The Ubiquitin Ligase MuRF1 Protects Against Cardiac Ischemia/Reperfusion Injury by Its Proteasome-Dependent Degradation of Phospho-c-Jun
2011; Elsevier BV; Volume: 178; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2010.11.049
ISSN1525-2191
AutoresHuihua Li, Jie Du, Yong-Na Fan, Mei-Li Zhang, De‐Pei Liu, Luge Li, Pamela Lockyer, Eunice Kang, Cam Patterson, Monte S. Willis,
Tópico(s)Mitochondrial Function and Pathology
ResumoDespite improvements in interventions of acute coronary syndromes, primary reperfusion therapies restoring blood flow to ischemic myocardium leads to the activation of signaling cascades that induce cardiomyocyte cell death. These signaling cascades, including the mitogen-activated protein kinase signaling pathways, activate cardiomyocyte death in response to both ischemia and reperfusion. We have previously identified muscle ring finger-1 (MuRF1) as a cardiac-specific protein that regulates cardiomyocyte mass through its ubiquitin ligase activity, acting to degrade sarcomeric proteins and inhibit transcription factors involved in cardiac hypertrophy signaling. To determine MuRF1's role in cardiac ischemia/reperfusion (I/R) injury, cardiomyocytes in culture and intact hearts were challenged with I/R injury in the presence and absence of MuRF1. We found that MuRF1 is cardioprotective, in part, by its ability to prevent cell death by inhibiting Jun N-terminal kinase (JNK) signaling. MuRF1 specifically targets JNK's proximal downstream target, activated phospho-c-Jun, for degradation by the proteasome, effectively inhibiting downstream signaling and the induction of cell death. MuRF1's inhibitory affects on JNK signaling through its ubiquitin proteasome-dependent degradation of activated c-Jun is the first description of a cardiac ubiquitin ligase inhibiting mitogen-activated protein kinase signaling. MuRF1's cardioprotection in I/R injury is attenuated in the presence of pharmacologic JNK inhibition in vivo, suggesting a prominent role of MuRF1's regulation of c-Jun in the intact heart. Despite improvements in interventions of acute coronary syndromes, primary reperfusion therapies restoring blood flow to ischemic myocardium leads to the activation of signaling cascades that induce cardiomyocyte cell death. These signaling cascades, including the mitogen-activated protein kinase signaling pathways, activate cardiomyocyte death in response to both ischemia and reperfusion. We have previously identified muscle ring finger-1 (MuRF1) as a cardiac-specific protein that regulates cardiomyocyte mass through its ubiquitin ligase activity, acting to degrade sarcomeric proteins and inhibit transcription factors involved in cardiac hypertrophy signaling. To determine MuRF1's role in cardiac ischemia/reperfusion (I/R) injury, cardiomyocytes in culture and intact hearts were challenged with I/R injury in the presence and absence of MuRF1. We found that MuRF1 is cardioprotective, in part, by its ability to prevent cell death by inhibiting Jun N-terminal kinase (JNK) signaling. MuRF1 specifically targets JNK's proximal downstream target, activated phospho-c-Jun, for degradation by the proteasome, effectively inhibiting downstream signaling and the induction of cell death. MuRF1's inhibitory affects on JNK signaling through its ubiquitin proteasome-dependent degradation of activated c-Jun is the first description of a cardiac ubiquitin ligase inhibiting mitogen-activated protein kinase signaling. MuRF1's cardioprotection in I/R injury is attenuated in the presence of pharmacologic JNK inhibition in vivo, suggesting a prominent role of MuRF1's regulation of c-Jun in the intact heart. Factors that occlude the coronary arteries, such as thrombi and acute alterations in atherosclerotic plaques, result in myocardial ischemia. Advances in therapeutic interventions that restore the blocked coronary have had profound effects in limiting the size of the infarct by reducing the amount of frank necrosis in proportion to how quickly reperfusion takes place.1Maxwell S.R. Lip G.Y. 