innate defense mechanism against virus infection within the cardiac myocyte requiring gp130-stat3...

Innate Defense Mechanism Against Virus Infection Withinthe Cardiac Myocyte Requiring gp130-STAT3 Signaling

Toshitaka Yajima, MD, PhD*; Hideo Yasukawa, MD, PhD*; Eun-Seok Jeon, MD, PhD;Dingding Xiong, MD, PhD; Andrea Dorner, PhD; Mitsuo Iwatate, MD, PhD; Miwako Nara, PhD;

Hanbing Zhou, BS; Daphne Summers-Torres, BS; Masahiko Hoshijima, MD, PhD;Kenneth R. Chien, MD, PhD; Akihiko Yoshimura, PhD; Kirk U. Knowlton, MD

Background—Little is known about innate immune mechanisms within the cardiac myocyte that determine susceptibilityto enterovirus infection, an important cause of myocarditis and subsequent heart failure. Although interferon (IFN)generally plays a key role in innate immunity, ablation of IFN receptors has little or no effect on acute coxsackievirusB3 infection in the heart. Interestingly, gp130-cytokine–mediated stimulation of neonatal ventricular myocytes has acytoprotective effect against virus infection in culture that can be inhibited by suppressors of cytokine signaling(SOCS)-3, a physiological inhibitor of gp130 signaling that does not affect IFN signaling. Therefore, we hypothesizedthat inhibition of gp130 signaling by SOCS3 would change cardiac myocyte susceptibility to virus infection withoutaffecting IFN signaling.

Methods and Results—We generated cardiac-specific SOCS3 transgenic mice. Despite an intact IFN-mediated antiviralresponse in adult transgenic myocytes, there was a marked increase in susceptibility to viral infection in the SOCS3transgenic mouse hearts. This indicated the presence of IFN-independent innate defense mechanisms within the cardiacmyocyte. Subsequently, we demonstrated that cardiac-specific gp130-knockout mice also had increased susceptibilityto viral infection. Furthermore, we demonstrated that the gp130-mediated increase in survival of infected myocytesoccurred through a signal transducers and activators of transcription-3–dependent mechanism that did not affect viralreplication. This was accompanied by a persistent expression of full-length dystrophin after coxsackievirus B3 infection.In addition, we found that both SOCS3 transgenic and gp130-deficient mice had a decrease in �-sarcoglycan.

Conclusions—SOCS3-mediated regulation of gp130 signaling can affect susceptibility to viral infection in the heart.Increased cardiac cell survival through gp130–signal transducers and activators of transcription-3 signaling appears toplay an important role in preserving nondividing cardiac myocytes until specific immune responses begin to clear thevirus. (Circulation. 2006;114:2364-2373.)

Key Words: heart failure � immune system � infection � molecular biology � myocarditis � signal transduction

Viral myocarditis and dilated cardiomyopathy can becaused by common viral pathogens such as enterovi-

ruses and adenoviruses that can have a direct myocytopathiceffect and can activate a potent T-cell immune response.Virus-mediated cardiac disease can be a debilitating andpotentially lethal disease in adults and children and is one ofthe main indications for cardiac transplantation.1,2

Although our understanding of the pathogenesis of virus-mediated cardiomyopathy has improved, relatively little isknown regarding factors that determine susceptibility to viralinfection. What factors determine why most individuals

Clinical Perspective p 2373

infected with cardiomyopathic viruses do not develop signif-icant cardiac disease whereas other individuals develop se-vere virus-mediated heart disease?

Activation of the innate immune response can be mediatedby cytokines such as the interferons (IFNs) and interleukin-6and is one of the earliest defenses against infectious patho-gens. Cytokines activate transcription of the cytokine-responsive genes, including suppressors of cytokine signaling(SOCS). This is often mediated through Janus kinase (JAK)

Received May 27, 2006; revision received August 23, 2006; accepted September 15, 2006.From the Department of Medicine (T.Y., E-S.J., D.X., A.D., M.N., H.Z., D.S-T., K.U.K.) and Institute of Molecular Medicine (H.Y., M.I., M.H.,

K.R.C.), University of California, San Diego, La Jolla; Cardiovascular Research Institute and the Third Department of Medicine, Kurume UniversitySchool of Medicine (H.Y.), Kurume, Japan; Department of Medicine, Sungkyunkwan University School of Medicine (E.-S.J.), Seoul, South Korea;Charite-Campus Benjamin Franklin, Cardiology and Pneumology (A.D.), Berlin, Germany; Medical Institute of Bioregulation, Division of Molecular andCellular Immunology (A.Y.), Kyushu University, Fukuoka, Japan.

*The first 2 authors contributed equally to this article.The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.642454/DC1.Correspondence to Kirk U. Knowlton, MD, Department of Medicine, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA

92093–0613K. E-mail [email protected]© 2006 American Heart Association, Inc.

Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.106.642454

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http://circ.ahajournals.org/

signal transducers and activators of transcription (STAT)signaling pathways. When induced, SOCS molecules inhibitJAK-mediated phosphorylation of the cytokine receptor,inhibiting activation of STAT signaling.3–8 This negativefeedback via SOCS tightly regulates the duration and inten-sity of the cytokine-induced JAK-STAT signaling.3,9–11 Littleis known of the importance of the innate immune responseagainst viral infection within the cardiac myocyte.

