Determining the regulatory role of microprotein ALN on SERCA2a and heart contractility

Project Summary/Abstract Cardiovascular disease is the leading cause of morbidity and mortality worldwide. Cardiomyocyte calcium (Ca2+) dysregulation is a central feature of disease and is largely driven by defects in the activity of a critical Ca2+ pump called the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a). SERCA2a itself is dynamically regulated by

the small protein phospholamban (PLN), which inhibits SERCA2a via direct binding and reducing its Ca2+ affinity. Heart disease is associated with SERCA2a/PLN dysfunction, which further worsens Ca2+ disequilibrium and disease progression. However, our knowledge of Ca2+ regulation in the heart remains incomplete, therefore few

advances have been made to restore cardiomyocyte Ca2+ homeostasis in disease conditions. Our lab recently discovered the microprotein another-regulin (ALN) as a SERCA2a-inhibiting microprotein that is enriched in cardiomyocytes. My preliminary data indicates that the activity of ALN may be controlled by

phosphorylation on position Serine 19, whereby in vitro co-immunoprecipitation experiments in HEK293 cells show that this phosphorylation event results in the dissociation of ALN from SERCA2a. Studies in isolated adult mouse cardiomyocytes stimulated with various G-protein-coupled receptor agonists indicate that ALN is

phosphorylated downstream of Gq activation, thus presenting ALN as a novel regulator of Ca2+ in the heart. This preliminary evidence has led to our central hypothesis that ALN uniquely integrates neuroendocrine signaling in cardiomyocytes via SERCA2a regulation of Ca2+ signaling and heart contractility. This

proposal will test this hypothesis by determining how ALN phosphorylation affects ALN-SERCA2a binding affinity and SERCA2a enzymatic activity (Aim 1). This proposal also explores neuroendocrine and Gq pathway activation upstream of ALN phosphorylation and its resulting regulation of SERCA2a activity (Aim 2).

The completion of this proposal will lead to advances in our knowledge of cardiomyocyte Ca2+ handling and Ca2+ dysregulation that occurs during disease. Such findings may lead to the development of novel therapeutics aimed at restoring Ca2+ homeostasis, an area which is currently lacking in cardiovascular care. The Aims herein expect

to show that phosphorylation of ALN on Serine 19 downstream of Gq signaling causes its dissociation from SERCA2a and relief of its inhibitory effects, thus resulting in increased cardiomyocyte Ca2+ cycling and contractility. The trainee leading this proposed project is currently a third-year graduate MD/PhD student at

Cincinnati Children’s Hospital Medical Center in the laboratory of Dr. Cat Makarewich. The candidate will use this proposal to gain meaningful experience in conducting cardiovascular research, leading to a strong base of knowledge for the treatment and prevention of chronic cardiovascular disease.