FRZDMD · Contribution of calcium-independent isoforms of phospholipase A2 in the pathogenesis of Duchenne muscular dystrophy

Contribution of calcium-independent isoforms of phospholipase A2 in the pathogenesis of Duchenne muscular dystrophy.

The goal of this project was to determine the role of isoforms of phospholipase A2 (PLA2) in cytosolic calcium imbalances and cell death of muscle fibers during Duchenne muscular dystrophy (DMD). DMD is a muscle degenerating disease that affects 1 in 3,500 boys worldwide. It has been identified has being an X-linked disease. The patients are detected as having DMD in their toddlerhood, they have to be in wheelchairs in their 10’s, and they usually die from respiratory failure in their 20’s or 30’s. The only treatment currently given to patients is a regimen of corticoids (mainly prednisolone and deflazort), which alleviates the symptoms and limits the muscle degradation process. But it is not a cure and it does not prevent an early death. DMD patients are missing a structural protein known as dystrophin. This protein normally links the actin filaments to the extracellular matrix via a complex of dystrophin-associated proteins. The relationship between the absence of dystrophin and the degeneration of muscles of the body is not understood. Neither gene therapy for the insertion of dystrophin in the genome of patients, nor the injections of cells expressing dystrophin in affected muscles to try to correct the defect and restore dystrophin expression have been working so far. However it is known that inflammation and fibrosis are two main issues in DMD. The cortico-steroid treatments given to DMD patients help decrease the inflammation process. But these treatments have severe secondary effects. In our project, we decided to study the role of phospholipases A2 in DMD. The rationale behind it was that 1) PLA2s activation results in an increase inflammation, and 2) it was observed a few decades ago that PLA2 activity is increased by more than 10 fold in DMD patients compared to control subjects (Lindhal, 1995). So it appeared to us that these enzymes could be potential interesting targets to decrease inflammation in DMD. As phospholipases A2 are a family of more than 30 different enzymes, we wondered which isoform(s) could be involved in DMD. There are 3 main classes of PLA2s: cytosolic, independent of calcium, and secreted. The PLA2s independent of calcium have been the most studied in muscles. In particular one isoform, iPLA2b, has been shown by two research groups to play a role in calcium entry (Work of Profs. Bolotina and Prof. Ruegg). We conducted our research project in immortalized control and DMD cells provided to us by the Genethon in Paris. We confirmed that the DMD cells had in culture features of DMD, including a calcium entry defect. We performed quantitative polymerase chain reaction (PCR) and observed that some PLA2 appeared over-expressed in the DMD cells. Interestingly, our PCR experiments showed that a class of PLA2 not studied in muscles had expression level differences between the control and DMD cells. This class of PLA2 was the secreted PLA2s (sPLA2). We investigated the role played by sPLA2 in calcium entry using pharmacological inhibitors, and noticed in parallel that in the presence of a sPLA2 pharmacological inhibitor DMD cells appeared to differentiate faster than without it. This effect was not detected in control cells. We next targeted individual sPLA2s using siRNAs and identified potential candidates responsible for the increased myotube sizes in absence of sPLA2s. Our experiments show that this effect might be through regulation of myogenin at the promoter level and the secretion of hypertrophic factors. This work will need to be continued and expanded, and we do not know at the present time neither what are the effects of sPLA2 in patients, nor if downregulating any sPLA2 expression in DMD patients would be beneficial or not. Our data show that inhibiting sPLA2s in DMD cells in Petri dishes could make bigger myotubes. But would these myotubes be less fragile and improve patients’ condition, or would they use all satellite cells and thus increase the DMD condition? These questions remain and further experiments would be needed to answer them. However it is clear that the current treatments are insufficient and that there is no cure to the disease. With that perspective in mind, this research project opened the possibility of an involvement of some sPLA2 in the DMD pathology. If confirmed, these data could lead to new treatment strategies in DMD patients. At the moment, further experiments and research are needed to fully understand the roles played by sPLA2 and if their inhibition would benefit or not DMD patients.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз