Abstract Body: Introduction: Cardiomyocyte hypertrophy is regulated by the spatial compartmentalization of G protein-coupled receptor (GPCR) signaling. We previously demonstrated that β1-adrenergic receptors (β1AR) at the Golgi trigger pro-hypertrophic PLCε activity and drive PI4P hydrolysis. In contrast, internalized β2ARs can signal from endosomes through a Gi-dependent mechanism that counteracts this pathway. Although long-acting β2-agonists (LABAs) widely used clinically, it remains unclear how differences in their onset kinetics and membrane interactions influence the duration of this protective endosomal signal.
Hypothesis: We proposed that Salmeterol, through its unique membrane-anchoring and partial agonist properties, promotes a more sustained endosomal β2AR signal compared to full agonists such as Formoterol, leading to persistent inhibition of Golgi-localized PLCε activity.
Methods: To monitor Golgi PI4P dynamics in neonatal rat ventricular myocytes (NRVMs), we used the FAPP-PH-GFP biosensor in live-cell imaging experiments. Cells were pre-treated with Salmeterol or Formoterol (100 nM) followed by Angiotensin II (AngII, 100 nM) stimulation. To confirm the trafficking platform, we performed high-resolution confocal imaging in HEK293 cells expressing FLAG-tagged β2AR together with endosomal markers.
Results: Initial validation in HEK293 cells confirmed robust and rapid β2AR internalization into endosomal compartments following agonist stimulation, supporting the feasibility of intracellular signaling from this location. In NRVMs, AngII treatment promoted a significant loss of the perinuclear PI4P "ring," a hallmark of PLCε activation. In acute time-course experiments (n=3-5 per group), pre-treatment with either Salmeterol or Formoterol significantly attenuated AngII-induced PI4P hydrolysis at 30 minutes, confirming that endosomal β2AR signaling effectively opposes Golgi-localized pro-hypertrophic stimuli. Current experiments are focused on determining the temporal window of this protection to evaluate whether the unique lipophilicity of Salmeterol allows for sustained Golgi PI4P integrity compared to the rapid-acting agonist Formoterol.
Conclusions: In conclusion, these we propose that the duration of β2AR endosomal signaling is a critical determinant of its cardioprotective capacity. Agonists that promote prolonged endosomal engagement may offer a more effective strategy for limiting Golgi-dependent pathological hypertrophic stimuli.