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.

The structure of the left ventricle is spatially and temporally heterogeneous. The heart’s electrical system helps synchronize these events, but mechanical events play an important role as well. However, experimental evidence of the regional mechanical processes that underpin a “normal contraction” remain largely unknown. Despite the differences in deformation between the anterior and posterior walls, sarcomere lengths during filling and ejection are the same in these regions. One explanation for this disconnect is the myocyte’s force production is different despite similar sarcomere lengths, a relationship known as Length Dependent Activation (LDA). Indeed, a modeling study suggested LDA is a critical regulator of mechanical synchrony in the left ventricle. We recently found the serine/threonine kinase GSK-3β localizes to the z-disc and can modulate LDA, and thus hypothesized it may play a role in maintaining normal cardiac synchrony. We used inducible cardiomyocyte specific GSK-3β KO mice and GSK-3βfl/fl/Cre- littermate Con treated with tamoxifen in which GSK-3β was KO for three weeks. We assessed regional differences in GSK-3β protein levels and found that Con mice had substantial regional heterogeneity: 50% more cytosolic GSK-3β in the posterior wall compared to the anterior wall; however, the myofilament-bound GSK-3β was the opposite, with 50% less GSK-3β in the posterior wall. This regional heterogeneity was lost in KO mice, in which protein levels were homogenously low. There were no differences detected by m-mode echo between Control and KO mice. However, strain analysis revealed a significant interaction (p=0.037) between genotype and region (Anterior vs. Posterior), in which regional differences in Systolic Strain Rate were ablated in the knockout. These data suggest that the regional differences in GSK-3β sarcomeric levels were important for maintaining regional functional heterogeneity. We further observed that sarcomeric GSK-3β levels (but not cytosolic) decreased in a mouse myocardial infarction model, which these findings suggest could worsen mechanical dyssynchrony post-infarct. Perhaps a possible therapy for mechanical dyssynchrony could involve regional restoration of sarcomeric GSK-3β levels to restore synchrony.