Abstract
Context
Atrial fibrillation (AF) is the most common sustained arrhythmia in clinical practice. The increasing evidence supports links between inflammation and AF. There is evidence showing that obesity is a major cause of adipose tissue (AT) inflammation. Ghrelin (GHRL), through its growth hormone secretagogue receptor (GHS-R) present on adipose tissue macrophages (ATMs), could modulate AT inflammation.
Objective
Our study aimed to evaluate the role of adipose tissue macrophages (ATMs) and their GHS-R in adipose tissue samples of right atrial appendages (RAA) biopsies.
Subjects and Method
We obtained RAA biopsies from 10 obese patients, undergoing cardiac surgery for coronary artery bypass graft (CABG) and developing postoperative atrial fibrillation (POAF). The epicardial tissue samples were examined using immunohistochemistry to visualize and quantify CD68 and GSH-R expression of the ATMs.
Results
Histologically, the mean adipocyte diameter (MAD) of epicardial adipose tissue (EAT) was larger in EAT samples with inflammation as compared to EAT without inflammation (84.2 µm vs. 79.6 µm). The expression of CD68 was lower in EAT without inflammation as compared to EAT with inflammation in adipose tissue samples. Similarly, the expression of GSH-R was lower in EAT samples without inflammation as compared to EAT samples with inflammation in adipose tissue.
Conclusions
Increased epicardial fat area, macrophage infiltration, and GHS-R expression in epicardial ATMs appeared to be associated with postoperative atrial fibrillation in obese patients.
Keywords: growth hormone secretagogue receptor (GHS-R), adipose tissue macrophages (ATMs), epicardial adipose tissue, right atrial appendages, postoperative atrial fibrillation
INTRODUCTION
Obesity is a major cause of adipose tissue (AT) inflammation. It is a pro-inflammatory condition in which hypertrophied adipocytes and adipose tissue-resident immune cells (primarily macrophages) both contribute to a state of chronic low-grade systemic inflammation (1, 2).
Ghrelin (GHRL), a gut peptide consisting of 28 amino acids, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS-R1a) (3). Extensive studies have demonstrated the omnipresence of ghrelin and its receptors in many peripheral organs including the brain, pituitary, pancreas, thyroid, intestine, adrenal gland, kidney, heart, and blood vessels (3-5). Moreover, the ghrelin and its receptor GHS-1Ra participate in the regulation of appetite, energy, body weight, lipid, and glucose metabolism, and exert protective effects by preventing chronic inflammation and antiapoptotic effect in lungs, kidneys, and heart (6). Ghrelin improves cardiovascular functions by increasing cardiac performance, lowering blood pressure, and protecting against cardiac ischemia and heart failure. In AT, ghrelin through its receptor GHS-R1a may promote adipose tissue inflammation, increased macrophage infiltration, and promoted macrophage polarization to M1 (7). In a previous paper, we showed that adipocyte diameter, ATM accumulation, and immunohistochemical expression of the ghrelin receptor in AT are associated with obesity (8). Ghrelin through its growth hormone secretagogue receptor (GHS-R1a), present on ATMs, may have a pro-inflammatory effect, and deletion of GHS-R1a would decrease the macrophage infiltration in adipose tissue (7, 8). The effect of ghrelin and its receptors on pericardial adiposity is less known and could provide new therapeutic opportunities for ghrelin in cardiovascular medicine.
Postoperative atrial fibrillation (POAF) is a common complication after cardiac surgery in obese patients. Atrial fibrillation (AF) is the most frequent cardiac arrhythmia in clinical practice and is often associated with structural alterations including fibrosis, fatty infiltration, and increased epicardial adiposity (9, 10). Epicardial adipose tissue (EAT) is a visceral adipose tissue that secretes a variety of adipokines (11). Among other cardiac structural changes in obese POAF patients, excess EAT, which promote inflammation, was associated with disturbances in the electrical activity of the heart (12).
As a cardiovascular hormone, ghrelin and its receptors widely exist in the cardiovascular system (13). The immunohistochemistry expression of ghrelin peptide in human cardiac adipocytes (CA) and cardiomyocytes-(CM) may suggest that ghrelin may exert paracrine/autocrine effects in the cardiovascular system (14). Also, pieces of evidence indicated that ghrelin can be synthesized by human vascular endothelial cells (15).
