- Email: [email protected]
NFAT Signalling Pathway
Accepted Manuscript Nicotine induces cardiomyocyte hypertrophy through TRPC3-mediated Ca signaling pathway
2+ / NFAT
Na Li, MD, PhD, Biao Si, MD, Ji-Feng Ju, MD, Meng Zhu, MD, Feng You, MD, Dong Wang, MD, Jie Ren, MD, Yan-Song Ning, MD, Feng-Quan Zhang, MD, Kai Dong, MD, PhD, Jing Huang, MD, PhD, Wen-Qian Yu, MD, PhD, Tong-Jian Wang, MD, PhD, Bin Qiao, MD, PhD PII:
S0828-282X(15)01686-4
DOI:
10.1016/j.cjca.2015.12.015
Reference:
CJCA 1965
To appear in:
Canadian Journal of Cardiology
Received Date: 17 August 2015 Revised Date:
11 November 2015
Accepted Date: 2 December 2015
Please cite this article as: Li N, Si B, Ju J-F, Zhu M, You F, Wang D, Ren J, Ning Y-S, Zhang F-Q, Dong K, Huang J, Yu W-Q, Wang T-J, Qiao B, Nicotine induces cardiomyocyte hypertrophy through 2+ TRPC3-mediated Ca / NFAT signaling pathway, Canadian Journal of Cardiology (2016), doi: 10.1016/ j.cjca.2015.12.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Nicotine
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TRPC3-mediated Ca2+/ NFAT signaling pathway
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Na Li1, MD, PhD, Biao Si1, MD, Ji-Feng Ju1, MD, Meng Zhu1, MD, Feng You1, MD,
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Dong Wang1, MD, Jie Ren1, MD, Yan-Song Ning1, MD, Feng-Quan Zhang1, MD,
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Kai Dong1, MD, PhD, Jing Huang1, MD, PhD, Wen-Qian Yu1, MD, PhD, Tong-Jian
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Wang1,*, MD, PhD, Bin Qiao1,*MD, PhD
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China
induces
cardiomyocyte
hypertrophy
through
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Institute of Cardiovascular Disease, General Hospital of Jinan Military Region, Jinan,
*Correspondence should be addressed to:
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Dr. Bin Qiao, M.D., Ph.D.
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Institute of Cardiovascular Disease, General Hospital of Jinan Military Region, Jinan,
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China
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Tel: +86 13905311870;
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Fax: +86 0531 51636718
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Email: [email protected]
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Dr.Tong-Jian Wang, M.D., Ph.D.
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Institute of Cardiovascular Disease, General Hospital of Jinan Military Region, Jinan,
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China
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Email: [email protected]
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Short title: Nicotine induces cardiomyocyte hypertrophy.
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Brief Summary
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We demonstrate that nicotine could significantly promote cardiomyocyte
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hypertrophy through TRPC3-mediated Ca2+ influx and calcineurin-NFAT signaling
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activation. Our findings implicate the pro-hypertrophic effect of nicotine on
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cardiomyocytes
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calcium-dependent regulatory loop driving cardiomyocyte hypertrophy, which could
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become a potential target for prevention and treatment of cardiac hypertrophy.
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Background: Nicotine is thought to be an important risk factor for development of
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cardiovascular diseases. However, the effects of nicotine on cardiomyocyte
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hypertrophy are poorly understood. The present study was designed to explore the
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role of nicotine in cardiomyocyte hypertrophy and its underlying mechanism.
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Methods: We used primary cardiomyocytes isolated from Wistar rats to examine the
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effects of nicotine on intracellular Ca2+ mobilization and hypertrophy determined by
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immunofluorescence, qPCR and western blot analysis. Luciferase reporter assay was
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used to examine the activity of NFAT signaling.
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Results: We found that nicotine caused cardiomyocyte hypertrophy, which was
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accompanied with an increased intracellular Ca2+. Nicotine-enhanced intracellular
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Ca2+ concentration ([Ca2+]i) was significantly abolished by SOCE and TRPC
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inhibitors. Knockdown of TRPC3 significantly decreased nicotine-induced SOCE and
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hypertrophy. Moreover, calcineurin-NFAT is involved in TRPC3-mediated Ca2+
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signaling and cardiomyocyte hypertrophy. Notably, upregulation of TRPC3 by
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nicotine requires TRPC3-mediated Ca2+ influx and calcineurin-NFAT signaling
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activation.
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Conclusion: Our findings demonstrate that the pro-hypertrophic effect of nicotine on
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cardiomyocytes
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calcium-dependent regulatory loop, which could become a potential target for
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prevention and treatment of cardiac hypertrophy.
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Key words: Nicotine; cardiac myocytes; hypertrophy; TRPC3; calcium; NFAT
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Introduction Cigarette smoking, as an important risk factor, is responsible for development of
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diverse diseases such as obesity, atherosclerosis, and cardiovascular disease [1-3].
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Nicotine is the most addictive component of cigarette smoke and has been found to
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have a wide range of effects [4]. However, the role of nicotine in cardiovascular
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disease is not completely understood.
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Cardiac hypertrophy is a severe cardiovascular disease, characterized by an
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increase in left ventricular mass and shape, which has been established as an
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important risk factor for cardiac mortality [5, 6]. Therefore, understanding the
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molecular mechanisms involved in cardiac hypertrophy is of great importance.
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However, the molecular mechanisms contributing to the initiation of cardiac
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hypertrophy are just beginning to be understood. Previous studies have revealed that
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intracellular calcium signaling plays a central role to induce cardiac hypertrophy [7,
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8]. Myocytes treated by agonists such as angiotensin II (Ang II) or endothelin-1 (ET-1)
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result in a remarkable increase in intracellular Ca2+, and the augmentation in
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intracellular Ca2+ is thought to be closely involved in Ca2+-calmodulin-dependent
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phosphatase calcineurin (Cn)-NFAT activation, and then leads to initiate the genes
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expression in cardiac hypertrophy[9, 10]. Recent studies show that calcineurin
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activation is dependent on the Ca2+ influx through transient receptor potential (TRP)
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proteins [11, 12].
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TRP channels belong to the nonselective cation influx channels that are grouped
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into 7 families, responsible for receptor activated Ca2+ entry and store operated Ca2+
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entry (SOCE)[11,13]. Overexpression of TRP channels can initiate cardiac
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hypertrophy because of increased Ca2+ influx and calcineurin activation [12, 14].
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Once activated, TRP channels induce signal transduction through cytoplasmic Ca2+
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elevations or endoplasmic reticulum Ca2+ release to activate signaling events [15, 16].
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Most recently, some studies suggest that TRP channels are required for cardiac
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hypertrophy [12]. However, it remains unknown how TRPC channels and associated
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Ca2+ influx functioned in the development of cardiomyocyte hypertrophy.
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In this study, we aimed to clarify whether nicotine contributes to cardiac 4
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hypertrophy by regulating TRPC expression and Ca2+ signaling, and to explore the
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effects of nicotine on TRPC expression, SOCE and cardiac hypertrophy in cultured rat
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cardiomyocytes to elucidate the potential mechanisms of cardiac hypertrophy.
