Mitochondrial Dynamics and VMP1-Related Selective Mitophagy in Experimental Acute Pancreatitis

Abstract

Mitophagy and zymophagy are selective autophagy pathways early induced in acute pancreatitis that may explain the mild, auto limited, and more frequent clinical presentation of this disease. Adequate mitochondrial bioenergetics is necessary for cellular restoration mechanisms that are triggered during the mild disease. However, mitochondria and zymogen contents are direct targets of damage in acute pancreatitis. Cellular survival depends on the recovering possibility of mitochondrial function and efficient clearance of damaged mitochondria. This work aimed to analyze mitochondrial dynamics and function during selective autophagy in pancreatic acinar cells during mild experimental pancreatitis in rats. Also, using a cell model under the hyperstimulation of the G-coupled receptor for CCK (CCK-R), we aimed to investigate the mechanisms involved in these processes in the context of zymophagy. We found that during acute pancreatitis, mitochondrial O₂ consumption and ATP production significantly decreased early after induction of acute pancreatitis, with a consequent decrease in the ATP/O ratio. Mitochondrial dysfunction was accompanied by changes in mitochondrial dynamics evidenced by optic atrophy 1 (OPA-1) and dynamin-related protein 1 (DRP-1) differential expression and ultrastructural features of mitochondrial fission, mitochondrial elongation, and mitophagy during the acute phase of experimental mild pancreatitis in rats. Mitophagy was also evaluated by confocal assay after transfection with the pMITO-RFP-GFP plasmid that specifically labels autophagic degradation of mitochondria and the expression and redistribution of the ubiquitin ligase Parkin1. Moreover, we report for the first time that vacuole membrane protein-1 (VMP1) is involved and required in the mitophagy process during acute pancreatitis, observable not only by repositioning around specific mitochondrial populations, but also by detection of mitochondria in autophagosomes specifically isolated with anti-VMP1 antibodies as well. Also, VMP1 downregulation avoided mitochondrial degradation confirming that VMP1 expression is required for mitophagy during acute pancreatitis. In conclusion, we identified a novel DRP1-Parkin1-VMP1 selective autophagy pathway, which mediates the selective degradation of damaged mitochondria by mitophagy in acute pancreatitis. The understanding of the molecular mechanisms involved to restore mitochondrial function, such as mitochondrial dynamics and mitophagy, could be relevant in the development of novel therapeutic strategies in acute pancreatitis.

Keywords: DRP1; Parkin1; VMP1; autophagy; mitochondrial dynamics; mitochondrial function; mitophagy; pancreatitis.

FIGURE 1

Experimental pancreatitis produces mitochondrial dysfunction in rats. (A) Representative traces obtained during the assessment of pancreatic mitochondrial O₂ consumption in rest (state 4) and active (state 3) metabolic state, and after the addition of 2 μM oligomycin (state 4o) and 2 μM m-CCCP (state 3u) to the reaction chamber, in the control animal. Pancreatic mitochondria respiration in the control and CAE-treated animals in state 4 (B) and state 3 (C), ***p < 0.001 with respect to control group by ANOVA-Dunnett test, n = 5. (D) As a marker of mitochondrial coupling, respiratory control rate (RCR) was calculated as state 4/state 3. *p < 0.05 with respect to the control group by ANOVA-Dunnett test, n = 5; **p < 0.01 with respect to the control group by ANOVA-Dunnett test, n = 5; ***p < 0.001 with respect to the control group by ANOVA-Dunnett test, n = 5. (E) ATP production rate in pancreatic mitochondria from the control and CAE-treated rats. Malate plus glutamate were used as substrates. **p < 0.01 with respect to the control group by ANOVA-Dunnett test, n = 5; ***p < 0.001 as compared with the control group, ANOVA-Dunnett test. (F). As a marker of mitochondrial efficiency, ATP/O rate was calculated as ATP production rate/state3 oxygen consumption ratio, *p < 0.05 with respect to the control group by ANOVA-Dunnett test, n = 5; **p < 0.01 with respect to the control group by ANOVA-Dunnett test, n = 5.

