the myocardial jak/stat pathway: from protection to failure

Pharmacology & Therapeutics 120 (2008) 172–185

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The myocardial JAK/STAT pathway: From protection to failure

Kerstin Boengler a, Denise Hilfiker-Kleiner b, Helmut Drexler b, Gerd Heusch a, Rainer Schulz a,⁎a Institut für Pathophysiologie, Universitätsklinikum Essen, Germanyb Abteilung für Kardiologie und Angiologie, Medizinische Hochschule Hannover, Germany

⁎ Corresponding author. Institut für Pathophysiologiefax: +49 201 7234481.

E-mail address: [email protected] (R. Schul

0163-7258/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.pharmthera.2008.08.002

a b s t r a c t
a r t i c l e i n f o
Keywords:
Signal transducer and activator of transcription
Proteins of the interleukin-transduction involves activa

JAK/STATPreconditioningPostconditioningAgingMitochondriaHypertrophyIschemiaReperfusion

6 (IL-6) family bind to receptors in the plasma membrane. Subsequent signaltion of the janus kinase (JAK) and signal transducer and activator of transcription

(STAT) proteins. STAT proteins are translocated into the nucleus, where they bind to the promoter region oftarget genes and are thereby involved in regulating the transcription of target genes.In the first part, the present review focusses on the role of STAT3 in ischemia/reperfusion injury and incardioprotection by ischemic pre- and postconditioning. In the heart, ischemia induces an increase in IL-6cytokines, which is associated with activation of STAT3. Genetic modification of the myocardial STAT3 proteincontent shows a protective role of STAT3 on infarct size after ischemia/reperfusion injury. Thecardioprotection by both early and late ischemic preconditioning as well as by ischemic postconditioning
involves an activation of STAT3 and is dependent on STAT3 protein level. Whereas the infarct-sparing effect oflate preconditioning is clearly mediated by an increase in transcription-mediated protein synthesis, earlypreconditioning is independent of gene transcription, suggesting a role of STAT3 independent oftranscriptional regulation. Possibly, STAT3 plays a role in modifying mitochondrial function, organellescentral for the cardioprotection by pre- and postconditioning.In the second part, the role of STAT3 in physiological stresses such as aging and pregnancy, as well as inpathophysiological situations such as myocardial infarction and heart failure is summarized. Furthermore,the requirements for the use of STAT3 as a target for treatment strategies of cardiovascular diseases isdiscussed.
© 2008 Elsevier Inc. All rights reserved.

Abbreviations:AngII, angiotensin IIAT1, AT1 receptorBcl-xL, Basal cell lymphoma-extra largeCNTF, ciliary neurotrophic factorCOX2, cyclooxygenase 2CT-1, cardiotrophin-like cytokineeNOS, endothelial nitric oxide synthaseERK, extracellular signal related kinaseGAS elements, interferon gamma activation sitesGCSF, granulocyte colony stimulating factorGp130, glycoprotein 130IFN, interferonIL, InterleukiniNOS, inducible nitric oxide synthaseIP, ischemic preconditioningJAK, Janus kinaseLIF, leukemia inhibitory factorLRb, long form of the leptin receptorMnSOD, manganese superoxide dismutaseMPTP, mitochondrial permeability transitionporeMT, metallothioneinPIAS, protein inhibitor of STATPPCM, postpartum cardiomyopathyROS, reactive oxygen species
SOCS, suppressors of cytokine signalingSTAT, Signal transducer and activator oftranscriptionTAC, thoracic aorta constrictionTNFα, tumor necrosis factor αTyk2, tyrosine kinase 2
, Wetsdeutsches Herzzentrum, Universitätsklinikum Essen, Hufelandstr. 55, 45122 Essen, Germany. Tel.: +49 201 7234521;

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http://dx.doi.org/10.1016/j.pharmthera.2008.08.002

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173K. Boengler et al. / Pharmacology & Therapeutics 120 (2008) 172–185

Contents

1. STAT proteins as signaling molecules of the JAK–STAT pathway in the heart. . . . . . . . . . . . . . . . . 1732. Role of STAT proteins in ischemia/reperfusion injury. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1743. STAT proteins in cardioprotection by ischemic and pharmacological preconditioning . . . . . . . . . . . . 1754. Does STAT3 have an impact on mitochondrial function? . . . . . . . . . . . . . . . . . . . . . . . . . . 1765. STAT3 in ischemic postconditioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1766. JAK/STAT proteins regulate myocardial apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1777. The expression of STATs in aged cells or organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1788. Consequences of STAT3 activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1789. STAT3 in exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

10. STAT3 in pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17911. STAT3 in heart failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17912. Does STAT3 represent a therapeutic target for the treatment of cardiovascular diseases? . . . . . . . . . . . 180Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
1. STAT proteins as signalingmolecules of the JAK–STAT pathway in the heart

Signal transducer and activator of transcription (STAT) proteins arepart of the Janus kinase (JAK)–STAT pathway, which mediates thetransduction of stress signals from the plasma membrane to thenucleus (Fig. 1). The JAK–STAT pathway is initiated by binding of aligand to its receptor in the plasma membrane and the subsequenthomo- or heterodimerization of the receptor. In the heart, interleukin(IL)-6-type cytokines such as IL-6, IL-11, leukemia inhibitory factor(LIF), oncostatin M, ciliary neurotrophic factor (CNTF), and cardio-trophin-like cytokine (CT-1) transduce their signals via glycoprotein130 (gp130) predominantly to STAT3 (Fischer & Hilfiker-Kleiner,2007). The receptor dimerization, in turn, induces phosphorylationand activation of JAK proteins, which are associated with theintracellular domain of the receptor. JAK proteins phosphorylate thereceptor, thereby creating docking sites for cytosolic STAT proteins viatheir SH2 domains. Next, STAT proteins become phosphorylated on aspecific tyrosine residue (Tyr705 for STAT3) by activated JAK kinases,and undergo homo- or heterodimerization by interaction of thephosphotyrosine residue of one STAT monomer and the SH2 domainof the other monomer. The STAT dimers dissociate from the receptorand translocate into the nucleus, where they bind to specific DNAsequences and regulate the expression of target genes. In the heart,STAT proteins regulate the expression of genes encoding proteinsinvolved in angiogenesis, inflammation, apoptosis, extracellularmatrix composition and cellular signaling (Hilfiker-Kleiner et al.,2004; Hilfiker-Kleiner et al., 2005; Snyder et al., 2008). Thephosphorylation of a specific serine residue in the transactivationdomain of STAT proteins (Ser727 for STAT3) generally promotestranscriptional activity. The ability of STATs to function as transcrip-tional activators is controlled by PIAS proteins (protein inhibitors ofSTAT), which negatively regulate DNA-binding of activated STATproteins and positively by co-activators such as p300/CREB-bindingprotein (Wang et al., 2005) and CR-6-interacting factor 1, Crif1 (Kwonet al., 2008). The activation and nuclear translocation of STATs occurswithin 15min, but STAT proteins are also rapidly inactivated, resultingin a half-life of nuclear phosphorylated STAT between 15 and 30 min(Haspel et al., 1996). Upon dephosphorylation by nuclear phospha-tases, STAT proteins shuttle back into the cytosol through the nuclearpore. The JAK/STAT pathway is not only controlled via phosphoryla-tion of the signaling proteins, but also by negative regulators. Uponactivation, STATs bind to the promoter region of SOCS genes(suppressors of cytokine signaling) and up-regulate the transcriptionof these target genes. SOCS proteins (mainly SOCS 1 and 3) negativelyregulate the JAK-STAT pathway by either directly binding to JAK, bybinding to the receptor and to JAK, or by competing with STATs for the

docking sites at the receptor (for review see (Cooney, 2002)). Anothernegative feedback mechanism of the JAK/STAT pathway comprisesSHP-2 proteins (src homology 2 domain containing protein tyrosinephosphatase), which dephosphorylate the receptor, JAK or STATproteins (for review see (Heinrich et al., 2003). The details of theJAK-STAT signaling pathway have been extensively reviewed else-where (Horvath, 2000; Kisseleva et al., 2002; O'Shea et al., 2002; Lim &Cao, 2006; Fischer & Hilfiker-Kleiner, 2007).

