Plasma concentration and expression of adipokines in epicardial and subcutaneous adipose tissue are associated with impaired left ventricular filling pattern

The study included 55 male individuals diagnosed with coronary artery disease (CAD) who underwent planned coronary artery bypass graft at the Department of Cardiac Surgery, Medical University of Bialystok, Poland. Exclusion criteria were cardiac arrhythmias, heart valve disease, myocardial infarction in the last 30 days, chronic morbidity (other than type 2 diabetes, hypertension or hyperlipidemia), additional cardiac surgery that was not planned beforehand, age over 80 years or under 30, body mass index (BMI) over 40 kg/m². Patients were divided into three groups: I. The non-obese controls, without obesity and diabetes mellitus, with BMI < 26 kg/m², normal fasting blood glucose (< 100 mg/dL) and normal glycated hemoglobin (< 6%) (n = 14); II. patients with overweight or obesity (BMI > 26 kg/m²) without diabetes mellitus, with normal fasting blood glucose and normal glycated hemoglobin (n = 27); III. patients with overweight or obesity (BMI > 26 kg/m²) and with T2DM (n = 14). The study was approved by the Ethics Committee of Medical University of Bialystok (decision number R-I-002/358/2010) and adhered to the principles of the Declaration of Helsinki. All the individuals agreed to participate in the study and gave informed consent.

In all patients an assessment of clinical and anthropometrical parameters was recorded. On the day of the surgery, a fasting blood sample was collected to measure serum glucose, insulin, triacylglycerol, total cholesterol, HDL-cholesterol and LDL-cholesterol concentrations. An evaluation of morphological and functional heart parameters by transthoracic echocardiography was done before the surgery, including two-dimensional, M-mode and Doppler imaging. EAT thickness was measured on the free wall of the right ventricle (RV) from both parasternal long- and short-axis views [13]. Right after chest opening, samples of subcutaneous adipose tissue from the sternal region and epicardial adipose tissue adjacent to the right coronary artery were collected, snap frozen in liquid nitrogen, and then stored in − 80 °C upon further analysis.

Plasma adipokine concentrations were measured using enzyme-linked immunosorbent assays (ELISA). Leptin concentrations were assessed with Human Leptin ELISA kit (Merck Millipore, MA, US), adiponectin with Human Adiponectin Elisa kit (Merck Millipore, MA, US), resistin with Human Resistin Elisa kit (Merck Millipore, MA, US). IL-6 was measured with IL-6 High Sensitivity ELISA kit (eBioscience, CA, US), TNF-α with Human TNF-α High Sensitivity ELISA kit (eBioscience, CA, US). Apelin levels were measured using Apelin-36 (Human)—EIA kit (Phoenix Pharmaceuticals, CA, US), and visfatin using Enzyme-linked Immunosorbent Assay Kit For Visfatin kit (Uscn Life Science Inc, TX, US).

Adipose tissue samples for analysis of adipokine expression were pulverized and reduced with crystalline Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Then, RNA and protein were isolated using NucleoSpin RNA/Protein kit (Macherey–Nagel, Germany).

Adipokine mRNA expression in adipose tissue samples was measured by real-time polymerase chain reaction. First, using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, MA, US) a reverse transcriptase PCR was undertaken. Then, in thermocycler Chromo4 (Bio-Rad Laboratories, CA, US) using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich, MO, US) a real-time PCR-amplification was performed. Forward and reverse primers are listed in Table 1. Cyclophilin A was used as a housekeeping gene [14].

To analyze protein expression of adipokines, Western blots were performed using lysates from tissues. First, an electrophoresis was performed in TGS buffer (BioRad), in which Precision Plus Protein Western C Standards (BioRad) were used. Then the proteins were transferred to polyvinylidene difluoride transfer membrane with semi-dry technique and incubated with primary antibodies: leptin (Abcam, Cambridge, UK, cat. no ab9826), adiponectin (Abcam, cat. no. ab75989), TNF-α (Abcam, cat. no. ab66579), IL-6 (Abcam, cat. no. ab93356), resistin (Abcam, cat. no. ab124681), apelin (Abcam, cat. no. ab125213). As a secondary antibody a goat anti-rabbit IgG-HRP was used (Santa Cruz Biotechnology, cat. no. sc-2004). Bands were visualized by chemiluminescence (Molecular Imager ChemiDoc XRS+, BioRad). Expression of adipokines was normalized to GAPDH (GAPDH Antibody, Santa-Cruz Biotechnology, cat. no. sc-25778) protein levels for each sample.

