Dichotomous Actions of NF-κB Signaling Pathways in Heart

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  • Dichotomous Actions of NF-B Signaling Pathways in Heart

    Rimpy Dhingra & James A. Shaw & Yaron Aviv &Lorrie A. Kirshenbaum

    Received: 26 April 2010 /Accepted: 4 May 2010 /Published online: 25 May 2010# Springer Science+Business Media, LLC 2010

    Abstract Despite the substantial progress in heart researchover the past two decades heart failure still remains a majorcause of morbidity and mortality in North America and isreaching pandemic proportions worldwide. Though theunderlying causes are varied, the functional loss ofcontractile myocytes through apoptosis, necrosis, andautophagy has emerged a central unifying theme to explaindiminished cardiac performance in individuals with heartfailure. At the molecular level, there has been considerableinterest in understanding the signaling pathways thatregulate cell death in the heart with specific interest in theextrinsic and intrinsic cell death pathways. The cellularfactor nuclear factor-B (NF-B) is a key transcriptionfactor involved in the regulation of a wide range of genesinvolved in cellular process including inflammation, im-mune cell maturation, cell proliferation, and, most recently,cell survival. NF-B signaling is important for the normalcellular growth and is a major target of inflammatorycytokines. Several studies have highlighted a protective roleof NF-B in the heart under certain circumstancesincluding hypoxic or ischemic myocardial injury. The

    diverse nature and involvement of NF-B in regulation ofvital cellular processes including cell survival notably in thepost-mitotic heart has sparked considerable interest inunderstanding the signaling pathways involved in regulat-ing NF-B in the heart under normal and pathologicalconditions. However, whether NF-B is adaptive, maladap-tive or is a homeostatic response to cardiac injury maysimply depend on the context and timing of its activation.In this forum we discuss NF-B signaling pathways andtherapeutic opportunities to modulate NF-B activity inheart failure.

    Keywords Nuclear Factor-B . Apoptosis . Cell Death .

    Heart Failure . Ventricular Myocytes


    The Role of Apoptosis in Heart Disease

    From a historical perspective the adult myocardium hasgenerally been viewed as a non-proliferative organ with alimited and meager capacity for de novo myocyte regener-ation and/or self-renewal after injury cited from (c.f.) [62,98]. After birth cardiac myocytes are believed to exit thecell cycle. As a result, growth of the post-natal heart occursby hypertrophic rather than hyperplasic processes. There-fore, the loss of functional cardiac myocytes throughapoptotic processes during ischemic or hypoxic injury hasbeen postulated to be an underlying cause of ventricularremodeling and ventricular failure. There remains consid-erable debate as to whether differentiated adult ventricularmyocytes readily traverse the cell cycle, the frequency ofsynthetic events and whether or not DNA synthesiscoincides with a concomitant increase in cell number.

    R. Dhingra : J. A. Shaw :Y. Aviv : L. A. Kirshenbaum (*)The Institute of Cardiovascular Sciences,St. Boniface General Hospital Research Centre,Department of Physiology, Faculty of Medicine,University of Manitoba,Rm. 3016, 351 TachAvenue,Winnipeg, MB R2H 2A6, Canadae-mail: Lorrie@sbrc.ca

    L. A. KirshenbaumThe Institute of Cardiovascular Sciences,St. Boniface General Hospital Research Centre,Department of Pharmacology and Therapeutics,Faculty of Medicine, University of Manitoba,Winnipeg, MB R2H 2A6, Canada

