There is a fundamental gap in understanding of the mechanisms that regulate cardiac sarcoplasmic reticulum (SR) calcium release through Ryanodine Receptor Type-2 (RyR2). A fuller understanding of the regulation of RyR2 is needed to better comprehend mechanisms of calcium dysregulation in cardiovascular pathology. Striated Muscle Preferentially Expressed Protein Kinase (SPEG), a binding partner of RyR2, is a protein with dual kinase domains that is downregulated in heart failure. Cardiac loss of SPEG results in decreased SR stores through increased diastolic calcium leak from RyR2. Although its kinase activity has not been extensively studied, there is evidence that the internal kinase domain, SPEG1, is catalytically active. As previous phosphorylation events have been shown to modify RyR2 activity, it is possible that SPEG acts as a regulator of RyR2 through its kinase activity. The overall objective of this application is to determine what role SPEG’s kinase activity has on SR calcium handling and more specifically whether SPEG’s kinase activity is important in the regulation of RyR2. Preliminary data generated in the applicant’s laboratory revealed a significant decrease in phosphorylation at the RyR2-S2367 site in a cardiac specific SPEG knock-out mouse. Phosphorylation of this site, located in the critical clamp region of RyR2, has not been reported in the literature. The central hypothesis is that phosphorylation of the RyR2-S2367 site results in decreased diastolic calcium leak through stabilization of RyR2. The rationale for this research is that an understanding of the effects of this novel phosphorylation site and the mechanism of this phosphorylation event has the potential to lead to novel approaches to modulate RyR2 activity. The hypothesis will be tested with the following specific aims 1) Determine whether SPEG primarily phosphorylates RyR2 at the S2367 site, 2) Test the hypothesis that phosphorylation at the S2367 site inhibits diastolic RyR2 opening, and 3) Determine whether SPEG1 kinase activity is therapeutic in heart failure. For the first aim, we will determine whether RyR2-S2367 is phosphorylated by SPEG using both an in vitro kinase assay and overexpression of the SPEG1 kinase domain with adeno-associated virus. Under aim 2, we will utilize recombinant RyR2 and knock-in mouse models with RyR2-S2367 phospho-mimetic and phospho-resistant mutations to study the effects of this phosphorylation event on calcium release using both single channel RyR2 studies and calcium imaging with isolated cardiomyocytes. For aim 3, we will determine whether overexpression of the SPEG1 kinase domain with adeno-associated virus improves ejection fraction and SR calcium handling in a non-ischemic mouse model of heart failure. This research is significant in that it will advance understanding of a novel RyR2 phosphorylation site and the physiological importance of SPEG’s kinase activity. This knowledge has the potential to aid in the identification...