Depressed expression of MuRF1 and MAFbx in areas remote

ORIGINAL CONTRIBUTIONDepressed expression of MuRF1and MAFbx in areas remote of recent myocardial infarction:a mechanism contributing to myocardial remodeling?Viviane M.Conraads •Christiaan J.Vrints •Inez E.Rodrigus •Vicky Y.Hoymans •Emeline M.Van Craenenbroeck •Johan Bosmans •Marc J.Claeys •Paul Van Herck •Axel Linke •Gerhard Schuler •Volker AdamsReceived:12August 2009/Revised:10September 2009/Accepted:16September 2009/Published online:27October 2009ÓSpringer-Verlag 2009Abstract Ventricular remodeling following myocardial infarction (MI)includes myocardial hypertrophy,a process requiring increased protein synthesis and sarcomere assembly.The anti-hypertrophic effect of MuRF1/MafBx,both muscle-specific E3-ubiquitin ligases,has been dem-onstrated in animal experiments and in cultured cardio-myocytes.We assessed MuRF1/MAFbx expression in myocardium remote of recently (\2weeks)infarcted regions (MI),compared with patients undergoing coronary artery bypass surgery,with normal systolic function and without previous infarction (control or Con).Left ventric-ular myocardial biopsies were obtained from the contra-lateral normal zone in MI (n =14)patients and from theCon (n =12)group.MuRF-1/MAFbx expression was assessed using RT-PCR and Western blot (WB).In addi-tion,the myocardial expression of TNF-a was measured (RT-PCR)and troponin I,b -myosin and phosphorylated Akt/Akt (pAkt/Akt)were quantified (WB).MuRF1and MAFbx expression (mRNA and protein level)were sig-nificantly reduced in biopsies from MI patients.TNF-a was significantly higher in MI and exhibited a negative corre-lation with MuRF1and MAFbx.The expression of tropo-nin I and cardiomyocyte size were increased in MI in comparison to Con,whereas b -myosin expression was not altered.When compared with Con,pAkt/Akt was elevated.The results of the present study suggest that the atrogenes MuRF1/MAFbx are involved in regulating the hypertro-phic response,characteristic of the early post-infarction remodeling phase.Reduced expression of MuRF1and MAFbx in the myocardium might permit hypertrophy,which is supported by the elevation of troponin I.A reg-ulatory role of TNF-a needs to be confirmed in further experiments.Keywords Heart failure ÁInfarction ÁRemodeling ÁAtrogenes ÁUbiquitin–proteasome systemIntroductionUnfavorable left ventricular remodeling often follows acute myocardial infarction (MI)and is a leading cause of chronic heart failure.The classical pattern of post-infarc-tion remodeling involves three phases;early compensatory concentric hypertrophy,a subsequent dilative phase lead-ing to eccentric hypertrophy,and the end-stage of pro-gressive wall thinning and dilation [1].Myocyte hypertrophy,apoptosis and cell necrosis,and alterations inV.M.Conraads (&)ÁC.J.Vrints ÁV.Y.Hoymans ÁE.M.Van Craenenbroeck ÁJ.Bosmans ÁM.J.Claeys ÁP.Van HerckDepartment of Cardiology,Antwerp University Hospital,Wilrijkstraat 10,2650Edegem,Belgium e-mail:Viviane.Conraads@uza.beV.M.Conraads ÁC.J.Vrints ÁV.Y.Hoymans ÁE.M.Van CraenenbroeckCenter of Cell Therapy and Regenerative Medicine,Antwerp University Hospital,Wilrijkstraat 10,2650Edegem,BelgiumI.E.RodrigusDepartment of Cardiac Surgery,Antwerp University Hospital,Wilrijkstraat 10,2650Edegem,BelgiumA.Linke ÁG.Schuler ÁV.Adams Department of Cardiology,University Leipzig-Heart Centre Leipzig,Leipzig,GermanyBasic Res Cardiol (2010)105:219–226DOI 10.1007/s00395-009-0068-5the extracellular matrix explain morphological and func-tional myocardial adaptations[20,25].On a molecular basis,abnormal calcium handling,resulting in depressed myocyte contractility and relaxation[16]and recurrence of the fetal genotype are pivotal[33].Reorganization of the contractile apparatus