LNK/SH2B3 loss of function increases susceptibility to murine and human atrial fibrillation

Abstract

Aims: The lymphocyte adaptor protein (LNK) is a negative regulator of cytokine and growth factor signalling. The rs3184504 variant in SH2B3 reduces LNK function and is linked to cardiovascular, inflammatory, and haematologic disorders, including stroke. In mice, deletion of Lnk causes inflammation and oxidative stress. We hypothesized that Lnk-/- mice are susceptible to atrial fibrillation (AF) and that rs3184504 is associated with AF and AF-related stroke in humans. During inflammation, reactive lipid dicarbonyls are the major components of oxidative injury, and we further hypothesized that these mediators are critical drivers of the AF substrate in Lnk-/- mice.

Methods and results: Lnk-/- or wild-type (WT) mice were treated with vehicle or 2-hydroxybenzylamine (2-HOBA), a dicarbonyl scavenger, for 3 months. Compared with WT, Lnk-/- mice displayed increased AF duration that was prevented by 2-HOBA. In the Lnk-/- atria, action potentials were prolonged with reduced transient outward K+ current, increased late Na+ current, and reduced peak Na+ current, pro-arrhythmic effects that were inhibited by 2-HOBA. Mitochondrial dysfunction, especially for Complex I, was evident in Lnk-/- atria, while scavenging lipid dicarbonyls prevented this abnormality. Tumour necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β) were elevated in Lnk-/- plasma and atrial tissue, respectively, both of which caused electrical and bioenergetic remodelling in vitro. Inhibition of soluble TNF-α prevented electrical remodelling and AF susceptibility, while IL-1β inhibition improved mitochondrial respiration but had no effect on AF susceptibility. In a large database of genotyped patients, rs3184504 was associated with AF, as well as AF-related stroke.

Conclusion: These findings identify a novel role for LNK in the pathophysiology of AF in both experimental mice and humans. Moreover, reactive lipid dicarbonyls are critical to the inflammatory AF substrate in Lnk-/- mice and mediate the pro-arrhythmic effects of pro-inflammatory cytokines, primarily through electrical remodelling.

Keywords: Atrial fibrillation; Inflammation; Isolevuglandins; Oxidative stress; Pro-inflammatory cytokines.

Graphical Abstract

Figure 1

In the setting of structurally normal hearts, Lnk^−/− mice display increased AF susceptibility that is prevented by the dicarbonyl scavenger 2-HOBA, while atrial inflammation is not affected. (A) Representative B- and M-mode images in WT and Lnk^−/− mice with (B) quantification of ejection fraction (EF) and fractional shortening (FS; n = 9, 10). Summary data for (C) total AF duration and (D) sustained AF (≥15 s) incidence in WT littermate controls, and Lnk^−/− mice treated with vehicle, 2-HOBA, or 4-HOBA (n = 19, 30, 18, 13). (E) Representative density plots and quantification of total atrial leucocytes by flow cytometry (CD45⁺; n = 26, 24, 14). Each data point represents the atria from 2 mice. Summary data for atrial (F) antigen-presenting cells (MHCII⁺/CD11b⁺; n = 22, 20, 9), (G) natural killer cells (NK1.1⁺; n = 26, 24, 14), (H) B cells (CD19⁺; n = 23, 24, 14), and (I) T cells (CD3⁺; n = 26, 24, 14). Statistical tests: (B) Student’s t-test; (C, E–G, I) Kruskal–Wallis followed by Dunn’s MCT; (D) Fisher’s exact test; and (H) one-way ANOVA with a Tukey’s post hoc test. EDD, end diastolic diameter; ESD, end systolic diameter; ns, not significant; SAX, short axis; SSC-A, side scatter area. *P < 0.05; **P < 0.01.

Figure 2

Superoxide and IsoLG-adducted proteins are increased in Lnk^−/− atria. (A) Representative images of dihydroethidium (DHE) staining (n = 5–6; scale bars = 50 µm). (B) Quantification of IsoLG adducts in atrial leucocytes (CD45⁺) by flow cytometry using D11 ScFv (n = 14, 17, 12). Each data point represents atria from two mice. (C) Representative images of D11 ScFv immunostaining, demonstrating IsoLG-adducted proteins in the Lnk^−/− atria (LA: n = 3, 3, 2; RA: n = 4, 4, 3; scale bars = 50 µm). Statistical test: Kruskal–Wallis with Dunn’s MCT post hoc. LA, left atria; ns, not significant; RA, right atria.