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Materials and Methods
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Ethics statement
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This work was approved by the Clinical Research Ethics Committee of General
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Hospital of Jinan Military Region. All the procedures were performed according to
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the “Guide for the Care and Use of Laboratory Animals” published by National
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Institutes of Health.
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Cell isolation and culture
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Cardiomyocytes were obtained from 2-day-old Wistar rats as previously
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described [17]. The detailed procedures of isolation and culture are described in the
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online supplementary materials.
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Cell size measurement
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The isolated cardiomyocytes were incubated with 100 nM Ang-II or increased
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concentrations of nicotine with or without BAPTA-AM, an intracellular calcium
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chelator. Cells were then subjected to immunofluorescence analysis. For detailed
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procedures and data analysis, see online supplementary materials.
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Measurement of protein synthesis
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Cultured cardiomyocytes were transfected with si-TRPC3 or control siRNA, and
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treated with nicotine or not as indicated. 3[H]-leucine incorporation assay was used to
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evaluate the protein synthesis speed. The detailed procedures are described in the
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online supplementary materials.
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Quantitative real-time PCR 5
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The qPCR amplifications were carried out using SYBR Green detection chemistry. For detailed procedures and analysis, see online supplementary materials.
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Western blot analysis Western blot analysis was performed using standard procedures. The antibodies
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used in the western blot analysis and the detailed procedures are listed in the online
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supplementary materials.
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Measurement of Ca2+ concentration
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Cardiomyocytes were loaded with 5 µM Fura-2 (Life technology, NY, USA), and
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intracellular calcium levels was determined by the ratio of fluorescence at 340 nm and
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380 nm using Metafluor 7.0 software (Molecular Devices, CA, USA). The detailed
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procedures are described in the online supplementary materials.
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Immunofluorescence and confocal imaging
Cardiomyocytes were infected with adenovirus coding GFP-NFATc3. The
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localization of GFP-NFATc3 was determined with a Laser Scanning Confocal
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Imaging System (Carl Zeiss, Oberkochen, Germany) as described previously [18].
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The detailed procedures are described in the online supplementary materials.
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Luciferase Reporter Assay
Cardiomyocytes were co-transfected with 500 ng of pNFAT-Luc or TRPC3-Luc
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with 50 ng of pRL-SV40 control plasmid. The cells were incubated with nicotine, and
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luciferase activity was measured using the Dual Luciferase Reporter assay (Promega,
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Madison, WI, USA).
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Statistical analysis
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All data are expressed as mean ± SEM. A two-tailed Student’s t-test or
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Mann-Whitney test was used to determine differences between groups. All the
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statistical analyses were performed using SPSS 17.0 software, and the graphs were 6
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generated using GraphPad Prism 6.0 (Graphpad Software Inc, CA, USA). P<0.05
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was considered statistically significant.
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Results
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Nicotine induces cardiomyocyte hypertrophy
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We firstly retrospectively analyzed the patients with cardiac hypertrophy in our
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hospital, and found that the incidence is closely associated with cigarette smoking
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(Supplementary Table S1). Figure 1A showed the morphological change of
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cardiomyocytes exposed to nicotine and Ang-II. Cell size was increased after nicotine
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or Ang-II treatment. Furthermore, three important hypertrophic markers, ANP, BNP
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and β-MHC, were detected by qPCR and western blotting analysis. Figure 1B and C
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showed a dose-dependent increase of ANP, BNP and β-MHC expression induced by
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nicotine in cardiomyocytes. The 3[H]-leucine incorporation was significantly greater
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in Ang II and nicotine-treated cells than that in the control group (Figure 1D).
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Moreover, concurrent treatment with nicotine and Ang-II promotes more severe
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cardiomyocyte hypertrophy than either nicotine or Ang II on their own
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(Supplementary Figure 1A-E). These results suggest that nicotine can induce
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hypertrophic responses of cardiomyocytes.
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Nicotine initiates an intracellular calcium wave and calcium chelation inhibits
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nicotine-induced cardiomyocyte hypertrophy To assess the changes in calcium signaling in cardiomyocytes treated by nicotine,
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the ability of nicotine to increase [Ca2+]cyt in cardiomyocytes was evaluated. Nicotine
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induced a transient increase of [Ca2+]cyt in a dose dependent manner, and 100 nM of
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nicotine had the most significant effect (Figure 2A and B). To directly elucidate the
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function of calcium signaling in nicotine-induced cardiomyocyte hypertrophy,
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cardiomyocytes were pretreated with the calcium chelator. A significant decrease in
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nicotine-induced mRNA and protein expression of ANP, BNP and β-MHC was
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observed with calcium chelation (Figure 2C-F). Collectively, these results
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demonstrate a critical role of Ca2+ signaling in nicotine-induced cardiomyocyte
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hypertrophy.
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Nicotine activates SOCE and induces Ca2+ entry through TRPC in
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cardiomyocyte hypertrophy
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We further determined whether intracellular calcium was released from
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sarcoplasmic reticulum stores, extracellular influx, or both. Figure 3A shows that
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repeated nicotine (100 nM) could consistently increase [Ca2+]i in cardiomyocytes.
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Nicotine evoked a Ca2+ response with two components: a Ca2+ release from
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intracellular stores and Ca2+ influx from extracellular medium (Figure 3B). Next, we
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examined whether nicotinic acetylcholine receptor (nAChR) inhibitor α-bungarotoxin
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(αBtx) or phospholipase C (PLC) inhibitor U73122 could inhibit nicotine-induced
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Ca2+ increase. As shown in Figure 3C, both αBtx and U73122 inhibited
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nicotine-induced Ca2+ release and extracellular Ca2+ influx. SOCE was activated by
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depletion of intracellular Ca2+ stores using thapsigargin (TG, 1 µM) without
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extracellular Ca2+. The peak increase in [Ca2+]i was significantly higher in
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nicotine-induced cardiomyocytes compared to the control cells (Figure 3D). The
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TG-mediated Ca2+ increase was higher in nicotine-treated cardiomyocytes (Figure
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3D). Nicotine-evoked Ca2+ entry could be blocked with inhibitors of SOCE, Gd3+ and
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2-aminoethoxydiphenyl borate (2APB) (Figure 3E). Cells were pre-treated with
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SOCE blockers, ML9 (5 mM) and 2APB (50 mM), and then nicotine was added to
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induce hypertrophy. As shown in Supplementary Figure S2A and B, ML9 and 2APB
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significantly inhibited expression of hypertrophic markers, suggesting that nicotine
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promotes cardiomyocytes hypertrophy through a significant increase of SOCE. To
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evaluate what types of channels are involved in the induction of Ca2+ influx, the
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effects of Nifedipine (a dihydropyridine L-type Ca2+ channel antagonist), KB-R7943
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(Na+/Ca2+ exchanger (NCX) inhibitor) and SKF-96365 (TRPC blocker) were
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evaluated. Ca2+ influx was not inhibited with the Nifedipine or KB-R7943 but was
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inhibited with SKF-96365 (Figure 3F). These results demonstrate that TRPC channels
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are responsible for the Ca2+ transient in nicotine-treated cardiomyocytes.
