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In vivo Evaluation of Cardiac Functions of SPRED Proteins

SPRED 蛋白心脏功能的体内评估

基本信息

批准号：
247435065
负责人：
Professor Dr. Kai Schuh
金额：
--
依托单位：
Lehrstuhl für Physiologie I - Schwerpunkt vegetative Physiologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2016-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/247435065?language=en
关键词：
vivo Evaluation Cardiac Functions SPRED

项目摘要

SPRED (Sprouty-related proteins with an EVH1 domain) proteins are inhibitors of the pro-hypertrophic Ras/ERK-MAPK signaling pathway and are expressed in the heart. To investigate physiological SPRED functions, we generated global SPRED2 knockout (KO) mice and establish conditional heart-specific KO mice. Global SPRED2 KO mice show an increased heart weight/body weight ratio and a reduced survival probability. In these mouse models, we systematically investigate cardiac performance. Echocardiographic measurements revealed increased wall thickness and, accordingly, decreased inner diameters of the left ventricle of SPRED2 KO mice. Hemodynamic studies demonstrated an increased stroke volume, an elevated ejection fraction and an augmented peak rate of pressure rise and, therefore, also an increase in cardiac output and index in SPRED2 KOs. In line with the cardiac output increase, high resolution ECGs showed an accelerated basal heart rate characterized by a shortened TP segment, which is likely caused by premature depolarization of pacemaker cells. The resulting positive chronotropic and inotropic effect is not beneficial for the SPRED2 KO, since the ECG recordings also demonstrated various forms of spontaneous arrhythmias, e.g. different forms of AV blocks and extrasystoles. Electrophysiological overdrive pacing of atria at decreasing pulse pause frequencies provoked arrhythmias in SPRED2 KO mice and increasing pacing rates elicited atrial fibrillation in KO mice. Furthermore, atrial pacing at a rate of 700 bpm demonstrated impaired sinus node function by prolonged sinus node recovery times in global SPRED2 KOs. Western blot analysis of heart lysates revealed an up-regulated Ras/ERK-MAPK signaling due to the loss of SPRED-mediated Ras inhibition in SPRED2 KO hearts and excluded a compensatory up-regulation of the related SPRED1. Based on these preliminary findings confirming the pivotal role of SPREDs in the heart and indicating an important role of these MAPK inhibitors in the development of cardiac hypertrophy and electrical conduction, we aim to unravel the underlying molecular mechanisms in a cardiac-specific background using conditional SPRED1 and SPRED2 KO mice. These mouse models enable us to investigate both the specific and distinct cardiac functions of the different SPRED isoforms and, furthermore, by breeding of SPRED2 KOs with SPRED1 KOs, we will be able to examine also a possible mechanistic compensation of SPRED2 deficiency by SPRED1. Functional and systematic analysis of cardiac performance will again be carried out systematically by invasive hemodynamics, high-resolution long-term ECG recordings in conscious mice, electrophysiological investigations by intracardiac pacing, echocardiography, non-invasive blood pressure measurements, pathological analysis of cardiac sections (diameter of cardiomyocytes, fibrosis), and biochemical characterization of typical markers of cardiac hypertrophy and failure.

SPRED(带有EVH1结构域的Sprouty相关蛋白)蛋白是促肥大的RAS/ERK-MAPK信号通路的抑制剂，在心脏中表达。为了研究生理刺激功能，我们建立了全局SPRED2基因敲除(KO)小鼠，并建立了条件性心脏特异性KO小鼠。全球SPRED2 KO小鼠的心脏重量/体重比增加，存活概率降低。在这些小鼠模型中，我们系统地研究心脏性能。超声心动图测量显示，SPRED2KO小鼠的左心室壁厚度增加，相应地减少了内径。血流动力学研究显示SPRED2KO患者每搏输出量增加，射血分数增加，压力升高峰值速度增大，因此心输出量和指数也增加。随着心输出量的增加，高分辨率心电图显示以TP段缩短为特征的基础心率加快，这可能是起搏细胞过早去极化所致。由此产生的正性变时性和变力性效应对SPRED2KO是不利的，因为心电记录也显示了各种形式的自发性心律失常，例如不同形式的房室传导阻滞和早搏。在SPRED2 KO小鼠中，以较低的脉冲停顿频率进行电生理超速起搏可引起心律失常，而较高的起搏频率则可引起KO小鼠的心房颤动。此外，在SPRED2KO中，以700次/分的频率心房起搏显示出窦房结功能受损，因为窦房结恢复时间延长。心脏裂解产物的Western印迹分析显示，由于SPRED2 KO心脏失去了spred介导的RAS抑制，Ras/ERK-MAPK信号上调，并排除了相关SPRED1的代偿性上调。基于这些初步的发现，证实了SPRED在心脏中的关键作用，并表明这些MAPK抑制剂在心肌肥大和电传导的发展中发挥了重要作用，我们的目标是在心脏特有的背景下，利用条件SPRED 1和SPRED2 KO小鼠来揭示潜在的分子机制。这些小鼠模型使我们能够研究不同SPRED2亚型的特定和不同的心脏功能，此外，通过培育带有SPRED1KO的SPRED2 KO，我们还将能够研究SPRED2缺陷的可能的机制补偿。对心脏性能的功能和系统分析将再次通过有创血流动力学、清醒小鼠的高分辨率长期心电记录、心内起搏的电生理调查、超声心动图、无创血压测量、心脏切片的病理分析(心肌细胞直径、纤维化)以及心肌肥大和衰竭的典型标志物的生化特征来系统地进行。