Ischemic Postconditioning Protects Aged Myocardium Against Ischemia/Reperfusion Injury by Regulating Mitochondrial Dynamics via piR‐4913

Figures

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  FIGURE 1. IPostC attenuates I/R injury by inhibiting excessive mitochondrial fission in aged hearts. Aged rats were subjected to Sham, I/R, and IPostC protocols (n = 6). (A) Assessment of cardiac hemodynamics, including left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVDP), and the maximum rate of pressure rise/fall (±dP/dtmax) recorded via the BL‐420N system. (B, C) Representative M‐mode echocardiograms and quantitative analysis of left ventricular ejection fraction (EF) and fractional shortening (FS). (D) Serum levels of lactate dehydrogenase (LDH), creatine kinase‐MB (CK‐MB), and cardiac troponin I (cTn‐I) measured by an automated biochemical analyzer. (E, F) Representative transmission electron microscopy (TEM) images showing mitochondrial ultrastructure in the myocardium. Scale bar = 2 µm. (G) Western blot analysis of mitochondrial fission‐related proteins (Drp1, Fis1) and fusion‐related proteins (Mfn1, Mfn2, OPA1) in aged myocardium. (H) Representative images of mitochondrial morphology labeled with MitoTracker Red. Scale bar = 5 µm. Data are mean±SEM. ** p < 0.01, *** p < 0.001.

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  FIGURE 2. Identification and characterization of piR‐4913 in IPostC‐mediated protection against I/R injury in aged hearts. (A) Sequence length distribution of all detected piRNAs in aged cardiomyocytes. (B) First‐nucleotide bias of piRNAs across different length cohorts. (C) Hierarchical clustering heatmap of the union of differentially expressed piRNA clusters identified in different groups. Expression values are presented as row‐wise Z‐scores calculated from log10(TPM+1)‐transformed TPM values. Red and blue indicate up‐ and down‐regulated piRNAs, respectively (n = 3). (D) Volcano plots showing DE‐piRNAs in aged cardiomyocytes under H/R and HPostC conditions. (E) Venn diagram illustrating the intersection of DE‐piRNAs across treatment groups. (F) Heatmap displaying the expression profiles of 12 candidate DE‐piRNAs in the aged myocardium. Red: upregulated; Green: downregulated. (G, H) GO and KEGG pathway enrichment analyses of the 12 candidate DE‐piRNAs. (I, J) qRT‐PCR validation of the 12 candidate piRNAs in aged cardiomyocytes (HPostC) and aged myocardium (IPostC) (n = 3–6). (K) Interaction between candidate piRNAs and PIWI proteins assessed by RNA immunoprecipitation (RIP) assay (n =…

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  FIGURE 3. piR‐4913 inhibition mediates the cardioprotective effects of IPostC by regulating mitochondrial dynamics. (A) Western blot analysis of mitochondrial fission proteins (Drp1, Fis1) and fusion proteins (OPA1, MFN1, MFN2) in aged cardiomyocytes transfected with piR‐4913 antagomir under H/R conditions (n = 3). (B) Representative images and quantification of mitochondrial morphology using MitoTracker Red (n = 3). Scale bars = 5 µm. (C, D) Western blot analysis of fission/fusion proteins and representative MitoTracker Red staining in aged cardiomyocytes transfected with piR‐4913 agomir under HPostC conditions (n = 3). (E, F) Representative images and quantitative analysis of myocardial infarct size in aged rats following piR‐4913 antagomir treatment and I/R injury, determined by Evans blue and TTC double staining. (G) Histological changes in myocardial tissues visualized by HE staining (light microscopy). (H–J) Evans blue/TTC double staining and HE staining of myocardial infarction and pathological changes following piR‐4913 agomir treatment under IPostC conditions in aged rats. Data are expressed as mean±SEM. ** p < 0.01, *** p < 0.001.

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  FIGURE 4. piR‐4913 interacts with Drp1 and promotes its mitochondrial translocation during HPostC. (A) Representative silver‐stained SDS‐PAGE gel showing proteins pulled down by biotinylated sense or antisense piR‐4913 probes in aged cardiomyocytes. Rectangular boxes indicate gel regions excised for mass spectrometry (MS) identification of the piR‐4913 interactome. (B) MS analysis showing the identification peptide peaks and enrichment scores of Drp1. (C) Validation of the interaction between piR‐4913 and Drp1 by Western blot analysis following RNA pull‐down. (D) RNA immunoprecipitation (RIP) assay using anti‐Drp1 or control IgG antibodies; piR‐4913 enrichment was quantified by qRT‐PCR (n = 3). (E) Representative RNA‐FISH images showing the subcellular co‐localization of piR‐4913 (red) and Drp1 (green) in aged cardiomyocytes. Nuclei were counterstained with DAPI. (F–I) Effects of piR‐4913 agomir transfection on Drp1 dynamics under HPostC conditions: (F) Representative confocal images showing co‐localization of Drp1 (green) with mitochondria (MitoTracker Red) (n = 6). (G) Subcellular fractionation and Western blot analysis of Drp1 expression in mitochondrial and cytosolic fractions (n = 3).…

