Materials
ITO-coated glass slides (75 × 25 mm) were obtained from Sigma (St. Louis, MO, USA). These ITO-coated glass slides had surface resistivity of 8–12 Ω with nominal transmittance > 83% and thickness of 1.1 mm. The ITO was washed by sonication (15 minutes each) using acetone, ethanol, and 2-propanol (Sigma).
NRVM isolation and electrical stimulation
Cardiomyocytes were obtained from 1-day-old neonatal rat heart ventricles. Sprague Dawley rats were used following a protocol approved by the Institutional Animal Care and Use Committee of the Korea Institute of Toxicology (IACUC 17-1-0190) and the Guidelines for the Care and Use of Laboratory Animals of the National Research Council. The ventricle was minced in 0.2% solution of trypsin in Hank’s balanced salt solution (HBSS, Thermo Scientific, Grand Island, NY, USA) and then subjected to a series of digestions (15 minutes, 37°C, 500 x g) with a mixture of 0.05% collagen type II and 0.06% pancreatin in HBSS (enzyme solution). The first digest was discarded. The cell suspension from the 4th digestion was centrifuged (330 x g, 10 minutes) and then washed with HBSS for resuspension. Subsequently, the cells were resuspended in high glucose DMEM (Gibco BRL, Gaithersburg, MD, USA) containing 15% fetal bovine serum (FBS, Gibco) and then plated onto a 100-mm cell culture dish at 37°C for 2 hours. The cells remaining in suspension were collected for further culture on ITO. Before seeding with cells, the ITO slides were coated with 2% gelatin for 30 minutes. Cell number and viability were determined with a hemocytometer and trypan blue staining, respectively. After cardiomyocyte seeding (5 x104 cells/well) on ITO slides, stimulation was applied at 5 V with a pulse duration of 10 ms at a pacing frequency of 2 Hz for 21 days. The cells were maintained in a defined medium. The control cells received no stimulation. During the experimental period, the medium was replaced with fresh DMEM supplemented with 10% FCS (Gibco) every two days.
Cell viability assay
Cell viability was analyzed using a LIVE/DEAD Viability Assay Kit (Invitrogen, Grand Island, NY, USA). Briefly, cells in 5 mL of cardiomyocyte culture media were mixed with 1 µL of calcein AM solution (LIVE) and 5 µL of ethidium homodimer-1 solution (DEAD) followed by incubation at 37°C for 40 minutes in a 5% CO2 incubator. NRVM staining was analyzed under a Ti-2000 fluorescence microscope (Nikon, Japan).
Measurement of NRVM alignment
Cell morphology was assayed with phase contrast microscopy. Cultured plates were removed from the incubator on an inverted microscope (Eclipse TS100, Nikon). Live images were captured using a digital camera (U3, Nikon) and analyzed with NIS software (Nikon). Cell area and circularity were determined by tracing the outline of 100 individual cells from four different areas of the coverslip using ImageJ software and standard analysis plugins [8]. The average value was calculated (n = 30).
Immunochemistry
Cells were fixed in 4% PFA at 4°C for 15 minutes and permeabilized with 0.1% Triton X-100 in PBS (Welgene, South Korea) for 5 minutes. After treatment with blocking solution containing 5% normal goat serum for 30 minutes, cells were stained with primary cardiac-specific antibodies against sarcomeric α-actin (Sigma) and connexin43 (BD Biosciences, Bedford, MA) at 4°C overnight. The cells were washed three times with PBS and then stained with TRITC-conjugated secondary antibodies (Molecular Probes Inc., Eugene, OR, USA) at room temperature for one hour. After washing with PBS, nuclei were stained with DAPI (Invitrogen). All images were analyzed using LSM 510 and 710 META confocal microscopes (Carl Zeiss Inc., Oberkochen, Germany)
Gene expression
Total RNA was isolated from the cells cultured in the presence or absence of electrical stimuli using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions and used for cDNA synthesis with the SuperScript II Reverse Transcription Kit (Invitrogen). Quantitative real-time PCR (qRT-PCR) analysis was performed using SYBR Green gene expression assays (Roche, Mannheim, Germany) with GAPDH as an internal control. The primer sequences used in qRT-PCR are shown in Supplementary Table 1. The gene expression level was calculated using the ΔΔCt method (n = 3).
Statistical analysis
All data are presented as mean values ± standard deviations. Comparisons between two groups were performed using Student’s t-test. A P value of less than 0.05 was considered statistically significant. Data graphs were fit with GraphPad Prism version 7.