Loss Of Specific Force Research Articles

Reduction of circulating sphingosine-1-phosphate worsens mdx soleus muscle dystrophic phenotype.

What is the central question of the study? What are the consequences of reducing circulating sphingosine-1-phosphate (S1P) for muscle physiology in the murine model of Duchenne muscular dystrophy (DMD)? What is the main result and its importance? Reduction of the circulating S1P level in mdx mice aggravates the dystrophic phenotype, as seen by an increase in fibre atrophy, fibrosis and loss of specific force, suggesting that S1P signalling is a potential therapeutic target in DMD. Although further studies are needed, plasma S1P levels have the intriguing possibility of being used as a biomarker for disease severity, an important issue in DMD. Sphingosine-1-phosphate (S1P) is an important regulator of skeletal muscle properties. The dystrophin-deficient mdx mouse possesses low levels of S1P (∼50%) compared with wild type. Increased S1P availability was demonstrated to ameliorate the dystrophic phenotype in Drosophila and in mdx mice. Here, we analysed the effects produced by further reduction of S1P availability on the mass, force and regenerative capacity of dystrophic mdx soleus. Circulating S1P was neutralized by a specific anti-S1P antibody (S1P-Ab) known to lower the extracellular concentration of this signalling lipid. The S1P-Ab was administered intraperitoneally in adult mdx mice every 2days for the duration of experiments. Soleus muscle properties were analysed 7 or 14days after the first injection. The decreased availability of circulating S1P after the 14day treatment reduced mdx soleus fibre cross-sectional area (-16%, P<0.05), an effect that was associated with an increase in markers of proteolytic (MuRF1 and atrogin-1) and autophagic (p62 and LC3-II/LC3-I ratio) pathways. Moreover, an increase of fibrosis was also observed (+26%, P<0.05). Notably, the treatment also caused a reduction of specific tetanic tension (-29%, P<0.05). The mdx soleus regenerative capacity was only slightly influenced by reduced S1P. In conclusion, neutralization of circulating S1P reduces the mass and specific force and increases fibrosis of mdx soleus muscle, thus worsening the dystrophic phenotype. The results confirm that active, functional S1P signalling might counteract the progression of soleus mdx pathology and validate the pathway as a potential therapeutic target for muscular dystrophies.

Diaphragm weakness and proteomics (global and redox) modifications in heart failure with reduced ejection fraction in rats

Inspiratory dysfunction occurs in patients with heart failure with reduced ejection fraction (HFrEF) in a manner that depends on disease severity and by mechanisms that are not fully understood. In the current study, we tested whether HFrEF effects on diaphragm (inspiratory muscle) depend on disease severity and examined putative mechanisms for diaphragm abnormalities via global and redox proteomics. We allocated male rats into Sham, moderate (mHFrEF), or severe HFrEF (sHFrEF) induced by myocardial infarction and examined the diaphragm muscle. Both mHFrEF and sHFrEF caused atrophy in type IIa and IIb/x fibers. Maximal and twitch specific forces (N/cm2) were decreased by 19 ± 10% and 28 ± 13%, respectively, in sHFrEF (p < .05), but not in mHFrEF. Global proteomics revealed upregulation of sarcomeric proteins and downregulation of ribosomal and glucose metabolism proteins in sHFrEF. Redox proteomics showed that sHFrEF increased reversibly oxidized cysteine in cytoskeletal and thin filament proteins and methionine in skeletal muscle α-actin (range 0.5 to 3.3-fold; p < .05). In conclusion, fiber atrophy plus contractile dysfunction caused diaphragm weakness in HFrEF. Decreased ribosomal proteins and heighted reversible oxidation of protein thiols are candidate mechanisms for atrophy or anabolic resistance as well as loss of specific force in sHFrEF.

Human skeletal muscle fibre contractile properties and proteomic profile: adaptations to 3 weeks of unilateral lower limb suspension and active recovery.

