Stimulated disruption of Cav‐1/eNOS association plays a vital role in adaptation of H9c2 cells against high glucose concentration via Akt /HSP90 /eNOS activation.

Abstract

Hyperglycemia is linked to reduced eNOS activity and Nitric Oxide (NO) production but the role of heat shock protein (HSP‐90)/Akt/Caveolin‐1 (Cav‐1) in this regulatory mechanism is unknown. In this study we examined the mechanism involved in the regulation of eNOS on high glucose (HG‐25mM) treatment in H9c2 (cardiac myoblast) cells. We examined the levels of Cav‐1, eNOS, p‐eNOS, p‐Akt and HSP‐90 in control (5mM) and HG treated cells at 24h, 48h and 72h in the sucrose gradient (5–40%) fractions to observe the status of these proteins in caveolar rich raft fractions and in the cytosol. We have observed increased expression of HSP‐90, p‐eNOS and p‐Akt at 24h and 48h of HG treatment. No significant alteration in Cav‐1 was observed at 24h of HG treatment however there was an increase in Cav‐1 levels at 48h. When the treatment was increased to 72h the HSP‐90, p‐eNOS, p‐Akt levels decreased while Cav‐1 levels increased and significant association of Cav‐1/eNOS was observed. These results were validated by immunoprecipitation assay at all time points to observe the association of Cav‐1/eNOS and HSP‐90/eNOS. Thus this study showed that earlier time points of HG treatment stimulate an adaptive mechanism by disruption Cav‐1/eNOS association and by phosphorylation of Akt and eNOS and increased expression of HSP‐90 which were reversely regulated on prolonged treatment resulting in reduced eNOS activation and NO production.