PDK1调控小鼠心脏的电生理机制研究

发布时间：2018-05-26 00:57

本文选题：3-磷酸肌醇依赖性蛋白激酶1 + 叉头框蛋白O　；参考：《南京医科大学》2015年博士论文

【摘要】：背景慢性心力衰竭(chronic heart failure,CHF)是一种复杂的临床疾病,发病率高,有临床症状患者的5年存活率与恶性肿瘤相仿,这类患者中约一半因恶性室性心律失常的发生而猝死。虽然心律失常是发生心脏性猝死(SCD)的重要原因,与多种离子通道有关,但是其中的潜在的电生理机制仍旧不清楚。3-磷酸肌醇依赖性蛋白激酶1(PDK1)是蛋白激酶家族AGC中的一员,它位于该信号通路的上游,参与调控AGC家族下的多种调节因子,其中包括蛋白激酶B(PKB/Akt),磷酸化核糖体S6蛋白激酶(P70S6K),血清和糖皮质激素诱导的蛋白激酶(SGK)以及叉头框蛋白O(FoxO)等。PDK1参与多种细胞生理的调控,诸如代谢、生长及生存等。此外,目前多个研究发现,该信号通路的多种上游及下游因子参与心脏心力衰竭和病理性重构的发生与发展。例如在小鼠上,当心脏特异性敲除PDK1后,可以发现心肌细胞体积缩小,心脏结构改变以及严重的心力衰竭,且于出生后的11周左右突然死亡。其中是否有心律失常的参与还不清楚。AGC家族蛋白激酶的上游因子磷脂酰肌醇3-激酶(PI3K)的特异性阻断剂在临床上常常用于抗肿瘤的治疗,但是心律失常是其中的副作用之一。最近的研究就发现PI3K特异性阻断剂可以影响多种离子通道,延长动作电位时程(APD)。Fox O1是AGC家族蛋白激酶的主要下游调控因子之一,最近的研究发现它可能是钠通道负向调控因子。而且,钠通道功能的下降与临床上传导阻滞(AVB),3型长QT间期综合征(LQT3)和Brugada综合征(Brs)有关,这类患者的主要死亡原因之一就是心律失常。因此,PDK1作为该信号通路的主要上游因子,是否参与心脏钠通道以及其他离子通道的调控,乃至多种病理情况下心律失常的发生,值得进一步的研究。因此,本研究构建心脏特异性敲除pdk1的小鼠模型,运用全细胞膜片钳和westernblot技术,研究心脏动作电位,多种离子通道之间变化及可能原因,探讨pdk1参与心律失常发生的潜在机制。实验内容1.心脏特异性敲除pdk1小鼠的鉴定与电生理改变的初步探索目的:心脏特异性敲除pdk1小鼠模型的鉴定。方法:采用westernblot方法,检测小鼠心室肌细胞pdk1的表达情况;运用体表心电图检测基因敲除后小鼠心率、qrs、qtc的变化。结果:基因敲除后的8周,心室肌pdk1的表达显著下降。且心电图表现为心率下降(362.22±12.69vs.422.31±20.10,p0.05),qrs(12.81±0.30msvs.18.93±1.17ms)、qtc(82.69±4.08msvs.113.91±8.20ms)时程的延长,11周即出现了一定程度的心脏传导异常。结论:心脏特异性敲除pdk1小鼠模型构建是有效的,且出现了初步的电生理异常。2.小鼠心肌细胞的分离目的:建立稳定的急性分离小鼠心室肌细胞的方法。方法:采用酶解法分离小鼠心室肌细胞,使用改良langendorff装置和灌流液,经主动脉逆行性灌流ii型胶原酶消化心肌,得到单个心室肌细胞。结果:经过ii型胶原酶的消化,在pdk1基因敲除以及正常小鼠上均可获得长杆状,边缘及横纹清晰的存活心室肌细胞。结论:稳定的急性分离小鼠心室肌细胞的方法已构建成功。3.pdk1对小鼠心脏钠通道的调控目的:探索心脏pdk1敲除后对于心室肌细胞钠通道的影响以及可能的机制方法:运用全细胞膜片钳方法,比较pdk1基因敲除后心室肌钠通道电流以及相关动力学的变化。运用westernblot技术,检查pdk1下游信号通路的改变。结果:心脏特异性敲除pdk1后,小鼠心室肌细胞钠电流密度下降(-23.86±1.10pa/pfvs.-36.34±1.45pa/pf,p0.05),且11周大小鼠有着类似的改变(-24.11±1.23pa/pfvs.-36.76±2.07pa/pf,p0.05),但是钠通道动力学仅有轻度变化。westernblot检测发现小鼠pdk1信号通路下游308位点akt磷酸化下降,24位点foxo1磷酸化下降,且细胞核中foxo1表达上升,钠通道蛋白表达下降。结论:pdk1敲除可以通过抑制钠通道蛋白的表达来抑制心室肌细胞钠通道电流的密度,潜在的机制可能是通过调控下游信号通路的磷酸化,通过提高钠通道负向调控因子foxo1在细胞核的表达来调控的。4.pdk1对乳大鼠心室肌钠通道的调控目的:排除pdk1敲除对心功能的影响因素,进一步证实pdk1敲除导致钠电流下调的机制。方法:在培养的乳大鼠心室肌细胞上,运用pdk1特异性阻断剂gsk2334470,akt特异性阻断剂mk2206,foxo1特异性阻断剂as1842856或联合使用,分别运用全细胞膜片钳技术和westernblot技术,观察钠通道电流的变化以及相关信号通路的变化。结果:运用pdk1特异性阻断剂gsk2334470和akt特异性阻断剂mk2206后,乳大鼠心室肌细胞的钠电流密度下降从-36.23±1.25pa/pf分别下降至-27.52±1.49pa/pf和25.48±2.06pa/pf。相反,这种抑制效应可以被foxo1特异性阻断剂所抵消。westernblot实验中运用了pdk1特异性阻断剂和foxo1特异性阻断剂探索了钠通道的表达变化,此外细胞核中foxo1的变化与pdk1敲除的小鼠类似。结论:实验结果进一步说明PDK1可以通过对于下游调控因子的FoxO1的调控来调节心室肌细胞钠通道功能。5.PDK1对小鼠心室肌细胞电生理的影响目的:进一步探索PDK1在心律失常的发生中发挥的作用。方法:运用全细胞膜片钳技术,检测小鼠心室肌细胞动作电位时程的变化,EAD的诱发,以及复极相关主要离子通道的功能变化。结果:心脏特异性敲除PDK1后,小鼠心室肌细胞APD显著延长,L型钙电流(49%)以及Ito电流都显著抑制(89%),但EAD诱发几率和正常小鼠相比无显著差异。结论:PDK1敲除延长动作电位时程主要是通过抑制瞬时外向钾电流来完成的,它可能在心脏心律失常发生发展中发挥了重要作用。
[Abstract]:Background chronic heart failure (chronic heart failure, CHF) is a complex clinical disease with high incidence and 5 year survival rates in patients with clinical symptoms similar to malignant tumors. About half of these patients die of sudden death due to malignant ventricular arrhythmia. Subchannel related, but the potential electrophysiological mechanism remains unclear..3- phosphoric acid inositol dependent protein kinase 1 (PDK1) is a member of the protein kinase family AGC, which is located upstream of the signal pathway and participates in the regulation of a variety of regulatory factors under the AGC family, including protein kinase B (PKB/Akt), phosphorylated ribosome S6 protein kinase (P). 