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Effects of nicotine on the expression of TRPC isoforms in cardiomyocytes We further examine whether nicotine affects the gene expression of TRPCs. Only
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TRPC3 mRNA expression increased in response to nicotine stimulation, while the
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expression levels of TRPC1, 4, 5, 6 and 7 remained unchanged (Figure 4A). And
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nicotine promotes TRPC3 mRNA expression with the increase of the incubation time
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(Figure 4B). TRPC3 protein level had increased significantly after treatment with
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nicotine (Figure 4C). Taken together, these findings demonstrated that nicotine could
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promote TRPC3 expression in cardiomyocytes.
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TRPC3 exerts an important role in SOCE and cardiomyocytes hypertrophy
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Furthermore, we investigate whether knockdown of TRPC3 could attenuate
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nicotine-induced SOCE or hypertrophy in cardiomyocytes. We used siRNA to inhibit
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endogenous TRPC3 proteins selectively. The inhibitory effect was determined after
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siRNA transfection (Supplementary Figure S3A and B). The peak increase in [Ca2+]i
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was significantly decreased in siRNA-treated cardiomyocytes (Figure 5A and B). We
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then examined whether overexpression of TRPC3 could increase calcium signaling or
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hypertrophy. The TRPC3 expression was confirmed after TRPC3 vector transfection
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(Supplementary Figure S3C and D). TRPC3 overexpression remarkably increased
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nicotine-induced SOCE (Supplementary Figure S3E). Inhibition of TRPC3 expression
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could significantly attenuate nicotine-induced expression of hypertrophic markers
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(Figure 5C), while TRPC3 overexpression increased nicotine-induced hypertrophic
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markers expression (Supplementary Figure S3F). The 3[H]-leucine incorporation was
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significantly lower in TRPC3 knockdown group (Figure 5D). Taken together, these
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results demonstrate that TRPC3 molecules play an important role in nicotine-induced
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hypertrophic response.
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Role of TRPC3 in NFAT translocation and activation induced by nicotine in
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We tested the effect of nicotine on Ca2+-induced NFAT translocation (Figure 6A).
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Exposure to nicotine significantly increased the intensity of nuclear GFP fluorescence
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(Figure 6B). We then evaluated the efficacy of nicotine on NFAT activation in
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cardiomyocytes transfected with an NFAT-luciferase reporter. Nicotine increased
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NFAT
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nicotine-induced NFAT activation was abolished by nicotine receptor nAChR
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inhibitor (αBtx) and PLC inhibitor U73122, indicating that nAChR-mediated PLC
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activation is involved in nicotine-induced NFAT activation (Figure 6D). We then
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tested whether IP3 or DAG was involved in nicotine-induced NFAT activation.
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Treatment with RHC80267, a DAG lipase inhibitor, significantly increased
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nicotine-mediated nuclear NFAT translocation and activity. However, treatment with
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xestospongin C (XestC), an IP3R blocker, did not have any effects on NFAT
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translocation and activity (Supplementary Figure 4A and B). We next explored
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whether SOCE could be associated with nicotine-induced NFAT activation. ML9 (5
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mM) and 2APB (50 mM) could significantly attenuate nicotine-induced NFAT
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activation, suggesting that nicotine-activated SOCE is responsible for NFAT
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activation (Figure 6D). Nicotine increased the nuclear translocation of GFP-NFAT4
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and NFAT activity, both of which were almost completely abolished by knockdown of
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TRPC3 (Figure 6E and F). However, overexpression of TRPC3 could significantly
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increase nicotine-induced NFAT activity (Supplementary Figure S5A and B),
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demonstrating that Ca2+ influx through TRPC3 mediates nicotine-induced NFAT
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activation. To further investigate whether calcineurin-NFAT signaling were involved
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in nicotine-induced hypertrophy, calcineurin inhibitor cyclosporine A (CsA) was used.
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The data showed that CsA could significantly attenuate nicotine-induced hypertrophic
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markers expression (Supplementary Figure S6A-C). Moreover, CsA attenuated
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nicotine-induced protein synthesis and cardiomyocytes size (Supplementary Figure
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S6D and E). These results indicate that calcineurin-NFAT signaling pathway was
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involved in nicotine-induced cardiomyocyte hypertrophy.
in
a
dose-dependent
manner
(Figure
6C).
Furthermore,
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Nicotine upregulates TRPC3 expression via calcineurin-NFAT signaling 10
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inhibitor cyclosporine A (CsA, 200 nM) (Figure 7A). We further found that CsA or
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NFAT kockdown could remarkably inhibit nicotine-induced TRPC3 promoter activity,
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suggesting that nicotine promoted TRPC3 promoter activity through NFAT signaling
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(Figure 7B). We demonstrated that calcineurin inhibition or NFAT silencing prevented
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nicotine-induced TRPC3 expression (Figure 7Cand D), suggesting that nicotine
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promoted TRPC3 expression through calcineurin-NFAT signaling. We further showed
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that in the cardiomyocytes, overexpression of TRPC3 resulted in enhanced NFAT
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activation. And in line with our hypothesis, CsA blocked the NFAT activation
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secondary to exogenous TRPC3 overexpression (Figure 7E), demonstrating a
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regulation loop between NFAT signaling and TRPC3 expression.
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Discussion
Cardiac hypertrophy is a severe cardiovascular disease, which is an adaptative
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reaction in response to a wide range of physiological and pathophysiological stimuli
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[19]. Treatment of the cardiac hypertrophy is thought to be an important therapeutic
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target for prevention of heart failure. Here, we demonstrate that nicotine can induce
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cardiomyocyte hypertrophy. Nicotine stimulation significantly increases [Ca2+]i and
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SOCE that are greatly associated with nicotine-induced cardiomyocyte hypertrophy.
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We further demonstrate that nicotine promotes the TRPC3 expression, and reveal a
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deleterious positive feedback mechanism, in which TRPC3-mediated Ca2+ influx
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stimulates
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nicotine-associated cardiomyocyte hypertrophy.
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Previous studies reported that chronic smoke exposure could induce the
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enlargement of the cardiac chambers and myocardial hypertrophy [20, 21]. Our
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retrospective data also revealed that cardiac hypertrophy was closely associated with
323
cigarette smoking. The chronic effects of nicotine on whole organ or behavior were
324
mainly evaluated using animal or human study. Nevertheless, very few studies
325
focused on the effects of nicotine on isolated cells in vitro. We noticed that some
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previous studies reporting that nicotine could induce cardiomyocyte apoptosis, while 11
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found high dose of nicotine, particularly at concentrations of 500 nM or higher, could
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result in cell death of cultured cardiomyocytes. Meanwhile, we noticed that the high
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concentrations of nicotine used in those studies were far exceeding the physiological
331
concentration in cigarette smokers. Previous studies showed that the average peak
332
concentration of nicotine was 35 ng/ml (about 200 nM), which then gradually
333
declined to a final concentration of 7 ng/ml [24]. Therefore, our study mainly aimed
334
to investigate the effect of nicotine on cardiomyocytes at clinically relevant
335
concentrations.
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Several studies have shown that Ca2+-dependent signaling play an important role
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both in cardiac contractility and hypertrophy [10, 25]. We observe that
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nicotine-induced hypertrophy was calcium-dependent. Upon GPCRs activation,
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intracellular Ca2+ is released from internal stores [26]; however, little is known about
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the role of Ca2+ entry on [Ca2+]i increase in nicotine-induced hypertrophy. We
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demonstrate that nicotine acts through nAChR and PLC signaling cascade and
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induces [Ca2+]i increase through calcium entry and release. SOCE was activated by
343
depletion of intracellular Ca2+ using thapsigargin without extracellular Ca2+ [27].
