Gene therapy with EphB1 improves cardiac function

Simone-Franziska Glaser (Frankfurt am Main)1, A. Fischer (Frankfurt am Main)1, A. Debes (Frankfurt am Main)1, M. Klangwart (Frankfurt am Main)1, S. Hille (Kiel)2, O. J. Müller (Kiel)3, S. Dimmeler (Frankfurt am Main)1

1Goethe Universität Frankfurt am Main Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration Frankfurt am Main, Deutschland; 2Universitätsklinikum Schleswig-Holstein Kiel, Deutschland; 3Universitätsklinikum Schleswig-Holstein Innere Medizin III mit den Schwerpunkten Kardiologie, Angiologie und internistische Intensivmedizin Kiel, Deutschland


Pathological cardiac hypertrophy is one of the major causes of heart failure and often induced by aortic valve stenosis. Pressure-overload induces remodeling of the cardiac tissue and the vasculature. The hypertrophic response of cardiomyocytes has been extensively studied, however the crosstalk with other cardiac cell types is less well explored. Using single-nuclei RNA sequencing, we identified significant changes in the transcriptome and a strikingly reduced communication in the hypertrophic human heart. Particularly, the communication between endothelial cells and cardiomyocytes was impaired. Within the cohort of significantly dysregulated receptors on cardiomyocytes, the EPH-receptor family exhibited a striking downregulation. We further showed that the expression of EPH-receptor-B1 (EPHB1) is important to maintain cardiac function. Thus, disruption of EphB1 signaling or blocking EphB1 activation was sufficient to induce a hypertrophic and stress response in cardiomyocytes in vitro. To determine whether EphB1 overexpression protects against cardiac hypertrophy, we first used an AAV6 approach to overexpress EphB1 in cardiomyocyte in vitro. EphB1 significantly reduced PE-induced hypertrophy (0.78±0.01-fold, p=0.0002 vs mock). Next, we tested the therapeutic benefit in vivo by using AAV9 particles encoding EphB1 in a mouse model of pressure-overload induced hypertrophy (TAC surgery). Injection of AAV9-EphB1 at day 7 after surgery significantly increased cardiomyocyte EphB1 mRNA levels (17.6±4.2-fold, p=0.001; n=9-10) and improved the ejection fraction two- and four-weeks post-surgery (1.25±0.08 and 1.20±0.07 fold, respectively, p=0.06 vs mock). Additionally, we observed a reduction in cardiomyocyte hypertrophy (-12.4±3.8% vs mock, p=0.02; assessed by WGA staining of tissue sections) and reduced wall thickness (-27.4±5.3%, p=0.07, n=3-4) in AAV9-EphB1 compared to mock-AAV9 injected mice after TAC. AAV9-EphB1 treated mice further showed reduced fibrosis as assessed by Sirius red staining (-22.6±6.8%; n=3-4). Ongoing experiments currently identify the down-stream pathways that mediate EphB1 effects.  
In summary, our data demonstrate that the therapeutic application of Ephb1 gene therapy seven days post-surgery improves cardiac function, and reduces cardiac hypertrophy and fibrosis.  
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