Recently, we showed that coxsackievirus B3 (CVB3)infection is associated with activation of JAK-STAT signal-ing in the heart with an induction of SOCS mRNA. Inaddition, we demonstrated that SOCS1-mediated inhibition ofJAK-STAT signaling in the intact heart markedly increasedsusceptibility to viral infection.12 This demonstrated theimportant role for JAK-STAT signaling within the cardiacmyocyte in the prevention of viral infection.

A potential explanation for this finding is that SOCS1increases susceptibility to viral infection by inhibiting IFNsignaling in the heart. However, although CVB3 infection ofglobal IFN-�/� (type I) receptor–knockout mice resulted in amarked increase in mortality, there was surprisingly littleincrease in viral infection of the heart in either IFN-�/� (typeI) or -� (type II) receptor–knockout mice.13 This raised thequestion of which innate signaling mechanisms against viralinfection are important in the heart.

Among SOCS family proteins, there are clear differencesin the effect of SOCS1 and SOCS3 on the innate immuneresponse. Endogenous SOCS1 plays a key role in the negativeregulation of IFN-�,7,8,14 whereas SOCS3 predominantlyinhibits gp130 signaling.15–18 SOCS1 binds directly to theactivation loop of all 4 JAKs, whereas SOCS3 initially bindsto the cytokine receptor, thus inhibiting JAK activation of asubset of receptors such as gp130.19 Therefore, overexpres-sion of SOCS1 inhibits activation of all 4 JAKs, whereas thatof SOCS3 does not. Adenovirus-mediated overexpression ofSOCS1 in neonatal rat cardiac myocytes inhibits both IFNand gp130 signaling, whereas that of SOCS3 inhibits onlygp130 signaling and has no effect on IFN signaling.12 In theintact adult heart, it is not clear whether the inhibition of IFNor gp130 signaling is primarily responsible for the SOCS-mediated increase in susceptibility to viral infection.

To dissect the mechanisms by which SOCS overexpressionin the heart could increase susceptibility to viral infection, wegenerated a transgenic mouse that overexpressed SOCS3 inthe cardiac myocyte. We hypothesized that SOCS3 overex-pression would increase susceptibility to viral infection with-out an effect on IFN signaling. Once we confirmed this, wefurther hypothesized that a mechanism by which SOCS3increased susceptibility to viral infection was via inhibition ofgp130 signaling. Our findings indicate that overexpression ofSOCS3 in transgenic mouse myocytes effectively inhibitedgp130 signaling. Furthermore, we found that cardiac-specificknockout of gp130 increased susceptibility to viral infection.The protective effect of gp130 signaling in isolated myocytesis mediated via STAT3 and is associated with a persistentexpression of full-length dystrophin even after CVB3 infec-tion. In addition, �-sarcoglycan protein is decreased inSOCS3 transgenic (SOCS3 Tg) hearts and in cardiac-specificgp130–knockout mice. These findings demonstrate the im-

portance gp130 signaling as a novel anti-viral mechanismwithin the cardiac myocyte and the minor contribution madeby activation of IFN signaling during the early stages ofenteroviral infection in the cardiac myocyte.

MethodsMiceTo generate SOCS3 Tg mice, the fragment carrying the �-myosinheavy chain promoter and myc-tagged SOCS3 cDNA was microin-jected into (C57BL/6�Balb/c) F1 (CB6F) zygotes. The cardiac-specific gp130-knockout mice were generated as described previous-ly.20 The SOCS3 Tg, homozygous floxed gp130 (gp130F/F) and themyosin light chain-2v Cre mice (MLC-2vCre/�)21 have been back-crossed for 5 generations into a Balb/c background (Charles RiverLaboratories, Wilmington, Mass) to perform experiments in aninbred line that is susceptible to viral infection.

VirusesThe CVB3 used in this study was the cardiotropic H3 strain(Woodruff variant) of CVB3 (CVB3-H3).22 The CVB3 that ex-presses green fluorescent protein (GFP) (CVB3-GFP) was generatedas described previously.23

AntibodiesWestern blot and immunofluorescence staining were performed asdescribed previously24,25 with anti-STAT1, anti–phospho-STAT1,anti-STAT3, and anti–phospho-STAT3 antibodies (New EnglandBioLabs Inc, Beverly, Mass); rabbit polyclonal anti-CVB3 antibody(a gift from Dr A. Henke, Friedrich Schiller University, Jena,Germany),26 anti–c-myc (A-14) antibody (Santa Cruz Biotechnol-ogy, Inc, Santa Cruz, Calif), anti-GFP antibody (Medical & Biolog-ical Laboratories, Nagoya, Japan), and anti–coxsackie-adenovirusreceptor (CAR) antibody.27

EchocardiogramAvertin (2.5%) was administered intraperitoneally at 0.015 mL/gbody weight. Recordings were performed as described previously.28

Left ventricular end-systolic (LVESD) and end-diastolic (LVEDD)dimensions were measured from the left ventricular M-mode record-ing. Percent fractional shortening (%FS) of the LV was calculated asfollows

(1) %FS�100�LVEDD-LVESD

LVEDD.

