Abstract Body: In the heart, Ca/Calmodulin Kinase IIδ (CaMKIIδ) functions to maintain electromechanical and calcium homeostasis. Post-translational modifications (PTMs) of CaMKIIδ render its activity autonomous of Ca/CaM. In hypertrophic cardiomyopathy (HCM), increased levels of autonomous CaMKIIδ are linked to disease progression. We have shown that the pathogenic role of CaMKIIδ in HCM is mutation-specific: autonomous CaMKIIδ is elevated in cardiac troponin T (cTnT)-R92W mice, but not in cTnT-R92L mice. These results pose a clinically relevant and critical question: what is the trigger for CaMKIIδ dysregulation in HCM, such that it is only found with certain mutations? We hypothesize that CaMKIIδ dysregulation results from distinct modifications in thin filament (TF) biophysical properties altered by point mutations. cTnT-R92W TFs display faster calcium dissociation kinetics compared to wild type TFs, whereas cTnT-R92L TFs have slower dissociation kinetics, thus we propose accelerated calcium dissociation kinetics as a mutation-specific trigger of CaMKIIδ dysregulation. To test this, we will use mass spectrometry following CaMKIIδ immunoprecipitation to assess the levels of phosphorylation at Thr-287 and oxidation at Met-281/282 in mice expressing the R94H, I79N, or R92W mutations in cTnT, which exhibit differing calcium dissociation kinetics. Preliminary data shows that CaMKIIδ PTMs can be readily detected. We will also use the CaMKIIδ biosensor CaMKAR in cardiomyocytes isolated from these mice to evaluate CaMKIIδ activity. We have shown that genetic inhibition of CaMKIIδ in cTnT-R92W mice improves diastolic function and atrial remodeling. We also aim to test the efficacy of the small molecule CaMKIIδ inhibitor ruxolitinib (rux) in cTnT-R92W mice. Two-week treatment of these mice with rux improves systolic and diastolic function, and has no effect in nontransgenic controls, implicating small molecule inhibition of CaMKIIδ as a potential therapeutic option in HCM. To continue these studies, we will explore changes in calcium handling in the three mouse models to further establish potential mechanistic pathways for CaMKIIδ dysregulation in HCM and will extend rux treatment to longer timepoints to further evaluate its efficacy as an HCM therapeutic.