Four JAKs and seven STATs have been identified (Darnell, 1997).The ubiquitously expressed JAK proteins JAK1, JAK2, and TYK2 arepresent in cardiomyocytes (Pan et al., 1999), whereas the highest levelof JAK3 protein is found in the thymus (Gurniak & Berg, 1996). Allseven STATs (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6)are expressed in the heart (Xuan et al., 2001), where they are detectedin endothelial cells (Bartoli et al., 2000; Buttmann et al., 2007), insmooth muscle cells (Dumler et al., 1999), in cardiac fibroblasts (Wanget al., 2002b; Zurney et al., 2007), and in cardiomyocytes (McWhinneyet al., 1998; Pan et al., 1999; Hilfiker-Kleiner et al., 2004; Zurney et al.,2007).

The functional protein domains are conservedwithin themembersof the STAT family. STAT proteins contain an amino-terminal domaininvolved in dimerization or tetramerization, a coiled-coil domainimportant for protein–protein interactions, a DNA-binding domain, alinker, a SH2 domain predominantly mediating receptor binding ofSTATs, and a carboxyterminal transactivation domain (for review see(Lim & Cao, 2006).

Alternative splicing of the 3' end of the transcript has beendescribed for STAT1, 3, 4, 5A, and 5B, resulting in β isoforms with atruncated C-terminal transactivation domain. STAT1β does notcontain the C-terminal 38 amino acids of STAT1α, and thereforelacks the transactivation domain (Schindler et al., 1992). STAT1α andSTAT1β exert different functions regarding transcriptional activationon DNA and chromatin templates (Zakharova et al., 2003). STAT3βlacks the 55 C-terminal amino acids, but has gained seven uniqueamino acids. Therefore, STAT3β contains the phosphorylation sitetyrosine 705, but lacks the phosphorylation site serine 727 (Schaeferet al., 1995). Whereas both STAT3α and STAT3β are activated bysimilar cytokines and growth factors, and formed homodimers andheterodimers with STAT1, activated STAT3β reveals greater stabilityand DNA-binding activity than STAT3α. In contrast, STAT3α istranscriptionally more active than STAT3β suggesting that STAT3βacts as a dominant negative STAT3 isoform (Caldenhoven et al., 1996;Schaefer et al., 1997). However, more recent data show that STAT3β iscapable of inducing the transcription of target genes (Maritano et al.,2004). Furthermore, the analysis of target genes of the STAT4 isoformsSTAT4α and STAT4β (lacking the C-terminal 44 amino acids) revealedthat, whereas 98 genes were induced by both isoforms upon IL-12

Table 1Phenotypes of STAT knockout mice

STATknockout

Phenotype References

STAT1 Sensitive to infection by microbial pathogensand viruses, impaired responsiveness to IFNalpha or gamma

(Meraz et al., 1996),(Durbin et al., 1996)

STAT2 Susceptibility to viral infections, defects in type IIFN signaling

(Park et al., 2000)

STAT3 Embryonically lethal (Takeda et al., 1997)STAT4 Defects in IL-12 signaling, impaired T helper cell

1 development(Thierfelder et al., 1996),(Kaplan et al., 1996)

STAT5A Lactation defects (Liu et al., 1997)STAT5B Defective growth hormone signaling (Udy et al., 1997)STAT6 Defects in IL-4 and IL-13 signaling, impaired T

helper cell 2 development(Kuperman et al., 1998),(Akimoto et al., 1998)

Fig. 1. Signal transduction of the JAK/STAT pathway. STATs are activated by binding ofligands (cytokines or growth factors) to receptors with or without tyrosine kinaseactivity. Binding of the ligand induces dimerization of the receptor, followed byactivation of receptor-associated JAK proteins. JAK phosphorylates the receptor, therebycreating docking sites for STAT proteins. The STATs become phosphorylated, dimerize,dissociate from the receptor and translocate into the nucleus, where they bind to thepromoter region of target genes. Upon dephosphorylation, STAT proteins shuttle backinto the cytosol. Inactivation of the pathway occurs via inhibition of the JAK and/orreceptor phosphorlyation by SOCS or SHP-2 proteins. DNA-binding of STATs iscontrolled by PIAS proteins. JAK: janus kinase; STAT: signal transducer and activatorof transcription; PIAS: protein inhibitor of STAT; SHP-2: src homology 2 domaincontaining protein tyrosine phosphatase; SOCS: suppressors of cytokine signaling.Processes activating the JAK/STAT pathway are shown in solid lines, processes inhibitingthe JAK/STAT signal transduction cascade are represented in dashed lines.

174 K. Boengler et al. / Pharmacology & Therapeutics 120 (2008) 172–185

stimulation, 32 genes were specifically induced by STAT4α and 29only by STAT4β, confirming a role in transcriptional activation ofSTAT4β (Hoey et al., 2003). C-terminal truncated proteins have beendescribed for both STAT5A and STAT5B and the truncated forms arecapable of dimerizing with the wildtype proteins and display atyrosine phosphorylation more stable than the wildtype proteins(Wang et al., 1996).

Although the STAT proteins are structurally related, they partici-pate in different cellular processes, as observed in STAT knockout mice(Table 1). STAT1 and STAT2 knockout mice display defects ininterferon signaling and have an increased susceptibility to viralinfections (Durbin et al., 1996; Meraz et al., 1996; Park et al., 2000).STAT3 knockout mice are not viable, demonstrating that STAT3 is notonly important for interferon signaling (Takeda et al., 1997). Theablation of STAT4 and STAT6 demonstrated that both proteins areinvolved in T-helper cell polarization (Kaplan et al., 1996; Kupermanet al., 1998). Whereas STAT4 mediates IL-12 signaling, STAT6participates in the transduction of IL-4 and IL-13 signals (Thierfelderet al., 1996; Akimoto et al., 1998; Kuperman et al., 1998). Mice deficientin STAT5 isoforms have defects in lactation (Liu et al., 1997), in growthhormone signaling (Udy et al., 1997), in IL-2-induced T-cell prolifera-tion, in the number of natural killer cells, and females displayinfertility (Teglund et al., 1998; Moriggl et al., 1999).

Studies analyzing the role of STAT proteins in cardiac function havemainly focused on STAT1 and STAT3 and to a lesser extent on STAT5Aand STAT6. In the heart, the JAK-STAT pathway is involved in ischemia/reperfusion injury, hypertrophy, and post-partum cardiomyopathy(Booz et al., 2002; Bolli et al., 2003; Hilfiker-Kleiner et al., 2004, 2007;Terrell et al., 2006; Barry et al., 2007). Inmore chronic scenarios such ashypertrophy and cardiomyopathy, the JAK/STAT pathway is involved inregulating the transcription of target genes. In contrast, in more acutescenarios such as ischemia/reperfusion injury, the time frames are tooshort to exclusively explain the functions of JAK/STAT proteins bytranscriptional regulation of target genes and the subsequent de novo

protein synthesis. In these processes, non-transcriptional effects of theJAK/STAT proteins such as posttranslational modifications of down-stream proteins are obviously important.

The role of STAT, especially STAT3, in chronic and acutepathophysiological processes will be discussed in the present review.

2. Role of STAT proteins in ischemia/reperfusion injury

Since hypoxia and myocardial ischemia increase the levels of IL-6and gp130 (Yamauchi-Takihara et al., 1995; Kukielka et al., 1995;Chandrasekar et al., 1999), the involvement of the JAK-STAT pathwayin ischemia/reperfusion injury has been investigated. In rat myocar-dium subjected to 25 min ischemia an increase in STAT3 phosphor-ylation was observed compared to sham operated rats, which wasfurther augmented at 30 min of subsequent reperfusion (McCormicket al., 2006). Such enhanced STAT3 tyrosine phosphorylation inischemic rat myocardium persisted for at least 6 h (Negoro et al.,2000).

