Role Of Mitochondrial Calcium Uniporter In Zn²⁺ Induced Cardiomyocyte Protection

Objective To explore the function and mechanism of mitochondrial calcium uniporter（MCU）in zinc ion(Zn²⁺)-induced cardiomyocyte protection.Methods H9c2 cells were treated with tunicamycin（TM）as an endoplasmic reticulum stress（ERS）inducer to construct an ERS model.The cells were pretreated with zinc chloride（Zn Cl₂）,MCU inhibitor ruthenium red（RR）,and MCU si RNA.MTT was used to detect the effect of different concentrations of TM（1μg/ml,3μg/ml,5μg/ml）on cell viability.The intracellular Ca²⁺and mitochondrial reactive oxygen species（ROS）were observed by laser confocal microscopy.The expression of GRP 78,GRP 94 and MCU was detected by western blot.Microplate method was used to detect LDH in the extracellular fluid of cardiac cells.Fluorescence microscopy to determine the opening of m PTP by measuring the mitochondrial membrane potential.Detection of Ca²⁺in mitochondria using a fluorescent plate reader.Results 1 The activity of H9c2 cardiomyocytes was reduced by different concentrations of TM.MTT results showed that different concentrations of TM（1μg/ml,3μg/ml,5μg/ml）significantly reduced the activity of H9c2 cardiomyocytes.2 Different concentrations of TM（1μg/ml,3μg/ml,5μg/ml）induced ERS.Different concentrations of TM all increased the expression of GRP 78 and GRP 94 expression was increased by different concentrations of TM,with the most pronounced expression at 3μg/ml.3 Different concentrations of TM（1μg/ml,3μg/ml,5μg/ml）increased MCU and intracellular Ca²⁺.The expression of MCU was enhanced by different concentrations of TM,and the most pronounced expression was observed at 3μg/ml.The increase in intracellular Ca²⁺was also most evident at 3μg/ml TM.Therefore,TM concentration of 3μg/ml was used in subsequent experiments.4 Zn²⁺inhibited ERS-induced myocardial injury via MCU.Compared with the control,TM increased LDH in the extracellular fluid,Zn Cl₂decreased LDH in the extracellular fluid of H9c2 myocardium,and RR significantly enhanced the effect of Zn Cl₂.5 Zn²⁺inhibited TM-induced ERS via MCU.Compared with the control,TM upregulated protein expression of GRP 78 and MCU,Zn Cl₂ significantly inhibited the effect of TM,and RR significantly enhanced the effect of Zn Cl₂.6 MCU si RNA further enhanced the effect of Zn²⁺inhibition of ERS.Compared with the control group,TM upregulated the expression of GRP 78 and MCU,Zn Cl₂ significantly inhibited the effect of TM,and MCU si RNA significantly enhanced the effect of Zn Cl₂.This result is consistent with the MCU inhibitor RR.7 Zn²⁺inhibited TM-induced m PTP opening via MCU.Compared with the control,TM caused a significant decrease in mitochondrial TMRE red fluorescence intensity,ERS caused a decrease in mitochondrial membrane potential and m PTP opening,and Zn Cl₂ significantly inhibited the TM-induced decrease in red fluorescence intensity,while RR further enhanced the effect of Zn Cl₂.8 Zn²⁺inhibited the ERS-induced increase in mitochondrial Ca²⁺via MCU.Compared with the control,TM caused a significant increase in the fluorescence intensity of the mitochondrial Ca²⁺probe Rhod-2AM,and Zn Cl₂ significantly inhibited the effect of TM,while RR further enhanced the effect of Zn Cl₂.9 Different concentrations of TM（3μg/ml,5μg/ml）increased mitochondrial ROS.Confocal microscopy results showed that different concentrations of TM significantly enhanced the red fluorescence intensity of the mitochondrial reactive oxygen species Mito SOX compared to the control,with 3μg/ml of TM being the most pronounced.10 Zn²⁺inhibited the ERS-induced increase in mitochondrial ROS via MCU.Fluorescence microscopy results showed that TM significantly increased the red fluorescence intensity of mitochondrial reactive oxygen species Mito SOX,Zn Cl₂ significantly inhibited the effect of TM,and RR further enhanced the effect of Zn Cl₂.Conclusions 1 TM induces ERS in cardiomyocytes,resulting in decreased cell activity,increased MCU expression,and increased Ca²⁺in cells and mitochondria.2 Zn²⁺inhibits ERS-induced increase in mitochondrial Ca²⁺and ROS via MCU,prevents the opening of m PTP,and attenuates cardiomyocyte injury.Figure 12;Table 3;Reference 150...