Cardioprotective effects of peroxisome proliferator activated receptor gamma activators on acute myocarditis: anti-inflammatory actions associated with nuclear factor kappaB blockade

Abstract

Objective: To test the hypothesis that activation of peroxisome proliferator activated receptor gamma (PPAR-gamma) reduces experimental autoimmune myocarditis (EAM) associated with inhibitor kappaB (IkappaB) alpha induction, blockade of nuclear factor kappaB (NF-kappaB), and inhibition of inflammatory cytokine expression.

Methods: EAM was induced in Lewis rats by immunisation with porcine cardiac myosin. PPAR-gamma activators 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) and pioglitazone (PIO) were administered to rats with EAM.

Results: Enhanced PPAR-gamma expression was prominently stained in the nuclear and perinuclear regions of infiltrating inflammatory cells. Administration of 15d-PGJ2 and PIO greatly reduced the severity of myocarditis and suppressed myocardial mRNA and protein expression of inflammatory cytokines in rats with EAM. In addition, treatment with PPAR-gamma activators enhanced IkappaB concentrations in the cytoplasmic fractions and nuclear fractions from inflammatory myocardium. Concurrently, NF-kappaB was greatly activated in myocarditis; this activation was blocked in the 15d-PGJ2 treated and PIO treated groups.

Conclusions: PPAR-gamma may have a role in the pathophysiology of EAM. Because an increase in IkappaB expression and inhibition of translocation of the NF-kappaB subunit p65 to the nucleus in inflammatory cells correlated with the protective effects of PPAR-gamma activators, these results suggest that PPAR-gamma activators act sequentially through PPAR-gamma activation, IkappaB induction, blockade of NF-kappaB activation, and inhibition of inflammatory cytokine expression. These results suggest that PPAR-gamma activators such as 15d-PGJ2 and PIO may have the potential to modulate human inflammatory heart diseases such as myocarditis.

Figure 1

Immunohistochemical staining for peroxisome proliferator activated receptor γ (PPAR-γ). (A) In normal control, marginal or trivial immunoreactivity for PPAR-γ was detected in myocardium. (B) In rats with experimental autoimmune myocarditis, PPAR-γ was strongly stained in infiltrating inflammatory cells. The expression of PPAR-γ was prominently located in the nuclear and perinuclear regions of inflammatory cells (arrows). (C, D) Administration of PPAR-γ activators 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) and pioglitazone greatly suppressed PPAR-γ expression (arrows). Original magnification ×400.

Figure 2

Myocardial PPAR-γ protein expression by western blotting. (A, C) Western blot analysis. (B, D) Densitometric analysis of relative protein concentrations. 15d-PGJ₂, rats with myocarditis treated with 15d-PGJ₂; Con-15d-PGJ₂, normal rats treated with 15d-PGJ₂; Control: normal rats; Con-PIO, normal rats treated with PIO; Con-Vehicle, normal rats treated with vehicle; Myocarditis: rats with myocarditis treated with phosphate buffered saline (PBS); PIO, rats with myocarditis treated with PIO. Values were derived from five animals and determined as a percentage of controls. *p < 0.01 v Control; †p < 0.01 v Myocarditis; NS, no significant difference v Con-Vehicle.

Figure 3

Ribonuclease protection assay for mRNAs of type 1 (Th1) and type 2 (Th2) T helper cells and of proinflammatory cytokines. In control, the myocardial mRNA expression of cytokines was detected only for macrophage inhibitory factor (MIF) and interferon γ (IFN-γ). In myocarditis rats treated with vehicle, mRNAs of Th1 cytokines (such as interleukin (IL) -18), Th2 cytokines (such as IL-6), and proinflammatory cytokines (such as MIF, IFN-γ, IL-1β, and IL-1Ra) were greatly upregulated, and mRNA expression of IL-1α, IL-12p35, IL-12p40, and IL-10 was slightly upregulated compared with controls. Treatment with 15d-PGJ₂ and PIO greatly reduced the expression of cytokine mRNAs. L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are housekeeping genes. A representative finding of three distinct experiments is shown.

Figure 4

Myocardial IL-1β and tumour necrosis factor α (TNF-α) protein expression by western blotting. (A) Western blot analysis. (B) Densitometric analysis of relative protein concentrations. In rats with myocarditis treated with PBS, IL-1β and TNF-α protein expression was greatly increased; 15d-PGJ₂ and PIO treatment decreased IL-1β and TNF-α protein expression. Values are derived from five animals and determined as the percentage of control values. *p < 0.01 v control; †p < 0.01 v vehicle.

Figure 5

Electrophoretic mobility shift assay for nuclear factor κB (NF-κB) and western blot analysis of inhibitor IκB (IκB) α in cytoplasmic fraction of myocardium. (A) Representative electrophoretic mobility shift assay of NF-κB activity in myocardium. Specificity was determined by addition of p65 antibody (supershift) or unlabelled (cold) NF-κB oligonucleotide to the myocardium. Four separate experiments yielded similar results. (B) Densitometric analysis of DNA binding activity in the region of NF-κB (p65) was quantified. In acute myocarditis, NF-κB was greatly activated (enhanced DNA binding activity by 5.6-fold versus normal controls). This activation was inhibited in the 15d-PGJ₂ and PIO treated groups. (C) Representative western blots. (D) Densitometric analysis (n  =  5). IκBα concentration decreased greatly in the cytoplasmic and nuclear fractions in rats with EAM treated with PBS. Treatment with 15d-PGJ₂ and PIO enhanced the cytoplasmic concentrations of IκBα by 2.2-fold and 2.1-fold, and increased the nuclear concentrations of IκBα by 1.7-fold and 1.5-fold, respectively, versus myocarditis. * p < 0.01 v control; †p < 0.01 v myocarditis.