    J. of Cardiovasc. Trans. Res. (2010) 3:344354DOI 10.1007/s12265-010-9195-5

  • Notwithstanding the acknowledged ability of adult myo-cytes to synthesize DNA, these limited events alone do notappear to be adequate to functionally restore diminishedventricular performance in patients with heart failure post-myocardial infarction. Given that myocyte number candirectly influence ventricular performance, the ultimatetherapeutic goal in reducing morbidity and mortality inpatients with heart failure would be to preserve the numberof existing myocytes by suppressing the cell death process.Before the landmark study by Wencker et al. [93], celldeath was known to occur during heart failure, but it wasunknown to what extent the loss of myocytes wascontributing to the cardiac pathology and decline inhemodynamic parameters. By generating a transgenicmouse with inducible caspase 8 activity targeted to theheart, the authors demonstrated that an extremely low levelof apoptosis was sufficient to induce ventricular remodelingand heart failure. Notably, the proportion of apoptotic cellswas below what had been reported in previous studiessuggesting that a low level of apoptosis is sufficient toinduce heart failure. Moreover, in another study, a caspaseinhibitor was used to reduce apoptosis and improve survivalin Gq model of heart failure [37]. This study along withother studies highlight the importance of apoptotic celldeath as an underlying mechanism for loss of contractilemyocytes and heart failure [29, 64, 66].

    Characterization of NF-B

    Nuclear factor-B (NF-B) was originally discovered byDavid Baltimores laboratory in 1986 [75]. Using Blymphocytes, the newly named NF-B was characterizedas a factor that bound to the immunoglobulin kappa lightchain enhancer DNA sequence, coincident with increasedkappa chain gene expression [75]. Since these observations,NF-B activity has been implicated in a large array ofbiological processes including immunity, inflammation,proliferation, tumorigenesis, cardiac hypertrophy, and cellsurvival [7, 46, 70, 86]. Likewise, defects in NF-Bsignaling have been associated with a variety of humandiseases including cancers, rheumatoid arthritis, inflamma-tory bowel disease as well as AIDS and Alzheimersdisease reviewed in [5, 28] (Fig. 1).

    NF-B itself is comprised of two subunits in either ahomo- or heterodimeric complex. In the NF-B familythere are five protein subunits: p50 (precursor p105), p52(precursor p100), p65 (RelA), RelB, and c-Rel, which areencoded by the following genes, respectively: NFKB1,NFKB2, RELA, RELB, and REL [4, 32, 33]. Each of theseproteins contains a Rel homology domain, which is usedfor dimerization and DNA binding. The most well-characterized NF-B dimer is comprised of the p50 andp65 subunits [14, 21]. Our laboratory has focused our

    studies on the p65 subunit of NF-B because many of thebiological actions attributed to NF-B are linked to p65[61, 72]. Structurally, p65 is a 551 amino acid proteincomprised of an N-terminal Rel homology domain (Rel), anuclear localization sequence (NLS), and a C-terminaltransactivation domain [89]. Because it can bind to DNAand activate transcription, p65-containing NF-B dimersare transcription factors, and indeed, NF-B is generallythought to exert its influence on the cell through activationof target genes. For example, these include proinflamma-tory genes (interleukin-1beta (IL-1), IL-6, tetrahydrofuran, granulocyte-macrophage colony-stimulating factor,monocyte chemotactic protein-1, proangigenic factorsvascular endothelial growth factor (VEGF) adhesion mol-ecules); anti-apoptotic proteins B-cell lymphoma 2; Bcl-X,cIAP (inhibitor of apoptosis); FLICE-inhibitory proteins(FLIP); and inducible enzymes inducible nitric oxidesynthase, cylooxygenase 2, and matrix metallopeptidase 9c.f. [45].

    However, in some situations, NF-B has been shown torepress rather than activate target gene transcription [1, 3].Post-translational modifications of p65 subunit have beenreported that impart functional changes to NF-B regula-tion, beyond the classical NF-B signaling described in thenext section. Phosphorylation on specific serine residuesincluding S276, S311, S468, S529, S535, and S536 haveeach been shown to increase the transcriptional activity ofp65 subunit [6, 52]. Notably, one of the most well-characterized phosphorylation sites for the transcriptionpotential of p65 subunit is arguably S536. Phosphorylationat this residue occurs after stimulation with tumor necrosisfactor (TNF) and lipopolysaccharide (LPS) and contrib-utes to the activation of NF-B target genes [19, 20, 22].Phosphorylation at S529 has also been shown to be inducedby TNF, and the loss of this phosphorylation site wasassociated with less NF-B-dependent transcription afterstimulation [91]. Interestingly, basal phosphorylation of p65on S468 is maintained by glycogen synthase kinase-3,which inhibits its NF-Bs transcriptional activity [12].

