The PI3K-PKB/Akt-GSK-3β Signaling Pathway Mediates Fibroblast Growth Factor-2 Cardioprotection

Ischemic heart disease(IHD) is an important cause of human death in the world. Exploration the protective methods and mechanisms of ischemic myocardial damage have become a research focus and hotspot in the field of cardiovascular with great theory and application of value.IHD and its complications(such as reperfusion injury) exist in different degrees of myocardial apoptosis. Myocardial apoptosis is one of the main pathological mechanisms of myocardial ischemia-reperfusion injury(IRI), and is closely related to the occurrence and development of cardiac IRI. The number of myocardial apoptosis determines the extent of myocardial injury, can cause cardiac dysfunction, and even heart failure. It can be significantly reduced myocardial infarct size and the incidence of cardiac dysfunction in myocardial ischemia-reperfusion by inhibiting myocardial apoptosis.Previous studies have shown that Fibroblast growth factors(FGFs) can promote cell proliferation and migration, promote angiogenesis, improve myocardial blood supply, fight against myocardial necrosis and apoptosis, protect the ischemic heart from injury. It may provide a new idea for the treatment of IHD. There are 22 members in the heparin growth factor FGFs, which Fibroblast growth factor-2(FGF-2, also known as basic fibroblast growth factor(b FGF)) gets much attention. FGF-2 is a multifunctional growth factor, can promote cell proliferation, differentiation, migration and angiogenesis. Recent research has proved that FGF-2 is an important endogenous cytoprotective substance with a significant cardioprotective effect. Our group and other researchers have found that the cardioprotective effect of FGF-2 is relevant to the combination of FGF receptor 1(FGFR1), the activation of protein kinase C(PKC), mitogen-activated protein kinase(MAPKs) and other signaling pathways. However, PKC and MAPK types of diversity and its sub-category feature inconsistency make this protein kinase mediating the cardioprotective effect of FGF-2 in the complexity of situation, and the FGF-2 cardiprotection is difficult to completely explain with PKCs, MAPKs activation. So, it is very necessary and important to look for the other mechanisms of FGF-2 myocardial protective effect.Glycogen synthase kinase-3(GSK-3) is a multifunctional serine /threonine kinase and is widely expressed in a variety of tissues. GSK-3 may be distributed in the cell cytoplasmic matrix, mitochondria and nucleus, the GSK-3 activity in the cell mitochondria and nucleus is much higher than in the cytoplasmic matrix. Human GSK-3 has GSK-3α and GSK-3β subtypes. GSK-3α/β has two closely related to its function and very important phosphorylation sites--Tyr279/Tyr 216 and Ser21/Ser9. GSK-3α/β can be actived at cell resting state by modulating Tyr 279/Tyr 216 phosphorylation, but GSK-3α/β can be inhibited by modulating Ser21/Ser9 phosphorylation. A lot of studies have proved that GSK-3β plays an important role in myocardial protection.Various signaling pathways converged on GSK-3β, made GSK-3βSer9 phosphorylation, inhibited GSK-3β activity, thereby reducing myocardial infarct size, inhibiting cardiomyocyte apoptosis, improving heart function, fighting against arrhythmia and realization cardioprotective effects.After combination of FGF receptor 1(FGFR1), it usually activates PLC-PKC, Ras-MAPKs and PI3K-PKB/Akt signaling pathway. Recent research has shown that PI3K-PKB/Akt-GSK-3β signaling pathway plays an important role in myocardial protection. Some studies have proved that ischemic