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  FIGURE 5. piR‐4913 stabilizes Drp1 by promoting O‐GlcNAcylation and inhibiting its ubiquitin‐mediated degradation. (A) Prediction of potential post‐translational modifications (PTMs) of Drp1 using the UniProt database. (B) Immunoprecipitation (IP) analysis of Drp1 phosphorylation, ubiquitination, SUMOylation, and O‐GlcNAcylation levels in aged cardiomyocytes transfected with piR‐4913 agomir under HPostC conditions. (C) IP and immunoblotting of O‐GlcNAcylation and ubiquitination levels in aged cardiomyocytes co‐transfected with Flag‐Drp1 and/or piR‐4913 agomir. (D) RNA pull‐down assay using biotinylated piR‐4913 or negative control (NC) probes, followed by Western blot analysis of OGT and OGA. (E, F) Co‐immunoprecipitation analysis of the physical interaction between Drp1 and OGT in aged cardiomyocytes transfected with piR‐4913 agomir or piR‐4913 antagomir under HPostC or H/R conditions. (G, H) Effect of OGT knockdown on Drp1: (G) Drp1 O‐GlcNAcylation levels in si‐OGT‐transfected cells; (H) Western blot analysis of OGT and Drp1 expression in cells transfected with si‐OGT and/or piR‐4913 agomir. (I) Western blot analysis of Drp1 levels in aged cardiomyocytes pretreated with MG132 (proteasome…

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  FIGURE 6. piR‐4913 modulates Drp1 mitochondrial translocation via OGT‐mediated O‐GlcNAcylation. (A) Western blot analysis of Drp1 and OGT protein levels in cytosolic and mitochondrial fractions of aged cardiomyocytes transfected with piR‐4913 agomir under HPostC conditions (n = 3). (B) Immunoprecipitation (IP) analysis of Drp1 O‐GlcNAcylation levels in the cytoplasm versus mitochondria. (C–E) Effects of OGT knockdown on piR‐4913‐induced Drp1 redistribution: (C) Representative immunofluorescence images showing the co‐localization of Drp1 (green) with mitochondria (MitoTracker Red, top) and with Fis1 (red, bottom) (n = 3). (D) Western blot analysis of Drp1 and OGT expression in cytosolic and mitochondrial fractions following transfection with piR‐4913 agomir and/or si‐OGT (n = 3). (E) IP analysis of Drp1 O‐GlcNAcylation levels in the indicated groups. Data are presented as mean±SEM. *** p < 0.001.

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  FIGURE 7. O‐GlcNAcylation at Thr604 governs Drp1 stability and mitochondrial translocation. (A) Bioinformatic prediction of potential O‐GlcNAcylation sites on Drp1 using the O‐GlcNAcylation database. (B) Immunoprecipitation (IP) analysis of O‐GlcNAcylation levels in WT‐Drp1 and various mutants (T585A, T595A, T604A, and T605A) in aged cardiomyocytes under HPostC conditions. (C) IP analysis of O‐GlcNAcylation levels in WT‐Drp1 and various mutants (T585A, T604A) in aged cardiomyocytes transfected with piR‐4913 agomir under HPostC conditions. (D) In silico structural modeling of the Drp1 T604A mutation using AlphaFold3 and conformational comparison with WT‐Drp1 via PyMOL analysis. (E, F) IP analysis of Drp1 ubiquitination and O‐GlcNAcylation levels. (G–J) Effects of the T604A mutation on Drp1 dynamics and stability in aged cardiomyocytes co‐transfected with piR‐4913 agomir: (G) Representative immunoblots and quantitative analysis of Drp1 protein half‐life following cycloheximide treatment. (H) Western blot analysis of Drp1 distribution in cytosolic and mitochondrial fractions (n = 3). (I) IP analysis of Drp1 O‐GlcNAcylation levels in mitochondrial fractions. (J) Representative…

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  FIGURE 8. The Drp1‐T604A mutation alleviates I/R injury by restoring mitochondrial function in the aged myocardium. Aged rats were injected intramyocardially with AAV9‐negative control (AAV9‐NC), AAV9‐Drp1WT, or AAV9‐Drp1T604A and subsequently subjected to IPostC. (A) Quantitative analysis of mitochondrial ATP production in the aged myocardium (n = 6). (B, C) Flow cytometric analysis of mitochondrial ROS levels using the MitoSOX fluorescent probe (B) and mitochondrial membrane potential (MMP) using the JC‐1 probe (C) (n = 6). (D) Representative M‐mode echocardiograms (left) and quantitative assessment of left ventricular ejection fraction (EF) and fractional shortening (FS) (right) (n = 6). (E) Serum levels of cardiac injury biomarkers (LDH, CK‐MB, and cTn‐I) measured by an automated biochemical analyzer (n = 6). (F, G) Representative images of Evans Blue/TTC‐stained heart sections (F) and statistical quantification of myocardial infarct size (G) (n = 3). (H) Histopathological changes in the myocardium visualized by HE staining. Scale bar = 50 µm. Data are presented as mean±SEM. ** p < 0.01, *** p < 0.001.