involves a switch of sarcomeric pro-teins to a fetal isoform(i.e.,downregulation of a-myosin heavy chain(MHC)in parallel with an increase in b-MHC expression).This process requires both de novo protein synthesis and degradation of pre-existing proteins.The ubiquitin–proteasome system(UPS)plays a crucial role in protein quality control.It masters the degradation of misfolded as well as long-lived proteins,such as components of the contractile apparatus of striated mus-cles[25,35].Proteins linked to four or more ubiquitin molecules are unfolded by the proteasome and cleaved into short peptides and amino acids.A series of energy-consuming reactions,involving ubiquitin-activating enzymes(E1),ubiquitin-conjugating enzymes(E2)and ubiquitin ligases(E3),is required to tag targeted proteins. Two E3ligases have been identified to play a critical role in the development of skeletal muscle atrophy[14].Both muscle ringfinger1(MuRF1)and muscle atrophy F-box (MAFbx)are expressed specifically in striated(skeletal and cardiac)muscle and are central players in the UPS regulated turnover of sarcomeric proteins.In the absence of human data,in vitro and animal experiments have provided controversial results with regard to the function of the UPS in left ventricular hypertrophy and remodel-ing.Indeed,several proteins that control hypertrophy (e.g.,calcineurin)have in fact been identified as targets of the UPS[9,21,22].On the other hand,hypertrophy itself requires increased protein synthesis,and hence,rigid quality control[25].In a rat model of chronic heart failure(7weeks after ligation of left coronary artery),we previously demon-strated increased myocardial expression of both MuRF1 and MAFbx and a clear increase in troponin I ubiquitina-tion and degradation in the remote area of the anterior wall [3].Myocardial overexpression of TNF-a and its ability to stimulate the in vitro expression of both MuRF1and MAFbx,also denominated as atrogenes(genes involved in atrophy),provided a new plausible mechanism in the process of adverse ventricular remodeling.The possible role of these atrogenes in the pathological remodeling process of human myocardium is largely unknown.In contrast to chronic heart failure,the early phase of post-infarction remodeling involves compensatory hypertrophy in non-injured contralateral areas.This pro-cess tends to normalize wall stress by maintaining a con-stant ratio of wall thickness to chamber radius[4,5,24].In the present study,we aimed to assess the myocardial expression of MuRF1/MAFbx,TNF-a,troponin I and b-myosin at the mRNA and protein level in human myo-cardial biopsies obtained from the non-infarcted region within2weeks after MI.In addition,Akt(protein kinase B),which has been implicated in the downstream tran-scriptional regulation of the presently studied atrogenes, was investigated.Thesefindings were compared with the results obtained in myocardial tissue from patients with coronary artery disease,referred for surgical revasculari-zation,but with normal systolic function and without a previous infarction.MethodsStudy designPatient enrollment took place at the Departments of Car-diology and Cardiac Surgery of the Antwerp University Hospital.Two patient groups were selected;patients post-recent ST-segment elevation MI(MI group)and a control group(Con group),consisting of patients with normal left ventricular systolic function,referred for coronary artery bypass surgery(CABG).Because of the limited amount of tissue that could be obtained,each group was divided into two subgroups, from which biopsies were processed either for mRNA (RT-PCR)or protein analysis(Western blotting).In the MI group(n=14,5anteroseptal,9inferoposterior infarctions),2–3left ventricular myocardial biopsies (retrograde aortic approach,Boston Scientific,T RexÒbiopsy forceps,104cm—7F)were obtained from the non-infarcted contralateral area during diagnostic left heart catheterization and coronary angiography.Patients were eligible for the study if MI had occurred within a maxi-mal time frame of2weeks before biopsy sampling and if only one significant coronary artery lesion([70%steno-sis)was involved supplying the infarcted region.In addition,all of these patients had been adequately treated with thrombolysis at the time of presentation,and clinical signs of heart failure were excluded.In the Con group (n=12),2–3transmural left ventricular biopsies were taken during surgery(Tru-cut biopsy needle,14G41/2’’). All of these patients presented with three-vessel disease and angina,but without prior acute coronary syndrome or infarction that resulted in depressed left ventricular function.The harvested tissue was immediately snap frozen in liquid nitrogen and stored at-80°C.A small part of the tissue was transferred in formaldehyde and embedded in paraffin.Left ventricular systolic function was assessed during left ventricular angiography.Technicians were blinded to patient information.The study complies with the Declaration of Helsinki and was approved by the localethics committee and written informed consent was obtained from all patients.RNA isolation and quantification of mRNA expression Total RNA was isolated from frozen cardiac biopsies (RNeasy,Qiagen,Hilden,Germany)and reverse tran-scribed into cDNA using random hexamers and Sensiscript reverse transcriptase(Qiagen,Hilden,Germany).An ali-quot of the cDNA was used for quantitative RT-PCR(IQ5 cycler,BioRad,Munich,Germany).The expression of specific genes was normalized to the expression of18S-rRNA.The following primers/probes and conditions were used:18S-rRNA:50-ATACAGGACTCTTTCGAGGCC C-30and50-CGGGACACTCAGCTAAGAGCAT-30at61°C annealing;MuRF1-primer:50-AGAACATCATCGACATC TACA-30and50-ACTCACTTTTCTTCTCATCCA-30at 55°C annealing;MuRF1TaqMan-probe:50-GAGGTGCC CACCTGCTCCAT-30;MAFbx-primer:50-GAAGAGCGG CAGTTTCGT-30and50-TGCCACTCAGGGATGTGA-30 at55°C annealing;MAFbx TaqMan-probe:50-TCCAA CAGCCGGACCACGTA-30;TNF-a-primer:50-CTTCTCC TTCCTGATCGTGGC-30and50-GGGTTTGCTACAAC ATGGGC-30at58°C annealing;TNF-a TaqMan-probe: 50-CGCCACCACGCTCTTCTGCCT-30.Quantification of protein expressionFrozen tissue samples were homogenized in lysis buffer [10]and protein expression was quantified by Western blot using specific antibodies to MuRF1,MAFbx(gen-erated in rabbits by our group as recently described[3]), troponin I and slow b-MHC(both obtained from Sigma, Deisenhofen,Germany),Akt and p-Akt(Ser473)(both obtained from Cell Signaling,Beverly,MA,USA).After incubation with a horseradish peroxidase-conjugated sec-ondary antibody,specific bands were visualized by enzymatic chemiluminescence(Super Signal West Pico, Pierce,Bonn,Germany)and densitometry was quantified using a1D scan software package(Scanalytics,Rockville, USA).Loading differences were controlled by re-probing the blot with an antibody against GAPDH(Hytest,Turku, Finland).Cardiac myocyte sizeMyocardial biopsies were formalinfixed and embedded into paraffin.Sections were cut(4-l m thick)and stained with hematoxylin and eosin to visualize tissue morphol-ogy.At least50randomly selected cells per experi-ment were used to determine cell surface area(Analysis software package,Soft-Imaging System GmbH,Mu¨nster, Germany).Slides were anonymized to the investigator.Statistical analysisValues are given as mean±SEM for all variables.Inter-group comparisons were performed with Mann–Whitney U test.Correlations were determined using Spearman’s rank correlation test.A probability value of\0.05was consid-ered statistically significant.All the