Since STAT3 is central for the initial stages of cardiomyogenesis(Foshay et al., 2005) and complete STAT3 knockout causes embryoniclethality (Takeda et al., 1997), studies designed to characterize thefunction of STAT3 in ischemia/reperfusion injury in geneticallyengineered mice had to be based on organ-specific overexpressionor knockout of STAT3. Transgenic mice with a cardiac-specific 10-foldoverexpression of a constitutively active STAT3 (Osugi et al., 2002) hadreduced infarct size both after 1 h ischemia and 2 h reperfusion as wellas after 30 min ischemia and 24 h reperfusion compared to non-transgenic mice, supporting the cardioprotective role of STAT3(Oshima et al., 2005). In turn, a reduction of the cardiomyoycyteSTAT3 protein level using a Cre/lox system under control of the α-myosin heavy chain promoter (STAT3 knockout mice) was associatedwith larger infarcts after 1 h ischemia and 24 h reperfusion than inwildtype littermates (Hilfiker-Kleiner et al., 2004), but interestinglynot after 30 min ischemia and 2 h reperfusion (Boengler et al., 2008a).The ablation of STAT3 by a MLC2v driven Cre-recombinase did notaffect infarct size after 30 min ischemia and 45 min reperfusioncompared to isolatedwildtypemouse hearts (Smith et al., 2004). Thus,with longer duration of ischemia, STAT3 contributed to the reductionof irreversible tissue injury. A close cell–cell interaction betweenendothelial cells and cardiomyoyctes was shown in isolated mousehearts, in which ablation of only endothelial STAT3 was sufficient todecrease the activation of cardiomyocyte STAT3 thereby enhancingLDH release — an indicator of irreversible tissue injury — following20 min ischemia and 1 h reperfusion compared to control hearts(Wang et al., 2007).

In addition to STAT3, also STAT1 modulates ischemia/reperfusioninjury. Isolated hearts subjected to 35min ischemia and 2 h reperfusionhad an increase in both total and phosphorylated STAT1. The


overexpression of STAT1 increased while the expression of antisenseSTAT1 decreased neonatal rat cardiomyocyte death compared to controltransfected cells after 4 h ischemia and 16 h reperfusion. The STAT1-associated increase in cardiomyocyte death was diminished by co-expression of STAT3, demonstrating that STAT1 was involved inenhanced and STAT3 in reduced cell death (Stephanou et al., 2000b).Also, epigallocatechin-3-gallate, the major constituent of green tea,decreased STAT1 phosphorylation and protected cardiomyocytes fromischemia/reperfusion injury (Townsend et al., 2004b). Furthermore,administration of interferon gamma 5 min prior to reperfusion to rathearts in vivo induced STAT1 phosphorylation and increased the infarctsize after 25 min ischemia and 2 h reperfusion compared to ischemia/reperfusion alone (McCormick et al., 2006).

On the STAT1 protein level, the carboxyterminal transactivationdomain of STAT1 was involved in mediating ischemia/reperfusioninjury. Mice lacking the aminoterminal part of STAT1 displayed largerinfarcts after 35 min ischemia and 1 h reperfusion than control mice(Stephanou et al., 2002).

The role of the JAK/STAT pathway for ischemia/reperfusion injurywas also studied using the JAK-specific tyrosine kinase inhibitor AG-490. AG-490 has an IC50 of about 10 µM, and maximal inhibition isachieved at concentrations ranging between 50 and 100 µM (Xuanet al., 2001). AG-490 has been demonstrated to block specifically JAK2in acute lymphoblastic leukaemia (Meydan et al., 1996); however, itsspecificity for JAK2 in the myocardium is uncertain. Whereas AG-490inhibited both JAK1 and JAK2 phosphorylation induced by ischemicpreconditioning (IP) in mouse myocardium (Xuan et al., 2001), it hadno effect on the LIF-induced JAK1 phosphorylation in neonatal ratcardiomyocytes (Negoro et al., 2000). AG-490 efficiently inhibitedSTAT3 phosphorylation in rat hearts in vivo (Gross et al., 2006),however, the phosphorylation status of other kinases downstream ofJAK such as Akt may have been affected as well (Ueda et al., 2006). Inin-situ mouse hearts, 40 µg/g AG-490 administered intraperitoneallyhad no effect on infarct size following 24 h ischemia withoutreperfusion (Xuan et al., 2001). In in-situ mouse hearts, 1 µg/g AG-490 applied intravenously did also not modify infarct size after 30 minischemia and 2 h reperfusion (Boengler et al., 2008a), and even 3 µg/gAG-490 administered intravenously had no impact on infarct size inrat myocardium after 30 min ischemia and 2 h reperfusion (Nicolosiet al., 2008). In contrast, in isolated rat hearts undergoing 20 minischemia and 1 h reperfusion and pretreated with 50 µM AG-490(molecular weight 294.3 g/mol) 10 min prior to ischemia creatinekinase release decreased and left ventricular functional recovery uponreperfusionwas improved over that in untreated hearts (Hwang et al.,2005). The contribution of the JAK-STAT pathway to the pathogenesisof ischemia/reperfusion injury was confirmed in isolated rat hearts inwhich already 5 µMAG-490 reduced infarct size after 30min ischemiaand 2 h reperfusion (Mascareno et al., 2001). Thus, while AG-490reduced ischemia/reperfusion injury in isolated hearts, it did notmodify ischemia/reperfusion injury in in vivo hearts, permitting nofinal conclusion on the importance of a pharmacological modulationof the JAK/STAT pathway for the extent of ischemia/reperfusion injury.

3. STAT proteins in cardioprotection byischemic and pharmacological preconditioning

Since the first description in 1986 (Murry et al., 1986), multiplestudies have been performed aiming to unravel the molecularmechanisms of ischemic preconditioning (IP), a phenomenon describ-ing the infarct size reduction by one or several short non-lethalperiods of ischemia/reperfusion preceding a prolonged, lethal episodeof ischemia/reperfusion. The protection by early or classic precondi-tioning occurs immediately, whereas the protection by late precondi-tioning or second window of protection manifests several hours afterthe preconditioning ischemia. The complex signal transductioncascade of IP has not been elucidated yet in detail, but it involves

activation of receptors in the plasma membrane, which transducetheir signals via activation of multiple protein kinases (Schulz et al.,2001; Yellon & Downey, 2003; Shi et al., 2004; Cohen & Downey,2008) and uncoupling of mitochondria (Murphy, 2004; Murphy &Steenbergen, 2007). Reactive oxygen species (ROS) are produced inpart by activation of NADPH-oxidase (Bell et al., 2005) or byuncoupling of oxidative phosphorylation during ischemia/reperfu-sion. While excessive ROS formation is detrimental to cardiomyocytespotentially by opening of the mitochondrial permeability transitionpore (Halestrap et al., 2007), small amounts of ROS function as triggermolecules of IP's cardioprotection. ROS contribute to the cardiopro-tection potentially by activating protein kinases such as protein kinaseC (Baines et al., 1997) or p38 MAP kinase and nuclear translocation ofNFкB (Das et al., 1999). ROS also play a role as second messengers inpharmacological preconditioning with diazoxide (Pain et al., 2000;Heinzel et al., 2005).

The essential role of STAT3 for the cardioprotection by IP has beendemonstrated in mice in which STAT3 was ablated by MLC2vpromoter driven activation of the Cre-recombinase. Whereas inthese animals STAT3 had no impact on ischemia/reperfusion injuryper se, the infarct size reduction by IP was abolished (Smith et al.,2004). This finding was confirmed in cardiomyocytes isolated fromSTAT3 knockout mice which — in contrast to cardiomyocytes fromwildtype mice — displayed no increase in cell viability by IP after 26 hsimulated ischemia. Furthermore, not only ischemic but also pharma-cological preconditioning with tumour necrosis factor α, diazoxide oradenosine was ineffective in reducing cell death in cardiomyocytesisolated from STAT3-deficient mice (Smith et al., 2004). The findingthat ischemic and pharmacological preconditioning were abolished inSTAT3 knockout mice and in STAT3-deficient cardiomyocytes suggestsan activation of the cytoprotective JAK-STAT pathway by thepreconditioning maneuver. Indeed, in isolated rat hearts STAT3phosphorylation increased after three preconditioning cycles of5 min ischemia and 5 min reperfusion (Hattori et al., 2001).Furthermore, six cycles of 4 min ischemia and 4 min reperfusion inin-situ mouse hearts induced a tyrosine phosphorylation of the JAKproteins JAK1 and JAK2, but not of TYK2. Thirty minutes after thepreconditioning cycles, the tyrosine and serine phosphorylations ofboth STAT1 and STAT3 were increased, and the proteins weretranslocated into the nucleus as demonstrated by the decrease incytosolic and the increase in nuclear protein levels. Not only theprotein content, but also the STAT DNA-binding was enhanced by IP(Xuan et al., 2001; Xuan et al., 2007). In addition to STAT1 and STAT3,also STAT5A and STAT6 became activated by IP in isolated rat hearts. IPby four cycles of 5 min ischemia and 10 min reperfusion induced atyrosine phosphorylation of STAT5A and STAT6 after 30 min ischemiaand 2 h reperfusion (Yamaura et al., 2003). However, only STAT5Aappears to be important for cardioprotection since the infarct sizereduction by IP was lost in STAT5A knockout mice, but not in STAT6knockout mice (Yamaura et al., 2003).