70S6K), serum and glucocorticoid induced protein kinase (SGK) and fork head frame protein O (FoxO), such as.PDK1, are involved in a variety of cell physiological regulation, such as metabolism, growth and survival. In addition, a number of studies have found that a variety of upstream and downstream signals of the signal pathway participate in the development and development of heart failure and pathological remodeling. For example, in mice, when cardiac specific knockout of PDK1, myocardial cell volume, cardiac structural changes, and severe heart failure can be found and suddenly died around 11 weeks after birth. The involvement of arrhythmia is not clear about the specificity of the upstream factor of the.AGC family protein kinase, phosphatidylinositol 3- kinase (PI3K) Sexual blockers are often used in clinical antitumor treatment, but arrhythmia is one of the side effects. Recent studies have found that PI3K specific blockers can affect a variety of ion channels, prolonging action potential time (APD).Fox O1 is one of the main downstream regulators of AGC family protein kinase. Recent studies have found that it is possible It can be a negative regulator of sodium channel. Moreover, the decrease of sodium channel function is associated with clinical conduction block (AVB), type 3 long QT interval syndrome (LQT3) and Brugada syndrome (Brs). One of the main causes of death in these patients is arrhythmia. Therefore, PDK1 is the main upstream factor of the signal pathway, whether it is involved in the heart sodium channel. The regulation of the other ion channels and the occurrence of arrhythmia in a variety of pathological conditions is worth further study. Therefore, this study constructs a mouse model of cardiac specific knockout PDK1, using whole cell patch clamp and Westernblot technique to study the cardiac action potential, the changes of multiple ion channels and possible causes, and discuss the PDK1 parameter. Potential mechanisms for the occurrence of arrhythmias. Experimental content 1. identification of PDK1 mice with cardiac specific knockout and preliminary exploration of electrophysiological changes: identification of cardiac specific knockout PDK1 mouse models. Methods: Westernblot method was used to detect the expression of PDK1 in ventricular myocytes of mice; the detection of gene knockout with body surface electrocardiogram was used to detect gene knockout. The changes of heart rate, QRS, QTc in mice. Results: the expression of PDK1 in ventricular muscle decreased significantly at 8 weeks after knockout, and the electrocardiogram showed a decrease of heart rate (362.22 + 12.69vs.422.31 + 20.10, P0.05), QRS (12.81 + 0.30msvs.18.93 + 1.17ms), QTc (82.69 + 4.08msvs.113.91 + 8.20ms) duration, and a certain degree of cardiac conduction in the 11 week. Conclusion: the construction of cardiac specific knockout PDK1 mouse model is effective, and the primary electrophysiological abnormalities of.2. mouse cardiomyocytes are isolated: a stable and acute method for acute isolation of murine ventricular myocytes is established. Method: the isolated mouse ventricular myocytes were separated by enzymatic method, and the improved Langendorff device and perfusion fluid were used. Pulse retrograde perfusion II collagenase digested the myocardium and obtained single ventricular myocytes. Results: through the digestion of II collagenase, the survival of the ventricular myocytes with long rod-shaped, marginal and transverse lines could be obtained on PDK1 gene knockout and normal mice. Conclusion: a stable and acute method of separating ventricular myocytes from mice has been constructed successfully.3.pdk1 The aim of the control of sodium channel in the heart of mice: To explore the effect of PDK1 knockout on the sodium channel of ventricular myocytes and the possible mechanism: using the whole cell patch clamp method to compare the changes of ventricular sodium channel current and related kinetics after PDK1 knockout. Westernblot technique was used to examine the downstream signal pathway of the PDK1. Results: after the cardiac specific knockout of PDK1, the sodium current density of ventricular myocytes in mice decreased (-23.86 + 1.10pa/pfvs.