Resveratrol Improves Heart Function by Moderating Inflammatory Processes in Patients with Systolic Heart Failure

The effects of resveratrol (RES) in heart failure have already been evaluated in animal models; however, in human clinical trials, they have not been confirmed yet. The aim of this study was to assess the effects of resveratrol treatment in systolic heart failure patients (heart failure with reduced ejection fraction or HFrEF). In this human clinical trial, 60 outpatients with NYHA (New York Heart Association) class II-III HFrEF were enrolled and randomized into two groups: receiving either 100-mg resveratrol daily or placebo for three months. At the beginning and at the end of the study echocardiography, a six-minute walk test, spirometry, quality of life questionnaire, lab test and RNA profile analysis were performed. The systolic and diastolic left ventricular function, as well as the global longitudinal strain, were improved significantly in the resveratrol-treated group (RES). Exercise capacity, ventilation parameters and quality of life also improved significantly in the RES group. In parallel, the cardiac biomarker levels (N-terminal prohormone of brain natriuretic peptide (NT-proBNP) and galectin-3) decreased in the treated group. The level of inflammatory cytokines decreased significantly after RES supplementation, as a consequence of the decreased expression level of leucocyte electron transport chain proteins. The main findings of our trial are that RES treatment added to the standard heart failure therapy improved heart function and the clinical condition by moderating the inflammatory processes in patients with HFrEF.


Introduction
Heart failure (HF) is a complex multifactorial syndrome caused by structural and/or functional cardiac abnormalities, resulting in a reduced cardiac output or elevated intracardiac pressures [1]. Heart failure prevalence is continuously rising throughout the world. In developed countries, approximately 2% of the adult population has heart failure.

Eligibility
The main inclusion and exclusion criteria are listed in Table 1. Briefly, 60 stable outpatients (ages: 66.7 ± 2.01 years, 17 women and 43 men) with HFrEF in NYHA (New York Heart Association) class II or III were enrolled between 01/03/2016 and 30/11/2017 into our study (ejection fraction (EF) <40% and ischemic/non-ischemic origin: 34/26). They were randomized into two groups (resveratrol group and placebo group). one-hundred milligrams of resveratrol was administered orally (2 × 50 mg) for 3 months in the RES group (n = 30) and placebo capsule in the other group (n = 30).

Eligibility
The main inclusion and exclusion criteria are listed in Table 1. Briefly, 60 stable outpatients (ages: 66.7 ± 2.01 years, 17 women and 43 men) with HFrEF in NYHA (New York Heart Association) class II or III were enrolled between 01 Narch 2016 and 30 November 2017 into our study (ejection fraction (EF) <40% and ischemic/non-ischemic origin: 34/26). They were randomized into two groups (resveratrol group and placebo group). one-hundred milligrams of resveratrol was administered orally (2 × 50 mg) for 3 months in the RES group (n = 30) and placebo capsule in the other group (n = 30).
The resveratrol and the matching placebo capsules were purchased from Argina Nutraceuticals Ltd. (Fót, Hungary). The resveratrol capsule is commercially available and has an official license for being marketed.
All the involved patients were in the evidence-based drug treatment of HFrEF, including angiotensin-converting enzyme (ACE) inhibitors (or ARBs-angiotensin receptor blockers), beta-blockers, mineralocorticoid receptor antagonists and ivabradine. None of the patients received ARNI (angiotensin receptor-neprilysin inhibitor). The preventive drug regime and the used doses were based on the actual European Society of Cardiology (ESC) heart failure guideline [1]. The patients had baseline visits and follow-up visits after 1 month and after 3 months of treatment. During visits, the compliance of patients was checked, and at the final visit (3-month follow-up visit), they were asked to return the remained capsules. During the whole study period, subjects were in stable clinical conditions and received unchanged medical therapy ( Table 2). The baseline characteristics of the randomized patients are detailed in Table 2. There were no significant differences in epidemiological characteristics between the placebo and RES-treated groups at the baseline.   Values are expressed as mean ± SEM. There were no significant differences in patient characteristics between the RES (resveratrol) and placebo-treated groups at the baseline. ACEI: angiotensin-converting enzyme inhibitors, ARB: angiotensin receptor blocker, BMI: body mass index, BP: blood pressure, COPD: chronic obstructive pulmonary disease, CRT-P/D: cardiac resynchronization therapy-pacemaker/defibrillator, ICD: implantable cardioverter defibrillator, MRA: mineralocorticoid receptor antagonist and NT-proBNP: N-terminal prohormone of brain natriuretic peptide.
On the day of randomization and 3-months-later physical examination, blood pressure and weight measurement, echocardiography, lab test, six-minute walk test (6MWT), spirometry and quality of life questionnaire (QoL test) were performed. At the 1-month follow-up visit, only a physical examination, blood pressure, weight measurement and lab test were done (Table 3). Table 3. Timetable of the study.

