Interactions moléculaires des calpaïnes 1 et 3 avec la région N1 de la titine humaine

Calpains are papain-like cystéine protease first identified several years ago. Because they are present in the cytosol of mammalian cells and because they are activated in response to Ca2+ mobilization, they are thought to be involved mainly in cell signalling pathways. They could participate in cellular responses such as apoptosis, proliferation, extracellular matrix adhesion and motility, that have relevance to pathophysiological issues in dystrophies, ischemia, neuronal diseases, tumor progression. The objective of our investigations is to elucidate activation mechanisms of calpains since association between calpain 3 and titin was shown to be essential to regulate activity of the protease. Here we consider molecular intercations between calpains (1 and 3) and the N1 line region of human titin. We first showed that calcium binding in this region induced a spontaneous agregation. These structural changes could affect the amount of calpain 1 bound to the immunoglobulin-like domain I4 of titin. We further defined the immunoglobulin-like I5 as the calpain 3 binding site that sized in the extreme vincinity of the calpain 1 binding site. Nevertheless, it sounds that calcium dependant changes on titin have no effects on calpain 3 binding. Only structural changes on calpain 3 itself could influence its association with the N1 line region of titin. In light of these results, titin appears to be a strong modulator of calpainolytic activity.Les calpaïnes sont des protéases à cystéine de type papaïne identifiées depuis plusieurs décennies. Parce que leur expression est cytosolique et parce que leur activité est contrôlée par la concentration de Ca2+, il est maintenant admis qu'elles jouent un rôle essentiel dans la signalisation intracellulaire. Elles participent ainsi au contrôle de l'apoptose, de la prolifération, de l'adhérence à la matrice extracellulaire et de la mobilité cellulaire. Il n'est donc pas étonnant qu'elles soient impliquées dans les pathologies lourdes (dystrophies musculaires, ischémies, dégénérescences neuronales, mobilité cellulaire invasive). L'objectif de nos recherches est d'élucider les mécanismes d'activation de différentes calpaïnes (Capn1,Capn2, Capn3) et les voies de signalisation qu'elles mettent en jeu. En effet, l'association de la calpaïne 3 avec la titine est un élément essentiel dans la régulation et l'activation de la protéase. Dans ce cadre, ce travail visait à caractériser les interactions moléculaires des calpaïnes 1 et 3 avec la région N1 de la titine humaine. Nous avons pu montré que cette région pouvait fixer très fortement du calcium permettant la polymérisation des molécules de titine. Cette structuration calcium dépendante conditionne la fixation de la µ-calpaïne sur le domaine I4 de la titine. La seconde partie du travail a permis de caractériser la colocalisation de la calpaïne 3 avec la calpaïne 1 au niveau du domaine I5. A l'inverse de la µ-calpaïne , l'association de la calpaïne 3 ne semble pas dépendre de la structure de la titine mais plutôt des changements structuraux pouvant intervenir sur la protéase elle-même quand elle fixe du calcium. A la lumière de ces résultats, la titine apparaît comme un régulateur de l'action calpainolytiqu

MicroRNA and ROS Crosstalk in Cardiac and Pulmonary Diseases

Reactive oxygen species (ROS) affect many cellular functions and the proper redox balance between ROS and antioxidants contributes substantially to the physiological welfare of the cell. During pathological conditions, an altered redox equilibrium leads to increased production of ROS that in turn may cause oxidative damage. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level contributing to all major cellular processes, including oxidative stress and cell death. Several miRNAs are expressed in response to ROS to mediate oxidative stress. Conversely, oxidative stress may lead to the upregulation of miRNAs that control mechanisms to buffer the damage induced by ROS. This review focuses on the complex crosstalk between miRNAs and ROS in diseases of the cardiac (i.e., cardiac hypertrophy, heart failure, myocardial infarction, ischemia/reperfusion injury, diabetic cardiomyopathy) and pulmonary (i.e., idiopathic pulmonary fibrosis, acute lung injury/acute respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, lung cancer) compartments. Of note, miR-34a, miR-144, miR-421, miR-129, miR-181c, miR-16, miR-31, miR-155, miR-21, and miR-1/206 were found to play a role during oxidative stress in both heart and lung pathologies. This review comprehensively summarizes current knowledge in the field

Purification and partial characterization of antithrombin III from bovine skeletal muscle and possible role of thrombin in postmortem apoptosis development and in efficiency of low voltage electrical stimulation

International audienceThrombin/antithrombin III (AT-III) proteolytic system is well known for its function in blood coagulation. Thrombin is expressed in skeletal muscle but nothing is known about the presence of AT-III in the tissue. In postmortem muscle this system has been and is still totally ignored. We therefore successfully attempted to purify AT-III from bovine skeletal muscle and characterized the purified protein (identified as AT-III by N-ter sequencing and mass spectrometry finger print) for its physicochemical and inhibitory properties. As the human blood serpin, muscle AT-III is thermolabile and stable only at alkaline pH (pH 910). The muscle serpin inhibits strongly thrombin in a heparin dependent manner and trypsin. Phosphatidylserine (PS) externalization demonstrated in the present work suggested that prothrombin can be activated to thrombin through binding of the activator complex on the external PS groups. PS externalization is concomitant with shrinkage of muscle fibers indicating that muscle cells are engaged in the cell death program known as apoptosis few minutes after death. We then discussed the potential role of this proteolytic system in postmortem apoptosis development as well as in the control of low voltage electrical stimulation efficiency