The involvement of the JAK-STAT pathway in ischemic andpharmacological preconditioning was further analyzed using theJAK/STAT inhibitor AG-490. The treatment of isolated rat hearts with5 µM AG-490 prior to the preconditioning cycles abolished STAT3phosphorylation and infarct size reduction by IP (Hattori et al., 2001).Additionally, the application of AG-490 during the early reperfusionabolished the cardioprotection by IP and pharmacological precondi-tioning with TNFα, both in isolated rat hearts and in isolated mousecardiomyocytes (Lecour et al., 2005). The cardioprotection by thelanthanide cation gadolinium was lost when the JAK/STAT pathwaywas blocked with 3 µg/g AG-490 prior to ischemia (Nicolosi et al.,2008). Similarly, in mice hearts in-situ 40 µg/g AG-490 intraperito-neally not only abrogated the protection by early, but also that by latepreconditioning (Xuan et al., 2001). The treatment of isolated rathearts with the coronary effluent of preconditioned rat heartsincreased STAT3 phosphorylation, demonstrating that the JAK-STAT


pathway is also involved in transferred cardioprotection (Huffmanet al., 2008). Thus, independent from the animal model and theexperimental condition, AG-490 abolished IP's protection.

Since the administration of the transcription inhibitor actinomycinD prior to ischemia did not abolish the cardioprotection by early IP inisolated rat or rabbit hearts (Rowland et al., 1997; Matsuyama et al.,2000) or in rabbit hearts in vivo (Thornton et al., 1990) the earlycardioprotective effect is independent of increased mRNA levels. Theenhanced protein level induced by IP appears therefore to be regulatedat the translational level (Rowland et al.,1997;Matsuyama et al., 2000).In contrast, the infarct-sparing effect of late preconditioning, whichoccurs 24–72 h after the preconditioning stimulus (Kuzuya et al., 1993;Marber et al., 1993) clearly involves protein synthesis. Therefore, aninvolvement of STAT proteins functioning in cardioprotection asactivators of target gene transcription is likely to occur in late ratherthan in early IP. Since late preconditioning is mediated in part by theinducible nitric oxide synthase (iNOS) (Bolli et al., 1997; Takano et al.,1998; Guo et al., 1999), iNOS expression was analyzed after inhibitionof the JAK-STAT pathway by AG-490 (Xuan et al., 2001). Pre-treatmentof mouse hearts with 40 µg/g AG-490 intraperitoneally inhibited theup-regulation of iNOS protein and the increase in iNOS activity 24 hafter the preconditioning stimulus. Three STAT consensus sequences,the so-called interferon gamma activation sites (GAS elements), arepresent in the mouse iNOS promoter (Singh et al., 1996), allowingSTAT3 to directly control the transcription of the iNOS gene. In IL-6knockout mice, the preconditioning-induced activation of JAK1, JAK2,STAT1, and STAT3 was reduced, and the increases in iNOS andcyclooxygenase 2 (COX2) protein expression 24 h after the precondi-tioning stimulus were abrogated (Dawn et al., 2004). STAT3 phosphor-ylation, DNA binding and late preconditioning-induced up-regulationof COX2 expressionwere also abrogated in eNOS knockout mice (Xuanet al., 2007), confirming the role of eNOS-derived NO as trigger of latepreconditioning.

However, since the analysis of differentially expressed genesbetween wildtype and STAT3 knockout mice revealed multiple targetgenes, among them factors involved in angiogenesis, inflammation,apoptosis, extracellular matrix composition and cellular signaling(Hilfiker-Kleiner et al., 2004, 2005; Snyder et al., 2008), the role ofSTAT3 in cardioprotection can not be attributed to the regulation ofiNOS and COX2 expression alone.

Stimulation of the JAK-STAT pathway and transfection of constitu-tively active STAT3 in neonatal rat cardiomyocytes reduced the hypoxia/reoxygenation-induced formation of detrimental amounts of ROS(radical burst). The decrease in ROS was achieved via up-regulation ofthe mitochondrial free radical scavenging enzyme manganese super-oxide dismutase (MnSOD), which is located in themitochondrial matrix(Negoro et al., 2001). Since the STAT3 binding sites are present in theMnSOD promoter region, it is likely that the increased mRNA level ofMnSOD was achieved via binding of STAT3 to the nuclear MnSODpromoter. Additionally, inmicewith a cardiac-specific overexpression ofconstitutively active STAT3, ROS generation at early reperfusion wasreduced compared to that in wildtype mice. The reduction of ROS wasattributed to an increase in themRNAandprotein level of the free radicalscavenging enzymes metallothionein 1 and 2 (MT1, MT2), whereas themRNA level of MnSOD was similar between STAT3 overexpressing andnon-transgenic mice (Oshima et al., 2005).

Taken together, the available information suggests that activationof STAT3 is essential for the cardioprotection by ischemic andpharmacological preconditioning. Especially in late preconditioning,STAT3 exerts its protective function by modifying the transcription oftarget genes.

4. Does STAT3 have an impact on mitochondrial function?

Mitochondria have been proposed to be involved in the signaltransduction cascade of IP's cardioprotection. Mitochondria generate

ROS — partially by uncoupling of oxidative phosphorylation duringischemia/reperfusion—which, if present in small amounts, trigger IP'scardioprotection (Baines et al., 1997; Das et al., 1999; Pain et al., 2000;Forbes et al., 2001; Heinzel et al., 2005). Additionally, IP's cardiopro-tection includes inhibition of the opening of the mitochondrialpermeability transition pore (MPTP), suggesting that mitochondriaalso act as end-effectors of IP (Hausenloy et al., 2002; Javadov et al.,2003). MPTP opens when exposed to high concentrations of ROS(radical burst) and calcium at a normal intracellular pH.MPTP openingprobability is reduced by inactivating glycogen synthase kinase 3 β(GSK3β) (Juhaszova et al., 2004) and by overexpressing Bcl-xL (Pacher& Hajnoczky, 2001). Since mitochondria are central for IP's cardio-protection, a possible involvement of STAT3 for mitochondrialfunction has been investigated. STAT3 directly influences mitochon-drial respiration since expression of a constitutively active STAT3 inpro-B lymphocytes restored the oxygen consumption in Tyk2-deficient cells treated with interferon beta (Potla et al., 2006).

Increased STAT3 levels not only reducenecrosis but also decrease theamount of apoptosis induced by ischemia/reperfusion (Negoro et al.,2000;Hilfiker-Kleiner et al., 2004). STAT3modifies the expression of theanti-apoptotic factor Bcl-xL, which is present in the outer mitochondrialmembrane (Zhang et al., 2007; Lin et al., 2005) and known to stabilizemitochondrial membranes (Gabriel et al., 2003). However, in cardio-myocytes, LIF-stimulated Bcl-xL expressionwas mediated by binding ofSTAT1 rather than of STAT3 to the GAS consensus element in the Bcl-xLpromoter region (Fujio et al., 1997).

Loss of cardioprotection by IP with genetic ablation of STAT3involves posttranslational protein modifications converging at thelevel of mitochondria (Murphy, 2004; Hausenloy & Yellon, 2006).Therefore, it is possible that STAT3 exerts effects independent of thetranscriptional control of nuclear target genes. Indeed, in opioid-induced cardioprotection inhibition of the JAK-STAT pathway by AG-490 reduced the phosphorylation of GSK3β (Gross et al., 2006).Inhibition of GSK3β — reflected by increased phosphorylation of theprotein— is suggested to protect by blockingMPTP opening (Juhaszovaet al., 2004). Activation of the JAK/STAT pathway by treatment ofneonatal rat cardiomyocytes for 6 h with IL-6 followed by 18 hcultivation in maintenance medium resulted in a polarization of themitochondrial membrane potential and increased the mitochondrialCa2+-concentration compared to untreated cells (Smart et al., 2006).Furthermore, IL-6 inhibited reperfusion-induced mitochondrial depo-larization and swelling. In hippocampal neurons, stimulation withleptin, which regulates energy metabolism and is also suggested to beimplicated in cardiovascular disease (Heusch, 2006), induced astabilization of the mitochondrial membrane potential in a STAT3-dependent manner (Guo et al., 2008).

