Reagents and antibodies
DOX, 5-bromo-2’-deoxyuridine (BrdUrd), lipid peroxidation (malondialdehyde, MDA) assay kit, catalase (CAT) and total superoxide dismutase (SOD) assay kits were from Sigma-Aldrich Chemicals (Men,, USA). Protein A/G plus agarose was from Santa Cruz Biotechnology (Dallas, TX, USA). MitoSOX Red was from Invitrogen (Carlsbad, CA, USA). CardioTACS in situ apoptosis detection kit was from Trevigen (Gaithersburg, MD, USA). Lipofectamine 3000 and Trizol Reagent were from Invitrogen (Carlsbad, CA, USA). Annexin V-APC/7-AAD Cell Assay Kit was from BD Pharmingen (San Diego, CA, USA). Simple ChIP Enzymatic Chromatin IP Kit, and antibodies anti-total p53 (1:1000, #2524), anti-acetyle-p53 (Lys382) (1:1000, #2525), anti-phospho-p53 (Ser392) (1:1000, #9281), anti-Sirt6 (1:1000, #12486), anti-caspase-3 (1:1000, #9662), anti-caspase-8 (1:1000, #9746), anti-RIP1 (1:1000, #3493), anti-RIP3 (1:1000, #15828), anti-histone H3 (1:2000, 9715), anti-H3K9Ac (1:1000, #9649), anti-Myc-Tag (9B11) (1:1000, #2276), anti-Acetylated-Lysine (1:100, #9441), anti-β-actin (1:2000, #4970) and the negative control for mouse IgG (#5415) were from Cell Signaling Technology (Danvers, MA, USA). The antibodies anti-Fas (1:1000, #ab82419) and anti-FADD (1:1000, #ab24533) were from Abcam (MA, USA). The antibodies anti-HMGB1 (1:1000, #MAB16901-SP) and anti-FasL (1:1000, #AB16982) were from R&D Systems (Minneapolis, MN, USA) and Sigma-Aldrich Chemicals (St Louis, MO, USA), respectively. The membrane protein extraction kit was a Mem-PER™ Plus Membrane Protein Extraction Reagent kit from Thermo Scientific (Catalog number: 89842, Rockford, IL, USA). The cytosol and nuclear protein extraction kit was a ReadyPrep™ Protein Extraction Kit (cytoplasmic/nuclear) from Bio-Rad (cat# 163–2089; Bio-Rad, Hercules, CA, USA). BCA protein assay kit was from Beyotime Institute of Biotechnology (Beijing, China). The PrimeScript RT reagent Kit with gDNA Eraser was from Takara Biotechnology (Dalian, China). All pairs of PCR primers were synthesized by the Chengdu Qingke Biotechnology (Chengdu, China). All other chemicals were of analytical grade.
Sirt6+/− (Sirt6 heterozygous) mice on a129Sv background were purchased from the Jackson laboratory (Bar Harbor, ME, USA). Sirt6 heterozygous mice (Sirt6+/−) were backcrossed with wild-type (WT) C57BL/6J mice on a C57BL/6J background to obtain Sirt6+/− mice and littermates (Wang et al. 2016). The mice were confirmed by PCR genotyping analysis (data not shown).
All procedures involving experimental animals were approved by the Animal Care and Use Committee of Sichuan University and performed in accordance with the Guidelines for Animal Experiments from the Committee of Medical Ethics, National Health Department of China.
In vivo DOX-induced cardiotoxicity mouse model
Male Sirt6+/− and WT mice (8 to 12 weeks old) were treated with a 5-week protocol, which consisted of intraperitoneal injection (i.p.) of DOX at 8 mg/kg (body weight) or saline once a week for 4 weeks, and maintenance for another a week. All drug doses and injection regiments were based on previous reports with some modification (Wang et al. 2014).
Cardiac echocardiography and hemodynamics
After the 5-week treatment, mice (n = 10) were anesthetized with isoflurane (2%) in oxygen and positioned on a heated pad in a recumbent position (Wang et al. 2016; Wang et al. 2014). 2-D short-axis images were obtained by using a Vivid7 ultrasound imaging system (GE Health Medical, Milwaukee, WI, USA) operating at 12-MHz. Echocardiographic image acquisition and measurements were acquired from grayscale M-mode images at the mid-papillary level in the parasternal short-axis view, and 2-D mode images were obtained in the parasternal long- and short-axis views (Wang et al. 2016; Wang et al. 2014). After the echocardiography assessment, the mice were ventilated with a rodent ventilator (Harvard Apparatus, Holliston, MA, USA), then maintained with isoflurane (2%) in oxygen for anesthesia. An incision in the neck was made and a pressure–volume (P-V) catheter 1.2 F transducer (4.5 mm electrode spacing, Serial No. 112B-B057, SCIsense Inc., ON, Canada) was inserted into the left ventricle (LV) through the right carotid artery. After the P-V catheter was stabilized for several minutes, the signals were recorded by using an eight-channel physiological recorder (iWorx 308, iWorx/CB Sciences, Inc., Dover, NH, USA). The LV performance, including the LV peak systolic, end-diastolic pressures and heart rate were detected, while LV peak-negative developed pressure (dP/dtmin), LV pressure at peak positive developed pressure (dP/dtmax) was calculated (Wang et al. 2016; Wang et al. 2014). After hemodynamic measurements, mice were euthanized by pentobarbital overdose (i.p.), and hearts were removed and cut into three pieces. For histology, heart slices were fixed in 4% paraformaldehyde, paraffin-embedded, and cut into 4-µm serial sections for immunohistochemistry and the other two slices were snap-frozen in liquid nitrogen and stored at -80°C (Wang et al. 2014).
