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Protective effects of Hsp70 on the structure and function of SERCA2a expressed in HEK-293 cells during heat stress

Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada

Submitted 10 December 2008 ; accepted in final form 24 February 2009

Heat shock protein 70 (Hsp70) can physically interact with and prevent thermal inactivation of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) 1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. This study examined whether Hsp70 could physically interact with and prevent thermal inactivation of SERCA2a, the SERCA isoform expressed in heart. HEK-293 cells were cotransfected with cDNAs encoding human Hsp70 and rabbit SERCA2a (S2a/Hsp70). Cells cotransfected with SERCA2a cDNA and pMT2 (S2a/pMT2) were used as control. One-half of the cells was heat shocked at 40°C for 1 h (HS), and one-half was maintained at 37°C before harvesting the cells and isolating microsomes. Western blot analysis showed that Hsp70 and SERCA2a were colocalized in the microsomal fraction. The levels of Hsp70 were approximately fivefold higher (P < 0.05) in S2a/Hsp70 compared with S2a/pMT2 and approximately twofold higher (P < 0.05) following HS in all cells. Coimmunoprecipitation demonstrated that Hsp70 directly binds to SERCA2a. Following HS, maximal SERCA2a activity was reduced (52%, P < 0.05) in S2a/pMT2 but was increased (33%, P < 0.05) in S2a/Hsp70. Thermal inactivation of SERCA2a in S2a/pMT2 was associated with decreased (49%, P < 0.05) binding capacity for fluorescein isothiocyanate (FITC) and increased carbonyl (42%, P < 0.05) and nitrotyrosine (40%, P < 0.05) levels in SERCA2a. By contrast, the HS-induced increase in maximal SERCA2a activity observed in S2a/Hsp70 corresponded with no change (P > 0.05) in FITC-binding capacity and reductions in carbonyl (40%, P < 0.05) and nitrotyrosine (23%, P < 0.05) levels in SERCA2a compared with S2a/pMT2. These results show that Hsp70 forms a protective interaction with SERCA2a during HS actually reducing oxidation and nitrosylation of SERCA2a thus increasing its maximal activity.

coimmunoprecipitatoin; carbonyl; nitrotyrosine; nucleotide binding site

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