CARDEB1 · Role of Calcineurin alternative splicing variant CnAbeta1 in stem cell biology and cardiac development

The heart is the first organ to form and it is essential for blood distribution during embryogenesis. Congenital heart malformations are the most common birth defect in humans, present in 1 % of the human population worldwide. The calcineurin splicing variant CnAbeta1 is expressed in stem cells, precursors and developing tissues and its expression decreases during tissue maturation. We aim to determine the role of CnAbeta1 during different stages of embryonic development. Aims:1. Identify the role of CnAbeta1 in embryonic stem (ES) cell biology:We will knock down CnAbeta1 expression in ES cells using siRNA constructs and study their self-renewal and differentiation capacity. 2. Study embryonic development of transgenic mice in which CnAbeta1 expression is reduced ubiquitously:We will generate transgenic mice in which CnAbeta1 expression will be knocked down ubiquitously by overexpression of a specific CnAbeta1 shRNA. We will identify morphological defects and study target tissues using immunohistochemical and biochemical approaches.3. Determine the role of CnAbeta1 in cardiac development by using conditional knockdown mice in which CnAbeta1 expression is reduced specifically in the heart:We will overexpress CnAbeta1 shRNA specifically in the heart from E8.0 onwards by using Cre/Lox technology. We will study the effects of CnAbeta1 knockdown by immunostaining and biochemical approaches and study cardiac function in the adult heart by using echocardiography and telemetry. Transcriptional and phosphoprotein profiles will be analyzed using DNA and protein microarrays.4. Study the role of CnAbeta1 in the epicardium during embryonic development and in adult heart:We will overexpress CnAbeta1 shRNA specifically in the epicardium using Cre/Lox technology. Mice will be analyzed as in Aim 3. We will study the ability of epicardium-derived cells (EPDC) to migrate and undergo EMT in the absence of CnAbeta1.