Skeletal muscle mitochondrial adaptations induced by long-term cigarette smoke exposure.

Because patients with chronic obstructive pulmonary disease (COPD) are often physically inactive, it is still unclear whether the lower respiratory capacity in the locomotor muscles of these patients is due to cigarette smoking per se or is secondary to physical deconditioning. Accordingly, the purpose of this study was to examine mitochondrial alterations in the quadriceps muscle of 10 mice exposed to 8 mo of cigarette smoke, a sedentary mouse model of emphysema, and 9 control mice, using immunoblotting, spectrophotometry, and high-resolution respirometry in permeabilized muscle fibers. Mice exposed to smoke displayed a twofold increase in the oxidative stress marker, 4-HNE, (P < 0.05) compared with control mice. This was accompanied by significant decrease in protein expression of UCP3 (65%), ANT (58%), and mitochondrial complexes II-V (∼60%-75%). In contrast, maximal ADP-stimulated respiration with complex I and II substrates (CON: 23.6 ± 6.6 and SMO: 19.2 ± 8.2 ρM·mg-1·s-1) or octanoylcarnitine (CON: 21.8 ± 9.0 and SMO: 16.5 ± 6.6 ρM·mg-1·s-1) measured in permeabilized muscle fibers, as well as citrate synthase activity, were not significantly different between groups. Collectively, our findings revealed that sedentary mice exposed to cigarette smoke for 8 mo, which is typically associated with pulmonary inflammation and emphysema, exhibited a preserved mitochondrial respiratory capacity for various substrates, including fatty acid, in the skeletal muscle. However, the mitochondrial adaptations induced by cigarette smoke favored the development of chronic oxidative stress, which can indirectly contribute to augment the susceptibility to muscle fatigue and exercise intolerance.NEW & NOTEWORTHY It is unclear whether the exercise intolerance and skeletal muscle mitochondrial dysfunction observed in patients with COPD is due to cigarette smoke exposure, per se, or if they are secondary consequences to inactivity. Herein, while long-term exposure to cigarette smoke induces oxidative stress and an altered skeletal muscle phenotype, cigarette smoke does not directly contribute to mitochondrial dysfunction. With this evidence, we demonstrate the critical role of physical inactivity in cigarette smoke-related skeletal muscle dysfunction.

Relative lipid oxidation associates directly with mitochondrial fusion phenotype and mitochondria-sarcoplasmic reticulum interactions in human skeletal muscle.

Disturbances in skeletal muscle lipid oxidation might induce ectopic fat deposition and lipotoxicity. Nevertheless, the cellular mechanisms that regulate skeletal muscle lipid oxidation have not been fully determined. We aimed to determine whether there was an association between relative whole body lipid oxidation and mitochondrial size or mitochondria-sarcoplasmic reticulum interactions in the skeletal muscle. Twelve healthy men were included [mean (standard deviation), 24.7 (1.5) yr old, 24.4 (2.6) kg/m2]. The respiratory quotient (RQ) was used to estimate relative lipid oxidation at rest and during exercise (50% maximal oxygen consumption, 600 kcal expended). A skeletal muscle biopsy was obtained from the vastus lateralis at rest. Transmission electron microscopy was used to determine mitochondrial size and mitochondria-sarcoplasmic reticulum interactions (≤50 nm of distance between organelles). Protein levels of fusion/fission regulators were measured in skeletal muscle by Western blot. Resting RQ and exercise RQ associated inversely with intermyofibrillar mitochondrial size (r = -0.66 and r = -0.60, respectively, P < 0.05). Resting RQ also associated inversely with the percentage of intermyofibrillar mitochondria-sarcoplasmic reticulum interactions (r = -0.62, P = 0.03). Finally, intermyofibrillar mitochondrial size associated inversely with lipid droplet density (r = -0.66, P = 0.01) but directly with mitochondria fusion-to-fission ratio (r = 0.61, P = 0.03). Our results show that whole body lipid oxidation is associated with skeletal muscle intermyofibrillar mitochondrial size, fusion phenotype, and mitochondria-sarcoplasmic-reticulum interactions in nondiabetic humans.

Super-Resolution Reconstruction for Two- and Three-Dimensional LA-ICP-MS Bioimaging.

