Endogenous HAX-1 Regulates SERCA Activity and Oxidation Dependent Stability

HS-associated protein X-1 (HAX-1) has been identified as a novel binding partner of both SERCA2a and phospholamban (PLN) in the heart. We observed that the expression levels of HAX-1 are decreased in human failing hearts and experimental ischemia/reperfusion injury. To elucidate the impact of such reduction in endogenous HAX-1, a mouse model with inducible cardiac-specific ablation of HAX-1 was generated. Ablation of HAX-1 resulted in a hypercontractile phenotype in whole hearts and isolated cardiomyocytes. The underlying mechanisms included enhanced Ca-transient kinetics and SR Ca uptake affinity through relief of the PLN inhibitory effects. Despite the increases in Ca-cycling, HAX-1 ablation diminished contractile recovery and increased infarct size and apoptosis in hearts subjected to ex vivo ischemia/reperfusion. This was associated with enhanced degradation of SERCA2a by the calcium sensitive protease, calpain. Modulation of SERCA2a degradation by HAX-1 was also observed in isolated cardiomyocytes and SR microsomes. The ROS scavenger thioredoxin1 in cardiomyocytes and reducing conditions in SR microsomes attenuated degradation of SERCA2a and abolished the effects of HAX-1 deficiency, suggesting the involvement of a redox mechanism. Indeed, SERCA2a oxidation was assessed by two complementary free cysteine labelling methods and was found to be uniquely increased in HAX-1 deficient hearts. The SERCA2a inhibitor thapsigargin, which stabilizes a specific conformation of the enzyme, eliminated the HAX-1 variations in free cysteine labelling of SERCA2a. This suggests that HAX-1 alters the conformation of SERCA2a thereby changing the reactivity/availability of specific cysteines to oxidation. Additionally, HAX-1 levels inversely correlated to ROS production at the ER/SR compartment, quantified by genetically encoded HyPer indicators. These findings indicate that endogenous HAX-1 prevents SERCA2a cysteine oxidation, by a change in structure and/or a change in ROS, resulting in decreased susceptibility of SERCA2a to degradation. Thus, restoring the decreased HAX-1 levels in the stressed heart may hold therapeutic potential.