Statistical Analysis
SPSS statistical software, version 25 (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.) was used to conduct a descriptive analysis and to describe the sample. After using the Kolmogorov-Smirnov test to check the normality of the data distribution, differences of the mean values within the groups were analyzed by repeated-measures ANOVA with a Greenhouse-Geisser correction. Differences between the groups were calculated by one-way ANOVA test. Data are expressed as mean ± S.E.M. Significance level was defined as p < 0.05. The homogeneity of the groups was tested by Levene's F-test. The nonparametric Friedman test (post-hoc analysis with Wilcoxon signed-rank test) was applied to analyze the potential changes of the quality of life of the patients.

Blood Pressure and Body Weight Measurement
Body weight and blood pressure were measured during randomization at 1-month and at 3-month follow-up visits. For body weight measurements, a high-quality calibrated digital scale was used (CAS PB-150 digital scale; CAS Corporation, Tustin, CA, USA). Blood pressures were measured using an automated electronic device after a 5-min resting; the location of the cuff was the upper arm at the Antioxidants 2020, 9, 1108 6 of 19 level of the heart (OMRON M2 Intellisense, Tokyo, Japan). The technique of the measurement was based on the actual hypertension guideline [27].

Laboratory Test
Laboratory analysis performed on blood samples that were extracted from the cubital vein after a 12-h-long fasting period. From blood samples, NT-proBNP; fasting lipid levels (total cholesterol, low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL) -cholesterol and triglycerides); troponin T; glycosylated hemoglobin-HgbA1c; serum albumin; renal and liver function; serum iron parameters and quantitative and qualitative blood cell counts were measured in the Department of Laboratory Medicine, University of Pecs, Pecs, Hungary.

Echocardiography
Transthoracic echocardiography was performed for noninvasive evaluation of cardiac structure and function at baseline and after the 3-month-long treatment. We used a GE Vivid E9 ultrasound imaging device (GE Healthcare, Chicago, IL, USA). Structures were visualized from parasternal long axis; short axis and apical four, two and five-chamber views. M-mode (one-dimensional mode), 2D-mode, 3D-mode, PW (pulsed-wave), CW (continuous wave) and tissue Doppler mode imaging were used to determine the left ventricular ejection fraction (EF, %), the diameters (mm) and volumes (mL) of the left ventricle and left atrium, the diastolic function (E/A and E/E'), the global longitudinal strain of the left ventricle (GLS, %), the wall thicknesses of the left ventricle (mm) and the right ventricular parameters. The EF was measured by two different methods ("Simpson" and "Quinone"). The investigators were blinded to the protocol; thus, they did not know whether the patient was taking resveratrol or placebo.

