CFTR corrector C17 is effective in muscular dystrophy, in vivo proof of concept in LGMDR3.

Limb-girdle muscular dystrophy R3 (LGMDR3) is caused by mutations in the SGCA gene coding for α-sarcoglycan (SG). Together with ß- γ- and δ-SG, α-SG forms a tetramer embedded in the dystrophin associated protein complex crucial for protecting the sarcolemma from mechanical stresses elicited by muscle contraction. Most LGMDR3 cases are due to missense mutations, which result in non-properly folded, even though potentially functional α-SG. These mutants are prematurely discarded by the cell quality control. Lacking one subunit, the SG-complex is disrupted. The resulting loss of function leads to sarcolemma instability, muscle fiber damage and progressive limb muscle weakness. LGMDR3 is severely disabling and, unfortunately, still incurable. Here, we propose the use of small molecules, belonging to the class of cystic fibrosis transmembrane regulator (CFTR) correctors, for recovering mutants of α-SG defective in folding and trafficking. Specifically, CFTR corrector C17 successfully rerouted the SG-complex containing the human R98H-α-SG to the sarcolemma of hind-limb muscles of a novel LGMDR3 murine model. Notably, the muscle force of the treated model animals was fully recovered. To our knowledge, this is the first time that a compound designated for cystic fibrosis is successfully tested in a muscular dystrophy and may represent a novel paradigm of treatment for LGMDR3 as well as different other indications in which a potentially functional protein is prematurely discarded as folding-defective. Furthermore, the use of small molecules for recovering the endogenous mutated SG has an evident advantage over complex procedures such as gene or cell transfer.

Chronic Systemic Curcumin Administration Antagonizes Murine Sarcopenia and Presarcopenia.

Curcumin administration attenuates muscle disuse atrophy, but its effectiveness against aging-induced, selective loss of mass or force (presarcopenia or asthenia/dynopenia), or combined loss (sarcopenia), remains controversial. A new systemic curcumin treatment was developed and tested in 18-month-old C57BL6J and C57BL10ScSn male mice. The effects on survival, liver toxicity, loss of muscle mass and force, and satellite cell responsivity and commitment were evaluated after 6-month treatment. Although only 24-month-old C57BL10ScSn mice displayed age-related muscle impairment, curcumin significantly increased survival of both strains (+20-35%), without signs of liver toxicity. Treatment prevented sarcopenia in soleus and presarcopenia in EDL of C57BL10ScSn mice, whereas it did not affect healthy-aged muscles of C57BL6J. Curcumin-treated old C57BL10ScSn soleus preserved type-1 myofiber size and increased type-2A one, whereas EDL maintained adult values of total myofiber number and fiber-type composition. Mechanistically, curcumin only partially prevented the age-related changes in protein level and subcellular distribution of major costamere components and regulators. Conversely, it affected satellite cells, by maintaining adult levels of myofiber maturation in old regenerating soleus and increasing percentage of isolated, MyoD-positive satellite cells from old hindlimb muscles. Therefore, curcumin treatment successfully prevents presarcopenia and sarcopenia development by improving satellite cell commitment and recruitment.

Are muscle fibres of body builders intrinsically weaker? A comparison with single fibres of aged-matched controls.

AIM: Skeletal muscles of Body Builders (BB) represent an interesting model to study muscle mass gains in response to high volume resistance training. It is debated whether muscle contractile performance improves in proportion to mass. Here, we aim to assess whether muscle hypertrophy does not occur at the expense of performance. METHODS: Six BB and Six untrained controls (CTRL) were recruited. Cross-sectional area (CSA) and maximum voluntary contraction (MVC) of quadriceps femoris muscle (QF) and CSA and architecture of vastus lateralis (VL) were determined. Moreover, a biopsy was taken from VL mid-portion and single fibres were analysed. RESULTS: QF CSA and MVC were 32% (n.s., P = .052) and 58% (P = .009) higher in BB than in CTRL, respectively. VL CSA was 37% higher in BB (P = .030). Fast 2A fibres CSA was 24% (P = .048) greater in BB than in CTRL, when determined in immunostained sections of biopsy samples. Single permeabilized fast fibres CSA was 37% (n.s., P = .052) higher in BB than in CTRL, and their force was slightly higher in BB (n.s.), while specific tension (P0 ) was 19% (P = .024) lower. The lower P0 was not explained either by lower myosin content or by impaired calcium diffusion. Conversely, the swelling caused by skinning-induced permeabilization was different and, when used to correct P0 , differences between populations disappeared. CONCLUSIONS: The results show that high degree of muscle hypertrophy is not detrimental for force generation capacity, as increases in fibre size and force are strictly proportional once the differential swelling response is accounted for.

