Animals and cell line
All animal experiments were performed in strict accordance with the guidelines for the Care and Use of Laboratory Animals formulated by the National Institutes of Health (NIH, Bethesda, MD). The protocols were approved by the Animal Care and Use Committee of Tongji Medical College of Huazhong University of Science and Technology (permission number: SCXK (E) 2016-0057).
Eight to ten week-old male Sprague-Dawley rats, weighing 200 - 300 g (Wuhan University Laboratory Animal Center, Wuhan, China) were used in this study. The human HEK293 cell line (American Type Culture Collection) was cultured in Dulbecco’s modified Eagle’s medium (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 100 mg∙ml-1 sodium pyruvate, in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. Adult rat cardiomyocytes were isolated by the Langendorff method and cultured as described previously22.
Construction and in vivo cardiac transfection of AAV9-ZsGreen-shRNA
AAV9-ZsGreen vectors carrying PIASy-shRNA or scramble-shRNA were commercially synthesized from Shenzheng BioWit Technologies Co., Ltd and its titer were 1.0×1012vg/ml. Briefly, three shRNAs targeting PIASy were designed and used to build the respective plasmids. After enzyme digestion and sequencing, the plasmids were transfected into H9c2 cells to pack with AAV9 harboring ZsGreen. The shRNA with the best interfering efficiency was selected based on Q-PCR results. Purification and titer determination of the AAV9 shRNA vector were performed following standard protocols.
Cardiac delivery of the AAV9 vectors in vivo was performed by intraventricular injection in 8-week-old male Sprague-Dawley rats according to a previous description23. The rats were anesthetized with pentobarbital sodium (70 mg/kg, iv.) and fixed in the supine position. An Insulin syringe was inserted through the thoracic wall into the left ventricular chamber, which was confirmed by blood withdrawal. Then, AVV9 suspension (50μL, 1.3*1011 vg) was slowly injected.
Grouping
Eighty rats were randomly assigned to four groups (n=20 per group), including Scramble-shRNA (AAV9 ZsGreen scramble shRNA transfer with sham operation), Scramble-shRNA + I/R (AAV9 ZsGreen scramble shRNA transfer with I/R), PIASy-shRNA (AAV9 ZsGreen PIASy shRNA transfer with sham operation) and PIASy-shRNA + I/R (AAV9 ZsGreen PIASy shRNA transfer with I/R) groups. Rats were subjected to I/R 14 days after intraventricular injection of AAV9 shRNA.
In vivo I/R and in vitro H/R models
An in vivo myocardial I/R model was established by surgical ligation of the left anterior descending coronary artery (LAD) as described previously21. Rats in the Scramble-shRNA and PIASy-shRNA groups underwent the same procedure except for LAD ligation. Two weeks after systemic delivery of AAV9 vectors, the animals were subjected to a 45 min of ischemia followed by 2h reperfusion. Fresh ventricular tissue samples in the peri-infarct region (area of approximately 3 mm surrounding the infarction induced by LAD ligation) were obtained, snap frozen in liquid nitrogen and stored at -80°C.
An in vitro simulated I/R model was established by exposing transfected HEK293 cells to hypoxia (serum-free DMEM in an incubator containing a humidified atmosphere with 1% air + 5% CO2 + 94% N2) for 3h followed by 2h of reoxygenation (DMEM with serum in 5% CO2 + 95% air), as well as isolated adult rat cardiomyocytes to hypoxia for 40 min followed by 30 min of reoxygenation (H/R).
Plasmids and transfection
The plasmid pTracer-SV40 containing the WT human Nav1.5 gene was a kind gift from Dr. Thomas ZIMMER at Friedrich Schiller University Jena. Flag-tagged Cav-3 (Human caveolin 3) was purchased from Origene Technologies. The Flag-hPIASy, HA-SUMO1, HA-SUMO2/3, ubc9, scramble pCMV6-entry plasmids were obtained from Addgene. Cav-3 mutations at the K38 and K149 positions (Flag-tagged) were generated with the QuickChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions, and verified by DNA sequencing. Plasmids, including the SUMO machinery (SUMO2/3, SUMO1, ubc9, and PIASy), Cav-3 (Wt, K38R and K149R mutants), and Nav1.5, were transfected into cells with Attractene from Qiagen, following the manufacturer’s instructions. HEK 293 cells transfected with various plasmids were exposed to hypoxia or hypoxia/reoxygenation to detect Cav-3 binding to SUMO2/3 and Nav1.5, respectively.
Immunofluorescence and histology
Two weeks after transfer of the AAV9 carrying reporter gene (ZsGreen) into the rat myocardium, six-micron cryosections were cut from rat heart, lung and liver tissues, and observed directly for green fluorescence protein expression by fluorescence microscopy (Olympus BX-51 Microscope). Transfected HEK293 cells and isolated adult rat cardiomyocytes exposed to H/R, as well as fresh frozen ventricular sections from the four animal groups were fixed for 10 minutes in 4% formaldehyde (Beyotime Biotechnology, Germany) and stained with primary antibodies targeting PIASy, Cav-3, Nav1.5 and SUMO2/3, respectively, followed by incubation with secondary antibodies. Nuclei were stained with 4'6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology) during the secondary antibody incubation step. Sections were mounted with Gelvatol for microscopy. Fresh ventricular tissues were processed by paraffin embedding, and 5-μm thick sections were stained with hematoxylin and eosin (H&E). An Olympus fluorescence microscope was used for imaging.