Little is known about the relationship between ATMs and GSH-R in adipose tissue inflammation. We studied the association between epicardial adipose tissue (EAT) area, GSH-R, and EAT inflammation in adipose tissue samples of human right atrial appendages (RAA) biopsies and developing postoperative atrial fibrillation (POAF) in obese patients by assessing immunohistochemical expression of CD68 and GHS-R in EAT. We hypothesized that the arrhythmogenic mechanisms could be related to the relationship between the abundance of atrial fatty deposits, epicardial inflammation, and ghrelin.
SUBJECTS AND METHODS
Patients
Samples of RAA containing epicardial adipose tissues were obtained from 10 obese patients, undergoing cardiac surgery for coronary artery bypass graft (CABG) and developing postoperative atrial fibrillation (POAF). The patients were recruited from the 1st of January to 31st of December 2017. Written informed consent was obtained from all participants before cardiopulmonary bypass. This study was approved by the Ethics Committee of the Institute of Cardiovascular Diseases (Iasi, Romania) and was performed according to the Declaration of Helsinki.
Histological examination and morphometry in periatrial EAT
Tissue sections from RAA were fixed in 10% neutral-buffered formalin and embedded in paraffin blocks.
The histological examination was performed using light microscopy (Olympus CX41, Tokyo, Japan).
Measurement of epicardial adipose tissue and cardiac adipocyte size was manually performed by using a color image analysis system: QuickPHOTO MICRO 3.0. Morphometry was applied on 10 random high-power fields (HPF, x400) of periatrial EAT. We calculated the mean adipocyte diameter (MAD) and the mean EAT area (contour EAT area).
Immunohistochemistry of CD 68 and GHS-R in periatrial EAT
IHC procedures were used (8) for assessing the GSH-R (GHSR Polyclonal Antibody, ThermoFisher Scientific, PA5-28752) and CD68 (Dako, IS613) expression of the macrophages in periatrial adipose tissue samples from obese POAF patients.
Immunohistochemistry (IHC) and morpho-metric examination were applied to each of the 10 samples. Cells immunostained with CD68 and GSHR were counted manually and the mean value was calculated.
The mean values for GSH-R and CD68 immunoreactivity were reported to the mean EAT area. We calculated the percentage of the inflammatory cells reported to the measured area. The results were expressed as mean frequency and mean percentage values reported to the adipose tissue area. The EAT inflammation was considered in samples with an increased number of macrophages (mean value > 6).
Statistical analysis
The statistical analysis was performed using IBM SPSS Statistics 21 Software. Continuous variables were reported as means ± SD and were compared using the Mann Whitney test. Categorical data were reported as frequencies and percentages and were compared using the Pearson chi-square test. Statistical significance was set at p < 0.05.
RESULTS
Histological examination and adipocyte size in periatrial epicardial adipose tissue (EAT) was assessed allowing us to compare the atrial structural changes between the two main groups of obese POAF patients (EAT with or without inflammation). EAT changes observed in both study groups consisted of increased EAT area, mainly by increasing of CA sizes.
Histologically, the mean adipocyte diameter (MAD) of EAT was larger in samples with inflammation as compared to EAT without inflammation (84.2 µm vs. 79.6 µm) (Table 1).
Table 1.
Mean adipocyte diameter (MAD), EAT area and immunohistochemical markers of macrophages in peri-atrial EAT samples from obese patients
| Peri-atrial EAT samples | Patients (N) |
MAD (µm) |
EAT area (μm2) |
CD68 (No/μm2) |
CD68 (%) |
GHS-R (No/μm2) |
GHS-R (%) |
|---|---|---|---|---|---|---|---|
| Without inflammation | 5 | 79.6±38.6 | 155.8±27.9 | 4.8±1.3 | 2.8±0.8 | 6±1.8 | 3.5±1.5 |
| With inflammation | 5 | 84.2±45.6 | 172.1±21.3 | 10.6±3.7 | 6.8±2.2 | 9.4±4.1 | 6.1±3.6 |
EAT=epicardial tissue inflammation; MAD= Mean Adipocyte Diameter.