    In this regard, a cell survival role for NF-B has beendescribed [7, 87, 91]. This has largely been substantiated bystudies in cells in which the p65 gene had been deleted or

    NF KBNF-

    InflammationGrowth InflammationGrowth

    Hypertrophy Proliferation Acute ChronicHypertrophy Proliferation Acute Chronic

    ApoptosisHeart Failure Cancer Immunity Autophagy ApoptosisHeart Failure Cancer Immunity Autophagy

    Fig. 1 Schematic representation of NF-B-regulated pathways

    J. of Cardiovasc. Trans. Res. (2010) 3:344354 345

  • functionally inactivated resulted in an increased incidenceof apoptosis. The fact that deletion of the p65 NF-Bsubunit is embryonic lethal with embryos dying fromexcessive apoptosis supports a crucial role for NF-B incell survival during development [7]. Moreover, inability ofthe p50 subunit to prevent apoptosis or rescue theembryonic lethality [7] of the p65/ animals illustratesthe lack of functional redundancy of p50 and p65 subunits.A cytoprotective role for the NF-B p65 subunit is furtherillustrated by the observation that mouse embryonicfibroblast derived from p65/ embryos were found to bemore sensitive to death signals triggered by TNF or UVtreatment than wild-type controls [91]. Replacement of afunctional p65 gene into p65/ cells was sufficient to avertcell death equivalent to that of wild-type p65+/+ cells.Accordingly, work from our laboratory was among the firstto establish that functional loss of NF-B signalingincreased susceptibility to TNF-induced apoptosis andcell death in post-natal ventricular myocytes [61].

    NF-B Signaling

    Under basal conditions, NF-B is predominantly localizedto the cytoplasm. Research over the last number of yearshas identified two major signaling pathways for NF-Bactivation and translocation to the nucleus, where it mayexert its influence over cellular gene expression, Fig. 2. Themost well described of these pathways is known as theclassical or canonical pathway and the other is known asthe non-canonical or alternative pathway. In the canonicalpathway, under basal non-stimulated conditions the NF-Binhibitor protein inhibitor of B (IB) binds to and retainsthe p50/p65 NF-B heterodimer in the cytoplasm. The

    association of NF-B with IB masks the NLS of the p65subunit thereby preventing NF-Bs nuclear targeting [31].Importantly, the p65 subunit has been observed in thenucleus of unstimulated cells including cardiac myocytessuggesting some level of basal shuttling exists which maybe important for basal gene regulation [3, 15].

    In response to biological signals that lead to NF-Bactivation, IB is phosphorylated at serines 32 and 36which signal its ubiquitination and degradation by the 26Sproteasome [56]. The loss of IB activity unmasks thep65 NLS, allowing NF-B to translocate to the nucleus[31]. Accordingly, serine to alanine substitution mutationsat these critical serine residues of IB results in a superrepressor protein that cannot be degraded and preventsNF-B activation by retaining it in the cytoplasm [10].Phosphorylation of IB is primarily achieved by thekinase activity of the IKK complex, which has three coresubunits. IKK and IKK subunits both have kinaseactivity, and IKK, NF kappa B essential modulator(NEMO), is characterized as an essential regulatory subunit[69]. Importantly, knockout and loss-of-function experi-ments have implicated IKK as the major kinase in thecanonical pathway. IKK/ mice die at embryonicday 14.5 from apoptotic liver degeneration, similar top65/ mice that die at approximately the same time withsimilar liver apoptosis [84]. Furthermore, embryonic cellsderived from both of these lines were less resistant toTNF-induced cell death and displayed reduced NF-Btarget gene activation, implying a critical role for the betasubunit that cannot be complemented by IKK. IKK/

