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Phosphoinositide-3-Kinase/Akt Graves et al. Journal of Biomedical Science 2012, 19:59 http://www.jbiomedsci.com/content/19/1/59 RESEARCH Open Access Phosphoinositide-3-kinase/akt - dependent 2+ signaling is required for maintenance of [Ca ]i, 2+ ICa, and Ca transients in HL-1 cardiomyocytes Bridget M Graves1, Thomas Simerly2, Chuanfu Li1, David L Williams1 and Robert Wondergem2,3* Abstract The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on 2+ 2+ cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca ]i,Ca transients and 2+ membrane Ca current, ICa, in cultured murine HL-1 cardiomyocytes. LY294002 (1–20 μM), a specific PI3K inhibitor, 2+ 2+ dramatically decreased HL-1 [Ca ]i,Ca transients and ICa. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the 2+ contribution of specific isoforms to HL-1 [Ca ]i regulation. Pharmacologic inhibition of each of the individual PI3K 2+ 2+ isoforms significantly decreased [Ca ]i, and inhibited Ca transients. Triciribine (1–20 μM), which inhibits AKT 2+ 2+ downstream of the PI3K pathway, also inhibited [Ca ]i, and Ca transients and ICa. We conclude that the PI3K/Akt 2+ pathway is required for normal maintenance of [Ca ]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB 2+ signaling sustains [Ca ]i required for excitation-contraction coupling in cardiomyoctyes. Keywords: Calcium, Fura-2, Phosphoinositide-3-kinase/Akt, HL-1 cardiomyocytes, Whole-cell voltage clamp, Electrophysiology Background report, we demonstrated that preventing sepsis-induced The phosphoinositide 3-kinases (PI3K) are a conserved changes in myocardial Akt activation correlates with family of signal transduction enzymes that are involved prevention of cardiac dysfunction [5]. in regulating cellular proliferation and survival [1,2]. The PI3K/Akt/PKB may play a role in cardiomyocyte cal- PI3Ks and the downstream serine/threonine kinase Akt cium regulation; however, the precise mechanisms by (also known as protein kinase B; PKB) regulate cellular which this occurs have not been fully elucidated. Yano activation, inflammatory responses, chemotaxis and and colleagues employed a transgenic mouse model apoptosis [1]. We [3] and others [4] have demonstrated over-expressing PI3K p110α in the heart [6], which that activation of PI3K/Akt dependent signaling attenu- resulted in increased left ventricular pressure, increased ates the pro-inflammatory phenotype and increases sur- levels of L-type Ca2+ channels, ryanodine receptors and vival outcome in sepsis. We have also reported that sarcoplasmic reticulum Ca2+-ATPase 2a [6]. In a subse- sepsis decreases myocardial Akt activation [5], which quent report, Lu et al. demonstrated that genetic abla- correlates with cardiac dysfunction in sepsis. In the same tion of PI3K p110α resulted in reduced numbers of voltage-dependent L-type Ca2+ channels in isolated car- 2+ * Correspondence: [email protected] diomyocytes, reduced inward Ca current and a defect 2Departments of Biomedical Science, James H. Quillen College of Medicine, in contractile function [7]. Taken together the results East Tennessee State University, Johnson City, TN 37614, USA 3Department of Physiology/Biomedical Science, James H. Quillen College of above indicate that PI3k/Akt signaling plays a critical Medicine, East Tennessee State University, P.O. Box 70,576, Johnson City, TN role in normal cardiac function and in maintaining 37614-1708, USA Full list of author information is available at the end of the article © 2012 Graves et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Graves et al. Journal of Biomedical Science 2012, 19:59 Page 2 of 9 http://www.jbiomedsci.com/content/19/1/59 cardiac function in sepsis [5-7]. This signaling most clamp technique [12]. Voltage-clamp currents were mea- likely involves regulation of cellular calcium. sured with a patch clamp amplifier (Axopatch 200B, Axon We conducted the present study to determine whether Instruments, Foster City, CA) with the lowpass, Bessell fil- direct inhibition of the PI3K, PI3K-specific isoforms or tering (−3dB)setat5kHz.Signalsfromthepatchclamp Akt-PKB signaling in HL-1 cardiomyocytes alters cal- amplifier were fed into a computer via a digital interface cium regulation. HL-1 is a proliferating atrial myocyte (Digidata 1322A) and processed by Clampex 8 software cell line established from a subcutaneous tumor of AT-1 (Axon Instruments). Ag/AgCl half-cells constituted