The rat H9C2 cardiomyocytes were cultured at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, high glucose, Gibco-Invitrogen, Carlsbad, USA), supplemented with 1% antibiotic-antimycotic (10,000 U/mL of penicillin, 10,000 mg/mL of streptomycin) and 10% fetal bovine serum (FBS, Gibco-Invitrogen), and were used between passages 15 to 25. To examine the autophagic status in response to bromide stimulation, we infected the cells with the adenovirus expressing GFP-RFP-LC3 for 24 h (hours), and treated with bromide or vehicle (equal molar sodium chloride) for another 24 h. The GFP-RFP-LC3-positive cells were examined by a Nikon fluorescence microscope (ECLIPSE, Ts2R-FL). Rat neonatal primary cardiomyocytes were isolated from the ventricles of Wistar rats aged 1–10 days (n = 10, purchased from Model Animal Research Center of Nanjing University, China) as previously described . All animal procedures in this research conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85–23, revised 1996) and was approved by the Laboratory Animal Care & Use Committee at Nanjing Medical University (Permit number SYXK2018–0012). Briefly, newborn male Wistar rats (1–10 days old) were sacrificed by decapitation, hearts from rats were minced and dissociated with 0.25% trypsin and collagenase II. Dispersed cells were seeded at 105 cells/well in 96-well plates with DMEM supplemented with 10% FBS and then cultured in a 5% CO2 incubator at 37 °C.
CCK-8 toxicity assay
CCK-8 assay was performed to analyze potential toxic effects of sodium bromide (NaBr; Sigma-Aldrich, Germany) on H9C2 cardiomyocytes. Briefly, 1 × 104 cells were seeded into each well of a 96-well plate and were cultured at 37 °C overnight. After synchronization with serum-free DMEM, cells were transferred into 100 μL serum-free DMEM containing either NaBr or equal amounts of sodium chloride (NaCl, positive control) at indicated concentrations (ranging from 10 μM to 600 μM) and incubated for another 24 h. Then, 10 μL CCK-8 reagent (Jiancheng, Nanjing, China) was added to each well and incubated at 37 °C for 4 h. Finally, a microplate reader was used to measure the absorbance at 450 nm.
The media of confluent cultures was replaced with DMEM plus 50% horse serum. After 2 h shock, the cells were washed twice with PBS and incubated with serum-free DMEM containing 400 μM NaBr or NaCl. Cell samples were collected at 4-h intervals. Total RNA was extracted and processed for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis.
Total RNA from cells was isolated using Trizol reagent (Invitrogen, Carlsbad, California, USA), reverse transcribed with the PrimeScript RT reagent kit (Takara, Tokyo, Japan), and analyzed by real-time quantitative PCR using 2 × ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The Primers for rat GAPDH were included for normalization. A complete list of Primers was shown in Table S4 and synthesized by Generay Biotech Co., Ltd. (Shanghai, China).
Western blotting analysis
For protein analysis, cells were lysed in RIPA buffer. The protein concentration was quantified with a BCA Protein Quantitation Assay Kit (Beyotime Biotech., Shanghai, China). Equal amounts of protein were loaded and separated by 10% SDS-PAGE and then transferred onto a PVDF membrane (Millipore Corp., Billerica, MA, USA). The membranes were incubated overnight with appropriate primary antibodies at 4 °C. Bound antibodies were then visualized using horseradish peroxidase-conjugated secondary antibodies. A quantitative analysis was performed by using ImageJ software (U.S. National Institutes of Health). For the antibody information, the antibodies against CRY1, HK2 and PKM2 were purchased from Proteintech (Chicago, IL, USA). Anti-RORα were obtained from Santa Cruz Biotechnology (CA, USA). The antibodies against LC3II/I, BMAL1, ULK1 and ATG5 were purchased from Bioworld Technology, Inc. (Nanjing, China). The antibody against β-ACTIN was derived from Servicbio Technology Co., Ltd. (Wuhan, China). The secondary antibodies were obtained from Santa Cruz Biotechnology (CA, USA). Uncropped images are shown in Fig. S5.
Groups of data were presented as the means ± standard deviation (SD). Data were analyzed by using one-way ANOVA followed by Fisher’s LSD post hoc test. Calculations were performed by using Origin 8 software (version 8.6, OriginLab, Northampton, MA, USA). A value of P < 0.05 was considered statistically significant. Circadian variations, including amplitude andvphase shift, were calculated by fitting a cosine-wave equation [y = baseline + (Amplitude × Cos (2 × π × (x-phaseshift)/24)] on clock gene expression, with a fixed 24-h period (detailed data for the oscillation of clock genes were presented in Supplementary Table 2, 3 and 4). Time series data were analyzed using one-way or two-way ANOVA followed by Bonferroni’s post hoc test. A p-value of less than 0.05 was considered to be statistically significant. Unless otherwise indicated, the statistics was performed using Student’s t-test when only two groups were compared.