preconditioning(IPC), ischemic postconditioning(IPost C), hydrogen sulfide and some drugs can protect the heart from IRI by activating PI3K-PKB/Akt-GSK-3β signaling pathway. It has been reported that FGF-2 can fight against chronic endoplasmic reticulum stress-induced neuronal apoptosis, promote human neural stem cells differentiation into motor neurons and make prostate cancer occurring epithelial-mesenchymal transitions(EMT) via the activation of PI3K-PKB /Akt-GSK-3β signaling pathway. However, whether PI3K-PKB /Akt-GSK-3β signaling pathway can mediate the cardioprotective effect of FGF-2? It remains unclear.Therefore, we hypothesize that FGF-2 may activate PI3K-PKB/Akt-GSK-3β signaling pathway to exert its cardioprotective effect. To test this hypothesis, we first observed the effect of FGF-2 on myocardial infarct size, cardiac function, myocardial apoptosis and PI3K-PKB/Akt-GSK-3β signaling pathway by using isolated rat heart ischemia/reperfusion injury model and rat cardiomyocytes hypoxia/reoxygenation injury model. Then, we elucidated the specific mechanism of GSK-3β-mediated FGF-2’s fighting against myocardial apoptosis by using rat cardiomyocytes hypoxia/reoxygenation injury model. This project is expected to reveal the new mechanisms and ways of FGF-2 cardiprotection, to further clarify the important endogenous cytoprotective substances in physiological and pathological significance and provide new ideas and new areas of intervention in IHD treatments.Part I: Exploring the new mechanisms of FGF-2 cardiprotection: the role of PI3K-PKB/Akt-GSK-3β signaling pathwaysObjective: To explore whether PI3K-PKB/Akt-GSK-3β signaling pathway is involved in FGF-2 myocardial protectionMethods: 1. To observe the effect of PI3K-PKB/Akt-GSK-3β signaling pathway on FGF-2’s fighting against isolated SD rat hearts IRI. All ex vivo Langendorff perfused heart were equilibrated with Krebs-Henseleit(K-H) buffer for 30 min, 47 SD rats were randomly divided into the following 7 groups, each group included 6 to 7 rats:(1)I/R group, the isolated hearts were subjected to normothermic global ischemia for 30 min, followed by 60 min of reperfusion in K-H buffer;(2)I/R+LY294002 group(LY294002), I/R with the selective PI3 K inhibitor LY294002, 15μM LY294002 was initiated 5 minutes before ischemia and remained during the entire period of reperfusion;(3)I/R+SH-5 group(SH-5), I/R with the PKB/Akt inhibitor SH-5,10μM SH-5 was started 5 minutes before ischemia and remained during the entire period of reperfusion;(4)I/R+SB216763 group(SB216763), I/R with the GSK-3βinhibitor SB216763, 3μM SB216763 was started 5 min before the onset of reperfusion and continued for 60 min;(5)I/R+FGF-2 group(FGF-2), I/R with FGF-2, FGF-2(10μg/heart each)was infused into the heart during the first 10 min of reperfusion;(6)I/R+FGF-2+LY294002 group(FGF-2+LY294002), I/R with FGF-2 group and LY294002 group;(7) I/R+FGF-2+SH-5 group(FGF-2+SH-5), I/R with FGF-2 group and SH-5 group. Myocardial infarct size were defined by 1% 2,3,5-triphenyltetrazolium chloride(TTC) staining. The lactate dehydrogenase(LDH) release in the coronary effluent was measured by Automatic biochemical analyzer. The parameters of cardiac function including left ventricular systolic pressure(LVSP), left ventricular diastolic pressure(LVDP), maximal rate of the increase/decrease of left ventricular pressure(±dp/dtmax) were recorded respectively by multileads physiologic recorder at preischemia and postreperfusion. Apoptosis of myocardial cells was detected by TUNEL and DAPI staining. The expression of Akt, p-Akt(Ser 473),GSK-3βand p-GSK-3β(Ser9) were analyzed by Western Blot. 2. To observe the effect of PI3K-PKB/Akt-GSK-3β signaling pathway on FGF-2’s fighting against H9c2 cardiomyocytes hypoxia/reoxygenation(H/R) injury.