Stefan Matecki

Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy

Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in “leaky” RyR2 channels and a diastolic SR Ca2+ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca2+ channel stabilizer S107 (“rycal”) inhibited the SR Ca2+ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca2+ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca2+ leak prevents fatal sudden cardiac arrhythmias in DMD.

l-Arginine Decreases Inflammation and Modulates the Nuclear Factor-κB/Matrix Metalloproteinase Cascade in Mdx Muscle Fibers

Duchenne muscular dystrophy (DMD) is a lethal, X-linked disorder associated with dystrophin deficiency that results in chronic inflammation, sarcolemma damage, and severe skeletal muscle degeneration. Recently, the use of L-arginine, the substrate of nitric oxide synthase (nNOS), has been proposed as a pharmacological treatment to attenuate the dystrophic pattern of DMD. However, little is known about signaling events that occur in dystrophic muscle with l-arginine treatment. Considering the implication of inflammation in dystrophic processes, we asked whether L-arginine inhibits inflammatory signaling cascades. We demonstrate that L-arginine decreases inflammation and enhances muscle regeneration in the mdx mouse model. Classic stimulatory signals, such as proinflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha, are significantly decreased in mdx mouse muscle, resulting in lower nuclear factor (NF)-kappaB levels and activity. NF-kappaB serves as a pivotal transcription factor with multiple levels of regulation; previous studies have shown perturbation of NF-kappaB signaling in both mdx and DMD muscle. Moreover, L-arginine decreases the activity of metalloproteinase (MMP)-2 and MMP-9, which are transcriptionally activated by NF-kappaB. We show that the inhibitory effect of L-arginine on the NF-kappaB/MMP cascade reduces beta-dystroglycan cleavage and translocates utrophin and nNOS throughout the sarcolemma. Collectively, our results clarify the molecular events by which L-arginine promotes muscle membrane integrity in dystrophic muscle and suggest that NF-kappaB-related signaling cascades could be potential therapeutic targets for DMD management.

Rapid Onset of Specific Diaphragm Weakness in a Healthy Murine Model of Ventilator-induced Diaphragmatic Dysfunction

Background: Controlled mechanical ventilation is associated with ventilator-induced diaphragmatic dysfunction, which impedes weaning from mechanical ventilation. To design future clinical trials in humans, a better understanding of the molecular mechanisms using knockout models, which exist only in the mouse, is needed. The aims of this study were to ascertain the feasibility of developing a murine model of ventilator-induced diaphragmatic dysfunction and to determine whether atrophy, sarcolemmal injury, and the main proteolysis systems are activated under these conditions. Methods: Healthy adult male C57/BL6 mice were assigned to three groups: (1) mechanical ventilation with end-expiratory positive pressure of 2–4 cm H2O for 6 h (n = 6), (2) spontaneous breathing with continuous positive airway pressure of 2–4 cm H2O for 6 h (n = 6), and (3) controls with no specific intervention (n = 6). Airway pressure and hemodynamic parameters were monitored. Upon euthanasia, arterial blood gases and isometric contractile properties of the diaphragm and extensor digitorum longus were evaluated. Histology and immunoblotting for the main proteolysis pathways were performed. Results: Hemodynamic parameters and arterial blood gases were comparable between groups and within normal physiologic ranges. Diaphragmatic but not extensor digitorum longus force production declined in the mechanical ventilation group (maximal force decreased by approximately 40%) compared with the control and continuous positive airway pressure groups. No histologic difference was found between groups. In opposition with the calpains, caspase 3 was activated in the mechanical ventilation group. Conclusion: Controlled mechanical ventilation for 6 h in the mouse is associated with significant diaphragmatic but not limb muscle weakness without atrophy or sarcolemmal injury and activates proteolysis.