statistics was per-formed using the GraphPad Instat software package Version3.01(GraphPad Software Inc.,La Jolla,USA). ResultsPatient characteristicsPatient demographics,left ventricular function,invasively measured left ventricular end-diastolic pressure(LVEDP) and medication are summarized in Table1.In both the Con and the MI group,LVEDP was significantly elevated, whereas left ventricular ejection fraction(LVEF)was within normal limits.Myocardial expression of MuRF1,MAFbx and TNF-aIn the MI group,myocardial mRNA transcription of MuRF1(Fig.1a,2.3?0.4vs.4.9?0.4arbitrary units; P\0.001)and MAFbx(Fig.1b,3.8?0.5vs.7.6?0.5 arbitrary units;P\0.001)was significantly lower com-pared with controls.There was a strong correlation between MuRF1and MAFbx mRNA expression(r=0.87, P\0.0001).Table1Patient characteristicsCon(n=12)MI(n=14)Age(years)66.1±2.659.6±2.2 Male1012LVEF(%)68±260±3 LVEDP(mmHg)24±326±2 Treatment(n)ACE-149Beta-blockers69 Diuretics31Statins74Data are expressed mean±SEMCon controls,MI post-recent myocardial infarction,LVEF left ven-tricular ejection fraction,LVEDP left ventricular end-diastolic pres-sure,ACE-I angiotensin-converting enzyme inhibitorIn parallel,protein expression of MuRF1and MAFbx was reduced by 64%(1.3±0.6vs.3.6±0.4arbitrary units,P \0.05,Fig.1c)and 86%(0.22±0.01vs.1.6±0.2,P \0.01,Fig.1d)respectively in MI versus Con patients.TNF-a mRNA transcription was strongly upregulated in the MI group (Fig.2a).There was a significant inverse relationship between TNF-a and both MuRF1and MAFbx transcription (Fig.2b,c).Fig.1Quantification of MuRF1(a )and MAFbx (b )mRNA expression as well as MuRF1(c )and MAFbx (d )protein expression inmyocardial biopsies obtained from controls (Con )and patients post-recent myocardialinfarction (MI ).The results are expressed as mean ±SEMIncreased expression of troponin IAlthough troponin I expression was significantly increased in biopsy specimens obtained from the non-infarcted region in MI patients (1.9±0.6vs.0.86±0.04arbitrary units,P \0.05,Fig.3a),b -MHC expression was compa-rable in both groups (3.6±1.2vs. 3.7±0.3arbitrary units,P =n.s.,Fig.3b).Downstream signaling pathwaysFor the MI group,the level of phosphorylation of Akt at Ser 473and the ratio of pAkt/total Akt were increased compared with controls (P =0.03,Fig.4).Cardiac myocyte sizeThe cell surface area of cardiac myocytes was significantly increased by 33%in the MI group when compared with controls (Fig.5;control:472±40l m 2vs.MI:631±42l m 2,P =0.02).DiscussionThe present study investigates the expression of the atr-ogenes MuRF1and MAFbx in human myocardial biopsies and their involvement in the early remodeling phase post-MI.Several interesting findings emerge from this study:1.The mRNA and protein expression of MuRF1and MAFbx were significantly downregulated in remote myocardial areas of patients that had suffered a recent MI.2.The reduced myocardial expression of MuRF1and MAFbx coincided with higher troponin I expression and increased myocyte size.3.TNF-a mRNA overexpression correlated inversely with MuRF1and MAFbxexpression.Fig.3Quantitative analysis of troponin I (a )and b -myosin (b )protein expression inmyocardial biopsies obtained from controls (Con )and patients post-recent myocardialinfarction (MI ).The results are expressed as mean ±SEMFig.4Quantitative analysis of the ratio pAkt/Akt in myocardial biopsies obtained from controls (Con )and patients post-recent myocardial infarction (MI ).The results are expressed as mean ±SEM4.The increase in pAkt/Akt may be involved in the downstream signaling pathway that affects MuRF1and MAFbx mRNA transcription.The pathophysiological role of MuRF1and MAFbx has been largely confined to diseases that involve peripheral skeletal muscle wasting [2,7,30]Only recently,in vitro and animal experiments have started exploring their pos-sible function in the process of ventricular remodeling.Calcineurin-dependent pathologic left ventricular hyper-trophy after thoracic aorta constriction (TAC)was pre-vented in transgenic mice overexpressing cardiac MAFbx [21].These authors later on demonstrated that MAFbx also controls physiologic cardiac hypertrophy by increasing FOXO1ubiquitination and upregulation of forkhead target genes [22].Agonist-mediated hypertrophy (i.e.;phenyl-ephrine,angiotensin II,endothelin-1)in neonatal rat ven-tricular myocytes is prevented by MuRF1by inhibiting the protein kinase C-mediated signaling response [6].Very recently,Willis et al.[36]demonstrated that MuRF1-/-mice,following TAC release,failed to show regression of TAC-induced cardiac hypertrophy.Besides,the degrada-tion of cardiac troponin I [18],MuRF1also binds to the sarcomeric protein titin,[14]whereas MAFbx interacts with calcineurin 1and alpha-actinin-2[21].On the otherhand,several investigators have demonstrated that the UPS is activated during cardiac hypertrophy [21]and that pre-cise control of the synthesis,assembly,and turnover of contractile proteins of the sarcomere is essential to main-tain structural integrity [12,13].We previously demonstrated in rats,which had devel-oped chronic heart failure 7weeks after left coronary artery ligation,that both MuRF1and MAFbx were upregulated and that MuRF1expression was clearly pro-portional to troponin I degradation in the area remote from the anterior wall [3].In contrast to chronic heart failure,the early phase of post-infarction remodeling involves com-pensatory hypertrophy in non-affected areas.The findings of the present study are concordant with the results of our animal model.Indeed,downregulated mRNA expression of both atrogenes,together with increased troponin I contents in areas remote of recently infarcted tissue,might provide a possible link between molecular biological changes and the clinical features of early post-infarction hypertrophy and remodeling.The observed increase in cardiomyocyte size in biopsies obtained after recent MI,compared with con-trols,supports this premise.Interestingly,despite a similar degree of diastolic dys-function and normal systolic performance in both the MI and Con group,MuRF1and MAFbx were significantly downregulated in the biopsies from the non-infarcted region of MI patients.These findings suggest that other than hemodynamic abnormalities initiate the signaling pathway that regulates the expression of these atrogenes.TNF-a ,as well as other pro-inflammatory cytokines,is implicated in the process of early post-infarction left ventricular remod-eling.Irwin et al.[17]demonstrated a significant increase in myocardial expression of TNF-a in an acute rat coronary artery ligation model.Notably,this increase was detected from day-1post-infarction and was most pronounced in the contralateral normal zone.In the semi-acute stages after infarction,TNF-a is thought to activate matrix metallo-proteinases and to provoke hypertrophic growth in cardiac myocytes,thereby setting the stage for compensatory ven-tricular remodeling [23].The fact that TNF-a upregulation correlated negatively with mRNA expression of both atr-ogenes in the present study supports this notion.At first glance,however,these findings contradict the results of our previous chronic heart failure rat model,in which a strong and positive correlation of TNF-a with MuRF1and MAFbx expression was demonstrated [3].Several reasons might account for the observed discrepancies.First,acute cardiac injury versus a more chronic condition could be relevant.The role of TNF-a in cardiovascular diseases is extremely complex.Depending