Abstract Body: Introduction: Elevated oxidative stress linked with heart failure is a major cause for the downregulation of sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase 2a (SERCA2a). NADPH oxidase (NOX) complexes are a key source of reactive oxygen species (ROS) in cardiomyocytes. p22^phox, a transmembrane partner of NOX1-4, plays an essential role in mediating ROS production in multiple NOX complexes. We investigated the role of p22^phox in oxidative stress and SERCA2a levels during pressure overload (PO).
Question: Does loss of p22^phox alleviate heart failure through reduction of oxidative stress and stabilization of SERCA2a levels during PO?
Methods/approach: Cardiac-specific p22^phox knockout (p22^phox-cKO) mice were subjected to sham operation or transverse aortic constriction (TAC). The p22^phox interactome was analyzed by co-immunoprecipitation and mass spectrometry. Protein cysteine oxidation was probed by biotinylated-iodoacetamide (BIAM) labelling.
Results: The p22^phox-cKO mouse heart showed a 30% reduction in Dityrosine levels compared to WT after 4-week TAC (p<0.05) and 50% lower total tissue H₂O₂ levels compared to WT after 1-week TAC (p<0.01). Unexpectedly, p22^phox-cKO mice had higher mortality (+20%, p<0.05), lung congestion (2-fold, p<0.05), and fibrosis (>40%, p<0.01) and a lower (40%, p<0.01) left ventricle ejection fraction than WT after TAC. p22^phox directly interacted with SERCA2a. Lack of p22^phox led to oxidation of SERCA2a (50% reduction in BIAM labelling, p<0.01) at cysteine 498 and promoted proteasome-mediated degradation of SERCA2a. The relative SERCA2a ATPase activity was unaltered in both WT and p22^phox-cKO mice.
Furthermore, in the absence of p22^phox, SERCA2a interacted strongly with HMG-CoA reductase degradation protein 1 (Hrd1), an endoplasmic reticulum-associated degradation (ERAD)-specific E3 ubiquitin ligase. Knockdown of Hrd1 in the presence of p22^phox knockdown restored SERCA2a to 70% of WT levels (ns and p<0.5 vs p22^phox knockdown). SERCA2a C498S knock-in mice exhibited better cardiac function and stable SERCA2a protein levels after TAC compared to WT mice.
Conclusion: The loss of p22^phox exacerbated heart failure through increased SERCA2a cysteine 498 oxidation and degradation through ERAD.