RNA extraction and quantitative real-time PCR
Total RNA was extracted from ventricular tissue samples using TRIzol Reagent (Invitrogen) and treated with DNase I to remove genomic DNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on a Bio-Rad thermocycler with a SYBR green kit (Invitrogen) following the manufacturer’s instructions. The primers used for amplification were: Nav1.5, forward 5’-CCTTCACTGCCATCTACAC-3’ and reverse 5’-GCCTGAAATGACCGATAT-3’; CAVEOLIN-3, forward 5’-GACATTGTGAAGGTGGATTT-3’ and reverse 5’-GTAGACAGCAGGCGGTAG-3’; GAPDH, forward 5’-AAGGGCTCATGACCACAGTC-3’ and reverse 5’-GGATGCAGGGATGATGTTCT-3’ (Wuhan BioBuffer Biotechnology, Co. Ltd., Wuhan, China).
Co-immunoprecipitation and Western blot
The following primary antibodies were used for Western blot and/or co-immunoprecipitation (Co-IP): anti-SUMO1 (ab11672), anti-SUMO2/3 (ab3754), anti-Caveolin-3 (rabbit polyclonal,ab2912), and anti-Sodium Potassium ATPase antibody (ab198366), purchased from Abcam; anti-phosphor-Tyrosine (#9411), anti-phospho-Threonine Antibody (#9381), anti-GFP (#2555), and anti-β-actin (#4970), from Cell Signaling Technology; anti-Caveolin-3 (mouse polyclonal, sc-55518) and anti-PIASy (sc-166706, sc-50348), obtained from Santa Cruz Biotechnology; anti-Nav1.5 (#ASC-005), from Alomone Labs; anti-myc-tag (No. 66004-1-Ig) and anti-HA-tag (No. 66006-1-Ig), from Proteintech. Horseradish peroxidase (HRP) conjugated anti-mouse IgG (cs7076) and anti-rabbit IgG (cs7074) secondary antibodies were from Cell Signaling Technology; anti-mouse AlexaFluor488 (ab150113) and anti-rabbit AlexaFluor647 (ab150115) secondary antibodies were from Abcam. N-Ethylmaleimide (NEM) was purchased from Sigma-Aldrich.
Forty-eight hours after transfection, HEK293 cells were washed with PBS. Cells and tissue samples (stored at −80℃) were lysed for 30 minutes on ice in RIPA lysis buffer (Beyotime, Shanghai, China) containing 0.1 mM PMSF and a protease inhibitor cocktail (Roche). Equal amounts of protein in cell or tissue lysates were separated by 10% SDS-PAGE and electro-transferred onto PVDF membranes. Then, the membranes were blocked with 5% BSA (bovine serum albumin) for 1 hour at room temperature, and sequentially incubated with respective primary antibodies overnight and HRP-conjugated secondary antibodies (1:3000) for 1 hour. The blots were evaluated by ECL (enhanced chemiluminescence), with β-actin (A2228; 1:5000 dilution; Sigma-Aldrich) as a loading control. Cytosolic and membrane fractions were prepared with Mem-PER Plus Membrane Protein Extraction Kit (Pierce Protein Biology) according to the manufacturers’ instructions.
For immunoprecipitation, 50μg of clarified cell and tissue lysate samples were immunoprecipitated with 1µg of anti-Cav-3 or anti-Nav1.5 overnight, and then incubated with 15μL protein G/A-agarose (Santa Cruz, CA) for another 4h at 4°C. Agarose beads were sedimented and washed 5 times with cell lysis buffer, and bound proteins were released with 30µl of 2×SDS-loading buffer. The immunoprecipitated proteins were further analyzed by Western blot as described above.
For the detection of SUMOylation, 20 mmol/L NEM was added to the lysis buffer for protein extraction. Importantly, the samples were not boiled for SUMOylation determination. Individual bands were quantified by densitometry with the ImageJ software (version 5, NIH).
Electrocardiography (ECG)
ECG was performed continuously during the experiments. Surface ECG parameters were analyzed under stable baseline conditions at least 5 minutes after anesthesia induction and before jugular vein preparation. P duration, PR interval, QRS duration, and corrected QT interval (QTc) were measured on standard limb lead II. ECG recordings were evaluated with the LabChart software (version 7.3, AD Instruments Pty Ltd, Australia). The durations of ventricular arrhythmias, and episodes of ventricular tachycardia (VT), sustained VT and ventricular fibrillation (VF) within every consecutive 5-min block after ligation were determined. VT is 3 or more consecutive ventricular premature beats; sustained VT is an episode of VT lasting 10 sec or more. We used Curist-Walker scoring systems to grade the severity of arrhythmias occurring in rats under the experimental conditions of myocardial I/R24.
Statistical Analysis
The results were presented as mean ± SEM and analyzed by Student’s t-test and one-way analysis of variance (one-way ANOVA) followed by the Newman–Keuls post hoc test. For non-normally distributed parameters, such as episodes of ventricular arrhythmias, durations of sustained VT and VF, the Kruskal Wallis test was used. For ECG wave changes, two-way ANOVA was employed for analysis. Percent survival was analyzed with the Kaplan-Meier method. The GraphPad Prism 6.0 software package (version 5 for Windows, San Diego, CA) was used for all statistical analyses. Statistical significance was set at p<0.05.