The data are presented as the mean (± standard deviations) values.
Immunohistochemistry and morphometry of CD 68 and GHS-R in periatrial EAT
The GHS-R expression of ATMs was lower in periatrial EAT without inflammation as compared to EAT with inflammation (Table 1, Fig. 1). Similarly, the expression of GSH-R was lower in EAT without inflammation samples as compared to EAT with inflammation in adipose tissue samples (Table 1, Fig. 2).
Figure 1.
Epicardial adipose tissue (EAT) (HE x400). A. EAT without inflammation; B. EAT with inflammation.
Figure 2.
GSH-R and CD68 expression of resident macrophages in peri-atrial epicardial adipose tissue (EAT) from obese patients. Magnification x 400.
DISCUSSION
Our study suggested that obese POAF patients developed EAT structural changes. The first observation, also noted by Kostin et al. (9), was EAT area enlargement in obese POAF patients with EAT inflammation. The second important result of our study was the increase in CA size, especially in obese POAF patients with inflammatory specimens. In a previous study, we identified enlarged CAs as a histological predictive risk factor in atrial fibrillation (12).
Another most significant result of our study was the variable presence of EAT inflammatory cells, probably reflecting the local effects of proinflammatory cytokines released from epicardial adipose tissue. According to Weisberg SP et al. (16), epicardial fat is a source of anti-inflammatory adipokines (adiponectin and omentin) and proinflammatory cytokines (TNF-α, IL-6, leptin, chemerin, visfatin, resistin)(17). Dysregulation of local pro-inflammatory and anti-inflammatory mediators in peri-atrial adipose tissue could play an important role in the pathogenesis of AF (18).
Our study aimed to immunohistochemically assess the relationship between CD68 and GSH-R expression of ATMs in epicardial adipose tissues collected from obese POAF patients. The specimens from obese patients showed an increased macrophage accumulation and CD68 immunohistochemical expression in periatrial adipose tissue. Previous studies demonstrated that the increased inflammatory cell population was associated with increased expression of CD68 in obese adipose tissue (1, 2). Weisberg et al. observed that ATMs infiltration correlated with adiposity and represented an important mechanism in the development of chronic inflammation of obesity (16). Adipose tissue also contributes to inflammation, secreting adipokines with pro-inflammatory and anti-inflammatory properties which play an important role in controlling low-grade systemic inflammation (19).
There is little information about the immunohistochemical expression of GSH-R in human adipose tissues (8). Our current data showed positive immunohistochemical expression of GSH-R associated with CD68-macrophage infiltration in epicardial adipose tissue. The ghrelin and its receptor could play a role in adipose tissue inflammation and previous studies demonstrated that exogenous administration of ghrelin may suppress the expression of anti-inflammatory cytokines in macrophages (13). Few are known about the role of the GHS-R receptor in mediating inflammatory effects of ghrelin. Recent studies found that the ablation of the action of ghrelin by deletion of GHS-R1a could promote an anti-inflammatory process in experimental-induced adipose inflammation (20).
Taken together these results could suggest that ATMs that express GSH-R may amplify the inflammation in obese POAF patients. The immunohistochemical examination might be a valuable test to assess the macrophage functions and therapy targeting GSH-R may prevent adipose tissue inflammation and obesity-associated metabolic complications.
Study limitations
Our study has some limitations: First, our data should be considered preliminary because of the limited number of cases assessed. Second, the inflammatory phenotype of ATMs has not been evaluated and the functions of the detected macrophages were not known. To further clarify the role of GSH-R in EAT inflammation and POAF more studies are needed.
In conclusion, our data suggested that the accumulation of CD68-macrophages of peri-atrial epicardial fat should be a trigger factor for heart arrhythmia in obese patients. Also, the study reported an increased immunoreactivity of GHS-R associated with increased macrophage infiltration of periatrial adipose tissue. The immunohistochemical detection of GHS-R in different adipose depots could suggest increased adipose inflammation and may contribute to POAF in obese cardiac patients.
Conflict of interest
The authors declare that they have no conflict of interest.
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