Parkin plays a crucial role in acute viral myocarditis by regulating mitophagy activity

Figures

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  Figure 1. During VMC, Parkin and BNIP3-mediated mitophagy is acutely upregulated. (A) Representative photographs of M-mode echocardiography of the left ventricle (LV) were shown. Quantitative data of ejection fraction (EF) and fractional shortening (FS). n = 5. (B) Histological alterations in mouse hearts were analyzed by HE staining and the scores of inflammatory cell infiltration were quantified. n = 5. (C) The levels of TNF-α and IL-1β in mouse hearts were determined by qPCR. (D) Mitochondrial morphology changes in mouse hearts were analyzed by TEM. Scale bar = 500 nm. n = 5. (E) Western blot analysis of p62, Parkin, BNIP3 and LC3A/B-I/II expression in heart tissues. (F) TEM images to detect mitophagy in heart tissues and the quantitative analysis of mitophagosomes in the indicated groups. The zoomed-in images represent high-magnification views of the outlined areas. Green arrows indicate mitophagosomes, red arrows indicate mitophagic/autophagic multi-lamellar vesicles and white arrows indicate normal mitochondria. Scale bar = 400 nm. n = 5. Data represent the mean ± SEM. **P < 0.01, and ***P < 0.001 vs. Sham group. #P < 0.05, ##P < 0.01, ##P < 0.001 vs. VMC D7 group.

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  Figure 2. CVB3 induced Parkin-mediated mitophagy and blocked autophagy flux in NRCMs. NRCMs were infected with CVB3 at a multiplicity of infection (MOI) of 10. (A) Western blot analysis of p62, Parkin, BNIP3, LC3A/B-I/II and VP1 expression in NRCMs infected with CVB3. n = 3. (B) Immunofluorescence staining to examine the colocalization Parkin (green) and Mito Tracker (red). Scale bar = 50 μm. Intensity profiles were obtained using ImageJ software, along the dashed line. (C) Immunofluorescence staining to examine the colocalization VP1 (green) and Mito Tracker (red). Scale bar = 50 μm. Quantification of mean optical density values of VP1. (D) Microscopy of cells double stained with Lyso Tracker for lysosomes (red) and Mito Tracker for mitochondria (green). Scale bar = 50 μm. Count of cells with Lyso Tracker on mitochondria (merged signal, yellow) in 400 μm2. n = 6. (E) The levels of TNF-α and IL-1β in NRCMs were determined by qPCR. n = 6. Data represent the mean ± SEM. *P < 0.05, **P < 0.01 vs. CVB3 0 h group.

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  Figure 3. Knockdown of Parkin alleviated mitophagy and inflammation of cardiomyocytes. (A) Western blot analysis of Parkin silencing effects in NRCMs infected with CVB3. n = 3. (B) Western blot analysis of PINK1, Parkin, p62 and LC3A/B-I/II expression in NRCMs treated in CVB3 with si-Parkin or si-Ctrl. (C) The levels of TNF-α, IL-1β and IL-6 in NRCMs were determined by qPCR. n = 5. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. CVB3+si-Ctrl group. (D) Microscopy of cells double stained with Lyso Tracker for lysosomes (red) and Mito Tracker for mitochondria (green). Scale bar = 50 μm. Count of cells with Lyso Tracker on mitochondria (merged signal, yellow) in 400 μm2. n = 5. (E) Immunofluorescence staining to examine the colocalization VP1 (green) and Mito Tracker (red). Scale bar = 50 μm. n = 3.

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  Figure 4. Parkin silencing worsened mortality and cardiac dysfunction during VMC. (A) Sham mice and VMC mice were treated with AAV9-shNC and AAV9-shParkin by intravenous injection into the tail two weeks ago, and after an additional one week, mice were euthanized. (B) The survival rate was monitored daily until day 7. n = 15-25. (C) Histological alterations in mouse hearts were analyzed by HE staining and the scores of inflammatory cell infiltration were quantified. (D) The levels of TNF-α, IL-1β and IL-6 in mouse hearts were determined by qPCR. (E) The levels of viral mRNA in mouse hearts were determined by qPCR. (F) Representative photographs of M-mode echocardiography of the left ventricle (LV) were shown. Quantitative data of ejection fraction (EF) and fractional shortening (FS). Data represent the mean ± SEM. n = 5. ns P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001 vs. Sham+AAV9-shNC group. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. VMC+AAV9-shNC group. &&&P < 0.001 Sham+AAV9-shParkin group vs. VMC+AAV9-shParkin group.

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  Figure 5. Parkin silencing reduced the mitophagy activity during VMC. (A-E) Western blot analysis of PINK1, Parkin, p62 and LC3A/B-I/II expression in heart tissues from Sham mice and VMC mice with Parkin silencing. n = 4. Data represent the mean ± SEM. ns P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001 vs. Sham+AAV9-shNC group. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. VMC+AAV9-shNC group. &P < 0.05 and &&P < 0.01 Sham+AAV9-shParkin group vs. VMC+AAV9-shParkin group. (F) TEM images showing the changes of mitophagosomes and autolysosomes in the hearts. Approximately 15-20 random fields with 2000-2500 mitochondria were analyzed per heart sample. Scale bar = 500 nm. n = 5. The zoomed-in images represent high-magnification views of the outlined areas. Red arrows indicate (1) normal mitochondria, (2) electron lucent vacuoles, (3) autolysosomes, (4) damaged mitochondria.

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  Figure 6. Parkin silencing exacerbated apoptosis without activating the NF-κB-related pathway. (A) Cardiomyocyte apoptosis in heart tissues were measured by TUNEL staining. Scale bar = 50 μm. n = 3. (B) Western blot analysis of Caspase-3, cleaved-Caspase-3, Bcl2 and Bax expression in NRCMs treated in CVB3 with si-Ctrl or si-Parkin. n = 3. (C) Western blot analysis of NF-κB p65 and phospho-NF-kB p65(Ser536) expression in each group of mouse hearts. Data are presented as mean ± SEM. n = 6. ns P > 0.05 and **P < 0.01 vs. Sham+AAV9-shNC group. #P < 0.05 vs. VMC+ AAV9-shNC group. (D) Western blot analysis of NF-κB p65 and phospho-NF-kB p65(Ser536) expression in NRCMs. ***P < 0.001 vs. CVB3+si-Ctrl group. (E) Immunofluorescence staining to examine the colocalization NF-κB p65 (red) and nucleus (DAPI, blue). Scale bar = 50 μm. n = 3.