MicroRNA-100-5p Exacerbates Myocardial Ischemia-Reperfusion Injury Through Downregulation of PRMT5

Abstract

Acute myocardial infarction (AMI) rates are rising due to the increasing prevalence of cardiac metabolic disorders, particularly diabetes mellitus (DM). While revascularization procedures such as coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) restore blood flow, they can also exacerbate myocardial ischemia-reperfusion (I/R) injury, for which current cardioprotective strategies remain insufficient. MicroRNAs are critical regulators of gene expression, but the role of miR-100-5p in I/R injury remains unclear. This study demonstrates that miR-100-5p is significantly upregulated in myocardial tissue during I/R injury, leading to the downregulation of protein arginine methyltransferase 5 (PRMT5). This reduction in PRMT5 impairs its capacity to methylate and inactivate Phosphatase and tensin homolog (PTEN), resulting in disruption of the PI3K-AKT signaling pathway, increased cardiomyocyte apoptosis, and aggravated myocardial damage. Using antisense oligomers to inhibit miR-100-5p, we restored PRMT5 expression, reactivated PI3K-AKT signaling, and reduced cardiomyocyte death, thereby mitigating myocardial injury. Our findings identify the miR-100-5p/PRMT5/PI3K-AKT axis as a key regulatory pathway in myocardial I/R injury and highlight miR-100-5p as a potential therapeutic target to protect the heart during revascularization procedures.

Keywords: PI3K‐AKT pathway; PRMT5; PTEN; cardiomyocyte death; miR‐100‐5p; myocardial I/R injury.

FIGURE 1

Decreased PRMT5 Expression and Elevated Levels of miR‐100‐5p in I/R Injury. (A) Quantitative PCR analysis of miR‐100‐5p and PRMT5 levels in myocardial tissue after 12 and 24 h of reperfusion (n = 4/group). (B) Western blot analysis of PRMT5 protein levels in myocardial tissue from I/R injury models and Sham controls (n = 4/group). (C) Immunofluorescence and in situ hybridization analyses showed the co‐localization of PRMT5 and miR‐100‐5p in myocardial tissues. (D and E) Quantitative PCR analysis of miR‐100‐5p and PRMT5 in AC16 cells subjected to H/R treatment (n = 3/group). (F) Western blot analysis of PRMT5 expression in AC16 cells subjected to H/R treatment (n = 3/group). Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01.

FIGURE 2

miR‐100‐5p Suppresses PRMT5 Expression and Enhances Cardiomyocyte Injury. (A) Quantitative PCR analysis of PRMT5 mRNA expression in AC16 cells transfected with 20 or 40 nM miR‐100‐5p or scramble control (mimic‐Ctrl) (n = 3/group). (B) Western blot analysis of PRMT5 protein levels in AC16 cells following transfection with 20 or 40 nM miR‐100‐5p or scramble control (n = 3/group). (C) Dual luciferase reporter assay assessing luciferase activity from the wild‐type and mutant (MT) PRMT5 3′‐UTR in AC16 cells with or without miR‐100‐5p or control microRNA transfection (n = 6/group). (D) Western blot analysis of PRMT5 levels in AC16 cells subjected to H/R with or without PRMT5 transfection. (E) BrdU assay measuring cell proliferation in AC16 cells after H/R treatment, with or without PRMT5 overexpression (n = 6/group). (F) Caspase 3/7 apoptosis assay assessing apoptosis rates in AC16 cells after H/R treatment, with or without PRMT5 overexpression (n = 6/group). (G) DCFDA assay measuring ROS levels in AC16 cells after H/R treatment, with or without PRMT5 overexpression (n = 6/group). (H) Western blot analysis examining the phosphorylation status of PI3K and AKT in AC16 cells after H/R treatment, with or without PRMT5 overexpression. (I) Quantitative PCR analysis of anti‐apoptotic factors Bcl‐2 and Bcl‐xL, and pro‐apoptotic factor BAX in AC16 cells after H/R treatment, with or without PRMT5 overexpression (n = 6/group). Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01. Nor, normoxia.