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SOCE is closely associated with nicotine-induced cardiomyocyte hypertrophy. To
345
evaluate what types of channels are involved in Ca2+ influx, the effects of Nifedipine,
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KB-R7943 and SKF-96365 were examined. Our data show that TRPC3 is responsible
347
for nicotine-induced calcium increase. Importantly, TRPC3 plays an important role in
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nicotine-induced hypertrophic response.
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Previous report indicated that Ca2+ entry contributed to NFAT translocation, and
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NFAT activation was closely associated with pathological cardiomyocyte hypertrophy
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[9, 28]. We show that Ca2+ influx through TRPC3 is contributing to activate Cn-NFAT
352
signaling to induce cardiomyocyte hypertrophy. Interestingly, we also showed that
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nicotine was able to increase TRPC3 expression. Some previous studies revealed that
354
Ang II-mediated TRPC6 transcription appears to be controlled by the Ca2+-dependent
355
calcineurin/NFAT pathway, involving Ca2+ influx through TRPC6 itself [29]. We also
356
found that nicotine increased the TRPC3 expression through calcineurin-NFAT 12
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signaling, and overexpression of TRPC3 resulted in enhanced NFAT activation,
358
suggesting a regulation loop between NFAT signaling and TRPC3 expression.
359 360
Conclusions We have demonstrated for the first time that nicotine promoted cardiomyocyte
362
hypertrophy, and Ca2+ influx through TRPC3 is contributing to activate
363
calcineurin-NFAT signaling. Moreover, there is a positive regulatory loop in
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nicotine-induced cardiomyocyte hypertrophy (Figure 7F), which could become a
365
potential target for prevention and treatment of cardiac hypertrophy.
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Funding Sources
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This study was supported by the National Natural Science Foundation of China
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(81501721)
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Disclosures
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The authors have no conflicts of interest to disclose.
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channels in disease. Physiol Rev. 2007; 87:165-217. 16. Abramowitz J, Birnbaumer L. Physiology and pathophysiology of canonical transient receptor potential channels. FASEB J. 2009; 23:297-328. 17. Zhang H, Makarewich CA, Kubo H, Wang W, Duran JM, Li Y, et al.
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angiotensin receptor-induced NFAT activation in cardiac fibroblasts. J Biol
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19. van de Vrie M, Heymans S, Schroen B. MicroRNA involvement in immune activation during heart failure. Cardiovasc Drugs Ther. 2011; 25:161-70. 20. Minicucci MF, Azevedo PS, Polegato BF, Paiva SA, Zornoff LA. Cardiac
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25. Frey N, McKinsey TA, Olson EN. Decoding calcium signals involved in cardiac growth and function. Nat Med. 2000; 6:1221-7. 15
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28. Hunton DL, Lucchesi PA, Pang Y, Cheng X, Dell'Italia LJ, Marchase RB.
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Capacitative calcium entry contributes to nuclear factor of activated T-cells
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29. Nijenhuis T, Sloan AJ, Hoenderop JG, Flesche J, van Goor H, Kistler AD, et al.
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Angiotensin II contributes to podocyte injury by increasing TRPC6 expression vi
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a an NFAT-mediated positivefeedback signaling pathway. Am J Pathol. 2011;
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179:1719-32.
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Figure legends
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Figure 1.
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(A) Representative immunostaining images in cardiomyocytes and statistical graph
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showeing relative cell size. *P<0.05. (B) and (C) The mRNA and protein expression
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of ANP, BNP and β-MHC were measured using qPCR and western blotting analysis.
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*P<0.05. (D) Cardiomyocytes were treated with Ang II and nicotine and 3[H]-leucine
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incorporation assay was performed. *P<0.01.
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Nicotine induces hypertrophy of rat neonatal cardiac myocytes
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Figure 2.
Nicotine induces cardiomyocyte hypertrophy through a transient
486
increase in cytosolic calcium
487
(A) Average relative [Ca2+]cyt transients, and (B) peak relative [Ca2+]cyt response in
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cardiomyocytes with increased concentration of nicotine. *P<0.01. (C-F) Intracellular
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calcium chelation (BAPTA-AM) abolished nicotine-induced expression of ANP, BNP,
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and β-MHC in cardiomyocytes. *P<0.01.
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Nicotine induces SOCE and Ca2+ influx through TRPC
Figure 3.
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(A) Representative Ca2+ traces in cardiomyocytes with repeated nicotine stimulation.
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(B) Nicotine induced [Ca2+]i in cardiomyocytes. Nicotine (100 nM) was applied 3-4
495
min in the absence of extracellular Ca2+ and then Ca2+ was added as indicated. (C)
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Traces of calcium in cardiomyocytes treated with nicotine with or without αBtx (100
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nM) and U73122 (50 mM). (D) Thapsigargin-stimulated SOCE was measured upon a
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change from Ca2+-free conditions to 2 mM Ca2+. (E) Traces of calcium in
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cardiomyoctes treated with nicotine or TG, and for cells treated with 2APB or Gd3+.
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(F) Nifedipine, KB-R7943 and SKF-96365 have distinct effect on Ca2+ transients.
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Figure 4.
Effects of nicotine on the expression of TRPC homologs in
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cardiomyocytes.
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(A) Detection of mRNA expression of TRPCs in cardiomyocytes treated with nicotine.
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(B) The expression of TRPC3 was increased with the increase of incubation time. (C)
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Western bloting analysis of TRPC3 expression in nicotine-treated cardiomyocytes. 17
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Figure 5. Inhibition of TRPC3 expression attenuates SOCE and hypertrophy in
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cardiomyocytes treated with nicotine.
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(A) SOCE was significantly decreased in siRNA-treated cardiomyocytes compared to
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the control. (B)The results of siRNA and nsRNA-treated cardiomyocytes are
512
presented. *P<0.01. (C) mRNA and protein expression of ANP, BNP and β-MHC
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were significantly attenuated by knockdown of TRPC3. (D) The
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incorporation induced by nicotine was significantly abolished by knockdown of
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TRPC3. *P<0.01.
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[H]-leucine
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Figure 6. Role of TRPC3 in NFAT translocation and activation in
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cardiomyocytes
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(A, B) The nuclear GFP fluorescence intensity to cytoplasmic GFP fluorescence
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intensity ratio was significantly higher in cardiomyocytes exposed to nicotine than
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that in control. * P<0.01. (C, D) NFAT activation in cardiomyocytes was determined
522
by transfecting with an NFAT-luciferase reporter. NFAT-luc reporter stimulation by
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increased concentration of nicotine, and in the presence of nicotine receptor nAChR
524
inhibitor (αBtx) or a PLC inhibitor U73122, or SOCE blockers, ML9 (5 mM) and
525
2APB (50 mM). *P<0.05. (E, F) The translocation of NFAT and NFAT activation
526
were evaluated by knockdown of TRPC3. Average results are from three independent
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experiments. *P<0.05.
