Ultrasonography validation for early alteration of diaphragm echodensity and function in the mdx mouse model of Duchenne muscular dystrophy

The mdx mouse model of Duchenne muscular dystrophy is characterized by functional and structural alterations of the diaphragm since early stages of pathology, closely resembling patients’ condition. In recent years, ultrasonography has been proposed as a useful longitudinal non-invasive technique to assess mdx diaphragm dysfunction and evaluate drug efficacy over time. To date, only a few preclinical studies have been conducted. Therefore, an independent validation of this method by different laboratories is needed to increase results reliability and reduce biases. Here, we performed diaphragm ultrasonography in 3- and 6-month-old mdx mice, the preferred age-window for pharmacology studies. The alteration of diaphragm function over time was measured as diaphragm ultrasound movement amplitude. At the same time points, a first-time assessment of diaphragm echodensity was performed, as an experimental index of progressive loss of contractile tissue. A parallel evaluation of other in vivo and ex vivo dystrophy-relevant readouts was carried out. Both 3- and 6-month-old mdx mice showed a significant decrease in diaphragm amplitude compared to wild type (wt) mice. This index was well-correlated either with in vivo running performance or ex vivo isometric tetanic force of isolated diaphragm. In addition, diaphragms from 6-month-old dystrophic mice were also highly susceptible to eccentric contraction ex vivo. Importantly, we disclosed an age-dependent increase in echodensity in mdx mice not observed in wt animals, which was independent from abdominal wall thickness. This was accompanied by a notable increase of pro-fibrotic TGF-β1 levels in the mdx diaphragm and of non-muscle tissue amount in diaphragm sections stained by hematoxylin & eosin. Our findings corroborate the usefulness of diaphragm ultrasonography in preclinical drug studies as a powerful tool to monitor mdx pathology progression since early stages

Ergogenic effect of bcaas and l-alanine supplementation: Proof-of-concept study in a murine model of physiological exercise

Background: Branched-chain amino acids (BCAAs: leucine, isoleucine, valine) account for 35% of skeletal muscle essential amino acids (AAs). As such, they must be provided in the diet to support peptide synthesis and inhibit protein breakdown. Although substantial evidence has been collected about the potential usefulness of BCAAs in supporting muscle function and structure, dietary supplements containing BCAAs alone may not be effective in controlling muscle protein turnover, due to the rate-limiting bioavailability of other AAs involved in BCAAs metabolism. Methods: We aimed to evaluate the in vivo/ex vivo effects of a 4-week treatment with an oral formulation containing BCAAs alone (2:1:1) on muscle function, structure, and metabolism in a murine model of physiological exercise, which was compared to three modified formulations combining BCAAs with increasing concentrations of L-Alanine (ALA), an AA controlling BCAAs catabolism. Results: A preliminary pharmacokinetic study confirmed the ability of ALA to boost up BCAAs bioavailability. After 4 weeks, mix 2 (BCAAs + 2ALA) had the best protective effect on mice force and fatigability, as well as on muscle morphology and metabolic indices. Conclusion: Our study corroborates the use of BCAAs + ALA to support muscle health during physiological exercise, underlining how the relative BCAAs/ALA ratio is important to control BCAAs distribution

"Of Mice and Measures": A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies

A comprehensive study on the effect of bentonite fining on wine charged model molecules

In bottled wines, haze and turbidity are phenomena to be avoided. Since bentonite fining is a common process to clarify wines removing heat unstable proteins, a theoretical study on the adsorption of three Charged Model Molecules (CMMs, egg albumin, polyphenols and riboflavin) was carried out to deep comprehend this chemical phenomenon. Four bentonites were adopted and finely characterized together with the potential release of Na+ and Ca2+ cations, revealing suitable for RT albumin removal within 120 min. Better results in terms of adsorbed quantity were achieved by adopting 12%v/v EtOH/H2O solvent and by swelling bentonites for 24 h before use. With the most performing sample (Na/Ca_0.27), a comprehensive study on simultaneous adsorption of the three CMMs was performed, resulting in polyphenols adsorption increase due to their interactions with albumin. Notwithstanding the majority of albumin and riboflavin was successfully removed, ca. 40\u201350% of tested poly- phenols was preserved

Data on protein abundance alteration induced by chronic exercise in mdx mice model of Duchenne muscular dystrophy and potential modulation by apocynin and taurine