FIGURE 2

Mitochondrial dynamics during experimental AP in rats. (A) Panel (i) shows typical examples of Western blots of pancreatic homogenates samples. ß-Actin was used as loading control. Bars in panel (ii) represent densitometric analysis of OPA1/ß-actin ratio. Bars in panel (iii) represent densitometric analysis of DRP1/ß-actin ratio blot measurements. **p < 0.01 as compared with the control group by ANOVA-Dunnett test; ***p < 0.001 as compared with the control group by ANOVA-Dunnett test, n = 4. (B) Representative transmission electron micrographs shows the ultrastructure of acinar pancreatic tissue. A typical normal appearance of cytosolic acinar cell, with mitochondria (mt) and zymogens granules (ZG), included in a compact and defined area of endoplasmic reticulum (ER) is observed in control animals (a). No lysosomal structures are observed in this tissue (a,b). The ultrastructure corresponding to animals treated with CAE (b–i) showed a disorganization and swelling of endoplasmic reticulum (compatible with reticulum stress) and it is evident that an increase in lysosomal structures is a time dependent manner (b–i). Scale bars: 1 μm in panels (a,h); 0.5 μm in panels (b,c), 0.4 μm in panels (d,e,f,g,i).

FIGURE 3

Experimental pancreatitis induces autophagy and mitophagy in rats. (A) Changes in mitochondrial autophagy proteins expression during experimental AP. Panel (i) shows typical Western blots of pancreatic homogenates samples. ß-Actin was used as loading control. Bars in panel (ii) represent densitometric analysis of PG62/ß-actin ratio. Bars in panel (iii) represent densitometric analysis of LC3-II/LC3-I ratio blot measurements. Bars in panel (iv) represent densitometric analysis of VMP1/ß-actin ratio. *p < 0.05 as compared with the control group, ANOVA-Dunnett test; **p < 0.01 as compared with the control group, ANOVA-Dunnett test; ***p < 0.001 as compared with the control group, ANOVA-Dunnett test; n = 4. (B) Representative transmission electron micrographs of pancreas from control (a,b) and CAE 60 min treated animals (c–f). (a,b): control animals displayed normal mitochondria morphology. Typical organization of mitochondria (mt) clearly displayed the inner membrane (im) comprizing of cristae and well defined mitochondrial outer membrane (om). (c–f): 1 h after CAE administration, mitochondria displayed several abnormalities, such as loss and/or disruption of cristae, clearer matrix, and swelling [black arrows, in panels (c,e)]. On the other hand, “isolation membranes” (IM) can be observed that partially surround mitochondria (e,f) and visible autolysosomes (AL) with remains of mitochondrial structures in their interior (d,f), both structures compatible with mitophagy. Scale bars: 1 μm in panel (a); 0.2 μm in panel (b); 0.5 μm in panels (c,f); 200 nm in panel (d); and 0.4 μm in panel (e).

FIGURE 4

Mitochondrial dynamics in AR42J cells under CCK-R hyperstimulation. (A) OPA1 and DRP 1 protein expression of AR42J pancreatic acinar cells treated with vehicle and CAE. Panel (i) shows typical examples of Western blots of pancreatic homogenates samples. ß-tubulin was used as loading control. Bars in panel (ii) represent densitometric analysis of OPA1/ß-tubulin ratio. Bars in panel (iii) represent densitometric analysis of DRP1/ß-tubulin ratio blot measurements. *p < 0.05 as compared with control group, ANOVA-Dunnett test, n = 4. (B) Red-MitoTracker probe was used to mark the mitochondrial population. Control, CAE 15 min, CAE 30 min, and CAE 60 min representative images are shown. Populations of shortened mitochondria [panel (B), CAE 30 min] and elongated mitochondria [panel (B), CAE 60 min], morphologies compatible with changes in mitochondrial dynamics (mitochondrial fission and fusion), are observed in cells treated with CAE. Scale bar represents 15 μm.

