PINK1/Parkin-mediated mitophagy in mechanical ventilation-induced diaphragmatic dysfunction

Abstract

Background: Mechanical ventilation (MV) often leads to ventilation-induced diaphragm dysfunction (VIDD). Although the development of this disorder had been linked to oxidative stress, mitochondrial energy deficiency, autophagy activation, and apoptosis in the diaphragm, it remains unclear whether the activation of mitophagy can induce VIDD. With our research, our endeavor is to uncover whether PTEN-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy affects the MV-caused diaphragmatic dysfunction.

Methods: Sprague-Dawley rats were subjected to MV treatment for 6 h (MV-6h), 12 h (MV-12h), or 24 h (MV-24h). Post MV, the diaphragm muscle compound action potential (CMAP) and cross-sectional areas (CSAs) of the diaphragm of these rats were measured. The levels of proteins of interest were examined to assess muscle health, mitochondrial dynamics, and mitophagy in the diaphragm. The co-localization of PINK1 with the mitochondrial protein marker tom20 was examined, as well as transmission electron microscopy analysis to detect changes in diaphragm mitochondrial ultrastructure.

Results: MV-12h and MV-24h treatments resulted in a decrease in CSA of diaphragm and CMAP amplitude. In addition, the expressions of F-box (MFAbx), muscle-specific ring finger 1 (MURF1), PINK1, and p62 were elevated in rats treated with MV for 12 h and 24 h, while mfn2 expression was reduced. Rats following MV-24h treatment displayed an increase in mitochondrial dynamic protein (Drp1) and Parkin expression and microtubule-associated protein 1 light chain 3/1 (LC3II/I) ratio. Moreover, decreased SOD and GSH activity and membrane potential were observed after MV-12h and MV-24h treatment, while H₂O₂ activity increased after MV-24h treatment. In addition, a strong co-localization between PINK1 and tom20 was identified.

Conclusion: These results reveal that MV leads to various changes in mitochondrial dynamics and significantly increases the mitophagy levels, which subsequently cause the variation in diaphragmatic function and muscle atrophy, indicating that mitophagy could be one of the possible mechanisms by which MV induces diaphragmatic dysfunction.The reviews of this paper are available via the supplemental material section.

Keywords: diaphragmatic dysfunction; mechanical ventilation; mitochondrial autophagy.

Figure 1.

CMAP analysis of the diaphragm. (a) The amplitude of CMAP of each group; and (b) The time course of CMAP of each group. The measurement data were expressed as mean ± standard deviation. The experiment was performed at least three times. Data among multiple groups were analyzed by one-way ANOVA, with Tukey’s post hoc test. ^*p < 0.05 compared with blank group.

Figure 2.

The CSAs of diaphragm muscle fibers in each group. (a) Representative image of the control group; (b) Representative image of the MV-6h group; (c) Representative image of the MV-12h group; (d) Representative image of the MV-24h group; and (e) The statistical results of the CSAs in each group. The measurement data were expressed as mean ± standard deviation. The experiment was performed at least three times. Data among multiple groups were analyzed by one-way ANOVA, with Tukey’s post hoc test. ^*p < 0.05 compared with blank group.

Figure 3.

Protein expression of MFABX and MURF1 in each group. (a) Protein bands of MFABX and MURF1 by Western blot; (b) Protein expression of MFABX in each group; and (c) Protein expression of MURF1 in each group. The measurement data were expressed as mean ± standard deviation. The experiment was performed at least three times. Data among multiple groups were analyzed by one-way ANOVA, with Tukey’s post hoc test. ^*p < 0.05 compared with blank group.

Figure 4.

Production of SOD, GSH, and H₂O₂ in each group. (a) SOD content; (b) GSH content; and (c) H₂O₂ content. The measurement data were expressed as mean ± standard deviation. The experiment was performed at least three times. Data among multiple groups were analyzed by one-way ANOVA, with Tukey’s post hoc test. ^*p < 0.05 compared with blank group.

Figure 5.

Comparison of the mitochondria membrane potential of each group (^*p < 0.05). (a) The red fluorescence of each group; (b) The green fluorescence of each group; and (c) The membrane potential of each group; the ratio was calculated by red/green fluorescence ratio. The measurement data were expressed as mean ± standard deviation. The experiment was performed at least three times. Data among multiple groups were analyzed by one-way ANOVA, with Tukey’s post hoc test. ^*p < 0.05 compared with blank group.

Figure 6.

Morphologic abnormalities of mitochondria and autophagy in the diaphragm. (a) Representative TEM results in the control group showed normal ultrastructure and the absence of autophagosomes; (b) Representative TEM images from the MV-6h group, showing mitochondria are swollen and fragmented, with disorganized cristae (green arrows); (c) Representative TEM images from the eMV-12h group, showing autophagosomes and swollen mitochondria (green arrows); and (d) Representative TEM images from the MV-24h group, showing lipid droplets (yellow arrows). M, mitochondria; N, nucleus.

Figure 7.

The level of protein of mitophagy in each group (^*p < 0.05). (a) The protein expression of Drp1, Mfn2, and PINK1 in each group. The Western blot protein bands are shown in the left panel and the corresponding quantitative results are shown in the right panel; and (b) the protein expression of PINK1, Parkin, P62 and LC3II/I in each group. The Western blot protein bands are shown in the left panel and the corresponding quantitative results are shown in the right panel. The measurement data are expressed as mean ± standard deviation. The experiment was performed at least three times. Data among multiple groups were analyzed by one-way ANOVA, with Tukey’s post hoc test. ^*p < 0.05 compared with blank group.

Figure 8.

The co-localization of LC3 and PINK1 with tom20, respectively. (a) In the control group, autophagosomes were rarely observed; (b) the MV-24h group showed a comparatively high amount of green LC3 fluorescence mention autophagosome; (c) the control group has a small amount of overlapping areas that represent the co-localization of PINK1 and mitochondria; and (d) there were lots of co-located areas of PINK1 and tom20 in the MV-24h group.