Isolation of Adult Ventricular MyocytesThe heart was perfused retrogradely for 3 minutes using a calcium-free buffer (heart solution) containing (in mmol/L) 120 NaCl, 5.4KCl, 1.2 MgSO4, 1.2 NaH2PO4, 20 NaHCO3, 5.6 D-glucose, 20 of2,3-butanedione monoxime (Sigma-Aldrich, St Louis, Mo), 5 tau-rine, and 1 pyruvate. The enzymatic digestion was achieved byperfusing a collagenase type 2 solution (Worthington Biochemical,Lakewood, NJ; final concentration, 1 mg/mL in heart solution) at37°C for 10 to 20 minutes. Isolated myocytes were allowed to pelletby gravity in heart solution containing 5 mg/mL bovine serumalbumin (Sigma) (washing solution). The myocytes were resus-pended in a series of washing solutions with increasing concentrationof CaCl2 (0.125, 0.25, and 0.5 mmol/L). The myocytes were platedon laminin-precoated plates (10 mg/mL, Sigma) and cultured in aminimal essential media (Gibco BRL) with 0.5 mmol/L CaCl2 for 12hours.

Statistical AnalysisData are expressed as mean�SE unless otherwise noted. Statisticalsignificance was evaluated with the unpaired Student t test forcomparisons between 2 means. For multiple comparisons, 1-way or2-way analysis of variance using the Tukey-Kramer post hoc test wasused. For survival rate after CVB3 inoculation, the differences

Yajima et al Innate Antiviral Defense in the Cardiac Myocyte 2365

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between 2 groups were analyzed by log-rank (Mantel-Cox) test.Values of P�0.05 were considered significantly different.

The authors had full access to the data and take full responsibilityfor their integrity. All authors have read and agree to the manuscriptas written.

Results

Marked Increase in Susceptibility to CVB3-MediatedCardiomyopathy in SOCS3 Tg MiceWe generated transgenic mice overexpressing a myc-taggedSOCS3 under the control of the cardiac myocyte–specific�-myosin heavy chain promoter. Transgene expression wasconfirmed by immunoblotting with an anti-myc antibody in 3separate founder lines. There was no evidence of embryoniclethality, and the mice grew to adulthood without increasedmortality. Global cardiac structure and function were normalin the mice (data not shown). The mouse line that had acomparable level of expression of the transgene to the SOCS1Tg mice12 was chosen for this study (Figure 1A).

To determine whether cardiac overexpression of SOCS3affects the susceptibility to CVB3-mediated cardiomyop-athy, we inoculated 6-week-old female Balb/c mice,SOCS3 Tg mice, and their wild-type (WT) littermates withCVB3. Mortality in the SOCS3 Tg mice was 80%, whichcompared with 10% in the infected control mice at day 6after infection (Figure 1B). Significant abnormalities in thehearts of SOCS3 Tg mice were found at day 5 afterinfection compared with WT littermate mice infected withCVB3. These include (1) a marked increase in the percentarea of Evans blue dye staining, a marker for viralinfection and disruption of the sarcolemma12,24 (Figure1C); (2) an increase in virus titer in the heart but not in theliver (Figure 1D); (3) a significant decrease in left ventric-ular fractional shortening with an increase in LVESD(Figure 1E); and (4) alterations in muscle fiber structurewithout significant infiltration of mononuclear cells (Fig-ure 1F). Incidentally, hemorrhage in the myocardium(Figure 1F, right) and left ventricular mural thrombus (not

Figure 1. Increased susceptibility toCVB3-mediated cardiomyopathy inSOCS3 Tg mice. A, The expression ofmyc-tagged SOCS3 in the heart wasconfirmed in SOCS3 Tg mice by immuno-blotting with anti-myc antibody and com-pared with the level of SOCS1 Tg–medi-ated expression.12,14 B, Cardiac-specifictransgenic overexpression of SOCS3leads to early mortality in CVB3-infectedmice. Kaplan-Meier survival curves dem-onstrate that the survival rate in infected(104 pfu of the virus) SOCS3 Tg mice wassignificantly lower than in infected WTlittermates (n�16 mice for each group;P�0.0001). C, Evans blue dye uptake inthe cardiac cells was markedly increasedin the surviving SOCS3 Tg mice on day 5after virus inoculation (red stain, left andmiddle). The percent Evans blue–positivearea in SOCS3 Tg vs WT littermates was38.3�9.9% vs 2.2�0.7%, respectively(right; mean�SE; n�5 mice for eachgroup; *P�0.0001). D, Increased viral titerin the heart but not the liver of SOCS3 Tgmice on day 5 after viral inoculation(mean�SE; n�6; *P�0.0001, SOCS3 Tgmice vs WT littermates). E, Echocardiog-raphy was performed 4 days after virusinoculation (n�4 mice in each group).Left, LVESD was significantly elevated inSOCS3 Tg (solid bar) compared with WT(open bar). The percent fractional short-ening (%FS; right), a parameter of car-diac function, was significantly decreasedin SOCS3 Tg vs WT mice. (mean�SE;*P�0.0001, SOCS3 Tg mice vs WT litter-mates). F, Hematoxylin and eosin stainsof representative WT littermate andSOCS3 Tg mouse heart 5 days after virusinoculation. Note the hemorrhage in themyocardium (right). Scale bars�1 mm(C), 100 �m (F, right).