We have recently shown that apart from thewell known localizationof the protein in the cytosol, associated with receptors at the plasmamembrane or in the nucleus, STAT3 is localized also in mitochondriaisolated from the rat left ventricle (Boengler et al., 2008b). Whether ornot themitochondrial STAT3 level is affected by IP remains unknown atpresent. In pigs, addition of diazoxide — a substance acting onmitochondrial ATP-dependent potassium channels — to a cardioplegicsolution reduced cardiomyocyte damage compared to cardioplegiaalone, and diazoxide induced tyrosine phosphorylation of STAT3 andnuclear DNA binding suggesting that factors influencing mitochondrialfunction also control nuclear STAT3 activation (Hsieh et al., 2007).

STAT3 regulates the transcription of genes encoding proteins,which are imported into the mitochondria thereby modifyingmitochondrial function such as the maintenance of the mitochondrialmembrane potential.

5. STAT3 in ischemic postconditioning

Ischemic postconditioning describes the reduction of infarct size byseveral very brief (5–60 s) cycles of intermittent ischemia/reperfusion

Fig. 2. Infarct size (in % of the area at risk, AAR) in young C57Bl6/J micewithout and withAG-490 and in young STAT3-deficient mice undergoing ischemia/reperfusion alone orischemic postconditioning (3×10 s or 5×5 s). Modified from Boengler et al. (2008a).

Fig. 3. Role of STAT3 in the cardioprotection by ischemic pre- and postconditioning.Ischemic preconditioning (IP) and ischemic postconditioning by three cycles of 10 sischemia and reperfusion (iPoco3×10) transduce their signals via STAT3. STAT3 functionsin regulating the expression of target genes and may also be involved in modulatingmitochondrial (mito) function, both processes contributing to cardioprotection. The infarctsize reduction is abrogated in STAT3 knockout mice (STAT3 KO), by pharmacologicalinhibition of STAT3 with AG-490, and in aged mice, in which the STAT3 protein level isreduced. In contrast, the cardioprotection by ischemic postconditioningwith five cycles of5 s ischemia and reperfusion (iPoco5×5) is independent of STAT3 signaling. iNOS:inducible nitric oxide synthase; COX2: cyclooxygenase 2; MnSOD:manganese superoxidedismutase; MT1, MT2: metallothionein 1 or 2, respectively; Bcl-xL: basal cell lymphoma-extra large.


following a sustained ischemic insult (Zhao et al., 2003). Ischemicpostconditioning was first described in a dog model, however,ischemic postconditioning can also be recruited clinically in humansto reduce myocardial ischemia/reperfusion injury (Staat et al., 2005;Thibault et al., 2008). Since ischemic pre- and postconditioning sharesome, but not all signaling elements (Heusch et al., 2006), we recentlyinvestigated whether or not STAT3 also contributes to the cardiopro-tection by ischemic postconditioning (Boengler et al., 2008a). In wild-type mice, ischemic postconditioning by three cycles of 10 s ischemiaand 10 s reperfusion (iPoco3x10) or five cycles of 5 s ischemia and 5 sreperfusion (iPoco5x5) immediately after 30 min ischemia signifi-cantly reduced infarct size, as assessed 2 h after reperfusion. Thecardioprotective effect of iPoco3x10was associatedwith an increase inSTAT3 phosphorylation. The pre-treatment of wildtype mice with AG-490 abrogated the increase in STAT3 phosphorylation and the infarctsize reduction by iPoco3x10. Additionally, the cardioprotection byiPoco3x10 was lost in mice with a cardiomyocyte-specific knockout ofSTAT3, demonstrating that STAT3 was not only important for ischemicpre- but also for ischemic postconditioning. However, iPoco5x5reduced infarct size also in STAT3 knockout mice, indicating that thecardioprotection by ischemic postconditioning involves signalingcascades independent of STAT3 (Fig. 2). Thus, ischemic postconditio-ning's cardioprotection is dependent on the postconditioning protocolin wildtype and in STAT3-deficient hearts. A scheme demonstratingthe putative roles of STAT3 in cardioprotection is presented in Fig. 3.

Studies designed to identify factors important for the cardiopro-tection by ischemic pre- and postconditioning resulted in thedetection of multiple proteins localized at different cellular compart-ments (sarcolemma, cytosol, mitochondrion, nucleus) with distinctfunctions such as ion channels, receptors, protein kinases ortranscription factors (Vinten-Johansen, 2007; Downey et al., 2008).While several factors act in concert to induce cardioprotection(Suleman et al., 2008), inhibition of one protein is often sufficient toabolish the cardioprotection, making it difficult to establish ahierarchy regarding the relevance of different proteins in cardiopro-tection. STAT3 represents one factor, which — if knocked out —

eliminates the infarct size reduction by ischemic pre- and postcondi-tioning. The fact that STAT3 influences a variety of cellular functions byregulating the transcription of multiple target genes or acts indepen-dently of nuclear transcription within mitochondria makes it likelythat STAT3 indeed is a key player in the signal transduction cascade ofcardioprotection.

6. JAK/STAT proteins regulate myocardial apoptosis

The cardioprotection by ischemic pre- and postconditioningreduces both necrotic and apoptotic cell death (Zhao & Vinten-Johansen, 2002, 2007). Previous studies on the role of JAK/STATproteins in ischemia/reperfusion-induced apoptosis have focusedpredominantly on STAT1 and STAT3 (Stephanou & Latchman, 2005).The induction of apoptosis in neonatal rat cardiomyocytes bysimulated ischemia was associated with an activation of STAT1. Theoverexpression of STAT1 in neonatal rat cardiomyocytes increased andthe expression of antisense STAT1 decreased ischemia-induced celldeath compared to control transfected cells (Stephanou et al., 2000b).The enhancement of cardiomyocyte death by ischemia/reperfusionwas dependent on the phosphorylation of STAT1 at Ser727 (Stephanouet al., 2001; Soond et al., 2008). STAT1 exerted its pro-apoptoticfunction by enhancing the transcription of the pro-apoptotic p53-target genes Bax, Noxa and Fas (Townsend et al., 2004a) and byinhibiting the promoters of the anti-apoptotic genes Bcl-2 and Bcl-x(Stephanou et al., 2000a). In contrast to STAT1, STAT3 plays a role indecreasing apoptotic cell death of cardiomyocytes. Treatment ofneonatal rat cardiomyocytes with hydrogen peroxide resulted in asubpopulation of cardiomyocytes susceptible and in a subpopulationof cells resistant to apoptosis. The apoptosis-resistant cardiomyocytesdisplayed higher levels of STAT3 (Lu et al., 2008). In isolated rat hearts,the perfusion with AG-490 enhanced the number of apoptoticcardiomyocytes (Hattori et al., 2001). The anti-apoptotic function ofSTAT3 was confirmed in a rat model of acute myocardial infarction,where AG-490 suppressed STAT3 phosphorylation, increased Baxexpression and the number of apopotic nuclei (Negoro et al., 2000).Additionally, mice with a cardiomyocyte-specific deletion of STAT3displayed higher apoptosis rates induced by ischemia/reperfusion(Hilfiker-Kleiner et al., 2004) and by lipopolysaccharide (Jacoby et al.,2003) than their wildtype counterparts.


Both STAT1 and STAT3 are implicated in regulating apoptosis ofcardiomyocytes. However, STAT1 and STAT3 exert opposing roles inprogrammed cell death; whereas STAT1 mediates a pro-apoptotic,STAT3 mediates an anti-apoptotic response.

