Zn2 + alleviates ischemia/reperfusion injury in H9c2 cells by modulating mitochondrial biogenesis and dynamics via MCU

Background

Zinc is an essential nutrient implicated in cardiovascular health. This study investigates whether Zn²⁺ protects H9c2 cells by regulating mitochondrial biogenesis, dynamics, and calcium homeostasis via the mitochondrial calcium uniporter (MCU).

Methods

The I/R model were established using simulated ischemia and reoxygenation as previous reported, and cells were then treated with MCU siRNA. Biochemical kits, inductively coupled plasma mass spectrometry (ICP-MS), RT-qPCR, and transmission electron microscopy were used to assess the effects of Zn²⁺ on cell viability, cytotoxicity, Zn²⁺ and ATP content, NAD⁺/NADH ratio, mtDNA copy number, and mitochondrial morphological changes following myocardial I/R. Confocal microscopy and fluorescence microscopy were used to observe the fluorescence changes of Zn²⁺, mitochondrial membrane potential, protein expression, and mitochondrial Ca²⁺. The effects of Zn²⁺ on protein expression levels were evaluated using molecular docking and Western blot analysis.

Results

Compared to the Control group, the I/R group exhibited decreased cell viability, and increased cytotoxicity. Intracellular and mitochondrial Zn²⁺ levels were reduced, accompanied by mitochondrial dysfunction and an increase in mitochondrial Ca²⁺ content. The expression levels of mitochondrial biosynthesis proteins SIRT1, PGC-1α, NRF1, and TFAM, mitochondrial fusion proteins OPA1, MFN1, and MFN2, as well as MCUb gene and protein expression were downregulated. Conversely, the expression of mitochondrial fission proteins DRP1 and FIS1, along with MCU, MICU1, and MICU2 proteins, was upregulated. Exogenous Zn²⁺ treatment reversed these alterations. MCU silencing by siRNA further enhanced the protection effects of Zn²⁺.

Conclusions

I/R induced damage in H9c2 cells and mitochondrial dysfunction. Zn²⁺ protected H9c2 cells against I/R injury by regulating mitochondrial biogenesis, mitochondrial dynamics, and Ca²⁺ homeostasis via the MCU, with this protective effect potentially associated with the entire MCU complex.