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

NEW FINDINGS: 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. ABSTRACT: 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 2 days for the duration of experiments. Soleus muscle properties were analysed 7 or 14 days after the first injection. The decreased availability of circulating S1P after the 14 day 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.

Alterations of Extracellular Matrix Mechanical Properties Contribute to Age-Related Functional Impairment of Human Skeletal Muscles.

Aging of human skeletal muscles is associated with increased passive stiffness, but it is still debated whether muscle fibers or extracellular matrix (ECM) are the determinants of such change. To answer this question, we compared the passive stress generated by elongation of fibers alone and arranged in small bundles in young healthy (Y: 21 years) and elderly (E: 67 years) subjects. The physiological range of sarcomere length (SL) 2.5-3.3 µm was explored. The area of ECM between muscle fibers was determined on transversal sections with picrosirius red, a staining specific for collagen fibers. The passive tension of fiber bundles was significantly higher in E compared to Y at all SL. However, the resistance to elongation of fibers alone was not different between the two groups, while the ECM contribution was significantly increased in E compared to Y. The proportion of muscle area occupied by ECM increased from 3.3% in Y to 8.2% in E. When the contribution of ECM to bundle tension was normalized to the fraction of area occupied by ECM, the difference disappeared. We conclude that, in human skeletal muscles, the age-related reduced compliance is due to an increased stiffness of ECM, mainly caused by collagen accumulation.

Fibre and extracellular matrix contributions to passive forces in human skeletal muscles: An experimental based constitutive law for numerical modelling of the passive element in the classical Hill-type three element model.

The forces that allow body movement can be divided into active (generated by sarcomeric contractile proteins) and passive (sustained by intra-sarcomeric proteins, fibre cytoskeleton and extracellular matrix (ECM)). These are needed to transmit the active forces to the tendon and the skeleton. However, the relative contribution of the intra- and extra- sarcomeric components in transmitting the passive forces is still under debate. There is limited data in the literature about human muscle and so it is difficult to make predictions using multiscale models, imposing a purely phenomenological description for passive forces. In this paper, we apply a method for the experimental characterization of the passive properties of fibres and ECM to human biopsy and propose their clear separation in a Finite Element Model. Experimental data were collected on human single muscle fibres and bundles, taken from vastus lateralis muscle of elderly subjects. Both were progressively elongated to obtain two stress-strain curves which were fitted to exponential equations. The mechanical properties of the extracellular passive components in a bundle of fibres were deduced by the subtraction of the passive tension observed in single fibres from the passive tension observed in the bundle itself. Our results showed that modulus and tensile load bearing capability of ECM are higher than those of fibres and defined their quantitative characterization that can be used in macroscopic models to study their role in the transmission of forces in physiological and pathophysiological conditions.

Evaluation of chest ultrasound integrated teaching of respiratory system physiology to medical students.

Ultrasound imaging is a widely used diagnostic technique, whose integration in medical education is constantly growing. The aim of this study was to evaluate chest ultrasound usefulness in teaching respiratory system physiology, students' perception of chest ultrasound integration into a traditional lecture in human physiology, and short-term concept retention. A lecture about respiratory physiology was integrated with ultrasound and delivered to third-year medical students. It included basic concepts of ultrasound imaging and the physiology of four anatomic sectors of the body of a male volunteer, shown with a portable ultrasound device (pleural sliding, diaphragmatic movement, inferior vena cava diameter variations, cardiac movements). Students' perceptions of the integrated lecture were assessed, and attendance recorded. After 4 mo, four multiple-choice questions about respiratory physiology were administered during the normal human physiology examinations, and the results of students who attended the lesson and those of who did not were compared. One hundred thirty-four students attended the lecture. Most of them showed encouragement for the study of the subject and considered the ultrasound integrated lecture more interesting than a traditional one and pertinent to the syllabus. Exposed students achieved a better score at the examination and committed less errors than did nonexposed students. The chest ultrasound integrated lecture was appreciated by students. A possible association between the exposure to the lecture and short-term concept retention is shown by better performances of the exposed cohort at the examination. A systematic introduction of ultrasound into physiology traditional teaching will be promoted by the Ultrasound-Based Medical Education movement.

Ablation of S1P3 receptor protects mouse soleus from age-related drop in muscle mass, force, and regenerative capacity.

