In vivo Evaluation of Cardiac Functions of SPRED Proteins

SPRED（带有EVH1结构域的芽生相关蛋白）蛋白是促肥厚性Ras/ERK-MAPK信号通路的抑制剂，在心脏中表达。为了研究SPRED的生理功能，我们构建了全局SPRED2敲除（KO）小鼠，并建立了条件心脏特异性KO小鼠。全球SPRED2 KO小鼠显示心脏重量/体重比增加，生存概率降低。在这些小鼠模型中，我们系统地研究了心脏性能。超声心动图测量显示，SPRED2 KO小鼠左心室壁厚度增加，内径相应减小。血流动力学研究表明，SPRED2型ko患者卒中容量增加，射血分数升高，峰值血压上升率增加，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基础心率加快，其特征是TP段缩短，这可能是由起搏器细胞过早去极化引起的。由此产生的正性变时性和正性肌力效应对SPRED2 KO不利，因为心电图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心动过速。在降低脉冲暂停频率时，电生理心房过度起搏引起SPRED2 KO小鼠心律失常，而增加起搏频率引起KO小鼠心房颤动。此外，心房起搏速度为700 bpm时，在整体SPRED2 KOs中，窦房结恢复时间延长，表明窦房结功能受损。心脏裂解物的Western blot分析显示，由于SPRED2 KO心脏中SPRED2介导的Ras抑制丧失，Ras/ERK-MAPK信号上调，排除了相关SPRED1的代偿上调。基于这些初步发现，证实了SPRED1在心脏中的关键作用，并表明这些MAPK抑制剂在心脏肥厚和电传导的发展中发挥重要作用，我们旨在通过条件SPRED1和SPRED2 KO小鼠揭示心脏特异性背景下的潜在分子机制。这些小鼠模型使我们能够研究不同SPRED2异构体的特定和独特的心脏功能，此外，通过SPRED2 KOs与SPRED1 KOs的杂交，我们将能够研究SPRED1对SPRED2缺陷的可能机制补偿。通过有创血流动力学、有意识小鼠的高分辨率长期心电图记录、心内起搏电生理调查、超声心动图、无创血压测量、心脏切片病理分析（心肌细胞直径、纤维化）以及典型心脏肥大和衰竭标志物的生化表征，将再次系统地进行心脏功能和系统分析。

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.