Investigation of Pitx2 signaling targets in the remodeling of the cardiac inflow tract, the conduction system and during cardiac regeneration

• Applicant: Dr. Peter Christian Kahr
• Subject Area: Cardiology, Angiology ; Developmental Biology
• Term: from 2014 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 252779258

Pitx2, a homeobox transcription factor, is a key player in cardiac morphogenesis. Early in embryogenesis, it is the final member of the signaling cascade that orchestrates the left-right asymmetrical development of the heart. The precise localization of the sinus node and the confluence of the pulmonary veins into the left atrium is controlled by Pitx2. In addition, Pitx2 expression continues beyond early embryogenesis and even into postnatal stages. Currently, knowledge of the signaling network that helps Pitx2 to mediate its effects is scarce. Therefore, we are investigating Pitx2 function and networks in 3 experimental settings: In order to investigate the effect of Pitx2 on atrial septation we are planning to investigate mice with conditional knock-out of Pitx2 in a cell lineage that is marked by transcription factor Mef2c, a marker of the anterior second heart field. Thereby, normal function of Pitx2 in the posterior second heart field and extracardiac tissues will be maintained. Because of delays in re-deriving the Mef2cCRE/+ mice, and crossing them with Pitx2flox/flox mice, we have not yet been able to accomplish morphological or molecular studies on these mice. During the next weeks, we will be able to investigate embryos at post-conceptional days 10.5 to 12.5 using suggested strategies. In a second project we are investigating the function of Pitx2 during cardiac regeneration in adult mice. Physiologically, levels of Pitx2 are almost undetectable in the ventricular myocardium of postnatal hearts. Nevertheless, Pitx2 becomes up-regulated after myocardial injury, and Pitx2 knock-out mice demonstrate markedly impaired cardiac regeneration after resection of the ventricular apex. At the same time, we could demonstrate that overexpression of Pitx2 results in reduced scar size after myocardial infarction at adult stages. Together, we have then investigated the molecular alterations that mediate the effects of Pitx2 in myocardial regeneration. Using RNA-seq and ChIP-seq, we found that Pitx2 influences myocardial ROS production in the MI scar area and borderzone. As next steps, we are planning to test Pitx2 interaction with selected candidate genes in vitro and in vivo. Thirdly we are investigating the role of Pitx2 signaling in the cardiac conduction system. Although it is known that Pitx2 regulates the embryonic patterning of the conduction system by inducing or for example node-specific gene programs, our observation of a Pitx2c transgenes activity (Pitx2c-Cre-ERT2) in all structures of the specialized conduction system is a novelty. Besides studies on Pitx2 function in the adult conduction system, we believe that this Cre line would be a very valuable tool for a wide range of studies focusing on the conduction system. Therefore it is our aim to characterize the phenotype of Pitx2cCreERT2/+ mice using morphological and electrophysiological tools and to investigate the contribution of the conduction system to cardiac regeneration.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. James F. Martin