MiR-144-5p/CCL12 Signaling Axis Modulates Ischemic Preconditioning-Mediated Cardio-protection by Reducing Cell Viability, Enhancing Cell Apoptosis, Fibrosis, and Pyroptosis

Abstract

Ischemic postconditioning (IPost) represents short periods of nonlethal ischemia-reperfusion performed at the onset of reperfusion. Studies have shown that IPost involves various biological processes such as cell proliferation, apoptosis, and pyroptosis and can activate complex signaling pathways. CCL12 is a critical mediator in the inflammatory process after tissue injury. In the present study, we examined the potential actions of CCL12-mediated signaling pathways in cardioprotection after IPost using a cardiomyocyte model. By applying the bioinformatics analysis, we found that CCL12 was upregulated in the rat heart tissues after I/R injury, and the expression level of CCL12 was restored in rats with IPost. The in vitro studies showed that CCL12 and CCR2 expression levels were upregulated in the hypoxia/reoxygenation (H/R)-induced H9C2 cells, which was attenuated in the H/R + hypoxia post-conditioning (PostC) group. The functional assays showed that H/R treatment reduced cell viability, increased cell apoptosis, and promoted fibrosis and pyroptosis of H9C2 cells, which was attenuated in the H/R + PostC group. Overexpression of CCL12 impaired the protective action of hypoxia post-conditioning in the H9C2 cells. Further mechanistic studies showed that miR-144-5p could directly target the 3' untranslated region of CCL12. Overexpression of miR-144-5p markedly repressed the expression levels of CCL12 and CCR2 in H9C2 cells, while miR-144-5p inhibition had the opposite effects. Furthermore, the inhibition of miR-144-5p reduced the cell viability, increased cell apoptosis, and enhanced fibrosis and pyroptosis of H9C2 cells after H/R or H/R + PostC treatment. In conclusion, CCL12 was downregulated in cardiomyocytes following ischemic postconditioning, and CCL12 overexpression impaired the cardioprotective actions of ischemic postconditioning by reducing cell viability, enhancing cell apoptosis, fibrosis, and pyroptosis. Further mechanistic evidence revealed that CCL12 was a direct target of miR-144-5p, and miR-144-5p/CCL12/CCR2 signaling may represent a critical pathway in mediating the cardioprotective effects of ischemic postconditioning.

Keywords: Apoptosis; Cardiomyocytes; Fibrosis; Ischemic postconditioning; Pyroptosis; Viability.