7. The expression of STATs in aged cells or organs

The expression levels of STAT proteins are not constant throughoutthe life span of an organism, but decrease with age in different organsand cell types. The total STAT1 level and STAT1 phosphorylationinduced by interferon gamma were reduced in macrophages isolatedfrom old (18–24 months) compared to young Balb/c mice. Thisreductionwas accompanied by an inhibition of STAT1 gene expressionin response to interferon gamma (Yoon et al., 2004). In the aged rathippocampus the IL-4 concentration was reduced, leading to adecrease in JAK1 and STAT6 phosphorylation (Maher et al., 2005).Whereas the ability of natural killer cells to produce interferon gammawas similar between young and aged cells, nuclear translocation ofSTAT5A and STAT5B was decreased in aged cells (Albright et al., 2004).However, in cultured senescent human fibroblasts (WI-38 cells)angiotensin II was still capable of inducing nuclear STAT3 transloca-tion (Wang et al., 2006). Furthermore, a downregulation of STAT3, butnot of STAT1 was observed in the aged rat brain (De-Fraja et al., 2000).In aged mouse myocardium (C57Bl/6 mice older than 13 months),total and phosphorylated STAT3 levels were reduced. The age-relateddecrease of STAT3 resulted in an impairment of the signaling cascadeof ischemic postconditioning, since a postconditioning stimulus ofthree cycles of 10 s ischemia and 10 s reperfusion (iPoco3×10) inducedlower levels of phosphorylated STAT3 in aged compared to youngmice. The defect in STAT3 phosphorylation by iPoco3×10 in agedmyocardium also attenuated cardioprotection, since the infarct sizereduction by iPoco3×10was lost in agedmouse hearts (Boengler et al.,2008a). However, a postconditioning stimulus of five cycles of 5 sischemia and 5 s reperfusion, which was capable to reduce infarct sizein STAT3 knockout mice, reduced infarct size also in aged mice hearts.Therefore, in aged mouse hearts only a stronger stimulus than inyoung mice was effective in reducing infarct size. Apart from ischemicpostconditioning, also the cardioprotection by IP was abolished in themyocardium of mice older than 13 months (Boengler et al., 2007).These results demonstrate that the age-related decrease in themyocardial STAT level interferes with the capacity of the heart torespond to the cardioprotective stimuli of ischemic pre- andpostconditioning. Apart from aging, the STAT3 expression wasreduced in a rat model of diabetes (Gross et al., 2007) and in patientswith end-stage heart failure (Podewski et al., 2003), pathophysiolo-gical conditions associated with a loss of cardioprotection (Ferdinandyet al., 2007).

8. Consequences of STAT3 activation

The IL-6-gp130-JAK/STAT signaling pathway is involved in theresponse of the heart tomultiple physiological and pathophysiologicalforms of stress and is a key player in the development of cardiachypertrophy and heart failure (for detailed review see (Booz et al.,2002; Terrell et al., 2006; Fischer & Hilfiker-Kleiner, 2007)).

In isolated cardiomyocytes subjected to hypoxic stress, elevatedlevels of IL-6 were detected (Yamauchi-Takihara et al., 1995). Also in amodel of long-term intermittent hypoxia in rat hearts, the develop-ment of eccentric hypertrophy was associated with increased levels ofIL-6, STAT1 and STAT3 (Chen et al., 2007). IL-6 is causally involved inthe progression of hypertrophy since mice overexpressing both IL-6and IL-6 receptor developed ventricular hypertrophy and ventricularwall thickening at the age of five months (Hirota et al., 1995).Furthermore, IL-6 was upregulated in the viable border zone adjacentto infarcted tissue (Gwechenberger et al., 1999). However, nodifferences in infarct size, mortality rates, left ventricular remodeling,

and left ventricular dysfunction were detected in IL-6 knockout micecompared to wildtype mice, presumably caused by a compensation ofIL-6 loss by other members of the IL-6 family (Fuchs et al., 2003). IL-6transduces a hypertrophic signal to STAT3 also in satellite cells,demonstrating that the role of IL-6 and STAT3 for hypertrophy is notrestricted to cardiac myocytes (Serrano et al., 2008).

LIF, another IL-6 type cytokine, has been shown to be central forcardiogenesis during embryonic development (Bader et al., 2000).However, LIF was also expressed in the adult heart in response tohemodynamic overload and it induced sarcomeric protein synthesis(Wang et al., 2001). In a canine model of pacing-induced heart failure,a positive correlation between the LIF mRNA level and left ventricularmass index was observed (Jougasaki et al., 2003). A stimulation of theJAK/STAT pathway by LIF treatment of cardiac fibroblasts for 48–72 hreduced the collagen content and matrix metalloproteinase activity,suggesting involvement of LIF in extracellular matrix remodeling aftermyocardial injury (Wang et al., 2002a). Indeed, overexpression of LIFthrough LIF-plasmid injection in the mouse thigh muscle induced areduction in infarct size, decreased fibrosis and stimulated neovascu-larization in the border zone following myocardial infarction in rathearts. Furthermore, overexpression of LIF in acute myocardialinfarction increased the number of cardiomyocytes in the cell cycleand enhanced the mobilization of bone marrow cells to the heart (Zouet al., 2003). The overexpression of LIF by adenovirus-mediated genetransfer into myocardium bordering the ischemic area after leftanterior coronary artery ligation resulted in a decreased rate ofapoptosis and enhanced the cardiomyocyte cross-sectional area (Berryet al., 2004). Stimulation of cardiac myocytes with LIF resulted inincreased cell size, characterized by an enhancement of cell lengthrather than cell width (Wollert et al., 1996). After LIF stimulation,cardiac myocytes transfected with a STAT3 expressing adenovirus hadincreased levels of atrial natriuretic factor (ANF) mRNA and protein,representing a reactivation of embryonic genes known to beassociated with a hypertrophic response (Kunisada et al., 1998). Incardiomyocytes transfected with the JAK inhibitor SOCS3, the LIF-induced cardiomyocyte hypertrophy and reduction of cardiomyocyteapoptosis were attenuated (Yasukawa et al., 2001).

Apart from promoting survival and hypertrophy, LIF reducescontractile function and increases anaerobic metabolism in isolatedrat cardiomyocytes, at least in part by a decreased expression ofcomponents in the adenosine triphosphate synthase complex andinsulin-like growth factor binding proteins 1 and 6 (Florholmen et al.,2004).

The mRNA of cardiotrophin 1 (CT-1), another member of the IL-6-type cytokines, increased in the early stage of hypertrophy inspontaneously hypertensive rats (Ishikawa et al., 1999) and in acanine model of pacing-induced heart failure where the CT-1 mRNAlevel positively correlated with the left ventricular mass index(Jougasaki et al., 2000). Additionally, the CT-1 mRNAwas upregulatedalready 1 day after myocardial infarction, and this effect persisted for56 days after infarction compared to sham operated rats. Theincreased CT-1 mRNA level persisted not only in the infarcted areabut also in the non-infarcted area of the heart (Aoyama et al., 2000).Similar to LIF stimulation, treatment of cardiomyocytes with CT-1induced an increase in cell length rather than in cell width (Wollertet al., 1996). CT-1 stimulated the expression of STAT3 in cardiacmyocytes, and this effect was blocked by AG-490 or simvastatin (Wuet al., 2006). Transfection of cardiomyocytes with PIAS3— an inhibitorof STAT3 — attenuated the CT-1-mediated hypertrophy, however, theCT-1-induced decrease in apoptosis was independent of STAT3(Railson et al., 2002). Whereas these data suggest that CT-1 signalingis mediated by STAT3, recent studies indicate that the hypertrophicresponse induced by CT-1 may be also transduced via MEK5-ERK5rather than by STAT3 (Takahashi et al., 2005).