(1) To observe the effect of FGF-2 on normal cardiomyocytes Akt and GSK-3β expression. The H9c2 cardiomyocytes were treated with 10 ng/ml FGF-2 for 0,5,10,20,30,60 minutes respectively. Akt, p-Akt(Ser473), GSK-3βand p-GSK-3β(Ser9) expression were analyzed by Western Blot.(2) To observe the effect of H/R on myocardial cell injury, Akt and GSK-3β expression. H9c2 cardiomyocytes were randomly divided into 3 groups:(1)Normal group(Control), cardiomyocytes were cultured in DMEM for 3~4 h;(2)Hypoxia group(H), cardiomyocytes anoxia for 90m;(3)H/R, cardiomyocytes anoxia for 90 min and reoxygeonation for 120 min. At the end of experiment, cardiomyocytes viability was measured by CCK-8.Then, the cells were subjected to anoxia for 0,0.5,1,2 h, and to reoxygeonation for 0.5,1,2,12, 24 h after 2 h of anoxia respectively. The expression of Akt, p-Akt(Ser 473),GSK-3βand p-GSK-3β(Ser9) were analyzed by Western Blot.(3)To observe the effect of FGF-2 on myocardial cell injury, Akt and GSK-3β expression induced by H/R. H9c2 cardiomyocytes were randomly divided into 3 groups:(1)Normal group(Control);(2)H/R;(3)H/R+FGF-2. The viability of cardiomyocytes was measured by CCK-8.The apoptosis rate of cardiomyocytes was detected by TUNEL and Armexin-V/PI assays with flow cytometry. Akt, p-Akt(Ser473), GSK-3βand p-GSK-3β(Ser9) expression were analyzed by Western Blot.(4)To observe the effect of PI3K-PKB/Akt-GSK-3β signaling pathway on FGF-2’s fighting against myocardial cell injury induced by H/R. Firstly, H9c2 cardiomyocytes were randomly divided into 5 groups:(1) Normal group(Control);(2) H/R group(H/R);(3) H/R+FGF-2 group(FGF-2);(4)H/R+FGF-2+LY294002 group(FGF-2+LY294002);(5) H/R+FGF-2+SH-5 group(FGF-2+SH-5). The cells were incubated with 10ng/ml FGF-2 for 30 min, and then subjected to anoxia for 90 min, followed by 120 min of reoxygenation.10μmol/L LY294002 or 5μmol/L SH-5 was added at 1h before anoxia. At the end of experiment, the viability of cardiomyocytes was measured by CCK-8. The apoptosis rate of cardiomyocytes was detected by TUNEL and Armexin-V/PI assays with flow cytometry. The expression of Akt, p-Akt(Ser 473),GSK-3βand p-GSK-3β(Ser9) were analyzed by Western Blot. Then, to observe the effect of GSK-3β-WT,GSK-3β-S9 A and GSK-3β-K85 R mutant on the expression of cardiomyocytes GSK-3βand p-GSK-3β(Ser9). First, V5 Tag labeled pc DNA3/GSK3β-WT, pc DNA3/GSK-3β-S9 A, pc DNA3/GSK-3β-K85 R plasmid were amplified, extracted and sequenced. Second, H9c2 cardiomyocytes were transfected with pc DNA3/GSK3β-WT, pc DNA3/GSK-3β-S9 A, pc DNA3/GSK-3β-K85 R plasmid by Lipofecter. The expression of V5 Tag was analyzed by Western Blot after the plasmids were successfully transfected for 48 h. Third, The cells were randomly divided into 5 groups:(1) control group(Con);(2) empty vector group(Vec);(3)GSK-3β-WT group(WT);(4)GSK-3β-S9 A group(S9A);(5)GSK-3β-K85 R group(K85R). GSK-3βand p-GSK-3β(Ser9) expression were analyzed by Western Blot. Finally, to observe the effect of FGF-2 on the expression of apoptosis-related proteins in the H/R cardiomyocytes. After H9c2 cardiomyocytes were successfully transfected with pc DNA3/GSK3β-WT, pc DNA3/GSK-3β-S9 A and pc DNA3/GSK-3β-K85 R plasmid, the cells were divided into 6 groups:(1) normal group(Control);(2) H/R group(H/R);(3)H/R+GSK-3 β-K85 R group(H/R+K85R);(4) H/R+GSK-3 β-S9 A group(H/R+S9A);(5) H/R+FGF-2 group;(6) H/R+GSK-3β-S9A+FGF-2group(H/R+S9A+FGF-2). The expression of cytoplasmic and mitochondria Bax, Bcl-2, 32 KD and 17 KD