Exploring Cardiac H9c2 Cell Response

Embryonic rat cardiac H9c2 (2-1) cells (ECACC, Salisbury, UK) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Aurogene, Rome, Italy), containing 5.5 mM d-glucose and supplemented with 10% heat-inactivated fetal bovine serum (FBS; AU-S181H Aurogene, Rome, Italy), 1% L-Glutamine (L-Glu; AU-X0550 Aurogene, Rome, Italy), and 1% penicillin/streptomycin solution (P/S; AU-L0022 Aurogene, Rome, Italy), at 37 °C under an atmosphere of 5% CO₂.
After reaching 80% confluence, H9c2 cells were trypsinized, seeded at a specific cell density for each assay, and then exposed to NG, high glucose (HG; 33 mM d-glucose), or NG + 27.5 mM mannitol (M; as an osmotic control) for 48 h [40 (link)]. Cells were then treated for 6 days [41 (link)] in NG or HG medium with the following substances:-

CHR 0.399 mg/mL (CHR), dissolved in NaCl;

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SBECD 7.3 m/m%, dissolved in NaCl;

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Binary system SBECD + 0.095 mg/mL CHR (SBECD + CHR), dissolved in NaCl;

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DMSO 2.5% as a vehicle of OTX008;

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OTX008 (0.75–1.25–2.50 µM);

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Binary system OTX008 (2.5–1.25–0.75 µM)-SBECD (OTX008-SBECD), dissolved in NaCl;

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Ternary system CHR (0.324 mg/mL)-OTX008 (2.5–1.25–0.75 µM)-SBECD (CHR-OTX008-SBECD), dissolved in NaCl.

Three independent experiments were conducted, each performed in triplicate (N = 9).