It is generally assumed that muscle fibres go through atrophy following disuse with a loss of specific force and an increase in unloaded shortening velocity. However, the underlying mechanisms remain to be clarified. Most studies have focused on events taking place during the development of disuse, whereas the subsequent recovery phase, which is equally important, has received little attention. Our findings support the hypotheses that the specific force of muscle fibres decreased following unilateral lower limb suspension (ULLS) and returned to normal after 3weeks of active recovery as a result of a loss and recovery of myosin and actin content. Furthermore, muscle fibres went through extensive qualitative changes in muscle protein pattern following ULLS, and these were reversed by active recovery. Resistance training was very effective in restoring both muscle mass and qualitative muscle changes, indicating that long-term ULLS did not prevent the positive effect of exercise on human muscle. Following disuse, muscle fibre function goes through adaptations such as a loss of specific force (PO /CSA) and an increase in unloaded shortening velocity, which could be a result of both quantitative changes (i.e. atrophy) and qualitative changes in protein pattern. The underlying mechanisms remain to be clarified. In addition, little is known about the recovery of muscle mass and strength following disuse. In the present study, we report an extensive dataset describing, in detail,the functional and protein content adaptations of skeletal muscle in response to both disuse and re-training. Eight young healthy subjects were subjected to 3weeks of unilateral lower limb suspension (ULLS), a widely used human model of disuse skeletal muscle atrophy. Needle biopsies samples were taken from the vastus lateralis muscle Pre-ULLS, Post-ULLS and after 3weeks of recovery during which heavy resistance training was performed. After disuse, cross-sectional area (CSA), PO /CSA and myosin concentration (MC) decreased in both type 1 and 2A skinned muscle fibres. After recovery, CSA and MC returned to levels comparable to those observed before disuse, whereas Po/CSA and unloaded shortening velocity reached a higher level. Myosin heavy chain isoform composition of muscle samples did not differ among the experimental groups. To study the mechanisms underlying such adaptations, a two-dimensional proteomic analysis was performed. ULLS induced a reduction of myofibrillar, metabolic (glycolytic and oxidative) and anti-oxidant defence system protein content. Resistance training was very effective in counteracting ULLS-induced alterations, indicating that long-term ULLS did not prevent the positive effect of exercise on human muscle.

Quantitative and qualitative adaptations of muscle fibers to glucocorticoids.

The aim of this study was to understand the effects of short-term glucocorticoid administration in healthy subjects. Five healthy men received dexamethasone (8 mg/day) for 7 days. Vastus lateralis muscle biopsy and knee extension torque measurement were performed before and after administration. A large number of individual muscle fibers were dissected from the biopsy samples (pre-administration: n = 165, post-administration: n = 177). Maximal knee extension torque increased after administration (∼ 13%), whereas both type 1 and type 2A fibers had decreased cross-sectional area (type 1: ∼ 11%, type 2A: ∼ 17%), myosin loss (type 1: ∼ 18%, type 2A: ∼ 32%), and loss of specific force (type 1: ∼ 24%, type 2A: ∼ 33%), which were preferential for fast fibers. Short-term dexamethasone administration in healthy subjects elicits quantitative and qualitative adaptations of muscle fibers that precede (and may predict) the clinical appearance of myopathy in glucocorticoid-treated subjects.

The Effect of Nutritional Status in the Pathogenesis of Critical Illness Myopathy (CIM).

The muscle wasting and loss of specific force associated with Critical Illness Myopathy (CIM) is, at least in part, due to a preferential loss of the molecular motor protein myosin. This acquired myopathy is common in critically ill immobilized and mechanically ventilated intensive care patients (ICU). There is a growing understanding of the mechanisms underlying CIM, but the role of nutritional factors triggering this serious complication of modern intensive care remains unknown. This study aims at establishing the effect of nutritional status in the pathogenesis of CIM. An experimental ICU model was used where animals are mechanically ventilated, pharmacologically paralysed post-synaptically and extensively monitored for up to 14 days. Due to the complexity of the experimental model, the number of animals included is small. After exposure to this ICU condition, animals develop a phenotype similar to patients with CIM. The results from this study show that the preferential myosin loss, decline in specific force and muscle fiber atrophy did not differ between low vs. eucaloric animals. In both experimental groups, passive mechanical loading had a sparing effect of muscle weight independent on nutritional status. Thus, this study confirms the strong impact of the mechanical silencing associated with the ICU condition in triggering CIM, overriding any potential effects of caloric intake in triggering CIM. In addition, the positive effects of passive mechanical loading on muscle fiber size and force generating capacity was not affected by the nutritional status in this study. However, due to the small sample size these pilot results need to be validated in a larger cohort.