2366 Circulation November 28, 2006



shown) were observed only in the infected SOCS3 Tgmice, a finding that is likely secondary to the extent ofmyocardial damage. Generally, the phenotype of infectedSOCS3 Tg mice was very similar to that of infectedSOCS1 Tg mice.12

Effect of SOCS3 Overexpression on IFN-MediatedAntiviral Activity in Isolated Transgenic AdultCardiac MyocytesTo determine the functional effect of exogenously adminis-tered cytokines on viral replication in adult cardiac myocytes,we isolated ventricular cardiac myocytes from the adult heartand stimulated them for 12 hours with IFN-�, IFN-�, orcardiotroponin (CT)–1. The myocytes were then infected for24 hours with a recombinant CVB-3 GFP.23 IFN-� had apartial but significant antiviral effect in adult myocytesisolated from WT Balb/c mice. However, IFN-� and CT-1had no significant effect on the percent of infected myocytes(Figure 2).

We then sought to determine the effect of overexpressionof SOCS3 on the cytokine-stimulated antiviral effect. TheIFN-�–mediated antiviral effect was preserved in myocytesfrom SOCS3 Tg mice, whereas the effect was completelyabolished in those from SOCS1 Tg mice, demonstrating thelack of inhibition of IFN antiviral effects by transgenicoverexpression of SOCS3 (Figure 2). Incidentally, there wasno significant difference in the level of CAR protein in theSOCS3 Tg myocytes as assessed by immunoblot (data notshown).

Effect of SOCS Overexpression on IFN-� andCT-1 Signaling in Isolated Transgenic AdultCardiac MyocytesBecause IFN-� and CT-1 had no effect on coxsackieviralreplication in the isolated adult myocytes, we sought to deter-mine whether downstream signaling mechanisms were intactand whether SOCS1 or SOCS3 overexpression would affectdownstream signaling for these cytokines.12 First, we stimulatedadult cardiac myocytes from WT Balb/c mice with CT-1 orIFN-� and found that CT-1 stimulated STAT3 but not STAT1phosphorylation. However, IFN-� induced both STAT1 andSTAT3 phosphorylation 15 minutes after stimulation (Figure 3).As expected, in myocytes from SOCS1 Tg mice, STAT1 andSTAT3 were not phosphorylated after CT-1 or IFN-� stimula-tion. However, overexpression of SOCS3 did not prevent IFN-�–stimulated STAT1 or STAT3 phosphorylation, but as hypoth-esized, it inhibited CT-1–mediated STAT3 phosphorylation.Myocytes from cardiac-specific gp130-knockout mice, gener-ated by crossing mice with a homozygous floxed gp130 allelewith the MLC-2v Cre mice as previously described,20 had apattern of STAT phosphorylation in response to CT-1 and IFN-�similar to that of the SOCS3-overexpressing Tg myocytes, thusdemonstrating that cardiac IFN-� signaling in SOCS3 Tg mice isintact despite complete inhibition of gp130 signaling (Figure 3).

Knockout of Cardiac gp130 IncreasesSusceptibility to CVB3 InfectionBecause SOCS3 overexpression did not affect the IFN-�antiviral effects or IFN-�–mediated STAT phosphorylation,we sought to identify IFN-independent, SOCS3-inhibitable,

Figure 2. Effect of SOCS3 overexpres-sion on IFN-mediated antiviral effect inisolated adult cardiac myocytes. Isolatedadult ventricular myocytes from WTBalb/c, SOCS3 Tg, and SOCS1 Tg micewere stimulated with IFN-� (500 U/mL),IFN-� (100 ng/mL), and CT-1 (1 nmol/L)immediately after plating for 12 hoursbefore CVB3-GFP infection at a multi-plicity of infection of 2000. Virus infectionefficiency (%infected cells) defined aspercent GFP-positive rod-shaped cellswas quantified by immunofluorescencestaining with anti-GFP antibody 24 hoursafter infection (upper row representsphase-contrast images; rows 2 to 4,immunofluorescent images of CVB3-GFPstain). Bar graphs are quantified from 3independent experiments and areexpressed as mean�SE. *P�0.01, IFN-�stimulated cells vs control.