Here we present original data related to the research paper entitled "Proteome analysis in dystrophic mdx mouse muscle reveals a drastic alteration of Key Metabolic and Contractile Proteins after chronic exercise and the potential modulation by anti-oxidant compounds" (Gamberi et al., 2018) [1]. The dystrophin-deficient mdx mouse is the most common animal model for Duchenne muscular dystrophy. The mdx mice phenotype of the disorder is milder than in human sufferers and it can be worsened by chronic treadmill exercise. Apocynin and taurine are two antioxidant compounds proved to be beneficial on some pathology related parameters (Schroder and Schoser, 2009) [2]. This article reports the detailed proteomic data on protein abundance alterations, in tibialis anterior muscle of mdx mice, induced by chronic exercise protocol. A selected group of mdx mice was also treated with apocynin and taurine during this protocol. Detailed MS data, comparison between mdx vs wild type, exercised mdx vs wild type, and complete analysis of spot variation are provided. Furthermore, in wild type mice subjected to the same exercise protocol, the abundance of key proteins, resulted modified in exercised mdx, were analyzed by western blot. (C) 2018 The Authors. Published by Elsevier Inc

Proof-of-concept validation of the mechanism of action of Src tyrosine kinase inhibitors in dystrophic mdx mouse muscle: in vivo and in vitro studies

Src tyrosine kinase (TK), a redox-sensitive protein overexpressed in dystrophin-deficient muscles, can contribute to damaging signaling by phosphorylation and degradation of β-dystroglycan (β-DG). We performed a proof-of-concept preclinical study to validate this hypothesis and the benefit-safety ratio of a pharmacological inhibition of Src-TK in Duchenne muscular dystrophy (DMD). Src-TK inhibitors PP2 and dasatinib were administered for 5 weeks to treadmill-exercised mdx mice. The outcome was evaluated in vivo and ex vivo on functional, histological and biochemical disease-related parameters. Considering the importance to maintain a proper myogenic program, the potential cytotoxic effects of both compounds, as well as their cytoprotection against oxidative stress-induced damage, was also assessed in C2C12 cells. In line with the hypothesis, both compounds restored the level of β-DG and reduced its phosphorylated form without changing basal expression of genes of interest, corroborating a mechanism at post-translational level. The histological profile of gastrocnemius muscle was slightly improved as well as the level of plasma biomarkers. However, amelioration of in vivo and ex vivo functional parameters was modest, with PP2 being more effective than dasatinib. Both compounds reached appreciable levels in skeletal muscle and liver, supporting proper animal exposure. Dasatinib exerted a greater concentration-dependent cytotoxic effect on C2C12 cells than the more selective PP2, while being less protective against H2O2 cytotoxicity, even though at concentrations higher than those experienced during in vivo treatments. Our results support the interest of Src-TK as drug target in dystrophinopathies, although further studies are necessary to assess the therapeutic potential of inhibitors in DMD

BCAAs and Di-Alanine supplementation in the prevention of skeletal muscle atrophy: preclinical evaluation in a murine model of hind limb unloading

Skeletal muscle atrophy occurs in response to various pathophysiological stimuli, including disuse, aging, and neuromuscular disorders, mainly due to an imbalance of anabolic/catabolic signaling. Branched Chain Amino Acids (BCAAs: leucine, isoleucine, valine) supplements can be beneficial for counteracting muscle atrophy, in virtue of their reported anabolic properties. Here, we carried out a proof-of-concept study to assess the in vivo/ex vivo effects of a 4-week treatment with BCAAs on disuse-induced atrophy, in a murine model of hind limb unloading (HU). BCAAs were formulated in drinking water, alone, or plus two equivalents of L-Alanine (2 ALA) or the dipeptide L-Alanyl-L-Alanine (Di-ALA), to boost BCAAs bioavailability. HU mice were characterized by reduction of body mass, decrease of soleus – SOL – muscle mass and total protein, alteration of postural muscles architecture and fiber size, dysregulation of atrophy-related genes (Atrogin-1, MuRF-1, mTOR, Mstn). In parallel, we provided new robust readouts in the HU murine model, such as impaired in vivo isometric torque and ex vivo SOL muscle contractility and elasticity, as well as altered immune response. An acute pharmacokinetic study confirmed that L-ALA, also as dipeptide, enhanced plasma exposure of BCAAs. Globally, the most sensitive parameters to BCAAs action were muscle atrophy and myofiber cross-sectional area, muscle force and compliance to stress, protein synthesis via mTOR and innate immunity, with the new BCAAs + Di-ALA formulation being the most effective treatment. Our results support the working hypothesis and highlight the importance of developing innovative formulations to optimize BCAAs biodistribution