When analyzing the receptor level of the IL-6-gp130-JAK/STATpathway, mice with a ventricular knockout of gp130 displayed no

Fig. 4. Role of STAT3 in hypertrophy and heart failure. CT-1, LIF and IL-6 transduce theirsignals via gp130 receptor homodimers or gp130/LIF receptor heterodimers. Down-stream, STAT3 is induced either directly or indirectly via ERK activation. Induction ofSTAT3 enhances cardiomyocyte hypertrophy and reduces apoptosis. Leptin, whichtransduces its signal via the leptin receptor, induces hypertrophy in a STAT3-dependentmanner. Stimulation of the AngII/AT1 receptor pathway e.g. by LIF or IL-6 enhanceshypertrophy via STAT3. Since STAT proteins bind to the angiotensinogen promoter,STAT3 is part of the cardiac autocrine AngII loop. LIF-R: LIF receptor, AT1: AT1 receptor,LRb: long form of the leptin receptor, ERK: extracellular signal related kinase. Inhibitoryprocesses are represented in dashed lines.


change in cardiac function at baseline compared to control mice.However, following thoracic aorta constriction (TAC) gp130 knockoutmice developed a dilated cardiomyopathy and had increasedcardiomyocyte apoptosis, whereas in control mice TAC was associatedwith compensatory hypertrophy (Hirota et al., 1999). The TAC-mediated increase in STAT3 phosphorylation observed in controlmice was attenuated in gp130 knockout mice (Hirota et al., 1999). Theoverexpression of dominant negative gp130 under control of the αmyosin heavy chain promoter had no impact on heart to body weightratio or cardiomyocyte cross-sectional area under control conditions.Yet, with subjection of the transgenic mice to TAC the pressureoverload-induced hypertrophy was attenuated compared to wildtypemice, and this was associatedwith a decreased activation of STAT3 andreduced mRNA level of hypertrophic marker genes such as brainnatriuretic factor (Uozumi et al., 2001).

The JAK/STAT pathway has also been reported to interfere with therenin–angiotensin system (RAS), which is involved in the pathophy-siology and progression of hypertrophy and heart failure. AngiotensinII (AngII), the major peptide of the RAS, activates STAT1 and STAT2within 30 min and STAT3 within 120 min in cultured rat neonatalcardiomyocytes (Kodama et al., 1998). Furthermore, DNA-bindingactivities of STAT3 and STAT6 are induced by AngII in culturedcardiomyocytes (Mascareno et al., 1998). A stimulation of Rac1-mediated STAT3 phosphorylation by AngII has also been described inatrial myocytes and fibroblasts. The AngII-induced increase in collagensynthesis and atrial fibrosis were attenuated by oral administration oflosartan, an AT1 antagonist (Tsai et al., 2008). Ligation of the coronaryartery in rats increased STAT3 phosphorylation, and this increasedSTAT3 phosphorylation was attenuated by the AT1 antagonistcandesartan (Omura et al., 2001). AngII not only induces STATphosphorylation, but also binding of STAT proteins to the angiotensi-nogen promoter, suggesting that the JAK/STAT pathway is part of thecardiac autocrine AngII loop (Mascareno et al., 1998; Fukuzawa et al.,2000).

However, it was also observed that LIF-mediated STAT3 phosphor-ylation in cardiac myocytes was attenuated by AngII pretreatment.Preincubation of cardiac myocytes with the AT1 antagonist CV11974before the addition of AngII and LIF blocked the inhibitory effect ofAngII on LIF-induced STAT3 phosphorylation (Tone et al., 1998).

Obesity is associated with enhanced levels of the energymetabolism regulating hormone leptin, and leptin has been suggestedto be implicated in cardiovascular disease (Heusch, 2006). Plasmalevels of leptin are elevated in patients with congestive heart failure(Leyva et al., 1998). Treatment of neonatal rat cardiomyocytes withleptin induces an increase in cell surface area (Rajapurohitam et al.,2003), and the development of hypertrophy is mediated by enhancedSTAT3 phosphorylation and DNA binding (Abe et al., 2007). Incontrast, administration of another member of the IL-6 family, ciliaryneurotrophic factor (CNTF), to leptin-deficient mice reduces theobesity-associated cardiac hypertrophy (Raju et al., 2006).

Taken together, the cardiac gp130-JAK/STAT pathway is activatedby various forms of stress and stimulation of the pathway is associatedwith the development of cardiac hypertrophy.

9. STAT3 in exercise

Physiological cardiac hypertrophy occurs in response to growth,exercise or pregnancy where cardiomyocyte hypertrophy is paralleledby a proportional growth of the vasculature and the capillary networkand is usually not accompanied by cardiac fibrosis (Olson, 2004).Regular exercise induces anti-inflammatory effects with elevatedlevels of anti-inflammatory cytokines such IL-6 which suppressoxidative stress and the production of pro-inflammatory cytokines,i.e. TNFα. In addition, exercise-induced production and release of IL-6from myofibers may contribute to abrogate an atherogenic lipidprofile, which is often associated with chronic cardiac diseases

(Petersen & Pedersen, 2006). However, there is little information ona direct role for cardiac gp130 and/or STAT3 during exercise.

10. STAT3 in pregnancy

Reversible physiological growth of the heart also occurs in womenduring pregnancy, where ventricular hypertrophy, diastolic dysfunc-tion and longer QT-interval dispersion develop as a result of volumeoverload (Eghbali et al., 2005). STAT3 is activated in the normal heartduring pregnancy. However, pregnancy induced hypertrophy, theproportional growth of the cardiac vasculature and survival is normalin STAT3-KO females suggesting that STAT3 is not essential in theheart during pregnancy (Hilfiker-Kleiner et al., 2007).

By contrast, the physiological stress of nursing resulted in apostpartum cardiomyopathy (PPCM) in STAT3-KO females (Hilfiker-Kleiner et al., 2007), a disorder in which initial left ventricular systolicdysfunction and symptoms of heart failure occur between the latestages of pregnancy and the early postpartum period. Unbalancedoxidative stress causes a conversion of the nursing hormone prolactininto an anti-angiogenic and pro-apoptotic 16 kDa form, which causes amassive loss in cardiac capillaries (Hilfiker-Kleiner et al., 2007).Subsequently STAT3-KO hearts become ischemic and develop PPCM(Hilfiker-Kleiner et al., 2007), indicating a major role for STAT3 in theprotection of the maternal heart form peripartum associated adverseremodeling. A schematic representation of the role of STAT3 inhypertrophy and heart failure is shown in Fig. 4.

11. STAT3 in heart failure

The effect of STAT3 on heart failure development was assessed inSTAT3 knockoutmice. Micewith a cardiomyocyte-specific knockout ofSTAT3 did not develop gross cardiac abnormalities of function andmorphology at young age (Jacoby et al., 2003). However, already threemonths old STAT3-KO mice displayed small morphological changessuch as a modest increase in interstitial fibrosis and a decrease in


myocardial capillary density. At an age of six months, STAT3-KO micedeveloped severe cardiac fibrosis, an important determinant ofpathologic hypertrophy in heart failure (Hilfiker-Kleiner et al., 2004).Moreover, adult STAT3-KO mice displayed increased expression ofgenes well known to inhibit angiogenesis and/or alter extracellularmatrix composition, which is consistent with the profibrotic pheno-type of the mice (for review see (Hilfiker-Kleiner et al., 2005)). Incultured mouse fibroblasts, LIF stimulation for 48–72 h increasedfibroblast proliferation and decreased the collagen content, confirm-ing a role of the JAK/STAT pathway in preventing excessive remodeling(Wang et al., 2002a).

The STAT3-KO mice also exhibited an increased susceptibility tobacterial infection (Jacoby et al., 2003). Ultimately, the lack ofcardiomyocyte STAT3 led to age-related heart failure (Jacoby et al.,2003; Hilfiker-Kleiner et al., 2004) indicating that STAT3, similar togp130, was required for protection of the aging heart. Mice with acardiomyocyte-specific overexpression of STAT3 developed concentrichypertrophy and increased capillary density accompanied byenhanced expression of the pro-angiogenic factors vascular endothe-lial growth factor and VE-cadherin (Osugi et al., 2002).