JAK-dependent signaling pathways that regulate cardiac cellsusceptibility to viral infection. We focused on cardiac gp130signaling as the best-known physiological target of SOCS3.Before cardiac-restricted gp130-knockout mice were gener-ated, both the mice with homozygous floxed gp130 allele(gp130F/F)20 and the mice that express Cre under the directionof the myosin light chain-2v promoter (MLC-2vCre/�)21 werebackcrossed for 5 generations into a Balb/c background. Thegp130-knockout efficiency in the MLC-2vCre/�, gp130F/F car-diac myocytes was �80% compared with the WT controlmyocytes (Figure 4A). Knockout of gp130 did not affect thelevel of CAR expression (Figure 4B). However, 5 days afterinfection with CVB3, we found that the Evans blue dye areain 6-week-old female gp130-knockout mice was increasedcompared with WT littermates (Figure 4C), although thedifference was not as dramatic as that observed in SOCS3 Tgmice (Figure 1C).

Subsequently, we examined the relationship between the virusdistribution and Evans blue dye area by immunofluorescencestaining. Generally, the myocytes that were positive for viralcapsid proteins also were positive for Evans blue dye in both WTand gp130-knockout mice (Figure 4D, yellow in merged panel).

gp130 Signaling Has No Effect on CVB3Replication but Has a Strong Cytoprotective EffectThrough STAT3 Activation and Maintenance ofFull-Length Dystrophin ExpressionBecause CT-1 did not have a direct antiviral effect in isolatedadult cardiac myocytes (Figure 2), we sought to determinehow gp130 signaling could affect viral pathogenesis. Wehave previously demonstrated that CT-1 has a strong cyto-protective effect in CVB3-infected neonatal rat cardiac myo-cytes.12 Because of the inherent time-dependent cytopathiceffect that occurs after isolation of uninfected adult myocytes,the subsequent experiments were performed in culturedneonatal rat cardiac myocytes.12 First, we determined that thecytoprotective effect of CT-1 against CVB3 infection wasselective to cardiac myocytes by demonstrating that it had nocytoprotective effect on nonmyocytes such as HeLa and 293

cells even though there was activation of STAT3 signaling(data not shown). Then, we found that there was no detectablechange in CAR expression level 24 hours after CT-1 stimu-lation in the cardiac myocytes (Figure 5A). Subsequently, wefound that CT-1 did not directly affect viral replication usinga 1-step growth curve of the virus titer in CT-1–stimulated ornonstimulated neonatal myocytes (Figure 5B). These observationsare consistent with the result from adult myocytes that CT-1stimulation had no effect on infection efficiency (Figure 2).

We have previously demonstrated that one of the mecha-nisms by which CVB3 infection leads to death of cardiacmyocytes is disruption of the sarcolemmal membrane. This isassociated with dystrophin cleavage by the virus protease2A.24 In addition, we have demonstrated that dystrophincleavage has an important role in viral propagation in theheart.29 On the basis of this concept, we examined whetherthere is a difference in dystrophin cleavage in the myocytesstimulated with or without CT-1 after CVB3 infection (Figure5C). Cleaved dystrophin bands were identified in CVB3-infected myocytes in both groups 12 hours after infection.However, both cleaved and noncleaved bands were barelydetected 24 hours after infection in control myocytes,whereas uncleaved dystrophin was observed until at least 36hours after infection in CT-1–stimulated myocytes. Thisindicated that activation of gp130 signaling prevented cleav-age of all dystrophin by CVB3 infection (Figure 5C).

Finally, we sought to determine the gp130 downstreamsignaling pathway that is involved in the cytoprotective effectagainst CVB3 infection. Activation of gp130 signaling hasbeen associated with activation of PI3K, MEK, and STAT3.30

To inhibit PI3K-Akt and MEK–extracellular signal–regu-lated kinase (ERK)-1/2 pathways, we added Wortmannin andPD98095, respectively, to the culture media before CT-1stimulation. To inhibit the STAT3 pathway, we overex-pressed dominant-negative STAT3 (dnSTAT3) using recom-binant adenovirus vector. The specific inhibition of thePI3K-Akt, MEK-ERK1/2, and STAT3 pathways in our ex-perimental conditions was confirmed by Western blot usinganti–phospho-Akt, ERK1/2, and STAT3 antibodies, respec-tively (Figure 5D, top). In these conditions, cells wereinfected with CVB3 in the presence or absence of CT-1stimulation. Although the inhibition of PI3K-Akt and MEK-ERK1/2 signaling pathways had no effect on CT-1–mediatedcytoprotective effect against CVB3 infection (Figure 5D,bottom left), inhibition of the STAT3 pathway significantlyabolished the cytoprotective effect (Figure 5D, lower right,white bar), indicating that the gp130-mediated cytoprotectiveeffect against CVB3 is mediated through STAT3 signaling.