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Figure 7. Nicotine upregulates TRPC3 expression via calcineurin-NFAT signaling
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(A) NFAT activation in cardiomyocytes was determined by nicotine and in the
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presence of calcineurin inhibitor cyclosporine A (CsA, 200 nM). (B) TRPC3 promoter
532
activity was evaluated by calcineurin inhibitor CsA and NFAT silencing. (C, D) The
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effects of CsA and NFAT silencing on TRPC3 expression in nicotine-treated
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cardiomyocytes were examined by qPCR and western blot analysis. (E) NFAT activity
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was evaluated in cardiomyocytes transfected with TRPC3 constructor or empty vector
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in the presence or absence of CsA. (F) Schematic of nicotine-induced NFAT 18
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2+ / NFAT
Na Li, MD, PhD, Biao Si, MD, Ji-Feng Ju, MD, Meng Zhu, MD, Feng You, MD, Dong Wang, MD, Jie Ren, MD, Yan-Song Ning, MD, Feng-Quan Zhang, MD, Kai Dong, MD, PhD, Jing Huang, MD, PhD, Wen-Qian Yu, MD, PhD, Tong-Jian Wang, MD, PhD, Bin Qiao, MD, PhD PII:
S0828-282X(15)01686-4
DOI:
10.1016/j.cjca.2015.12.015
Reference:
CJCA 1965
To appear in:
Canadian Journal of Cardiology
Received Date: 17 August 2015 Revised Date:
11 November 2015
Accepted Date: 2 December 2015
Please cite this article as: Li N, Si B, Ju J-F, Zhu M, You F, Wang D, Ren J, Ning Y-S, Zhang F-Q, Dong K, Huang J, Yu W-Q, Wang T-J, Qiao B, Nicotine induces cardiomyocyte hypertrophy through 2+ TRPC3-mediated Ca / NFAT signaling pathway, Canadian Journal of Cardiology (2016), doi: 10.1016/ j.cjca.2015.12.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Nicotine
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TRPC3-mediated Ca2+/ NFAT signaling pathway
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Na Li1, MD, PhD, Biao Si1, MD, Ji-Feng Ju1, MD, Meng Zhu1, MD, Feng You1, MD,
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Dong Wang1, MD, Jie Ren1, MD, Yan-Song Ning1, MD, Feng-Quan Zhang1, MD,
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Kai Dong1, MD, PhD, Jing Huang1, MD, PhD, Wen-Qian Yu1, MD, PhD, Tong-Jian
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Wang1,*, MD, PhD, Bin Qiao1,*MD, PhD
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1
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China
induces
cardiomyocyte
hypertrophy
through
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Institute of Cardiovascular Disease, General Hospital of Jinan Military Region, Jinan,
*Correspondence should be addressed to:
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Dr. Bin Qiao, M.D., Ph.D.
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Institute of Cardiovascular Disease, General Hospital of Jinan Military Region, Jinan,
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China
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Tel: +86 13905311870;
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Fax: +86 0531 51636718
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Email: [email protected]
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Dr.Tong-Jian Wang, M.D., Ph.D.
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Institute of Cardiovascular Disease, General Hospital of Jinan Military Region, Jinan,
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China
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Email: [email protected]
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Short title: Nicotine induces cardiomyocyte hypertrophy.
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Brief Summary
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We demonstrate that nicotine could significantly promote cardiomyocyte
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hypertrophy through TRPC3-mediated Ca2+ influx and calcineurin-NFAT signaling
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activation. Our findings implicate the pro-hypertrophic effect of nicotine on
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cardiomyocytes
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calcium-dependent regulatory loop driving cardiomyocyte hypertrophy, which could
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become a potential target for prevention and treatment of cardiac hypertrophy.
dependent
on
enhanced
TRPC3
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Background: Nicotine is thought to be an important risk factor for development of
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cardiovascular diseases. However, the effects of nicotine on cardiomyocyte
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hypertrophy are poorly understood. The present study was designed to explore the
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role of nicotine in cardiomyocyte hypertrophy and its underlying mechanism.
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Methods: We used primary cardiomyocytes isolated from Wistar rats to examine the
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effects of nicotine on intracellular Ca2+ mobilization and hypertrophy determined by
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immunofluorescence, qPCR and western blot analysis. Luciferase reporter assay was
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used to examine the activity of NFAT signaling.
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Results: We found that nicotine caused cardiomyocyte hypertrophy, which was
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accompanied with an increased intracellular Ca2+. Nicotine-enhanced intracellular
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Ca2+ concentration ([Ca2+]i) was significantly abolished by SOCE and TRPC
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inhibitors. Knockdown of TRPC3 significantly decreased nicotine-induced SOCE and
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hypertrophy. Moreover, calcineurin-NFAT is involved in TRPC3-mediated Ca2+
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signaling and cardiomyocyte hypertrophy. Notably, upregulation of TRPC3 by
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nicotine requires TRPC3-mediated Ca2+ influx and calcineurin-NFAT signaling
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activation.
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Conclusion: Our findings demonstrate that the pro-hypertrophic effect of nicotine on
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cardiomyocytes
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calcium-dependent regulatory loop, which could become a potential target for
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prevention and treatment of cardiac hypertrophy.
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Key words: Nicotine; cardiac myocytes; hypertrophy; TRPC3; calcium; NFAT
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Introduction Cigarette smoking, as an important risk factor, is responsible for development of
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diverse diseases such as obesity, atherosclerosis, and cardiovascular disease [1-3].
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Nicotine is the most addictive component of cigarette smoke and has been found to
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have a wide range of effects [4]. However, the role of nicotine in cardiovascular
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disease is not completely understood.
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Cardiac hypertrophy is a severe cardiovascular disease, characterized by an
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increase in left ventricular mass and shape, which has been established as an
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important risk factor for cardiac mortality [5, 6]. Therefore, understanding the
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molecular mechanisms involved in cardiac hypertrophy is of great importance.
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However, the molecular mechanisms contributing to the initiation of cardiac
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hypertrophy are just beginning to be understood. Previous studies have revealed that
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intracellular calcium signaling plays a central role to induce cardiac hypertrophy [7,
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8]. Myocytes treated by agonists such as angiotensin II (Ang II) or endothelin-1 (ET-1)
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result in a remarkable increase in intracellular Ca2+, and the augmentation in
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intracellular Ca2+ is thought to be closely involved in Ca2+-calmodulin-dependent
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phosphatase calcineurin (Cn)-NFAT activation, and then leads to initiate the genes
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expression in cardiac hypertrophy[9, 10]. Recent studies show that calcineurin
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activation is dependent on the Ca2+ influx through transient receptor potential (TRP)
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proteins [11, 12].
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TRP channels belong to the nonselective cation influx channels that are grouped
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into 7 families, responsible for receptor activated Ca2+ entry and store operated Ca2+
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entry (SOCE)[11,13]. Overexpression of TRP channels can initiate cardiac
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hypertrophy because of increased Ca2+ influx and calcineurin activation [12, 14].
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Once activated, TRP channels induce signal transduction through cytoplasmic Ca2+
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elevations or endoplasmic reticulum Ca2+ release to activate signaling events [15, 16].
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Most recently, some studies suggest that TRP channels are required for cardiac
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hypertrophy [12]. However, it remains unknown how TRPC channels and associated
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Ca2+ influx functioned in the development of cardiomyocyte hypertrophy.
