Reduction of Na/K-Atpase Potentiates Marinobufagenin-Induced Cardiac Dysfunction and Myocyte Apoptosis Changxuan Liu

Marshall University Marshall Digital Scholar Biochemistry and Microbiology Faculty Research Spring 5-11-2012 Reduction of Na/K-ATPase potentiates marinobufagenin-induced cardiac dysfunction and myocyte apoptosis Changxuan Liu Yan Bai Yiliang Chen Yu Wang Yoann Sottejeau See next page for additional authors Follow this and additional works at: http://mds.marshall.edu/sm_bm Part of the Medical Biochemistry Commons Recommended Citation Liu C, Y Bai, Y Chen, Y Wang, Y Sottejeau, L Liu, X Li, JB Lingrel, D Malhotra, CJ Cooper, JI Shapero, ZJ Xie, and J Tian. 2012. Reduction of Na/K-ATPase potentiates marinobufagenin-induced cardiac dysfunction and myocyte apoptosis. J. Biol. Chem. 287:16390-8. This Article is brought to you for free and open access by the Faculty Research at Marshall Digital Scholar. It has been accepted for inclusion in Biochemistry and Microbiology by an authorized administrator of Marshall Digital Scholar. For more information, please contact [email protected]. Authors Changxuan Liu, Yan Bai, Yiliang Chen, Yu Wang, Yoann Sottejeau, Lijun Liu, Xiaomei Li, Jerry B. Lingrel, Deepak Malhorta, Christopher Cooper, Joseph I. Shapiro M.D., Zi-jian Xie, and Jiang Tian This article is available at Marshall Digital Scholar: http://mds.marshall.edu/sm_bm/113 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 20, pp. 16390–16398, May 11, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Reduction of Na/K-ATPase Potentiates Marinobufagenin-induced Cardiac Dysfunction and Myocyte Apoptosis* Received for publication, September 14, 2011, and in revised form, March 23, 2012 Published, JBC Papers in Press, March 26, 2012, DOI 10.1074/jbc.M111.304451 Changxuan Liu‡§, Yan Bai‡, Yiliang Chen‡, Yu Wang‡§, Yoann Sottejeau‡, Lijun Liu‡, Xiaomei Li§, Jerry B. Lingrel¶, Deepak Malhotraʈ, Christopher J. Cooperʈ, Joseph I. Shapiro‡ʈ, Zi-jian Xie‡ʈ, and Jiang Tianʈ1 From the ‡Department of Physiology and Pharmacology, University of Toledo, Toledo, Ohio 43614 the §Renal Division and Institute of Nephrology, Peking University First Hospital, Beijing 100034, China, ¶Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267, and the ʈDepartment of Medicine, University of Toledo, Toledo, Ohio 43614 Background: Na/K-ATPase decrease has been reported in patients with heart failure and is related to cardiac dysfunction. Results: Reducing Na/K-ATPase activates caspase 9 and induces cardiac dilation when treated with marinobufagenin. Conclusion: Reduction of Na/K-ATPase potentiates marinobufagenin-induced cardiac myocyte apoptosis. Significance: Decreased Na/K-ATPase content together with increased cardiotonic steroids levels is a novel mechanism that may account for cardiac dysfunction. Decreases in cardiac Na/K-ATPase have been documented in aging (3, 4), diabetes with hypertension (5–7), and neurological patients with heart failure. Reduction of Na/K-ATPase ␣1 also disorders (8, 9). In heart tissue biopsies from patients with contributes to the deficiency in cardiac contractility in animal dilated cardiomyopathy, the Na/K-ATPase amount is de- models. Our previous studies demonstrate that reduction of cel- creased by up to 40% (1). Reduction of cardiac Na/K-ATPase is lular Na/K-ATPase causes cell growth inhibition and cell death also related with decreased cardiac contractile function in in renal proximal tubule cells. To test whether reduction of humans and in animals (1, 10, 11). Previous reports showed that Na/K-ATPase in combination with increased cardiotonic ste- exposure of cells to low concentrations of cardiotonic steroids roids causes cardiac myocyte death and cardiac dysfunction, we (CTS)2 stimulates cell growth (12–14). Our recent work also examined heart function in Na/K-ATPase ␣1 heterozygote shows that binding of ouabain to Na/K-ATPase in pig renal ؉ ؊ knock-out mice (␣1 / ) in comparison to wild type (WT) litter- proximal tubule cells stimulates Src/Akt/mTOR signaling mates after infusion of marinobufagenin (MBG). Adult cardiac pathways and results in a stimulation of cell growth. Interest- ؉ ؊ myocytes were also isolated from both WT and ␣1 / mice for ingly, the stimulatory effects of CTS on cell growth are abol- in vitro experiments. The results demonstrated that MBG infu- ished when cellular Na/K-ATPase is reduced by siRNA (15). sion increased myocyte apoptosis and induced significant left In addition to the reduction of Na/K-ATPase, studies have ؉ ؊ ventricle dilation in ␣1 / mice but not in their WT littermates. found that levels of endogenous CTS increase in patients and in Mechanistically, it was found that in WT myocytes MBG activated experimental animals with volume expanded hypertension and the Src/Akt/mTOR signaling pathway, which further increased chronic renal disease (16–18). It was also found that endoge- phosphorylation of ribosome S6 kinase (S6K) and BAD (Bcl-2-as- nous ouabain-like compounds increased in patients with left sociated death promoter) and protected cells from apoptosis. In ventricular dysfunction due to