Tolerance of Immature Cardiomyoblasts toward Mitochondrial Complex III Inhibition Depends on NRF2 but Not the Unfolded Protein or Heat Shock Response

Objectives: Inhibition of electron transport along the mitochondrial respiratory chain leads to cellular stress characterized by ATP depletion, as well as excessive generation of reactive oxygen species. Adult cardiomyocytes are highly susceptible to oxidative stress and energy depletion and rapidly undergo cell death upon mitochondrial dysfunction. In contrast, embryonic cardiomyocytes in the mouse heart have recently been proposed to be resistant toward mitochondrial complex-III inhibition by orchestrating a variety of cell protective responses.

Methods: To functionally characterize the molecular mechanisms mediating such stress tolerance, we used H9c2 cells as an in vitro model for immature cardiomyoblasts and treated them with the mitochondrial complex-III inhibitor antimycin A (AMA). Metabolic activity, cell density, proliferation rates, cell death, as well as RNA, and protein expression were determined after 24 and 48 hours.

Result: H9c2 cells are indeed largely resistant to AMA bot not rotenone (complex-I inhibitor) or oligomycin (complex-V inhibitor) treatment, despite comparable induction of oxidative stress. In contrast, HL-1 cells, resembling a differentiated cardiomyocyte cell line, rapidly undergo cell-cycle arrest and death upon AMA treatment. In H9c2 cells, AMA induces various cellular stress mechanisms, including the mitochondrial unfolded protein response (UPR), integrated stress response (ISR), heat shock response (HSR), and antioxidative defense. The siRNA-mediated knock down of the key UPR transcription factors, ATF4 and CHOP, does not impair growth of H9c2 cells upon AMA treatment, neither does pharmacological inhibition of the UPR. Similarly, siRNA knock down of HSF1, the key transcription factor regulating the HSR, has no negative effect on cell growth under AMA conditions. In contrast, knock down of NRF2, an important transcriptional regulator of genes involved in detoxification of reactive oxygen species, reduces growth of H9c2 cells upon AMA treatment.

Conclusion: These data indicate that NRF2 mediated antioxidative defense mechanisms are essential for growth and survival of immature cardiomyoblasts during mitochondrial stress conditions, whereas UPR and HSR are dispensable. Considering reactivation of embryonic stress response signaling in adult cardiomyocytes as a potential cardioprotective strategy to prevent cell death of the latter, our data suggest that enforcing antioxidative mechanisms should be given high priority.

Publication History

Article published online:
21 February 2021