-Adrenergic Receptor Subtype-Specific Signaling in Cardiac Myocytes from 1 and 2 Adrenoceptor Knockout Mice

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. 577 at A PE T Jornals on M ay 0, 2017 m oharm .aspeurnals.org D ow nladed from KO mice, we have been able to identify distinct signaling pathways for the 1AR, 2AR, and the 3AR. Materials and Methods Mice. 1AR-KO (Rohrer et al., 1996), 2AR-KO (Chruscinski et al., 1999) and 1 2AR-KO (Rohrer et al., 1999) mice were all constructed by gene targeting as described. Preparation of Cultured Neonatal Mouse Ventricular Myocytes. Spontaneously beating neonatal cardiac myocytes were prepared from hearts of 1to 2-day-old mouse pups (from wild-type mice and from 1AR-KO, 2AR-KO, and 1/ 2AR-KO mice). Briefly, hearts were quickly excised, the atria were cut off, then the ventricles were minced and digested at 37°C for 45 min in calcium-free HEPES-buffered Hanks’ solution, pH 7.4, plus 100 g/ml collagenase type II and 1X pancreatin (Invitrogen, Carlsbad, CA). To reduce the contribution of nonmyocardial cells, cells are preplated for 1 h. The myocyte-enriched cells remaining in suspension were plated in 35-mm tissue culture dishes for the contraction studies, or 12-well plates for cAMP accumulation assay. Culture dishes are precoated with 1.5% gelatin for 30 min. Myocytes were cultured in Dulbecco’s modified Eagle’s medium containing 10% horse serum, 5% fetal bovine serum, and antibiotics (1 gentamycin; Roche Molecular Biochemicals, Indianapolis, IN). Although the culture technique includes a preplating step that effectively decreases fibroblast contamination, myocytes are cultured in presence of 1 cytosine-Darabinofuranoside (Sigma, St. Louis, MO) to block the cardiac fibroblast proliferation. Contraction rate and cAMP accumulation assays are performed in culture media containing serum and buffered with 20 mM HEPES, pH 7.4. Measurement of Spontaneous Rate of Cardiac Myocyte Contraction. Measurement of spontaneous contraction rate was carried out as described previously (Johnson and Mochly-Rosen, 1995) with some modifications. Briefly, about 3 10 cardiac cells were cultured in 35-mm Petri dishes (Corning, Palo Alto, CA, as described above) to obtain a uniformly beating syncytium. On day 4, the culture dishes were placed in a temperature-regulation apparatus positioned on the stage of an inverted microscope (Nikon, Tokyo, Japan) connected to a video camera. Cells were equilibrated at 37°C for 10 min before monitoring the contraction rate. Contraction rates of cells within the syncytium were determined at 2to 5-min intervals for 10 min before and 30 min after the addition of isoproterenol or CL 316243. All assays were recorded on videotape. Measurement of cAMP Accumulation. To measure intracellular cAMP, myocytes were cultured in 12-well plates (5 10 cardiac cells per well). Cells were incubated for 30 min at 37°C with 1 mM isobutyl methylxanthine (IBMX; Sigma) immediately before the addition of the agonist isoproterenol. Cells were treated with different concentrations of isoproterenol for 5 to 15 min at room temperature. The assay was terminated by the aspiration of the incubation buffer and addition of 1 ml of 100% ethanol to each well. The cell lysates were then collected, boiled for 5 min, cooled, and stored at 80°C. Aliquots were dried in a spin-vacuum, and cAMP in the residue was determined using a radioimmunoassay (Amersham Pharmacia Biotech, Piscataway, NJ). PTX Treatment. For contraction and cAMP measurement, cells were preincubated with PTX (0.75 mg/ml) at 37°C for at least 3 h as described previously (Xiao et al., 1995). Successful inactivation of inhibitory G proteins (Gi/Go) in PTX-treated cells was verified by the loss of the ability of adenosine (at 10 6 M) to decrease the basal contraction rate or to reverse the positive chronotropic effect of AR stimulation by isoproterenol in WT myocytes. PKI Treatment. Cells were preincubated with myr PKI14–22 (20 M) at 37°C for 10 min before isoproterenol exposure. Drugs. Isoproterenol, adenosine, and PTX were obtained from Sigma, CL-316243 was a kind gift of Wyeth Ayerst Laboratories (Philadelphia, PA), and myr PKI14–22 was obtained from Calbiochem (San Diego, CA). Data Analysis. Time course experiments, two-way analysis of variance corrected for repeated measures was used to test for significance (p 0.05) between groups. If the analysis of variance was significant, a t test using Bonferroni’s method was used to compare responses at multiple time points of interest where maximal effects of treatments were observed. Analysis was done using Prism (GraphPad Software, Inc., San Diego, CA).