We investigated the effects of S1P3 deficiency on the age-related atrophy, decline in force, and regenerative capacity of soleus muscle from 23-mo-old male (old) mice. Compared with muscle from 5-mo-old (adult) mice, soleus mass and muscle fiber cross-sectional area (CSA) in old wild-type mice were reduced by ~26% and 24%, respectively. By contrast, the mass and fiber CSA of soleus muscle in old S1P3-null mice were comparable to those of adult muscle. Moreover, in soleus muscle of wild-type mice, twitch and tetanic tensions diminished from adulthood to old age. A slowing of contractile properties was also observed in soleus from old wild-type mice. In S1P3-null mice, neither force nor the contractile properties of soleus changed during aging. We also evaluated the regenerative capacity of soleus in old S1P3-null mice by stimulating muscle regeneration through myotoxic injury. After 10 days of regeneration, the mean fiber CSA of soleus in old wild-type mice was significantly smaller (-28%) compared with that of regenerated muscle in adult mice. On the contrary, the mean fiber CSA of regenerated soleus in old S1P3-null mice was similar to that of muscle in adult mice. We conclude that in the absence of S1P3, soleus muscle is protected from the decrease in muscle mass and force, and the attenuation of regenerative capacity, all of which are typical characteristics of aging.

S1P3 receptor influences key physiological properties of fast-twitch extensor digitorum longus muscle.

To examine the role of sphingosine 1-phosphate (S1P) receptor 3 (S1P3) in modulating muscle properties, we utilized transgenic mice depleted of the receptor. Morphological analyses of extensor digitorum longus (EDL) muscle did not show evident differences between wild-type and S1P3-null mice. The body weight of 3-mo-old S1P3-null mice and the mean cross-sectional area of transgenic EDL muscle fibers were similar to those of wild-type. S1P3 deficiency enhanced the expression level of S1P1 and S1P2 receptors mRNA in S1P3-null EDL muscle. The contractile properties of S1P3-null EDL diverge from those of wild-type, largely more fatigable and less able to recover. The absence of S1P3 appears responsible for a lower availability of calcium during fatigue. S1P supplementation, expected to stimulate residual S1P receptors and signaling, reduced fatigue development of S1P3-null muscle. Moreover, in the absence of S1P3, denervated EDL atrophies less than wild-type. The analysis of atrophy-related proteins in S1P3-null EDL evidences high levels of the endogenous regulator of mitochondria biogenesis peroxisome proliferative-activated receptor-γ coactivator 1α (PGC-1α); preserving mitochondria could protect the muscle from disuse atrophy. In conclusion, the absence of S1P3 makes the muscle more sensitive to fatigue and slows down atrophy development after denervation, indicating that S1P3 is involved in the modulation of key physiological properties of the fast-twitch EDL muscle.

Effects of cysteinyl-leukotriene receptors' antagonism by montelukast on lung mechanics and olfactory system histology in healthy mice.

CONTEXT: At variance with steroid administration, the possible effects of leukotrienes inhibition on basal respiratory mechanics and olfactory system function are still unclear. OBJECTIVE: To investigate if interference with the leukotrienes activity may influence basal lung mechanics in healthy mammals, as well as the olfactory system. MATERIALS AND METHODS: We measured lung mechanics by the end-inflation occlusion method in control and in montelukast i.p. treated anaesthetised healthy mice (10 mg/kg/die for a week). A study of olfactory system histology was also conducted. RESULTS: Elastance and resistive properties of the lung were not affected by montelukast, while a significant increment of lung hysteresis was observed. The analysis of olfactory system histology revealed no significant effects of montelukast compared to controls. DISCUSSION AND CONCLUSIONS: Leukotrienes' antagonism does not affect respiratory mechanics in basal conditions, except for a hysteresis increment, which might counteract the increase in expiratory flow in asthmatic subjects assuming montelukast.

Pressure-flow relationship in swine ureter: effect of nitric oxide synthase inhibition by L-NAME.

INTRODUCTION: The purpose of this report is to elucidate the possible role of nitric oxide (NO) in modulating smooth muscle tone of swine ureteral wall and its hydraulic resistance to urinary flow. MATERIALS AND METHODS: We performed direct monitoring of perfusion pressure for different flows in isolated fresh swine ureter. Fluid used for perfusions was saline or L-NAME 200 mumol saline added, and relative pressure-flow relationships were obtained. RESULTS: The general shape of pressure-flow relationships indicates a decrease of ureteral hydraulic resistance with increasing flow. NO synthase inhibition by L-NAME caused a statistically significant increase of perfusion pressures for every tested flow, hence of ureteral hydraulic resistance. CONCLUSIONS: Our data show an effective role of NO in modulating ureteral flow resistance, and suggest possible pharmacological implications. A simple mathematical approach to define hydraulic ureteral flow resistance for low and high urinary flows is also suggested.

Time course of alterations after olfactory bulbectomy in mice.

