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Propofol mediates risk-safe cross-talk in h9c2 myofibroblasts Shravah, Jayant


Intro: Propofol, an IV anesthetic that activates cardioprotective signaling mechanisms against I/R injury. It activates the PI3K/Akt branch of the RISK pathway to increase Bcl-2 and, therefore the anti-apoptotic potential of cardiomyocytes. SAFE pathway is another branch of cardioprotective signaling. Dual activation of the RISK and SAFE pathways may induce crosstalk. NFκB pathway is also known to be involved in TNFα induced cardioprotection. It is unknown if the SAFE pathway is activated under propofol stimulation and if propofol stimulation activates cross-talk with the RISK pathway. The exact mechanism for increased Bcl- 2 under propofol stimulation is also unknown. NFκB pathway may also be activated in response to propofol stimulation. Methodology: H9c2 cells were serum-starved for 48 hours for all experiments. The effects of propofol on the SAFE pathway, namely Stat3 activation were investigated in a time-course experiment. Inhibitors, specific to the RISK (wortmannin and API-2) and SAFE (AG490 and stattic) pathways were used to identify mechanisms of cross-talk. Cells were inhibited for 30 minutes followed by 10 or 30 minutes (for phosphorylation studies) or 24 hours (for inhibitor Bcl-2 studies) stimulation with propofol. DMSO was used as a vehicle control. RNAi was used to knockdown Stat3. Effect of propofol on IκBα degradation was investigated in a time-course experiment with TNFα as the positive control. Western blot analysis was used to gauge protein phosphorylation and levels. Immunofluorescence was used to observe nuclear localization of NFκB in response to propofol stimulation. Results: Propofol activated the SAFE pathway through increased phosphorylation of Stat3 at both Tyr705 and Ser727 residues. Propofol also activated cross-talk between the RISK and SAFE pathways. Propofol modulation showed a trend towards increased Bcl-2. Formation of Bcl-2 was blocked by PI3K, Jak2, and Stat3 inhibition. Inhibition was relieved after addition of propofol. Stat3 knockdown was counteracted by an increase in NFκB (p65). Propofol increased degradation of IκBα but did not increase NFκB nuclear localization, which instead, showed perinuclear accumulation. Conclusion: Propofol activates the SAFE pathway and stimulates its cross-talk with the RISK pathway. Propofol modulation of Bcl-2 utilizes RISK and SAFE pathways. Propofol increases IκBα degradation but not NFκB nuclear localization.

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