Effects Of Zinc Deficiency On Endoplasmic Reticulum And Mitochondrial Of Cardiomyocytes And Its Mechanism

Objectives To investigate the effects of zinc deficiency on endoplasmic reticulum and mitochondria in cardiomyocytes to elucidate the signaling pathways and mechanisms of myocardial cell damage caused by zinc deficiency.Methods 1 The H9c2 cells derived from rat embryonic heart tissue were selected as experimental subjects.Zinc deficiency model was established by using zinc ions(Zn²⁺)chelating agent N,N,N’,N’-tetrakis Ethylenediamine（TPEN）.Mitochondrial fluorescent transition TMRE（100 nM）pre-stained cells,laser scanning confocal microscopy to observe the effect of TPEN on mitochondrial membrane potential（?Ψm）,and then determine mitochondrial permeability transition pore（mPTP）Opening situation;The binding immunoglobulin protein（BIP）was detected by Western blot to determine the optimal concentration of TPEN.2 Western blot was used to detect the effect of TPEN on endoplasmic reticulum stress（ERS）chaperone BIP and glucose regulated protein（GRP94）,and whether exogenous zinc（ZnCl₂）could inhibit this effect.3 The effect of zinc deficiency on the expression of ERS receptor inositol-requiring enzyme 1（IRE1）was observed.4 The effect of the IRE1 inhibitor STF083010 on IRE1 expression was observed to determine the optimal concentration.5 To observe whether STF083010 inhibits IRE1expression caused by zinc deficiency.6 The effect of TPEN on the fluorescence intensity of the endoplasmic reticulum red fluorescent probe ER Tracker was observed by laser scanning confocal microscopy.7 The effect of TPEN on the fluorescence intensity of the mitochondrial red fluorescent probe TMRE was observed.8 Cytotoxicity was measured using a lactate dehydrogenase（LDH）kit.Results 1 H9c2 cells were treated with TPEN at different concentrations（0.01,0.1,1,10,100μM）for 20 min.Compared with the Control group（100.0±4.1%）,1,10,100μM TPEN significantly reduced the red fluorescence intensity of TMRE,100μM.TPEN reduced the fluorescence intensity most obviously.10μM TPEN had little effect on TMRE fluorescence intensity（67.8±2.3%）,and the difference was statistically significant（P<0.05）,indicating that zinc deficiency can cause mitochondrial membrane potential to decrease,that is,induce mPTP opening;Western blot analysis showed that compared with the Control group（100±3.8%）,the expression of BIP was significantly increased after treatment with 10μM TPEN for 20 min（210.2±7.7%）,the difference was statistically significant（P<0.05）.It indicated that zinc deficiency caused ERS,and the concentration of TPEN in subsequent experiments was 10μM.2 Western blot results showed that compared with the Control group（100±2.2%）,TPEN treatment for 20 min significantly increased the expression of BIP（215.1±8.3%）,which was reversed by ZnCl₂（163.2±9.5%）.Academic significance（P<0.05）;Compared with the Control group（100±3.9%）,TPEN treatment for 20 min significantly increased the expression of GRP 94（194.1±6.2%）,which was also reversed by ZnCl₂（156.6±4.9%）,and the difference was statistically significant（P<0.05）,the above results indicate that zinc deficiency causes ERS,and supplementation with exogenous zinc（5μM ZnCl₂）can prevent ERS.3 Western blot results showed that compared with the Control group（100±1.5%）,the expression of IRE1was significantly increased after TPEN treatment for 20 min（188.4±3.9%）,and this effect was reversed by ZnCl₂（133.0±7.8%）.Statistical significance（P<0.05）,indicating that zinc deficiency may activate IRE1.4 After treated with different concentrations（0.1,1,5,10,30,45μM）of STF083010 for 30 min,compared with the Control group（100±8.1%）,1μM STF083010 treated cells significantly decreased the expression of IRE1（59.1±2.4%）,the difference was statistically significant（P<0.05）,indicating that STF083010 can inhibit the expression of IRE1.The concentration of STF083010 in the subsequent experiments was 1μM.5 Compared with the Control group（100±3.8%）,the expression of IRE1 was significantly increased after 20 min of TPEN treatment（204.2±7.3%）,and this effect was reversed by STF083010（152.2±3.3%）.The difference was statistically significant（P<0.05）,further indicating that zinc deficiency activates IRE1.6 Compared with the Control group（100.00±3.0）,TPEN significantly reduced the red fluorescence intensity of ER Tracker（59.15±3.2）,and STF083010 significantly inhibited the decrease of red fluorescence intensity caused by TPEN（79.4±3.8%）.The difference was statistically significant（P<0.05）,indicating that zinc deficiency caused damage to the endoplasmic reticulum through IRE1.7 Compared with the Control group（100.00±4.5）,TPEN significantly reduced the red fluorescence intensity of TMRE（61.35±2.7）,and STF083010significantly inhibited the decrease of TMRE fluorescence intensity induced by TPEN（79.6±2.2%）.The difference was statistically significant（P<0.05）,indicating that zinc deficiency leads to mPTP opening through IRE1.8 The results of LDH cytotoxicity kit showed that compared with the Control group（100.00±0.0）,TPEN significantly increased cytotoxicity（195.17±7.4）,STF083010 significantly inhibited the cytotoxicity of TPEN（150.0±4.0%）,the difference was statistically significant.The significance of the study（P<0.05）,indicating that zinc deficiency increases cytotoxicity through IRE1.Conclusions Zinc deficiency causes endoplasmic reticulum stress,which leads to the opening of mPTP through sensor IRE1,which increases cytotoxicity and ultimately leads to damage of cardiomyocytes.Supplementation of exogenous zinc can inhibit endoplasmic reticulum stress and protect cardiomyocytes.Figure 11;Table10;Reference 172.