Internalized β2-Adrenergic Receptors Inhibit Subcellular Phospholipase C-Dependent Cardiac Hypertrophic Signaling
Authors: Wei Wenhui , Smrcka Alan 
Published: May 2024

Chronic overstimulation of Gs-coupled b-adrenergic receptor (b-AR) signaling by catecholamines, induces pathological cardiac hypertrophy and ultimately heart failure. b1-ARs and b2-ARs are the two major subtypes of b-ARs present in the human heart and mediate the activity of sympathetic nervous system. It is known that b1-ARs and b2-ARs elicit significantly different or even opposite effects on cardiac functions such as contractility and heart failure, though they share overall sequence and structural homology, and both stimulate cAMP production. The molecular mechanisms underlying these distinct biological effects have not been fully elucidated. Previously, our laboratory demonstrated that a cAMP/EPAC/phospholipase Ce (PLCe)-mediated signaling pathway at the Golgi apparatus is important for regulation of cardiac hypertrophy. We also showed that this pathway is selectively stimulated by activation of b1-ARs localized at Golgi apparatus but not those at the plasma membrane (PM). Access to intracellular b1-ARs by catecholamines was mediated by the organic cation transporters (OCTs), and blockade of OCT3 inhibited catecholamine stimulated cardiomyocyte hypertrophy. In my current study using adult ventricular cardiac myocytes, I found that the activation of endosomal b2-ARs opposes stimulation of the prohypertrophic EPAC/PLCe pathway at the Golgi apparatus by either Angiotensin II (ATII), or the activation of Golgi localized b1-ARs by Dobutamine (Dob). b2-AR-dependent inhibition is at the level of PLCe since activation of b2-AR with salmeterol (Sal) blocks direct activation of EPAC/PLCe by the EPAC selective cAMP analog 8-(4-chlorophenylthio)-2′- O-methyl-cAMP (CPTOMe)-AM, and blocks PLCe activation by ATII which activates PLCe through a pathway that does not rely on EPAC. Blockade of internalization of b2-ARs with Dyngo-4A abolished this inhibitory signaling. Downstream of b2-ARs, I found that b2-AR-dependent PLCe inhibition is not mediated by PKA. Rather, blockade of Gi with PTX, or Gbg with gallein, inhibited the ability of b2-ARs to block Golgi PLCe activity. Additionally, inhibition of ERK activation by PD0325901 abolished b2-AR-mediated inhibition of Golgi PLCe activity. This supports a model where b2ARs internalized from the plasma membrane activate Gi releasing Gbg subunits leading to ERK activation and inhibition of PI hydrolysis at the Golgi apparatus, thereby inhibiting hypertrophic signaling by PLCe. In addition, we also showed PKD and HDAC phosphorylation downstream of PLCe signaling are significantly attenuated by b2ARs activation in mouse hearts. Consistent with the results from mice, b2ARs activation by Sal blunted cellular hypertrophy induced by b1-ARs activation by Dob in vitro measured by cell area and the expression of the hypertrophic marker, atrial natriuretic factor (ANF). Importantly, inhibition endosomal Gbg using a highly selective Gbg inhibitor (GRK2CT) fused to an endosomal targeting sequence (FYVE domain) prevent Sal mediated protective signaling in hypertrophy measurements in vitro. This indicates the inhibitory signaling downstream of b2ARs is indeed driven by endosomal Gbg. This study reveals a novel potential mechanism for b2-AR antagonism of the Epac/PLCe pathway that may contribute to the known protective effects of b2AR signaling on the development of heart failure. Elucidation of a mechanism for the anti-hypertrophic versus hypertrophic signaling balance from b1-ARs and b2-ARs gives critical insights into the development of new strategies for treatment of heart failure by targeting bAR subtypes. This work is supported by grant R35 GM127303-01 (A.V.S.). Preprint: https://www.biorxiv.org/content/10.1101/2023.06.07.544153v1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.