The co-chaperone Bcl2-associated athanogene-3 (BAG3) maintains cellular protein quality control through the regulation of heat shock protein 70 (HSP70). Cancer cells manipulate BAG3-HSP70-regulated pathways for tumor initiation and proliferation, which has led to the development of promising small molecule therapies, such as JG-98, which inhibit the BAG3-HSP70 interaction and mitigate tumor growth. However, it is not known how these broad therapies impact cardiomyocytes, where the BAG3-HSP70 complex is a key regulator of protein turnover and contractility. Here, we show that JG-98 exposure is toxic in neonatal rat ventricular myocytes (NRVMs). Using immunofluorescence microscopy to assess cell death, we found that apoptosis increased in NRVMs treated with JG-98 doses as low as 10 nM. JG-98 treatment also reduced autophagy flux and altered expression of BAG3 and several binding partners involved in BAG3-dependent autophagy, including SYNPO2 and HSPB8. We next assessed protein half-life with disruption of the BAG3-HSP70 complex by treating with JG-98 in the presence of cycloheximide and found BAG3, HSPB5, and HSPB8 half-lives were reduced, indicating that complex formation with HSP70 is important for their stability. Next, we assessed sarcomere structure using super-resolution microscopy and found that disrupting the interaction with HSP70 leads to sarcomere structural disintegration. To determine whether the effects of JG-98 could be mitigated by pharmacological autophagy induction, we cotreated NRVMs with rapamycin, which partially reduced the extent of apoptosis and sarcomere disarray. Finally, we investigated whether the effects of JG-98 extended to skeletal myocytes using C2C12 myotubes and found again increased apoptosis and reduced autophagic flux. Together, our data suggest that nonspecific targeting of the BAG3-HSP70 complex to treat cancer may be detrimental for cardiac and skeletal myocytes.

Background: Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3β (glycogen synthase kinase 3β) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3β’s in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. Methods: Inducible cardiomyocyte-specific GSK-3β knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. Results: Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3β. Agreeing with the localization of its targets, GSK-3β that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3β in neonatal rat ventricular cardiomyocytes. One of GSK-3β’s z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3β. Last, GSK-3β myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. Conclusions: We identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in patients with heart failure, indicating z-disc localized GSK-3β is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

No 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.

No abstract