Abstract 616: Glycogen Synthase Kinase 3b Localizes to and Modulates Sarcomere Function in Health and Disease

Abstract

Ischemic damage to a region of myocardium renders it weaker than the surrounding tissue, leading to contractile heterogeneity. This heterogeneity causes abnormal stress and strain patterns, further reducing systolic function. We hypothesized glycogen synthase kinase 3β (GSK-3β) may be a therapeutic target for improving function in patients with ischemic damage, as we previously discovered its activity is reduced by abnormal mechanical stress and it can phosphorylate myofilament proteins. We measured GSK-3β levels in LV whole tissue lysate from patients with ischemic cardiomyopathy (ICM) and non-failing rejected donor hearts (NF), but detected no differences. However, we found GSK-3β present at the myofilament, and ICM patients had a ~70% reduction in this localized pool compared to NF controls. Furthermore, GSK-3β phosphorylated at Y216 (pY216), an understudied site, was highly enriched in the myofilament. We used immunofluorescence, co-immunoprecipitation, and adenoviral constructs of Y216 phospho-mimetic or -blocked, in human, mouse, or neonatal rat ventricular myocytes to confirm Y216 phosphorylation results in GSK-3β binding to the myofilament. pY residues can dock to SH2 domains. The only SH2-containing protein that associates with the myofilament is tensin-1 (discovered by us in the myofilament by mass spectrometry). Here we further found tensin-1 localizes to the cardiac z-disk. GSK-3β and tensin-1 co-IP together in myocytes and in HEK cells (in the absence of the myofilament), but this interaction is reduced if Y216 phosphorylation is blocked. To identify the functional consequences of this interaction, we utilized an inducible myocyte specific GSK-3β knock-out mouse. Skinned myocytes from GSK-3β KO mice had significantly depressed calcium sensitivity compared to WT mice. Furthermore, treating skinned myocytes isolated from ICM patients with GSK-3β significantly increased calcium sensitivity, but had no effect in NF cells. These findings identify a novel functional role for GSK-3β in the ischemic human heart. GSK-3β is a promiscuous kinase, but we have discovered a specific regulatory mechanism (Y216 phosphorylation) that could allow precise therapeutic intervention to improve contractile function in ICM patients.