EXCEED THE SPACE PROVIDED. Pathologic release of norepinephrine (NE) from cardiac sympathetic nerve endings (cSNE) during myocardial ischemia leads to arrhythmias and death. The mechanisms responsible remain obscure. The overall objective of this grant is to address the important biological issue of whether a local cardiac renin-angiotensin system (RAS) plays a significant role in NE release from cSNE in myocardial ischemia. Our preliminary results demonstrate that cardiac mast cells are a source of extra-renal renin-like protein capable of cleaving angiotensinogen to ANG I. We hypothesize that degranulation of cardiac mast cells in ischemia is pivotal for local RAS activation and ANG II formation, proximal to cardiac SNE.
In Specific Aim I, we will characterize the molecular identity of mast-cell derived renin in SNE isolated from rat, human, wild-type and mast cell deficient mouse hearts, and from the human mast cell line, HMC-1 cells. We will also investigate in ischemic cardiac models from guinea-pigs and humans, whether locally synthesized ANG II acts in a paracrine mode on AT1 receptors (AT1R) to elicit NE release. To confirm the role of mast cells in this cardiac RAS, parallel experiments will be conducted in mast-cell deficient mice. Because ANG II is a known stimulant of the Na+/H + exchanger (NHE),Iwe hypothesize that in ischemia, NHE stimulation by ANG II will lead to an increase in intracellular Na _, thereby triggering excessive NE release via the NE transporter. We will test this hypothesis in isolated cSNE expressing native ANG II receptors, and in human neuroblastoma cells transfected with cloned rat AT1A receptor (SH-SY5Y-AT_A). The signal transduction pathways mediating the effects of ANG II via AT1R on NE release will be studied in Specific Aim II. Pilot experiments demonstrate that ANG II stimulates the PLC-PKC pathway in cSNE, regulating NHE activity and NE release. Hence, we will study the roles of key elements of ANG II receptor-mediated signaling pathways involved in NHE activation and associated NE release. The experiments will focus on PLC, Cai, PKC, and calmodulin. The overall premise for conducting these experiments is that if we understand the physiological basis for the formation of ANG II in the heart, and how ANG II stimulates NE release, then we will also have identified targets that potentially can be exploited for therapies for ischemic arrhythmias and associated cardiac dysfunctions. PERFORMANCE SITE ========================================Section End===========================================
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