Heart failure (HF) affects an estimated 4.7 million Americans, with approximately 550,000 new cases diagnosed annually and estimated annual costs ranging from $10 to $40 billion. One of the characteristics of progression to heart failure i reduced cardiac output due to decreased contractility and cardiac hypertrophy. We have discovered novel and exciting roles for phospholipase C (PLC) scaffolding in regulation of cardiac function and disease. Our overall model is that PLCs localized in different compartments in the cell cooperate to generate signals required for cardiac contraction and hypertrophy. We have shown that PLCe scaffolded at the nuclear envelope (NE) hydrolyses the novel substrate phosphatidylinositol 4-phosphate (PI4P) at the Golgi apparatus to generate local diacylglycerol (DAG) required for activation of PKD in the nucleus and activation of hypertrophic gene expression. Our major focus will be to understand this mechanism at a more detailed level and to understand how compartmentalized PLCs specify different aspects of cardiac cell function in the following specific aims.
Aim 1. Subcellular compartmentation of PLCe in cardiac cells. We propose that scaffolding of PLCe to either the type 2 ryanodine receptor (Ryr2) or mAKAP specifies distinct subcellular functions in cardiac cells. We have shown that PLCe binds to mAKAP and have extensively characterized this complex. Here we will examine the scaffolding to Ryr2.
Aim 2. Subcellular Signaling pathway networks that control PLC-dependent cardiac hypertrophy. Here we will identify the mechanisms involved in communicating signals from cell surface GPCRs to perinuclear PI4P hydrolysis, and we will identify GPCR driven pathways that cooperate with perinuclear PLCe to regulate hypertrophy. I. We propose that signals generated at the plasma membrane cooperate with perinuclear PI4P hydrolysis. II. To examine signals that regulate nuclear we will monitor perinuclear PI4P hydrolysis, PKD activation and myocyte hypertrophy in the presence of various targeted G protein inhibitors. III. We will examine the role of Golgi and PM G?y signaling in an in vivo model of heart failure.
Aim 3. Understanding how Nuclear Envelope-scaffolded PLCe accesses PI4P in the Golgi Apparatus. PLCe bound to mAKAP at the nuclear envelope hydrolyzes PI4P in the closely associated Golgi apparatus in cardiac myocytes. We will use a combination of high resolution transmission electron microscopy and super resolution Stochastic Optical Reconstruction microscopy (STORM) to localize the key components in primary myocytes and heart tissue. We will use both STORM imaging and Immuno-localization EM to localize these components. Understanding the topological organization of these components at the cis-Golgi-nuclear envelope interface to develop a detailed model of how PLCe at the nuclear envelope hydrolyzes Golgi PI4P to lead to nuclear PKD activation.
Heart failure (HF) affects an estimated 4.7 million Americans, with approximately 550,000 new cases diagnosed annually and estimated annual costs ranging from $10 to $40 billion. One of the characteristics of progression to heart failure is reduced cardiac output due to decreased contractility and cardiac hypertrophy. The proposed experiments to understand new roles for phospholipase C in the heart will address fundamental mechanisms of heart failure and function that could lead to the development of novel therapies for heart failure.
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