The Mechanism Of Parkin-dependent Mitochondrial Autophagy In HUA-induced Myocardial Injury

Objective:To explore the mechanism of Parkin-dependent mitophagy in HUA-induced myocardial injury,and to provide experimental basis for clarifying the mechanism of HUA related cardiovascular diseases in the future.Materials and methods:1.To study the process and possible mechanism of mitophagy induced by HUA in H9C2 cardiocytes.1).To observe the effect of HUA on mitophagy of cardiomyocytes: The mitophagy of ca-rdiomyocytes was determined by observing the co-localization of mitochondria and lysoso-mes with Zeiss LSM 880 confocal microscope under the effect of HUA.2).Determination of the effect of HUA on the level of reactive oxygen species in cardio-myocytes: Inversion fluorescence microscope and flow cytometry were used to quantitati-vely detect the level of reactive oxygen species in cardiomyocytes under the effect of HUA.3).Determine the effect of HUA on mitochondrial membrane potential in cardiomyocytes:Invert fluorescence microscope and flow cytometry were used to quantitatively detect the level of mitochondrial membrane potential in cardiomyocytes under the effect of HUA.4).Determine the effect of HUA on the level of ATP in cardiomyocytes: The level of ATP in cardiomyocytes under the action of HUA was quantitatively detected by using ATP kit.5).To observe the effect of HUA on the expression of mitophagy related signaling proteins(Parkin,Drp1)in cardiomyocytes.6).To observe whether HUA affects mitophagy of cardiomyocytes through oxidative stress and calcineurin/Ca MKII,to observe the expression of mitophagy-related signaling proteins(Parkin,LC3,P62)in cardiomyocytes under the pre-action of relevant signaling pathway inhibitors/blockers,and to clarify the relationship between the various pathways.Results:1.In H9C2 cardiomyocytes treated with high uric acid,confocal analysis showed that the degree of mitochondrial autophagy was significantly increased in the high uric acid group.2.Fluorescence microscopy and flow cytometry showed that reactive oxygen species increased significantly in H9C2 cardiomyocytes after hyperuric acid stimulation(P<0.01).3.Fluorescence microscopy and flow cytometry showed that the membrane potential of H9C2 cardiomyocytes decreased significantly after hyperuric acid stimulation(P<0.01).4.ATP detection kit showed that the ATP level of H9C2 cardiomyocytes decreased significantly after hyperuric acid stimulation(P<0.01.).5.Western blotting result has proved that high uric acid in H9C2 cardiomyocytes can increase the LC3 B protein involved in mitochondrial autophagy-after Ⅱ,Parkin,the expression of P62,a significant increase in H9C2 cardiomyocytes of calmodulin Ca MKII delta protein expression levels,shows that high uric acid activation of the mitochondrial autophagy and calmodulin.However,after the use of calmodulin inhibitor KN-93,reactive oxygen species inhibitor NAC and mitochondrial autophagy related inhibitor 3-MA,the expression of mitochondrial autophagy related signaling proteins(Parkin,LC3,P62)in cardiomyocytes was significantly decreased,indicating that high uric acid activated mitochondrial autophagy in cardiomyocytes through oxidative stress and the calmodulin pathway.Conclusions:HUA-induced oxidative damage activates ROS/ Ca MKIδ /Parkin signaling pathway to activate mitochondrial autophagy in cardiomyocytes,thus becoming a new mechanism of hyperuricemia related cardiovascular diseases.