Cell culture
HL-1, a mouse atrial cardiomyocyte, is used in this study and obtained from Dr. William C. Claycomb (Louisiana State University Health Sciences Center, New Orleans, LA). HL-1 cells can be serially passaged, maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. The cells were cultured in Claycomb medium supplemented with 10% FBS (Gibco, Scotland, UK), 2 mM L-glutamine (Gibco, Scotland, UK), 0.1 mM norepinephrine and antibiotics (100 µg/ml penicillin and 100 µg/ml streptomycin) at 37 °C under a 5% CO2 air atmosphere. H2O2 (200 µM) was added to induce oxidative stress for 24 h.
Lentiviral transduction for gene knockdown
Lentiviral particles containing shRNAs pLKO.1 (#TRCN0000031289) was used to knockdown DPP4 in HL-1 cardiomyocytes. Lentivirus containing scrambled shRNA, pLKO-shScr (#TRCN00001) was used as non-targeting control and served as wild-type. HL-1 cardiomyocytes were transduced with lentiviral vectors with MOI of 15, along with 8 ug/mL Polybrene (hexadimethrine bromide, Sigma Aldrich, MO, USA) in medium supplemented with 1% FBS for 24 hr and then replaced with fresh medium. Transduced HL-1 cardiomyocytes were treated with culture media containing puromycin at final concentration of 5 ng/ml, for selection of transduced cells.
RNA extraction and reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated from cells using TRIzol (Thermo Fisher Scientific, MA, USA). Total RNA was reverse transcribed with Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA) and SYBR Green was used for performing quantitative real time PCR. The expression level of each individual transcript was normalized to HPRT gene and expressed relative to the mean expression values of wild-type samples.
Detection of cell viability
Cell viability was determined by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Cells were treated with MTT at 0.5 mg/ml. The purple formazan crystals were dissolved in DMSO. Solutions were then loaded into a 96 well plate, and the absorbance was determined on an automated microplate spectrophotometer at 570 nm.
Detection of ROS production
Intracellular ROS and mitochondria superoxide generation were detected in HL-1 cardiomyocytes by labeling with fluorescence dye dihydroethidium (DHE) and MitoSOX (Thermo Fisher Scientific, MA, USA), respectively. Cells were loaded with 10 µM dye at 37℃ for 30 min, washed with PBS. By using fluorescence microscopy, DHE and MitoSOX were monitored at 510 nm excitation and 580 nm emission, respectively. Fluorescence intensity was calculated by averaging fluorescence intensity of numerous outlined cells using ImageQuant (Molecular Dynamics, Inc., Sunnyvale, CA, USA).
Detection of mitochondrial membrane potential
Mitochondrial membrane potential was detected in HL-1 cardiomyocytes by labeling with fluorescence dye tetramethylrhodamine (TMRM) (Thermo Fisher Scientific, MA, USA). Cells were loaded with 10 µM dye at 37℃ for 30 min, washed with PBS. By using fluorescence microscopy, TMRM was monitored at 548 nm excitation and 574 nm emission, respectively. Fluorescence intensity was calculated by averaging fluorescence intensity of numerous outlined cells using ImageQuant (Molecular Dynamics, Inc., Sunnyvale, CA, USA).
Detection of mitochondrial bioenergetic function
A Seahorse metabolic flux analyzer was used for measuring the rate of oxidative phosphorylation, according to methods previously described [24]. For measuring oxygen consumption rate (OCR), Seahorse XF Cell Mito Stress Test kit (Agilent Technologies, Inc., Santa Clara, CA, USA) including different pharmacological inhibitors were used to probe the function of individual components in the respiratory chain. A seeding density of 30,000 cells/well was used to detect the changes in OCR due to exposure with H2O2. The cells were incubated in XF24 culture microplates with culture medium for 24 h as well as the sensor cartridge hydrated in XF Calibrant at 37 °C in a non-CO2 incubator overnight. Prior to all bioenergetic assays, the culture medium was replaced with unbuffered medium. To estimate the basal oxygen consumption rate (OCR) coupled to ATP synthesis, 1uM oligomycin was injected to inhibit the ATP synthase. The decreased OCR in response to oligomycin indicated the cells were using mitochondria to generate ATP. To determine the maximal OCR that the cells could sustain, 0.5 uM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) was injected, which made the mitochondrial inner membrane permeable to protons. The deduction between maximal OCR and the basal OCR is the spare capacity of mitochondria. Finally, 0.5uM antimycin A and rotenone was injected to inhibit electron flux. The remaining OCR could be ascribed to oxygen consumption due to the formation of mitochondrial ROS and non-mitochondrial sources.
Protein extraction
A nucleus isolation kit (Abcam,USA) and the multiple centrifugation method were used to perform protein extraction of nuclear fractions, according to methods previously described [25]. The total fraction or the nucleus fraction of protein were isolated by an extraction buffer (Thermo Fisher Scientific Inc., IL, USA) containing a cocktail protease and phosphatase inhibitor (Sigma, St. Louis, MO, USA). A BCA protein assay kit (Thermo Fisher Scientific Inc.) was used to determine the protein concentration.
Determination of protein expression
A Western blotting technique was performed to detect protein expression. Equal quantities of proteins were first denatured for 10 min in boiling sample buffer (31.3 mM Tris-HCl at pH 6.8, 25% glycerol, 10% sodium dodecyl sulfate (SDS), 10% 2-mercaptoethanol, and 0.00125% bromophenol blue). Then, proteins were separated using SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Perkin-Elmer Life Sciences, Boston, MA, USA). The membranes were blocked with 5% fat-free milk dissolved in Tris-buffered saline with Tween 20 (TBST) and incubated overnight with the primary antibodies of bax, cleaved Poly (ADP-ribose) polymerase (PARP), nuclear factor erythroid 2–related factor 2 (Nrf2), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1ɑ), lamin (Abcam, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, CA, USA) at 4 °C. Subsequently, the membranes were washed several times with TBST, which was followed by incubation with horseradish-peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.) for 1 hour. After washing several times, the protein signals were detected using an enhanced chemiluminescence system (Millipore, Bedford, MA, USA). The blots were scanned and quantified using Imagequant (Molecular Dynamics, Inc., Sunnyvale, CA, USA).
Statistical analyses
All values are represented as mean ± standard error. The results were analyzed using one-way ANOVA, followed by Bonferroni post hoc tests. We considered that p < 0.05 to be significant.