Abstract 225: Ischemic Cardiomyopathy Perturbs GSK-3β Myofilament Localization and Reduces Function

In ischemic cardiomyopathy (ICM), regions with ischemic damage are weaker than surrounding tissue, leading to contractile heterogeneity (ischemia-induced dyssynchrony, IID) that worsens function and mortality. IID is distinct from conduction abnormality-induced dyssynchrony that is treated with Cardiac Resynchronization Therapy (CRT). Our previous work found CRT reactivates glycogen synthase kinase 3β (GSK-3β) and restores myofilament function. Pacing an infarct cannot strengthen it, so CRT is ineffective in IID. However, we hypothesized GSK-3β may modulate myofilament function in ICM and could be leveraged to treat IID. We measured GSK-3β in whole tissue and myofilament-enriched samples from human rejected donor (Control), ICM, and dilated cardiomyopathy (no IID, DCM) LV. GSK-3β was detected in all the myofilament samples, but there was a 71±12% reduction in ICM. In whole tissue, there was very little phospho-Y216 GSK-3β, however it was highly enriched in the myofilament. Immunofluorescence on adult human myocytes showed weak co-localization of total GSK-3β and α-actinin at the z-disc compared to a strong correlation with p-Y216 GSK-3β. Furthermore, co-IP of GSK-3β and the myofilament show total GSK-3β had low-affinity to myofilament proteins, while p-Y216 GSK-3β binds with a high affinity. This led us to hypothesize Y216 phosphorylation modulates GSK-3β binding to the myofilament. To remove the confounding effect of antibodies, we created adenoviral constructs of myc-tagged wild-type, Y216F (unphosporylatable) and Y216E (constitutively phosphorylated) GSK-3β and transfected them into rat neonatal ventricular myocytes (NRVMs). The Y216E construct alone associated with the myofilament in co-IP experiments. We then performed skinned myocyte functional studies on human Control and ICM LV. The ICM myocytes were desensitized to calcium compared to Control and this was restored with exogenous GSK-3β treatment, but had no effect on Control myocytes. While GSK-3β is a promiscuous kinase in the myocyte, we have identified a specific regulatory mechanism involving Y216 phosphorylation, a site with a largely unknown role, that could allow precise therapeutic intervention to improve contractile function in the ICM heart.