Baseline characteristics of the study groups are presented in Table 2. Importantly, there were no significant differences in patients’ age, disease status, or drug use. Non-T2DM patients with obesity presented increased Homeostasis Model Assessment-Insulin Resistance (HOMA2-IR), but their glycated hemoglobin (HbA1_c) and serum glucose levels were normal. The presence of diabetes was associated with increased waist-to-hip ratio, hyperglycemia, hypertriglyceridemia, and high HbA1_c.

Results of echocardiographic examination are summarized in Table 3. Importantly, EAT thickness in non-obese controls was lower than in patients with obesity, but the differences were insignificant. Both systolic and diastolic heart function, as well as all heart structure parameters but left atrium diameter (LA), including left ventricular mass (LVM), left ventricular mass index (LVMi), interventricular septum diastolic diameter (IVSD), relative wall thickness (RWT), and posterior wall thickness in diastole (PWTd) were similar in analyzed groups.

Notably, there were no significant differences in the frequency of heart failure (HF) revealed by clinical and echocardiographic examination. The majority of patients in each study group was classified in class II of the New York Heart Association (NYHA) and class II in The Canadian Cardiovascular Society (CCS) Functional Classification. None of the patients was presented with symptoms of HF at rest. In 41 (69.5%) patients a left ventricular hypertrophy (LVH) was diagnosed, as defined by LVMi ≥ 51 g/m^2.7 [15]. LVH was detected in 7 (50%) non-obese controls, 22 (81%) patients with obesity, and 9 (64%) patients with obesity and diabetes. Importantly, impaired left ventricle (LV) relaxation, as defined by decrease in the ratio of peak velocity blood flow in early diastole to peak velocity flow in late diastole caused by atrial contraction (E/A ratio) under 0.8, was observed in 7 non-obese controls (50%), 13 patients with obesity (48%), and 9 patients with obesity and diabetes (64%) patients. None of the patients presented pseudonormal LV filling pattern. In patients with obesity and diabetes there was only one individual with restrictive filling pattern described by E/A = 2.25. Six non-obese controls (43%), 13 patients with obesity (48%), and 3 patients with obesity and diabetes (21%) had E/A ratio between 0.8 and 1.5, but presented signs of impaired LV filling, including prolonged deceleration time (DT), prolonged isovolumic relaxation time (IVRT) and/or increased LA. One patient in each study group did not meet echocardiographic criteria for heart failure, but all three presented clinical signs of heart failure.

Decreased plasma levels of adiponectin, but increased levels of leptin were found in patients with obesity (Fig. 1). Concentrations of resistin, visfatin, IL-6 and apelin were similar across the groups. TNF-α was detected in only 9 plasma samples (17%), therefore it was not included in the comparison.

Messenger RNA expression of all seven adipokines did not differ between the groups in SAT and EAT fat depots (data not shown). Protein expression of TNF-α, apelin and resistin was below the detection threshold in both fat depots. In patients with obesity and diabetes there was approximately 1.3-fold higher protein expression of leptin, and ~ 1.7-fold higher protein expression of IL-6 in SAT, as compared to non-obese controls. Visfatin protein expression in EAT was 1.3-fold higher in patients with obesity than in non-obese controls (Figs. 2 and 3). Protein expression of other adipokines in SAT and EAT depots did not differ between the groups. The presence of diabetes did not influence adipokine protein expression in either adipose tissue depot in patients with obesity.