    mice exhibit skeletal and skin developmental abnormalitiesand do not survive after birth [44]. However, in IKK/

    mouse embryonic fibroblasts, there was no change in IB








    I BI B NFB ActivationC t lCytoplasm NucleusNucleus


    Fig. 2 Model of classical NF-Bactivation and transcriptional reg-ulation of the target genes. TheIB binds to and retains NF-Bin the cytoplasmic preventingnuclear localization of the NF-B. Phosphorylation of IB byIKK signaling complex (coresubunits IKK and IKK andIKK) triggers the ubiquitinationand subsequent proteasomal deg-radation of IB permittingtranslocation of NF-B to thenucleus affecting gene transcrip-tion (see text for details)

    346 J. of Cardiovasc. Trans. Res. (2010) 3:344354

  • phosphorylation after TNF stimulation, and a considerableamount of IKK activity was retained in these cells,revealing that IKK activity is dispensable with regard tothe cytokine-induced NF-B response [82]. Concordantwith these findings, IKK and not IKK appears to be theprincipal IKK required for NF-B activation in the heart[26].

    The activation of the IKK signaling module is complexand partially dependent on the initial stimulus. Forexample, ligands such as TNF, LPS, and IL-1 have eachbeen shown to stimulate IKK-NF-B activity, but the arrayof adapter proteins recruited to the receptor in each caseslightly differs [80]. In the case of TNF, ligand bindinginduces receptor trimerization and the recruitment of TNFreceptor-associated Factor2 (TRAF2), TRAF5, and receptorinteracting protein (RIP1) via tumor necrosis factor receptortype 1-associated DEATH domain [42, 43]. The IKKcomplex may also be recruited to the receptor via RIP1NEMO interactions [69]. It has been suggested that thismay induce a conformational change in the IKK complex,leading to autophosphorylation and activation; however,some evidence also supports the role of TAK1 as an IKKkinase in the canonical pathway [38].

    Termination of NF-B activation is mediated by anegative feedback loop mediated by NF-B-dependenttranscription of IB [81]. Furthermore, activation of thedeubiquitinases such as A20 and cylindromatosis (CYLD)by NF-B has been shown to contribute to the attenuationof the signal, possibly by inhibiting IKK activation [11, 94].As previously mentioned, post-translational modificationsof p65 may also be involved in signal terminationpotentially through association with histone modifyingproteins discussed below.

    One fundamental difference between the canonical andnon-canonical pathways is that the non-canonical pathwayis regulated by proteolytic cleavage of precursor proteins,rather than by IB proteins. Aside from the absence of IB-mediated regulation, the non-canonical pathway featuresp52-containing dimers, such as p52-RelB, as opposed top50-containing dimers in the canonical pathway [9].Briefly, the phosphorylation-dependent cleavage of theprecursor protein p100 to p52 allows p52-containing NF-B dimers to translocate to the nucleus and affect genetranscription [79]. The processing of p100 is mediated byIKK and the upstream kinase NIK, which may, in fact,target both IKK and p100 [76]. Interestingly, IKK isactivated in a similar manner to IKK involving recruit-ment of adaptor proteins such as B cell-activating factor(BAFF) and/or receptor activator for nuclear factor Bligand to cell surface receptors reviewed in [38]. Becausethe subsequently generated NF-B dimers contain p52 andnot p50, the non-canonical NF-B target genes are distinctfrom the canonical target genes [27]. Genes known to be

    activated by canonical NF-B signaling include A1 [90],Bcl-2 [16], IL-6 [50], c-FLIP [58], manganese superoxidedismutase [23], and cIAPs [92]. In this regard, NF-Bactivity is usually characterized as anti-apoptotic andproinflammatory. Interestingly, TNF is associated withthe activation of both the extrinsic apoptotic pathway andwith the activation of NF-B signaling. Activation of NF-B-dependent anti-apoptotic genes such as FLIP, Bcl-2, theIAPs, and A20 antagonize TNF-induced apoptosis [58].