FIGURE 5

Mitophagy is induced in AR42J cells under CCK-R hyperstimulation. (A) Representative confocal microscopy images of AR42J cells transfected with plasmid encoding for RFP-LC3 (control, CAE 15 min, CAE 30 min, and CAE 60 min). (B) AR42J cells treated with CAE and transfected with plasmids encoding for RFP-GFP-pMITO. The GFP signal is quenched at the lower pH of lysosomes, while RFP can be consistently visualized. Yellow fluorescence (RFP merged with GFP) indicates normal mitochondria population, whereas red fluorescence (RFP) indicates population of mitochondria undergoing mitophagy. (C) Quantification of the percentage of the mitochondrial population found in mitophagy. ***p < 0.001 compared with the control group, ANOVA-Dunnett test. (D) Representative confocal micrographs showing mitochondria detected with Red-MitoTracker and lysosome detected with Blue-LysoTracker in treated pancreatic AR42J cells (control, CAE 15 min, CAE 30 min, and CAE 60 min). Scale bar represents 15 μm. (E) Lysosomal area quantification per cell (in arbitrary units). ***p < 0.001 compared with the control group, ANOVA-Dunnett test.

FIGURE 6

Mitochondrial depolarization induces parkin-dependent mitophagy during CCK-R hyperstimulation. (A) Mitochondrial inner membrane potential evaluated by flow cytometry using TMRM probe (i) Overlaid histograms of mitochondrial event versus TMRM fluorescence intensity. Control (light blue), CAE 30 min (orange), CAE 60 min (red), and autofluorescence (full black). (ii) % TMRM⁺ cells quantification. **p < 0.01 compared with the control group, ANOVA-Dunnett test. (B) Mitochondrial mass evaluated by flow cytometry used MTDR. (i) Overlaid histograms of mitochondrial events versus MTDR fluorescence intensity. Control (light blue), CAE 30 min (orange), CAE 60 min (red), and autofluorescence (full black). (ii) % MTDR⁺ cells quantification. **p < 0.01 compared with the control group, ANOVA-Dunnett test. (C) Parkin expression by Western Blot assay in AR42J cells treated with CAE at different times. Panel (i) shows typical Western Blots of pancreatic homogenates samples. ß-actin was used as the loading control. Bars in panel (ii) represent densitometric analysis of parkin/ß-actin ratio. *p < 0.05 as compared with the control group, ANOVA-Dunnett test, n = 4. (D) Representative confocal microscopy images of AR42J cells treated with CAE showing intracellular changes distribution of mitochondria (detected with Red-MitoTracker probe) and Parkin (detected by immunofluorescence in green).

FIGURE 7

Identification of the novel VMP1 pathway mediating selective mitophagy in experimental pancreatitis. (A) AR42J transfected cells with plasmid encoding for GFP-VMP1 and treated with CAE. Red-MitoTracker was used to show mitochondrial population. (B) Western Blot of isolated autophagosomes by magnetic beads linked to anti V5 or VMP1 antibodies from AR42J CAE-treated cells. The presence of the mitochondrial protein VDAC is observed only in the cells treated with CAE. (C) VMP1 inhibition attenuates mitophagy during pancreatitis, evaluated by flow cytometry using MTDR. AR42J cells were transfected with GFP-shVMP1 plasmid 48 h before CAE treatment. Overlaid histograms of AR42J events versus MTDR fluorescence intensity. shVMP1-transfected population and not transfected in panel (i) control, (ii) CAE 30 min, and (iii) CAE 60 min. (iv) MTDR fluorescence quantification, *p < 0.01 as compared to the same treatment without transfection; ^#p < 0.01 as compared to the control group Two-way ANOVA-Tukey test.

FIGURE 8

Proposed mechanism: Mitophagy as a cellular rescue mechanism during pancreatitis. During AP, mitochondrial failure is able to induce phenotypic changes in acinar cells (OPA1, DRP1, Parkin1, and VMP1 expressions) that triggers mitochondrial remodeling processes. These changes include fusion events (through OPA1) which allow internal rearrangement of their structure; and fission events (through DRP1) that originate new functional mitochondria as well as damaged and depolarized mitochondria. The latter are labeled by Parkin1, and through the VMP1-dependent autophagic pathway, are selectively detected and degraded by mitophagy within the lysosomes.