Zn2+ protects H9C2 cardiomyocytes by alleviating MAMs-associated apoptosis and calcium signaling dysregulation

Abstract

Purpose

This study aims to investigate whether zinc ion (Zn²⁺) alleviates myocardial ischemia-reperfusion injury (MIRI) through the MAM-associated signaling pathway and to explore its impact on ERS and calcium overload.

Methods

H9C2 cells were cultured in a DMEM supplemented with 10 % fetal bovine serum and 1 % antibiotic solution. A MIRI model was established through simulated ischemia and reoxygenation with Zn²⁺ treatment in a complete medium. Cells were then treated with the MCU inhibitor ruthenium red (RR), the MCU activator spermine (SP), and siRNAs targeting Bap31, MCU, VDAC1, and FUNDC1. Cell viability was assessed using MTT and CCK-8 assays. Lactate dehydrogenase (LDH) levels were measured with a commercial kit. Western blot was performed to detect protein expression levels. Cell apoptosis, intracellular zinc, calcium levels, mitochondrial membrane potential, and protein fluorescence changes were observed using laser scanning confocal microscopy.

Results

Compared to the control group, cell viability was significantly reduced in the I/R group, accompanied by increased expression of apoptosis and calcium overload-related proteins increased cell injury, apoptosis, calcium overload, and a decrease in mitochondrial membrane potential. Zn²⁺ treatment reversed the detrimental effects of I/R in the I/R + Zn²⁺ group. When Bap31, VDAC1, FUNDC1, or MCU were silenced using siRNA, the protective effect of Zn²⁺ was further enhanced (P < 0.05).

Conclusions

Ischemia-reperfusion (I/R) leads to cardiomyocyte injury and apoptosis. Zn²⁺ downregulates the expression of key apoptotic proteins through the Bap31/Fis1 pathway and regulates MCU activity through the IP3R1-GRP75-VDAC1 and IP3R2/FUNDC1 pathways to alleviate calcium overload and ultimately protect cardiomyocytes after I/R.