The resolution of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) elemental bioimaging is usually constrained by the diameter of the laser spot size and is often not adequate to explore in situ subcellular distributions of elements and proteins in biological tissue sections. Super-resolution reconstruction is a method typically used for many imaging modalities and combines multiple lower resolution images to create a higher resolution image. Here, we present a super-resolution reconstruction method for LA-ICP-MS imaging by ablating consecutive layers of a biological specimen with offset orthogonal scans, resulting in a 10× improvement in resolution for quantitative measurement of dystrophin in murine muscle fibers. Layer-by-layer image reconstruction was also extended to the third dimension without the requirement of image registration across multiple thin section specimens. Quantitative super-resolution reconstruction, combined with Gaussian filtering and application of the Richardson-Lucy total variation algorithm, provided superior image clarity and fidelity in two- and three-dimensions.

Normative data of muscle fiber diameter of vastus lateralis during childhood: a field test.

INTRODUCTION: Currently, our knowledge of standard data for muscle morphology in children is largely limited to the 1969 article by Brooke and Engel (BE). In 2016, we reported normal muscle morphology from vastus lateralis biopsies in ambulant children with cerebral palsy (CP). This report compares our normal biopsy results against BE standard value criteria. METHODS: Single-blind prospective cross-sectional study design. RESULTS: Results of biopsies taken in ambulant children with CP were normal according to morphometry and light and electron microscopy; however, only 5 of 10 fulfilled the BE standard value criteria. DISCUSSION: This short report highlights the requirement for contemporary age-specific normative data from a larger number of biopsies, including typically developing children. Review of the literature suggests that biopsy material may be available from typically developing children who were control patients in research trials. This morphometric data could contribute to expanding the normative data set. Muscle Nerve 59:590-590, 2019.

Adult expression of PGC-1α and -1ß in skeletal muscle is not required for endurance exercise-induced enhancement of exercise capacity.

Exercise has been shown to be the best intervention in the treatment of many diseases. Many of the benefits of exercise are mediated by adaptions induced in skeletal muscle. The peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family of transcriptional coactivators has emerged as being key mediators of the exercise response and is considered to be essential for many of the adaptions seen in skeletal muscle. However, the contribution of the PGC-1s in skeletal muscle has been evaluated by the use of either whole body or congenital skeletal muscle-specific deletion. In these models, PGC-1s were never present, thereby opening the possibility to developmental compensation. Therefore, we generated an inducible muscle-specific deletion of PGC-1α and -1ß (iMyo-PGC-1DKO), in which both PGC-1α and -ß can be deleted specifically in adult skeletal muscle. These iMyo-PGC-1DKO animals were used to assess the role of both PGC-1α and -1ß in adult skeletal muscle and their contribution to the exercise training response. Untrained iMyo-PGC-1DKO animals exhibited a time-dependent decrease in exercise performance 8 wk postdeletion, similar to what was observed in the congenital muscle-specific PGC-1DKOs. However, after 4 wk of voluntary training, the iMyo-PGC-1DKOs exhibited an increase in exercise performance with a similar adaptive response compared with control animals. This increase was associated with an increase in electron transport complex (ETC) expression and activity in the absence of PGC-1α and -1ß expression. Taken together these data suggest that PGC-1α and -1ß expression are not required for training-induced exercise performance, highlighting the contribution of PGC-1-independent mechanisms.

Chronic disuse and skeletal muscle structure in older adults: sex-specific differences and relationships to contractile function.

In older adults, we examined the effect of chronic muscle disuse on skeletal muscle structure at the tissue, cellular, organellar, and molecular levels and its relationship to muscle function. Volunteers with advanced-stage knee osteoarthritis (OA, n = 16) were recruited to reflect the effects of chronic lower extremity muscle disuse and compared with recreationally active controls (n = 15) without knee OA but similar in age, sex, and health status. In the OA group, quadriceps muscle and single-fiber cross-sectional area were reduced, with the largest reduction in myosin heavy chain IIA fibers. Myosin heavy chain IIAX fibers were more prevalent in the OA group, and their atrophy was sex-specific: men showed a reduction in cross-sectional area, and women showed no differences. Myofibrillar ultrastructure, myonuclear content, and mitochondrial content and morphology generally did not differ between groups, with the exception of sex-specific adaptations in subsarcolemmal (SS) mitochondria, which were driven by lower values in OA women. SS mitochondrial content was also differently related to cellular and molecular functional parameters by sex: greater SS mitochondrial content was associated with improved contractility in women but reduced function in men. Collectively, these results demonstrate sex-specific structural phenotypes at the cellular and organellar levels with chronic disuse in older adults, with novel associations between energetic and contractile systems.

Dual effects of exercise in dysferlinopathy.

