Abstract 11592: Glycogen Synthase Kinase 3β Localizes to the Z-Disc to Maintain Length Dependent Activation

Abstract

Glycogen Synthase Kinase 3β (GSK-3β) can modulate myofilament function in vitro . However, it’s in vivo role, mechanism, and translational relevance are unknown, which we investigated here using inducible cardiomyocyte specific GSK-3β KO mice. Compared to tamoxifen-treated GSK-3β fl/fl Cre- mice (Con), skinned myocytes from KO mice had reduced calcium sensitivity at long sarcomere lengths (SL = 2.2 μm), but there were no differences at short SL (~1.9 μm). Thus, myocytes from KO mice did not sensitize to calcium with stretch, a mechanism called length dependent activation (LDA) that underlies the organ-level Frank-Starling law. LDA has been attributed to (1) phosphorylation of myofilament proteins, (2) altered lattice-spacing, and (3) changes to titin’s elastic properties. Using mass spectrometry and small energy x-ray diffraction we ruled out the first two mechanisms, however we did find that GSK-3β KO myocytes had decreased passive tension – indicating the loss of LDA was due to loss of titin as a length sensor. Interestingly, immunofluorescence showed that GSK-3β localized to the z-disc when phosphorylated at Y216 and, via mass spectrometry, phosphorylated primarily z-disc proteins including several sites on the structural protein Ablim-1, which we showed also localizes to the z-disc. These data suggested that GSK-3β’s effect on titin is likely through altering its ability to anchor to the z-disc through targeting of these z-disc proteins. To provide further evidence that GSK-3β is specifically altering LDA, we genetically removed a downstream effecter of LDA, cardiac myosin binding protein-C (cMyBP-C). In vitro treatment with exogenous GSK-3β was able to increase calcium sensitivity at long SLs in both KO and WT mice but had no effect on cMyBPC KO mice or mice lacking the c-terminal domains of cMyBPC that are known to be important for LDA. Lastly, we found that human heart failure patients had less myofilament GSK-3β compared to non-failing patients, and that these same samples had a depressed LDA. This work has identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA and indicates that z-disc localized GSK-3β may be a possible therapeutic target to restore the Frank-Starling mechanism in heart failure patients.