Six-Minute Walk Test (6MWT)
The 6-min walk test (6MWT) is a submaximal exercise test that measures the walking distance for 6 min. The test was performed on a 30-m-long section of the corridor in our department according to the guidelines of the American Thoracic Society (ATS). The patients were at rest comfortably for 10 min prior to the test. The supervisor was a study nurse, who was blinded to the protocol. After 6 min or if the patient could not go any further, the test was stopped, and the distance, as well as the reason for stopping (dyspnea, fatigue, chest pain, etc.), were recorded [28].

Spirometry
Spirometry is a routine method to evaluate the lung function, and it has many uses, including chest surgery or the grading of chronic obstructive pulmonary disease (COPD), etc. It is well-known that heart failure can influence the pulmonary function, which may include combined obstructive (e.g., significant FEV1 reduction) and restrictive changes [29].
We used a PISTON PDD 301/s spirometer (Piston Ltd., Budapest, Hungary) for measuring the basic resting lung parameters of the patients at baseline and 3 months later.
During the test, the patient wore a soft nose clip to prevent air escaping through the nose, as well as a mouthpiece filter to prevent the spread of microorganisms. During the test, patients were asked to take the deepest breath they could and then exhale into the sensor as hard and as long as possible. Generally, a period of quiet breathing in and out from the sensor precedes the test, and the rapid breathing in (forced inspiratory part) comes before the forced exhalation. We determined the most common parameters in the percentage of the reference values, such as forced vital capacity (FVC); forced expiratory volume (FEV 1 ); FEV 1 /FVC ratio; peak expiratory flow (PEF); forced expiratory flow 25-75% (FEF 25-75); maximal expiratory flows (MEF 25%, MEF 50% and MEF 75%) and inspiratory vital capacity (IVC).

Quality of Life Questionnaire (QoL Test)
Quality of life (QoL) in heart failure patients is severely compromised by the symptoms of the disease. Quality of life is also a good predictor of mortality and the need for hospitalization. For the assessment of QoL, we used the "Euro QoL five-dimension" (EQ-5D) questionnaire. Among health-related quality of life (HR QoL) questionnaires, EQ-5D has gained widespread use due to its simplicity to administer, score and interpret. This scoring system consists of five dimensions (mobility, self-care, usual activities, pain/discomfort and anxiety/depression), each with three levels of response or severity (no problems-2 point, some problems-1 point or extreme problems-0 point). In addition to the index-based scoring system, the visual analog scale (VAS) component of the EQ-5D enables the patient to place their current health state on a range from 0 (worst imaginable health state) to 100 (best imaginable health state) [30].

RNA Isolation, RNA-Seq Library Preparation and Sequencing
QIAamp RNA Blood Mini Kit was used for purification of total RNA from the whole blood. During the QIAamp procedure for purification of RNA from blood, erythrocytes are selectively lysed, and leukocytes are recovered by centrifugation. The leukocytes are then lysed using highly denaturing conditions that immediately inactivate RNases, allowing the isolation of intact RNA. After homogenization of the lysate by a brief centrifugation through a QIA shredder spin column, ethanol is added to adjust the binding conditions, and the sample is applied to the QIAamp spin column. RNA is bound to the silica membrane during a brief centrifugation step. Contaminants are washed out, and total RNA is eluted in 30 µL or more of RNase-free water.
RNA concentrations were measured using Qubit 3.0 (Invitrogen, Carlsbad, CA, USA). The RNA quality was verified on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA). High-quality (RIN > 8) RNA samples were processed for library preparation. Further sample preparation was conducted by Illumina TruSeq ® Stranded Total RNA sample preparation guide.

RNA-Sequencing Data Processing and Analysis
Paired-end fastq files were imported into the CLC Genomics Workbench (CLC Bio, version 12, Hilden, Germany). Adaptor sequences and bases with low quality were trimmed. Reads containing more than 2 ambiguous bases were discarded. Preprocessed sequencing reads were aligned to the gene regions and transcripts of the GRCh38 human genome.
Differential expression of genes at the end of the study was assessed in each experimental group compared to baseline expression values while controlling cross-checking of the patient IDs of paired data. Differentially expressed genes (DEGs) were filtered out, using a threshold of fold change (FC) absolute value > 1.5 and false discovery rate (FDR)-adjusted p-value < 0.05. DEGs were visualized on a heat map using CLC Genomics Workbench 12. Hierarchical clustering was conducted, using Euclidean distances and complete linkages.