Both SOCS3 Overexpression and gp130 Deficiencyin the Cardiac Myocyte Reduced �-Sarcoglycan inthe Intact HeartWe have previously demonstrated that the susceptibility toCVB3-mediated cardiomyopathy can be increased in theabsence of dystrophin-sarcoglycan complex in mdx mice.29 Inthis study, we found that CT-1 stimulation improved myocytesurvival with an increase in full-length dystrophin. Therefore,we sought to determine whether inhibition of gp130 signalingby SOCS3 Tg overexpression or knockout of the gp130

Figure 3. Effect of SOCS3 Tg overexpression on IFN-� andCT-1 signaling in isolated adult cardiac myocytes. Adult cardiacmyocytes were isolated directly from WT Balb/c, SOCS3 Tg,SOCS1 Tg, and gp130-knockout (gp130-KO) mice as describedin Methods. After serum starvation for 12 hours, the isolatedcultured myocytes were stimulated with IFN-� (100 ng/mL) orCT-1 (1 nmol/L) for 15 minutes, followed by protein extraction.Activation of STAT1 and STAT3 was estimated by immunoblot-ting with anti–phospho-STAT1 (P-STAT1), STAT1, anti–phospho-STAT3 (P-STAT3), and STAT3 antibodies. Representative West-ern blots from 3 independent experiments are shown.




receptor affected expression of dystrophin or �-sarcoglycan.We found no detectable changes in the cellular localization ofdystrophin (data not shown) and �-sarcoglycan (Figure 6A)in the heart of SOCS3 Tg and gp130-knockout mice; how-ever, the expression level of �-sarcoglycan by Western blotanalysis was decreased in both SOCS3 Tg and gp130-knockout mice compared with WT littermate controls. Wewere not able to detect clear differences in dystrophin levelsby immunoblot (Figure 6B, left). Based on serially dilutedheart protein samples, the protein expression level of�-sarcoglycan in the heart of SOCS3 and gp130-knockoutmice was approximately half of that in the hearts of WTlittermates (Figure 6B, right).

DiscussionIn the present study, we focused on innate defense mech-anisms in the cardiac myocyte that are important determi-nants of susceptibility to viral infection in viral myocardi-tis. Like SOCS1, SOCS3 overexpression in the transgeniccardiac myocyte has a marked effect on the susceptibilityof the heart to CVB3 infection; however, SOCS3 overex-pression does not affect IFN-receptor signaling or the IFNantiviral effect in isolated myocytes. Finally, we showed

that cardiac-specific knockout of gp130 increases suscep-tibility to CVB3, demonstrating a role for gp130 signalingvia activation of STAT3.

It has previously been demonstrated that there is a potentcardiac myocyte– dependent innate immune response thatcan be inhibited by cardiac-specific overexpression ofSOCS1.12 In addition, transgenic overexpression ofSOCS1 in pancreatic islet cells affects susceptibility toCVB infection.31 A likely target for the SOCS1 protein thatcould affect susceptibility to viral infection in thesemodels would be the IFN, JAK-STAT signaling cascade.The data from the present study, however, challenge thatnotion by demonstrating that both SOCS1 and SOCS3overexpression increases susceptibility to viral infectiondespite the fact that SOCS3 overexpression does notinhibit IFN signaling or IFN-mediated antiviral effects.

Although endogenous SOCS3 expression predominantlyinhibits gp130 signaling,15 some have indicated that over-expression of SOCS3 can have inhibitory effect on IFNsignaling.32 Differences in these results may be secondaryto differences in cell types studied, mechanisms of over-expression, or levels of expression of SOCS3. To improveour understanding of the mechanisms by which SOCS3

Figure 4. Increased susceptibility to viralinfection in gp130-deficient hearts. A,Knockout efficiency of gp130 in the iso-lated adult cardiac myocytes from theknockout mice (gp130-KO) was con-firmed by immunoblotting with anti-gp130 antibody using sarcomeric�-actinin as an internal control. B,Expression level of CAR in cardiac myo-cytes was examined by immunoblottingwith anti-CAR antibody using glyceralde-hyde phosphate dehydrogenase as aninternal control. Lane 1 shows positivecontrol, protein from HeLa cells; lanes 2and 3, protein extracted from isolatedadult cardiac myocytes of WT litter-mates; and lanes 4 and 5, proteinextracted from isolated adult cardiacmyocytes of gp130-KO mice. C, Evansblue dye uptake in the cardiac cells wasincreased in gp130-KO mice on day 5after the virus inoculation (red stain, leftand middle). The percent Evans blue–positive area in gp130-KO mice vs WTlittermate was 12.2�6.0% vs 1.7�0.9%,respectively (right; mean�SE; n�7 micefor each group; *P�0.0004). D, Dual im-munostaining of the infected heart ofgp130-KO mice and WT littermates. Left,Immunofluorescence staining with anti-CVB3 antibody (green); center, Evansblue (EB) dye (red) uptake in the samefield; and right, merged image.




overexpression in the heart alters susceptibility to infec-tion, we isolated adult cardiac myocytes from transgenicmice overexpressing SOCS3 or SOCS1 and demonstratedthat SOCS3 overexpression in the adult cardiac myocytehad no significant effect on IFN-mediated antiviral effectsor IFN-� STAT1 phosphorylation. As expected, IFN sig-

naling in the SOCS1-overexpressing Tg cardiac myocyteswas clearly inhibited as measured by IFN-�–mediatedantiviral effects and IFN-�–mediated STAT1 and STAT3phosphorylation. At first glance, these data may seemcontrary to previously published data demonstrating thatglobal knockout of either the IFN-�/� receptor13 or IFN-