on the magnitude and the acute versus chronic exposure to increased TNF-a ,the consequences at the level of the myocardium vary from adaptive to a path-ological response [27,29,32].Indeed,in instancesofFig.5Two representative hematoxylin–eosin staining of paraffin-embedded myocardial biopsies for the evaluation of myocyte surface area.Original magnification 940.a MI patient,b controlischemia,post-infarction remodeling and hemodynamic overload,TNF-a elicits myocyte hypertrophy and mainte-nance of cardiac contractility.As such,this process allows adaptation to an increased workload [26].On the other hand,however,chronic overexpression to TNF-a results in the development of the typical phenotype of dilated car-diomyopathy [19].Secondly,one cannot exclude that interspecies disparities explain the apparent different path-ophysiological roles of MuRF1and MAFbx.Furthermore,patients in the present study were pharmacologically trea-ted.Substances such as ACE inhibitors [28],as well as statins [34]have been demonstrated to modulate myocar-dial TNF-a expression.Finally,whereas the harvest of tissue in an animal experiment is straightforward,sampling sufficient human myocardial biopsies is a delicate matter.The lack of suf-ficient tissue amounts precluded the investigation of both mRNA and protein expression in the same patients.On the other hand,however,concordant findings in mRNA and protein expression in two subgroups with the same clinical presentation,strengthens our findings.Several forkhead transcription factors,including FOXO3,are inactivated by Akt [11].Both in vitro and in vivo,FOXO3has been demonstrated to regulate cardio-myocyte size by interfering with multiple atrophy-related genes,including MuRF1and MAFbx [31]Akt phosphor-ylation leads to nuclear exclusion and inhibition of FOXO3,thereby reducing transcription of both atrogenes and allowing cardiac hypertrophy.It has been demon-strated that TNF-a induces rapid and concentration-dependent activation of Akt in cardiac myocytes,leading to hypertrophy [8,15].Although partly speculative,on the basis of the present findings,we would like to propose the following hypo-thetical working model in Fig.6.LimitationsAlthough not studied previously,we cannot exclude dif-ferent expression profiles of the atrogenes assessed in endocardial (MI group)versus transmural (Con)tissue samples.Because patients allocated to the MI group had(as per protocol)single vessel disease,these were obvi-ously not candidates for surgery and hence,percutaneous pre-elevation of endocardial biopsies was the only option.The observed increase in the ratio of pAkt/Akt in MI versus Con is striking and fits well with the previously identified regulating role of the pAkt/FOXO3pathway on atrogene expression [31].Nevertheless,the present findings should be interpreted with caution since stress factors and drugs might have affected our results.Owing to tissue restrictions,we decided to quantify b -MHC only and found no significant difference between both patient groups.Analysis of a MHC and the ratio of both isoforms might have provided interesting additional information.In summary,our data suggest that the atrogenes,MuRF1and MAFbx,are involved in regulating the hypertrophic response,characteristic of the early post-infarction remodeling phase.Although a causal role remains to be confirmed,the inverse relation between TNF-a expression and both atrogenes in biopsies taken from the contralateral normal wall supports the premise that this pro-inflamma-tory cytokine might be the acute injury related mediator of the observed changes.Acknowledgment We would like to thank Nicole Urban and Tina Fischer for excellent technical assistance;and Siegfried Labeit for providing the MuRF1specific