The anti-ageing molecule sirt1 mediates beneficial effects of cardiac rehabilitation

Fifty-three consecutive patients affected by HF were recruited from the Cardiac Rehabilitation Unit. All patients completed the CRP. As only three patients were women, they were excluded from the analysis. Therefore, the final study population consisted of 50 elderly male patients (mean age 68.6 ± 6.3 years). None of the patients had experienced a myocardial infarction (MI) in the 12 months preceding the study. All patients were in clinically stable condition, and classified as in NYHA II and III class with a preserved Ejection Fraction (EF) [10 patients with HF mid-range EF; 40 with HF preserved EF]. All definitions were based on the ESC and ACCF/AHA criteria, in which the term “stable” defines treated patients with symptoms and signs that have remained generally unchanged for at least 1 month [22].

The clinical and demographic features of the study population are listed in Table 1. Information on comorbidities and concomitant medications were gathered from all patients. No racial/ethnic-based differences were present. At baseline, no differences in medical therapy were found, and no changes occurred during the study period.

Data are expressed as mean (SD) or number of subjects (%). BMI, Body Mass Index; SBP, Systolic Blood Pressure; DBP, Diastolic Blood Pressure; HR, Heart Rate; bpm, beat/minutes; CAD, Coronary Artery Disease; PTCA, Percutaneous Transluminal Coronary Angioplasty; CABG, Coronary Artery Bypass Graft; COPD, Chronic Obstructive Pulmonary Disease; ARBs, Angiotensin II Receptor Blockers.

Biochemical, echocardiographic and cardiopulmonary stress test features of patients before (P) and at the end of the CRP (RP) are shown in Table 2. A CRP significantly reduced cholesterol and increased creatinine levels (both P < 0.0001).

Cardiopulmonary stress test revealed a reduction in maximum systolic blood pressure (P = 0.002), and increased maximum heart rate (P = 0.034), rate-pressure product (P < 0.0001), test duration (P < 0.0001), and VO2 max (P < 0.0001) with consequent significantly higher exercise tolerance, one of the most crucial target in the HF treatment, after CRP.

The activity of Sirt1 and of its molecular targets, Cat and SOD before and at the end of the CRP were evaluated.

The CRP enhanced Sirt1 activity measured in PBMCs from patients (RP vs P, P = 0.02) (Fig. 1, Panel a). Likewise, Cat and SOD activities measured in serum were greater in RP than in P (P < 0.005 and P < 0.05, respectively) (Fig. 1, Panels b and c).

To investigate the possible role of Sirt1, Cat and SOD in modulating the beneficial effects of the CRP, an in vivo-in vitro model was set up by conditioning human endothelial cells (ECs) with sera from patients at time 0 (Patient serum-conditioned ECs, P-ECs) and at the end of the CRP (Rehabilitated Patient serum-conditioned ECs, RP-ECs). Moreover, the antioxidant response in such conditioned cells was evaluated after the induction of stress using H₂O₂.

Sirt1 and Cat activities were higher in RP-ECs than in P-ECs (both, P < 0.0001) (Fig. 2, Panels a and b). Conversely, SOD activity decreased in RP-ECs (P < 0.05) compared with P-ECs (Fig. 2, Panel c).

In the presence of H₂O₂-induced oxidative stress, Sirt1 and Cat activities were higher in RP-ECs than in P-ECs (P < 0.0001 and P < 0.05, respectively) (Fig. 2, Panels a and b), whereas SOD activity did not change (Fig. 2, Panel c).

These results showed that a CRP induced Sirt1 and Cat activation in both the absence and presence of oxidative stress, suggesting the role of Sirt1 in stimulating the antioxidant response.

The senescence in endothelial cells (ECs) conditioned with patients’ sera was measured. ECs conditioned with sera from patients at the end of a 4-week CRP (RP-ECs) showed a significantly reduced senescence compared to that conditioned with sera from patients before CRP (P-ECs), in both the absence and presence of induced oxidative stress (both, P < 0.0001; Fig. 3).

