Cell culture

Rat embryonic cardiomyocyte cell line H9c2 was obtained from ATCC (Rockville, USA) and cultured in DMEM containing 10 % FBS (Gibco, Carlsbad, CA, USA) at 37 °C and additional 5% CO2.

MTT assay

Cell viability was measured using the MTT assay kit (ab211091, Abcam, USA). In brief, approximately 5 × 104 cells were placed into each well of 96-well plates and cultured overnight. To evaluate the effect of CoCl2, cells were treated with CoCl2 at different concentrations (400, 500, 600, 800, 1000, and 1200 µM) for 24 or 48 h. To evaluate the effect of nesfatin-1, cells were treated with nesfatin-1 at different concentrations (10, 20, 40, 60, 80, 100, and 120 nM) after incubation with 800 µM CoCl2 for 48 h for another 24 h. Subsequently, 5 mg ml− 1 MTT reagent was added to each well, and the plates were incubated for 4 h. The absorption at 570 nm was detected with an ELISA plate reader.

TUNEL staining assay

The apoptotic cells were visualized using TUNEL Apoptosis Detection Kit (Yeasen, Shanghai, China). Approximately, 1 × 105 cells per well were seeded into 24-well plates and treated with 800 µM CoCl2 for 48 h before treated with nesfatin-1 at different concentrations (20, 60, and 80 nM) for another 24 h. Then cells were incubated with Alexa Fluor 488-12-dUTP Labeling Mix for 20 min, and the nuclei were counterstained by DAPI solution. The apoptotic cells were counted under a fluorescence microscope (Olympus, Tokyo, Japan) (× 40).

Annexin V apoptosis assay

Cell apoptosis was detected using the Annexin V-APC and 7-AAD Apoptosis Detection reagent (BD Biosciences) as previously described [21]. Briefly, 2 × 105 cells per well were plated into 6-well plates and treated with 800 µM CoCl2 for 48 h before treatment with nesfatin-1 at different concentrations (20, 60, and 80 nM) for another 24 h. Afterward, cells were washed and incubated Annexin V-conjugated APC and 7-AAD. Cell apoptosis was detected using a flow cytometer (Beckman, Pasadena, USA).

Western blot

Total proteins were extracted from the cultured cells using RIPA lysis buffer containing 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM Tris pH 7.5, and 150 mM NaCl (Beyotime Institute of Biotechnology, Shanghai, China). Approximately equal amounts of protein samples (25 µg) were separated by 12% SDS-PAGE and transferred onto PVDF membranes. After blocking in 5% nonfat milk, the membranes were incubated overnight at 4 °C with primary antibodies against p38 (ab31828, 1:1000, Abcam, USA), p-p38 (ab195049, 1:1000, Abcam, USA), ERK1/2 (ab184699, 1:1000, Abcam, USA), p-ERK1/2 (ab223500, 1:1000, Abcam, USA), JNK1/2 (#9255, 1:1000, Cell Signaling Technology, USA), p-JNK1/2 (#4668, 1:1000, Cell Signaling Technology, USA), Notch1 (#3608, 1:1000, Cell Signaling Technology, USA), Hes 1 (#11,988, 1:1000, Cell Signaling Technology, USA), Jagged 1 (#70109, 1:1000, Cell Signaling Technology, USA), Bax (ab32503, 1:1000, Abcam, USA), Bcl2 (ab32124, 1:1000, Abcam, USA), c-caspase-9 (ab2324, 1:1000, Abcam, USA), c-caspase-3 (ab32042, 1:1000, Abcam, USA) and β-actin (ab8226, 1:10,000, Abcam, USA). On the next day, the membranes were then incubated with HRP-conjugated secondary antibody for 2 h. The antibody-bound proteins were detected using an ECL chemiluminescence detection kit (Thermo Fisher Scientific, Waltham, MA, USA).

Measurement of intracellular ROS

The intracellular ROS in H9c2 cells after different treatments was detected using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe (Sigma Chemicals Co., USA) as previously reported [22]. Briefly, 5 × 105 cells per well were seeded into 24-well plates and treated with 800 µM CoCl2 for 48 h, followed by 20, 60, and 80 nM nesfatin-1 for 24 h. After addition of 25 µM DCF-DA solution, the plates were incubated for 30 min, and the fluorescence of DCF was detected using a microplate reader (Bio-Rad, USA).

Detection of LDH, MDA, SOD, GSH, and CAT

Approximately 2 × 105 cells per well were plated into 24-well plates and treated with 800 µM CoCl2 for 48 h, followed by 20, 60, and 80 nM nesfatin-1 for 24 h. The levels of released LDH, MDA, SOD, GSH, and CAT in the supernatant were detected using specific detection kits.

Detection of mitochondrial membrane potential (MMP)

The MMP was detected as previously described [23]. In brief, 5 × 105 cells per well were plated into 6-well plates and treated with 800 µM CoCl2 for 48 h, followed by 20, 60, and 80 nM nesfatin-1 for 24 h. The cells were incubated with 2.5 nM tetramethylrhodamine ethyl ester (Sigma-Aldrich, St Louis, MO, USA; Merck KGaA, Darmstadt, Germany) for 25 min. After cells were washed with PBS twice, the fluorescence at 490 nm (excitation)/590nm (emission) was detected using a flow cytometer (Beckman, Pasadena, USA).

Statistical analysis

The data were presented as mean  ±  SD (standard deviation). Data analysis was performed in SPSS ver. 22.0 (IBM Corp, Armonk, NY, USA). The difference between two groups was tested using Student’s t test . p  <  0.05 was considered significant.