Crosstalk Between ?-Adrenergic Receptor And PI3K Signaling Pathway: Implication For Regulation Of Cardiac Growth And Proliferation

§1 p-Adrenergic Receptor-PI3K Signaling Crosstalk in Mouse heart:Elucidation of Immediate Downstream Signaling CascadesObjective:?-adrenergic receptors (?AR) are known to mediate critical of heart functions such as heart rate and contractility. Sustained PAR activation leads to cardiac hypertrophy and prevents left ventricular (LV) atrophy during LV unloading. The basic knowledge about mechanisms by which PAR activation promotes myocardial growth and prevents cardiomyocyte atrophy has been acquired from studies using animal models of chronic systemic?AR agonist administration. However, the immediate signaling pathway(s) following acute?AR stimulation have not been well investigated. The current study was to examine the early cardiac signaling mechanism(s) following PAR stimulation, as well as the relationship of which to PAR-mediated cardiac hypertrophy and/or atrophy.Methods:Experiments were conducted on adult male C57BL/6 mice and?1/?2AR double knockout (?1/?2 DKO) mice. Animals were injected intraperitoneally with saline, isoproterenol (ISO) (1.25mg/kg) dobutamine (1.7mg/kg, the same molar equivalent with ISO), formoterol (2,1mg/kg, the same molar equivalent with ISO) or saline for 30min (unless otherwise specified). Left ventricular lysates were used for in vitro kinase assays (to measure PI3K activity) and Western blotting (for measuring the levels of Akt, ERK1/2, GSK-3?, mTOR, p70S6K, S6, FoxO1 and FoxO3a). Quantitative determination of muscle-specific E3 ligases atrogin-1 and MuRFl mRNA levels in LV tissue was performed by real-time quantitative-PCR.Results:Acute?AR stimulation induced significant increases in PI3K activity and activation of Akt and ERK1/2 in the heart, but not in lungs or livers. In contrast, the same treatment did not elicit these changes in?1/?2 DKO mice. We further showed the specificity of (32AR in this crosstalk as treatment with formoterol, a?2AR-selective agonist, but not dobutamine, a predominantly?1AR agonist, activated cardiac Akt and ERK1/2. Acute?2AR stimulation also significantly increased the phosphorylation of mTOR, p70S6K, ribosomal protein S6, GSK-3a/?, and FoxO1/3a. Moreover, acute PAR stimulation time-dependently decreased the mRNA levels of atrogin-1 and MuRF1 in mouse heart.Conclusions:Our results indicate that acute?AR stimulation in vivo affects multiple cardiac signaling cascades, including the PI3K signaling pathway, ERK1/2, atrogin-1 and MuRF1. These data 1) provide convincing evidence for the crosstalk between?AR and PI3K signaling pathways; 2) confirm the?2AR specificity in this crosstalk in vivo; and 3) identify novel signaling factors involved in cardiac hypertrophy and LV unloading. Understanding of the intricate interplay between P2AR activation and these signaling cascades should provide critical clues to the pathogenesis of cardiac hypertrophy and enable identification of targets for early clinical interaction of cardiac lesions. §2?-Adrenergic Receptors Regulate Cardiomyocyte Proliferation through PI3K/Akt Pathway in Neonatal MouseObjective:During early cardiac development PAR, their ligands and enzymes required for ligand biosynthesis can be detected with no excitation-contraction coupling. In adult mice, chronic PAR activation leads to cardiac hypertrophy. We have shown PI3K activities are highly regulated during cardiac development, high in fetal and neonatal periods but low in the adult. We further demonstrated that altering?2AR leads to corresponding changes in cardiac-specific PI3K signaling pathway, thus establishing the?AR-PI3K crosstalk in a cardiac context. The goal of the current study was to elucidate the biological consequences, especially regulation in cardiomyocyte proliferation, resulting from such crosstalk, and to identify signaling kinases that might be important in this regulation.Methods:Newborn C57BL/6 mice were treated with propranolol (10 mg/kg, ip), a nonspecific?AR antagonist, for 1 hour followed by injection with EdU (5-ethynyl-2'-deoxyuridine,50 mg/kg, ip) for 4 hours to monitor cell proliferation. Paraffin-embedded heart sections were processed for immunofluorescence detection of 1) Edu using a Click-iT(?) EdU assay kit, and 2) cardiomyocytes using a rabbit monoclonal anti-a-actinin-2 antibody. Confocal microscopic results were analyzed by using Image J software. To identify the signaling kinases, mice were injected with saline or propranolol for 30 minutes. Cardiac tissue lysates were subjected to in vitro kinase assay to measure PI3K activity, and Western blotting to measure the levels of Akt, ERK1/2, GSK-3a/?, FoxOs, mTOR, p70S6K and S6.Results?AR blockade significantly decreased the number of proliferating cardiomyocytes per field (19.0±3.1 vs.30.4±2.2 in controls, p<0.05) and the proliferation index (# proliferating cardiomyocytes/# total nuclei [DAPI staining],6.2±1.0% vs.11.7±0.9% in controls, p<0.05).?AR blockade by propranolol induced a significant reduction in cardiac PI3K activity, and the phosphorylation of some downstream effectors of PI3K, including Akt (Ser473 and Thr308), GSK-3a (Ser21), GSK-3p (Ser9), FoxO1 (Thr24) and FoxO3a (Thr32, Ser253). But interestingly, acute PAR blockade had no functional change to the activities of mTOR/p70S6K/S6 axis, neither the activation of ERK1/2.Conclusions:Our study not only established the?AR-PI3K cross talk in neonatal mouse heart but also indentified control of cardiomyocytes proliferation as the biological significance for such crosstalk. Further delineation of the cardiac?AR-PI3K crosstalk should contribute to our understanding of the perinatal cardiac development, especially the mechanism underlying the switch from hyperplasia to hypertrophy, and may have important implication in identifying new strategy for treatment of acute cardiac infarction and heart failure.