ULTRASTRUCTURAL FEATURES OF HUMAN SPERM CELLS CRYOPRESERVED BY DIFFERENT METHODS

Cryopreservation of human spermatozoa has been recognized as a key strategy for management of male fertility. Nevertheless, current protocols of sperm freezing are neither optimal nor standardized between different labs (1). In this study we compare the ultrastructural features of human normospermic sperm samples (according to WHO parameters 2010) from 5 different freezing techniques in order to identify the best methods of cryopreservation. After informed con- sent, 21 normospermic patients (from the Medically Assisted Procreation PMA Center of the Fondazione IRCCS Policlinico San Matteo in Pavia) were recruited and both traditional and improved analysis of sperm quality were applied, in order to define critical steps of cryopreservation. Cryopreservation of human spermatozoa has been related to decreased motility associated with impaired velocity and viability of sperm pre-freeze and post-thaw. For all applied methods there was a significant reduction of progressive and total motility (P) as a result of freezing. To investigate ultrastructural details, 5 additional cryopreserved samples by the best two freezing methods were analyzed with elec- tron microscopy (TEM). Preliminary data showed the minimal differences between the protocols, with a large number of queues detached and large quantities of cyto- plasmic debris after of the first protocol. Spermatozoa appear to be better preserved in the second analyzed method, despite both procedures induced deteriorations at ultrastructural level (2). Other non-routine analysis will be performed to determine whether the cooling time to +4°C may affect the procedure; Comet Assay (to assess the degree of sperm DNA fragmentation) (3) and flow cytometry (to study light scat- ters patterns and membrane integrity) (4) will be applied

β‐Dystroglycan Restoration and Pathology Progression in the Dystrophic mdx Mouse: Outcome and Implication of a Clinically Oriented Study with a Novel Oral Dasatinib Formulation

ROS‐activated cSrc tyrosine kinase (TK) promotes the degradation of β‐dystroglycan (β‐ DG), a dystrophin‐glycoprotein complex component, which may reinforce damaging signals in Duchenne muscular dystrophy (DMD). Therefore, cSrc‐TK represents a promising therapeutic target. In mdx mice, a 4‐week subcutaneous treatment with dasatinib (DAS), a pan‐Src‐TKs inhibitor approved as anti‐leukemic agent, increased muscle β‐DG, with minimal amelioration of morphofunctional indices. To address possible dose/pharmacokinetic (PK) issues, a new oral DAS/hydroxypropyl(HP)‐β‐cyclodextrin(CD) complex was developed and chronically administered to mdx mice. The aim was to better assess the role of β‐DG in pathology progression, meanwhile confirming DAS mechanism of action over the long‐term, along with its efficacy and tolerability. The 4‐week old mdx mice underwent a 12‐week treatment with DAS/HP‐β‐CD10% dissolved in drinking water, at 10 or 20 mg/kg/day. The outcome was evaluated via in vivo/ex vivo disease‐relevant readouts. Oral DAS/HP‐β‐CD efficiently distributed in mdx mice plasma and tissues in a dose‐related fashion. The new DAS formulation confirmed its main upstream mechanism of action, by reducing β‐DG phosphorylation and restoring its levels dose‐dependently in both diaphragm and gastrocnemius muscle. However, it modestly improved in vivo neuromuscular function, ex vivo muscle force, and histopathology, although the partial recovery of muscle elasticity and the decrease of CK and LDH plasma levels suggest an increased sarcolemmal stability of dystrophic muscles. Our clinically oriented study supports the interest in this new, pediatric‐suitable DAS formulation for proper exposure and safety and for enhancing β‐DG expression. This latter mechanism is, however, not sufficient by itself to impact on pathology progression. In‐depth analyses will be dedicated to elucidating the mechanism limiting DAS effectiveness in dystrophic settings, meanwhile assessing its potential synergy with dystrophin-based molecular therapies

Effect of exercise on telomere length and telomere proteins expression in mdx mice

In Duchenne muscular dystrophy (DMD), telomere shortening has been postulated to contribute to the failure of regenerative activity promoting the premature senescence of satellite cells. The aim of the present study was to investigate the telomere length and the expression of telomeric repeat-binding factor-1 (TRF1), poly (ADP-ribose) polymerase-1 (PARP1) and mouse telomerase reverse transcriptase (MTERT) in gastrocnemius, tibialis anterior and diaphragm muscles of the murine model of DMD, the mdx mouse and whether a chronic protocol of forced exercise impacts on them. Our results confirmed a telomere shortening in mdx muscles, more evident in the diaphragm, in which exercise induced a greater shortening than in wild-type mice. Moreover, we showed for the first time in mdx an increased TRF1 and PARP1 expression and an augmented activity of MTERT, further enhanced by exercise. These results reinforce the hypothesis that a deregulation of mechanisms involved in telomere length occurs and may pave the way for the test of compounds targeting proteins modulating telomere maintenance as a novel strategy to treat dystrophinopathies