Materials
Cell culture reagents including Dulbecco's modified Eagle's medium (DMEM) high glucose, Fetal Bovine Serum (FBS), antibiotic-antimycotic solution (Anti-Anti 100X), and 0.25% trypsin-EDTA solution were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Western blot reagents were acquired from Bio-Rad (Hercules, CA, USA). Antibodies against b-actin, troponin I, and atrial natriuretic peptide were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The autophagy assay kit was supplied by ABCAM (Cambridge, UK). Annexin-V-FLUOS staining kit was provided by Roche (Nonnenwald, Germany). Annexin-V-FITC was provided by Biolegend (San Diego, CA, USA), Annexin-V buffer and Propidium Iodine were supplied by BD (New Jersey, USA). Sterile plastic material for tissue culture and 8-well tissue culture chambers were obtained from Sarstedt (Nümbrecht, Germany). MitoTracker Green FM and LysoTracker were from Invitrogen (Carlsbad, CA, USA). ZnO nanopowder < 50 nm particle size (BET), >97% (cat. No. 677450), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Cell culture
Cells derived from embryonic rat ventricular tissue (H9c2) were obtained from American Type Culture Collection (ATCC). Cells were cultured in DMEM high glucose supplemented with 10% FBS and Anti-Anti solution (1X). Cells were maintained at 37°C under a humidified atmosphere with 5% CO2.
ZnO NPs characterization
For the hydrodynamic diameter, zeta potential, and polydispersity index measurements, ZnO NPs powder was suspended both in DMEM medium supplemented with 10% FBS and in 0.9 % NaCl-HEPES buffer (NHB). Both suspensions were vortexed at 60 Hz for 10 min for subsequent reading on the Zetasizer Nano-ZS90 equipment.
ZnO NPs internalization
ZnO NPs internalization was detected by TEM. Cells were cultured with ZnO NPs (2.5, 5, 10, and 20 mg/cm2) for 24 h. After treatment, H9c2 cells were fixed with 2.5% glutaraldehyde-paraformaldehyde in phosphate buffer solution (PBS) (pH 7.2) for 45 min. After fixation cells were placed in 1% osmium tetroxide for 1 h. Then, cells were dehydrated through graded series of alcohols and embedded in Epon 812 epoxy resin. Then 60 nm-thin sections were obtained with a diamond knife on Ultracut-R ultramicrotome, mounted on copper-grids, and impregnated with heavy metals, lead nitrate, and uranyl acetate. Grids were examined in a JEOL 10/10 transmission electron microscope at 60 kV and AMT Camera System.
Cell morphology
For morphological analysis, H9c2 cells (20 × 103) were seeded on 8-well tissue culture chambers, exposed to 2.5, 5, 10, and 20 mg/cm2 over 48 h, then stained with hematoxylin/eosin (H/E), analyzed with a microscope Olympus BX51 with 20x objective and photographed with a camera Q Imaging MicroPublisher 5.0 RTV.
Cell proliferation and viability
Proliferation and viability were evaluated by crystal violet staining and MTT reduction respectively, as previously described by our work group [39]. H9c2 cells (3 × 103/well) were cultured in 96-well plates and exposed to different concentrations of ZnO NPs (2.5, 5, 10, and 20 mg/cm2) over 24, 48, and 72 h. Non exposed cells were used as negative control. After exposure, cells were fixed with 1.1% glutaraldehyde for 10 min, washed twice with water, and stained with 0.1% crystal violet solution for 20 min. Incorporated crystal violet was solubilized with 100 ml/well acetic acid (10%) and optical density at 590 nm was measured in a microplate spectrophotometer (Benchmark Plus, BIO-RAD). To assay viability, 20 ml/well of MTT (5 mg/ml) were added, and cells were incubated for 4 h at 37°C. Then the medium was removed, formazan crystals were dissolved with acid isopropanol (0.04 N HCl) and optical density was read at 570 nm.
Oxidative stress
The cellular redox state was measured by oxidation of 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) into 2,7- dichlorodihydrofluorescein (DCF) which is a redox indicator probe [40]. H9c2 cells (200 × 103) were cultured without or with ZnO NPs (5, 10, 20 mg/cm2) for 1, 3, and 6 h in 56-mm glass Petri dishes. After treatment, cells were trypsinized and incubated with H2DCFDA (10 mM) for 30 min and washed twice with PBS. Fluorescence was quantified in a FACSAria flow cytometer (Becton-Dickinson, CA, USA).