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Hypoxia/reoxygenation stimulates Jun kinase activity through redox signaling in cardiac myocytes.Circ Res. 1997; 80: 336-344Crossref PubMed Scopus (164) Google Scholar, 3Seko Y. Takahashi N. Tobe K. Kadowaki T. Yazaki Y. Hypoxia and hypoxia/reoxygenation activate p65PAK, p38 mitogen-activated protein kinase (MAPK), and stress-activated protein kinase (SAPK) in cultured rat cardiac myocytes.Biochem Biophys Res Commun. 1997; 239: 840-844Crossref PubMed Scopus (114) Google Scholar, 4Knight R.J. Buxton D.B. Stimulation of c-Jun kinase and mitogen-activated protein kinase by ischemia and reperfusion in the perfused rat heart.Biochem Biophys Res Commun. 1996; 218: 83-88Crossref PubMed Scopus (150) Google Scholar, 5Mizukami Y. Yoshioka K. Morimoto S. Yoshida K. A novel mechanism of JNK1 activation Nuclear translocation and activation of JNK1 during ischemia and reperfusion.J Biol Chem. 1997; 272: 16657-16662Crossref PubMed Scopus (162) Google Scholar, 6Yin T. Sandhu G. Wolfgang C.D. 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Tissue-specific pattern of stress kinase activation in ischemic/reperfused heart and kidney.J Biol Chem. 1997; 272: 19943-19950Crossref PubMed Scopus (331) Google Scholar, 9Sugden P.H. Clerk A. “Stress-responsive” mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium.Circ Res. 1998; 83: 345-352Crossref PubMed Scopus (441) Google Scholar These findings led to the hypothesis that inhibiting JNK activity would reduce apoptosis after I/R injury. Several studies have provided evidence to prove this concept true by using a nonpeptide ATP competitive JNK inhibitor10Ferrandi C. Ballerio R. Gaillard P. Giachetti C. Carboni S. Vitte P.A. Gotteland J.P. Cirillo R. Inhibition of c-Jun N-terminal kinase decreases cardiomyocyte apoptosis and infarct size after myocardial ischemia and reperfusion in anaesthetized rats.Br J Pharmacol. 2004; 142: 953-960Crossref PubMed Scopus (144) Google Scholar and a peptide inhibitor of c-Jun.11Milano G. Morel S. Bonny C. Samaja M. von Segesser L.K. Nicod P. Vassalli G. A peptide inhibitor of c-Jun NH2-terminal kinase reduces myocardial ischemia-reperfusion injury and infarct size in vivo.Am J Physiol Heart Circ Physiol. 2007; 292: H1828-H1835Crossref PubMed Scopus (89) Google Scholar In these studies, inhibition of JNK or the more specific inhibition of c-Jun was found to be cardioprotective in vivo. We recently found that muscle ring finger-1 (MuRF1) blocks pathological cardiac hypertrophy by inhibiting pro-hypertrophic signaling by directly binding and inhibiting the transcription factor serum response factor.12Willis M.S. Ike C. Li L. Wang D.Z. Glass D.J. Patterson C. Muscle ring finger 1, but not muscle ring finger 2, regulates cardiac hypertrophy in vivo.Circ Res. 2007; 100: 456-459Crossref PubMed Scopus (146) Google Scholar We have also found that MuRF1 is necessary for the development of cardiac atrophy and cardiac hypertrophy reversal.13Willis M.S. Rojas M. Li L. Selzman C.H. Tang R.H. Stansfield W.E. Rodriguez J.E. Glass D.J. Patterson C. Muscle ring finger 1 mediates cardiac atrophy in vivo.Am J Physiol Heart Circ Physiol. 2009; 296: H997-H1006Crossref PubMed Scopus (84) Google Scholar MuRF1 is a cardiac ubiquitin ligase localized to the cytoplasm14Arya R. Kedar V. Hwang J.R. McDonough H. Li H.H. Taylor J. Patterson C. Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy.J Cell Biol. 2004; 167: 1147-1159Crossref PubMed Scopus (100) Google Scholar and the M-line of the sarcomere,15Mrosek M. Labeit D. Witt S. Heerklotz H. von Castelmur E. Labeit S. Mayans O. Molecular determinants for the recruitment of the ubiquitin-ligase MuRF-1 onto M-line titin.FASEB J. 2007; 21: 1383-1392Crossref PubMed Scopus (80) Google Scholar where it targets local proteins such as troponin I, β/slow myosin heavy chain, and myosin binding protein-C for degradation.16Kedar V. McDonough H. Arya R. Li H.H. Rockman H.A. Patterson C. Muscle-specific RING finger 1 is a bona fide ubiquitin ligase that degrades cardiac troponin I.Proc Natl Acad Sci U S A. 2004; 101: 18135-18140Crossref PubMed Scopus (271) Google Scholar, 17Fielitz J. Kim M.S. Shelton J.M. Latif S. Spencer J.A. Glass D.J. Richardson J.A. Bassel-Duby R. Olson E.N. Myosin accumulation and striated muscle myopathy result from the loss of muscle RING finger 1 and 3.J Clin Invest. 2007; 117: 2486-2495Crossref PubMed Scopus (199) Google Scholar, 18Cohen S. Brault J.J. Gygi S.P. Glass D.J. Valenzuela D.M. Gartner C. Latres E. Goldberg A.L. During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation.J Cell Biol. 2009; 185: 1083-1095Crossref PubMed Scopus (438) Google Scholar This degradation occurs through the coordinated placement of polyubiquitin chains on recognized substrates, which are subsequently degraded by the proteasome.19Willis M.S. Schisler J.C. Patterson C. Appetite for destruction: e3 ubiquitin-ligase protection in cardiac disease.Future Cardiol. 2008; 4: 65-75Crossref PubMed Scopus (20) Google Scholar MuRF1 exerts its regulation of a number of cellular processes through its ubiquitin ligase activity and is involved in monitoring the protein quality control of the cardiomyocyte.20Willis M.S. Schisler J.C. Portbury A.L. Patterson C. Build it up-tear it down: protein quality control in the cardiac sarcomere.Cardiovasc Res. 2009; 81: 439-448Crossref PubMed Scopus (122) Google Scholar While previous studies have identified several good candidates using in vitro approaches, the identification of the physiological in vivo targets of MuRF1 is still ongoing. Activation of MAPK signaling pathways occurs in response to increased oxidative stress, inflammatory mediators, and stretch, including focal adhesion kinase and stretch activated channels in cardiac myocytes.21Lammerding J. Kamm R.D. Lee R.T. Mechanotransduction in cardiac myocytes.Ann N Y Acad Sci. 2004; 1015: 53-70Crossref PubMed Scopus (142) Google Scholar In the present study, we identify a role of cardiac MuRF1 in the protection against I/R injury by inhibiting JNK signaling by its specific interaction with and subsequent degradation of JNK's proximal effector c-Jun. MuRF1 does this by preferentially recognizing and ubiquitinating the activated (phosphorylated) c-Jun, which is targeted for degradation by the 26S proteasome to effectively inhibit downstream signaling. With use of in vivo models of ischemia reperfusion injury, we identify that increasing MuRF1 inhibits cardiomyocyte apoptosis induced by I/R injury by blocking JNK signaling through c-Jun, resulting in significant cardioprotection. These findings represent a novel mechanism by which the cardiac ubiquitin ligase MuRF1 coordinates the ubiquitin proteasome system to regulate the JNK signaling pathway in response to stress-mediated stimuli. The MuRF1 Tg+ mice used in this study were previously described.22Willis M.S. Schisler J.C. Li L. Rodriguez J.E. Hilliard E.G. Charles P.C. Patterson C. Cardiac muscle ring finger-1 increases susceptibility to heart failure in vivo.Circ Res. 2009; 105: 80-88Crossref PubMed Scopus (80) Google Scholar All animal protocols were reviewed and approved by the University of North Carolina Institutional Animal Care Advisory Committee and were in compliance with the rules governing animal use as published by the National Institutes of Health. The full-length and truncated forms of MuRF1 and c-Jun were generated by PCR and subcloned into mammalian expression plasmid pCMV-TB3, pcDNA3.1, pEGFP-C1, or glutathione S-transferase (GST) fusion protein expression plasmid pGEX-KG as previously described.14Arya R. Kedar V. Hwang J.R. McDonough H. Li H.H. Taylor J. Patterson C. Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy.J Cell Biol. 2004; 167: 1147-1159Crossref PubMed Scopus (100) Google Scholar, 16Kedar V. McDonough H. Arya R. Li H.H. Rockman H.A. Patterson C. Muscle-specific RING finger 1 is a bona fide ubiquitin ligase that degrades cardiac troponin I.Proc Natl Acad Sci U S A. 2004; 101: 18135-18140Crossref PubMed Scopus (271) Google Scholar A luciferase reporter plasmid, AP1-Luciferase reporter, was from BD Clontech (Palo Alto, CA, generously provided by Dr. Chiwing Chow). MuRF1 small interfering RNA (siRNA) was generated using BD knockout RNA interference and cloned into pSIREN vector as previously described.14Arya R. Kedar V. Hwang J.R. McDonough H. Li H.H. Taylor J. Patterson C. Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy.J Cell Biol. 2004; 167: 1147-1159Crossref PubMed Scopus (100) Google Scholar The following primary and secondary antibodies were used: anti-Flag (M2, Sigma-Aldrich, St. Louis, MO); anti-HA (12CA5; Roche Diagnostics Corp., Basel, Switzerland); anti-GST (Amersham Pharmacia Biosciences, Piscataway, NJ); anti-Myc (9E10, Santa Cruz Biotechnology Inc., Santa Cruz, CA); anti-ubiquitin, anti-His, and anti-β-actin (Chemicon International Inc., Temecula, CA); anti-JNK, anti-phospho-JNK, anti-c-Jun, and anti-phospho-c-Jun (Cell Signaling Technology, Beverly, MA); and anti-mouse–conjugated and anti-rabbit–conjugated antibodies (Invitrogen Corp., Carlsbad, CA). Cycloheximide, MG132, E1, Ubc5C, and ubiquitin were purchased from Calbiochem (San Diego, CA). HEK293T and the H9C2 cardiomyocyte cell lines were obtained from American Type Culture Collection (Manassas, VA) and cultured as previously described.23Li H.H. Kedar V. Zhang C. McDonough H. Arya R. Wang D.Z. Patterson C. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex.J Clin Invest. 2004; 114: 1058-1071Crossref PubMed Scopus (352) Google Scholar Cycloheximide and MG132 were added at a final concentration of 50 μg/ml and 10 μmol/L, respectively. The luciferase reporter constructs were co-transfected using Lipofectamine 2000 (Invitrogen Corp.) with expression vectors carrying AP-1-Luc, c-Jun, MuRF-1 WT, and mutants into HEK293T cells or with MuRF1 siRNAs into H9C2 cells, and luciferase activity was measured as previously described.23Li H.H. Kedar V. Zhang C. McDonough H. Arya R. Wang D.Z. Patterson C. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex.J Clin Invest. 2004; 114: 1058-1071Crossref PubMed Scopus (352) Google Scholar The data represent the mean ± SEM of three independent experiments run in duplicate and normalized for β-gal activity. Cells were transduced after 24 hours with adenovirus and then challenged with a simulated I/R (SI/R) as previously reported.24Cai Z. Zhong H. Bosch-Marce M. Fox-Talbot K. Wang L. Wei C. Trush M.A. Semenza G.L. Complete loss of ischaemic preconditioning-induced cardioprotection in mice with partial deficiency of HIF-1 alpha.Cardiovasc Res. 2008; 77: 463-470Crossref PubMed Scopus (203) Google Scholar Briefly, cells were placed in an “ischemia buffer” for 60 minutes in a 5% CO2 to 2% O2 incubator (37°C) containing 118 mmol/L NaCl, 24 mmol/L NaHCO3, 1.0 mmol/L NaH2PO4, 2.5 mmol/L CaCl2-2H2O, 1.2 mmol/L MgCl2, 20 mmol/L sodium lactate, 16 mmol/L KCl, and 10 mmol/L 2-deoxyglucose (pH adjusted to 6.2). After 60 minutes, reperfusion was obtained by replacing the ischemic buffer with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a 5% CO2 incubator with room air. Cell viability was assessed by trypan blue exclusion assay and by flow cytometry using the LIVE/DEAD Fixable Dead Cell Stain Kit (Molecular Probes, Invitrogen Corp.) according to the manufacturer's instructions. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed according to the manufacturer's instructions (Apoptosis Detection System, fluorescein, catalog #G3250, Promega, Madison, WI). Western blot analysis of apoptosis was performed by using an anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA, Cat, #9661), which recognizes the endogenous levels of the large fragment (17/19 kDa) of activated caspase 3 resulting from cleavage adjacent to Asp175 as previously described.24Cai Z. Zhong H. Bosch-Marce M. Fox-Talbot K. Wang L. Wei C. Trush M.A. Semenza G.L. Complete loss of ischaemic preconditioning-induced cardioprotection in mice with partial deficiency of HIF-1 alpha.Cardiovasc Res. 2008; 77: 463-470Crossref PubMed Scopus (203) Google Scholar, 25Schwencke C. Schmeisser A. Walter C. Wachter R. Pannach S. Weck B. Braun-Dullaeus R.C. Kasper M. Strasser R.H. Decreased caveolin-1 in atheroma: loss of antiproliferative control of vascular smooth muscle cells in atherosclerosis.Cardiovasc Res. 2005; 68: 128-135Crossref PubMed Scopus (42) Google Scholar, 26Matsuoka S. Moriyama T. Ohara N. Tanimura K. Maruo T. Caffeine induces apoptosis of human umbilical vein endothelial cells through the caspase-9 pathway.Gynecol Endocrinol. 2006; 22: 48-53Crossref PubMed Scopus (8) Google Scholar The MAPK inhibitors U0126, SB203580, and SP600125 were used as previously described.27Xie P. Guo S. Fan Y. Zhang H. Gu D. Li H. Atrogin-1/MAFbx enhances simulated ischemia/reperfusion-induced apoptosis in cardiomyocytes through degradation of MAPK phosphatase-1 and sustained JNK activation.J Biol Chem. 2009; 284: 5488-5496Crossref PubMed Scopus (79) Google Scholar, 28Bennett B.L. Sasaki D.T. Murray B.W. O'Leary E.C. Sakata S.T. Xu W. Leisten J.C. Motiwala A. Pierce S. Satoh Y. Bhagwat S.S. Manning A.M. Anderson D.W. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase.Proc Natl Acad Sci U S A. 2001; 98: 13681-13686Crossref PubMed Scopus (2220) Google Scholar, 29Davidson S.M. Morange M. Hsp25 and the p38 MAPK pathway are involved in differentiation of cardiomyocytes.Dev Biol. 2000; 218: 146-160Crossref PubMed Scopus (97) Google Scholar, 30Yue T.L. Gu J.L. Wang C. Reith A.D. Lee J.C. Mirabile R.C. Kreutz R. Wang Y. Maleeff B. Parsons A.A. Ohlstein E.H. Extracellular signal-regulated kinase plays an essential role in hypertrophic agonists, endothelin-1 and phenylephrine-induced cardiomyocyte hypertrophy.J Biol Chem. 2000; 275: 37895-37901Crossref PubMed Scopus (176) Google Scholar The JNK inhibitor SP600125 (Anthra[1,9-cd]pyrazol-6(2H)-one; 1,9-pyrazoloanthrone; SAPK Inhibitor II) was purchased from EMD Chemicals, Inc. (Calbiochem, La Jolla, CA, product #420119). SP600125 (6 mg/kg dose dissolved in 100 μL dimethyl sulfoxide) was administered intraperitoneally to four wild-type and three MuRF1 Tg+ mice 2 hours before left anterior descending (LAD) coronary artery ligation and reperfusion as previously described.31Zhang J. Li X.X. Bian H.J. Liu X.B. Ji X.P. Zhang Y. Inhibition of the activity of Rho-kinase reduces cardiomyocyte apoptosis in heart ischemia/reperfusion via suppressing JNK-mediated AIF translocation.Clin Chim Acta. 2009; 401: 76-80Crossref PubMed Scopus (44) Google Scholar Immunoprecipitations and Western blot analysis was performed as previously described.23Li H.H. Kedar V. Zhang C. McDonough H. Arya R. Wang D.Z. Patterson C. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex.J Clin Invest. 2004; 114: 1058-1071Crossref PubMed Scopus (352) Google Scholar Briefly, HEK293T cells were co-transfected with Myc-MuRF1 and Flag-c-Jun expression vectors using FuGENE 6 (Roche Diagnostics Corp.). Tagged proteins were immunoprecipitated for 2 hours at 4°C with either anti-Myc or anti-FLAG using protein A/G agarose beads, washed, and analyzed by immunoblotting as previously described.23Li H.H. Kedar V. Zhang C. McDonough H. Arya R. Wang D.Z. Patterson C. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex.J Clin Invest. 2004; 114: 1058-1071Crossref PubMed Scopus (352) Google Scholar GST pull-down assays were performed as described.23Li H.H. Kedar V. Zhang C. McDonough H. Arya R. Wang D.Z. Patterson C. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex.J Clin Invest. 