Messenger RNA expression of adipokines in fat depots was compared in the cohort of all patients. We found significantly higher gene expression of all adipokines in EAT than in SAT depot. We report 1.8-fold higher expression of adiponectin, 2.4-fold higher mRNA expression of leptin, 2.5-fold higher expression of TNF-α, 2.9-fold higher expression of apelin, 4.6-fold higher expression of resistin, 6.8-fold higher expression of visfatin, and 104.2-fold higher expression of IL-6 in the EAT, compared to the SAT depot (p < 0.001 for all comparisons) (Fig. 4a). The comparison of protein expression was done in a separate Western blot analysis of 12 patients, 4 randomly selected from each study group. Similarly to the first analysis, protein expression of TNF-α, apelin and resistin was below the detection threshold in both fat tissue depots. Therefore only leptin, adiponectin, visfatin and IL-6 protein expressions were analyzed. We found that the content of adiponectin protein in EAT was only 40% of that in SAT (p < 0.001). The protein expression of other adipokines did not differ (Fig. 4b). For representative blots see Additional file 1.

We hypothesized that adipokines are associated with heart structure and function. A simple analysis of correlation would not give reliable results, because age and BMI have an impact on both heart parameters and adipokines. Therefore we created a regression model. In the model we included three dependent variables: plasma concentration or tissue expression of an adipokine, patient’s age, and patient’s BMI. Then we performed a stepwise regression analysis in which we identified adipokines that were related to heart structure and function, independently of age and BMI. Results are presented in Table 4. Impaired LV diastolic function was associated with increased plasma concentration of leptin, resistin, IL-6, and adiponectin, as well as with elevated level of resistin mRNA, apelin mRNA, and adiponectin mRNA and protein in SAT, and leptin mRNA and protein in EAT. A moderate correlation was found between ejection fraction (EF) and apelin mRNA, IL-6 protein, and leptin protein expressions in SAT. Importantly, none of the plasma adipokines was related to LV systolic function. However, plasma IL-6 was related to TAPSE, a parameter describing systolic function of the RV. The relationship of adipokines and parameters describing heart size did not make a clear pattern. Visfatin protein expression in EAT was inversely related to LA, but correlated positively with PWTd. Protein expression of leptin in EAT correlated negatively with PWTd, the relation with IVSD was, however, positive. Messenger RNA expression of TNF-α in the SAT correlated positively with LVM and RVdD. EAT thickness correlated only with apelin mRNA expression in the SAT.

Adipokine profiles between two types of heart failure were compared. Individuals with a preserved EF of the LV (HFpEF), compared to patients with a reduced EF (HFrEF), had 0.7-fold lower EAT expression of TNF-α mRNA (p = 0.027), but 1.7-fold higher EAT expression of IL-6 mRNA (p = 0.020) and 1.7-fold higher EAT expression of visfatin mRNA (p = 0.009). Plasma adiponectin concentration was lower in HFpEF (5.52 ± 3.13 vs. 6.92 ± 2.98 µg/mL; p = 0.024). Plasma concentrations and tissue expressions of other adipokines did not differ between the two types of HF. Interestingly, in individuals with HFpEF thickness of EAT was higher (12.5 ± 2.4 vs. 10.2 ± 3.5 mm; p = 0.021). Importantly, there were no differences regarding age, BMI, blood pressure, drug use or presence of comorbidities between patients with HFpEF and HFrEF.

We report an increase in plasma leptin and a decrease in plasma adiponectin levels in patients with obesity or diabetes. Leptin protein content in SAT was also increased in individuals with obesity. We hypothesize that plasma leptin levels are, at least partly, a reflection of SAT leptin overexpression. High volumes of SAT in individuals with obesity add to that effect. Similarly to our results, Kouidhi et al. reported higher mRNA leptin levels in SAT in patients with obesity [17]. We also found increased IL-6 protein expression in SAT in individuals with obesity, what is consistent with other studies [18]. Moreover, an elevated visfatin protein, but not mRNA expression was noted in EAT in patients with obesity. Plasma concentrations and tissue expressions of other adipokines were not affected by obesity or T2DM. Possibly, underlying CAD heavily influenced adipokine concentrations and tissue expressions, and as a result we failed to demonstrate any changes caused by obesity and diabetes alone. It has been observed before, that CAD is associated with an increase in proinflammatory adipokines tissue content. CAD might even be described by a unique profile of cytokines in serum [19, 20].

