The Mechanism Underlying Cardiac Cell Injury Caused By Zinc Deficiency

ObjectiveTo test the effect of zinc deficiency on cardiomyocyte survival and to explore the underlying mechanisms.MethodsThe H9c2 cardiac cells were cultured for 24 h and were divided into the control group,zinc chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine(TPEN)group.MTT assay was used to evaluate cell viability.The morphological changes of cells were observed by optical microscope.The LDH levels of cells were determined with LDH Assay Kit.Intracellular reactive oxygen species were determined with DCFH-DA.Cellular protein carbonylation was determined using western-blot assay.Mitochondrial membrane potential(ΔΨ)was measured with confocal microscope and fluorescence microplate reader using JC-1.The phosphorylation of ERK and STAT3 was detected by western-blot assay.Results1.Compared to the control,10,50,and 100 μmol/L TPEN significantly decreased cell viability(P < 0.05)with the minimum effect at 10 μmol/L and thus we used this concentration in all the subsequent studies.2.TPEN caused the cell morphological changes,increased LDH,ROS generation,and protein carbonylation,suggesting that zinc deficiency damages cells by enhancing oxidative stress.3.TPEN reduced the JC-1 ratio,indicating that TPEN leads to the loss of ΔΨm and the opening of mPTP.The studies with fluorescence microplate reader further confirmed the above observation.4.Compared to the control,TPEN significantly inhibited ERK phosphorylation and decreased cell viability,which was potentiated by PD98059,inhibitors of ERK,whereas SNAP,which can activate ERK,prevented this,indicating that zinc deficiency induces the injury in H9c2 cardiac cells through inhibiting ERK activity.5.Compared to the control,TPEN significantly inhibited ERK phosphorylation and this was prevented by the ROS scavenger,MPG,suggesting that ROS may mediate the effect of zinc deficiency on ERK activity.6.Compared to the control,TPEN significantly increased STAT3(Tyr705)phosphorylation and this was reversed by the STAT3 inhibitor,Stattic,pointing to that zinc deficiency activate STAT3 pathway.7.Compared to the control,TPEN markedly reduced cell viability and this was worsened by Stattic,suggesting that the activation of STAT3 may serve as an intrinsic mechanism of cell protection.Conclusions1.Zinc deficiency leads to cardiac cells injury.2.Zinc deficiency damages cells by enhancing oxidative stress.3.TPEN deactivates ERK via ROS,which may lead to cell injury.4.The activation of STAT3 may serve as an endogenous mechanism by which cells are resistant to zinc deficiency.