Dysferlinopathy refers to a group of autosomal recessive muscular dystrophies due to mutations in the dysferlin gene causing deficiency of a membrane-bound protein crucially involved in plasma membrane repair. The condition is characterized by marked clinical heterogeneity, the different phenotypes/modes of presentation being unrelated to the genotype. For unknown reasons, patients are often remarkably active before the onset of symptoms. Dysferlin deficiency-related persistence of mechanically induced sarcolemma disruptions causes myofiber damage and necrosis. We postulate that limited myodamage may initially remain hidden with well-preserved resistance to physical strains. By subjecting dysferlin-deficient B6.A/J-Dysf(prmd) mice to long-term swimming exercise, we observed that concentric/isometric strain improved muscle strength and alleviated muscular dystrophy by limiting the accumulation of membrane lesions. By contrast, eccentric strain induced by long-term running in a wheel worsened the dystrophic process. Myofiber damage induced by eccentric strain increased with age, reflecting the accumulation of non-necrotic membrane lesions up to a critical threshold. This phenomenon was modulated by daily spontaneous activity. Transposed to humans, our results may suggest that the past activity profile shapes the clinical phenotype of the myopathy and that patients with dysferlinopathy should likely benefit from concentric exercise-based physiotherapy.

Multiple mitochondrial alterations in a case of myopathy.

Mitochondrial alterations are the most common feature of human myopathies. A biopsy of quadriceps muscle from a 50-year-old woman exhibiting myopathic symptoms was examined by transmission electron microscopy. Biopsied fibers from quadriceps muscle displayed numerous subsarcolemmal mitochondria that contained crystalloids. Numbering 1-6 per organelle, these consisted of rows of punctuate densities measuring ∼0.34 nm; the parallel rows of these dots had a periodicity of ∼0.8 nm. The crystalloids were ensconced within cristae or in the outer compartment. Some mitochondria without crystalloids had circumferential cristae, leaving a membrane-free center that was filled with a farinaceous material. Other scattered fibrocyte defects included disruption of the contractile apparatus or its sporadic replacement by a finely punctuate material in some myofibers. Intramitochondrial crystalloids, although morphologically striking, do not impair organelle physiology to a significant degree, so the muscle weakness of the patient must originate elsewhere.

Formation of gelsolin amyloid fibrils in the rough endoplasmic reticulum of skeletal muscle in the gelsolin mouse model of inclusion body myositis: comparative analysis to human sporadic inclusion body myositis.

Sporadic inclusion body myositis has a significant impact on the life of the elderly. Despite some similarities to other myopathies with established genetic defects, little is known about mechanisms of its development and no effective treatment is available. Therefore, there is a need for animal models that can faithfully reconstitute important aspects of this human disease. The authors recently expressed a mutant form of human gelsolin in mice under the control of a muscle-specific promoter. This induced myopathic changes reminiscent of human inclusion body myositis. In this study, immunogold labeling is used to further characterize this model. The study demonstrates a presence of gelsolin amyloid deposits within the rough endoplasmic reticulum. It further compares this mouse model to human sporadic inclusion body myositis.

Postmenopausal effects of resistance training on muscle damage and mitochondria.

The purpose of this study was to measure the effects of a 12-month progressive resistance training intervention on muscle morphology and strength gains in postmenopausal women. Skeletal muscle biopsies were obtained from the vastus lateralis of 5 independent community-dwelling women (mean age: 75.6 ± 4.28 years; mean height: 163 ± 5.34 cm; mean weight: 72 ± 17.5 kg) before 6 months and 12 months after progressive resistance training. Muscle strength (1 repetition maximum) was measured at the same time points. After 6 months of training, morphological analysis revealed evidence of increased proteolysis and tissue repair, and rudimentary fiber development. The percent of Z-bands with mild Z-band disruption increased from 43.9% at baseline to 66.7% after 6 months of training (p < 0.01). Mitochondrial volume also increased (percent of mitochondria = 0.86% at baseline, 1.19% at 6 months, and 1.04% at 12 months, p < 0.05), and there was a shift to larger sized mitochondria. The training did not result in statistically significant increases in muscle leg strength (p < 0.18). It appears that mild Z-band disruption acts as a precursor for increased protein synthesis and stimulates an increase in mitochondrial mass. Therefore, although a progressive resistance training program in this population did not increase muscle strength, it did demonstrate clinical applications that lend support to the importance of resistance training in older adults.

Injury of peripheral muscles in smokers with chronic obstructive pulmonary disease.