The Effect of Resveratrol on Laboratory Parameters
Laboratory parameters did not show significant changes in either group after one month of treatment compared to the baseline values (data not shown). There was no significant change in renal function, liver function and quantitative and quality blood counts in either group after the Antioxidants 2020, 9, 1108 8 of 19 three-month follow-up period compared to the baseline values. Cardiac troponin T, iron parameters and glycosylated hemoglobin (HgbA1c) were also not influenced significantly by the resveratrol treatment during the study period. The total cholesterol (4.74 ± 0.22 mmol/L vs. 4.52 ± 0.24 mmol/L, p < 0.05) and LDL-cholesterol (3.09 ± 0.23 mmol/L vs. 2.79 ± 0.23 mmol/L, p < 0.05) levels were significantly lower in the RES group by the end of the follow-up period; however, in the placebo group, no significant alterations could be seen compared to the baseline values (Table 4). Unfortunately, the HDL-cholesterol level was also slightly but significantly lower in the RES group (1.33 ± 0.06 mmol/L vs. 1.24 ± 0.07 mmol/L, p < 0.05 vs. RES-Baseline) after three months.

Inflammatory Parameters
The acute-phase proteins (CRP and ferritin) and white blood cell count did not show significant changes in either group during the three-month-long follow-up period ( Table 4). The levels of IL-1 (95.61 ± 17.74 pg/mL vs. 140.65 ± 24.04 pg/mL, p < 0.05) and IL-6 (5.42 ± 0.35 pg/mL vs. 6.76 ± 0.56 pg/mL, p < 0.05) measured by ELISA, however, were significantly lower in the RES-treated group at the end of the study period compared to the placebo group ( Figure 2).

Biomarkers of Heart Failure
There was no significant difference in the NT-proBNP level between the RES and placebo groups at the baseline (2998 ± 507 pg/mL vs. 3139 ± 446 pg/mL, NS); however, by the end of the treatment period, the NT-proBNP level was significantly lower in the RES group than in the placebo group (2760 ± 346 pg/mL vs. 4054 ± 577 pg/mL, p < 0.05). This change suggests that RES inhibited the elevation of the plasma NT-proBNP level, a parameter which showed the severity of heart failure. In parallel with NT-proBNP, the galectin-3 level also showed a significant difference between RES and the placebo group at the end of the study (5.61 ± 0.42 ng/mL vs. 6.98 ± 0.54 ng/mL, p < 0.05) (Figure 3).

Inflammatory Parameters
The acute-phase proteins (CRP and ferritin) and white blood cell count did not show significant changes in either group during the three-month-long follow-up period ( Table 4). The levels of IL-1 (95.61 ± 17.74 pg/mL vs. 140.65 ± 24.04 pg/mL, p < 0.05) and IL-6 (5.42 ± 0.35 pg/mL vs. 6.76 ± 0.56 pg/mL, p < 0.05) measured by ELISA, however, were significantly lower in the RES-treated group at the end of the study period compared to the placebo group ( Figure 2).

Biomarkers of Heart Failure
There was no significant difference in the NT-proBNP level between the RES and placebo groups at the baseline (2998 ± 507 pg/mL vs. 3139 ± 446 pg/mL, NS); however, by the end of the treatment period, the NT-proBNP level was significantly lower in the RES group than in the placebo group (2760 ± 346 pg/mL vs. 4054±577 pg/mL, p < 0.05). This change suggests that RES inhibited the elevation of the plasma NT-proBNP level, a parameter which showed the severity of heart failure. In parallel with NT-proBNP, the galectin-3 level also showed a significant difference between RES and the placebo group at the end of the study (5.61 ± 0.42 ng/mL vs. 6.98 ± 0.54 ng/mL, p < 0.05) (Figure 3).

