A Mechanism Study On The Protective Effects Of Ginkgo Biloba Extract On Cell Signal Transduction Pathway Of Aging Myocardial Sarcoplasmic Reticulum Calcium Transport Protein Modification

BACKGROUNDSenescence is an inevitable physiological process involved in decreased cardiac pump function, resulting in high mortality rates due to cardiovascular aging[1,2].Overseas studies suggest that a dramatic decline in cardiac pump function with advanced age has been seen and are considered to be result of the left ventricular diastolic dysfunction[3]. It has been found that cardiac diastolic function reduction appeared in the D-galactose induced myocardia aging model in our previous animal experiments[4]. The sarcoplasmic reticulum (SR), which is the most important of the Ca 2+ stores in mammalian cardiac cells, takes part in the regulation of the Ca cycle in cardiac systole and diastole.2+ The Ca²⁺ rapidly entrance is regulated by the sarcoplasmic reticulum Ca²⁺-ATP enzyme (SERCA, mainly SERCA2a in the heart). Therefore, sarcoplasmic reticulum Ca²⁺-ATP enzyme (SERCA2a) activity and the calcium handling impairment are significantly related with myocardial cells diastolic dysfunction. It is well known that SERCA2a is regulated by the SR protein, phospholamban (PLN). PLN can be phosphorylated by the protein kinase C (PKC; Ser10 site),cAMP-dependent protein kinases(PKA;Ser16 site) ,and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII; Thr17 site)pathways, the latter two of which are considered to be the main mediators in the regulation of cardiomyocyte diastolic function[5].Many studies show that Ginkgo biloba extract (EGB761) has multiple biological effects in anti-aging, including that upregulation or downregulation signaling pathways, gene transcription, cell metabolism[6], anti-apoptosis[7], protective nerves in Alzheimer's disease[8], reducing endothelial cell senescence[9] and so on. In the current literature and our previous study [10], Ginkgo biloba extract (EGB761) could inhibit non-enzymatic glycation and increase myocardial cells of SERCA2a, PLN protein content. However, it is not yet clear whether EGb761 upregulated/downregulated phospholamban phosphorylation signaling pathway, relieved the inhibitory of SERCA2a, reduced intracellular calcium overload, and improved diastolic function in myocardial cells.OBJECTIVEThe objective of this study was to investigate the cell signaling pathways of sarcoplasmic reticulum calcium transport protein modified and the role of Ginkgo biloba extract (EGB761) to the myocardial sarcoplasmic reticulum calcium transport protein modification pathway in the myocardial cell by D-galactose induced.METHODSThe primary rat cardiomyocytes were cultured with either 5g/l D-galactose or 5g/l D-galactose combined with 20ug.ml-1 Ginkgo biloba extract (EGB761). After treatment, cardiac sarcoplasmic reticulum calcium pump (SERCA) activity was measured by improved p-NPP, myocardial sarcoplasmic reticulum calcium uptake were assessed by Fluorescent calcium indicator Fura-2/AM. In addition, protein levels of phosphorylated phospholamban (p-PLN) were detected by western blot.RESULTSIn the myocardial aging cell induced by D-galactose, intracellular diastolic calcium concentration (([Ca²⁺] i) D) levels was elevated, the time of taking calcium back to sarcoplasmic reticulum(tβ) was significantly prolonged ,the amplitude of caffeine-induced calcium reserves (Δ[Ca²⁺ ]i) was also reduced, sarcoplasmic reticulum Ca²⁺-ATP enzyme (SERCA2a) activity decreased significantly, protein level of phosphorylation of phospholamban (p-PLN) at Ser16 site was significantly decreased (P<0.05) and the expression of Thr17 site was no significant differences compared with the control group(P> 0.05). However, after given Ginkgo biloba extract (EGB761) ,diastolic myocardial cells calcium concentration (([Ca2 +] i) D) and the time of taking calcium back to sarcoplasmic reticulum(tβ) were significantly reduced, caffeine-induced calcium reserves increased significantly, sarcoplasmic reticulum Ca²⁺-ATP enzyme(SERCA2a) activity was significantly increased, protein level of phosphorylation of phospholamban (p-PLN) at Ser16 site (by PKA signaling pathway regulation)was significantly increased (P<0.05), phosphorylation level of Thr17 sites (subject to CAMKII pathway regulation) was no significant differences compared with the control group(P> 0.05).CONCLUSION1. The myocardial sarcoplasmic reticulum Ca²⁺-ATP enzyme (SERCA2a) activity decreased, intracellular calcium was overload and phospholamban phosphorylation decreased by PKA signaling pathway regulation in the aging cell by the D-galactose induced . All results suggest that abnormal PKA signaling pathway regulation may be the important role in the diastolic dysfunction of the aging myocardial cell induced by the advanced glycation endproducts .2. EGb761 upregulated phospholamban phosphorylation through PKA signaling pathway, relieved the inhibitory of SERCA2a, reduced intracellular calcium overload and improved myocardial diastolic function, thereby delay the myocardial cells aging.