Compartmentalized Gαq-Signaling in Adult Cardiac Myocytes

Abstract

Gαq-coupled G-protein receptors (Gq-receptors), including α1-adrenergic and angiotensin receptors (α1-AR, AT-R), activate cardiac signaling pathways controlling hypertrophy, cell survival/death, and inotropy. Early studies in cell and animal models established the convention that Gq-signaling exacerbates heart failure (HF). However, clinically, AT-R antagonists improve HF outcomes, whereas α1-AR antagonists worsen HF, suggesting Gq-receptors are functionally unique. Our work indicates that α1-ARs are unique cardiac Gq-receptors by inducing cardioprotection and localizing to the nucleus in adult cardiac myocytes, suggesting that subcellular localization might form the mechanistic basis for unique Gq-receptor function. Here, we test the hypothesis that α1-ARs and AT-Rs localize to and initiate proximal signaling in distinct subcellular compartments. Using a novel fluorescent α1-ligand that improves the dynamics of defining receptor localization, we detected α1-ARs only at the nucleus in adult cardiac myocytes and isolated nuclei. Conversely, using a fluorescent AT-R ligand, we detected AT-Rs at the sarcolemma and the t-tubules. To measure proximal Gq-receptor signaling, we employed a fluorescent probe, GFP-PHD, that binds phosphatidylinositol-4,5-bisphosphate (PIP2) in the membrane and is released when phospholipase Cβ (PLCβ) cleaves PIP2. In adult cardiac myocytes, GFP-PHD localized to the sarcolemma and t-tubules, but not the nucleus, consistent with previous reports. Angiotensin, but not phenylephrine, induced movement of GFP-PHD off of the sarcolemma and t-tubules. The failure to detect GFP-PHD at the nucleus indicates that either PIP2 is excluded from nuclear membranes or that the probe fails to target the nucleus. Addition of a nuclear localization sequence to GFP-PHD (NLS-GFP-PHD) produced nuclear localization of GFP-PHD suggesting the presence of PIP2 in nuclear membranes in adult cardiac myocytes. Phenylephrine, and to a lesser extent, angiotensin induced movement of NLS-GFP-PHD off the nuclear membrane. In summary, our data demonstrate that α1-ARs and AT-Rs localize to and initiate proximal signaling in unique subcellular compartments.