    In some instances, however, NF-B activity has in somecases been shown to activate apoptosis-inducing genes, andhas been directly implicated in the apoptotic process. Thisis supported by the fact that NF-B upregulates the deathinducing ligands TNF [84, 85], FasL [47], and TNF-related apoptosis-inducing ligand [2], and other pro-apoptotic proteins such as p53 [96]. Some reports showthat NF-B activation is pro-apoptotic in cardiomyocytesand endothelial cells under oxidative stress [55, 68, 74].Because NF-B appears to have both anti- and pro-apoptotic capabilities, its role in regulating cell death isstill a source of debate; however, the majority of theevidence points to an anti-apoptotic role for NF-B. In lightof this, perhaps the detrimental effects of NF-B areactivated under more extreme circumstances such as excessNF-B activation, as in the case of A20 knockout mice[67], or chronic absence of NF-B, in the case of p65knockout mice [7]. Future studies will further characterizethe contexts in which NF-B signaling is pro- or anti-deathproperties.

    Role of NF-B in the Heart

    Not only is there considerable debate over the nature of NF-B signaling in a cell, there is also controversy over therole of NF-B in heart disease. Interestingly, NF-Bactivation has been observed in many cardiovasculardisease states including ischemic and dilated heart failure,ischemic preconditioning, and myocarditis [34, 95, 99].Furthermore, stimuli such as ischemiareperfusion andROS [24, 99], TNF [25] as well as -adrenergicstimulation [70] have been shown to activate NF-B inthe heart. Because of these observations, it is generallybelieved that NF-B is involved in heart disease. However,despite a sizable body of research, the exact role of NF-Bin the heart has yet to be fully elucidated. The criticalquestion to be asked is whether NF-B signaling representsan important cardioprotective pathway, or alternatively, ifNF-B contributes to cardiac pathology post myocardialinjury. The answer to this question may actually be both,and dependent upon the temporal and spatial activation ofNF-B.

    Because the hearts response to ischemiareperfusion isdynamic and complex, time is an important variable in

    J. of Cardiovasc. Trans. Res. (2010) 3:344354 347

  • experiments examining NF-B activity in the heart follow-ing ischemiareperfusion. For example, Chandrasekar andFreeman [17] observed two waves of NF-B activity in theheart after ischemiareperfusion. In their experiments, theyobserved NF-B activity following 15 min of ischemia and15 min of reperfusion, which lasted until 1 h of reperfusion.The second wave began at 3 h of reperfusion and lasteduntil 6 h [17, 18]. In another study using an MI model ofischemia, NF-B activation was found to be maximum at3 days in the infarcted zone but declined to basal levels byday 7 post-MI [78]. In Langendorff-perfused rat heartssubjected to global ischemia and reperfusion, it was foundthat NF-B activity was induced as early as after 5 min ofischemia with IB degradation observed at 4 min ofischemia and re-synthesis at 30 min. In this study, NF-BDNA binding was only slightly increased by reperfusion.Interestingly, the antioxidant pyrrolidine dithiocarbamateprevented ischemia-induced NF-B activity, further impli-cating ROS as an NF-B activator [49]. Finally, in primarycultures of cardiac myocytes, NF-B is activated by 1 h ofhypoxia [54], but inhibited by 24 h of hypoxia [3]. Clearly,the timing of NF-B activation in the heart followingischemia and reperfusion must be carefully considered ifNF-B signaling is to be therapeutically exploited. It isimportant to keep in mind, however, that NF-B signalingis generally cyclical in nature, in large part due to thecontinuous feedback loop of IB degradation and re-synthesis [40, 41].