Caspase-3 were analyzed by Western Blot.Results: 1.Studies in isolated hearts found that FGF-2 group myocardial infarct size decreased by 42.3%, coronary effluent LDH activity decreased by 32.8% LVSP increased by 25.8%, LVDP increased by 38.5%, ±dp/dtmax absolute value increased by 35.9% and 52.0% respectively, apoptosis index(AI) decreased by 52.3%, the ratio of p-Akt/Akt and p-GSK-3β/GSK-3β increased by 112.5% and 112.1% compared with IR group(all P <0.01). PI3 K inhibitor LY294002 and PKB/Akt inhibitor SH-5 could fight against the cardioprotective effect of FGF-2. Compared to FGF-2 group, FGF-2+LY294002 group myocardial infarct size increased by 40.2%, coronary effluent LDH activity increased by 25.6%, LVSP decreased by 12.6%, LVDP decreased by 17.2%, ±dp/dtmax absolute value decreased by 13.9% and 22.4% respectively, the ratio of p-Akt/Akt and p-GSK-3β/GSK-3β decreased by 27.7% and 27%(all P <0.05). Compared to FGF-2 group, FGF-2+SH-5 group myocardial infarct size increased by 29.5%, coronary effluent LDH activity increased by 20.0%, LVSP decreased by 10.1%, LVDP decreased by 13.4 %, ±dp/dtmax absolute value decreased by 9.9% and 17.5% respectively, the ratio of p-Akt/Akt and p-GSK-3β/GSK-3β decreased by 19.2% and 20.3%(all P <0.05). Compared to I/R group, SB216763 group myocardial infarct size decreased by 43.6%, coronary effluent LDH activity decreased by 41.1%, LVSP increased by 29.9%, LVDP increased by 46.6%, ±dp/dtmax absolute value increased by 40.7% and 58.7% respectively, the ratio of p-GSK-3β/GSK-3β increased by 129.2%(all P <0.01). Compared to I/R group, there was no significant difference of myocardial infarct size, coronary effluent LDH activity, LVSP, LVDP, ±dp/dtmax absolute value, the ratio of p-Akt/Akt and p-GSK-3β/GSK-3β in LY294002 group and SH-5 group(all P> 0.05).. 2.Hypoxia and H/R significantly decreased the viability of cardiomyocytes(vs. the normal group, P<0.01). FGF-2 significantly increased the viability of cardiomyocytes subjected to H/R, suppressed cardiomyocytes apoptosis induced by H/R(vs. H/R group, P<0.01). LY294002 or SH-5 could antagonise FGF-2 cardiprotection(vs. FGF-2 group, P<0.05). 3.Studies in the normal cardiomyocytes models found that FGF-2 dynamically regulated cardiomyocytes Akt and GSK-3β activity. Hypoxia transiently activated Akt, H/R activated Akt and then inhibited the activation of Akt. However, Hypoxia transiently inhibited the activation of GSK-3β, H/R inhibited the activation of GSK-3β and then activated GSK-3β. FGF-2significantly up-regulated the expression of p-Akt and p-GSK-3βin the H/R cardiomyocytes(vs. H/R group, P<0.01), whose role was reversed by LY294002 or SH-5(vs. FGF-2 group, P<0.05). 4.Sequence analysis showed that the gene sequence of GSK-3β-WT is correct, GSK-3β-S9 A mutant 9 serine is indeed replaced by alanine, GSK-3β-K85 R mutant 85 lysine is indeed replaced by arginine. GSK-3β-WT, GSK-3β-S9 A and GSK-3β-K85 R were effectively transfected into the cardiomyocytes. The ratio of p-GSK-3β/GSK-3β was significantly decreased in GSK-3β-WT, GSK-3β-S9 A and GSK-3β-K85 R group(vs. the control group, P <0.05), and GSK-3β-S9 A group was the lowest group compared to other groups5.H/R significantly promoted Bax from the cytoplasm into the mitochondria, Caspase-3 activation and inhibited the expression of Bcl-2(vs. the normal group, P <0.05). However, these effect of H/R were suppressed by FGF-2 or GSK-3β-K85R(vs. H/R group, P<0.05). The effect of FGF-2 on H/R was also reversed by GSK-3β-S9A(vs. H/R+FGF-2 group, P<0.05).Conclusion: 1.FGF-2 can fight against myocardial IRI and cardiomyocytes H/R injury, it has a good cardioprotective effect. 