Abstract Body: Introduction: Sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase 2a (SERCA2a) is a calcium channel responsible for the ATP-dependent re-uptake of calcium into SR/ER that facilitates the diastolic phase of the cardiac cycle. Several posttranslational modifications (PTMs) of SERCA2a, including cysteine oxidative PTMs, are linked to its downregulation/inactivation during heart failure. Therefore, understanding the molecular mechanisms underlying the cysteine oxidative PTMs that regulate SERCA2a levels and function is critical. In this regard, Thioredoxin 1 (Trx1) is a key antioxidant protein that is crucial for reduction of cysteine oxidative PTMs and helps in maintaining cellular redox balance. Trx1 can also transnitrosylate various protein cysteine residues to prevent further oxidation and protect against cellular damage. Our study explores Trx1's role in regulating SERCA2a's stability and activity through redox mechanisms and transnitrosylation.
Methods and Results: Co-immunoprecipitation using a Trx1-C35S trap mutant (the C35S mutation causes Trx1 to form a disulfide linkage to its target substrates) revealed that Trx1 binds to SERCA2a, with this interaction intensifying under oxidative stress (5-fold, p<0.05). Absence of Trx1 led to a decrease in SERCA2a protein levels in vitro (0.5-fold, p<0.001) and in a Trx1 knockout mouse model (0.3-fold, p<0.05), without affecting mRNA levels. N-ethyl maleimide (NEM) labeling and mass spectrometry showed greater oxidation of SERCA2a at cysteine 364 (C364) (10-fold lower NEM labeling) in the absence of Trx1. This caused proteasomal degradation of SERCA2a through the endoplasmic reticulum-associated degradation (ERAD) pathway, involving an E3 ubiquitin ligase, autocrine motility factor receptor (AMFR). SERCA2a C364S mutation (non-oxidizable) stabilized SERCA2a (1.5-fold vs. wild type (WT), p<0.05) in vitro, but C364S knock-in (KI) lowered cardiac ejection fraction in mice (55.56% in WT to 49.06% in C364S KI, p<0.05). After 3 weeks of pressure overload stress, oxidation and SERCA2a degradation were reduced in C364S KI mice, but these mice exhibited worse cardiac fibrosis, hypertrophy, and calcium overload than WT. Further studies showed that C364 undergoes Trx1-dependent S-nitrosylation.
Conclusion: Trx1-dependent reduction and nitrosylation of C364 is vital for SERCA2a’s function and stability under basal and oxidative stress conditions.