antibody.This work was supported by Fonds voor Wetenschappelijk onderzoek (FWO)-Flanders (VC,clinical postdoctoral fellow)and by the Deutsche Forschungsgeme-inschaft (LI946/3-1).Conflict of interest statementNone declared.References1.Abbate A,Biondi-Zoccai GG,Bussani R,Dobrina A,Camilot D,Feroce F,Rossiello R,Baldi F,Silvestri F,Biasucci LM,Baldi A (2003)Increased myocardial apoptosis in patients with unfavor-able left ventricular remodeling and early symptomatic post-infarction heart failure.J Am Coll Cardiol 41:753–7602.Adams V,Linke A,Gielen S,Erbs S,Hambrecht R,Schuler G (2008)Modulation of Murf-1and MAFbx expression in the myocardium by physical exercise training.Eur J Cardiovasc Prev Rehabil 15:293–2993.Adams V,Linke A,Wisloff U,Doring C,Erbs S,Krankel N,Witt CC,Labeit S,Muller-Werdan U,Schuler G,Hambrecht R (2007)Myocardial expression of Murf-1and MAFbx after induction of chronic heart failure:effect on myocardial contractility.Cardio-vasc Res 73:120–1294.Anversa P,Olivetti G,Capasso JM (1991)Cellular basis of ventricular remodeling after myocardial infarction.Am J Cardiol 68:7D–16D5.Anversa P,Olivetti G,Li P,Herman MV,Capasso JM (1993)Myocardial infarction,cardiac anatomy and ventricular loading.Cardioscience 4:55–626.Arya R,Kedar V,Hwang JR,McDonough H,Li HH,Taylor J,Patterson C (2004)Muscle ring finger protein-1inhibitsFig.6Working model for the regulation of cardiac hyperthrophy following myocardial infarctionPKC{epsilon}activation and prevents cardiomyocyte hypertro-phy.J Cell Biol167:1147–11597.Centner T,Yano J,Kimura E,McElhinny AS,Pelin K,Witt CC,Bang ML,Trombitas K,Granzier H,Gregorio CC,Sorimachi H, Labeit S(2001)Identification of muscle specific ringfinger proteins as potential regulators of the titin kinase domain.J Mol Biol306:717–7268.Condorelli G,Morisco C,Latronico MV,Claudio PP,Dent P,Tsichlis P,Frati G,Drusco A,Croce CM,Napoli C(2002)TNF-alpha signal transduction in rat neonatal cardiac myocytes:defi-nition of pathways generating from the TNF-alpha receptor.FASEB J16:1732–17379.Depre C,Wang Q,Yan L,Hedhli N,Peter P,Chen L,Hong C,Hittinger L,Ghaleh B,Sadoshima J,Vatner DE,Vatner SF, Madura K(2006)Activation of the cardiac proteasome during pressure overload promotes ventricular hypertrophy.Circulation 114:1821–182810.Dimmeler S,Assmus B,Hermann C,Haendeler J,Zeiher A(1998)Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells.Circ Res83:334–34111.Fang CX,Dong F,Thomas DP,Ma H,He L,Ren J(2008)Hypertrophic cardiomyopathy in high-fat diet-induced obesity: role of suppression of forkhead transcription factor and atrophy gene transcription.Am J Physiol Heart Circ Physiol295:H1206–H121512.Fielitz J,Kim MS,Shelton JM,Latif S,Spencer JA,Glass DJ,Richardson JA,Bassel-Duby R,Olson EN(2007)Myosin accu-mulation and striated muscle myopathy result from the loss of muscle RINGfinger1and3.J Clin Invest117:2486–2495 13.Fielitz J,van Rooij E,Spencer JA,Shelton JM,Latif S,van derNagel R,Bezprozvannaya S,de Windt L,Richardson JA,Bassel-Duby R,Olson EN(2007)Loss of muscle-specific RING-finger3 predisposes the heart to cardiac rupture after myocardial infarc-tion.Proc Natl Acad Sci USA104:4377–438214.Glass DJ(2005)Skeletal muscle hypertrophy and atrophy sig-naling pathways.Int J Biochem Cell Biol37:1974–198415.Hiraoka E,Kawashima S,Takahashi T,Rikitake Y,Kitamura T,Ogawa W,Yokoyama M(2001)TNF-alpha induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myo-cytes.Am J Physiol Heart Circ Physiol280:H1861–H1868 16.Houser SR,Margulies KB(2003)Is depressed myocyte con-tractility centrally involved in heart failure?Circ Res92:350–358 17.Irwin MW,Mak S,Mann DL,Qu R,Penninger JM,Yan A,Dawood F,Wen W,Shou Z,Liu P(1999)Tissue expression and immunolocalization of tumor necrosis factor-a in postinfarction