Abstract 343: Glycogen Synthase Kinase 3β Localizes to the Cardiac Myofilament via Py216 and Dynamically Modulates Calcium Sensitivity

Abstract

Localization of kinases to sub-cellular compartments allows them dynamic control over specific subsets of their targets. We previously found GSK-3β could modulate myofilament Ca 2+ sensitivity. However, whether GSK-3β modulates Ca 2+ sensitivity in vivo , if it localizes to the myofilament, and the consequences, are unknown. In myofilament enriched LV tissue from human non-failing and heart failure (HF) patients (n = 66) we found GSK-3β does localize to the myofilament and is altered by sex, age, and HF. To determine its in vivo functional role, we used myocyte specific inducible GSK-3β knock-out mice for skinned myocyte force-calcium experiments and found that GSK-3β reduction reduced calcium sensitivity. Further, we measured function in human samples and found myofilament GSK-3β levels directly correlated to Ca 2+ sensitivity. To establish how GSK-3β binds to the myofilament, we performed co-IP and IHC with phosphorylated forms of GSK-3β. GSK-3β phosphorylated at Y216 had a high affinity for the myofilament and localized to the z-disc. Mutating Y216 to a phospho-mimetic increased binding to the myofilament while mutating it to a phospho-null ablated binding.To identify GSK-3β’s myofilament targets, we performed mass spectrometry on myofilament phospho-enriched samples from GSK-3β KO and WT mice. As GSK-3β modulated Ca 2+ sensitivity, we expected to detect thin filament proteins. However, in agreement with its localization to the z-disc, GSK-3β primarily phosphorylated z-disc proteins.In the GSK-3β KO mice, strain analysis revealed the posterior wall contracted significantly earlier than the anterior wall, indicating baseline mechanical dyssynchrony. The WT mice had synchronous contraction, and interestingly there was significantly higher myofilament GSK-3β in the anterior wall compared to the posterior wall, a difference we hypothesize maintains synchrony. Thus, losing this fine control over Ca 2+ sensitivity as in the GSK-3β KO mice would induce mechanical dyssynchrony. Overall, these findings reveal that GSK-3β dynamically localizes to the myofilament to modulate Ca 2+ sensitivity through Y216 phosphorylation. The consequence of this “fine tuning” maintains chamber level mechanical synchrony.