Cardioprotective Effects of Dantrolene and Thymoquinone in H9c2 Cells: Does the Phenotype Matter?

 

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Objectives: Ischemia reperfusion (IR) injury is characterized by inflammation and oxidative damage. Possible strategies to reduce IR injury are for example preconditioning with hypoxia or volatile anesthetics or treatment with calcium inhibitors like dantrolene or antioxidants. Dantrolene inhibits Ca²⁺ release from the sarcoplasmic reticulum by antagonizing ryanodine receptors (RyR). A promising antioxidant is thymoquinone (TQ) which is the main active component of N. sativa seed oil. Both substances have already been studied in IR-Langendorff models but extensive in vitro studies to unravel the underlying mechanism are still missing. Therefore we investigated the cardioprotective effects of dantrolene and TQ in differentiated and undifferentiated H9c2 cells in an in vitro ischemia reperfusion model.

Methods: H9c2 cells were cultured to 90% confluence and were then either differentiated with 100 nM retinoic acid (RA) for 7 days in 1% FCS containing medium or left untreated. Differentiated and undifferentiated H9c2 cells were incubated with different concentrations of dantrolene and TQ for 3 hour before stressing the cells. To mimic oxidative stress, cells were subjected to different concentrations of H₂O₂, depending on the differentiation status. The cell viability was evaluated by performing a MTT assay.

Results: Differentiated H9c2 cells were more sensitive to H₂O₂ treatment than undifferentiated cells. The optimal concentrations of H₂O₂ to induce ~50–60% cell death were 850 µM for undifferentiated and 650 µM for RA-differentiated H9c2 cells. Dantrolene as well as TQ were able to reduce the detrimental effect of H₂O₂ in both differentiated and undifferentiated cells. Treatment with 5 µM dantrolene 3 hour prior to H₂O₂ treatment increased cell viability by 20% compared with the control group in both phenotypes. Interestingly, the protective effect of 5 µM TQ was only present in differentiated cells and resulted in ~15% increased cell viability compared with control. In undifferentiated H9c2 cells, no protective effect of TQ was observed.

Conclusion: Differentiated H9c2 cells were more sensitive to oxidative stress than undifferentiated cells. Treatment with dantrolene reduced the H₂O₂-induced cell death independently of the phenotype whereas TQ was only protective in differentiated cells. This indicates that protection by the antioxidant properties of TQ is strictly dependent on a cardiac phenotype while Ca²⁺ inhibition is not.