2.The activity of PI3K-PKB/Akt-GSK-3β signaling pathway mediates FGF-2’s fighting against myocardial IRI and cardiomyocytes H/R injury, this is a new molecular mechanism of FGF-2 cardiprotectionPart II: Effect of the phosphorylation of mitochondrial GSK-3β Ser9 on FGF-2 anti-myocardial apoptosisObjective: To explore the mechanisms of FGF-2’s fighting against myocardial apoptosis mediated by GSK-3β.Methods: 1.To observe the effect of GSK-3βon myocardial apoptosis induced by H/R. The cardiomyocytes were randomly divided into 5 groups:(1)Normal group(Normal);(2) H/R group;(3) H/R+Control si RNA group(Control si RNA);(4) H/R+GSK-3 β si RNA group(GSK-3 β si RNA);(5)H/R+SB216763 group(SB216763). The cardiomyocytes were successfully transfected with GSK-3β si RNA for 48 h or incubated with 3μM SB216763 for 30 min, and then subjected to anoxia for 90 min, followed by 120 min of reoxygenation. At the end of experiment, the apoptosis of myocardial cells was detected by TUNEL and DAPI staining. 2.To observe the effect of the phosphorylation of mitochondrial GSK-3β Ser9 on FGF-2’s fighting against myocardial apoptosis induced by H/R. The cardiomyocytes were successfully transfected with GSK-3β-S9 A or GSK-3β-K85 R plasmid, and then divided into 6 groups:(1)Normal group(Normal);(2)H/R group;(3)H/R + GSK-3β-K85 R group(GSK-3β-K85R);(4)H /R + GSK-3β-S9 A group(GSK-3β-S9A);(5)H/R + FGF-2 group(FGF-2);(6)H/R + GSK-3β-S9 A + FGF-2 group(GSK-3β-S9 A + FGF-2). The apoptosis rate of cardiomyocytes was detected by TUNEL and DAPI staining. 3.To observe the effect of FGF-2 on the phosphorylation of mitochondrial GSK-3β Ser9. Firstly, to observe the effect of FGF-2 on the expression of GSK-3β. The cardiomyocytes were randomly divided into 2 groups:(1)Normal group(Vehicle);(2)FGF-2 group. The expression of GSK-3β was analyzed by Immunofluorescence. Then, to observe the effect of FGF-2 on the expression of p-GSK-3β(Ser9). The cells were randomly divided into 4 groups:(1) Normal group(Vehicle);(2) LY294002 group;(3) FGF-2 group;(4)FGF-2+LY294002 group. The expression of p-GSK-3β(Ser9) was analyzed by Immunofluorescence. Finally, to observe the effect of FGF-2 on the expression of mitochondria and cytoplasmic matrix GSK-3β and p-GSK-3β(Ser9). The cardiomyocytes were randomly divided into 2 groups:(1) Normal group(Vehicle);(2) FGF-2 group. The expression of mitochondria and cytoplasmic matrix GSK-3β and p-GSK-3β(Ser9) were analyzed by Western Blot. 4.To observe the effect of GSK-3βsi RNA on apoptosis related proteins expression in the H/R cardiomyocytes. The cells were randomly divided into 3 groups:(1)Normal group(Normal);(2)H/R group;(3)H/R+GSK-3βsi RNA group. The expression of Bcl-2, mitochondria and cytoplasm Bax were analyzed by Western Blot.Results: 1.GSK-3 β si RNA and SB216763 effectively antagonised cardiomyocytes apoptosis induced by H/R(vs. H/R group, P<0.05). 2. FGF-2 and GSK-3β-K85 R significantly decreased the apoptosis rate of cardiomyocytes(vs. H/R group, P<0.05), but the cardioprotective effect of FGF-2 was suppressed by GSK-3β-S9A(vs. H/R+FGF-2 group, P<0.05). 3.FGF-2 had no obvious effects on the cells, especially the mitochondrial GSK3β protein expression. However, FGF-2 significantly promoted myocardial p-GSK-3β(Ser9), especially mitochondria p-GSK-3β(Ser9) expression.4. GSK-3β si RNA attenuated the inhibition of Bcl-2 protein expression, prevented Bax from the cytoplasm into the mitochondria induced by H/R(vs. H/R group, P<0.05).Conclusion: 1.The phosphorylation of mitochondrial GSK-3β Ser9 plays an important role in the FGF-2’s fighting against myocardial apoptosis.