FIGURE 3

miR‐100‐5p Exacerbated I/R‐Induced Heart Injury In Vivo. (A) Western blot analysis of PRMT5 expression in mouse hearts subjected to I/R, I/R combined with miR‐100‐5p overexpression, and Sham control (n = 4/group). (B) Mouse serum levels of LDH in the indicated groups (n = 6–7/group). (C) Representative images and quantification of TUNEL and cardiac troponin T (cTNT) co‐immunostaining to identify apoptotic cardiomyocytes. TUNEL‐positive (red) and cTnT‐positive (green) cells were merged to confirm cardiomyocyte‐specific apoptosis on Day 7. Quantification of TUNEL⁺/cTnT⁺ cells is shown (n = 4/group). Scale bar, 50 μm. (D‐E) Representative echocardiography assessment of ejection fraction (EF) and fractional shortening (FS) on (D) Day 1 and (E) Day 7 post‐I/R in the indicated groups (n = 8/group). (F) Representative histological sections of mouse hearts at 7 days post‐reperfusion. Top row: H&E staining. Middle and bottom rows: Picrosirius red staining of perivascular and interstitial fibrosis. Scale bar, 100 μm. (G) Quantification of perivascular (top) and interstitial (bottom) fibrosis in each group (n = 4/group). Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01.

FIGURE 4

miR‐100‐5p Enhances I/R Injury by Regulating PRMT5‐dependent PTEN Methylation and Activity. (A) Western blot analysis of PRMT5 and phosphorylated PTEN (p‐PTEN) levels in AC16 cells subjected to H/R. (B) Western blot analysis showing the effects of miR‐100‐5p transfection on PRMT5 and p‐PTEN levels in AC16 cells, with or without PRMT5 overexpression. (C) Quantitative PCR analysis of PRMT5 and PTEN mRNA levels in AC16 cells following miR‐100‐5p transfection and PRMT5 overexpression. (D) Interaction analysis assessing the binding between PRMT5 and PTEN under H/R conditions. (E) In vitro methylation assay was used to determine the methylation of PTEN by PRMT5. (F) Semi‐in vitro methylation assay assessing PTEN methylation in AC16 cell lysates treated with H/R and/or miR‐100‐5p and PRMT5. (G) BrdU proliferation assay measuring cell proliferation in AC16 cells after H/R treatment, with or without ASO of miR‐100. (H) Caspase 3/7 apoptosis assay evaluating apoptosis rates in AC16 cells after H/R treatment with or without ASO. (I) DCFDA assay measuring ROS levels in AC16 cells post‐H/R treatment, with or without ASO. (J) Western blot analysis examining the phosphorylation status of PTEN, PI3K, and AKT in AC16 cells treated with H/R and miR‐100‐5p transfection. (K) Quantitative PCR analysis of anti‐apoptotic factors Bcl‐2 and Bcl‐xL and the pro‐apoptotic factor BAX in AC16 cells transfected with miR‐100‐5p or PRMT5. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01. Nor, normoxia.

FIGURE 5

Attenuation of I/R‐Induced Heart Injury by Antisense Oligomers Targeting miR‐100‐5p In Vivo. (A) Quantitative PCR analysis of PRMT5 mRNA levels in mouse hearts subjected to sham, I/R, and I/R + miR‐100‐5p antagomir‐treated groups (n = 4/group). (B) Western blot analysis of PRMT5 expression in the indicated groups (n = 4/group). (C) Echocardiographic assessment of cardiac EF and FS levels in the indicated groups (n = 8/group). (D) Mouse serum LDH levels in the indicated groups (n = 6‐7/group). (E) Representative H&E staining images of mouse hearts in the indicated groups. Scale bar, 100 μm. (F) Representative co‐immunostaining for TUNEL (red) and cTnT (green) to identify apoptotic cardiomyocytes, along with quantification of TUNEL⁺/cTnT⁺ double‐positive cells (n = 4/group). Scale bar, 50 μm. (G) Quantitative analysis of TUNEL‐positive cardiomyocytes corresponding to (F) (n = 4/group). (H) Representative CD68 immunostaining images and quantification to assess macrophage infiltration in mouse hearts from the indicated groups (n = 4/group). Scale bar, 50 μm. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01.