BMI, General Hemodynamic and ECG Parameters
Body mass index (BMI) alterations were not significant in either the resveratrol (29.31±0.99 kg/m 2 vs. 29.06 ± 1.01 kg/m 2 , NS) or in the placebo group (31.04 ± 1.46 kg/m 2 vs. 31.34 ± 1.39 kg/m 2 , NS) after three months compared to the baseline (Table 5). Similarly, no significant alterations could be seen in the blood pressure, in the heart rate and in the ECG parameters after one month (data not shown) and at the end of the study (Table 5). Values are expressed as mean ± SEM. Baseline: measured values at randomization in the resveratrol or in the placebo group, 3rd month: patients treated with resveratrol or placebo for 3 months, BMI: body mass index, BP: blood pressure, BW: body weight, HR: heart rate, PQ: PQ interval on an

BMI, General Hemodynamic and ECG Parameters
Body mass index (BMI) alterations were not significant in either the resveratrol (29.31 ± 0.99 kg/m 2 vs. 29.06 ± 1.01 kg/m 2 , NS) or in the placebo group (31.04 ± 1.46 kg/m 2 vs. 31.34 ± 1.39 kg/m 2 , NS) after three months compared to the baseline (Table 5). Similarly, no significant alterations could be seen in the blood pressure, in the heart rate and in the ECG parameters after one month (data not shown) and at the end of the study (Table 5). Values are expressed as mean ± SEM. Baseline: measured values at randomization in the resveratrol or in the placebo group, 3rd month: patients treated with resveratrol or placebo for 3 months, BMI: body mass index, BP: blood pressure, BW: body weight, HR: heart rate, PQ: PQ interval on an electrocardiogram (ECG), QRS: the wide of the QRS complex on the ECG and QTc: heart rate-corrected QT interval on ECG.

The Effect of Resveratrol on Echocardiographic Parameters
The echocardiographic parameters of the patients did not differ significantly between the groups at the beginning of the study. The left ventricular EF measured by two different methods was improved significantly due to resveratrol administration by the end of the treatment period compared to the baseline (Quinone method: 29.19 ± 1.04% vs. 33.40 ± 1.20%, p < 0.01 and Simpson method: 30.06 ± 1.04% vs. 34.60 ± 1.44%, p < 0.01) ( Table 6 and Figure 4). In parallel with this result, the left ventricular stroke volume (SV) also increased, and the left ventricular end-systolic volume (LVESV) decreased significantly in the RES group. Global longitudinal strain (GLS,%) of the LV showed a significant improvement (−8.40 ± 0.62 % vs. −9.58 ± 0.73%, p < 0.05) in the treated group by the end of the follow-up compared to the baseline. In contrast, in the placebo group, no significant change could be observed.  19.94 ± 1.43, p < 0.05) was observed between the resveratrol and placebo groups after the three-month-long treatment.
In addition, the LAESV, LA long axis, RVIDd, RVIDs and RAEDV were also decreased significantly in the RES group compared to the RES baseline values, as well as LAEDV, RAESV, IVC and TAPSE, which also showed significant differences between the resveratrol and placebo groups after three months (Table 7).

The Effect of Resveratrol on Exercise Capacity
The six-minute walk distance increased significantly in the resveratrol group (from 275 ± 19 m to 298 ± 22 m, p < 0.05) by the end of the treatment period compared to the baseline; however, in the placebo group, there was no apparent change (NS).