Olfactory bulbectomy in rodents causes behavioral alterations, which result in a model of depression, validated for pharmacological screening of antidepressant drugs. To unravel the appearance and time course of the major behavioral effects which follow surgery, mice underwent olfactory bulb ablation or sham operation, and were analyzed after 1, 2, or 4 weeks. Bulbectomized (BX) mice were anosmic, and hyperactive when tested under stressful situations in the forced swimming test. Predatory aggression was upregulated in a time-dependent way: only after 4 weeks BX mice were faster than controls in attacking prey. At the same time, they were less aggressive against intruders; they did not differ from controls in open field exploration, but displayed a cognitive impairment in water maze. Behavioral tests thus indicated a marked hyperreactivity, a dissociation among different aggressive behaviors, and also a cognitive impairment induced by bulbectomy. Histological confirmation of the damage revealed that major modifications took place in the rostral pole of frontal lobes, with a significant increase in the width of the rostral migratory stream, 2 weeks after surgery, and in the subventricular zone, 4 weeks after surgery. These results suggest a base for the time-course of appearance of behavioral symptoms in BX mice.

Outcome of bovine herpesvirus 4 infection following direct viral injection in the lateral ventricle of the mouse brain.

A recombinant bovine herpesvirus 4 (BoHV-4EGFPDeltaTK), obtained by the insertion of an EGFP gene into the TK locus of DN 599 BoHV-4 strain, was injected into the lateral ventricle of the brain of mice and a clinical score was evaluated for 90 days. Although BoHV-4 was not neuro-pathogenic, BoHV-4EGFPDeltaTK transduction capability was analyzed. EGFP expression was localized in close proximity to the border of the ventricles and EGFP-positive cells were found to co-localize with ependymal cells. Although most of the cells had a polarized morphology, they were not neurons. EGFP-positive cells were seen to spread in tangentially oriented rows within the rostral migratory stream (RMS). Co-localization of EGFP signal with anti-GFAP antibody showed that they were glial cells. EGFP-positive cells were observed until 31 days post-injection and then disappeared completely. Virus isolation was possible at an early post-injection time (3 days), but then virus titer was below the detection limits at later times. Viral DNA, however, could be detected until 21 days post-injection. Thus, in this report we showed that (i) BoHV-4EGFPDeltaTK did not replicate in the mouse brain, (ii) is not pathogenic and (iii) gene transfer can be obtained in long-lived cells belonging to the RMS after BoHV-4EGFPDeltaTK injection within the lateral ventricle.

Animal models of depression: olfactory lesions affect amygdala, subventricular zone, and aggression.

Psychiatric or depressed patients show alterations in both olfactory projection areas and mucosa. In rodents, ablation of olfactory bulbs causes a depression-like syndrome, useful to test antidepressant agents. We studied in mice the behavioral symptoms and neuroanatomical correlates after mucosal damage or ablation of the olfactory bulb. Our results are based on a battery of tests exploiting anxious, aggressive, and depressive behavior, on morphological and immunohistochemical analysis. We found similar results in both sensory-damaged and bulbectomized animals, with a behavioral dissociation concerning different forms of aggression. These findings do not support a simple downregulation of social interactions in damaged mice. The most prominent modifications in the brains of sensory damaged and bulbectomized mice are detected in the subventricular zone (SVZ), the source area of neural stem cells, and in the content of cAMP-dependent protein kinase within the amygdala, suggesting a central role of this structure in the functional modulation of behavior.

Endocrine status affects bladder size and postvoid residual urinary volume in mice.

Urine is one of the major media for intraspecific chemical communication in mice. The urination pattern is dependent both on the mice's hormonal and social status. The urination pattern and the morphology of the urinary tract were examined in mice following hormonal manipulations. In the first experiment, we compared pairs of intact and castrated males: intact males urinated earlier when exposed to a new environment, with a greater number of drops that were smaller than those of castrated males. In the second experiment, groups of intact males, castrated, testosterone-supplemented castrated, and isolated intact males were compared. The micturition pattern of isolated intact males consisted of numerous small droplets of urine, with a high volume of urine retained in the bladder after voiding. This also applied to grouped intact males and testosterone-treated castrated mice, while castrated mice voided a larger fraction of bladder content. Bladder weight was higher in intact males and testosterone-treated castrated males, as compared to castrated males. In the third experiment, ovary-intact and testosterone-treated intact females were compared. Testosterone-treated ovary-intact females retained a larger quantity of urine in the bladder and also had a larger bladder compared to ovary-intact females. Testosterone thus induces the morphological modifications of the urinary tract necessary for the dominant male urination pattern, which is an increase in postvoid urinary residual volume and bladder weight. As evidenced from the comparison of histological sections from intact, castrated, and testosterone-treated castrated males, the increase in bladder weight was mainly due to the bladder muscular mass.