The histological sections were stained with hematoxylin-eosin (HE) solution, wheat germ agglutinin (WGA), and Masson trichrome and underwent TdT-mediated dUTP nick end-labeling (TUNEL) with standard protocols (Wang et al. 2016; Li et al. 2018). Cardiomyocyte size was measured from images of HE-stained sections and Alexa Fluor 488-conjugated WGA-stained sections and cardiomyocytes. About 100–200 random cardiomyocytes from each group (n = 10) were quantitatively assessed by using Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA), and cardiomyocyte cross-sectional areas were visualized under a Zeiss microscope (AX10 imager A2 model, Germany) and presented in µm2 (Li et al. 2018). Myocardial fibrotic area was measured in five randomly selected fields per section on Masson's trichrome-stained sections from each group (n = 4) and the proportion of fibrosis were calculated by using Image-Pro Plus as the ratio of fibrotic area to total left ventricular (LV) area (Li et al. 2018; Ghosh et al. 2018; Wang et al. 2016; Zhang et al. 2010).
TUNEL staining used to assess DOX-induced cell apoptosis was analyzed by using a CardioTACS in situ apoptosis detection kit with the manufacturer’s instructions (Wang et al. 2014). Five randomly areas from each slice were visualized for comparison of each experimental group. The apoptotic index (%) was calculated as the ratio between TUNEL-positive cardiomyocyte nuclei and total number of nuclei ×100 (Men et al. 2020; Wang et al. 2014).
Cardiomyocyte culture, RNA interference, adenoviral infection and treatment
Neonatal rat cardiomyocytes were isolated from 0- to 2-day-old newborn Sprague-Dawley rats, which were sacrificed by decapitation, and cardiac ventricles were obtained from all rat pups for trypsin and collagenase II digestion as described (Wang et al. 2016; Li et al. 2018). After digestion, cells were suspended with Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin, then pre-plated for 1 h to remove any non-myocyte cells, then plated in cell culture dishes and placed overnight in incubator maintained at 5% CO2 in air and ༞ 95% humidity. On the following day, cells were cultured for 24 h in DMEM with 100 µM BrdUrd to prevent non-myocyte cell proliferation.
The rat Sirt6-siRNA (siRNA No., siG150505110012) and the control sequence (NTC, no target control) were from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Sirt6-siRNA and NTC were transfected into cells with Lipofectamine 3000 by the manufacturer’s instructions (Wang et al. 2016). The replication-defective adenoviral vectors expressing Sirt6 (Ad-Sirt6) and control green fluorescent protein (Ad-EGFP) was constructed by using the AdEasy1 system instructions (Wang et al. 2014). In all siRNA-treated or adenovirus-treated experiments, cardiomyocytes were transfected with Sirt6 siRNA or control NTC siRNA oligonucleotides for 48 h or with Ad-Sirt6 or control Ad-EGFP adenovirus for 48 h, then treated with DOX (2 µM) for another 18 h.
Malondiaedehyde (MDA) content and SOD2 and CAT enzyme activity assays
Heart tissues or cardiomyocytes were harvested and homogenized in 0.9% saline and centrifuged at 4°C for 15 min at 12, 000 rpm. The supernatant was collected. The protein concentration of lysates was measured with a BCA assay kit. Analysis of MDA content and both SOD2 and CAT activities in lysates of tissues and cardiomyocytes was according to the manufacturers’ instructions (Wang et al. 2016; Wang et al. 2014).
Total RNA was extracted by the phenol-chloroform extraction method with Trizol Reagent. A 1 µg amount of total RNA was reverse-transcribed for cDNA synthesis with the PrimeScript RT reagent kit with gDNA Eraser. All relative mRNA levels were standardized to that of β-actin. The primer sequences are in supplemental Table 1 and RT-PCR procedures involved using a CFX96 Real-Time PCR Detection System (Bio-Rad Life Sciences).