The Effect of Resveratrol on the Respiratory Parameters
The treatment with resveratrol resulted in significantly improved FVC (75.38 ± 3.31% vs. 79.27 ± 3.07%, p < 0.05) by the end of the study period. Other expiratory parameters did not show any changes compared to the baseline values (Table 8). However, the inspiratory vital capacity (IVC) also increased significantly by resveratrol administration (83.85 ± 3.65% vs. 90.27 ± 3.39%, p < 0.05) compared to the baseline. Values are expressed as mean± SEM. * p < 0.05, 3rd month values of resveratrol group compared to the baseline values. Baseline: measured values at randomization in resveratrol or in placebo group; 3rd month: patients treated with resveratrol or placebo for 3 months. FEF 25-75%: forced expiratory flow 25-75%, FEV 1 : forced expiratory volume, FVC: forced vital capacity, IVC: inspiratory vital capacity, MEF: maximal expiratory flows at 25%, 50% or 75% of FVC, PEF: peak expiratory flow.

The Effect of Resveratrol on the Quality of Life in Patients with Systolic Heart Failure
For the assessment of QoL the "Euro QoL five-dimension" (EQ-5D) questionnaire was used. The main domains of the test did not differ significantly among the groups at the beginning of the study. Our nonparametric statistical analysis showed significant improvement in the mobility (Z = −2.236, p < 0.05), usual activities (Z = −2.646, p < 0.05) and anxiety/depression (Z = −2.236, p < 0.05) but did not reveal any significant difference in self-care and pain/discomfort of the patients in their resveratrol-treated group after three months compared to the baseline values. Summary, RES administration resulted in a significantly improved percentage of the subjective health state (54.86 ± 2.78% vs. 61.17 ± 2.72%; Z = −3.146, p = 0.002), but there was no significant change in the placebo group (49.43 ± 3.15% vs. 48.17 ± 3.34%; Z = −0.631, NS).

Sequence Analysis and Differential Expression
At the end of the study, the mRNA analysis of seven patients showed that, in the resveratrol group, 222 genes were differently expressed ((FC (fold change) abs. value >1.5 and FDR p-value < 0.05)) compared to the baseline values ( Figure 5A,B). dehydrogenase, subunit 1-complex I), MT-ND2 (NADH dehydrogenase, subunit 2-complex I), MT-ND4 (NADH dehydrogenase, subunit 4-complex I), MT-ND5 (NADH dehydrogenase, subunit 5-complex I) and MT-ND4L (NADH dehydrogenase, subunit 4L-complex I) ( Figure 5C). However, the genes of complex II and complex IV were not affected by resveratrol. Our results revealed that resveratrol had an inhibitory effect on ATP synthesis via the oxidative phosphorylation; however it did not lead to mitochondrial dysfunction in leukocytes, because it has no negative effect on the proteins affecting the mitochondrial quality control ( Figure 5A).

Discussion
Our present work firstly proved that resveratrol beneficially influences heart failure in a randomized double-blind clinical trial (RCT). The major findings of our trial are that the resveratrol  Figure 5C). However, the genes of complex II and complex IV were not affected by resveratrol. Our results revealed that resveratrol had an inhibitory effect on ATP synthesis via the oxidative phosphorylation; however it did not lead to mitochondrial dysfunction in leukocytes, because it has no negative effect on the proteins affecting the mitochondrial quality control ( Figure 5A).

