Effects of 4-hydroxy-2-nonenal on SERCA pump structure and function in skeletal and cardiac muscle

Abstract

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps are approximately 100 kDa transmembranous proteins that catalyze the ATP-dependent transport of cytosolic Ca2+ [Ca2+]i into the sarcoplasmic reticulum (SR), thereby maintaining low concentrations of resting [Ca2+]i. Reactive free- and non-radical oxygen and nitrogen species regulate SERCA function. Additionally, free radicals can oxidize lipids producing bioactive lipid-peroxidation end-products which are capable of modifying membranous proteins, resulting in protein inactivation. Here, in order to characterize the effect of 4-HNE, a lipid-peroxidation end-product, on SERCA structure and function, mouse WG and LV tissues were treated with 4-HNE and subsequently assayed for maximal Ca2+-dependent SERCA activity and SERCA post-translational structural modifications. Ca2+-dependent, maximal SERCA activity assays demonstrate a dose-dependent functional impairment of the SERCA pumps following 4-HNE treatment; interestingly, western blotting revealed significant increases in 4-HNE – SERCA1a adduction. Moreover, blots illustrated that the SERCA inhibition by 4-HNE may be independent of nitrosative stress as there were no evident increases in total protein nitrotyrosine or SERCA specific nitrotyrosine formation with 4-HNE treatment. Additionally, there were no observed increases in total protein glutathionylation or SERCA specific glutathionylation with 4-HNE treatment. The impairments in SERCA function are likely due to increased SERCA aggregation as revealed by a significant reduction in monomeric SERCA in both isoforms, confirmed by results illustrating a significant increase in higher molecular weight SERCA aggregates. Immunoprecipitation of SERCA revealed reduced FITC binding to the SERCA1a isoform with no changes to the SERCA2a isoform, illustrating isoform specific structural changes following 4-HNE treatment. These results demonstrate that the reactive lipid-peroxidation end-product, 4-HNE, can directly impair SERCA function by potentially causing isoform specific structural changes.