E2F transcription factor-1 deficiency reduces pathophysiology in the mouse model of Duchenne muscular dystrophy through increased muscle oxidative metabolism

E2F1 deletion leads to increased mitochondrial number and function, increased body temperature in response to cold and increased resistance to fatigue with exercise. Since E2f1−/− mice show increased muscle performance, we examined the effect of E2f1 genetic inactivation in the mdx background, a mouse model of Duchenne muscular dystrophy (DMD). E2f1−/−;mdx mice demonstrated a strong reduction of physiopathological signs of DMD, including preservation of muscle structure, decreased inflammatory profile, increased utrophin expression, resulting in better endurance and muscle contractile parameters, comparable to normal mdx mice. E2f1 deficiency in the mdx genetic background increased the oxidative metabolic gene program, mitochondrial activity and improved muscle functions. Interestingly, we observed increased E2F1 protein levels in DMD patients, suggesting that E2F1 might represent a promising target for the treatment of DMD.

Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation

Significance Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that follows prolonged controlled mechanical ventilation, impeding recovery from respiratory failure. The mechanisms underlying VIDD are still not fully understood. Using human samples and murine models of VIDD, we identify here a pathophysiological pathway involving structural and functional impairment of the ryanodine receptor (RyR1), the main sarcoplasmic reticulum (SR) Ca2+ release channel. We demonstrate that RyR1 defects, which contribute to diaphragm muscle weakness, induced by controlled mechanical ventilation are the result of oxidative stress associated to sympathetic nervous system activation. Thus, preventing RyR1-mediated SR Ca2+ leak may provide a novel therapeutic approach in controlled mechanical ventilation. Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that occurs following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure. However, the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here, we show in human subjects and a mouse model of VIDD that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR1) in the diaphragm. The RyR1 macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated, and depleted of the stabilizing subunit calstabin1, following MV. These posttranslational modifications of RyR1 were mediated by both oxidative stress mediated by MV and stimulation of adrenergic signaling resulting from the anesthesia. We demonstrate in the murine model that such abnormal resting SR Ca2+ leak resulted in reduced contractile function and muscle fiber atrophy for longer duration of MV. Treatment with β-adrenergic antagonists or with S107, a small molecule drug that stabilizes the RyR1–calstabin1 interaction, prevented VIDD. Diaphragmatic dysfunction is common in MV patients and is a major cause of failure to wean patients from ventilator support. This study provides the first evidence to our knowledge of RyR1 alterations as a proximal mechanism underlying VIDD (i.e., loss of function, muscle atrophy) and identifies RyR1 as a potential target for therapeutic intervention.

Prospective randomized crossover study of a new closed-loop control system versus pressure support during weaning from mechanical ventilation.

Background:Intellivent is a new full closed-loop controlled ventilation that automatically adjusts both ventilation and oxygenation parameters. The authors compared gas exchange and breathing pattern variability of Intellivent and pressure support ventilation (PSV). Methods:In a prospective, randomized, single-blind design crossover study, 14 patients were ventilated during the weaning phase, with Intellivent or PSV, for two periods of 24 h in a randomized order. Arterial blood gases were obtained after 1, 8, 16, and 24 h with each mode. Ventilatory parameters were recorded continuously in a breath-by-breath basis during the two study periods. The primary endpoint was oxygenation, estimated by the calculation of the difference between the PaO2/FIO2 ratio obtained after 24 h of ventilation and the PaO2/FIO2 ratio obtained at baseline in each mode. The variability in the ventilatory parameters was also evaluated by the coefficient of variation (SD to mean ratio). Results:There were no adverse events or safety issues requiring premature interruption of both modes. The PaO2/FIO2 (mean ± SD) ratio improved significantly from 245 ± 75 at baseline to 294 ± 123 (P = 0.03) after 24 h of Intellivent. The coefficient of variation of inspiratory pressure and positive end-expiratory pressure (median [interquartile range]) were significantly higher with Intellivent, 16 [11–21] and 15 [7–23]%, compared with 6 [5–7] and 7 [5–10]% in PSV. Inspiratory pressure, positive end-expiratory pressure, and FIO2 changes were adjusted significantly more often with Intellivent compared with PSV. Conclusions:Compared with PSV, Intellivent during a 24-h period improved the PaO2/FIO2 ratio in parallel with more variability in the ventilatory support and more changes in ventilation settings.