To investigate the possible role played by Sirt1 and its molecular target Cat in the modulation of cell senescence, P-ECs and RP-ECs, either exposed or not to oxidative stress, were treated with Sirt1 and Cat pharmacological inhibitors, EX-527 and 3-amino-1,2,4-triazole (ATZ) respectively.

As shown in Fig. 3, the inhibition of Sirt1 activity by EX-527 caused an increase of senescence in RP-ECs compared with baseline (P = 0.001), but not in P-ECs. Interestingly, in the presence of H₂O₂ oxidative stress, EX-527 induced a rise in senescence, in both P-ECs (P < 0.05) and RP-ECs (P = 0.001). Hence, Sirt1 inhibition abolished the anti-senescent effect of a CRP, suggesting Sirt1 as a modulator of endothelial cell senescence.

Also the inhibition of Cat activity by ATZ resulted in an increased senescence, in both P-ECs and RP-ECs (both, P < 0.0001), compared with baseline. In the presence of H₂O₂-induced oxidative stress, ATZ treatment caused a significant increase in the senescence rate in ECs conditioned with RP sera (P < 0.0001) compared with basal levels. Of note, senescence become higher in stressed RP-ECs compared with stressed P-ECs when Cat was inhibited (P = 0.001). These data suggest that Cat is, at least in part, responsible for the reduced senescence rate observed in ECs conditioned with RP sera.

A possible limitation of the present study could be the lack of a group of heart failing patients not undergone CR. However, the main outcome was represented by the investigation of the molecular changes occurred before and after a well-structured 4-week rehabilitation program in patients affected by chronic HF, who did not change their clinical characteristics and pharmacological therapy during the study period.

Another drawback is the lack of women in the study population. Actually, we did recruit only three women, and then we decided to exclude them because of the small number. This is in line with the fact that women are less inclined to take part in cardiac rehabilitation programs [34]. Therefore, further studies are necessary to better clarify the molecular effects of CR also in female patients.

A prospective longitudinal observational study was conducted on patients consecutively admitted to the Cardiac Rehabilitation Unit of “San Gennaro dei Poveri” Hospital in Naples, Italy. Patients’ information and consent forms were approved by the ethical committee of ASL of Salerno Registry of Observational Studies (RSO) n.10/14.

The study was performed in accordance with the Declaration of Helsinki Seventh Revision (2013) and its amendments. This report adheres to the standards for the reporting of observational trials and was written according to the STROBE guidelines for Observational Studies in Epidemiology - Molecular Epidemiology (STROBE-ME) [35].

Exclusion criteria included unstable angina pectoris, uncompensated HF, complex ventricular arrhythmias, pacemaker implantation and orthopaedic or neurological limitations to exercise.

All enrolled patients underwent a physical examination, collection of demographic and routine blood chemistry tests, chest X-ray, blood pressure measurement, electrocardiographic and echocardiographic examinations, cardiopulmonary stress test and a 6-min walking test with Borg index evaluation. For interval training, low muscle commitment calisthenics and respiratory exercises were performed.

The CRP consisted of 30-min sessions of aerobic exercise, 5 days a week. A daily training session comprised a warm-up (10 min), endurance training (15 min) and a cool-down (5 min) on a cycle ergometer at 50% of the VO2 max achieved on the cardiopulmonary stress test.

Samples isolated from patients before CR were indicated as P, while those collected from patients after CR were designated as RP.

Human Umbilical Vein Endothelial Cells (HUVECs, ECs) were purchased from Clonetics (Walkersville, MD). The cells were cultured in an endothelial growth medium, containing FBS at a concentration of 2% and bovine brain extract (with FGF-2 at a concentration of 100–500 pg/ml). The cells were subcultured by trypsinization, seeded on cell culture dishes coated with 0.1% gelatin and growth in an atmosphere of 5% CO2 at 37 °C. Pilot experiments to identify the concentration of hydrogen peroxide (H₂O₂ = 100–750 μM) that effectively induced a significant decrease in the survival of control cells, were conducted and a concentration of 500 μM was chosen. Moreover, we evaluated the effect of oxidative stress 12, 24, 48, and 72 h after a treatment with 500 μM H₂O₂. Finally, we chose the time of 48 h as representative of the most relevant change.

