MiR-21-3p inhibitor exerts myocardial protective effects by altering macrophage polarization state and reducing excessive mitophagy

Abstract

Chronic heart failure (CHF) is closely associated with inflammation and mitochondrial dysfunction in cardiomyocytes. This study attempts to investigate the effects of microRNA-21-3p (miR-21-3p) on macrophage polarization and mitophagy in CHF. Here we found miR-21-3p was upregulated in CHF and negatively correlated with carnitine palmitoyl transferase 1A (CPT1A). L-palmitoyl carnitine (L-PC) exacerbated isoproterenol (ISO)-induced myocardial structural disruption and fibrosis in rats, which was exacerbated by miR-21-3p. Mechanistically, miR-21-3p accelerated M1 macrophage polarization. Both miR-21-3p inhibitor and CPT1A overexpression suppressed mitophagy. The inhibition of CPT1A on mitophagy was reversed by miR-21-3p. MiR-21-3p targeted CPT1A mRNA and co-localized with CPT1A protein in cardiomyocytes. In the co-culture system of M1 macrophages and H9c2 cells, miR-21-3p mimics in H9c2 cells promoted M1 polarization, whereas miR-21-3p inhibitor reduced M1 phenotype. M1 macrophages exacerbated H9c2 cell damage. These findings support the potential therapeutic targeting of miR-21-3p to regulate inflammation and mitophagy by inducing CPT1A in CHF.

Fig. 1. L-PC treatment exacerbates the development of ISO-induced CHF in rats.

A Representative echocardiographic images. B EF, FS, and LVIDs. C Changes in serum CK-MB levels by ELISA. D Changes in serum LPS, IL-5, IL-8, and TNF-α levels by ELISA. E Representative images of hematoxylin-eosin-stained cardiac tissue. Scale bar, 100 μm (top) and 25 μm (bottom). F Representative images of cardiac tissue stained with Masson trichrome stain. Scale bar, 100 μm (top) and 25 μm (bottom). G IF staining analysis of LC3 (green) and DAPI (blue) fluorescence in cardiac tissue. Scale bar, 25 μm. H Western blotting analysis of LC3II/I and p62 in cardiac tissue. I Evaluation of CPT1A expression in cardiac tissue after L-PC treatment by qRT-PCR and western blotting. *P < 0.05 vs. control. &P < 0.05 vs. ISO. All experiments consisted of 6 biological replicates, each representing the average of 3 technical replicates.

Fig. 2. L-PC increases the level of miR-21-3p and miR-21-3p enhances the M1 phenotype in macrophages.

A qRT-PCR detection of miR-21-3p levels in serum of patients with CHF. *P < 0.05 vs. normal. Experiment A consisted of 12 biological replicates, each representing the average of 3 technical replicates. B qRT-PCR detection of miR-21-3p levels in serum and heart of rats. C Pearson correlation analysis of miR-21-3p levels and CPT1A levels in heart. D Flow cytometry analysis of the effect of L-PC on the enrichment of macrophage surface markers CD86 and CD206 in spleen tissues of ISO-induced model. *P < 0.05 vs. control. &P < 0.05 vs. ISO. E qRT-PCR detection of miR-21-3p levels in Mφ, M1 and M2 macrophages. *P < 0.05 vs. Mφ. F qRT-PCR detection of the secretion of cytokines IL-1β and TNF-α in M1 macrophages by miR-21-3p. G Flow cytometry analysis of the effect of miR-21-3p on the surface marker CD86 in M1 macrophages. *P < 0.05 vs. M1. Experiments B to G consisted of 6 biological replicates, each representing the average of 3 technical replicates.

Fig. 3. miR-21-3p binds to CPT1A and promotes excessive mitophagy in H9c2 cells.

