Malondialdehyde and 4-hydroxynonenal adducts are not formed on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) in diabetes

Abstract

Recently, we reported an elevated level of glucose-derived carbonyl adducts on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) in hearts of streptozotocin(STZ)-induced diabetic rats. We also showed these adduct impaired RyR2 and SERCA2 activities, and altered evoked Ca2+ transients. What is less clear is if lipid-derived malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) also chemically react with and impair RyR2 and SERCA2 activities in diabetes. This study used Western blot assays with adduct-specific antibodies and confocal microscopy to assess levels of MDA, 4-HNE, Nε-carboxy(methyl)lysine (CML), pentosidine and pyrraline adducts on RyR2 and SERCA2 and evoked intracellular transient Ca2+ kinetics in myocytes from control, diabetic and treated-diabetic rats. MDA and 4-HNE adducts were not detected on RyR2 and SERCA2 from control or 8 weeks diabetic rats with altered evoked Ca2+ transients. However, CML, pentosidine, and pyrraline adducts were elevated 3–5 fold (p<0.05). Treating diabetic rats with pyridoxamine (a scavenger of reactive carbonyl species, RCS) or aminoguanidine (a mixed reactive oxygen species-reactive carbonyl species scavenger) reduced CML, pentosidine and pyrraline adducts on RyR2 and SERCA2 and blunted SR Ca2+ cycling changes. Treating diabetic rats with the superoxide dismutase mimetic tempol had no impact on MDA and 4-HNE adducts on RyR2 and SERCA2, and on SR Ca2+ cycling. From these data we conclude that lipid-derived MDA and 4-HNE adducts are not formed on RyR2 and SERCA2 in this model of diabetes, and are therefore unlikely to be directly contributing to the SR Ca2+ dysregulation.

Albendazole induces oxidative stress and DNA damage in the parasitic protozoan Giardia duodenalis

“…Membranes were washed and incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG (Thermo, USA). Chemiluminescence detection was performed with the Amersham ECL detection kit according to manufacturer’s instructions ( Hofštetrová et al, 2010 ; Moore et al, 2013 ).…”

Calcium Signaling in the Ventricular Myocardium of the Goto-Kakizaki Type 2 Diabetic Rat

“…This finding supports the idea that altered activity of sarcolemmal NCX during Ca 2+ efflux contributes to the decrease in Ca 2+ transient-observed GK myocytes. Previous experiments in ventricular myocytes from the streptozotocin-induced diabetic rats have reported reduced caffeine-evoked Ca 2+ transients [ 82 – 91 ], SERCA2 activity, and Ca 2+ uptake [ 83 , 88 , 92 – 94 ] and decreased SR Ca 2+ channel (ryanodine receptor) activity [ 87 , 95 ] suggesting decreased SR Ca 2+ content, Ca 2+ uptake, and Ca 2+ release mechanisms in ventricular myocytes from the streptozotocin-induced diabetic rat.…”

Reactive carbonyl species and their roles in sarcoplasmic reticulum Ca2+ cycling defect in the diabetic heart

“…The procedures used for these assays are detailed elsewhere [30, 43, 66]. We also began using adduct-specific antibodies to determine relative levels of MGO, GO, 4-HNE and MDA adducts on RyR2 and SERCA2a in hearts from control, diabetic and drug-treated diabetic animals [71], to gain insights into the role these RCS are playing in the pathophysiology of the disease.…”

“…The lack of immuno-reactive MDA and 4-HNE adducts were not due to the absence of RyR2 and SERCA2a proteins in our SR preparations, since these proteins were readily detected using RyR2 and SERCA2a antibodies (Figure7A and 7B, 5 th autoradiogram down). In a recent study, we showed that these antibodies detected MDA and 4-HNE adducts on proteins of M w between 30 kDa and 80 kDa in our SR preparation [71]. …”