Next-Generation SINE Compound KPT−8602 Ameliorates Dystrophic Pathology in Zebrafish and Mouse Models of DMD

Duchenne muscular dystrophy (DMD) is a progressive, X-linked childhood neuromuscular disorder that results from loss-of-function mutations in the DYSTROPHIN gene. DMD patients exhibit muscle necrosis, cardiomyopathy, respiratory failure, and loss of ambulation. One of the major driving forces of DMD disease pathology is chronic inflammation. The current DMD standard of care is corticosteroids; however, there are serious side effects with long-term use, thus identifying novel anti-inflammatory and anti-fibrotic treatments for DMD is of high priority. We investigated the next-generation SINE compound, KPT-8602 (eltanexor) as an oral therapeutic to alleviate dystrophic symptoms. We performed pre-clinical evaluation of the effects of KPT-8602 in DMD zebrafish (sapje) and mouse (D2-mdx) models. KPT-8602 improved dystrophic skeletal muscle pathologies, muscle architecture and integrity, and overall outcomes in both animal models. KPT-8602 treatment ameliorated DMD pathology in D2-mdx mice, with increased locomotor behavior and improved muscle histology. KPT-8602 altered the immunological profile of the dystrophic mice, and reduced circulating osteopontin serum levels. These findings demonstrate KPT-8602 as an effective therapeutic in DMD through by promotion of an anti-inflammatory environment and overall improvement of DMD pathological outcomes

Postmortem muscle cells die through apoptosis

International audienceSeveral reports suggested the activation of caspases in postmortem muscle implicating the onset of a caspase-dependent cell death process after animal bleeding. It has been further well established that apoptosis and necrosis are the two major cell death pathways. The questions addressed in the present work were as follows: (a) in postmortem muscle, do cells die as in vivo? and (b) if so, by which dying process this goal is achieved? Selected hallmarks of apoptosis (phosphatidylserine externalization (PS), cell shrinkage, actin degradation) were analyzed in postmortem rat muscles and compared to usual cell behavior in apoptotic and necrotic processes. Results presented clearly demonstrate a rapid PS externalization and cell shrinkage extending during the first 24 h postexsanguination together with a progressive degradation of cytoskeletal and thin filaments of actin. It was therefore concluded that, in postmortem muscle, cells commit suicide soon after animal bleeding through apoptosis

Next-Generation SINE Compound KPT−8602 Ameliorates Dystrophic Pathology in Zebrafish and Mouse Models of DMD

Duchenne muscular dystrophy (DMD) is a progressive, X-linked childhood neuromuscular disorder that results from loss-of-function mutations in the DYSTROPHIN gene. DMD patients exhibit muscle necrosis, cardiomyopathy, respiratory failure, and loss of ambulation. One of the major driving forces of DMD disease pathology is chronic inflammation. The current DMD standard of care is corticosteroids; however, there are serious side effects with long-term use, thus identifying novel anti-inflammatory and anti-fibrotic treatments for DMD is of high priority. We investigated the next-generation SINE compound, KPT−8602 (eltanexor) as an oral therapeutic to alleviate dystrophic symptoms. We performed pre-clinical evaluation of the effects of KPT−8602 in DMD zebrafish (sapje) and mouse (D2-mdx) models. KPT−8602 improved dystrophic skeletal muscle pathologies, muscle architecture and integrity, and overall outcomes in both animal models. KPT−8602 treatment ameliorated DMD pathology in D2-mdx mice, with increased locomotor behavior and improved muscle histology. KPT−8602 altered the immunological profile of the dystrophic mice, and reduced circulating osteopontin serum levels. These findings demonstrate KPT−8602 as an effective therapeutic in DMD through by promotion of an anti-inflammatory environment and overall improvement of DMD pathological outcomes

A stromal progenitor and ILC2 niche promotes muscle eosinophilia and fibrosis-associated gene expression.

Despite the well-accepted view that chronic inflammation contributes to the pathogenesis of Duchenne muscular dystrophy (DMD), the function and regulation of eosinophils remain an unclear facet of type II innate immunity in dystrophic muscle. We report the observation that group 2 innate lymphoid cells (ILC2s) are present in skeletal muscle and are the principal regulators of muscle eosinophils during muscular dystrophy. Eosinophils were elevated in DMD patients and dystrophic mice along with interleukin (IL)-5, a major eosinophil survival factor that was predominantly expressed by muscle ILC2s. We also find that IL-33 was upregulated in dystrophic muscle and was predominantly produced by fibrogenic/adipogenic progenitors (FAPs). Exogenous IL-33 and IL-2 complex (IL-2c) expanded muscle ILC2s and eosinophils, decreased the cross-sectional area (CSA) of regenerating myofibers, and increased the expression of genes associated with muscle fibrosis. The deletion of ILC2s in dystrophic mice mitigated muscle eosinophilia and impaired the induction of IL-5 and fibrosis-associated genes. Our findings highlight a FAP/ILC2/eosinophil axis that promotes type II innate immunity, which influences the balance between regenerative and fibrotic responses during muscular dystrophy