    These observations suggest an optimal window fortherapeutic NF-B interventions that exploit NF-Binhibition or activation to regulate cell survival that needto be further resolved experimentally. As previouslystated, the role of NF-B in the heart is not completelyunderstood at present. On the basis that cardiac ischemiaand subsequent reperfusion injury induces NF-B activ-ity and inflammatory cytokine expression [39], severalgroups have tested a variety of NF-B inhibitors in thecontext of ischemiareperfusion. There are a variety ofcompounds known to inhibit the NF-B signaling path-way, including proteasome inhibitors, IKK inhibitors, andNF-B decoy oligonucleotides reviewed in [13, 57]. In apig model of ischemic heart disease, the proteasomeinhibitor PS-519, administered just prior to ischemia/reperfusion, attenuated NF-B activation and reducedinfarct size compared to controls [71]. However, othergroups have observed that the effects of proteasomeinhibitors in the heart extend beyond NF-B, althoughthis type of compound has consistently been cardiopro-tective in these studies, but it is unlikely that this effect ismediated solely through NF-B [51]. In this regard, Bay65-1942 has recently been identified as selective inhibitorIKK. This compound competes with ATP for IKKbinding site thereby preventing IKK-mediated IB

    phosphorylation and degradation [30]. This compound,when delivered either prior to the onset of ischemia, or atthe time of reperfusion, resulted in smaller infarcts,improved myocardial function, and a reduction in TNFand IL-6 release [60].

    Another type of NF-B inhibitor, NF-B decoys, whichare oligonucleotides that compete with NF-B target genesfor NF-B binding, have also been characterized ascardioprotective during MI [55]. Finally, mice withcardiac-specific overexpression of a non-phosphorylatableIB super repressor protein demonstrated significantreduction in infarct size after ischemiareperfusion, againsuggesting a pathological role for NF-B [24]. Furtherevidence in support of this idea is that TNF, an inducerand target of NF-B, is increased in the heart post MI, andcontributes to infarct size and the decline in cardiacfunction [65, 85]. This statement is based on the fact thatantibodies directed against TNF reduced infarct size [8] asdid genetic knockout of TNF [53]. However, the role ofTNF is also unclear, and like NF-B, some studiessupport an alternate, protective role for them both [48, 59,61]. For example, using an in vivo murine model ofmyocardial infarction, Kurrelmeyer et al. [48, 59, 61],demonstrated greater infarct size and diminished cardiacperformance in hearts in which the TNFR1 had beenablated, supporting a cytoprotective role for TNF signal-ing pathway in the heart post MI. However, it was unclearin that study whether NF-B activation downstream ofTNF receptor was functionally impaired and contributedto the increased pathology.

    Despite the above evidence supporting the notion thatNF-B inhibition is potentially therapeutic, there is agrowing body of evidence that suggests that NF-B iscardioprotective. In this context, NF-B signaling has beenshown to not only avert apoptotic cell death in the heart, butalso to attenuate infarct size and, importantly, to play a rolein cardiac preconditioning, which is a reduced susceptibilityto ischemic damage after previous short exposures toischemia [48, 59, 61]. Indeed, NF-B inhibition has beenshown to abrogate the protective effects of myocardialpreconditioning, and thus, NF-B may have a protectiverole in the heart under certain circumstances [59, 61].Interestingly, because high (anti-rheumatic) doses of ASAhave been shown to inhibit NF-B through inhibition ofIKK [97], it may be important to limit the dose of ASA inpatients at high risk for MI due to the potential loss ofpreconditioning.

    Because of the established anti-apoptotic properties ofNF-B signaling, researchers have asked whether theseeffects are seen in the heart. Cardiac-specific over-expression of a IB mutant that retains NF-B in thecytoplasm and attenuates NF-B signaling demonstratedlarger infarcts 24 h post LAD ligation, and substantially

    348 J. of Cardiovasc. Trans. Res. (2010) 3:344354

  • increased myocardial apoptosis at 3 and 6 h, suggesting aprotective role for NF-B [59]. A study involving theknockout of both TNFR1 and TNFR2 revealed that thesemice had larger infarcts and increased apoptosis comparedto wild types, suggesting that TNF may activate aprotective pathway, such as NF-B [48]. In support ofthis, pre-treatment of hearts or cardiac myocytes withTNF conferred protection from ischemia and hypoxia[63]. Moreover, another study showed that cardiac-specific overexpression of the anti-apoptotic protein Bcl-

