The Effect Of AngⅡ-NOX2-ROS-JP2 Signal Pathway On Cardiomyocytes T-Tubule Remodeling And Excitation-Contraction Coupling

Objective:To explore the effect of excessively activated NOX2-ROS-JP2 signal pathway which induced by pathological concentration of AngⅡ on cardiomyocytes t-tubule remodeling, excitation -contraction (E-C) coupling.Method:The hearts of mice were perfused by Langendorff device, we got the myocytes by using protease and collagnase. After added the virus, the myocytes were cultured in 5% CO2 incubator at 37℃ for 48 hours (hrs).The myocytes were divided into Control group, Empty virus group, AT1 virus group, Empty+AngⅡ500nM group, AT1+AngⅡ 500nM group, AT1+AngⅡ+apocynin300μM group, apocynin300μM group, we would add and subtract some groups slightly according to different experimental purposes. The cardiomyocytes were stained with Di-8-ANEPPS in Ca2+ free tyrode solution at room temperature for 30 mins. The structures of Transverse-tubules (T-tubules) were visualized with confocal microscope. The TT spectrum represents the t-tubule structure of each ventricle. The power value (TT power) was processed by IDL6.4 and Clampfit10.2 program. The cardiomyocytes were stained with 2’,7’-dichlorodihydrofluorescein diacetate (DCF) in Ca2+ free tyrode solution at room temperature for 30 mins. We observed it with confocal microscope as well. The quantitative analysis of fluorescent intensity was proceeded by Image J. In Ca2+ transit experiment, cultured cardiomyocytes were loaded with Fluo-4 AM in 1.8 mmol/L Ca2+ tyrode solution for 30 min. The Ca2+ images were acquired by using confocal microscope in line-scan mode along the longitudinal axis of myocytes. SR Ca2+ content was assessed by measuring caffeine-induced Ca2+ transient. Ca2+ transient was processed by IDL program. SR content was detected by Image J. The cell proteins were collected on ice. Protein concentrations were determined by using the Pierce BCA assay. The expression of JP2 protein was detected by western blot.Result:The effect of AngⅡ on cardiomyocytes t-tubule remodeling in C57BL/6 mice: Compared with Control group, the t-tubule structure of AT1 group wasn’t changed obviously, the t-tubule structure of Empty virus+AngII500nM group and AT1+AngⅡ 500nM group were obviously damaged (P<0.05,P<0.01). The fluorescent intensity of Empty virus group and AT1 group were not obviously changed. It was enhanced significantly in Empty virus+AngⅡ500nM group and AT1+AngⅡ500nM group (P<0.05,P<0.01)The effect of apocynin on cardiomyocytes t-tubule remodeling, excitation-contraction coupling in C57BL/6 mice:The t-tubule structure of Empty virus+AngII500nM group and AT1+AngⅡ500nM group were obviously damaged (P<0.05, P<0.01), it was improved in AT1+AngⅡ+apocynin300μM group compared with AT1+AngⅡ500nM group (P<0.01). The transit and SR content levels in AT1 group were normal, they were significantly reduced in AT1+AngII500nMgroup (P<0.01) and were reversed in AT1+AngⅡ+apocynin300μM group (P<0.05). The fluorescent intensity of Empty virus+AngII500nM group and AT1+AngII500nM group were obviously enhanced (P<0.05,P<0.01), it could be attenuated in AT1+AngⅡ+apocynin300μM group (P<0.01). In the expression of protein level, the JP2 level of Empty virus group, AT1 group and apocynin300μM group were not changed obviously compared with Control group. The JP2 level of AT1+AngⅡ500nM group was descended significantly (P<0.01). And it was significant increased in AT1+AngⅡ+apocynin300μM group compared with AT1+AngⅡ500nM group (P<0.05).The effect of AngⅡ on cardiomyocytes t-tubule remodeling, excitation-contraction coupling in NOX2-/- mice:In cell T-tubule system, Ca2+ imaging, ROS content and the expression of JP2, no significant changes between Control group and the other groups.Conclusion:The pathological concentration of AngⅡ generates NOX2, which degrades JP2 protein, leading to cardiomyocytes t-tubule pathological remodeling and excitation-contraction uncoupling.