Discussion
Our present work firstly proved that resveratrol beneficially influences heart failure in a randomized double-blind clinical trial (RCT). The major findings of our trial are that the resveratrol treatment improved heart function, exercise tolerance, several spirometry parameters and quality of life and decreased the level of cholesterol and inflammatory cytokines in systolic heart failure patients.
It is well-established that resveratrol has various beneficial effects on the cardiovascular system. It has a marked antioxidant effect due to its scavenger capability and due to enhancing the antioxidant enzyme production (SOD, CAT and eNOS), as well as decreasing the amount of prooxidant enzymes (NOXs and MPO) [31][32][33]. Therefore, resveratrol also has a marked anti-inflammatory and antiplatelet effect [34][35][36]. Resveratrol also has beneficial effect on metabolic parameters; it can decrease the cholesterol level and improve the insulin resistance [37,38]. However, these data are derived almost completely from preclinical studies.
It was published previously by our workgroup that resveratrol in a murine post-infarct heart failure model improves the heart function and decreases myocardial fibrotic remodeling via its anti-inflammatory effect and via blocking the profibrotic intracellular signaling routes [25]. Other workgroups showed similar results in various animal heart failure models; however, in a human clinical trial, the effect of RES has not been confirmed yet [13,26].
In this one-center double-blind RCT, the baseline characteristics of symptomatic systolic heart failure (HFrEF) patients were well-balanced due to the used randomization method (adaptive minimization) [39]. Guideline-directed medical treatment (GDMT) was administered in the maximal tolerated dose, and almost every patient was given ACEI or ARB and beta-blockers, and only one-quarter of the patients was not on MRA treatments (Table 2).
In our heart failure patients, resveratrol supplementation improved the systolic left ventricular function expressed as the ejection fraction (EF). The EF was measured in two different ways, by using the Quinones and the Simpson methods. We chose the Quinones method instead of the Teichholz's method, because according to the literature, it is better correlation with volumetric methods [40]. Nowadays, however, the volumetric Simpson method is by far the most commonly used method for quantifying left ventricular function. In the case of both methods, a similar increase could be seen in the resveratrol-treated group; however, in the placebo group, no change was observable.
Global longitudinal strain (GLS) is another measure of LV global function that also correlates with the extent of myocardial fibrosis in patients with HFrEF [41]. In the placebo-treated group, the GLS value remained unchanged by the end of the treatment period. The resveratrol treatment, however, caused an improvement of the GLS value in parallel with other measures indicating systolic heart function. Moreover, GLS can show the extent of myocardial remodeling, especially myocardial fibrosis. In parallel with the improvement of systolic heart function, diastolic heart function also got better (i.e., the E/e' ratio decreased) in the resveratrol group. In the placebo group the diastolic function was unchanged.
Natural polyphenols could improve heart function in a wide variety of experimental heart failure models [42][43][44]. However, in human clinical trials, there are only a few data regarding the effect of resveratrol treatment on heart function. Our workgroup proved previously that a low-dose resveratrol treatment (10 mg/day) improved the diastolic function in patients with coronary artery disease (CAD), while, in the case of systolic LV function, only a favorable tendency was present (NS) [45]. In another small trial also conducted in CAD patients using a higher dose (100 mg/day) of resveratrol, both the systolic and diastolic function improved.
Not only the heart function but, also, the exercise tolerance determined by the 6MWT showed significant improvement in resveratrol-treated patients by the end of the three-month-long treatment period. This result is in accordance with the preclinical results carried out in several murine models [46]. However, there are no clinical data regarding the effect of resveratrol on the physical exercise capacity in heart failure patients yet. In an interesting trial, Voduc N. and his coworkers proved that resveratrol itself in healthy people has no positive effect on exercise capacity and on the maximal oxygen consumption (VO 2 max) [47]; moreover, Glieman L. et al. demonstrated that, in aged men, the resveratrol treatment blunted the positive cardiovascular effects of exercise and moderated the increase of VO 2 max caused by the training program [48]. Therefore, the increase of exercise capacity can be predominantly the consequence of the improvement of heart function in heart failure patients.