dilated myocardium (19). ؉ ؊ ␣1 / myocytes, the basal level of phospho-BAD is higher com- Because myocyte death may play an important role in develop- pared with WT myocytes, but MBG failed to induce further activa- ment of myocardium dilation and heart failure (20), the current tion of the mTOR pathway. Reduction of Na/K-ATPase also caused study tested whether reduction of Na/K-ATPase potentiates the activation of caspase 9 but not caspase 8 in these cells. Using CTS-induced myocyte apoptosis and cardiac dysfunction. cultures of neonatal cardiac myocytes, we demonstrated that inhibition of the mTOR pathway by rapamycin also enabled MBG to EXPERIMENTAL PROCEDURES activate caspase 9 and induce myocyte apoptosis. Animals—All animal experimentation described in this article was conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals Reduction of Na/K-ATPase has been documented in patients Committee. All procedures were approved by the Institutional and experimental animals with congestive heart failure (1, 2), Animal Care and Use Committee at the University of Toledo. The Na/K-ATPase heterozygous (␣1ϩ/Ϫ) mice and their wild * This work was supported, in whole or in part, by National Institutes of type littermates were generated as described previously (11). Health, NHLBI Grants HL-105649, HL-109015, and HL-36573. This work was also supported by American Heart Association Grant 0980027N. 1 To whom correspondence should be addressed: Dept. of Medicine Cardio- 2 The abbreviations used are: CTS, cardiotonic steroids; MBG, marinobufage- vascular Div., University of Toledo, 3000 Arlington Ave., MS 1008, Toledo, nin; mTOR, the mammalian target of rapamycin; RIPA, radioimmune OH 43614. Tel.: 419-383-3923; Fax: 419-383-3821; E-mail: Jiang.tian@ precipitation assay; PI, propidium iodide; BAD, Bcl-2-associated death utoledo.edu. promoter. 16390 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 20•MAY 11, 2012 Na/K-ATPase and Cardiac Myocyte Apoptosis Genomic DNA was obtained from tail biopsies and used for 16,000 ϫ g for 5 min to remove dead cells. The supernatant PCR-based genotyping. Adult mice at 2ϳ3 months were used was transferred to a new tube for lactate dehydrogenase meas- for the study. All mice were kept in a 12-h dark/light cycle and urement using a commercial kit from Roche Applied Science. fed standard chow ad libitum. Experimental mice were anes- Rat neonatal myocytes grown on glass coverslips were used for thetized and an osmotic mini-pump from Alzet (Cupertino, annexin V staining and TUNEL assay. The annexin V staining CA) was placed under the skin to constantly infuse MBG at the kits was from Invitrogen. The manufacturer’s instructions were dose of 10 ␮g/kg/day. Mice infused with solvent only (50% followed for annexin V staining with minor modifications. dimethyl sulfoxide and 50% saline) were used as control. Dopp- Briefly, rat neonatal myocytes were stained with annexin V and ler imaging study was performed at the end of the 4th week of PI in annexin V assay buffer for 15 min. The cells were washed infusion as described before (21). Mice were sacrificed at the twice with PBS and slightly fixed with 2% paraformaldehyde for end of 4th week after echocardiographic measurements. Blood 15 min. These cells were then examined using a Leica confocal was collected, and the heart was then removed. The whole heart microscope (Buffalo Grove, IL) on the same day. TUNEL assay and isolated left ventricle were weighed. Afterward, half of the was performed on rat neonatal myocytes using a commercial kit left ventricle was snap frozen in liquid nitrogen, whereas the from Millipore (Billerica, MA) following the manufacturer’s other half was fixed in 4% formalin solution for histology anal- instructions. Treated cells were first fixed and permeabilized ysis. Tissue homogenates for biochemistry analysis were with cold methanol. TUNEL-positive cells were stained with obtained by grinding tissues in liquid nitrogen and then homog- streptavidin-FITC after terminal deoxynucleotidyl transferase enized in radioimmune precipitation assay buffer (50 mM Tris- incubation and examined using a Leica confocal microscope. PI HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% Nonidet is used for counterstaining the nuclei. P-40, 1 mM EDTA). RIPA buffer was supplemented with 1 mM Statistics—Continuous data are presented as the mean Ϯ S.E. PMSF, 1 mM sodium orthovanadate, and 1% protease inhibitor Statistical analysis was performed using the Student’s t test, and cocktails from Sigma before use. significance was accepted at p Ͻ 0.05. Adult Cardiac Myocyte Preparation from Mice—Ca2ϩ-toler- ant adult mouse cardiac myocytes were prepared from isolated RESULTS hearts as described previously by O’Connell et al. (22) with MBG Infusion Induces Myocyte Apoptosis, Cardiac Dysfunc- minor modifications. Briefly, mice were heparinized and anes- tion, Ventricular Dilation, and Reduced Wall Thickness in thetized with Nembutal (100–140 mg/kg) via intraperitoneal ␣1ϩ/Ϫ Mice—Decreased Na/K-ATPase and increased endoge- injection.

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