Concurrent muscle and bone deterioration in a murine model of cancer cachexia

Cachexia is defined as an excessive, involuntary loss of fat and lean tissue. We tested the validity of the Lewis lung carcinoma (LLC) as a model of cancer cachexia and examined its effect on the two major lean tissue components, skeletal muscle and bone. LLC cells (0.75 × 106) were injected into the left thigh of C57BL/6 mice. Control mice received an equal volume injection of growth media. Tumors were observed in all LLC-injected animals 21 and 25 days post inoculation. LLC-injected animals showed significant reductions in fat and lean mass despite having the same average daily caloric intake as media-treated mice. Global bone mineral density (BMD) had fallen by 5% and 6% in the LLC animals at 21 and 25 days, respectively, compared to a BMD increase of 5% in the 25-day media-treated animals. Extensor digitorum longus (EDL) muscles (isolated from the noninjected hindlimb) showed earlier and quantitatively greater losses in mass, physiological cross-sectional area (pCSA), and tetanic force compared to soleus muscles from the same hindlimb. By the 25th day post-LLC inoculation, EDL force/pCSA was reduced by 19% versus media treatment. This loss in specific force was not trivial as it accounted for about one-third of the reduction in EDL absolute force at this time point. Muscle strips dissected from the diaphragm of LLC mice also exhibited significant reductions in force/pCSA at day 25. We conclude that LLC is a valid model of cachexia that induces rapid losses in global BMD and in limb and respiratory muscle function.

A Novel Approach to Study Mechanical Ventilator-Induced Diaphragm Muscle Weakness

Mechanical ventilation (MV) is widely used clinically, despite being lifesaving, several evidences point out that prolonged MV induces diaphragm muscle weakness. Until now, most studies have focused on the impact of early MV (up to 18h) in rats . Long-term changes remain unknown. In our lab, we have developed a unique model allowing to keep a rat mechanically ventilated for up to 3 weeks. Therefore, in the present study, we looked at the effect of 2 weeks of MV and immobilization in rats. The rats were divided into 4 groups according to MV and immobilization duration : control, 1-4 days, 5-8 days and 9-14 days. Cross-sectional area (CSA) and specific tension (force normalized to CSA) were measured in maximally activated (pCa 4.5) skinned single fibres set at a sarcomere length of 2.6 μm. The specific tension was significantly reduced in the second, third and fourth group compared to control (p<0.001). On the other hand, the CSA remained unchanged in the second group compared to control and was significantly reduced in groups 3 and 4 (p<0.02 and p<0.001 respectively). This tends to indicate that early loss in specific force is likely to be due to post-translational modifications of the contractile proteins rather than up-regulated proteolysis. Further experiments to analyze myosin speed and force using a modified single fibre in vitro motility assay as well as myosin content using a PLA™ based assay will be presented at the meeting.