Measurement of mitochondria reactive oxygen species production
Superoxide production was detected in living cardiomyocytes by using MitoSOX-red, a mitochondrion-specific hydroethidine-based fluorogenic dye following the manufacturer’s instructions (Wang et al. 2016). Briefly, after treatment with siRNA or adenovirus, neonatal rat cardiomyocytes (NRCMs) were stained with MitoSOX-red reagent (5 µM) with or without DOX (2 µM) for 18 h in the dark at 37°C, washed twice with phosphate buffered saline (PBS) and nuclei were stained with DAPI in anti-fade mounting buffer and visualized by Zeiss microscopy. Fluorescence intensity was analyzed by using Image J (Wang et al. 2016).
Apoptosis detected by flow cytometry
Primary cardiomyocytes (5×105/mL) were cultured in six-well plates for 24 h, then transfected with NTC-siRNA or Sirt6-siRNA for 48 h before incubation with or without DOX for 18 h or were overexpressed with Ad-EGFP or Ad-Sirt6 viruses and washed with PBS. Cells with no drugs were the control. Early/late apoptotic cells were measured by using the FACSAria SORP Cell Analyzer (BD, Biosciences, San Jose, CA, USA) with the Annexin V-APC/7-AAD Cell Assay Kit.
Western blot assay and immunoprecipitation
Western blot assay was used to determine protein expression as described (Wang et al. 2016; Li et al. 2018) including Sirt6, p53, caspase-8, caspase-3, RIP1, RIP3, Fas, FasL and FADD in cardiac tissues and cardiomyocytes. Total membrane proteins and cytosolic proteins were extracted to examine the expression of membrane Fas and FasL following the protein extraction kit as described (Hou et al. 2018). Nuclear proteins were extracted to detect the protein levels of high mobility group box 1 (HMGB1) and H3K9Ac by using a nuclear extraction kit (Wang et al. 2016). β-actin, Na/K-ATPase α1, and Histone H3 were used as a normalization control in whole cell homogenates, isolated membrane proteins and nuclei proteins, respectively.
The interacting partners of Sirt6 and p53 in myocardial cells were assessed by co-immunoprecipitation (Wang et al. 2016). Cardiomyocytes were harvested and suspended in 1 mL NP40 lysis buffer. Lysates were cleared by centrifugation at 13,000 rpm for 10 min. Protein (500 µg) was immunoprecipitated with protein A/G plus agarose, then incubated with 2.5 µg anti-p53 antibody or non-immune mouse IgG control at 4℃ overnight, and immunoprecipitated proteins were detected by western blot methods as described (Wang et al. 2016).
HEK293T cells were harvested at 48 h post-transfection with overexpression vectors of Flag-tagged Sirt6 (#13817, Addgene) and Myc-p53 (#MG50534, Sino Biological Inc. Beijing, China). Cell extracts were prepared, then pulled down with an anti-Flag antibody together with protein A/G plus agarose. Immunoprecipitated proteins were analyzed by western blot assay with anti-Myc antibody as described previously (Amir et al. 2009)
Detection of acetyl-p53 level in nuclear protein
NRCMs at 5×106 cells/10 cm plate were cultured for 24 h, then transfected with NTC-siRNA or Sirt6-siRNA for 48 h before incubation with or without DOX for 18 h, or NRCMs were overexpressed with Ad-EGFP or Ad-Sirt6 viruses before incubation with or without DOX for 18 h. The nuclear proteins were extracted and immunoprecipitated with anti-acetylated-lysine antibody or non-immune mouse IgG control and protein A/G plus agarose. The amount of p53 in the nuclear extracts and immunoprecipitates were determined by immunoblotting with antibody p53 (Ghosh et al. 2018).
Chromatin immunoprecipitation (ChIP) assays
ChIP assay was performed as described previously by using the Simple ChIP Enzymatic Chromatin IP kit (Wang et al. 2016). Briefly, cells were cross-linked with 1% formaldehyde and digested with Micrococcal Nuclease into 150–900 bp DNA fragments, then a mouse anti-p53 antibody was added to the diluted soluble chromatin solution for 18 h at 4°C on a rotator, and a mouse IgG was used as a negative control; 1% of the total chromatin sample before immunoprecipitation (input). After being reverse crosslinked, eluted DNA was amplifed with specific Fas and FasL promoter primers (for a 174-bp fragment of Fas promoter: forward 5’-ATGAGTTGTGTGGGTGTCAG-3’, reverse 5’-TAATGGAGGCAACAGAGCTAC-3’; for a 100 bp fragment of FasL promoter: forward 5'-CACTTACAGAACGGCACTAC-3' and reverse 5'-ACTAGAGTCCACCAATCCCA-3'). The relative abundance of the studied DNA fragments in immunoprecipitated DNA was calculated with the ΔΔCt method.
The data are expressed as mean ± SEM. Intergroup comparisons were analyzed by unpaired Student t test and three or more groups were analyzed by one-way ANOVA followed by the Newman–Keuls tests. P < 0.05 was considered statistically significant.