Combined Strategies for Maintaining Skeletal Muscle Mass and Function in Aging: Myostatin Inactivation and AICAR-Associated Oxidative Metabolism Induction

Myostatin (mstn) blockade, resulting in muscle hypertrophy, is a promising therapy to counteract age-related muscle loss. However, oxidative and mitochondrial deficit observed in young mice with myostatin inhibition could be detrimental with aging. The aim of this study was (a) to bring original data on metabolic and mitochondrial consequences of mstn inhibition in old mice, and (b) to examine whether 4-weeks of AICAR treatment, a pharmacological compound known to upregulate oxidative metabolism, may be useful to improve exercise capacity and mitochondrial deficit of 20-months mstn KO versus wild-type (WT) mice. Our results show that despite the enlarged muscle mass, the oxidative and mitochondrial deficit associated with reduced endurance running capacity is maintained in old mstn KO mice but not worsened by aging. Importantly, AICAR treatment induced a significant beneficial effect on running limit time only in old mstn KO mice, with a marked increase in PGC-1α expression and slight beneficial effects on mitochondrial function. We showed that AICAR effects were autophagy-independent. This study underlines the relevance of aged muscle remodelling by complementary approaches that impact both muscle mass and function, and suggest that mstn inhibition and aerobic metabolism activators should be co-developed for delaying age-related deficits in skeletal muscle.

Conséquences de la ventilation mécanique sur le diaphragme

INTRODUCTION Mechanical ventilation is associated with ventilator-induced diaphragmatic dysfunction (VIDD) in animal models and also in humans. BACKGROUND The main pathophysiological pathways implicated in VIDD seems to be related to muscle inactivity but may also be the consequence of high tidal volumes. Systemic insults from side effects of medication, infection, malnutrition and hypoperfusion also play a part. The diaphragm is caught in the cross-fire of ventilation-induced and systemic-induced dysfunctions. Intracellular consequences of VIDD include oxidative stress, proteolysis, impaired protein synthesis, autophagy activation and excitation-contraction decoupling. VIDD can be diagnosed at the bedside using non-invasive magnetic stimulation of the phrenic nerves which is the gold standard. Other techniques involve patients participation such as respiratory function tests or ultrasound examination. CONCLUSION AND PERSPECTIVES At this date, only spontaneous ventilatory cycles and perhaps phrenic nerve stimulation appear to diminish the severity of VIDD in humans but several pathways are currently being examined using animal models. Specific pharmacological options are currently under investigation in animal models.

Pressions respiratoires maximales chez l’enfant : les exigences méthodologiques

Resume Introduction Dans le suivi des enfants presentant une pathologie respiratoire, la mesure des pressions maximales respiratoires contre resistance occluse est utilisee depuis longtemps pour evaluer la force des muscles respiratoires. Elle represente le test principal dans les premiers stades de la pathologie, pour diagnostiquer une implication des muscles respiratoires dans l’atteinte du systeme respiratoire et evaluer le degre de cette atteinte. Etat des connaissances L’interpretation de cet examen reste difficile du fait de la grande variabilite des mesures et des valeurs de reference proposees. Le but de cet article est de presenter sous forme d’une revue de la litterature les differentes normes disponibles, les differents facteurs determinants ainsi que les avantages et les limites de cette mesure. Perspectives L’utilisation, par l’ensemble des centres qui effectuent la mesure des pressions maximales respiratoires chez l’enfant, d’un meme cahier des charges methodologique tel celui presente dans cette revue, pourrait etre le point de depart a l’obtention ulterieure de normes identiques pour tous. Conclusion L’âge, le sexe ou le niveau d’aptitude physique semblent etre les facteurs determinants les plus importants des pressions respiratoires maximales. Mais d’autres facteurs d’ordre methodologique, comme la cooperation ou le niveau d’entrainement de l’enfant a la realisation de la manœuvre respiratoire, le type de materiel ou le protocole utilises vont influencer egalement les resultats des mesures. Ces facteurs doivent etre pris en consideration pour diminuer le plus possible la variabilite des pressions maximales respiratoires chez l’enfant.

Ventilator‐induced diaphragmatic dysfunction in mdx mice

Patients with Duchenne muscular dystrophy (DMD) frequently undergo mechanical ventilation (MV) for treatment of hypoventilation, but the susceptibility of the dystrophic diaphragm to ventilator‐induced diaphragmatic dysfunction (VIDD) has not been examined.