β-Adrenergic Receptor Mediated Protection against Doxorubicin-Induced Apoptosis in Cardiomyocytes: The Impact of High Ambient Glucose

0013-7227/08/$15.00/0 Printed in U.S.A. Endocrinology 149(12):6449 – 6461 Copyright © 2008 by The Endocrine Society doi: 10.1210/en.2008-0292 ␤-Adrenergic Receptor Mediated Protection against Doxorubicin-Induced Apoptosis in Cardiomyocytes: The Impact of High Ambient Glucose Department of Pediatrics (N.Y., A.T., J.P.S., B.G.M., T.C.Z., J.F.P., Y.-T.T.), Women and Infant’s Hospital, The Warren Alpert Medical School of Brown University, Providence, Rhode Island 02905; and Division of Nephrology and Metabolism (D.S., M.E.), Department of Internal Medicine, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan Recent studies have demonstrated that the ␤2-adrenergic receptor (␤2AR)-G␣i signaling pathway exerts a cardiac antiapoptotic effect. The goals of this study were to determine the intracellular signaling factors involved in ␤2AR-mediated protection against doxorubicin-induced apoptosis in H9c2 cardiomyocyte and explore the impact of high ambient glucose on the antiapoptotic effect. Under physiological glucose environment (100 mg/dl), ␤2AR stimulation prevented doxorubicin-induced apoptosis, which was attenuated by cotreatment with wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, or transfection of a dominant-negative Akt. Inhibition of Src kinase with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine or cSrc small interfering RNA 32 also attenuated the antiapoptotic effect. Inhibition of platelet-derived growth factor receptor (PDGFR) with AG1296 reversed the ␤2AR-induced antiapoptotic effect. Transfection of T HE ␤-ADRENERGIC RECEPTORS (␤AR) are members of the G protein-coupled receptor (GPCR) family (1). At least three ␤AR subtypes, ␤1AR, ␤2AR, and ␤3AR, have been characterized at the gene level (2– 4). It has been shown that ␤1AR and ␤2AR modulate cardiac function differently and exert physiological responses by distinct signal transduction pathways (5). In particular, sustained ␤1AR stimulation promotes cardiomyocyte apoptosis, whereas sustained ␤2AR stimulation is known to protect cardiomyocytes from apoptotic insults (6). This finding is of great clinical interest because selective pharmacological activation of ␤2AR-mediated inotropy or its overexpression through gene therapy might be used as a novel therapeutic approach in the types of failing heart. Doxorubicin (Dox), an anthracycline antibiotic, has been established as an effective agent against a wide range of cancers (7). However, the severe cardiotoxicity of Dox is a First Published Online August 21, 2008 Abbreviations: ␤2AR, ␤2-Adrenergic receptor; ␤ARKct, ␤AR kinase-1 C terminus minigene; 2⬘,5⬘-DOA, 2⬘,5⬘-dideoxyadenosine; Dox, doxorubicin; EIA, enzymatic immunoassay; GPCR, G protein-coupled receptor; IGF-IR, IGF-I receptor; ISO, isoproterenol; PDGFR, platelet-derived growth factor receptor; PI3K, phosphoinositide 3-kinase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine; PTX, pertussis toxin; pY, phosphotyrosine; siRNA, small interfering RNA. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. an active Src cDNA (Y529F) alone was sufficient to render the cells resistant to apoptosis, and the resistance was blocked by wortmannin. Transfection of an active PI3K minigene (iSH2p110) alone also induced resistance to apoptosis, and the resistance was reversed by an Akt-inhibitor but not by AG1296. High ambient glucose (450 mg/dl) caused two major effects: 1) it significantly reduced ␤AR-induced PDGFR phosphorylation, Src kinase activity, and activation of PI3K signaling pathway; and 2) it partially attenuated ␤2AR-induced antiapoptotic effect. These data provide in vitro evidence supporting a signaling cascade by which ␤2AR exerts a protective effect against doxorubicin-induced apoptosis via sequential involvement of G␣i, G␤␥, Src, PDGFR, PI3K, and Akt. High ambient glucose significantly attenuates ␤2AR-mediated cardioprotection by suppressing factors involved in this cascade including PDGFR, Src, and PI3K/Akt. (Endocrinology 149: 6449 – 6461, 2008) major limiting factor for its effective use in the treatment of all malignancies (8). It is believed that mitochondria play a significant role in the toxicity as isolated heart mitochondria and myocytes have been shown to give rise to increased levels of oxygen radicals after Dox treatment (9). Mitochondria play a pivotal role in regulating apoptosis by mechanisms that result in the activation of caspases and subsequent cell death (10). Apoptosis has been attracting increasing attention as a possible mechanism underlying cardiomyopathy and chronic heart failure induced by Dox (11). Cardiomyopathy is also a common complication in patients with diabetes mellitus (12). Acute responses of cardiomyocytes to hyperglycemia include metabolic abnormalities, altered gene expression, and increased apoptosis (13– 15). It has been shown that the incidence of apoptosis increases in the hearts of patients with diabetes (16) and streptozotocin-induced diabetic animals (17). The present study was undertaken to delineate the signaling pathway(s) by which ␤2AR stimulation evokes protective effects against Dox-induced apoptosis and to examine whether the protective effects of ␤2AR are impaired under high glucose environment. For this purpose, we studied Doxinduced apoptosis in a ␤2AR-dominant fetal rat cardiomyocyte cell line, H9c2 (18). The results of this study provide evidence that the antiapoptotic signaling via ␤2AR is mediated by transactivation of the platelet-derived growth factor receptor (PDGFR) and phosphoinositide 3-kinase (PI3K). 6449 Naohiro Yano, Daisuke Suzuki, Masayuki Endoh, Andy Tseng, Joan P. Stabila, Bethany G. McGonnigal, Ting C. Zhao, James F. Padbury, and Yi-Tang Tseng 6450 Yano et al. • ␤AR-Mediated Cell Protection in Cardiomyocytes Endocrinology, December 2008, 149(12):6449 – 6461 to 70% confluence and synchronized overnight in serum-free medium before experiments. Immunoblotting and antibodies Furthermore, the antiapoptotic effect is significantly suppressed under high ambient glucose conditions by inhibition of these signaling pathways. Materials and Methods Reagents Unless otherwise specified, all materials were of reagent grade and were obtained from Sigma (St. Louis, MO). DNA fragmentation ELISA After a 6- or 10-h cell culture in medium without serum, the amount of apoptosis that had occurred was examined by using a DNA fragmentation detection ELISA kit (Roche Diagnostics, Mannheim, Germany). PI3K assay H9c2 cell lysates were prepared as described (19), and protein concentrations were determined with the bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL). PI3K activity was determined by an in vitro immunoprecipitation lipid kinase assay as described previously (19, 20). Cell culture H9c2 rat fetal cardiomyocytes were grown in DMEM (Invitrogen, Carlsbad, (...truncated)