Cardiomyocyte-specific IκB kinase (IKK)/NF-κB activation induces reversible inflammatory cardiomyopathy and heart failure

Abstract

Inflammation is a major factor in heart disease. IκB kinase (IKK) and its downstream target NF-κB are regulators of inflammation and are activated in cardiac disorders, but their precise contributions and targets are unclear. We analyzed IKK/NF-κB function in the heart by a gain-of-function approach, generating an inducible transgenic mouse model with cardiomyocyte-specific expression of constitutively active IKK2. In adult animals, IKK2 activation led to inflammatory dilated cardiomyopathy and heart failure. Transgenic hearts showed infiltration with CD11b(+) cells, fibrosis, fetal reprogramming, and atrophy of myocytes with strong constitutively active IKK2 expression. Upon transgene inactivation, the disease was reversible even at an advanced stage. IKK-induced cardiomyopathy was dependent on NF-κB activation, as in vivo expression of IκBα superrepressor, an inhibitor of NF-κB, prevented the development of disease. Gene expression and proteomic analyses revealed enhanced expression of inflammatory cytokines, and an IFN type I signature with activation of the IFN-stimulated gene 15 (ISG15) pathway. In that respect, IKK-induced cardiomyopathy resembled Coxsackievirus-induced myocarditis, during which the NF-κB and ISG15 pathways were also activated. Vice versa, in cardiomyocytes lacking the regulatory subunit of IKK (IKKγ/NEMO), the induction of ISG15 was attenuated. We conclude that IKK/NF-κB activation in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure by inducing an excessive inflammatory response and myocyte atrophy.

Fig. 1.

Expression of IKK2-CA in hearts of transgenic mice leads to cardiomyopathy and heart failure. (A) In double-transgenic IKK^MyHC mice, IKK2-CA is expressed under a bidirectional promoter together with luciferase, which can be used as a marker for the activation of the system. After administration of luciferin, luciferase activity can be detected by in vivo imaging in the left thoracal region of IKK^MyHC mice. (B) Western blot of heart extracts with antibodies detecting human IKK (transgene-specific, Upper) and murine and human IKK (Middle). GAPDH was used as loading control. (C) IKK complex of heart extracts from control and IKK^MyHC mice was precipitated with an antibody against NEMO and analyzed in an in vitro kinase assay by using GST-IκBα as substrate. (D) An electrophoretic mobility shift assay of nuclear heart extracts with NF-κB– and Oct-1–specific probes shows strong activation of NF-κB in the hearts of IKK^MyHC mice. Heart extracts from WT mice after i.p. injection of TNF-α, lipopolysaccharide (LPS), or PBS solution are shown as controls. (E) Left: Cardiomegaly of an IKK^MyHC heart. (Scale bar: 5 mm.) Right: Ventricular dilation (H&E stain). (Scale bar: 200 μm.) (F) Liver congestion (Upper) and generalized edema (Lower) in an IKK^MyHC mouse. (G) Disease-free survival of IKK^MyHC and control mice, weeks after doxycycline removal (n = 30 transgenic, n = 15 control mice). (H) Heart weight of IKK^MyHC mice, weeks after doxycycline withdrawal or during decompensated heart failure at any time point (HF), as a percentage of age-matched control mice (n ≥ 4 per group). (I) Expression of brain natriuretic peptide and ANP mRNA (quantitative PCR in ventricular heart tissue; fold up-regulation in comparison with age-matched control mice; n ≥ 4 per group).

Fig. 2.

Cardiomyocyte-specific IKK/ NF-κB activation induces inflammation and fibrosis. H&E stain (A) and immunofluorescent staining (B) of ventricular heart cryosections from control and IKK^MyHC mice show infiltration with inflammatory cells. (Scale bar: 100 μm.) (C) Quantitative PCR for Emr1/ F4/80 mRNA in ventricular tissue of IKK^MyHC and control hearts as a marker for macrophage infiltration (fold up-regulation vs. control; n ≥ 8 mice per group). (D) Quantitative PCR for mRNA transcripts encoding the inflammatory adhesion molecule ICAM-1 in ventricular tissue of IKK^MyHC and control hearts (fold up-regulation vs. control; n ≥ 8 mice per group). (E) Sirius red stain on paraffin sections of ventricular tissue of control (Left) and IKK^MyHC (Right) mice for the detection of fibrosis (counterstain, fast green). (Scale bar: 100 μm.)

Fig. 3.

