Inflammasome activation in end-stage heart failure-associated atrial fibrillation

Abstract

Aims: Inflammatory pathways are increasingly recognized as an important factor in the pathophysiology of both heart failure (HF) and atrial fibrillation (AF). However, there is no data about inflammation-related histological and molecular alterations in HF-associated AF. The objective of our study was to investigate inflammatory pathways and fibrosis in end-stage HF-associated AF.

Methods and results: Left atrial samples of 24 male patients with end stage ischemic HF undergoing heart transplantation were analysed. Twelve patients suffered from sustained AF while the others had no documented AF. The expression of inflammasome sensors and their downstream signalling were investigated by Western blot. No differences were observed in the expression of inflammasome sensors between the two groups, while cleaved caspase-1 increased tendentiously in the AF group (P = 0.051). Cleaved caspase-1 also showed significant correlation with the expression of interleukin-1β and its cleaved form in the total population and in the AF group (P < 0.05). The presence of myocardial and epicardial macrophages were assessed by ionized calcium-binding adaptor molecule 1 (Iba1) immunostaining. Number of macrophages showed a tendency towards elevation in the left atrial myocardium and epicardium of AF compared with SR group. The amount of total and interstitial fibrosis was determined on Masson's trichrome-stained sections. Histological assessment revealed no difference between AF and SR groups in the amount of either total or interstitial fibrosis.

Conclusions: This is the first study on inflammation-related differences between HF with SR or AF showing elevated inflammasome activity and enhanced macrophage infiltration in left atrial samples of patients with AF.

Keywords: Atrial fibrillation; Fibrosis; Heart failure; Inflammasome; Macrophages.

Figure 1

Inflammasome activation in heart failure‐associated atrial fibrillation. (A) Western blot detection of inflammasome markers in left atrial samples of ischemic HF patients with SR (blue) and AF (red). Samples excluded due to low‐quality homogenates are shown in parentheses. GAPDH is shown as loading control. No signal could be detected for NALP1 (not shown). (B) Analysis of normalized band intensities of inflammasome markers (P > 0.05, Student's t‐test; n = 9–10). (C) Correlation and regression analysis of inflammasome sensors and markers of their downstream signalling based on Western blot detection. Cleaved caspase‐1 showed correlation with interleukin‐1β and its cleaved form both in the total population (P = 0.005 and 0.004, respectively) and in AF group (P = 0.01), but not in SR group. No correlation was found with any inflammasome sensors (P > 0.05, Pearson‐correlation; n = 9–10). Continuous data passed the Shapiro–Wilk normality test.

Figure 2

Left atrial macrophage infiltration in heart‐failure associated atrial fibrillation. (A) The method for quantitative analysis of macrophage infiltration is demonstrated on a haematoxylin–eosin stained section. Left atrial macrophages were counted at several unit areas and averaged for the sample. Myocardial (yellow squares) and epicardial (red squares) regions were investigated separately (scale bar: 1000 μm). (B) Average macrophage number per unit area was higher at both the myocardium and the epicardium of the AF samples compared with the SR group. However, this difference was not significant (P > 0.05, Student's t‐test; n = 3–7). (C) Representative images about the presence of Iba1 positive macrophages in the myocardium (M) and epicardium (E) of AF and SR patients. Different amount of macrophage accumulation at the high‐magnification images indicates heterogeneous distribution of these cells within a given sample (scale bars: 60 μm). Continuous data passed the Shapiro–Wilk normality test.

Figure 3

Amount of left atrial fibrosis in heart failure with sinus rhythm or atrial fibrillation (A) Assessment of total and interstitial fibrosis after Masson's trichrome‐staining of left atrial samples (scale bars: 1000 μm). (B) Percentages of both total and interstitial fibrosis proved to be the same at the two groups (P > 0.05, Student's t‐test; n = 5–7). (C) Representative images of total (T) and interstitial (I) fibrosis from SR and AF groups (scale bars: 1000 μm). Continuous data passed the Shapiro–Wilk normality test.