MD1 deletion exaggerates cardiomyocyte autophagy induced by heart failure with preserved ejection fraction through ROS/MAPK signalling pathway

Figures

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  Figure 1. MD1 expression is significantly decreased during HFpEF. (A‐C) Representative Western blots for myocardial MD1 expression in normal control hearts and HFpEF hearts (n = 3 per group). (B‐D) MD1 expression in H9C2 cells. H9C2 cells were subjected to treatment with aldo (1 μM) at different time points and then collected for Western blot detection. *P < .05

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  Figure 2. MD1 deficiency aggravated HFpEF phenotype induced by aldosterone administration in mice. (A) Representative images of H&E‐stained heart sections. (B) The left ventricular cross‐sectional area in the indicated groups (n = 6 per group). (C) The HW/BW ratio (n = 6 per group). (D‐F) Results of Anp, Bnp and β‐Mhc mRNA levels (n = 6 per group). (G‐H) Alterations in LVEF and LVFS after aldosterone administration (n = 6 per group). (I) Results of –dp/dtmin (n = 6 per group). (J) Alterations in Tau index after aldosterone administration (n = 6 per group). (K) Alterations in EDPVR‐k after aldosterone administration (n = 6 per group). (L) The LW/BW ratio between the four groups (n = 6 per group). (M) Representative pressure‐volume loops. *P < .05 vs. Sham‐WT group. #P < .05 vs. HFpEF‐WT group

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  Figure 3. MD1 deletion accelerated HFpEF‐induced autophagy. (A) Representative images of transmission electron micrographs (TEM) indicating the formation of autophagosomes. (B) Representative LC3 immunofluorescence staining and (C) LC3 intensity (n = 6 per group). (D) Representative Western blots and quantitative results of the autophagy markers (E) LC3II/LC3I ratio (n = 3 per group) and (F) P62 protein expression (n = 3 per group). *P < .05 vs. Sham‐WT group. #P < .05 vs. HFpEF‐WT group

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  Figure 4. Elimination of MD1 deteriorated ROS in HFpEF mice. (A) Representative Western blots of MD1 protein expression in WT and MD1‐KO group (n = 3 per group). (B) Representative images and quantitative results of DHE‐stained heart sections (n = 6 per group). (C) Representative Western blots and quantitative results of the oxidative stress protein expression of (D) SOD1 (n = 3 per group) and (E) SOD2 between the four groups (n = 3 per group). Representative Western blots and quantitative results of the antioxidative stress protein expression of (F) Nrf2 (n = 3 per group) and (G) HO‐1 between the four groups (n = 3 per group). *P < .05 vs. Sham‐WT group. #P < .05 vs. HFpEF‐WT group

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  Figure 5. MD1‐KO aggravated HFpEF‐induced autophagy via activation of ROS/MAPK signalling pathway. (A) Representative Western blots and (B) quantitative results depicting the phosphorylation and total proteins of P38‐MAPK in the indicated groups 4 weeks after HFpEF model (n = 3 per group). (C) Representative Western blots and (D) quantitative results depicting the phosphorylation and total proteins of P38‐MAPK in the indicated groups in H9C2 cells infected with the siMD1 for 24 hour followed by treating with aldosterone for 18 hour (n = 3 per group). (E) Representative Western blots and (F) quantitative results of the phosphorylation and total proteins of P38‐MAPK, P62 and LC3 proteins. Cells pre‐treated with NAC (5 μM) for 1 hour and then infected with the siMD1 for 24 hour followed by treating with aldosterone (1 μM) for 18 hour. (n = 3 per group). (G) Representative Western blots and (H) quantitative results of the phosphorylation and total proteins of P38‐MAPK, P62 and LC3 proteins. Cells pre‐treated with SB203580 (10 μM) for 2 hour and then infected with the siMD1 for 24 hour followed by treating with aldosterone (1 μM) for 18 hour. (n = 3 per group). *P < .05

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  Figure 6. Autophagy inhibitor 3‐MA abolished the exacerbative effect induced by MD1‐knocking down in H9C2 cells. Cells pre‐treated with 3‐MA (5 μM) and infected with the siMD1 for 24 hour followed by treatment with aldosterone (1 μM) for 18 hour. (A) Representative LC3 immunofluorescence staining (LC3 stained with red and DAPI stained with blue) and (B) quantitative results of LC3 intensity. (C) Representative Western blots of autophagy‐related protein expression and quantitative results of the (D) LC3II/LC3I ratio and (E) P62 level. *P < .05

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  Figure 7. Autophagy inhibitor 3‐MA abolished the exacerbative effect of MD1‐KO in vivo. (A) Results of The HW/BW ratio (n = 6 per group). (B‐C) Alterations in LVEF and LVFS of the four groups (n = 6 per group). (D) Results of EDPVR‐k (n = 6 per group). (E) Results of Tau index (n = 6 per group). (F) The LW/BW ratio between the four groups (n = 6 per group). (G) Representative and (H) quantitative results of LC3 immunofluorescence staining. (I) Representative Western blot of autophagy‐related protein expression and quantitative results of the (J) LC3II/LC3I ratio and (K) P62 level in the heart of HFpEF‐WT or HFpEF‐MD1‐KO mice subjected to saline or 3‐MA (n = 3 per group). *P < .05 vs. Vehicle‐HFpEF group. #P < .05 vs. 3‐MA‐HFpEF group

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  Figure 8. Schematic summary. Mechanisms of MD1‐KO interact with ROS/MAPK signalling pathway in HFpEF‐induced autophagy