Fibrosis independent atrial fibrillation in older patients is driven by substrate leukocyte infiltration: diagnostic and prognostic implications to patients undergoing cardiac surgery

Abstract

Background: The objectives of the study were to characterize and quantify cellular inflammation and structural remodeling of human atria and correlate findings with molecular markers of inflammation and patient surrogate outcome.

Methods: Voluntary participants undergoing heart surgery were enrolled in the study and blood samples were collected prior to surgery, and right atrium samples were harvested intraoperatively. Blood samples were analyzed by flow cytometry and complete blood counts. Atrial samples were divided for fixed fibrosis analysis, homogenized for cytokine analysis and digested for single cell suspension flow cytometry.

Results: A total of 18 patients were enrolled and samples assessed. Isolated cells from the atria revealed a CD45+ population of ~ 20%, confirming a large number of leukocytes. Further characterization revealed this population as 57% lymphocytes and 26% monocyte/macrophages (MoΦ), with the majority of the latter cells being classical (CD14++/CD16-). Interstitial fibrosis was present in 87% of samples and correlated significantly with patient age. Older patients (> 65) had significantly more atrial fibrosis and cellular inflammation. AFib patients had no distinguishing feature of atrial fibrosis and had significantly greater CD45+ MoΦ, increased expression of MMP9 and presented with a significant correlation in length of stay to CCL-2/MCP-1 and NLR (neutrophil-to-lymphocyte ratio).

Conclusion: Atrial fibrosis is correlated with age and not determinate to AFib. However, severity of atrial leukocyte infiltration and markers of matrix degradation are determinant to AFib. This also correlated with CCL2 (or MCP-1) and NLR-indicative of marked inflammation. These data show the potential importance of diagnostic and prognostic assessments that could inform clinical decision making in regard to the intensity of AFib patient management.

Keywords: Atrial fibrillation; Cardiac fibrosis; Cardiac surgery; Cytokines; Human atrium; Inflammation; Leucocyte infiltration; Macrophages (MoΦ); NLR.

Fig. 1

Flow cytometry gating strategy (a): (i) Flow cytometry ungated dot plot of overall isolated cells. (ii) CD45 × CD14 gating was applied to exclude non-leukocyte events, resulting in a cleaner population of lymphocytes and MoΦ (iii) Followed by a CD11b x SSC positive gate (iv), then a FSC x SSC gate on MoΦ (v), enabling us to visualize the (vi) different macrophage sub-types with the aid of CD16 × CD14 plot. Leukocytes found in the atrium (b): (i) CD45+ cells per gram of tissue calculated from flow cytometry CD45+ data. (ii) Pie chart highlighting the percentage of isolated cells that were CD45+. (iii) Further characterization of the CD45+ population found in atrial samples. c Presence of MoΦ in a section of normal human atrial tissue stained with CD68 antibodies. Scale bar represents 400 µm

Fig. 2

a, b Flow cytometry dot plots from an individual patient’s atrium and blood respectively. c Bar graphs demonstrating the comparison between the different MoΦ phenotypes in atrium and blood. d CD16 MFI values in atrium and blood. e Individual patient data on CD16 MFI to further show that CD16 MFI shows a similar trend in all patients where CD16 MFI is lower in atrium than in blood. f MoΦ to lymphocyte ratio was significantly higher in atrial samples

Fig. 3

a H&E stain of normal human atrium. Scale bar represents 200 µm. b Sirius red stain showing collagen fibers stained red, and myocardial fibers stained green. Scale bar represents 100 µm. c graph showing fibrosis was positively correlated with patient age. d table representing salient differences between patients in SR and AFib. e CD45 MFI MoΦ shows no correlation to percent fibrosis. f CD45 MoΦ show statistically significant correlation to hospital length of stay

Fig. 4

Luminex analyses of atrial tissue isolates comparing SR and AFib patients with a normal atrial tissue used as reference value. MMP-9 was statistically significant in AFib patients with P < 0.05. Other cytokines highly expressed in AFib patients were MMP-2, MMP-7, VEGF and CCL-2/MCP-1

Fig. 5

Neutrophil to Lymphocyte ratio (NLR), Platelet to lymphocyte ration (PLR), and CCL-2/MCP-1 were positively correlated with hospital length of stay (LOS). MMP-9 a diagnostic indicator was negatively correlated to LOS

Fig. 6

Clinical overview of a potential algorithm to the modifiable and non-modifiable factors of the cardiac surgery patient’s risk for AFib. The modifiable factors are inflammatory, some having prognostic value determined through a LOS surrogate, such as NLR and CCL2, and some with diagnostic value determined as the onset of AFib, such as CD45 and MMP9. Utility of these biomarkers would be independent of on-modifiable factors such as age, sex, height and genetics, which can also contribute to the development of fibrosis in AFib patients. However, fibrosis is to be questioned as to whether it is an incipient cause or correlative pre-condition to AFib in patients, experimental models should be developed to answer this question definitively