| BackgroundChronic heart failure(CHF) is a common, but complex clinical disease, and reached epidemic proportions in the world. Patients with CHF have a low 5-year survival rate, which is similar to those of malignant tumor. Approximately half of the mortality in patients with CHF are sudden and unexpected, and presumably the consequence of lethal ventricular arrhythmias or cardiac sudden death(SCD). The pathogenesis and development of arrhythmia contributes a large portion of SCD, however, the underlying mechanisms of abnormal electrophysiological basis related multiple kinds of ion channels and sudden death are not clear.3-phosphoinositide-dependent protein kinase-1(PDK1), a key member of the AGC(protein kinase A, protein kinase G and protein kinase C) protein kinase family, acts as an upstream protein kinase by phosphorylating and activating many other AGC-family members, including protein kinase B(PKB)/Akt, p70 ribosomal S6 kinase(S6K), serum and glucocorticoid-induced protein kinase(SGK), and Forkhead box O(Foxo). PDK1 has an established role in regulating physiological processes relevant to metabolism, growth, proliferation and survival. Moreover, PDK1 and its upstream or downstream factors, including phosphatidyl inositol 3-kinase(PI3K), Akt and m TOR, are involved in heart failure and pathologic heart remodeling. Mice with PDK1 deletion have markedly reduced myocardium, smaller cardiomyocytes, thinner ventricles and enlarged atria, which eventually leads to heart failure and sudden death within 11 weeks; however, the underlying mechanisms of abnormal electrophysiological basis related deaths are not clear.In clinical, the specifical PI3 K inhibitor have been successfully utilized in the treatment of a variety of malignant conditions. However, cardiac arrhythmias caused by drug-induced long QT syndrome(LQT) are thought to result from drug side effects. Recently, the involvement of the AGC protein kinase family in regulating arrhythmia has drawn considerable attention. PI3 K signaling is associated with the alteration of ion channel function, which is established to play a role in the development of cardiac arrhythmia. Drug-induced increases in action potential and QT prolongation induce inhibition in multiple ion currents, including peak Na+ current, and are accompanied by decreased PI3 K signaling. Foxo1 transcription factor, one of the major PI3 K, Akt downstream effectors, binds to promoter sequences to regulate the expression of target genes, including the cardiac sodium channel gene(SCN5A). Further more, the SCN5 A mutations that reduce peak sodium current(such as mutations linked to Brugada syndrome), atrioventricular block(AVB) and QTc prolongation in patients with LQT3. Ventricular arrhythmias is the main causes of death in patients with SCN5 A mutations. Thus, the potential relationships between PDK1 signaling through Fox O1 and the function of Nav1.5 in the dysregulation of functions of ion channels and electrophysiological basis need further investigation.To understand the function of PDK1 in sodium channel activation and the dysregulation of electrophysiological basis, we prepared a conditional deletion of PDK1 in mouse cardiomyocytes through Cre-mediated excision. Using this conditional PDK1-deletion mouse, and using neonatal rat cardiomyocytes to exclude the possible impact of heart failure partly, we tested the hypothesis that PDK1 regulates APD and multiple kinds of ion channels activation in cardiomyocytes, thus facilitating the potential mechanism in the development of dysregulation of electrophysiology. Experimental contents Part I Verifications of Conditional PDK1 Knockout MiceObjective: Verifications and investigation of electrophysiology in conditional PDK1 knockout mice.Methods: The deletion of PDK1 in the myocardium was confirmed by Western blot analysis. The heart rate(HR), QRS and QT durations were measured were detected by electrocardiogram(ECG).Results: In western blot analysis, markedly reduced expression of PDK1 expression in hearts from PDK1F/F αMHC-Cre knockout mice. After 8 weeks of deletion, HR was lower in PDK1 knock mice(362.22±12.69 vs. 422.31±20.10, P<0.05), and both the QRS intervals(12.81±0.30 ms vs. 18.93±1.17 ms) and the QTc duration(82.69±4.08 ms vs. 113.91±8.20 ms)were significantly longer in the PDK1-deletion mice than the control mice. Interesting, an abnormal conduction was found in mice with PDK1 deletion at 11 weeks.Conclusion:The PDK1 deletion model of mice was successfully established, and ECG results indicated the association between PDK1 and dysregulated electrophysiology. Part II Isolation Adult Mice CardiomyocytesObjective: To establish an effective and stable way to isolate mice ventricular cardiomyocytes.Methods: ventricular cardiomyocytes were isolated using traditional enzymatic methods. We used the modified Langendorff and modified perfusion solutions for coronary perfusion with type II collagenase.Results: After perfused with type II collagenase, the hearts were palpably flaccid. Next, the ventricles were minced and gently agitated and he free cardiomyocytes were obtained for patch clamp experiments.Conclusion: The modified enzymatic method was effective in isolating mice ventricular cardiomyocytes. Part III The modulation of PDK1 on sodium channel in mice.Objective: PDK1-Fox O1 Pathway activation leads to reduced sodium channel density and expression in PDK1-deletion mice.Methods: In patch clamp experiment of sodium current recording, 8 weeks and 11 weeks PDK1F/F αMHC-Cre mice were used as experimental group, and PDK1F/F mice in the same age were used as control group. Next, we investigated the role of the PDK1-Fox O1 pathway in PDK1 knockout miceResults: Peak values of sodium currents were-23.86±1.10 p A/p F in PDK1F/F αMHC-Cre cells and-36.34±1.45 p A/p F in PDK1F/F cells(P<0.05). Similar results were observed in the 11 weeks olds mice(-24.11±1.23 p A/p F in PDK1F/F αMHC-Cre cells vs.-36.76±2.07 p A/p F, P<0.05) in PDK1F/F cells. These results suggest that the sodium current amplitude is decreased by about 33% in PDK1F/F αMHC-Cre mice. Further more, slight Change in the sodium channel kinetics were found in PDK1F/F αMHC-Cre mice. In western blot analysis, the phosphorylation of Akt(308T) and Foxo1(24T) and the expression of Nav1.5 in the myocardium of PDK1-knockout mice are decreased, while the nuclear localization of Foxo1 is increased.Conclusion: PDK1-deletion caused a decrease in sodium current amplitude that may be explained in part by electrical abnormalities via the PDK1-Foxo1 pathway, thus facilitating the potential mechanism in the development of dysregulation of electrophysiology. Part IV The modulation of PDK1 on sodium channel in neonatal rat cardiomyocytesObjective: To verify a decrease in sodium current amplitude by PDK1 deletion via the PDK1-Foxo1 pathway.Methods: PDK1 inhibitor GSK 2334470, or the Akt inhibitor MK 2206, or the Foxo1 inhibitor AS 1842856, or a combination of inhibitors were applied in cell culture for 48 h. Sodium current, Nav1.5 protein expression and related signal pathway were detected by patch clamp and Western Blot.Results: The amplitude of the current in untreated cells(-36.23±1.25 p A/p F) was suppressed by treatment with either GSK 2334470(-27.52±1.49 p A/p F) or MK 2206(25.48±2.06 p A/p F)(P<0.05), supporting the positive role of PDK1 and Akt. Conversely, the amplitude of sodium current was increased after application of AS 1842856(-42.85±1.51 p A/p F; P <0.05), which is consistent with the negative role of Foxo1. Furthermore, the sodium current had no obvious change when a combination of GSK 2334470 and AS 1842856(-33.41±1.50 p A/p F) or MK 2206 and AS 1842856(-35.09±4.29 p A/p F) were applied. In Western Blot analysis, the expression of Nav1.5 expression in rat cardiomyocytes was assessed after treatment with DMSO, GSK 234470, the Foxo1 inhibitor AS 1842856 or a combination of the two drugs. Nuclear Foxo1 expression in neonatal rat cardiomyocytes was similar to cardiomyocytes isolated from control and PDK1 deletion mice. Conclusion: PDK1-deletion downregulated sodium current amplitude that may be explained by electrical abnormalities via the PDK1-Foxo1 pathway. Part V the role of PDK1 in the dysregulation of electrophysiological basis Objective: To further explore the role of PDK1 in the pathogenesis of dysregulated electrophysiological basis. Methods: Using patch clamping technology, we explored action potential duration in both groups, the induced risk of EAD, and investigated the functions of transient outward potassium channel and L-type Ca2+ channel to explain the abnormal action potential duration. Results: Significant prolongation action potential duration was found in mice with PDK1 deletion. Further, the peak current of transient outward potassium current and L-type Ca2+ current were decreased by 84% and 49% respectively. In addition, dysregulation of channel kinetics lead to action potential duration prolongation further. However, no significant differences of the risk of EAD were detected between two groups. Conclusion: We have demonstrated that PDK1 participates in action potential prolongation in cardiac ablation of PDK1 mice. This effect is likely to be mediated largely through downregulation of transient outward potassium current. |
