-Adrenergic Receptor Subtype-Specific Signaling in Cardiac Myocytes

The sympathetic nervous system modulates cardiac contractility and rate by activating -adrenergic receptors ( AR) expressed on cardiac myocytes and specialized cells in the sinoatrial node and the conduction system. Recent clinical studies have suggested that -adrenergic receptors also play a role in cardiac remodeling that occurs in the pathogenesis of cardiomyopathy. Both 1 and 2 adrenergic receptors are expressed in human and murine hearts. We have examined the effect of AR activation on the spontaneous contraction rate of neonatal myocyte cultures from wild-type and receptor knockout (KO) mice ( 1AR-KO, 2AR-KO and 1 2AR-KO mice). Stimulation of the 1AR in 2AR-KO myocytes produces the greatest increase in contraction rate through a signaling pathway that requires protein kinase A (PKA) activation. In contrast, stimulation of the 2AR in 1AR-KO myocytes results in a biphasic effect on contraction rate with an initial increase in rate that does not require PKA, followed by a decrease in rate that involves coupling to a pertussis toxin sensitive G protein. A small isoproterenol-induced decrease in contraction rate observed in 1 2AR-KO myocytes can be attributed to the 3AR. These studies show that all three AR subtypes are expressed in neonatal cardiac myocytes, and the 1AR and 2AR couple to distinct signaling pathways. -Adrenergic receptors ( AR) have been among the most extensively studied members of the G protein-coupled receptor (GPCR) family. AR activation of adenylyl cyclase is one of the first hormone-activated GPCR pathways to be characterized, and these receptors are therapeutic targets for a variety of clinical conditions including hypertension, coronary artery disease, heart failure, and asthma. Three AR subtypes have been cloned ( 1AR, 2AR, and 3AR). The 1AR and 2AR are pharmacologically more similar to each other than they are to the 3AR. All three receptor subtypes are highly conserved across species, suggesting that both sequence similarities and differences between subtypes are physiologically important. The close structural and functional properties of the 1AR and 2AR are paradigmatic of many other GPCR families where two or more receptor subtypes respond to the same hormone and couple to the same effector systems. However, although 1AR and 2AR have very similar signaling properties when expressed in undifferentiated cell lines (Green et al., 1992), there is a growing body of experimental evidence suggesting that they have different signaling properties in differentiated cells in vivo (Aprigliano et al., 1997; Zhou et al., 1997). Moreover, these receptors may differ in other functional parameters such as desensitization (Michel et al., 1990). Thus, characterizing the functional differences between these highly homologous receptors in the context of differentiated cells will shed light on their physiologic function and the consequence of pharmacologic manipulation in vivo. We have been interested understanding the specific role of 1 and 2AR subtypes in regulating cardiac myocyte function. Both subtypes are expressed in human and murine hearts. Our previous in vivo studies on 1AR knockout ( 1AR-KO) mice, 2AR knockout ( 2AR-KO) mice and 1 2AR double knockout ( 1 2AR-KO) mice demonstrated that the 1AR is primarily responsible for catecholamineinduced changes in heart rate and contractility (Rohrer et al., 1996, 1999; Chruscinski et al., 1999). In an effort to understand the functional role of 2AR in the heart, we chose to study receptor subtype-specific signaling in cultured neonatal myocytes. This experimental system has several experimental advantages. These cells contract spontaneously and cultures can be maintained for up to 1 week. The contraction rate of cultured myocytes is responsive to catecholamines added to the culture medium. Thus, by studying receptormodulated contraction rate, we are able to examine the integrated response to all of the signaling pathways activated by the complement of adrenergic receptors natively expressed in these cells. By examining the effect of a nonselective AR agonist in myocytes from 1AR-KO, 2AR-KO, and 1 2ARABBREVIATIONS: AR, -Adrenergic receptor; GPCR, G protein-coupled receptor; KO, knockout; IBMX, isobutyl methylxanthine; PKI, protein kinase inhibitor; PTX, pertussis toxin; PKA, cAMP-dependent protein kinase A. 0026-895X/01/6003-577–583$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 3 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 797/925603 Mol Pharmacol 60:577–583, 2001 Printed in U.S.A.