    2 reduced infarct size and apoptosis following ischemiareperfusion. Notably, we have shown that Bcl-2 activatesNF-B in the heart which is indispensible for its anti-apoptotic activities [25]. Moreover, NF-B can reportedlymodulate HIF-1 expression in a manner dependent onIKK- [73]. Basal NF-B activity was shown to berequired for HIF-1 protein accumulation under hypoxiain the liver and brain of hypoxic animals [88] since IKK-deficiency resulted in defective induction of HIF-1alphatarget genes including VEGF. Thus, NF-B-mediated

    Fig . 3 IKK suppres seshypoxia-induced mito-defectsand cell death in postnatal ven-tricular myocytes. Ventricularmyocytes were infected withadenoviruses encoding wild-type IKK (IKKwt) or kinase-defective IKK (IKKmt). aMitochondrial permeabilitytransition pore opening wasdetected in ventricular myocyteswith the use of the membranepermeable dye calcein AM inthe presence of cobalt chloride,loss of green florescence isindicative of PTP opening.IKK-mediated NF-B activa-tion suppresses hypoxia-inducedmitochondrial permeability tran-sition pore opening, in contrastto a kinase-defective IKK mt.b Cell viability was determinedwith the use of vital dyes (cal-cein AM and ethidium homo-dimer), live (green) vs dead(red) cells, respectively. IKKmtoverexpression leads to celldeath under basal conditions aswell as hypoxia [77]. Hypoxia-induced cell death was avertedin IKK overexpressed cells,Labels for a and b, respectively,CNTL control, wt IKK wildtype, mt IKK kinase-defectivemutant, hypx hypoxia

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  • activation of HIF-1 in vasculature may promote angio-genesis as means of improving cell survival duringchronic ischemia.

    This raises the question as to how can these apparentdiscrepancies in the literature be resolved between thosewho state that NF-B is protective and those with theopposite viewpoint? The usual suspects are of courserelevant, for example: differences in experimental models,such as ischemia/reperfusion vs. ischemia alone, degree ofcoronary artery banding, localized ischemia vs. globalischemia, isolated hearts vs. whole animal experiments, aswell as species differences including mouse strains, andexperimental time points. Because of the varied and oftendivergent properties of NF-B in the heart, it is possiblethat the subtle differences in study time points may underliethe discordant and confounding results reports in literature.For example, in most studies investigators constitutivelyexpress transgenes as basis for their findings as a surrogatefor endogenous gene activity. However, because of thedynamic nature of NF-B signaling in the heart, thisapproach may be inadequate to fully address the true roleof NF-B activation under normal or pathological con-ditions such as hypoxia or MI. On the premise that NF-Bactivity may be protective or detrimental, the notion of aninducible transgene may allow researchers to dissect outwhen NF-B is protective, and when it is not, in relation tothe onset of ischemia or reperfusion. Insight into thediscrepancies involving the role of TNF in the heart may

    be ascertained from a study investigating the different NF-B responses from acute vs. chronic TNF exposure. Inresponse to acute TNF exposure, a normal NF-Bresponse was observed and comprised largely of p65/p50NF-B heterodimers entering the nucleus. In the context ofprolonged, chronic TNF elevation, as in TNF over-expressing transgenic animals or in heart failure, p50/p50NF-B homodimers were observed in addition to p65/p50NF-B heterodimers, suggesting a different pattern of NF-B gene expression [36].

