Heterozygous SOD2 deletion impairs SERCA function in soleus and heart muscles from female mice

Abstract

Background Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pumps actively transport intracellular Ca2+ back into the sarcoplasmic reticulum and are the primary facilitators of muscle relaxation, restoring intracellular [Ca2+] to resting levels. Like many proteins, SERCA pumps are subject to oxidation which may affect their functional capacity. Superoxide dismutase 2 (SOD2) is a critical antioxidant enzyme localised in the mitochondrial matrix which converts superoxide radicals into less reactive H2O2. The present study sought to explore the structural and functional consequences of halved SOD2 expression, particularly to SERCA isoforms, in skeletal and cardiac muscle. Specifically, we examined whether heterozygous SOD2 deletion would result in changes in levels of SERCA tyrosine nitration and S-glutathionylation. Tyrosine nitration is known to impair SERCA function, particularly SERCA2a (slow isoform) but not SERCA1a (fast isoform). Conversely, glutathionylation of SERCA may act as an adaptive modality to preserve function during moderate oxidative stress. Methods We obtained soleus, extensor digitorum longus (EDL), and left ventricle (LV) muscles from wild-type (WT) and SOD2 heterozygote (SOD2+/-) C57BL/6J female mice (n=8 per genotype, aged 6-7 months). Ca2+-dependent SERCA activity assays were performed along with immunoprecipitation experiments to examine SERCA2a and SERCA1a tyrosine nitration and S-glutathionylation. Results Heterozygous SOD2 deletion did not alter SERCA1a or SERCA2a expression in the soleus, EDL or LV compared with WT. However, relative to WT, SOD2+/- soleus muscles showed significant impairments in SERCA function, with a reduction in SERCA's affinity for Ca2+. This corresponded well with Western blot data showing significantly elevated SERCA2a tyrosine nitration and significantly reduced S-glutathionylation in the soleus. In contrast, we observed no change in SERCA function, SERCA1a tyrosine nitration, or S-glutathionylation in the EDL. Furthermore, there were no differences in SERCA1a tyrosine nitration in the soleus, which is consistent with the notion that the SERCA1a isoform is less susceptible to nitrosative modification. Together, these results suggest that the slow SERCA isoform is more susceptible to heterozygous SOD2 deletion, and this is further supported by a significant reduction in SERCA's affinity for Ca2+ in the LV where SERCA2a is the predominant isoform. Conclusions This is the first study showing the physiological effects of halved SOD2 expression on muscle SERCA function. Our results reinforce the idea that SERCA2a is more sensitive to nitrosative modification.