Figure 5. Cytoprotective effect of gp130-STAT3 signaling against CVB3 infection in neonatal rat cardiac myocytes via a STAT3-dependentmechanism. A, Expression level of CAR in the myocytes after CT-1 simulation was examined by immunoblotting with anti-CAR antibodyusing glyceraldehyde phosphate dehydrogenase as an internal control. Lane 1 shows positive control protein from HeLa cells; lane 2, myo-cytes without CT-1 stimulation; and lane 3, myocytes with 24 hours of CT-1 stimulation. B, One-step growth curve of CVB3 in the myocyteswith (solid line) or without (Control; dotted line) CT-1 stimulation. Myocytes were infected with CVB3 (multiplicity of infection, 100) for 1 hour,followed by washing 3 times with phosphate buffer saline. After the culture for indicated time on the x axis, cells were harvested to examinethe virus titer by plaque forming assay. C, Effect of CT-1 stimulation on transition of structural and nonstructural proteins in the myocytesafter CVB3 infection. Proteins from the myocytes stimulated with or without CT-1 (Control) were extracted at each time point indicated abovethe panel after CVB3 infection (multiplicity of infection, 100) and were subjected to immunoblotting with anti-dystrophin, sarcomeric �-actinin,STAT1, enterovirus VP1, and enterovirus proteinase 2A antibodies. D, Downstream signaling pathway of gp130 that is involved in the cyto-protective effect. All experiments were performed in culture media with 5% serum to prevent spontaneous cell death. This resulted in base-line activation of Akt and ERK1/2 signaling. The specific inhibition of PI3K-Akt and MEK-ERK1/2 signaling pathways after CT-1 stimulationwas accomplished in our experimental conditions using Wortmannin and PD98095, respectively (top left). To inhibit STAT3 activation specifi-cally, HA-tagged dnSTAT3 was overexpressed with the use of recombinant adenovirus vector. The vector containing the LacZ gene wasused as a control for adenoviral vector infection (top right). After inhibiting each downstream signaling pathway, cells were infected withCVB3 in the presence or absence of CT-1 for 24 hours. The number of cells that remained on the plate after CVB3 infection was quantifiedand reported as a percentage cell survival in the wells not infected with CVB3 (bottom right). *P�0.01, dnSTAT3-overexpressing cells vsLacZ-expressing control cells in the presence of CT-1 and CVB3 infection.




�33 markedly increases mortality after CVB3 infection.Careful analysis of both models, however, fails to demon-strate an increase in the extent of viral infection in thehearts of CVB3-infected mice. Thus, although it is clearthat IFNs are important innate immune molecules for viralinfection, our data with transgenic SOCS3 overexpressionin the heart strongly indicate that IFNs do not have asignificant effect on the early intracardiac myocyte innateimmune response to coxsackieviral infection.

This then raises the question of what mechanism isresponsible for the SOCS3-mediated increase in enteroviralsusceptibility in the cardiac myocyte. Our data demonstratethat transgenic SOCS3 overexpression in the isolated adultcardiac myocyte inhibited gp130 signaling. In addition, in-fection of mice with cardiac-specific knockout of gp130 alsodemonstrated an increase in susceptibility to viral infection.gp130 signaling is generally not known to have a directantiviral effect, an observation confirmed in our experimentsin neonatal and adult myocytes. Therefore, we wonderedwhat mechanism could be responsible for the increasedsusceptibility to viral infection in the absence of gp130signaling in the heart.

We have previously shown that the enteroviral protease2A is able to directly cleave the cytoskeletal protein

dystrophin.24 Furthermore, we have demonstrated that thepresence of intact dystrophin has an important role onsusceptibility to enteroviral infection.29 The data in thisarticle demonstrate that compared with unstimulated in-fected cells, stimulation of gp130 signaling with CT-1leads to an increase in myocyte survival and the continuedpresence of full-length dystrophin protein in CVB3-infected cardiac myocytes. The maintenance of full-lengthdystrophin in gp130-stimulated infected myocytes is sup-portive of a role for gp130 signaling in maintainingmembrane integrity in the presence of viral infection andthus decreasing susceptibility. This is supported in theintact heart by the increase in disruption of sarcolemmalintegrity in CVB3-infected hearts that lack the gp130receptor. It is interesting that �-sarcoglycan expressionlevel is decreased in both SOCS3 and gp130-knockoutmice even without infection (Figure 6B). Although furtherexperiments are needed, the decreased �-sarcoglycanmight be involved in the increased susceptibility to CVB3infection as observed in dystrophin-sarcoglycan– deficientmdx mice.29

ERK and Akt signaling have been shown to have a role inthe cytoprotective effect conferred by stimulation of gp130signaling.34,35 However, the cytoprotective effect that occurs