2004; 114: 1058-1071Crossref PubMed Scopus (352) Google Scholar Briefly, HEK293T cells were transfected with a Flag-c-Jun expression plasmid for 24 hours and then lysed for 30 minutes and pre-cleared with GST beads for 1 hour. Lysates were then incubated with either GST or GST-MuRF1 fusion proteins for 1 hour at 4°C. The bound glutathione-Sepharose beads were then washed four times with lysis buffer and analyzed by immunoblotting as previously described.23Li H.H. Kedar V. Zhang C. McDonough H. Arya R. Wang D.Z. Patterson C. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex.J Clin Invest. 2004; 114: 1058-1071Crossref PubMed Scopus (352) Google Scholar H9C2 cells were cultured and examined for MuRF1 or c-Jun expression by using immunostaining with anti-MuRF1 or anti-c-Jun antibodies, respectively, and appropriate secondary antibodies. Samples were observed using confocal microscopy (TCS SP2 laser-scanning spectral confocal system; Leica Microsystems, Wetzlar, Germany). To assess ubiquitination in vivo, HEK293T cells were transfected with expression vectors containing His-Ub, Flag-c-Jun, and Myc-MuRF-1 using FuGENE 6 (Roche Diagnostics Corp.). Protein lysates were immunoprecipitated and analyzed by Western blot using appropriate antibodies as previously described.23Li H.H. Kedar V. Zhang C. McDonough H. Arya R. Wang D.Z. Patterson C. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex.J Clin Invest. 2004; 114: 1058-1071Crossref PubMed Scopus (352) Google Scholar HEK293T cells were transfected with His-Ub, Flag-c-Jun, and Myc-MuRF-1 WT or RING deletion mutant Myc-MuRF1 (MuRF1 Δ RING) expression plasmids and lysed in Ni-agarose lysis buffer [50 mmol/L NaH2PO4, 300 mmol/L NaCl, 5 mmol/L imidazole, 0.05% Tween 20, 10 mmol/L N-ethylmaleimide, and Complete Protease Inhibitor (Roche Diagnostics Corp.)]. His-ubiquitin-conjugated proteins were purified by nickel chromatography and subjected to Western blot analysis using anti-His or anti-Flag antibody as previously described.32Li H.H. Willis M.S. Lockyer P. Miller N. McDonough H. Glass D.J. Patterson C. Atrogin-1 inhibits Akt-dependent cardiac hypertrophy in mice via ubiquitin-dependent coactivation of Forkhead proteins.J Clin Invest. 2007; 117: 3211-3223Crossref PubMed Scopus (210) Google Scholar Ubiquitination reactions in vitro were performed as previously described.23Li H.H. Kedar V. Zhang C. McDonough H. Arya R. Wang D.Z. Patterson C. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex.J Clin Invest. 2004; 114: 1058-1071Crossref PubMed Scopus (352) Google Scholar Briefly, the reaction mixture (final volume 30 μl) containing 50 mmol/L Tris-HCl (pH 7.4), 5 mmol/L MgCl, 2 mmol/L NaF, 10 nmol/L okadaic acid, 2 mmol/L ATP, 0.6 mmol/L dithiothreitol, 60 ng of E1, 600 ng of Ubc5C, 1 μg of purified GST–MuRF1 Wt or RING deletion mutant, 1 μg purified c-Jun, and 10 μg of ubiquitin was incubated at 30°C for 2 hours and terminated by boiling in SDS–sample buffer containing 0.1 M dithiothreitol for 5 minutes. Samples were resolved by SDS-polyacrylamide gel electrophoresis and analyzed by Western immunoblot. Hearts from MuRF1 Tg+ and wild-type mice were isolated and perfused as previously described.33Hampton T.G. Amende I. Fong J. Laubach V.E. Li J. Metais C. Simons M. Basic FGF reduces stunning via a NOS2-dependent pathway in coronary-perfused mouse hearts.Am J Physiol Heart Circ Physiol. 2000; 279: H260-H268PubMed Google Scholar, 34Hampton T.G. Amende I. Travers K.E. Morgan J.P. Intracellular calcium dynamics in mouse model of myocardial stunning.Am J Physiol. 1998; 274: H1821-H1827PubMed Google Scholar, 35Min J.Y. Hampton T.G. Wang J.F. DeAngelis J. Morgan J.P. Depressed tolerance to fluorocarbon-simulated ischemia in failing myocardium due to impaired [Ca(2+)] (i) modulation.Am J Physiol Heart Circ Physiol. 