Another-regulin regulates cardiomyocyte calcium handling via integration of neuroendocrine signaling with SERCA2a activity

Calcium (Ca²⁺) dysregulation is a hallmark feature of cardiovascular disease. Intracellular Ca²⁺ regulation is essential for proper heart function and is controlled by the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA2a). Another-regulin (ALN) is a newly discovered cardiomyocyte-expressed SERCA2a inhibitor, suggesting cardiomyocyte Ca²⁺-handling is more complex than previously appreciated. To study the role of ALN in cardiomyocytes, we generated ALN null mice (knockout, KO) and found that cardiomyocytes from these animals displayed enhanced Ca²⁺ cycling and contractility compared to wildtype (WT) mice, indicating enhanced SERCA2a activity. In vitro and in vivo studies show that ALN is post-translationally modified via phosphorylation on Serine 19 (S19), suggesting this contributes to its ability to regulate SERCA2a. Immunoprecipitation and FRET analysis of ALN-WT, phospho-deficient ALN (S19A), or phosphomimetic ALN (S19D) revealed that S19 phosphorylation alters the SERCA2a-ALN interaction, leading to relief of its inhibitory effects. Adeno-associated virus mediated delivery of ALN-WT or phospho-mutant ALN-S19A/D in ALN KO mice showed that cardiomyocyte-specific expression of phospho-deficient ALN-S19A resulted in increased SERCA2a inhibition characterized by reduced rates of cytoplasmic Ca²⁺ clearance compared to ALN-WT and ALN-S19D expressing cells, further supporting a role for this phosphorylation event in controlling SERCA2a-regulation by ALN. Levels of ALN phosphorylation were markedly increased in cardiomyocytes in response to Gα_q agonists (angiotensin II, endothelin-1, phenylephrine) and Gα_q-mediated phosphorylation of ALN translated to increased Ca²⁺ cycling in cardiomyocytes from WT but not ALN KO mice. Collectively, these results indicate that ALN uniquely regulates Ca²⁺ handling in cardiomyocytes via integration of neuroendocrine signaling with SERCA2a activity.