    Work from our laboratory has demonstrated a criticalrole of NF-B for averting hypoxia-induced cell death inpost-natal ventricular myocytes [3, 72, 77]. Adenovirus-mediated delivery of IKK to rat ventricular myocytesattenuated hypoxia-induced mitochondrial perturbations,caspase activity, and cell death [72]. Activation of the NF-B signaling pathway in ventricular myocytes suppressedbasal and hypoxia-inducible Bnip3 gene activity andsuppressed mitochondrial permeability transition poreopening and cell death during hypoxia, Fig. 3. Interestingly,we found that cells derived from p65 knockout mice orventricular myocytes rendered defective for NF-B signal-ing exhibited increased basal Bnip3 gene expression,mitochondrial PTP, and cell deathsupporting a criticalsurvival role for NF-B in ventricular myocytes [3].Another study from our lab demonstrated that NF-Bactively recruits histone deacetylase-1 (HDAC-1) to theBnip3 promoter providing a mechanism for transcriptional


    E2F-1 element NF-

    Bnip3 PromoterBnip3 Promoter Cell Survival




    E2F-1 element NF-E2F 1 element NF KB element

    Bnip3 PromoterBnip3 Promoter

    Bnip3Cell Death



    KB element





    Fig. 4 Transcriptional silencingof the Bnip3 promoter by NF-B-HDAC-1 inhibitory com-plexes. NF-B displaces E2F-1promoting cell survival in ven-tricular myocytes. a Binding ofE2F-1 to the Bnip3 promoterand Bnip3 gene transcriptionunder basal non-apoptotic con-ditions is inhibited in presenceof NF-B-HDAC1 complexhence promoting cell survival.b During hypoxia loss of p65-HDAC inhibitory complexesdis-inhibit the Bnip3 promoterallowing E2F-1 binding andBnip3 gene activation resultingin cell to death

    350 J. of Cardiovasc. Trans. Res. (2010) 3:344354

  • repression of the mitochondrial death factor Bnip3 and celldeath under basal conditions [77]. Moreover, we furthershowed that NF-B was sufficient to antagonize themitochondrial perturbations and cell death inducing prop-erties of the cellular factor E2F-1. These seminal findingsmay explain in part how cells, by selectively inhibitingE2F-1-dependent death gene transcription, avert apoptosisdownstream of the Rb/E2F-1 cell cycle pathway [77],Fig. 4. Collectively, these results strongly support acytoprotective role for NF-B in the heart.


    The exact role and nature of NF-B in heart is not fullyunderstood. In fact, NF-B may be both good and badand may simply depend on the context and timing ofactivation. For example, early activation of NF-B mediatespreconditioning, and IKK overexpression strongly inhibitshypoxia-induced death of cardiac myocytes. Furthermore,pre-treatment with TNF confers protection against ische-mia. Taken together, these results suggest a protective rolefor early NF-B activation [35]. However, the protectiveeffect of NF-B chemical inhibitors or NF-B decoys,delivered prior to ischemia/reperfusion, argue against this[55, 60]. Late NF-B activation by the elevated levels ofTNF in congestive heart failure, coupled with the alteredNF-B gene expression from chronic TNF exposure andthe cardio-depressant effects of TNF in the myocardiumsuggests that chronic NF-B activation may be detrimentalin the long run. Future studies designed to specifically targetthe temporal and spatial activation of NF-B as well ascombinatorial actions of the different RelA holds promise forthe design of novel therapeutic interventions as a means ofpreserving cardiac function and heart failure.

    Acknowledgments We are grateful to Dr. H. Weisman for criticalcomments on the manuscript. Pam Lowe for editorial assistance andmanuscript preparation; Floribeth Aguilar, Hongying Gang for technicalassistance. R.D. holds a post-doctoral fellowship from the ManitobaHealth Research Council. This work was supported by grants to L.A.Kfrom the CIHR and St. Boniface Hospital Research Foundation, L.A.K.holds a Canada Research Chair in Molecular Cardiology.


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    Dichotomous Actions of NF-B Signaling Pathways in HeartAbstractIntroductionThe Role of Apoptosis in Heart DiseaseCharacterization of NF-BNF-B SignalingRole of NF-B in the Heart


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