Abstract
Previous studies have demonstrated that β2-adrenergic receptors (β2ARs) can be phosphorylated by G protein-coupled receptor kinases (GRKs) and protein kinase A (PKA), affecting β2AR internalization and desensitization. However, the exact physiological function of β2ARs in cardiomyocytes is unknown. In this study, we showed that neonatal mouse cardiomyocytes had different contraction and internalization responses to sustained or repeated, transient agonist stimulation. Specifically, short-time stimulation (10 min) with epinephrine or norepinephrine increased the cardiomyocyte contraction rate, reaching a maximum at 5 min, followed by a slow decline. When the agonist was re-added after a 60-min wash-out period, the increase in the cardiomyocyte contraction rate was similar to the initial response. In contrast, when cardiomyocytes were exposed continuously to epinephrine or norepinephrine for 60 min, the second agonist stimulation did not increase the contraction response. These results indicated that continuous β2AR stimulation caused functional desensitization. Phosphorylation of β2ARs at serine (Ser)355/356 GRK phosphorylation sites, but not at Ser345/346 PKA phosphorylation sites increased with continuous epinephrine stimulation for 60 min. Accordingly, β2AR internalization increased. Interestingly, β2AR internalization was blocked by mutations at the GRK phosphorylation sites, but not by mutations at the PKA phosphorylation sites. Furthermore, inhibition of β2AR dephosphorylation by okadaic acid, a phosphatase 2A inhibitor, impaired the recovery of internalized β2ARs and reduced the cardiomyocyte contraction rate in response to epinephrine. Finally, epinephrine treatment induced the physical interaction of β-arrestin with internalized β2ARs in cardiomyocytes. Together, these data revealed the essential role of the Ser355/356 phosphorylation status of β2ARs in regulating receptor internalization and physiological resensitization in neonatal cardiomyocytes to contraction functions.
Highlights
When the agonist was removed for 60 min and re-added at the same concentration, the increase in the cardiomyocyte contraction rate was similar to the initial response (Fig 1A and 1C)
When the cells were exposed continuously to Epi or NE for 60 min beyond the initial 10-min stimulation, a second agonist stimulation did not increase the contraction response and, weakened the spontaneous contractions to below the basal level (Fig 1B and 1D). These results suggested that continuous β2-adrenergic receptors (β2ARs) stimulation caused functional desensitization of cardiac myocytes, which prevented contraction rate increases in response to a second agonist challenge
We report the functional resensitization of β2ARs as assessed by a neonatal cardiomyocyte contraction assay in vitro, as well as the receptor internalization response under repeated agonist stimulation
Summary
We found that the Ser355/356 phosphorylation sites on β2ARs have a crucial role in regulating β2AR internalization and resensitization in neonatal mouse cardiomyocytes. For measurements of the effect of repeated stimulation with agonist Epi or NE, the contraction response of mouse neonatal cardiomyocytes was recorded in real-time for 40 min. These results suggested that continuous β2AR stimulation caused functional desensitization of cardiac myocytes, which prevented contraction rate increases in response to a second agonist challenge.
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