Heat shock protein 70 protects against peroxynitrite-induced inactivation of the cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a).

Peroxynitrite (ONOO-) is a reactive nitrogen species implicated in oxidative injury and impaired calcium handling. We examined whether ONOO- inactivates the cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and whether heat shock protein 70 (Hsp70) preserves SERCA2a function during ONOO- exposure. Human embryonic kidney (HEK-293) cells were co-transfected with SERCA2a and either empty pMT2 vector (S2a-pMT2) or Hsp70 (S2a-Hsp70). Cells were untreated or exposed to ten nominal 250 μM additions of ONOO-, delivered once per minute for 10 min. Hsp70 abundance was greater in S2a-Hsp70 cells, although the magnitude of this difference varied by treatment (interaction, p = 0.0015). SERCA2a abundance was unaffected by transfection condition or ONOO- exposure. Co-immunoprecipitation showed that Hsp70 and SERCA2a were present in the same protein complex under the conditions examined. ONOO- reduced maximal SERCA2a activity by approximately 20% in S2a-pMT2 cells (p = 0.03), whereas activity was preserved in S2a-Hsp70 cells. ONOO- also increased SERCA2a-associated reactive carbonyl content by approximately 80% in S2a-pMT2 cells (p = 0.01), with no increase in S2a-Hsp70 cells. SERCA2a 3-nitrotyrosine content and FITC-binding capacity did not differ among groups. These findings show that Hsp70 overexpression preserves SERCA2a activity during acute ONOO- exposure and that this protection is associated with reduced SERCA2a carbonyl accumulation.