Ethical statement
This article does not describe any studies with human participants or animals performed by any of the authors.
Cell culture
The H9C2 cardiomyocytes were grown in Dulbecco’s modified essential medium (DMEM; high glucose; Gibco-Invitrogen, Carlsbad, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco-Invitrogen), 25 mmol/L HEPES (pH 7.4), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37 °C in a humidified atmosphere of 95% air and 5% CO2.
MTT assay
The effect of MC-LR (Taiwan Algal Science, Taiwan) on H9C2 cell viability was analyzed by using an MTT assay. Cells were seeded in 96-well plates (1 × 104 cells per well). MC-LR (0.1–100 μm) or vehicle (0.0001–0.1% DMSO) was added to cells and incubated for 24 h. After that, MTT (0.2 mg/ml) was added to each well and incubated for 4 h. The supernatant was removed and the formazan crystals were dissolved in DMSO. Cell viability was assessed by measuring the absorbance at 550 nm using a microplate reader.
Serum shock
To assess the rhythmic gene expression in H9C2 cells, serum shock experiments were performed (Balsalobre et al. 1998). In brief, medium was replaced with DMEM plus 50% horse serum (t = 0). After 2 h, H9C2 cells were washed once with PBS buffer and incubated with serum-free DMEM with or without 10 μM MC-LR. Cells were collected at the indicated time-points and subjected to reverse transcription-quantitative PCR (RT-qPCR) analysis.
RT-qPCR
Total RNA from H9C2 cells was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). 1 μg of total RNA was reverse-transcribed into complementary DNA. A primer for rat 18 s rRNA was included for normalization. mRNA levels were quantified by real-time RT-PCR using SYBR premix Ex Taq (Takara, Japan). Samples were amplified using the Mastercycler ep realplex2 system (Eppendorf, Hamburg, Germany). Primer sequences are available upon request. The fold change value was calculated by applying the following equation: fold change = 2-(ΔΔCt).
Statistical analysis
Groups of data are presented as mean ± standard error. Data were analyzed using two-way ANOVA followed by Fisher’s LSD post-hoc test. Calculations were performed using the statistical package SPSS for Windows version 12.5S (SPSS, Chicago, USA). A value of P < 0.05 was considered statistically significant.