Cardiomyocyte-specific IKK/NF-κB activation induces fetal reprogramming and myocyte atrophy. (A) Heart cryosections of control and IKK^MyHC animals were stained for α-smooth muscle actin (α-SMA) and dystrophin, and with DAPI. (Scale bars: 100 μm.) (B) Western blot of ventricular extracts of IKK^MyHC and control mice (8 wk after doxycycline withdrawal) against ANP, α-SMA, destrin, and GAPDH (loading control). (C) Quantitative PCR for Myh6 and Myh7 transcripts encoding the α- and β-isoforms of MyHC (shown are fold regulation vs. age-matched control mice and calculated Myh6/ Myh7 ratio; n ≥ 8 mice per group). (D) Control and IKK^MyHC heart paraffin sections were stained against the transgene (human IKK2, red), and with wheat germ agglutinin (WGA, green) and DAPI (blue). (Scale bars: 100 μm.)

Fig. 4.

Cardiac MRI of IKK^MyHC mice and reversibility of IKK-induced cardiomyopathy. A cohort of control and IKK^MyHC mice was examined in a first MRI under doxycycline to assess normal heart function, in a second MRI after 12 wk of doxycycline withdrawal to assess heart function during IKK-induced cardiomyopathy, and in a third MRI after 3 mo of doxycycline readministration. EDV, end-diastolic volume; ESV, end-systolic volume; EF, ejection fraction; LV, left ventricular (n = 8 mice for first and second MRI, n = 5 mice for third MRI; Fig. S3A and Movies S1, S2, S3, and S4).

Fig. 5.

Reversibility of IKK-induced cardiomyopathy on the cellular and molecular level. Doxycycline in drinking water was discontinued for 12 wk to activate transgene expression and induce cardiomyopathy. Mice were then either analyzed (diseased) or given doxycycline for 3 mo and then analyzed (reversed). (A and B) Quantitative PCR of ventricular tissue samples for F4/80 (macrophage infiltration), ICAM-1 (inflammation), ANP (ventricular remodeling), and brain natriuretic peptide (BNP; heart failure) mRNA transcripts (n ≥ 8 diseased, n ≥ 5 reversed state). Shown is the fold regulation vs. control mice at diseased time point. (C) Western blot for IKK, α-SMA, ANP, destrin, and GAPDH (loading control). (D) Quantitative PCR for α- and β-MyHC mRNA transcripts as a marker for the reversal of fetal reprogramming (n ≥ 8 diseased, n ≥ 5 reversed state).

Fig. 6.

Mechanisms, consequences, and significance of IKK-induced cardiomyopathy. (A) Cardiomyocytes were isolated from mice kept under doxycycline, cultivated without doxycycline (i.e., transgene induction) for 48 h, and analyzed by quantitative PCR for chemokine transcripts. Shown is fold regulation vs. control (n = 3 controls, n = 6 transgenic; Mann–Whitney test). (B) Proteomic analysis of supernatant of IKK^MyHC cardiomyocytes shows presence of factors involved in inflammatory cell recruitment. G-3-BP, galectin-3 binding protein; n.d., not detectable in control supernatant (Table S2). (C) In vivo blockade of NF-κB by transgenic expression of an IκBα superrepressor (3M+) prevents IKK-induced cardiomyopathy in IKK^MyHC mice. Shown is the heart weight/body weight ratio of mice in the acute disease state (n ≥ 3 mice per group). (D) Western blot of heart extracts of control and IKK^MyHC mice in the acute disease state expressing or not expressing IκBα-3M. (E) Immunofluorescent staining for CD45⁺ cells in heart cryosections (acute disease state) reveals absence of infiltrates in IKK^MyHC hearts expressing IκBα superrepressor (IKK-MyHC/3M+). (F) Quantitative PCR of mRNA isolated from heart tissue of control and IKK^MyHC animals, in the absence (3M−) or presence (3M+) of IκBα-3M (acute disease state; n ≥ 3 mice per group). (G) Western blot with an antibody against ISG15 reveals strong expression of ISG15 and widespread ISGylation of other proteins in the hearts of IKK^MyHC animals, both dependent on NF-κB; GAPDH, loading control. (H) WT mice of the susceptible ABY/SnJ strain were infected with CVB3, and nuclear heart extracts were obtained from the acute phase (8 d post infection) and analyzed in an NF-κB– and an Oct-1–specific EMSA. (I) Western blot against ISG15 and GAPDH (loading) with heart extracts from noninfected control and CVB3-infected mice (8 d post infection).