In vivo Evaluation of Cardiac Functions of SPRED Proteins

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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.