Final Report Summary - 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. Another 1 - 2 % of the population carries more subtle malformations that become evident with age. Heart development is exquisitely controlled by different signalling pathways inside the cells. A major player in this intracellular communication is the phosphatase calcineurin.Calcineurin (Cn) is a calcium / calmodulin-dependent serine / threonine phosphatase that activates different transcription factors controlling inflammation, muscle hypertrophy and differentiation, heart development and neural differentiation and function. The Cn enzyme is composed of two subunits: a catalytic A subunit (CnA) and a regulatory B subunit (CnB). Three CnA genes have been described in higher vertebrates: CnAalpha and CnAbeta are ubiquitously expressed, whereas CnAgamma is restricted to brain and testis. CnA isoforms share the same functional domains, including a catalytic domain, a CnB-interacting domain, a calmodulin binding region and an autoinhibitory domain which maintains the enzyme in an inactive conformation in the absence of calcium. Interestingly, an alternative version of CnAbeta can be produced in certain tissues. This alternative version (isoform) of CnAbeta is called CnAbeta1. As a result of intron retention, CnAbeta1 has a unique C-terminal domain that is not only different from the autoinhibitory domain present in all other naturally occurring Cn isoforms but is also different from any other known protein. We had previously observed that CnAbeta1 is expressed in stem cells, progenitor cells and developing tissues and its expression decreases during tissue maturation. In this project, we aimed to determine the role of CnAbeta1 during different stages of embryonic development. We found that CnAbeta1 is accumulated in Golgi-like organelles in the early embryo. To determine the role of CnAbeta1 in embryonic stem (ES) cells, we used small interference RNA that reduces CnAbeta1 expression. Blockade of CnAbeta1 expression in ES cells results in the downregulation of mesoderm differentiation markers followed by a decrease in the expression of cardiomyocyte differention markers. These results suggest that CnAbeta1 is necessary for the correct specification of ES cells towards the mesodermal embryonic lineage and for cardiomyocyte differentiation, at least in vitro.We developed a 'knockdown' transgenic mouse that overexpresses interference RNAs for CnAbeta1 specifically in cardiomyocytes. These mice express less CnAbeta1 in cardiomyocytes than the wild type control mice. We observed that CnAbeta1 knockdown mice showed deregulation of markers normally activated in heart failure, including alpha-skeletal actin and BNP. These results suggest that CnAbeta1 expression is necessary for normal cardiac homeostasis. We have now developed a knockout mouse in which CnAbeta's intron 12-13 has been deleted, such that they don't express CnAbeta1 in any tissue. These mice are currently under investigation and we expect to have results in the near future.To better study the role and mechanism of action of CnAbeta1 in the heart, we developed a transgenic mouse line that overexpresses CnAbeta1 in cardiomyocytes, such that the amount of CnAbeta1 in these cells is about four times that found in wild type mice. These mice grow and breed normally, showing no signs of cardiac hypertrophy. Interestingly, when we induced myocardial infarction, mice overexpressing CnAbeta1 showed improved cardiac function, reduced inflammation and reduced cardiac fibrosis. In addition these mice had a smaller infarct scar and developed less heart remodelling. These results indicate that overexpression of CnAbeta1 may have therapeutical potential for the treatment of myocardial infarction.We then investigated the mechanism of action of CnAbeta1 inside the cell. We found that unlike other Cn isoforms, CnAbeta1 uses its unique C-terminal domain to interact with the mTORC2 complex and activate the Akt signalling pathway and the transcription factor ATF4. This is a known cardioprotective pathway that had not been linked so far with calcineurin.Conclusions- CnAbeta1 is expressed in high amounts in embryonic stem cells, where it controls mesodermal specification and cardiomyocyte differentiation.- CnAbeta1 is necessary for cardiac homeostasis in the adult heart.- CnAbeta1 overexpression improves cardiac function after myocardial infarction, reducing scar formation, inflammation and cardiac fibrosis and therefore has therapeutic potential.
