group, n=10). In basal condition, resting tension, and contraction time (TPT) were greater, while relaxation time (RT 50) tended to be longer in RHT than C group. PRC and ISOP promoted a similar change in muscle function response intensity (Δ) in both groups. ECa 2+ shift did not change TPT in the C group and decreased TPT in the RHT animals; Δ was different between these groups. RT 50 increased in C and decreased in RHT, both without statistical significance; however, Δ was different. Conclusions: These results suggest that hypertrophied myocardial dysfunction may be attibuted to changes in intracellular calcium cycling. © Med Sci Monit, 2010.