Muscle injury has clinical relevance in diseased individuals because it is associated with muscle dysfunction in terms of decreased strength and/or endurance. This study was aimed at answering three questions: whether the presence of chronic obstructive pulmonary disease (COPD) is associated with peripheral muscle injury; whether muscle injury is associated with some of the relevant functional impairment in the muscles; and whether muscle injury can be solely justified by deconditioning. Twenty-one male COPD patients were eligible for the study. Seven healthy volunteers recruited from the general population were included as controls. Function of the quadriceps muscle was assessed through specific single-leg exercise (strength and endurance). Cellular (light microscopy) and subcellular (electron microscopy) techniques were used to evaluate muscle injury on biopsies from the vastus lateralis muscle. Signs of injury were found in muscles from both control and COPD patients, not only in cases showing severe airflow obstruction but also in the mild or moderate stages of the disease. Current smoking and presence of COPD were significantly associated with increased injury of the muscle as assessed by light and electron microscopy techniques. The authors conclude that peripheral muscle injury is evident in mild, moderate, and severe stages of COPD even in the absence of respiratory failure, hypercapnia, chronic steroid treatment, low body weight, or some coexisting disease. These findings support the theory that systemic factors with deleterious effect are acting on peripheral muscles of smokers with COPD, increasing the susceptibility of the muscle fibers to membrane and sarcomere injury.

Rectus Femoris Characteristics in Post Stroke Spasticity: Clinical Implications from Ultrasonographic Evaluation.

In stroke survivors, rectus femoris (RF) spasticity is often implicated in gait pattern alterations such as stiff knee gait (SKG). Botulinum toxin type A (BoNT-A) is considered the gold standard for focal spasticity treatment. However-even if the accuracy of injection is crucial for BoNT-A efficacy-instrumented guidance for BoNT-A injection is not routinely applied in clinical settings. In order to investigate the possible implications of an inadequate BoNT-A injection on patients' clinical outcome, we evaluated the ultrasound-derived RF characteristics (muscle depth, muscle thickness, cross-sectional area and mean echo intensity) in 47 stroke survivors. In our sample, we observed wide variability of RF depth in both hemiparetic and unaffected side of included patients (0.44 and 3.54 cm and between 0.25 and 3.16 cm, respectively). Moreover, our analysis did not show significant differences between treated and non-treated RF in stroke survivors. These results suggest that considering the inter-individual variability in RF muscle depth and thickness, injection guidance should be considered for BoNT-A treatment in order to optimize the clinical outcome of treated patients. In particular, ultrasound guidance may help the clinicians in the long-term follow-up of muscle quality.

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.

Nuclei of aged myofibres undergo structural and functional changes suggesting impairment in RNA processing.

Advancing adult age is associated with a progressive decrease in skeletal muscle mass, strength and quality known as sarcopenia. The mechanisms underlying age-related skeletal muscle wasting and weakness are manifold and still remain to be fully elucidated. Despite the increasing evidence that the progress of muscle diseases leading to muscle atrophy/dystrophy may be related to defective RNA processing, no data on the morpho-functional features of skeletal muscle nuclei in sarcopenia are available at present. In this view, we have investigated, by combining morphometry and immunocytochemistry at light and electron microscopy, the fine structure of myonuclei as well as the distribution and amount of RNA processing factors in skeletal myofibres of biceps brachii and quadriceps femoris from adult and old rats. Results demonstrate that the myonuclei of aged type II fibres show an increased amount of condensed chromatin and lower amounts of phosphorylated polymerase II and DNA/RNA hybrid molecules, clearly indicating a decrease in pre-mRNA transcription rate compared to adult animals. In addition, myonuclei of aged fibres show decreased amounts of nucleoplasmic splicing factors and an accumulation of cleavage factors, polyadenilated RNA and perichromatin granules, suggesting a reduction in the processing and transport rate of premRNA. During ageing, it seems therefore that in rat myonuclei the entire production chain of mRNA, from synthesis to cytoplasmic export, is less efficient. This failure likely contributes to the reduced responsiveness of muscle cells to anabolic stimuli in the elderly.

An examination of the relationships among myosin heavy chain isoform content, isometric strength, and mechanomyographic median frequency.

