Calcineurin is a calcium-activated protein phosphatase that is both necessary and sufficient for the development of a cardiac hypertrophic phenotype. In mammals, calcineurin-dependent dephosphorylation of nuclear factor of activated T cells (NFAT) transcription factors lead to the induction of hypertrophic genes. In contrast, Drosophila do not contain calcineurin-regulated isoforms of NFAT suggesting the idea that NFAT-independent mechanisms exist. Indeed, another transcription factor, myocyte enhancer factor 2 (MEF2), has also been implicated in calcineurin-mediated cardiac hypertrophy. In this competing renewal we propose to carry out a deficiency and EMS mutagenesis screen in Drosophila to identify novel NFAT-independent enhancer and suppressor genes of the calcineurin signaling pathway. To accomplish this goal we will use information already available from screens performed by Sullivan and Rubin and Gajewski and Schulz who have identified large regions of fly genome that act as suppressor and enhancer loci in a Drosophila screen sensitized by constitutive active calcineurin (CanAAct). Accordingly, we hypothesize that calcineurin-mediated cardiac hypertrophy can develop through an NFAT-independent pathway, and that a genetic screen in the fly will identify new molecular mechanisms that induce cardiac hypertrophy. We propose the following specific aims:
Aim 1. Identify novel Suppressor genes of Calcineurin-induced hypertrophic signaling in Drosophila. NFAT-independent calcineurin signaling genes will be identified by a deficiency screen of previously mapped suppressor loci using a sensitized fly expressing cardiac-specific CanAAct.
Aim 2. Identify novel Enhancer genes of Calcineurin-induced hypertrophic signaling in Drosophila. NFAT-independent calcineurin signaling genes will be identified with by a deficiency screen of previously mapped enhancer loci using a sensitized fly expressing cardiac-specific CanAAct.
Aim 3. Identify novel genetic modifiers of Calcineurin-induced hypertrophic signaling induced by EMS mutagenesis in Drosophila. EMS chemical mutagenesis screen to induce mutations across the entire Drosophila genome in a sensitized fly expressing actin- CanAAct. We anticipate that the proposed investigation will identify novel genes and pathways that lead to cardiac hypertrophy and will better define the role of calcineurin in the hypertrophic process. Furthermore, the proposed studies will lead to new targets to treat cardiac hypertrophy and heart failure in humans.
The proposed experiments will expand our knowledge of the molecular mechanisms of cardiac hypertrophy, which we believe will lead to new targets for the identification and treatment of patients with heart failure. Calcineurin is a nodal signaling pathway for cardiac hypertrophy. Simpler model systems, such as Drosophila, provide the unique ability to perform genetic screens to identify genetic modifiers of the Calcineurin pathway that otherwise would be very difficult to do in mammalian systems.
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