Specific Aims This is top down project that use genetically modified animals to test and understand how the regulatory loops with the cardiac myocyte can regulate gene expression to induce hypertrophy during heart failure. The design of the project with the genetically modified animals and the multi-scale model allows us to understand the mechanism of action of the b-blockers in altering regulatory loop organization and thus affecting hypertrophy. Heart failure is a complex process that is part regulated by sympathetic innervation (1). One component of heart failure is remodeling of the ventricles that contributes to the overall dysfunction. Sustained sympathetic activity results in p-adrenergic stimulation of the heart cells triggering many functional changes, including abnormal calcium recycling in the SR and induction of the fetal gene program. Studies in the Marks laboratory have shown that protein kinase A can phosphorylate the Ryanodyine receptor, which results in a calcium leak from the SR (2, 3). More recent studies have shown that PDE4D, a cAMP phosphodiesterase that is regulated by protein kinase A phosphorylation, has an important role in heart failure since PDE4D knock-out mice display a propensity for heart failure (4). This observation in animals agrees well with what is known in humans, where heart failure is characterized by a chronic hyperadrenergic state in which elevated levels of circulating catecholamines contribute to the many processes that lead to hypertrophy. This is the reason antagonists at the p-adrenergic receptors (p-blockers) can often be used therapeutically. Some of the molecular aspects of how the cAMP signal is propagated down stream have been controversial (5), data from several groups converge to show that spontaneous calcium release can occur and calcium levels may be increased (6). Prolonged increases of intracellular calcium at low levels can activate many lipid metabolizing enzymes such as phospholipase C and phospholipase A2 (7, 8). Additionally, Ca2+ through protein kinase C can regulate the shuttling of the histone-deacetylase, HDAC5, between the cytoplasm and the nucleus. Induction of the fetal gene program is crucially dependent on HDAC5 function in the nucleus (9, 10) that in turn is dependent on the shuttling events. Thus understanding the dynamics of regulated shuttling of HDAC5 is likely to be vital to understanding how the acute effects of calcium dysfunction in arrhythmias is connected to longer term effects such as hypertrophy and tissue remodeling. To address this question it is essential we define the regulatory loops (network motifs) induced by padrenergic stimulation of myocytes and the calcium leak, and study how the function of the regulatory loops change with the presence of a p-blocker. To define how the these regulatory loops that function in the cytoplasm can induce the long-term changes associated with hypertrophy that contribute to heat failure it is essential to study the dynamics of information transfer from cell body to the nucleus. In this project, the focus will be on understanding the dynamics of HDAC5 distribution between the nucleus and cytoplasm. HDAC5 dynamics is likely to be controlled by the regulatory loops that regulate its phosphorylation state. To address this issue, at the experimental level we will use control, PDE4D knock-out mice and mutant ryanodine receptor knock-in mice as model systems to conduct multivariable experiments to develop and validate a network such as that shown in Figure 1. For the development of predictive models, the stochastic reactiondiffusion method developed by Isaacson and Peskin (11) is ideally suited to model HDAC dynamics. This proposal will build on these experimental and mathematical modeling synergies to develop integrated analytical systems consisting of a integrated set of network models, differential equation models and 3D models along with multivariable experiments. The system will be used to examine how the presence and absence of the PKA-PDE4 feedback loop regulates local increases in PKA activity near the SR. Also, we will determine if this upstream regulatory loop can evoke sustained low-level calcium release that results in the formation of more downstream regulatory loops. These downstream regulatory loops may lead to the cytoplasmic trapping of HDAC5 resulting in the activation of transcription factors NKx2.5 and MEF2 among others, and thus inducing the fetal gene expression program. We will then test how (3-blockers can reconfigure pre-formed regulatory loops to block the maintenance of the fetal gene program required for cardiac growth and remodeling.
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