Age Increases Expression and Receptor-Mediated Activation of G[alpha]i in Human Atria

Recently, we demonstrated that β₂AR and several other Gα_s-coupled receptors in human atria also couple to Gα_i, a G protein that inhibits adenylyl cyclase (AC). The present study was undertaken to determine whether age increases expression of Gα_i in human atrium, and more specifically whether it results in an increase in receptor-mediated activation of Gα_i. Right atrial appendages were obtained from 14 mature adult (40-55 years) and 14 elderly (71-79 years) patients undergoing cardiac surgery. Immunoblotting of atrial membranes indicates that elderly atria have 82 ± 18% more Gα_i2 than atria from mature adults (P < 0.002); this increase in Gα_i with age is confirmed by pertussis toxin-catalyzed ADP-ribosylation as well as by photoaffinity labeling with [³²P]azidoanilido-GTP. We also find that receptor-mediated activation of Gα_i is greater in elderly atria and that both basal and receptor-mediated AC activities decrease in elderly atria. These decreases in AC activity can be reversed by disabling Gα_i with pertussis toxin, indicating that the age-dependent increases in Gα_i expression and activation have functional consequences. Because β₂ARs in human atria mediate contractility through cAMP-mediated phosphorylation of phospholamban, we conclude that an age-induced increase in Gα_i may have a role in depressing cardiac function in aged human atria.

Cardiac function in both humans and rodents decreases with age for reasons that are not yet clear. A role in this decrease may be played by alterations in signaling through G protein-coupled receptors (GPCRs): a class of receptors that includes β-adrenergic receptors (βARs) and that is important to cardiac function. 1 Age-dependent decreases in βAR responsiveness have been well documented in both humans and rodents. 2-8 In humans, the βAR agonist isoproterenol produces much smaller increases in heart rate, ejection fraction, and cardiac output in older healthy subjects than in younger healthy subjects. 9,10

At the molecular level, β-adrenergic signaling involves many proteins, including Gα_s, Gα_i, adenylyl cyclase (AC), protein kinase A, and G protein-coupled receptor kinases. 11 Although age-induced alterations in βAR signaling may result from changes in any of these molecules, we have focused on the inhibitory G protein Gα_i, a protein that mediates signaling through inhibitory receptors (such as muscarinic acetylcholine and adenosine receptors) that counter βAR signaling in heart. Moreover, Gα_i recently has been implicated in signaling through several cardiac Gα_s-coupled receptors that control cardiac contractility, including β₂-adrenergic, histaminergic, and glucagon receptors. 12 Finally, Gα_i levels are elevated in congestive heart failure, 13 which, like the aged heart, is marked by decreases in cardiac output, as well as decreased contractile response to agonists of βARs, vasoactive intestinal peptide receptors, and histamine receptors. 14

Although the effect of age on cardiac Gα_i has been extensively studied in rodents, the data have been inconsistent, even between studies in the same strain of rat. Some studies indicate an increase in Gα_i, whereas others report no change; the reasons for this variation are unclear. 6,8,15-20 Our recent studies in Fisher 344 rats demonstrate an age-induced increase in Gα_i, which causes a decrease in receptor-mediated AC activity. 21 These findings raise the question of whether a similar situation exists in human heart. The determination of age effects on Gα_i in human hearts is of increased importance because β₂ARs in human hearts mediate contractility via the AC/cAMP pathway, 22-24 whereas cardiac β₂ARs in rats, 25-28 cats, 29 sheep, 30 and dogs, 31 produce contractility via cAMP-independent pathways.

In humans, there have been only two studies on the effect of age on Gα_i levels, with inconsistent results. Brodde et al examined Gα_i levels in atrial appendages taken from surgical patients 3; they compared Gα_i expression in atrial samples from three groups-two younger groups (mean ages of 3.7 years and 37.9 years) and one older group (over the age of 50 years)-and showed that Gα_i levels increased with age. White et al examined Gα_i expression in ventricles from hearts unsuitable for transplant 4; they compared Gα_i expression in ventricles from patients ranging from 1 to 36 years (young group) and 37 to 71 years (old group), and found no change in Gα_i expression. These differences between the studies may be due to differences in the effect in atria versus ventricles, or they may be due to the inclusion of relatively younger patients in the older group of the study by White et al (more than half of the older group was under the age of 50 years). 4 Alternatively, the differences could be due to the state of the tissues used. The atrial tissues used by Brodde et al 3 were collected from the operating room and frozen immediately. In contrast, the ventricular tissues used by White et al 4 came from hearts that could not be used for transplants, and were kept on ice for up to 30 minutes after they were removed from the body. Clearly, additional studies are needed to establish the role of age in the expression of Gα_i in human hearts.

The goal of the present study was to establish whether Gα_i in human atria increases with advanced age, and if so, whether this leads to an increase in receptor-mediated activation of Gα_i resulting in enhanced AC inhibition. To this end, atria from mature adults (40-55 years) and elderly patients (71-79 years) were analyzed. We report that elderly human atria have higher expression of both Gα_i2 and Gα_i3, but not of Gα_s, Gα_o, Gβ₁, or Gβ₂. Consistent with these findings, photoaffinity labeling with [³²P]azidoanilido-GTP (AA-[³²P]GTP) indicates greater activation of Gα_i in elderly atria upon stimulation of β₂AR and other Gα_i-coupled receptors. Furthermore, the increase in activated Gα_i in older atria contributes to the age-induced decline in AC activity because a greater increase in receptor-mediated activation of AC in elderly atria is observed upon disabling of Gα_i with pertussis toxin (PTX). Finally, we show that, like stimulation of β₁AR, stimulation of β₂AR in live atrial tissue produces significant increases in the phosphorylation of phospholamban, demonstrating the importance of the AC/cAMP pathway in β₂AR-mediated atrial contractility in humans.