All animal experiments were approved by the medical ethics committee of Central Hospital of Baotou City, and performed in conformance with the Principles of Laboratory Animal Care (National Society for Medical Research) and in accordance with National Institutes of Health guidelines. Mice used in the study were C57BL/6 mice (8–10 weeks of age) purchased from Shanghai Laboratory Animal Center (Shanghai, China) and housed in a facility with a 12-h light/dark cycle at a controlled temperature and humidity with free access to food and water.

The TAC model was generated by tying a suture around the transverse aorta over a 27-gauge blunted needle to cause occlusion of the aorta. After withdrawing the needle, a stenotic aortic lumen was generated. Mice were euthanized by removing the heart under anesthesia. Sham-operated mice without TAC were used as controls.

For SIRT6 overexpression, lentiviral vectors were constructed by amplifying the cDNA of SIRT6 by PCR using specific primers (forward primer: 5′-CCGCTCGAGGAAGCGGCCTCAACAAGG-3′, and reverse primer: 5′-CGCGGATCCGTGGTTCCTTCAAGTTCCCC-3′) and subcloning into the pLVX-IRES-puro lentiviral vector using Xho I and BamH I restriction sites (underlined). High titer lentiviruses were generated by transfection of human embryonic kidney 293 cells with recombined vectors. Approximately 48 h post-transfection, cells were harvested, purified by centrifugation, and stored at −80°C for study.

Mice were injected intravenously via the tail vein with 100 μl pLVX-IRES-puro vector (vector) or vector expressing SIRT6 (pSIRT6) lentivirus plasmids (lentivirus at 100 million virus/100 μl PBS/mouse) at 2 weeks and 24 h before TAC or sham surgery. Myocardial tissue samples were collected at 28 days after surgery. Mice without lentivirus injection served as the control (Ctrl).

Echocardiographic measurements were obtained using a Vevo2100 imaging system (VisualSonics, Toronto, Canada) with a 22–25 MHz linear transducer probe as described previously (Balasubramanian et al., 2015). LV end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated using the Simpson method of disks. Ejection fraction (EF) was determined using the formula EF (%) = (EDV−ESV)/EDV × 100. Left ventricular end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD) were measured at one-dimensional mode according to the American Society of Echocardiography guidelines. LV fractional shortening (FS) was calculated with the following formula: FS (%) = (LVEDD − LVESD)/LVEDD × 100. The maximal rates of rise and fall in LV pressure (+dP/dt, −dP/dt) were recorded as previously described (Toldo et al., 2011). The investigator performing and reading the echocardiogram was blinded to the treatment allocation.

For quantitative real-time RT-PCR (qRT-PCR), total RNA was isolated from myocardial tissue samples using the TRIzol reagent (Invitrogen, USA) and reverse transcribed using the SuperScript III kit (Invitrogen) according to the manufacturer's instruction. The PCR primers used were as follows: SIRT6 forward, 5′-CCT GGT CAG CCA GAA CGT AG-3′, and reverse, 5′-GTG TCT CTC AGC TCC CCT CT-3′; GAPDH forward, 5′-CCA TCT TCC AGG AGC GAG AC-3′, and reverse, 5′-GCC CTT CCA CAA TGC CAA AG-3′. qRT-PCR reactions were performed using SYBR Green reagents (Invitrogen) and on a MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad Laboratories, USA). PCR conditions were 95°C for 10 min, then 40 amplification cycles of 95°C for 10 s and 72°C for 30 s. Reactions were performed in triplicate, and the level of mRNA was normalized to that of GAPDH using the 2^−ΔΔCt method.

For western blot analysis, mouse myocardial tissues from infarcted area of TAC groups and anterior left ventricular of sham operated groups were homogenized in radio-immunoprecipitation assay buffer (Beyotime, Shanghai, China) and protein concentration was determined using the BCA protein assay kit (Beyotime). Aliquots containing 20 μg of protein were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to nitrocelullose membranes (Pall Corporation, USA). Membranes were incubated in 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature, followed by incubation in primary antibodies at 4°C overnight. The primary antibodies used and their dilutions were as follows: anti-SIRT6, 1:2,000; anti-α-tubulin, 1:2,000; anti-TERT, 1:1,000; anti-TRF1, 1:1,000; anti-collagen I, 1:5,000; anti-FN, 1:1,000; anti-α-tubulin, 1:1,000; β-actin, 1:2,000 (all from Abcam, Cambridge, United Kingdom). After washing in TBST, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Signal detection was performed using the SuperSignal ECL system (ThermoScientific, Waltham, Massachusetts) and bands were analyzed by ImageJ software. Band intensity was normalized to that of α-tubulin or β-actin.

Infarct size was determined using the 2,3,5-triphenyltetrazolium chloride (TTC) staining. Briefly, mice were euthanized 28 days after surgery. The hearts were rapidly removed, frozen, and sliced into transverse sections (~2 mm). The heart slices were stained with TTC, fixed, and photographed using a digital camera. The infarct area (pale white) in the LV was determined using Meta Morph software (version 6.0, Universal Imaging Corporation). Infarct size was presented as percentage of total LV surface. Survival analysis was performed by daily cage inspection for up to 28 days after surgery.