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In this study, we aimed to clarify whether nicotine contributes to cardiac 4
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hypertrophy by regulating TRPC expression and Ca2+ signaling, and to explore the
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effects of nicotine on TRPC expression, SOCE and cardiac hypertrophy in cultured rat
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cardiomyocytes to elucidate the potential mechanisms of cardiac hypertrophy.
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Materials and Methods
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Ethics statement
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This work was approved by the Clinical Research Ethics Committee of General
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Hospital of Jinan Military Region. All the procedures were performed according to
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the “Guide for the Care and Use of Laboratory Animals” published by National
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Institutes of Health.
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Cell isolation and culture
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Cardiomyocytes were obtained from 2-day-old Wistar rats as previously
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described [17]. The detailed procedures of isolation and culture are described in the
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online supplementary materials.
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Cell size measurement
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The isolated cardiomyocytes were incubated with 100 nM Ang-II or increased
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concentrations of nicotine with or without BAPTA-AM, an intracellular calcium
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chelator. Cells were then subjected to immunofluorescence analysis. For detailed
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procedures and data analysis, see online supplementary materials.
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Measurement of protein synthesis
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Cultured cardiomyocytes were transfected with si-TRPC3 or control siRNA, and
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treated with nicotine or not as indicated. 3[H]-leucine incorporation assay was used to
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evaluate the protein synthesis speed. The detailed procedures are described in the
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online supplementary materials.
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Quantitative real-time PCR 5
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The qPCR amplifications were carried out using SYBR Green detection chemistry. For detailed procedures and analysis, see online supplementary materials.
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Western blot analysis Western blot analysis was performed using standard procedures. The antibodies
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used in the western blot analysis and the detailed procedures are listed in the online
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supplementary materials.
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Measurement of Ca2+ concentration
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Cardiomyocytes were loaded with 5 µM Fura-2 (Life technology, NY, USA), and
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intracellular calcium levels was determined by the ratio of fluorescence at 340 nm and
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380 nm using Metafluor 7.0 software (Molecular Devices, CA, USA). The detailed
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procedures are described in the online supplementary materials.
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Immunofluorescence and confocal imaging
Cardiomyocytes were infected with adenovirus coding GFP-NFATc3. The
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localization of GFP-NFATc3 was determined with a Laser Scanning Confocal
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Imaging System (Carl Zeiss, Oberkochen, Germany) as described previously [18].
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The detailed procedures are described in the online supplementary materials.
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Luciferase Reporter Assay
Cardiomyocytes were co-transfected with 500 ng of pNFAT-Luc or TRPC3-Luc
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with 50 ng of pRL-SV40 control plasmid. The cells were incubated with nicotine, and
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luciferase activity was measured using the Dual Luciferase Reporter assay (Promega,
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Madison, WI, USA).
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Statistical analysis
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All data are expressed as mean ± SEM. A two-tailed Student’s t-test or
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Mann-Whitney test was used to determine differences between groups. All the
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statistical analyses were performed using SPSS 17.0 software, and the graphs were 6
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generated using GraphPad Prism 6.0 (Graphpad Software Inc, CA, USA). P<0.05
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was considered statistically significant.
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Results
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Nicotine induces cardiomyocyte hypertrophy
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We firstly retrospectively analyzed the patients with cardiac hypertrophy in our
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hospital, and found that the incidence is closely associated with cigarette smoking
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(Supplementary Table S1). Figure 1A showed the morphological change of
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cardiomyocytes exposed to nicotine and Ang-II. Cell size was increased after nicotine
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or Ang-II treatment. Furthermore, three important hypertrophic markers, ANP, BNP
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and β-MHC, were detected by qPCR and western blotting analysis. Figure 1B and C
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showed a dose-dependent increase of ANP, BNP and β-MHC expression induced by
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nicotine in cardiomyocytes. The 3[H]-leucine incorporation was significantly greater
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in Ang II and nicotine-treated cells than that in the control group (Figure 1D).
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Moreover, concurrent treatment with nicotine and Ang-II promotes more severe
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cardiomyocyte hypertrophy than either nicotine or Ang II on their own
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(Supplementary Figure 1A-E). These results suggest that nicotine can induce
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hypertrophic responses of cardiomyocytes.
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Nicotine initiates an intracellular calcium wave and calcium chelation inhibits
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nicotine-induced cardiomyocyte hypertrophy To assess the changes in calcium signaling in cardiomyocytes treated by nicotine,
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the ability of nicotine to increase [Ca2+]cyt in cardiomyocytes was evaluated. Nicotine
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induced a transient increase of [Ca2+]cyt in a dose dependent manner, and 100 nM of
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nicotine had the most significant effect (Figure 2A and B). To directly elucidate the
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function of calcium signaling in nicotine-induced cardiomyocyte hypertrophy,
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cardiomyocytes were pretreated with the calcium chelator. A significant decrease in
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nicotine-induced mRNA and protein expression of ANP, BNP and β-MHC was
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observed with calcium chelation (Figure 2C-F). Collectively, these results
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demonstrate a critical role of Ca2+ signaling in nicotine-induced cardiomyocyte
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hypertrophy.
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Nicotine activates SOCE and induces Ca2+ entry through TRPC in
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cardiomyocyte hypertrophy
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We further determined whether intracellular calcium was released from
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sarcoplasmic reticulum stores, extracellular influx, or both. Figure 3A shows that
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repeated nicotine (100 nM) could consistently increase [Ca2+]i in cardiomyocytes.
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Nicotine evoked a Ca2+ response with two components: a Ca2+ release from
217
intracellular stores and Ca2+ influx from extracellular medium (Figure 3B). Next, we
218
examined whether nicotinic acetylcholine receptor (nAChR) inhibitor α-bungarotoxin
219
(αBtx) or phospholipase C (PLC) inhibitor U73122 could inhibit nicotine-induced
220
Ca2+ increase. As shown in Figure 3C, both αBtx and U73122 inhibited
221
nicotine-induced Ca2+ release and extracellular Ca2+ influx. SOCE was activated by
222
depletion of intracellular Ca2+ stores using thapsigargin (TG, 1 µM) without
223
extracellular Ca2+. The peak increase in [Ca2+]i was significantly higher in
224
nicotine-induced cardiomyocytes compared to the control cells (Figure 3D). The
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TG-mediated Ca2+ increase was higher in nicotine-treated cardiomyocytes (Figure
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3D). Nicotine-evoked Ca2+ entry could be blocked with inhibitors of SOCE, Gd3+ and
227
2-aminoethoxydiphenyl borate (2APB) (Figure 3E). Cells were pre-treated with
228
SOCE blockers, ML9 (5 mM) and 2APB (50 mM), and then nicotine was added to
229
induce hypertrophy. As shown in Supplementary Figure S2A and B, ML9 and 2APB
230
significantly inhibited expression of hypertrophic markers, suggesting that nicotine
231
promotes cardiomyocytes hypertrophy through a significant increase of SOCE. To
232
evaluate what types of channels are involved in the induction of Ca2+ influx, the
233
effects of Nifedipine (a dihydropyridine L-type Ca2+ channel antagonist), KB-R7943
234
(Na+/Ca2+ exchanger (NCX) inhibitor) and SKF-96365 (TRPC blocker) were
235
evaluated. Ca2+ influx was not inhibited with the Nifedipine or KB-R7943 but was
236
inhibited with SKF-96365 (Figure 3F). These results demonstrate that TRPC channels
237
are responsible for the Ca2+ transient in nicotine-treated cardiomyocytes.