dysfunctional myocardium.Circulation99:1492–149818.Kedar V,McDonough H,Arya R,Li HH,Rockman HA,Patt-erson C(2004)Muscle-specific RINGfinger1is a bonafide ubiquitin ligase that degrades cardiac troponin I.Proc Natl Acad Sci USA101:18135–1814019.Kubota T,McTiernan CF,Frye CS,Slawson SE,Lemster BH,Koretsky AP,Demetris AJ,Feldman AM(1997)Dilated car-diomyopathy in transgenic mice with cardiac-specific overex-pression of tumor necrosis factor-alpha.Circ Res81:627–635 uten A,Majos E,Mu¨hlich A,Wahlers T,Weider S,Fischer JH,Figulla HR,Bloch W(2009)Ischemia-reperfusion injury acti-vates early extracellular matrix processing and expression of endostatin in the heart with differential effects of temperature.Basic Res Cardiol104:559–56921.Li HH,Kedar V,Zhang C,McDonough H,Arya R,Wang DZ,Patterson C(2004)Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex.J Clin Invest114:1058–1071 22.Li HH,Willis MS,Lockyer P,Miller N,McDonough H,GlassDJ,Patterson C(2007)Atrogin-1inhibits Akt-dependent cardiac hypertrophy in mice via ubiquitin-dependent coactivation of forkhead proteins.J Clin Invest117:3211–322323.Mann DL(2002)Inflammatory mediators and the failing heart:past,present,and the foreseeable future.Circ Res91:988–998 24.Mann DL,Bristow MR(2005)Mechanisms and models in heartfailure:the biomechanical model and beyond.Circulation 111:2837–284925.Patterson C,Ike C,PWt Willis,Stouffer GA,Willis MS(2007)The bitter end:the ubiquitin–proteasome system and cardiac dysfunction.Circulation115:1456–146326.Sack MN,Smith RM,Opie LH(2000)Tumor necrosis factor inmyocardial hypertrophy and ischaemia:an anti-apoptotic per-spective.Cardiovasc Res45:688–69527.Sack M(2002)Tumor necrosis factor-alpha in cardiovascularbiology and the potential role for anti-tumor necrosis factor-alpha therapy in heart disease.Pharmacol Ther94:23–3528.Satoh M,Nakamura M,Akatsu T,Shimoda Y,Segawa I,Hira-mori K(2006)C-reactive protein co-expresses with tumor necrosis factor-alpha in the myocardium in human dilated car-diomyopathy.Eur J Heart Fail7:748–75429.Schulz R,Heusch G(2009)Tumor necrosis factor-alpha and itsreceptors1and2:Yin and Yang in myocardial infarction?Cir-culation119:1355–135730.Schulze PC,Fang J,Kassik KA,Gannon J,Cupesi M,MacGil-livray C,Lee RT,Rosenthal N(2005)Transgenic overexpression of locally acting insulin-like growth factor-1inhibits ubiquitin-mediated muscle atrophy in chronic left-ventricular dysfunction.Circ Res97:418–42631.Skurk C,Izumiya Y,Maatz H,Razeghi P,Shiojima I,Sandri M,Sato K,Zeng L,Schiekofer S,Pimentel D,Lecker S,Taegtmeyer H,Goldberg AL,Walsh K(2005)The FOXO3a transcription factor regulates cardiac myocyte size downstream of AKT sig-naling.J Biol Chem280:20814–2082332.Skyschally A,Gres P,Hoffmann S,Haude M,Erbel R,Schulz R,Heusch G(2007)Bidirectional role of tumor necrosis factor-alpha in coronary microembolization:progressive contractile dysfunction versus delayed protection against infarction.Circ Res 100:140–14633.Vanderheyden M,Mullens W,Delrue L,Goethals M,de BruyneB,Wijns W,Geelen P,Verstreken S,Wellens F,Bartunek J (2008)Myocardial gene expression in heart failure patients treated with cardiac resynchronization therapy responders versus nonresponders.J Am Coll Cardiol51:129–13634.Wallace CK,Stetson SJ,Kucuker SA,Becker KA,Farmer JA,McRee SC,Koerner MM,Noon GP,Torre-Amione G(2005)Sim-vastatin decreases myocardial tumor necrosis factor alpha content in heart transplant recipients.J Heart Lung Transplant24:46–51 35.Wang X,Robbins J(2006)Heart failure and protein qualitycontrol.Circ Res99:1315–132836.Willis MS,Rojas M,Li L,Selzman CH,Tang RH,StansfieldWE,Rodriguez JE,Glass D,Patterson C(2009)Muscle ring finger-1(MuRF1)mediates cardiac atrophy in vivo.Am J Physiol Heart Circ Physiol296:H997–H1006。