Figure 3

Inflammation promotes electrical remodelling in the atria of Lnk^−/− mice, which is largely prevented by 2-HOBA. (A) Representative ECG recordings. Red lines denote P-wave duration and blue lines the QT interval. Quantification of (B) P-wave duration as well as (C) QRS and (D) QT intervals (n = 24, 44, 30). (E) Representative action potential recordings from isolated atrial myocytes (n = 24, 36, 18). Experimental data shown for (F) action potential duration at 90% repolarization (APD₉₀), (G) the maximum phase 0 upstroke velocity (V_max), and (H) RMP. Statistical tests: (B, C) Kruskal–Wallis followed by Dunn’s MCT; (D) one-way ANOVA with a Tukey’s MCT; and (F–H) hierarchical cluster analysis by Sikkel et al. ns, not significant. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4

Alterations in I_Na and I_To are consistent with action potential changes in Lnk^−/− atrial myocytes. (A) Representative families of Na⁺ currents (I_Na) are illustrated with the voltage clamp protocol shown in the inset. Experimental data for (B, C) peak and (D, E) late I_Na (n = 16, 16, 23). Late I_Na is shown as percentage of peak I_Na at −30 mV. (F) L-type Ca⁺⁺ currents (I_Ca,L) are depicted along with the voltage clamp protocol used. (G, H) Summary data for I_Ca,L (n = 12, 23, 8). (I–K) Similar data are shown for I_To (n = 11, 13, 8). All statistical analyses (C, E, H, K) were performed using the hierarchical cluster analysis by Sikkel et al. ns, not significant. *P < 0.05; **P < 0.01. Data are shown as mean ± SEM for (B, G, J).

Figure 5

Inflammation promotes mitochondrial dysfunction and ultrastructural abnormalities in Lnk^−/− atria. Comparison of atrial oxygen flux between (A) WT and Lnk^−/− mice (n = 4, 4) and (B) vehicle- and 2-HOBA-treated Lnk^−/− mice (n = 4, 4). (C) Representative transmission electron microscopy images highlighting atrial mitochondria [LA: n = 3, 3, 2; RA: n = 3, 3, 3; scale bars = 1 µm (2700×) and 400 nm (6500×)]. White arrows denote heterogenous mitochondrial damage, while red arrows identify dense atrial granules. Quantification of mitochondrial cristae in the (D) left (n = 379, 348, 263) and (E) right atria (n = 345, 374, 328). Statistical tests: (A, B) Student’s t-test; and (D, E) Kruskal–Wallis with Dunn’s MCT post hoc. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. CI, Complex I; CII, Complex II; FAO, fatty acid oxidation; LA, left atria; mt, mitochondria; OXPHOS, oxidative phosphorylation; RA, right atria.

Figure 6

Plasma TNF-α and atrial IL-1β are elevated and promote IsoLG formation with electrical remodelling and bioenergetic dysfunction. Concentrations of pro-inflammatory cytokines in the (A–C) plasma (n = 13, 16, 13) and (D–F) atria (normalized to total protein) of Lnk^−/− mice (n = 10, 10, 10). Representative images demonstrating IsoLG adduct accumulation in atrial HL-1 cells following a 24 h exposure to (G) TNF-α or (H) IL-1β (n = 3 each; scale bars = 50 µm). Representative AP tracings from HL-1 cells treated with either (I) TNF-α or (K) IL-1β (n = 9–12) for 24 h. ATP production in atrial HL-1 cells following 24 h exposure to both (J) TNF-α (n = 5) and (L) IL-1β (n = 4). Statistical tests: (A–D) Kruskal–Wallis with Dunn’s MCT post hoc; (E, F) one-way ANOVA followed by a Tukey’s MCT; (J, L) Mann–Whitney U test. ns, not significant; *P < 0.05; **P < 0.01.

Figure 7

Inhibition of soluble TNF-α but not IL-1β prevents AF and electrical remodelling in Lnk^−/− mouse atria. Summary data of total AF duration and sustained AF (≥15 s) incidence for Lnk^−/− mice treated with (A, B) a soluble TNF-α inhibitor (XPRO1595; n = 15, 13) or (C, D) IL-1β inhibitor (anti-IL-1β antibody; n = 14, 9). Experimental data demonstrate the effect of (E) soluble TNF-α (n = 4, 4) and (F) IL-1β inhibition (n = 4, 5) on atrial oxygen flux. Action potential parameters in (G) XPRO1595- (n = 41, 40) and (H) anti-IL-1β antibody-treated (n = 40, 42) Lnk^−/− mice. Statistical tests: (A, C) Mann–Whitney U test; (B, D) Fisher’s exact test; (E, F) Student’s t-test; (G, H) hierarchical cluster analysis by Sikkel et al. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations are identical to Figures 3 and 5.