VMP1 dependent selective mitophagy and mitochondrial fragmentation are protective cellular mechanisms in pancreatitis

V. VANASCO, A. ROPOLO, D. GRASSO, S. ALVAREZ, M.I. VACCARO

Berlin

Congreso; 50th European Pancreatic Club Meeting 2018; 2018

European Pancreatic Club

VMP1 dependent selective mitophagy and mitochondrial fragmentation are protective cellularmechanisms in pancreatitisV. Vanasco, A. Ropolo, D. Grasso, S. Alvarez, M.I. VaccaroUniversity of Buenos Aires - CONICET, School of Pharmacy and Biochemistry, Institute of Biochemistry and MolecularMedicine, Buenos Aires, ArgentinaObjectivesWe analyzed mitophagy in Acute Pancreatitis (AP), its molecular mechanisms and its relationship withmitochondrial dynamics during selective autophagy in animal and cellular models.MethodsCerulein (CAE) rat model of mild AP and cell model using AR42J cells cultured in medium + 7.4 μM CAE. VMP1,LC3 and p62 expression were used as autophagy markers. Mitophagy was confocal analyzed through the dualpMITO-RFP-GFP specific probe and Parkin expression. Mitochondrial dynamics were determined by OPA1 andDRP1 expressions. TMRM probe was used to measure membrane potential, as parameter of mitochondrialfunction.ResultsIn the cell model, mitochondrial membrane potential was decreased at 30 min (25% compared to control p<0.01). Mitochondrial dynamics showed significant induction of the mitochondrial fission protein DRP1 at 30 min,while the mitochondrial fusion protein OPA1 was maximal at 1h. This was accompanied by an increase in theexpression of Parkin and recruitment of LC3-RFP. While, redistribution of EGFP-VMP1 showed the sequestrationof damaged mitochondria in autophagosomes. Confocal analysis using the specific probe confirmed mitophagythat was maximal at 30 min of CAE. Interestingly, inhibition of selective autophagy by downregulation of VMP1expression leads to a dramatic decreased in mitochondrial function and to death in the cell model. These resultsindicate that dysfunctional mitochondria are degraded by VMP1-dependent mitophagy and fragmentation as aprotective process, while functional mitochondria would avoid degradation by mitochondrial fusion andelongation. To evaluate the pathophysiological relevance of selective autophagy, we study mitophagy,mitochondrial dynamics and function in the rat model. Mitochondrial respiration and ATP production weredecreased by 35% at 1h (P<0.01). Dynamics studies showed that DRP1 expression was not detectable,whereas a time-course increased of OPA1 expression was observed after 1h. LC3II, VMP1 and Parkinexpressions as well as isolation of autophagosomes demonstrated VMP1-related mitophagy that was maximal at30 min; accompanied by the decrease of p62 expression as a function of time. Finally, all the parametersevaluated were found in control values when pancreatic morphology was recovered.ConclusionOur findings point to VMP1 dependent mitophagy and mitochondrial fragmentation as protective pancreatic cellmechanisms and provide novel evidence of the potential relevance of VMP1-mediated selective autophagy inacute pancreatitis.