Therefore, ECs were seeded and cultured for 48 h in a medium supplemented with either the patient’s serum (10%) at time 0 (Patient serum-conditioned ECs, P-ECs) and post CR (Rehabilitated Patient serum-conditioned ECs, RP-ECs), or FBS (10%) as a control and were exposed or not to oxidative stress induced by 500 μM H₂O₂. Four hours after H₂O₂ exposure, the growth medium was replaced with fresh medium containing FBS.

All experiments were performed at a population doubling level (PDL) of 8 to 12.

Crude nuclear samples were extracted by suspending the cells into 1 mL of lysis buffer (10 mM of Tris HCl at pH 7.5, 10 mM of NaCl, 15 mM of MgCl₂, 250 mM of sucrose, 0.5% NP-40, 0.1 mM of EGTA). Cells were spun through 4 ml of sucrose cushion (30% sucrose, 10 mM of Tris HCl at pH 7.5, 10 mM of NaCl, 3 mM of MgCl₂) at 1300 × g for 10 min at 4 °C. The isolated nuclei were suspended in 50–100 μl of extraction buffer (50 mM of HEPES KOH at pH 7.5, 420 mM of NaCl, 0.5 mM of EDTA Na₂, 0.1 mM of EGTA, 10% glycerol). After centrifugation at 15,000 rpm for 10 min, the protein concentration of the crude nuclear extract without protease inhibitor was determined by the Bradford method. Sirt1 activity in the nuclei was determined using the CycLex SIRT1/Sir2 Deacetylase Fluorometric Assay Kit (Ina, Nagano, Japan). The reaction was carried out by simultaneously mixing fluorescent-labeled acetylated peptide as substrate and 10 μl of the sample, trichostatin A, NAD, and lysyl endopeptidase. The intensity of the fluorescence at 440 nm was measured 60 min after the onset of the reaction. Values are reported as relative fluorescence/μg of protein (AU). All data are the means ± standard deviation (SD) of three independent experiments.

Catalase (Cat) and superoxide dismutase (SOD) activities were determined using the Catalase Assay Kit and the Superoxide Dismutase Assay Kit (Cayman Chemical, USA). Samples were previously diluted with buffer (1:10 for serum; 1:2 for cell lysate). The values were reported as U/μg of protein. All data are the means ± SD of three independent experiments.

To investigate if Sirt1 or Cat activity influenced changes in the senescence of the conditioned cells either exposed or not to H₂O₂, their respective activities were inhibited using EX-527 (Sigma, Milan-Italy) at a concentration of 5 μM for 1 h and 3-amino-1,2,4-triazole (ATZ) (Sigma, Milan-Italy) at a concentration of 10 mM for 3 h.

Cultured cells were washed in PBS and fixed with 2% formaldehyde and 2% glutaraldehyde for 10 min at room temperature. The cells were washed and then incubated at 37 °C in staining buffer with the following components: 40 mM citric acid/sodium phosphate (pH 6.0), 0.15 M NaCl, 2 mM MgCl₂, 5 mM potassium ferrocyanide, and 1 mg/mL X-gal (5-bromo-4 chloro-3-indolyl β-D-galactoside). After 4 h, the SA-β-gal rate was obtained by counting four random fields per dish and assessing the percentage of SA-β-gal-positive cells from 100 cells per field. Senescence values are shown as a percentage of the reference condition (FBS-conditioned ECs), which is 100%.

Continuous variables are expressed as mean ± SD and compared by paired or unpaired Student’s t test (normally distributed variables) or by two or three way ANOVA when appropriate, or as median ± interquartile range value and compared by the Mann–Whitney U test (not normally distributed). Normality of data distribution was evaluated using the Kolmogorov-Smirnov test. Non-normally distributed continuous variables were converted to their natural log functions. Categorical variables are expressed as a proportion and compared by the χ ² test, with risk ratios and 95% confidence intervals quoted.