Highly Reactive Isolevuglandins Promote Atrial Fibrillation Caused by Hypertension

Figures

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  Figure 1. Mechanism of IsoLG Scavengers The 1,4-dicarbonyl (red box) IsoLGs interact rapidly with lysines to form lactam adducts and crosslinking of proteins. The phenolic amine pyridoxamine and its structural analog 2-HOBA (blue box) react with IsoLGs at a rate several orders of magnitude more rapidly than they react with lysines, thus serving as scavengers to prevent adduct formation. 2-HOBA = 2-hydroxylbenzylamine; IsoLG = isolevuglandin.

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  Figure 2. Hypertension Promotes the Formation of Atrial IsoLG Protein Adducts and PAOs, Which Is Inhibited by 2-HOBA (A) During ang II–mediated hypertension (ang II), striking accumulation of IsoLG protein adducts is demonstrated in left (LA) and right (RA) atria using immunolabeling with an anti–IsoLG-lysyl adduct antibody (D11 ScFv; n = 2, 4 for sham and ang II–treated mice, respectively; scale bars = 50 μm) compared with control mice (sham). (B) Summary data are shown for quantitation of IsoLG adducts in LA and RA using liquid chromatography-tandem mass spectrometry assay (mean ± SEM; n = 5 each; ∗∗p < 0.01 between indicated groups, ns is nonsignificant, nonparametric Mann-Whitney U test). (C) Representative mass spectrometry traces are shown for IsoLG adduct quantitation in LA from sham, ang II, and ang II+2-HOBA–treated mice, along with the internal standard in red (Std). (D) Confocal images are shown for myocardium (red) and PAOs (green) on the left, and PAOs localized to the myocardium on the right, from control and hypertensive mice, with PAO burden expressed as G/R values (scale bars = 20 μm). (E) Summary data are illustrated for oligomer burden in LA and RA (n = 11, 16, 9, 5 per…

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  Figure 3. IsoLG Adducts and PAOs Develop at an Early Point During Hypertension, When Histological Abnormalities Are Absent For normotensive (sham), hypertensive (ang II), and 2-HOBA–treated hypertensive (ang II+2-HOBA) animals, columns from left to right display representative atrial images after exposure to hematoxylin and eosin (H&E), Masson’s trichrome, and Congo red stains. There was no evidence of myocardial structural abnormalities or amyloid, with minimal fibrosis that was similar between groups (see text). Scale bars = 50 μm. Abbreviations as in Figures 1 and 2.

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  Figure 4. 2-HOBA Prevented AF in Hypertensive Mice and Suppressed IsoLG Adduct and PAO Formation in Mechanically Stretched Atrial Cells (A) Total AF burden was increased in hypertensive (ang II) mice compared with controls (sham; n = 13, 22; ∗∗p < 0.01, nonparametric Mann-Whitney U test). During hypertension, cotreatment with 2-HOBA reduced AF burden, whereas the inactive structural analog 4-HOBA had no effect (ang II+2-HOBA, ang II+4-HOBA; n = 14, 7, respectively; ∗p < 0.05, nonparametric Mann-Whitney U test). Blood pressure normalization with hydralazine/hydrochlorothiazide (H/H) and cessation of ang II also led to a reduction AF (ang II+H/H, and ang II recovery; n = 7, 12, respectively; ∗p < 0.05, nonparametric Mann-Whitney test). (B) Summary data for systolic blood pressure are illustrated for the groups studied (∗p < 0.01 compared with sham, †p < 0.01 compared with ang II, 2-way analysis of variance for repeated measures). (C) Atrial HL-1 cells were subjected to either no stretch or stretch (10% at 1 Hz) for 48 h and analyzed by liquid chromatography-tandem mass spectrometry assay. Stretch caused robust development of IsoLG adducts, which was abrogated by 2-HOBA (n = 6 each; ∗∗p <…

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  Figure 5. ANP and BNP Form Cytotoxic Protein Oligomers in Hypertensive Atria (A) Western blotting is shown following incubation of ANP peptide (10 μmol/l) at 22oC for 24 h or 6 days, compared with incubation with IsoLGs (synthetic 15-E2-IsoLG, 1 μmol/l) for 24 h, demonstrating time-dependent oligomerization that is markedly accelerated by IsoLGs. (B) Similar results are shown for BNP (10 μmol/l) following 0 to 3 days of incubation in the absence and presence of IsoLGs. (C and D) ANP and BNP (30 μmol/l) were allowed to oligomerize for 1, 3, and 7 days. Oligomers were incubated with atrial HL-1 cells (0.45 μmol/l for 24 h), followed by quantitation of cellular ATP production expressed as % change from control untreated cells. Upon exposure to oligomers, there was a reduction in ATP production indicative of cytotoxicity that declined significantly with increased oligomerization time for ANP (mean ± SEM; n = 5 independent experiments; ∗p < 0.05, 1-way analysis of variance with Tukey’s multiple comparison test). (E) Immunofluorescent labeling with A11 (left) and ANP- or BNP-specific antibodies (middle) was performed in adjacent 5-μm atrial sections from a hypertensive mouse (scale bars = 50…

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  Figure 6. In Response to Hypertension, Oxidative Stress-Mediated IsoLGs Promote AF Susceptibility Hypertension and atrial cell stretch, as well as the rapid activation of atrial cells, causes oxidative stress and formation of highly reactive IsoLGs, that rapidly adduct and crosslink cellular proteins and other macromolecules. The generation of dysfunctional adducted proteins and protein oligomers promote atrial myocyte dysfunction to increase AF susceptibility. Abbreviations as in Figures 1 and 4.