The purpose of the present study was to determine if the combination of isometric knee extension strength and mechanomyographic (MMG) median frequency (MDF) could be used to estimate the percent (%) myosin heavy chain (MHC) Type II isoform content of the vastus lateralis. Five resistance-trained (mean +/- SD age = 23.2 +/- 3.7 years) and 5 aerobically trained (mean +/- SD age = 32.6 +/- 5.2 years) men volunteered to perform a 6-second isometric maximum voluntary contraction (MVC) of the dominant knee extensors at a joint angle of 90 degrees between the thigh and leg. During the isometric MVC, the surface MMG signal was detected from the vastus lateralis, and the MDF was calculated with the discrete Fourier transform. Following the isometric MVC and MMG measurements, muscle biopsies were taken from the vastus lateralis and analyzed for % MHC Type II isoform content. The results showed that neither isometric knee extension strength nor MMG MDF alone was significantly correlated with the % MHC Type II isoform content. The combination of isometric knee extension strength and MMG MDF, however, explained a significant proportion (i.e., 59.8%) of the variance in % MHC Type II isoform content, with a multiple correlation of R = 0.773 and a standard error of the estimate (SEE) of 15.4%. These findings indicated that a simple, time-efficient, and noninvasive test that simultaneously measures isometric strength and MMG MDF could be useful for estimating the % MHC Type II isoform content in well-trained men.

Acute effect of drop jumping on throwing performance.

The purpose of the present study was to investigate the acute effect of drop jumping on throwing performance. Eight men and 8 women, moderately trained subjects with basic shot put skills, performed 3 squat underhand front shot throws after a short standard warm-up. Three minutes later they performed 5 maximal consecutive drop jumps from 40 cm. Immediately after the drop jumps, they repeated the squat underhand front shot throws. On another day, their 6 repetition maximum (RM) muscular strength in leg press was assessed. Muscle biopsies were also obtained from vastus lateralis for the determination of fiber-type composition and fiber cross-sectional area. Throwing performance was significantly increased after drop jumping (8.25 +/- 1.1 m vs. 8.63 +/- 1.3 m, p < 0.01). The percentage of type II muscle fiber area was significantly related to the increase in throwing performance after drop jumping (r = 0.76, p < 0.01). The increase in throwing performance was significant in men (8.94 +/- 1 m vs. 9.60 +/- 0.9 m, p < 0.01) but not in women (7.56 +/- 1 m vs. 7.67 +/- 0.9 m, ns). Of note, the percentage of type II fiber area was higher in men than in women (M: 66.4 +/- 13%, F: 50.2 +/- 15%, p < 0.01). Leg press strength (6RM) was moderately related to the increase in throwing performance after drop jumping (r = 0.50, p < 0.05). These results suggest that drop jumping just before a throwing action induces an increase in performance in subjects with a high percentage of type II muscle fiber area and (to a lesser degree) in subjects with enhanced muscular strength.

Danon disease: a novel Lamp-2 gene mutation in a family with four affected members.

This is a report of a family with four members affected with Danon disease and variable clinical presentations, including cardiomyopathy, skeletal muscle pathology, and hepatopathy. Analysis by electron microscopy of the quadriceps muscle from the proband and his brother showed abnormal mitochondria, and immunohistochemistry revealed no expression of LAMP-2 protein. This defect is due to a yet undescribed mutation located at the second nucleotide in the intron 8 of the Lamp-2 gene (c.1093+2 T>A) that generated exon 8 skipping confirmed at RNA level in the proband.

A new form of childhood onset, autosomal recessive spinocerebellar ataxia and epilepsy is localized at 16q21-q23.

Childhood ataxias are a complex set of inherited disorders. Ataxias associated with generalized tonic-clonic epilepsy are usually included with the progressive myoclonus epilepsies (PME). Five disease entities, Unverricht-Lundborg disease, Lafora's disease, neuronal ceroid lipofuscinoses, myoclonic epilepsy with ragged red fibres and sialidoses, account for the majority of PME cases. Two rare forms of ataxia plus epilepsy, sensory ataxic neuropathy, dysarthria and ophthalmoparesis, and infantile onset spinocerebellar ataxia were described recently and found to be caused by defective mitochondrial proteins. We report here a large consanguineous family from Saudi Arabia with four affected children presenting with generalized tonic-clonic epilepsy, ataxia and mental retardation, but neither myoclonus nor mental deterioration. MRI and muscle biopsy of one patient revealed, respectively, posterior white matter hyperintensities and vacuolization of the sarcotubular system. We localized the defective gene by homozygosity mapping to a 19 Mb interval in 16q21-q23 between markers D16S3091 and D16S3050. Linkage studies in this region will allow testing for homogeneity of this novel ataxia-epilepsy entity.