Oxidative stress was also tested by ΔΨm changes based on the fluorescent dye rhodamine 123 (Rh123). To this, cell suspensions (1 × 106) were treated without and with ZnO NPs (5, 10, 20 mg/cm2) for 1 h. Then cells were washed with PBS and incubated with Rh123 (5 mg/mL) for 15 min. After incubation, cells were washed with PBS and analyzed in a FACSAria flow cytometer (Becton-Dickinson, CA, USA). Data were processed using FlowJo 8.7 software (Stanford University).
Mitophagy assay
To assay mitophagy (selective removal of damaged mitochondria by autophagosomes and lysosomes), H9c2 rat cardiomyoblasts (50 x 104) were cultured in 35-mm glass-bottomed Petri dishes (MatTek, Ashland, MA, USA) in absence or presence of ZnO NPs (2.5, 5, 10 and 20 μg/cm2) for 6 h. To detect nucleus, mitochondria, or lysosomes, cells were pre-incubated with 0.4 µM Bis-Benzimide H 33342 trihydrochloride (Hoechst), 0.5 µM MitoTracker Green (MTG), and 0.5 µM LysoTracker Red (LTR), respectively, for 30 min at 37°C in DMEM without phenol red. Epifluorescence images were taken with the EVOS FL (Thermo Fisher Scientific Waltham, MA, USA) cell imaging microscope at 60x magnification.
Cell death
The annexin-V-Fluos staining kit was used to determine cell death by microscopy. H9c2 cells (50 × 103/well) were cultured in 6-well plates and treated with 2.5, 5, 10, and 20 mg/cm2 ZnO NPs for 24 and 48 h. After exposure cells were analyzed by fluorescence microscopy in a Floid Cell Imaging Station (Life Technologies, Carlsbad, CA, USA). To this, the culture medium was discarded, cells were washed with PBS and subsequently incubated with 100 mL of annexin-V-Fluos labeling solution (20 mL propidium iodide plus 20 mL annexin-V-Fluos in 1 mL of incubation buffer) for 15 min and analyzed immediately.
Apoptosis and necrosis were also examined by flow cytometry using dual staining with annexin V-FITC and propidium iodide according to the manufacturer’s instructions. Briefly, 1 × 106 cells in 100 ml annexin buffer were stained with propidium iodide staining solution and FITC annexin V staining solution. Cells were incubated at room temperature in the dark for 15 minutes and acquired in a FACSAria flow cytometer (Becton-Dickinson, CA, USA). To flow cytometry, cells were washed with PBS, trysinized, incubated with 100 mL of annexin-V-Fluos labeling solution and immediately analyzed. Data were processed using FlowJo 8.7 software (Stanford University).
Protein expression
Western blot analysis was performed as described previously by our work group [41]. Cells were exposed to ZnO NPs (5, 10, 20 mg/cm2) for 48 h and total proteins were isolated with a lysis extraction buffer. Proteins were quantified using Bio-Rad protein assay dye reagent concentrate. Proteins (30 mg) were separated by electrophoresis using 8% SDS-polyacrylamide gels and transferred to PVDF membranes, blocked, and incubated with primary antibodies against troponin I, atrial natriuretic peptide and b-actin as a load control (diluted 1:2500, 1:000, and 1:2500, respectively) overnight. After, membranes were washed three times with TBS-T, incubated with the secondary antibody (diluted 1:2500) for 1 h, and washed again three times with TBS-T. Proteins were detected with the SuperSignal® system and the ChemiDocTM MP Imaging System (Bio-Rad). Densitometric analysis for protein quantification was carried out with the Image Lab™ V 4.0 Software (Bio-Rad).
Statistical Analysis
To quantify and compare the patterns of actin structures a non-parametric Kruskal-Wallis test was performed using the Prism 7.0a software (GraphPad software, Inc.). In order to determine statistical differences in all assays, Student’s t-tests were performed and p<0.05 was considered significant. For western blot analysis, multiple comparisons were based on one-way analysis of variance (ANOVA) followed by Turkey’s pairwise comparison as post hoc test in Prism 5.01.