2000; 278: H1446-H1456PubMed Google Scholar Briefly, mice were anesthetized with pentobarbital and heparinized. Hearts were then quickly removed and placed in ice-cold buffer, followed by aortic cannulation for retrograde perfusion with a phosphate-free Krebs-Henseleit buffer (Sigma-Aldrich, K3753) supplemented with calcium chloride and sodium bicarbonate according to the manufacturer's recommendations. Cardiac function was followed using a balloon placed in the left ventricle, monitored using a pressure transducer, and analyzed using EverBeat system acquisition software (Mouse Specifics, Inc., Boston, MA). The hearts were stabilized for a period of at least 20 minutes, followed by a 15-minute period of no-flow ischemia, followed by a 20-minute period of reperfusion. Recovery of function was determined as a percentage of pre-ischemic function. I/R injury was performed by ligation of the LAD coronary artery for 30 minutes, followed by 24 hours of reperfusion. Briefly, mice were anesthetized with pentobarbital (45 mg/kg), intubated, and placed on a ventilator (tidal volume 200 μl, respiratory rate 120/minute, 100% oxygen). The chest cavity was opened by an incision of the left fourth intercostal space, and the pericardial sac was removed to visualize the LAD coronary artery. A 7-0 silk suture was passed underneath the LAD artery ∼1 to 2 mm below the left auricle and tied around a 1-mm length of polyethylene tubing (OD = 0.61 mm; Intramedic PE-10, Clay Adams, Parsippany, NJ) to produce myocardial blanching and ECG ST-segment elevation. After 30 minutes, blood flow was restored and the chest wall was then closed. Postoperatively, a dose of buprenorphine (0.05 mg/kg) was given and the mice were allowed to recover at 37°C. Echocardiography on mice was performed at baseline and after 24 hours of reperfusion on a VisualSonics Vevo 770 ultrasound biomicroscopy system as previously described.12Willis M.S. Ike C. Li L. Wang D.Z. Glass D.J. Patterson C. Muscle ring finger 1, but not muscle ring finger 2, regulates cardiac hypertrophy in vivo.Circ Res. 2007; 100: 456-459Crossref PubMed Scopus (146) Google Scholar Total RNA was isolated from heart using TRIzol reagent (Invitrogen Corp.) according to the manufacturer's protocols. Gene expression studies were performed by a two-step reaction to determine mRNA expression as previously described.12Willis M.S. Ike C. Li L. Wang D.Z. Glass D.J. Patterson C. Muscle ring finger 1, but not muscle ring finger 2, regulates cardiac hypertrophy in vivo.Circ Res. 2007; 100: 456-459Crossref PubMed Scopus (146) Google Scholar, 13Willis M.S. Rojas M. Li L. Selzman C.H. Tang R.H. Stansfield W.E. Rodriguez J.E. Glass D.J. Patterson C. Muscle ring finger 1 mediates cardiac atrophy in vivo.Am J Physiol Heart Circ Physiol. 2009; 296: H997-H1006Crossref PubMed Scopus (84) Google Scholar The PCR reaction mix included 1 μl of mouse-specific TaqMan probes for MuRF1 (Mm01188690_m1), MuRF2 (Mm 01292965_m1), MuRF3 (Mm 00491308_m1), or 18S (Hs99999901_s1) in triplicate (Applied Biosystems, Foster City, CA). Rat MuRF1 (Rn00590197_m1), MuRF2 (Rn01493339_m1), and MuRF3 (Rn01470046_m1) were assayed in H9C2 cells and normalized to 18S in a similar manner. The relative expression of mRNA was determined using 18S as an internal sample loading control. Data are presented as means ± SEM. Differences between groups were evaluated for statistical significance using Student's t-test. P values less than 0.05 were regarded as significant. We used an adenovirus-dependent gene delivery system14Arya R. Kedar V. Hwang J.R. McDonough H. Li H.H. Taylor J. Patterson C. Muscle ring finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy.J Cell Biol. 2004; 167: 1147-1159Crossref PubMed Scopus (100) Google Scholar to investigate the effects of MuRF1 on H9C2 cardiomyocyte viability after SI/R injury. Challenging cardiomyocytes with a 1-hour simulated ischemia followed by a 24-hour reperfusion induced an approximately 35% decrease in control cell viability (Figure 1A). With increased MuRF1 expression, cardiomyocytes were significantly protected from SI/R ch
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