Novel Paradigms Governing β₁-Adrenergic Receptor Trafficking in Primary Adult Rat Cardiac Myocytes

The β₁-adrenergic receptor (β₁-AR) is a major cardiac G protein-coupled receptor, which mediates cardiac actions of catecholamines and is involved in genesis and treatment of numerous cardiovascular disorders. In mammalian cells, catecholamines induce the internalization of the β₁-AR into endosomes and their removal promotes the recycling of the endosomal β₁-AR back to the plasma membrane; however, whether these redistributive processes occur in terminally differentiated cells is unknown. Compartmentalization of the β₁-AR in response to β-agonists and antagonists was determined by confocal microscopy in primary adult rat ventricular myocytes (ARVMs), which are terminally differentiated myocytes with unique structures such as transverse tubules (T-tubules) and contractile sarcomeres. In unstimulated ARVMs, the fluorescently labeled β₁-AR was expressed on the external membrane (the sarcolemma) of cardiomyocytes. Exposing ARVMs to isoproterenol redistributed surface β₁-ARs into small (∼225-250 nm) regularly spaced internal punctate structures that overlapped with puncta stained by Di-8 ANEPPS, a membrane-impermeant T-tubule-specific dye. Replacing the β-agonist with the β-blocker alprenolol, induced the translocation of the wild-type β₁-AR from these punctate structures back to the plasma membrane. This step was dependent on two barcodes, namely, the type-1 PDZ binding motif and serine at position 312 of the β₁-AR, which is phosphorylated by a pool of cAMP-dependent protein kinases anchored at the type-1 PDZ of the β₁-AR. These data show that redistribution of the β₁-AR in ARVMs from internal structures back to the plasma membrane was mediated by a novel sorting mechanism, which might explain unique aspects of cardiac β₁-AR signaling under normal or pathologic conditions.