Physical inactivity and muscle weakness in the critically ill

Patients in the intensive care unit commonly develop muscle weakness. In part, this reflects loss of mechanical loading due to physical inactivity, bed rest, or immobilization. Mechanical unloading stimulates a complex adaptive response that results in muscle atrophy and loss of specific force. One element of this response is slowing of protein synthesis, which is regulated by signaling pathways downstream of mammalian target of rapamycin and insulin-like growth factor-1. In parallel, protein degradation is accelerated via three coordinate processes: calcium-dependent proteolysis, adenosine triphosphate-dependent proteolysis, and lysosomal proteolysis. Finally, unloading stimulates apoptosis of a subset of myonuclei within multinucleated muscle fibers. This helps to stabilize the relationship between nuclear number and cell volume during atrophy. Each of these responses is promoted by concurrent development of oxidative stress caused by increased production of reactive oxygen species in unloaded muscle fibers. Countermeasures that lessen the effects of unloading include physical activity, nutritional supplements, hormone therapy, and antioxidant administration. Targeted research is needed to define the role of mechanical unloading in intensive care unit-associated weakness and develop countermeasures to preserve muscle function, lessen illness, and hasten the recovery of critically ill patients.

Increased CaVβ1a expression with aging contributes to skeletal muscle weakness

Ca2+ release from the sarcoplasmic reticulum (SR) into the cytosol is a crucial part of excitation-contraction (E-C) coupling. Excitation-contraction uncoupling, a deficit in Ca2+ release from the SR, is thought to be responsible for at least some of the loss in specific force observed in aging skeletal muscle. Excitation-contraction uncoupling may be caused by alterations in expression of the voltage-dependent calcium channel alpha1s (CaV1.1) and beta1a (CaVbeta1a) subunits, both of which are necessary for E-C coupling to occur. While previous studies have found CaV1.1 expression declines in old rodents, CaVbeta1a expression has not been previously examined in aging models. Western blot analysis shows a substantial increase of CaVbeta1a expression over the full lifespan of Friend Virus B (FVB) mice. To examine the specific effects of CaVbeta1a overexpression, a CaVbeta1a-YFP plasmid was electroporated in vivo into young animals. The resulting increase in expression of CaVbeta1a corresponded to decline of CaV1.1 over the same time period. YFP fluorescence, used as a measure of CaVbeta1a-YFP expression in individual fibers, also showed an inverse relationship with charge movement, measured using the whole-cell patch-clamp technique. Specific force was significantly reduced in young CaVbeta1a-YFP electroporated muscle fibers compared with sham-electroporated, age-matched controls. siRNA interference of CaVbeta1a in young muscles reduced charge movement, while charge movement in old was restored to young control levels. These studies imply CaVbeta1a serves as both a positive and negative regulator CaV1.1 expression, and that endogenous overexpression of CaVbeta1a during old age may play a role in the loss of specific force.

Age Dependent Expression of CaVβ1a Subunit in Aging Skeletal Muscle

Ca2+ release from the sarcoplasmic reticulum (SR) into the cytosol is a crucial part of excitation-contraction (E-C) coupling. E-C uncoupling, a deficit in Ca2+ release from the SR, is thought to be responsible for at least some of the loss in specific force observed in aging skeletal muscle. E-C uncoupling may be caused by alterations in the expression of voltage-dependent calcium channel α1s (CaV1.1) and β1a (CaVβ1a) subunits. In addition to its classical role augmenting CaV1.1 trafficking and function, overexpression of CaVβ1a has recently been implicated by our laboratory as a negative regulator of CaV1.1 membrane expression. While previous studies have found CaV1.1 expression declines in old rodents, CaVβ1a expression had not been examined in aging models. We conducted western blot analyses to examine CaVβ1a expression in mouse skeletal muscle at four age groups across the animal's lifespan. Investigation of CaVβ1a expression was further segregated by muscle compartment location, fiber type composition, and mouse strain. Our results show a substantial increase of CaVβ1a expression both early and very late in life of FVB mice, regardless of fiber type or location. In order to examine the specific subcellular location where this increased quantity of CaVβ1a resides, we performed immunocytochemistry on dissociated single FDB fibers. Additional western blot analyses were performed on isolated subcellular fractions from young and old animals. Together with previous data from our laboratory showing decline in CaV1.1 as a result of both, aging and CaVβ1a artificial overexpression, these studies suggest a novel role of CaVβ1a, and that endogenous overexpression of CaVβ1a during old age may contribute to the coincident loss of specific force in skeletal muscle.