The analysis of cardiac biomarkers strengthens the findings regarding heart function and exercise tolerance. Galectin-3, which is secreted by macrophages, has been known for its significant role in mediating cardiac fibrosis and inflammation [49]. These data were also proved in a human clinical examination [50]. In our trial, the resveratrol treatment decreased the galectin-3 level, but in the placebo group, no change could be seen. This result supports the beneficial changes that occurred in the case of GLS, because GLS is directly proportional to the extent of interstitial fibrosis [41].
NT-proBNP is the most widely used biomarker in the diagnosis and risk stratification of heart failure. It shows a strong correlation with the severity of heart failure, although its value can be variable due to alterations in the volume status of patients. In our trial, the resveratrol supplementation decreased the level of NT-proBNP (NS), and in the placebo group, a worsening tendency could be seen, so by the end of the treatment period, a significantly lower natriuretic peptide level was achieved in the RES group than in the placebo group. In a study conducted in stable CAD patients, Militaru and coworkers found that resveratrol decreases the NT-proBNP level even without overt heart failure [51]. The continuously increasing level of heart failure biomarkers (NT-proBNP and galectin-3) in the placebo group can be a consequence of the progression of the disease. According to the literature, there was a linear relationship between NT-proBNP and the number of hospitalization, as well as mortality, due to heart failure [52].
The improvement of left ventricular heart function in the resveratrol-treated group is in-line with the decreased volume retention and with the decongestion of organs. It is known that extravascular fluid accumulation in the lungs is accompanied by the alteration of several spirometry measures, e.g., forced vital capacity (FVC) and inspiratory vital capacity (IVC) [53,54]. Moreover, Gehlback et al. showed that, after heart transplantation, not only the ventilation volumes but the airflow velocity (FEV1) was also better [54]. Due to resveratrol supplementation in the present trial, FVC and IVC increased significantly by the end of the treatment period. However, in the case of airway obstruction parameters, no significant changes could be seen in this study. FEV1 showed only a mild, nonsignificant improvement in the RES group.
The level of inflammatory cytokines, IL-1 and IL-6 decreased in the resveratrol-treated heart failure patients compared to the placebo group. Similar results were seen in the literature [55]. This favorable change is parallel with the level of galectin-3β that mediates inflammation. The levels of inflammatory cytokines produced by the activated leukocytes are inversely proportional to the systolic left ventricular function [56]. The increasing level of IL-1 and IL-6 in the placebo group can be explained by the slow progression of heart failure in our enrolled population. The correlation between inflammation and adverse cardiovascular outcomes in heart failure was documented. Essentially, heart failure progression was attributed to sustained proinflammatory cytokine signaling, based on the observation that proinflammatory cytokines were elevated and continuously worsened during the progression of the disease [57]. Interestingly, the mRNA profile analysis of leukocytes revealed a significant decrease in genes encoding several mitochondrial respiratory proteins. Resveratrol, however, did not interfere with the expression of various proteins playing a part in the mitochondrial quality control. Moderating the production of ETC proteins in leukocytes can decrease the oxidative phosphorylation in leukocytes, which is directly proportional to their activity. The decreased production of proinflammatory cytokines in this work can be a sign of the moderated activity of leukocytes. This anti-inflammatory effect can be an important aspect of the resveratrol treatment in heart failure patients, besides its well-known mitochondrial protective effects in cardiomyocytes.

Conclusions
In this human clinical trial, the positive effects of resveratrol were proved in systolic heart failure (HFrEF) patients added to the standard therapy. The resveratrol treatment improved several parameters of heart function, exercise tolerance and quality of life. Moreover, resveratrol exerted an anti-inflammatory effect measured by the decrease of levels of inflammatory cytokines (IL-1 and IL-6). According to our results, the decreased activity of leukocytes can be an important mechanism of resveratrol, and it can contribute to its cardioprotective effect in heart failure.

Limitations of the Study
This study has some potential limitations. Although the study was double-blinded and randomized, (1) the number of the enrolled patients was quite low (n = 30/group), and (2) the follow-up period was relatively short. Due to these limitations, the results, including RNA sequencing, have to be interpreted cautiously. In the future, adequately powered randomized, controlled trials reporting patient-relevant outcomes with long-term follow-up periods are required to properly prove the efficacy of resveratrol supplementation in systolic heart failure, as well as to understand the exact biochemical and cellular mechanism.