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Effects of nicotine on the expression of TRPC isoforms in cardiomyocytes We further examine whether nicotine affects the gene expression of TRPCs. Only
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TRPC3 mRNA expression increased in response to nicotine stimulation, while the
242
expression levels of TRPC1, 4, 5, 6 and 7 remained unchanged (Figure 4A). And
243
nicotine promotes TRPC3 mRNA expression with the increase of the incubation time
244
(Figure 4B). TRPC3 protein level had increased significantly after treatment with
245
nicotine (Figure 4C). Taken together, these findings demonstrated that nicotine could
246
promote TRPC3 expression in cardiomyocytes.
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TRPC3 exerts an important role in SOCE and cardiomyocytes hypertrophy
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Furthermore, we investigate whether knockdown of TRPC3 could attenuate
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nicotine-induced SOCE or hypertrophy in cardiomyocytes. We used siRNA to inhibit
251
endogenous TRPC3 proteins selectively. The inhibitory effect was determined after
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siRNA transfection (Supplementary Figure S3A and B). The peak increase in [Ca2+]i
253
was significantly decreased in siRNA-treated cardiomyocytes (Figure 5A and B). We
254
then examined whether overexpression of TRPC3 could increase calcium signaling or
255
hypertrophy. The TRPC3 expression was confirmed after TRPC3 vector transfection
256
(Supplementary Figure S3C and D). TRPC3 overexpression remarkably increased
257
nicotine-induced SOCE (Supplementary Figure S3E). Inhibition of TRPC3 expression
258
could significantly attenuate nicotine-induced expression of hypertrophic markers
259
(Figure 5C), while TRPC3 overexpression increased nicotine-induced hypertrophic
260
markers expression (Supplementary Figure S3F). The 3[H]-leucine incorporation was
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significantly lower in TRPC3 knockdown group (Figure 5D). Taken together, these
262
results demonstrate that TRPC3 molecules play an important role in nicotine-induced
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hypertrophic response.
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Role of TRPC3 in NFAT translocation and activation induced by nicotine in
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We tested the effect of nicotine on Ca2+-induced NFAT translocation (Figure 6A).
268
Exposure to nicotine significantly increased the intensity of nuclear GFP fluorescence
269
(Figure 6B). We then evaluated the efficacy of nicotine on NFAT activation in
270
cardiomyocytes transfected with an NFAT-luciferase reporter. Nicotine increased
271
NFAT
272
nicotine-induced NFAT activation was abolished by nicotine receptor nAChR
273
inhibitor (αBtx) and PLC inhibitor U73122, indicating that nAChR-mediated PLC
274
activation is involved in nicotine-induced NFAT activation (Figure 6D). We then
275
tested whether IP3 or DAG was involved in nicotine-induced NFAT activation.
276
Treatment with RHC80267, a DAG lipase inhibitor, significantly increased
277
nicotine-mediated nuclear NFAT translocation and activity. However, treatment with
278
xestospongin C (XestC), an IP3R blocker, did not have any effects on NFAT
279
translocation and activity (Supplementary Figure 4A and B). We next explored
280
whether SOCE could be associated with nicotine-induced NFAT activation. ML9 (5
281
mM) and 2APB (50 mM) could significantly attenuate nicotine-induced NFAT
282
activation, suggesting that nicotine-activated SOCE is responsible for NFAT
283
activation (Figure 6D). Nicotine increased the nuclear translocation of GFP-NFAT4
284
and NFAT activity, both of which were almost completely abolished by knockdown of
285
TRPC3 (Figure 6E and F). However, overexpression of TRPC3 could significantly
286
increase nicotine-induced NFAT activity (Supplementary Figure S5A and B),
287
demonstrating that Ca2+ influx through TRPC3 mediates nicotine-induced NFAT
288
activation. To further investigate whether calcineurin-NFAT signaling were involved
289
in nicotine-induced hypertrophy, calcineurin inhibitor cyclosporine A (CsA) was used.
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The data showed that CsA could significantly attenuate nicotine-induced hypertrophic
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markers expression (Supplementary Figure S6A-C). Moreover, CsA attenuated
292
nicotine-induced protein synthesis and cardiomyocytes size (Supplementary Figure
293
S6D and E). These results indicate that calcineurin-NFAT signaling pathway was
294
involved in nicotine-induced cardiomyocyte hypertrophy.
in
a
dose-dependent
manner
(Figure
6C).
Furthermore,
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Nicotine upregulates TRPC3 expression via calcineurin-NFAT signaling 10
ACCEPTED MANUSCRIPT Nicotine-induced NFAT activation could be significantly attenuated by calcineurin
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inhibitor cyclosporine A (CsA, 200 nM) (Figure 7A). We further found that CsA or
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NFAT kockdown could remarkably inhibit nicotine-induced TRPC3 promoter activity,
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suggesting that nicotine promoted TRPC3 promoter activity through NFAT signaling
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(Figure 7B). We demonstrated that calcineurin inhibition or NFAT silencing prevented
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nicotine-induced TRPC3 expression (Figure 7Cand D), suggesting that nicotine
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promoted TRPC3 expression through calcineurin-NFAT signaling. We further showed
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that in the cardiomyocytes, overexpression of TRPC3 resulted in enhanced NFAT
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activation. And in line with our hypothesis, CsA blocked the NFAT activation
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secondary to exogenous TRPC3 overexpression (Figure 7E), demonstrating a
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regulation loop between NFAT signaling and TRPC3 expression.
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Discussion
Cardiac hypertrophy is a severe cardiovascular disease, which is an adaptative
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reaction in response to a wide range of physiological and pathophysiological stimuli
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[19]. Treatment of the cardiac hypertrophy is thought to be an important therapeutic
313
target for prevention of heart failure. Here, we demonstrate that nicotine can induce
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cardiomyocyte hypertrophy. Nicotine stimulation significantly increases [Ca2+]i and
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SOCE that are greatly associated with nicotine-induced cardiomyocyte hypertrophy.
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We further demonstrate that nicotine promotes the TRPC3 expression, and reveal a
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deleterious positive feedback mechanism, in which TRPC3-mediated Ca2+ influx
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stimulates
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nicotine-associated cardiomyocyte hypertrophy.
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NFAT-dependent
TRPC3
expression,
which
is
involved
in
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Previous studies reported that chronic smoke exposure could induce the
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enlargement of the cardiac chambers and myocardial hypertrophy [20, 21]. Our
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retrospective data also revealed that cardiac hypertrophy was closely associated with
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cigarette smoking. The chronic effects of nicotine on whole organ or behavior were
324
mainly evaluated using animal or human study. Nevertheless, very few studies
325
focused on the effects of nicotine on isolated cells in vitro. We noticed that some
326
previous studies reporting that nicotine could induce cardiomyocyte apoptosis, while 11
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found high dose of nicotine, particularly at concentrations of 500 nM or higher, could
329
result in cell death of cultured cardiomyocytes. Meanwhile, we noticed that the high
330
concentrations of nicotine used in those studies were far exceeding the physiological
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concentration in cigarette smokers. Previous studies showed that the average peak
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concentration of nicotine was 35 ng/ml (about 200 nM), which then gradually
333
declined to a final concentration of 7 ng/ml [24]. Therefore, our study mainly aimed
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to investigate the effect of nicotine on cardiomyocytes at clinically relevant
335
concentrations.
