Mitofilin Mitigates Myocardial Damage in Acute Myocardial Infarction by Regulating Pyroptosis of Cardiomyocytes

Figures

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  Figure 1. Reduced mitofilin expression is associated with the upregulation of pyroptosis-related factors in an acute myocardial infarction (AMI) model. (A) The percentage of myocardial infarction (MI) area increased in the AMI group. (B,C) The malondialdehyde (MDA) level and the lactate dehydrogenase (LDH) level were increased upon AMI induction. (D,E) The levels of interleukin (IL)-1β and IL-18 were increased in the AMI group. (F) Mitofilin protein levels were significantly decreased in the AMI group, while the protein levels of cleaved-caspase-1, nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing (ASC), Gasdermin D (GSDND) were increased. **p < 0.01 and ***p < 0.001. ns: not significant.

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  Figure 2. Overexpression of mitofilin alleviates AMI. (A) The administration of adenovirus carrying mitofilin (ad-mitofilin) significantly increased the expression level of mitofilin in the myocardium. (B) The area of MI was partially reduced after the administration of ad-mitofilin. (C,D) MDA levels and LDH levels were partially reduced after the administration of ad-mitofilin. (E,F) IL-1β and IL-18 levels were partially reduced after the administration of ad-mitofilin. (G) After the administration of ad-mitofilin, the mitofilin protein level was significantly increased upon AMI induction, while the protein levels of cleaved- caspase-1, NLRP3, ASC, and GSDND protein levels were partially decreased. Sham vs AMI: **P < 0.01 and ***P < 0.001. AMI vs AMI+ad-mitofilin: ### P < 0.001.

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  Figure 3. Mitofilin knockdown exacerbates hypoxia/reoxygenation (H/R)-induced cell damage of H9C2 by regulating pyroptosis. (A) The expression level of mitofilin was strongly reduced after the administration of ad-si-mitofilin. (B,C) H/R induced the increase of MDA level and LDH level in H9C2 cells, while levels were further increased after the administration of ad-si-mitofilin. The presence of AC-YVAD-CMK (an inhibitor for caspase-1) suppressed the effect of mitofilin knockdown. (D,E) H/R induced the increase of IL-1β and IL-18 levels in H9C2 cells, which were further increased after the mitofilin knockdown. The presence of AC-YVAD-CMK suppressed the effect of mitofilin knockdown. (F) Mitofilin knockdown increased the protein levels of cleaved-caspase-1, NLRP3, ASC, and GSDND in H9C2 cells upon H/R induction, which was rescued by AC-YVAD-CMK. (G) H/R induction promoted cell death by increasing the percentage of cells with impaired membrane integrity [revealed by propidium iodide (PI) staining]. Mitofilin knockdown exacerbated cell death (with typical morphology of cell swelling and membrane rupture). AC-YVAD-CMK suppressed the effect of mitofilin knockdown. *P < 0.05, **P < 0.01, and ***P…

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  Figure 4. Mitofilin regulates the PI3K/AKT signaling pathway. (A) The levels of p-PI3K and p-AKT in H9C2 cells were decreased after H/R induction, which were further decreased after the mitofilin knockdown. (B) The levels of p-PI3K and p-AKT in H9C2 cells were rescued after mitofilin overexpression. Con vs H/R: **P < 0.01 and *** P < 0.001. H/R vs H/R+ad-si-mitofilin or ad-mitofilin: ### P < 0.001.

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  Figure 5. The protective effect of mitofilin in the H/R-induced H9C2 damage is dependent on PI3K activity. (A) Hypoxia/reoxygenation induced a decrease in the mitofilin expression level in H9C2 cells. The level of mitofilin protein was increased after mitofilin overexpression. (B,C) Hypoxia/reoxygenation induced increase of the MDA level and LDH level was partially reduced after mitofilin overexpression. The presence of GDC-0941 (a PI3K inhibitor) abrogated the rescue effect. (D,E) Hypoxia/reoxygenation induced the increase of IL-1β and IL-18 levels in H9C2 cells, and mitofilin overexpression reduced their levels. The presence of GDC-0941 abrogated the rescue effect. (F) Mitofilin overexpression suppressed the protein levels of cleaved-caspase-1, NLRP3, ASC, and GSDND in H9C2 cells upon H/R induction, and the effects were abrogated by GDC-0941. *P < 0.05, **P < 0.01, and ***P < 0.001. ##: H/R vs H/R+ad-mitofilin: P < 0.01.