Abstract P2070: The Microprotein Another-Regulin Regulates Serca2a In Response To Neurohormonal Signaling

Abstract

Objective: Calcium (Ca 2+ ) regulation is a key component of proper heart function and is largely controlled by the sarco/endoplsmic reticulum Ca 2+ ATPase 2a (SERCA2a). Ca 2+ dysregulation is a hallmark feature of heart failure, yet few advances have been made to reestablish Ca 2+ homeostasis during disease. Another-regulin (ALN) inhibits SERCA2a, though little is known about how ALN functions in heart physiology and disease. Here, we determine a mechanism by which ALN is regulated via phosphorylation at serine 19 (S19), resulting in dissociation from SERCA2a. Methods and Results: ALN phosphorylation at S19 was confirmed via Phos-tag gel migration assay and site directed mutagenesis (SDM), whereby serine to alanine (S19A) mutation abolished the migrational shift which was confirmed with a custom S19 phospho-specific antibody. Effects of S19 phosphorylation on ALN-SERCA2a affinity were investigated using co-immunoprecipitation (IP) and fluorescence resonance energy transfer (FRET) in HEK293 cells. In addition to phosphodeficient S19A, SDM was used to mutate S19 to phosphomimetic aspartate (S19D) and glutamate (S19E). IPs of SERCA2a and hemagglutinin (HA)-tagged ALN WT, S19A, S19D, or S19E revealed less interaction between SERCA2a and ALN-S19D/E, suggesting S19 phosphorylation causes ALN dissociation from SERCA2a. Acceptor sensitization and fluorescence lifetime measurements show that ALN-S19D decreases ALN/SERCA2a binding affinity. To investigate signaling upstream of ALN phosphorylation, adult mouse cardiomyocytes were stimulated with various agonists and levels of pALN measured. Phosphorylation was significantly induced with the neurohormonal stimuli endothelin-1 and phenylephrine compared to DMSO and the β-adrenergic stimulus isoproterenol, suggesting Gαq/PKC pathway involvement. In addition, pALN levels are elevated in whole heart lysates of mice subject to transverse aortic constriction which is a model of pathological hypertension. Conclusions: These results indicate that ALN is phosphorylated at S19 in response to neurohormonal stimuli, resulting in dissociation from SERCA2. Future directions will investigate the impact of ALN phosphorylation on SERCA2 activity and heart function.