Src tyrosine kinase mediates Zn2+-induced myocardial protection against endoplasmic reticulum stress and ischemia/reperfusion injury.

Objective

To investigate the role of Src tyrosine kinase in Zn2+-induced cardioprotection and the mechanisms involving endoplasmic reticulum stress (ERS), mitochondrial dysfunction, and mitochondrial calcium regulation.

Methods

H9c2 cells were used to establish in vitro models of ischemia/reperfusion (I/R) injury and ERS. Cell viability and cytotoxicity were assessed using MTT/CCK-8 assays and lactate dehydrogenase release. Co-immunoprecipitation was performed to examine the interactions of Src with MCU and Mfn1/Mfn2. Western blotting was used to detect Src phosphorylation, MCU complex components, CaMKIIδ/CREB signaling proteins, and ERS markers. Confocal microscopy was used to evaluate mitochondrial membrane potential, Zn2+ and Ca2+ levels, and reactive oxygen species generation.

Results

In 2-DG/TM-treated H9c2 cells, GRP78/94 expression was increased, cell viability was reduced, Src phosphorylation was suppressed, and intracellular and mitochondrial Zn2+ levels were decreased. These changes were accompanied by mitochondrial membrane potential loss, mPTP opening, Ca2+ overload, and ROS accumulation. Exogenous Zn2+ reversed these effects, whereas PP2 or Src siRNA reversed its protection. In the I/R model, Zn2+ improved cell viability, reduced LDH release, restored Zn2+ homeostasis, and alleviated Ca2+ overload. I/R decreased p-Src (Tyr416) and MCUb expression while increasing CaMKIIδ, MCU, MICU1, MICU2, and p-CREB (Ser133). These alterations were reversed by Zn2+ and attenuated by PP2.

Conclusions

Zn2+ protects H9c2 cells against ERS and I/R injury through a Src-dependent mechanism. Activated Src is associated with Mfn1/Mfn2 and preserves mitochondrial function during ERS. In addition, Zn2+ regulates the CaMKIIδ/CREB/MCU pathway through Src activation, thereby reducing mitochondrial Ca2+ overload, mPTP opening, ROS accumulation, and I/R-induced cardiomyocyte injury.