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Several studies have shown that Ca2+-dependent signaling play an important role
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both in cardiac contractility and hypertrophy [10, 25]. We observe that
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nicotine-induced hypertrophy was calcium-dependent. Upon GPCRs activation,
339
intracellular Ca2+ is released from internal stores [26]; however, little is known about
340
the role of Ca2+ entry on [Ca2+]i increase in nicotine-induced hypertrophy. We
341
demonstrate that nicotine acts through nAChR and PLC signaling cascade and
342
induces [Ca2+]i increase through calcium entry and release. SOCE was activated by
343
depletion of intracellular Ca2+ using thapsigargin without extracellular Ca2+ [27].
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SOCE is closely associated with nicotine-induced cardiomyocyte hypertrophy. To
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evaluate what types of channels are involved in Ca2+ influx, the effects of Nifedipine,
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KB-R7943 and SKF-96365 were examined. Our data show that TRPC3 is responsible
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for nicotine-induced calcium increase. Importantly, TRPC3 plays an important role in
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nicotine-induced hypertrophic response.
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Previous report indicated that Ca2+ entry contributed to NFAT translocation, and
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NFAT activation was closely associated with pathological cardiomyocyte hypertrophy
351
[9, 28]. We show that Ca2+ influx through TRPC3 is contributing to activate Cn-NFAT
352
signaling to induce cardiomyocyte hypertrophy. Interestingly, we also showed that
353
nicotine was able to increase TRPC3 expression. Some previous studies revealed that
354
Ang II-mediated TRPC6 transcription appears to be controlled by the Ca2+-dependent
355
calcineurin/NFAT pathway, involving Ca2+ influx through TRPC6 itself [29]. We also
356
found that nicotine increased the TRPC3 expression through calcineurin-NFAT 12
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signaling, and overexpression of TRPC3 resulted in enhanced NFAT activation,
358
suggesting a regulation loop between NFAT signaling and TRPC3 expression.
359 360
Conclusions We have demonstrated for the first time that nicotine promoted cardiomyocyte
362
hypertrophy, and Ca2+ influx through TRPC3 is contributing to activate
363
calcineurin-NFAT signaling. Moreover, there is a positive regulatory loop in
364
nicotine-induced cardiomyocyte hypertrophy (Figure 7F), which could become a
365
potential target for prevention and treatment of cardiac hypertrophy.
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Funding Sources
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This study was supported by the National Natural Science Foundation of China
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(81501721)
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Disclosures
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The authors have no conflicts of interest to disclose.
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Figure legends
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Figure 1.
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(A) Representative immunostaining images in cardiomyocytes and statistical graph
480
showeing relative cell size. *P<0.05. (B) and (C) The mRNA and protein expression
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of ANP, BNP and β-MHC were measured using qPCR and western blotting analysis.
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*P<0.05. (D) Cardiomyocytes were treated with Ang II and nicotine and 3[H]-leucine
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incorporation assay was performed. *P<0.01.
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Nicotine induces hypertrophy of rat neonatal cardiac myocytes
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Figure 2.
Nicotine induces cardiomyocyte hypertrophy through a transient
486
increase in cytosolic calcium
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(A) Average relative [Ca2+]cyt transients, and (B) peak relative [Ca2+]cyt response in
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cardiomyocytes with increased concentration of nicotine. *P<0.01. (C-F) Intracellular
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calcium chelation (BAPTA-AM) abolished nicotine-induced expression of ANP, BNP,
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and β-MHC in cardiomyocytes. *P<0.01.
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Nicotine induces SOCE and Ca2+ influx through TRPC
Figure 3.
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(A) Representative Ca2+ traces in cardiomyocytes with repeated nicotine stimulation.
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(B) Nicotine induced [Ca2+]i in cardiomyocytes. Nicotine (100 nM) was applied 3-4
495
min in the absence of extracellular Ca2+ and then Ca2+ was added as indicated. (C)
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Traces of calcium in cardiomyocytes treated with nicotine with or without αBtx (100
497
nM) and U73122 (50 mM). (D) Thapsigargin-stimulated SOCE was measured upon a
498
change from Ca2+-free conditions to 2 mM Ca2+. (E) Traces of calcium in
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cardiomyoctes treated with nicotine or TG, and for cells treated with 2APB or Gd3+.
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(F) Nifedipine, KB-R7943 and SKF-96365 have distinct effect on Ca2+ transients.
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Figure 4.
Effects of nicotine on the expression of TRPC homologs in
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cardiomyocytes.
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(A) Detection of mRNA expression of TRPCs in cardiomyocytes treated with nicotine.
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(B) The expression of TRPC3 was increased with the increase of incubation time. (C)
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Western bloting analysis of TRPC3 expression in nicotine-treated cardiomyocytes. 17
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Figure 5. Inhibition of TRPC3 expression attenuates SOCE and hypertrophy in
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cardiomyocytes treated with nicotine.
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(A) SOCE was significantly decreased in siRNA-treated cardiomyocytes compared to
511
the control. (B)The results of siRNA and nsRNA-treated cardiomyocytes are
512
presented. *P<0.01. (C) mRNA and protein expression of ANP, BNP and β-MHC
513
were significantly attenuated by knockdown of TRPC3. (D) The
514
incorporation induced by nicotine was significantly abolished by knockdown of
515
TRPC3. *P<0.01.
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Figure 6. Role of TRPC3 in NFAT translocation and activation in
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cardiomyocytes
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(A, B) The nuclear GFP fluorescence intensity to cytoplasmic GFP fluorescence
520
intensity ratio was significantly higher in cardiomyocytes exposed to nicotine than
521
that in control. * P<0.01. (C, D) NFAT activation in cardiomyocytes was determined
522
by transfecting with an NFAT-luciferase reporter. NFAT-luc reporter stimulation by
523
increased concentration of nicotine, and in the presence of nicotine receptor nAChR
524
inhibitor (αBtx) or a PLC inhibitor U73122, or SOCE blockers, ML9 (5 mM) and
525
2APB (50 mM). *P<0.05. (E, F) The translocation of NFAT and NFAT activation
526
were evaluated by knockdown of TRPC3. Average results are from three independent
527
experiments. *P<0.05.
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Figure 7. Nicotine upregulates TRPC3 expression via calcineurin-NFAT signaling
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(A) NFAT activation in cardiomyocytes was determined by nicotine and in the
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presence of calcineurin inhibitor cyclosporine A (CsA, 200 nM). (B) TRPC3 promoter
532
activity was evaluated by calcineurin inhibitor CsA and NFAT silencing. (C, D) The
533
effects of CsA and NFAT silencing on TRPC3 expression in nicotine-treated
534
cardiomyocytes were examined by qPCR and western blot analysis. (E) NFAT activity
535
was evaluated in cardiomyocytes transfected with TRPC3 constructor or empty vector
536
in the presence or absence of CsA. (F) Schematic of nicotine-induced NFAT 18
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