the cells that, in turn, were derived from the atria of a mouse electrodes, and agar bridges (4% w v-1 in external solution) transgenic for the simian virus 40 large T antigen under connected the reference electrodes to the bath solution. control of the atrial natriuretic factor promoter [8,9]. Series resistances were compensated following whole-cell These cells display spontaneous contractions in tissue access prior to recordings. Giga-ohm seals between pipettes 2+ culture, oscillations of [Ca ]i, and express functional L- and cell membranes were made with cells perfused with 2+ and T-type Ca channels [10]. HL-1 cells also express standard external solution. For ICa measurements the cells the PI3K/Akt-PKB signaling pathway, which mediates were perfused with an external solution in which an imper- interleukin-18 induced cellular hypertrophy [11]. Herein, meable cation was substituted for Na+,andCa2+ concentra- we report that inhibitors of PI3K/Akt-PKB decrease [Ca2+], tion was increased (below). diminish Ca2+ transients and inhibit membrane Ca2+ 2+ currents, ICa, in these murine cardiomyocytes. These data Intracellular Ca measurements indicate that PI3K/Akt-PKB is required for normal cardio- Cells were loaded with Fura-2/AM (TefLabs, Austin, myocyte calcium regulation. TX) by incubating them for 30 min at room temperature (22–23 °C) with a standard external salt solution con- Methods taining 2-μM Fura-2/AM. Cells were then washed with HL-1 cell culture the external salt solution and incubated at 37 °C with 5% HL-1 atrial cardiomyocytes were a gift of Dr. William CO2 for 30 min in the supplemented Claycomb media. Claycomb (Louisiana State University Medical Center). The coverslip was transferred to an acrylic chamber They were grown in 5% CO2 at 37 °C in Claycomb (Warner Instr. Co., Hamden, CT) on the microscope media (Sigma) supplemented with batch specific 10% stage and washed with the external salt solution for FBS (Sigma), 100 U/ml:100 μg/ml Penicillin/Strepto- 5 minutes before measurements. Temperature was mycin (Invitrogen), 0.1 mM norepinephrine (Sigma) and maintained throughout measurements at 37 °C by a 2 mM L-glutamine (Invitrogen). Before culturing cells, stage/inline temperature controller (Warner Instr. Co., flasks were treated overnight with 0.02% Bacto© gelatin Hamden, CT) Fluorescence was measured with an im- (Fisher Scientific):0.5% Fibronectin (Invitrogen). For aging system consisting of a xenon fiberoptic light electrophysiologic or calcium measurements cells were source (Perkin-Elmer, Waltham, MA), a filter wheel and plated at a density of 3X105 cells/35-mm culture dish on a Basler A311F VGA Camera connected to an Olympus glass cover slips (12 mm diameter), which had been IX71inverted fluorescence microscope. The filter wheel flamed briefly to enhance coating and then transferred and data acquisition were controlled by the InCyte2 soft- to a 35-mm culture dish where they were treated with ware (v. 5.29; Intracellular Imaging, Cincinnati, OH). gelatin/fibronectin overnight. [Ca2+] was determined by interpolation from a standard curve generated by Ca2+ calibration buffer kit #2 (Mo- Whole-cell voltage clamp measurements lecular Probes @ Life Technologies, Grand Island, NY) Cells were grown for 1–2 days on 12-mm diameter glass and Fura-2/K5-salt. After correction for the individual plastic coverslips, which were transferred to an acrylic background fluorescence, the ratio of the fluorescence at chamber (Warner, New Haven, CT) on the stage of an both excitation wavelengths (F340/F380) was monitored inverted microscope (Olympus IMT-2) equipped with simultaneously in 30–40 cells, identified by their fluores- Hoffman modulation contrast optics. Cells were superfused cence within a single view field. Images were collected at room temperature with a standard external salt solution. every 3.3 s. Each slide was perfused with standard exter- Patch pipettes (3–6MΩ in the bath solution) were fabri- nal salt solution for 6 min for control measurements, cated from glass capillaries (1.1-1.2 mm ID, 0.2 mm wall followed by 10 min with the experimental solution. At thickness, non-heparinized micro-hematocrit capillary 16 min, the slide was washed with standard external salt tubes; Fisher Scientific) with a Brown-Flaming horizontal solution for 5 min, and at 21 min data collection was micropipette puller (P-97, Sutter Instruments, Novato,
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