IL-6 cytokine levels are elevated in patients with unstable angina,after myocardial infarction, and in patients with heart failure(Manginas et al., 2005; Shu et al., 2007). IL-6 is a strong prognosticmarker for the morbidity and mortality in patients with heart failureor patients post-myocardial infarction (Tsutamoto et al., 1998; Oruset al., 2000; Rattazzi et al., 2003). More recently, the IL-6-gp130-JAK-STAT signaling cascade was explored in patients with end-stage heartfailure at the mRNA and protein level, indicating that this signalingcascade was altered at each stage of human heart failure (Eiken et al.,

Table 2Pharmacological stimulation of JAK/STAT activation

Stimulus Treatment Species Hear

Drug only Before isch During isch Reperfusion In vi

Erythropoietin X X Infant rabbit

X X Adult rabbit

Insulin X RatX Mouse

GCSF X Rat

X Neonatal rat

X Mouse X

Prostaglandin E2 X Neonatal rat

WT mice,EP(4)KO mice

X

Morphine X Rat X

X Rat X

DHA X Rat

X Rat XX Rat X

Abbreviations: CM: cardiomyocytes; DHA: dehydroascorbic acid; dnSTAT3tg: dominant necolony stimulating factor; isch: ischemia; KO: knockout; MI: myocardial infarction; WT: wi

2001; Podewski et al., 2003). Whereas the mRNA levels for gp130 andIl-6 were similar between patients with end-stage heart failure anddonor hearts, the LIF mRNA level was increased in heart failurepatients (Eiken et al., 2001). On the protein level, however, the cardiacexpression of IL-6 was reduced while the expression of LIF waselevated (Podewski et al., 2003). The expression of cardiotrophin-1(CT-1), another member of the IL-6 cytokine family, remainedunchanged. At the receptor level, gp130 expression was not different,but failing hearts displayed enhanced gp130 activation (tyrosinephosphorylation). The expression of JAK2, the next downstreamsignaling molecule of the gp130 receptor, was also not altered, butsurprisingly, its activation state (tyrosine phosphorylation) wasdiminished in failing hearts. Most strikingly, the effector signalingmolecule of the IL-6-gp130-JAK-STAT cascade, STAT3, was severelyreduced in expression and activation in failing hearts (Podewski et al.,2003). However, STAT1, 3β, 5 and 6 phosphorylation increased inpatients with dilated cardiomyopathy, whereas the total STAT proteinlevel was similar between failing and non-failing hearts (Ng et al.,2003).

In patients with ischemic heart disease only STAT1 and 5phosphorylation increased, the phosphorylation of STAT3 was similarto that in non-failing hearts (Ng et al., 2003).

12. Does STAT3 represent a therapeutictarget for the treatment of cardiovascular diseases?

A persistent activation of STAT transcription factors, in particularSTAT1, 3 and 5, has been described in a variety of malignanttransformations. Aberrant STAT signaling was observed in blood

t CM JAK/STATactivation

Effect References

vo In vitro

X JAK1 Increased recovery ofpostischemic ventriculardeveloped pressure

(Rafiee et al., 2005)JAK2STAT3STAT5A

X JAK2 Preservation of cardiacfunction, reduction of infarctsize, reduction of apoptosis

(Parsa et al., 2004)STAT3STAT5

X STAT3 Reduction of infarct size (Fuglesteg et al., 2008)X STAT3 Increase in cell viability in

WT onlyX JAK2 Infarct size reduction (Ueda et al., 2006)

STAT3X JAK2 Reduction of H2O2-induced

apoptosis, infarct sizereduction, preservation ofcardiac function in WT butnot in dnSTAT3tg mice

(Harada et al., 2005)STAT1STAT3STAT3

X STAT3 Reduction in protein synthesisand cell size in STAT3-silencedcells

(Frias et al., 2007)

STAT3 inWT only

No difference in infarct size,reduction of myocytecross-sectional area andinterstitial collagen in EP(4)KOafter MI

(Qian et al., 2008)

JAK2 Infarct size reduction (Gross et al., 2006;Gross et al., 2007)STAT3

JAK2 Infarct size reductionSTAT3

X STAT3 Increased cell viability afterhypoxia and hypoxia/reoxygenation, reduction ofapoptosis

(Guaiquil et al., 2004)

Infarct size reductionInfarct size reduction

gative STAT3 transgenic mice; EP(4): Prostaglandin E2 receptor 4; GCSF: granulocyteldtype.


malignancies such as leukemias, lymphomas and multiple myeloma, aswell as in solid tissue (head, neck, breast, and prostate cancer, for reviewsee (Buettner et al., 2002)), and constitutively active STAT3 has anestablished causal role for oncogenesis by dysregulating the expressionof target genes involved in cell proliferation, apoptosis and angiogenesis(for reviewsee (Turkson,2004). Different strategieswereused inorder toblock STAT3 activity, among them approaches in which dominantnegative mutants were expressed (Burke et al., 2001; Gariboldi et al.,2007), upstream kinases were inhibited e.g. by AG-490 (Turkson et al.,1999; Toyonaga et al., 2003; Huang et al., 2006; Levitzki & Mishani,2006), or STAT3 decoy oligonucleotides were used in order to preventSTAT3 from binding to its target DNA sequences (Xi et al., 2005; Zhanget al., 2007; Sun et al., 2008). Additionally, drugswere developed in orderto directly target and inhibit STAT3 (for review see (Kurdi & Booz, 2007)).

Whereas STAT3 activation is detrimental in oncogenesis, STAT3activation is important for mediating protective effects in the heartsuch as compensatory hypertrophy or a reduction of apoptosis.Additionally, STAT3 activation is central for the cardioprotection byischemic pre- and postconditioning. Therefore, in contrast to cancertherapy, treatment of cardiovascular diseases or stimulation ofcardioprotective signaling cascades would imply activation of STAT3signaling rather than its inhibition. Stimulation of STAT3 activity isexpected to be beneficial during the transition from compensatedhypertrophy to heart failure. STAT3-KO mice developed postpartumcardiomyopathy (PPCM), and the STAT3 protein level was reduced inthe left ventricle of women with PPCM (Hilfiker-Kleiner et al., 2007).In a preliminary clinical study, women with high risk for PPCM due toa previous pregnancy were treated with the dopamine-D2-receptoragonist bromocriptine, which blocks prolactin secretion. Serum levelsof bromocriptine-treated womenwere reduced to normal values, andbromocriptine-treated women displayed a preserved or increased leftventricular function compared to untreated women (Hilfiker-Kleineret al., 2007).

Several agonists of JAK/STAT signaling in the myocardium areknown (reviewed in (Kurdi & Booz, 2007)), among them agonists withtherapeutic potential (see Table 2). However, strategies inducingSTAT3 signaling e.g. by activation of gp130 signaling must take intoaccount that gp130 does not transduce its signals exclusively to theJAK/STAT pathway but that gp130 is also capable of activating othersignal transduction cascades such as phosphatidyl inositol 3-kinase/Akt or MEK/ERK (Fischer & Hilfiker-Kleiner, 2007). Furthermore,STAT3 as a transcriptional activator and co-activator controls thetranscription of several target genes and excessive up-regulation oftarget mRNAs by STAT3 activation could be detrimental for themyocardium. For example, induction of the STAT3 target gene iNOS viaIL-6 increases nitric oxide production and decreases cardiac contrac-tility (Yu et al., 2003), potentially via inhibition of mitochondrialenergy production (Tatsumi et al., 2000). A persistent activation ofSTAT3 should be avoided in view of the fact that this has been shownto be associated with malignant transformation (Turkson, 2004).Finally, the balance between STAT3 homo- and heterodimerizationmust be considered when targeting STAT3 activation, since the STATproteins differ in their DNA-binding site preferences (Ehret et al.,2001). Therefore, the STAT3 activation in cardiovascular disease mustbe carefully controlled and well defined treatment strategies aimingfor a balanced STAT3 signaling should be developed in order to protectthe heart from pathophysiological stress.

In summary, STAT3 is important for the cardioprotection byischemic and pharmacological pre- and ischemic postconditioning.The reduction of STAT3 activity — with increasing age, by pharmaco-logical inhibition and in STAT3 knockout mice — is associated with aloss of cardioprotection by pre- and postconditioning. STAT3 exerts itsprotective function via regulating the transcription of target genes andmay also control mitochondrial function. Increased STAT3 expressionand phosphorylation for longer period of times is associated with thedevelopment of hypertrophy. Patients with end-stage heart failure

display alterations in the gp130-JAK/STAT signaling cascade, amongthem a reduction in total and phosphorylated STAT3. Whereas severalmalignancies are associated with STAT3 activation and treatmentstrategies therefore aim at STAT3 inhibition, treatment of cardiovas-cular diseases rather implies a carefully controlled activation of STAT3.

Acknowledgments

This studywas supported by the German Research Foundation (R.S.843/7-1 and K.B. BO2955/1-1).

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the myocardial jak/stat pathway: from protection to failure

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