For histological analyses, mice were euthanized and the hearts were cut into transverse segments, fixed in 10% formaldehyde, and embedded in paraffin. Sections were cut at a thickness of 10 μm and either stained with Masson's trichrome to measure fibrotic areas using ImagePro software. The following antibodies were used for immunohistochemistry analyses: anti-Ly-6G (1:100, Abcam) to detect neutrophils and anti-CD68 (1:200, Abcam) to detect macrophages. Quantitative assessment of neutrophil and macrophage density was performed by counting the number of Ly6G- and CD68-immunoreactive cells, respectively, in a double-blind fashion from five different fields of the infarcted area at 400 × magnification. For immunofluorescent staining, tissue sections were incubated with anti-SIRT6 antibody (1:100, Abcam) overnight and secondary antibody for 90 min. DAPI solution was then added for 2 min to stain the nuclei. Images were captured using a Laser-Scanning Confocal Microscope (Olympus FluoView™ FV1000, Tokyo, Japan).

TNF-α, IL-1β, and IL-10 concentrations in mouse hearts were assessed by ELISA using a commercial kit (R&D System, Minneapolis, MN, USA), according to manufacturer's instruction. Tissues from infarcted area of TAC groups or anterior left ventricular of sham operated group were homogenized in ice-cold PBS buffer containing protease inhibitor cocktail (21 mmol/L leupeptin and 3.1 nmol/L aprotinin), and total proteins were extracted using a protein extraction kit (Pierce, Rockford, IL, USA). Results are expressed as pg/mg tissue.

Data are presented as the mean ± standard deviation. Data were analyzed with SPSS (version 22.0; SPSS, Chicago, USA) or GraphPad Prism (version 5.0; GraphPad, La Jolla, USA). Student's t-test was applied to determine statistically significant differences between groups. One-way analysis of variance followed by Tukey's post-hoc test was applied to determine the significance among groups. P < 0.05 was considered statistically significant.

The levels of SIRT6, TERT, and TRF1 were measured in myocardial tissues of mice exposed to TAC or sham surgery to explore the association of sirtuins and telomere function with TAC-induced heart failure. The results of qRT-PCR and western blotting showed that SIRT6 was significantly downregulated in TAC mice at the mRNA and protein levels compared with the levels in sham-operated mice (Figures 1A,B). Immunofluorescence analysis of myocardial tissues confirmed that SIRT6 was downregulated in TAC compared with sham-operated mice (Figure 1C). Western blot analysis showed that TAC significantly downregulated TERT and TRF1 proteins in myocardial tissues compared with their expression in sham-operated mice (Figure 1D).

The effect of SIRT6 was further analyzed by injection of a vector expressing SIRT6 lentivirus plasmids into mice before TAC, which resulted in the effective overexpression of SIRT6 mRNA and protein in TAC and sham-operated mice, as indicated by qRT-PCR and western blot analysis (Figures 2A,B). Kaplan-Meier survival analysis showed that mice overexpressing SIRT6 had significantly better survival rates after TAC than control or empty vector-injected mice (Figure 2C). The improved survival occurred in parallel with the upregulation of TERT and TRF1 proteins in TAC mice overexpressing SIRT6 compared with the respective controls (Figure 2D). These results suggested that SIRT6 overexpression protects mice against TAC-induced heart failure by modulating the expression of telomere regulatory proteins.

The protective effect of SIRT6 was further analyzed by measuring cardiovascular function after TAC. The results of echocardiographic and hemodynamic measurements showed that SIRT6 overexpression attenuated the effects of TAC on decreasing FS and EF and increasing LVEDD and LVESD (Figures 3A–D). SIRT6 overexpression also attenuated the effects of TAC on the maximal fall and rise of left ventricular pressure compared with that in vector mice (Figures 3E,F). Taken together, these results indicated that SIRT6 exerts a protective effect against TAC-induced cardiovascular function impairment.

Cardiac inflammation was assessed by measuring neutrophil and macrophage infiltration, which showed that SIRT6 overexpression significantly decreased neutrophil infiltration at 1 day after TAC and attenuated the increase in macrophages at 1 and 7 days after TAC, indicating that SIRT6 decreased cardiac inflammatory responses induced by aortic constriction (Figures 4A–D). In addition, assessment of the levels of TNF-α, IL-1β, and IL-10 by ELISA in the heart homogenate of control and pSIRT6-injected mice after TAC or sham surgery showed that SIRT6 significantly attenuated the TAC-induced upregulation of TNF-α and IL-1β and the downregulation of IL-10 (Figure 4E), suggesting that the effect of SIRT6 was mediated by the modulation of inflammatory factors.

Histological analysis of sections of the heart by Masson's trichrome staining showed that SIRT6 overexpression significantly attenuated the TAC-induced increase in the areas of fibrosis in mice exposed to TAC compared with sham-operated mice (Figures 5A,B). In addition, heart sections were stained with TTC to determine myocardial infarct size. The results showed that SIRT6 significantly reduced the size of the TAC-induced infarcted area (Figures 5C,D). Western blot analysis showed that SIRT6 significantly attenuated the TAC-induced upregulation of collagen I, fibronectin, and α-SMA in myocardial tissues (Figure 5E). Taken together, these results indicated that SIRT6 protects the myocardium against TAC-induced heart failure, improving heart function, reducing infarct size and cardiac fibrosis, and attenuating inflammatory responses, and these effects may be mediated by the modulation of telomere size and structure.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.