Figure 6. Decreased expression of�-sarcoglycan in the heart of SOCS3 Tgand cardiac-specific gp130-knockout(gp130-KO) mice. a, Immunofluores-cence staining of frozen heart sectionsfrom WT Balb/c, SOCS3 Tg, andgp130-KO with anti–�-sarcoglycan anti-body (Novocastra, Newcastle UponTyne, UK). Cellular localization of�-sarcoglycan was not changed witheither transgenic SOCS3 overexpressionor gp130 deficiency. Scale bar�200 �m.b, Effect of SOCS3 overexpression orgp130 deficiency on dystrophin and�-sarcoglycan expression in the heartwas examined by Western blot analysiswith anti-dystrophin (Vector Laboratories,Burlingame, Calif) and anti–�-sarcoglycan (Novocastra) antibodiesusing glyceraldehyde phosphate dehy-drogenase as an internal control (left, topand bottom). For semiquantification ofthe expression level of �-sarcoglycan,heart protein samples were seriallydiluted as indicated on the top of eachpanel (right, top and bottom). Detectionlimits of �-sarcoglycan by Western blotwere evaluated by comparison betweenSOCS3 Tg mice and WT littermates(right, top) and gp130-KO mice and WTlittermates (right, bottom).




in CVB3-infected cells after gp130 stimulation is dependenton STAT3 signaling.

Although these data demonstrate the importance ofgp130 signaling, it is possible that other non-IFN innatesignaling mechanisms that can be inhibited by SOCS3 alsoare important. This is suggested by the finding that theincreased susceptibility to CVB3 infection in the cardiac-specific gp130-knockout mice was not as dramatic as thatseen in the SOCS3 Tg mice (Figure 4). Although our datain isolated myocytes were obtained using CT-1 to stimu-late gp130 signaling, it possible that other gp130-activating cytokines such as interleukin-6 may play a rolein the innate immune response against viral infection in theintact heart.

The evidence that innate IFN signaling in the cardiacmyocyte has little effect on inhibiting the early stages ofadult cardiac myocyte CVB3 infection raises the questionof whether there are differences in IFN signaling in theadult cardiac myocyte compared with the other cell typesfrequently used to study IFN signaling. Our results andthose of others36 indicate that this is the case. Althoughexogenous IFN-� stimulation of isolated adult myocytesconfers a partial antiviral effect, transgenic overexpressionof SOCS3 has no impact on this effect, indicating that theincreased susceptibility to viral infection in SOCS3 Tgmice is independent of IFN-� signaling. IFN-� had nodirect antiviral effect on the isolated adult cardiac myo-cytes despite induction of STAT1 and STAT3 phosphory-lation. Taken together with these data, it is clear thatcaution should be exercised when the typical IFN antiviralparadigm is applied to adult cardiac myocytes.

Finally, these data conclusively demonstrate the presenceof a potent innate immune response within the cardiacmyocyte that can be inhibited by SOCS3. Surprisingly, IFNsignaling has little or no role in this innate immune response,whereas gp130 signaling makes an important contribution tothe innate immune response during the early stages ofcoxsackieviral infection in the heart.

AcknowledgmentsWe gratefully acknowledge Dr Darryl Belke for assistance withisolation of adult mouse. We also appreciate helpful discussions withDrs Ju Chen and Robert Ross.

Sources of FundingFunding for the research of this paper was supported by the NIH (DrKnowlton) and the American Heart Association and University ofCalifornia Discovery grant (Dr Yajima). Dr Yasukawa is supportedby the Ministry of Education, Science, Sports, Culture, and Tech-nology of Japan, Kato Memorial Foundation, and NovartisFoundation.

DisclosuresNone.

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CLINICAL PERSPECTIVEOne of the several causes of heart failure is viral infection of the heart that leads to dilated cardiomyopathy. Although thereare many unresolved clinical questions related to viral myocarditis, one that is frequently asked by patients and physiciansis, “What are the factors that determine why most individuals infected with cardiomyopathic viruses such as coxsackievirusdo not develop significant cardiac disease yet other individuals develop severe virus-mediated heart disease?” To addressthis question, it is necessary to understand the molecular mechanisms that can affect susceptibility to viral infection.Previously, considerable attention in regard to this issue has focused on the cellular immune response that occurs withmyocarditis. However, the data in the present study demonstrate that there is an antiviral defense mechanism within thecardiac myocyte that can affect susceptibility to viral myocarditis. Circulating cytokines such as interleukin-6 can affectmany aspects of the host immune response. Receptors for cytokines such as gp130, the receptor for interleukin-6, areexpressed on the surface of cardiac myocytes. The present article demonstrates that disruption of cytokine signaling byoverexpressing the suppressor of cytokine signaling-3, an inhibitor to the receptor for interleukin-6 in the heart, or bydisruption of the interleukin-6 receptor in the cardiac myocyte increases susceptibility to viral infection. This demonstratesthat alterations in this signaling cascade through genetic polymorphisms or changes in gene expression could affect aperson’s risk of developing viral myocarditis. It may also help to identify new targets for therapy of virus-mediated heartdisease.




R. Chien, Akihiko Yoshimura and Kirk U. KnowltonKennethIwatate, Miwako Nara, Hanbing Zhou, Daphne Summers-Torres, Masahiko Hoshijima,

Toshitaka Yajima, Hideo Yasukawa, Eun-Seok Jeon, Dingding Xiong, Andrea Dorner, MitsuoRequiring gp130-STAT3 Signaling

Innate Defense Mechanism Against Virus Infection Within the Cardiac Myocyte

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2006 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation doi: 10.1161/CIRCULATIONAHA.106.642454

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