PKA信号通路影响D-半乳糖诱导老化心肌细胞舒张功能障碍的机制

Mechanism of aging-associated diastolic dysfunction in cardiomyocytes induced by D-galactose via PKA pathway

目的探讨D-半乳糖诱导老化的心肌细胞舒张功能障碍的机制。方法实验用药物预处理后予以D-半乳糖建立鼠心肌细胞老化模型。分为4组:正常对照组(C组)、D-半乳糖组(D-G组)、cAMP依赖蛋白激酶A(PKA)预激动组(D-G+cAMP组)和PKA预抑制组(D-G+H-89组);测定心肌细胞肌浆网钙摄取能力,肌浆网Ca^(2+)-ATP酶(SERCA,心脏主要是SERCA2a)活性以及Western blot检测第16位丝氨酸磷酸化受磷蛋白(Ser^(16)phosphorylated phospholamban,Ser^(16)-PLN)的含量。结果与C组相比,D-G组、D-G+H-89组舒张期钙离子浓度[([Ca^(2+)]i)D]升高,钙离子回摄时间(tβ)延长,给予咖啡因刺激后,钙离子增幅(△[Ca^(2+)]i)减少;细胞内SERCA2a活性下降以及Ser^(16)-PLN表达下调(P<0.05)。与D-G组相比,D-G-H-89组([Ca^(2+)]i)D水平进一步升高,tβ进一步延长,给予咖啡因刺激后,△[Ca^(2+)]i明显降低,SERCA2a活性下降,Ser^(16)-PLN表达下调。而D-G+cAMP组([Ca^(2+)]i)D水平明显降低,tβ缩短,△[Ca^(2+)]i增高,细胞内SERCA2a明显提高,Ser^(16)-PLN表达增加。结论 D-半乳糖可能是通过抑制PKA通路,下调Ser^(16)-PLN的磷酸化水平,增加对SERCA2a活性的抑制,从而引起老化心肌细胞舒张功能障碍。

Objective To investigate the mechanism of aging-associated diastolic dysfunction in cardiomyocytes induced by D-galactose. Methods Aging cardiomyocytes model was established by Dgalactose after medication pretreatment. The cardiomyocytes were divided into four groups： normal group（ group C）,D-galactose group（ D-G group）,cAMP-dependent protein kniase A（ PKA） pre-agonist group（ D-G ＋ cAMP group） and PKA pre-inhibit group（ D-G ＋ H-89 group）. Cardiac sarcoplasmic reticulum calcium uptake ability of sarcoplasmic reticulum（ SR）,myocardial SERCA2 a activity and Ser^（16） phosphorylated phospholamban（ Ser^（16）-PLN） protein expression level were detected respectively. Results Compared with group C,increased diastolic [Ca^（2＋）]i,curtailment of the time from the maximum concentration of Ca^（2＋）to the baseline level and decreased reuptake of Ca^（2＋）stores in the SR were observed in D-G group and D-G ＋ H-89 group. In addition,the levels of Ser^（16）-PLN protein as well as SERCA2 a activity were significantly decreased（ P〈0. 05）. Compared with D-G group,the levels of diastolic[Ca^（2＋）]iincreased,the time from the maximum concentration of Ca^（2＋）to the baseline level prolonged in DG-89 group; The levels of diastolic[Ca^（2＋）]idecreased,the time from the maximum concentration of Ca^（2＋）to the baseline level shortened in D-G＋cAMP group. After stimulated by caffeine,reuptake of Ca^（2＋）,the level of SERCA2 a and Ser^（16）-PLN deeereased in D-G-H-89 group and increased in D-G-cAMP group.Conclusions D-galactose can induce aging-associated diastolic dysfunction in cardiomyocytes through inhibiting PKA pathway,decreasing the amount of Ser^（16）-PLN phosphorylation,and further inhibiting SERCA2 a activity.