A Prediction of potential binding sites of miR-21-3p to CPT1A using miRmap. B Validation of miR-21-3p binding to CPT1A by dual-LUC assay. *P < 0.05 vs. CPT1A-WT+NC mimics. C qRT-PCR analysis of miR-21-3p levels in H9c2 cells. D Evaluation of the effect of miR-21-3p on cell viability in H9c2 cells using CCK8 assay. E Analysis of the effects of miR-21-3p on the protein levels of LC3 II, LC3I, p62 and CPT1A in H9c2 cells using Western blotting. F Observation of the effect of miR-21-3p on the number of autophagosome-like structures in H9c2 cells by transmission electron microscopy. Scale bars: 2 μm and 500 nm. *P < 0.05 vs. control. & P < 0.05 vs. NC inhibitor. G qRT-PCR analysis of miR-21-3p and CPT1A mRNA levels in H9c2 cells. H Evaluation of the effect of CPT1A and miR-21-3p on cell viability in H9c2 cells using CCK8 assay. I Western blotting analysis of the effects of CPT1A and miR-21-3p on the protein levels of LC3 II, LC3I, p62, and CPT1A in H9c2 cells. J Observation of the effects of CPT1A and miR-21-3p on the number of autophagosome-like structures in H9c2 cells by transmission electron microscopy. Scale bars: 2 μm and 500 nm. K IF staining showing the subcellular localization of miR-21-3p and CPT1A in H9c2 cells. Scale bar: 25 μm. *P < 0.05 vs. oe-NC. &P < 0.05 vs. oe-CPT1A+NC mimic. All experiments consisted of 6 biological replicates, each representing the average of 3 technical replicates.

Fig. 4. MiR-21-3p regulates M1 macrophage polarization and H9c2 cell autophagy in the co-culture model.

A Establishment of a co-culture system between LPS/IFN-γ-induced M1 macrophages and H9c2 cells. Examination of the effect of H9c2 cells on the expression of the surface marker CD86 in macrophages by flow cytometry. *P < 0.05 vs. M1+H9c2 (NC). &P < 0.05 vs. M1+H9c2 (ISO+L-PC+NC). B Levels of CK-MB in the supernatant of H9c2 cells after co-culture by ELISA. C Levels of IL-6, IL-8 and TNF-α in the supernatant of H9c2 cells after co-culture by ELISA. D Observation of the punctate accumulation of LC3B in H9c2 cells after co-culture by immunofluorescence. Scale bar: 25 μm. E Western blotting analysis of LC3 II, LC3I and p62 in H9c2 cells after co-culture. *P < 0.05 vs. H9c2 (NC). & P < 0.05 vs. M1+H9c2 (NC). #P < 0.05 vs. M1+H9c2 (ISO+L-PC+NC). All experiments consisted of 6 biological replicates, each representing the average of 3 technical replicates.

Fig. 5. MiR-21-3p inhibitor restores the development of rat HF induced by ISO and L-PC.

A Representative images of hematoxylin-eosin staining of cardiac tissue sections. Scale bar, 100 μm (up) and 25 μm (down). B Representative images of Masson’s trichrome staining of cardiac tissue sections. Scale bar, 100 μm (up) and 25 μm (down). C Representative echocardiographic images. D EF, FS, and LVIDs. E Levels of CK-MB in rat serum by ELISA. F Levels of LPS, IL-6, IL-8, and TNF-α in rat serum by ELISA. G, H Flow cytometry analysis of CD86 expression in rat spleen tissue. I qRT-PCR determined levels of miR-21-3p in rat serum and heart. *P < 0.05 vs. control. &P < 0.05 vs. ISO+NC inhibitor. #P < 0.05 vs. ISO+L-PC+NC inhibitor. All experiments consisted of 6 biological replicates, each representing the average of 3 technical replicates.

Fig. 6. MiR-21-3p inhibitor inhibits excessive autophagy in heart tissue after ISO and L-PC treatment.

A Immunofluorescence images of LC3-labeled cells in the heart. Scale bar, 25 μm. B Western blotting analysis of LC3 II, LC3I and p62 expression in rat heart. C Western blotting analysis of CPT1A expression in rat heart. *P < 0.05 vs. control. &P < 0.05 vs. ISO+NC inhibitor. #P < 0.05 vs. ISO+L-PC+NC inhibitor. All experiments consisted of 6 biological replicates, each representing the average of 3 technical replicates.

Fig. 7

A graphical abstract illustrating that L-PC promotes mitophagy in cardiomyocytes and M1 macrophage polarization through the miR-21-3p/CPT1A axis.

Fig. 8

A graphical flow chart for the animal study.