A significantly higher mRNA expression of all adipokines, especially pro-inflammatory IL-6, was detected in the EAT, as compared to the SAT. Despite higher gene expression of adipokines, protein expressions of leptin, visfatin, and IL-6 were similar in the SAT and EAT. Moreover, despite higher adiponectin mRNA expression, its protein content in EAT was lower than in SAT. Previous studies have already demonstrated that EAT is an important source of proinflammatory cytokines [21‐24]. In contrary to our results, Bambace et al. reported lower adiponectin mRNA levels in EAT than in SAT [25]. Decreased abundance of anti-inflammatory adiponectin in EAT in the presence of heavily overexpressed IL-6 that we observed could be associated with pathophysiology of CAD. However, these results should be interpreted with caution, because of the discrepancy between protein and mRNA expressions. That disparity could lie in the elaborate process of protein production. Protein abundance is regulated by post-transcriptional, translational and protein degradation processes [26]. Tissue mRNA levels were reported to explain only 40% of variability in tissue protein levels [27]. Therefore, gene and protein expressions may differ in tissues. The discrepancy observed in our study casts in doubt results of other studies performed on adipose tissue where only mRNA expressions were investigated. Nevertheless, we clearly demonstrate that EAT in patients with CAD is characterized by a unique expression pattern of adipokines and notably overexpressed adipokines related to inflammation.

In the search for a relation between adipokines and heart disease, we created a regression model that included three independent variables: an adipokine plasma level or adipokine tissue expression, patient’s BMI, and patient’s age. Then we performed an analysis with a single echocardiographic heart parameter revealing a number of significant and independent relations. Protein content and mRNA expression of cytokines in SAT does not always reflect their plasma concentrations and hence it is difficult to conclude any effects on the heart. In order to create a clearer view on the observed relations, we focused on plasma and EAT adipokines only.

Impaired LV filling was associated with increased expressions of leptin in EAT, and elevated concentrations of resistin, leptin, and IL-6 in plasma. This is consistent with other studies [28‐31]. Surprisingly, adiponectin plasma concentration was also related to LV diastolic dysfunction. It has been established that adiponectin is a cardioprotective protein [32]. Adiponectin shares signaling pathways with natriuretic peptides and therefore it could be related to heart remodeling. Lindberg et al. observed that increased plasma adiponectin concentrations were strongly related to the risk of HF [33]. There was also a positive correlation between apelin plasma concentration and E/A ratio, indicating that higher apelin levels were associated with better LV diastolic function. Consistently with our observations, Cheng et al. found decreased serum apelin levels in patients with diastolic heart failure [34].

In accordance with other studies, echocardiographic traits of heart hypertrophy were positively associated with resistin in plasma, and there was a negative correlation with plasma adiponectin [35, 36]. Associations with leptin were ambiguous, as one of the indices correlated positively, whereas the other one correlated negatively with leptin in EAT.

In the analysis we found that impairment in LV systolic function was related to an increase in EAT TNF-α mRNA indicating its potential role in HF. Other studies have already linked serum TNF-α with HF [37].

Interestingly, there was a clear difference in mRNA profile of adipokines in EAT between patients with HF and preserved or reduced EF. Normal EF was associated with lower TNF-α mRNA, elevated IL-6, and elevated visfatin mRNA. As we discovered in regression analysis, TNF-α in EAT is negatively correlated with EF, but we found no such correlations for IL-6 and visfatin. Both adipokines were previously linked to heart failure, but studies did not differentiate between systolic and diastolic HF. Increased blood levels of IL-6 were associated to systolic heart dysfunction [38]. Reduced function of visfatin in a study on a knock-out mouse model, was associated with a rapid progression of heart failure [39].

It is important to note that we found independent relations between echocardiographic heart parameters and some of the adipokine expressions in EAT. We found leptin and visfatin in EAT to be linked with cardiac remodeling. Left ventricle diastolic dysfunction was associated with IL-6 and leptin expression in EAT. Decrease in LV contractility correlated with TNF-α EAT expression. In the comparison of HFpEF and HFrEF patients we found significant differences in EAT expression of as much as three adipokines. Our results support the hypothesis, that despite its relatively small mass, EAT could have an impact on heart function through production of adipokines acting directly on cardiomyocytes. Obesity and diabetes have very limited effect on expression of adipokines in EAT in CAD patients. Possibly, local inflammation plays more important role in EAT adipokine balance. To the best of our knowledge this is the first study to evaluate expressions of adipokines in EAT with relation to echocardiographic parameters.