-36.34 + 1.45pa/pf, P0.05), and the 11 weeks of mice had similar changes (-24.11 + 1.23pa/pfvs.-36.76 + 2.07pa/pf, P0.05), but the sodium channel dynamics only slightly changed.Westernblot to detect the PDK1 signal of mice. Akt phosphorylation at the 308 loci of the channel decreased, the 24 site FoxO1 phosphorylation decreased, and the expression of FoxO1 in the nucleus increased, and the expression of sodium channel protein decreased. Conclusion: PDK1 knockout can inhibit the density of sodium channel current in ventricular myocytes by inhibiting the expression of sodium channel protein. The potential mechanism may be through the regulation of phosphorus in the downstream signal pathway. Acidification, by improving the regulation of.4.pdk1 on the sodium channel of ventricular myocardium in milk rats by improving the expression of the negative control factor FoxO1 in the nucleus: exclude the influence factors of PDK1 knockout on the cardiac function, and further confirm the mechanism of the downregulation of sodium current caused by PDK1 knockout. Method: the use of PDK1 in cultured neonatal rat ventricular myocytes. Heterosexual blockers gsk2334470, Akt specific blocker mk2206, FoxO1 specific blocker as1842856 or combined use. Whole cell patch clamp technique and Westernblot technology were used to observe changes in sodium channel current and related signal pathways. Results: PDK1 specific blocker gsk2334470 and Akt specific blocker mk2206 The decrease of sodium current density in ventricular myocytes of milk rats decreased from -36.23 + 1.25pa/pf to -27.52 + 1.49pa/pf and 25.48 + 2.06pa/pf., respectively. This inhibitory effect could be counteracted by FoxO1 specific blockers in.Westernblot experiments and the PDK1 specific blockers and FoxO1 specific blockers were used to explore the expression of sodium channels. In addition, the change of FoxO1 in the nucleus is similar to that of PDK1 knockout mice. Conclusion: the experimental results further indicate that PDK1 can regulate the effect of.5.PDK1 on ventricular myocyte electrophysiology in ventricular myocytes by regulating the FoxO1 of the downstream regulatory factors in order to further explore the occurrence of PDK1 in the occurrence of arrhythmia. Methods: whole cell patch clamp technique was used to detect the changes of action potential time of ventricular myocytes in mice, the induction of EAD and the function changes of major ion channels related to repolarization. Results: after the specific knockout of PDK1, the APD of ventricular myocytes in mice was significantly longer, L type calcium current (49%) and Ito current were significantly inhibited (89%). However, there is no significant difference in EAD induced probability compared with normal mice. Conclusion: PDK1 knockout prolongs the action potential mainly by inhibiting transient outward potassium current, which may play an important role in the development of cardiac arrhythmias.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R541.6

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