GSK-3β Localizes to the Cardiac Z-Disc to Maintain Length Dependent Activation

Abstract

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.

Keywords: calcium; cardiac myocytes; connectin; mice; myofibrils.

Figure 1.. Genetic ablation of GSK-3β results in loss of length-dependent activation.

(A) Example western blots of GSK-3β in LV whole tissue lysis (top) and myofilament-enriched (bottom) samples from GSK-3β knockout (KO) and littermate control (CON) mice. (B) Quantification of GSK-3β in whole tissue (p=0.0022 n=6/group) and (C) myofilament-enriched (p=1.0 × 10⁻⁶, n-values: CON=10, KO = 9) samples. GSK-3β was normalized to either tubulin (whole tissue) or actin (myofilament) and further normalized to CON, shown as mean ± SEM. P-values were calculated by Mann-Whitney t-test for the whole tissue comparison. (D) Mean force as a function of calcium concentration and fitted curves for skinned myocytes from CON and (E) KO mice in which curves were measured at a sarcomere length (SL) of 1.9 μm and then 2.3 μm. Normalized curves are shown in the bottom right of each graph to emphasize shifts in calcium sensitivity. (F) Delta F_max and (G) EC₅₀ for CON and GSK-3β KO mice between SL 1.9 and 2.3 μm (p=0.0084 by unpaired T-test, n=12 myocytes from 4 CON and 3 KO mice).

Figure 2.. GSK-3β KO mice have increased titin compliance.

(A) Summary d10 (lattice spacing) collected by x-ray diffraction data from skinned papillary muscle fibers from CON (16 fibers from 6 animals) and GSK-3β KO mice (11 fibers from 4 animals). Fibers were measured at SL 2.1 and 2.4 μm. (effect of SL p=4.0 × 10⁻⁶ by repeated measures 2-way ANOVA with Sidak’s multiple pair-wise comparison test). (B) Passive tension measured in CON and GSK-3β KO mice at SLs from 1.6 to 2.6 μm. (p-values determined by unpaired parametric t-test, with the exception of data collected at 2.0, which was determined by Mann-Whitney T-test). Passive tension measured in (C) CON (n=3 mice/4–5 cells per mouse; p-values: 1.8=0.88^W, 2.0=0.022^W, 2.2=0.060^P, 2.4=0.011^W, 2.6=0.15^P) and (D) KO mice (n=3 mice/4–5 cells per mouse; p-values: 1.8=0.69^W, 2.0=0.080^W, 2.2=0.20^P, 2.4=0.043^P, 2.6=0.0073^P) at baseline and with GSK-3β treatment. P-values were determined by paired t-test, ^pParametric test, ^WNon-parametric Wilcoxon Test. (E) Representative images of fixed tissue from CON and KO mice in which the z-disc is designated with α-actinin (n=30 cells/animal from 3 animals/group). Scale bar = 10 um. (F) Summary data of resting SL (μm) (p=0.024 by Mann-Whitney t-test) shown as means ± SEM. (G) Representative Coomassie stained gel with quantification below of CON and GSK-3β KO mice in which titin isoforms N2BA, N2B, degraded titin (T2), and myosin are indicated by arrows (n= 5–6 mice/group). P-value was determined by Mann-Whitney-test.

Figure 3.. GSK-3β phosphorylates z-disc proteins.

(A) Volcano plot depicting phosphorylation sites in CON (n=4) and GSK-3β KO (n=5) mice identified by mass spectrometry. Cut-offs for p-value are displayed at p=0.05 and for a fold change of a 30% decrease. (B) Significantly downregulated and upregulated sites. Proteins shown to localize to the z-disc are color coded to their respective points on the volcano plot. (C) Representative immunofluorescence from CON and GSK-3β KO LV tissue in which samples were probed for the z-disc marker ⍺-actinin (red in merged image) and phosphorylated Serine/Threonine residues (green in merged image). A subset of CON tissue was pre-incubated with alkaline phosphatase to act as a positive control (Phosphatase). Scale bar = 5 μm (2 μm in enlarged images). Yellow arrows delineate z-discs. (D) Quantification of Z-disc area containing pSer/Thr staining (normalized to control). n=3 mice, 21–26 cells/group, 10 z-discs/cell. P-values were calculated via one-way ANOVA with Tukey’s multiple comparison test.

Figure 4.. GSK-3β associates with the myofilament which is mediated by Y216 phosphorylation.

(A) Representative immunofluorescence of human non failing (NF) ventricular myocytes. Total, pS9 and pY219 GSK-3β are depicted in green and counterstained with α-actinin (red). Scale bars = 10 μm. (B) Silver-stained gel depicting flow through (FT) and eluted immunoprecipitation (IP) for myofilament enriched NF sample co-immunoprecipitated with either total, pS9 or pY216 GSK-3β antibodies. Labels on the right correspond to hypothesized myofilament proteins and molecular weights. (C) Example western blot for cMyc for Con (un-transduced) neonatal rat ventricular cardiomyocytes (NRVMs) and those transduced with Myc-tagged WT GSK-3β (WT), Y216F GSK-3β, and Y216E GSK-3β. (D) Summary data from data in panel B, means ± SEM (n = 6/group). P-values were calculated via ordinary one-way ANOVA with Tukey’s multiple comparisons. (E) Representative silver-stained gel depicting co-immunoprecipitation of proteins pulled down (Myc co-IP) with a myc tag antibody in control, WT, Y216F, and Y216E groups. (F) Summary data of co-IP experiment, showing total lane intensity (mean ± SEM) (n = 4/group, statistics by non-parametric one-way ANOVA (Kruskal Wallis) with Dunn’s multiple comparisons). (G) Representative western blot from panel E, depicting total GSK-3β, pY216, and actin in myofilament enriched NRVMs.

Figure 5.. abLIM-1 localizes to the z-disc and modulates passive stiffness via titin.

(A) Graphic of abLIM-1 domains and phosphorylation sites. Sites depicted in black are unchanged in GSK-3β KO mice, and sites depicted in red were reduced. (B) Representative western blot of abLIM-1 in whole tissue lysis of CON and GSK-3β KO mice. (C) abLIM-1 normalized to total protein in CON and KO mice (n=5). Statistics were calculated via Mann-Whitney test (D) abLIM-1 (green) and α-actinin (pink) staining in CON and GSK-3β KO mouse LV. (E) Quantification (area under the curve) of Z1Z2 titin peptides non-specifically bound (IgG control) and pulled down by GST-tagged abLIM-1, at baseline (BL) and with GSK-3β pre-treatment (GSK) obtained by mass spectrometry. n values are as follows: IgG=5, BL=4, GSK-3β =5. P-values were calculated via one-way ANOVA and Tukey’s multiple comparison test (F) Representative western blot of abLIM-1 and total protein stain in scrambled and abLIM-1 EHTS (G) Quantification of abLIM-1/total protein, normalized to scrambled (n=5, p=0.032). P-value was calculated by Mann-Whitney test. (H) Cross-sectional area of scrambled and abLIM-1 EHTs. P-value was calculated via unpaired Mann-Whitney test (I) Twitch forces in EHTs treated with either scrambled (blue) or abLIM-1 (red) siRNA. Twitch forces were measured at 72 steps between a -10% (of total EHT length) slack to +10% stretch. Twitch forces are normalized to 0% stretch. (n values: Scrambled =6, abLIM-1= 7). p =0.0043 refers to the difference between groups at 10% stretch, calculated by unpaired t-test (J) Representative traces of force (μN) at 0% (light blue = scrambled, orange = abLIM-1) and 10% (dark blue = scrambled, red = abLIM-1) stretch in a scrambled and abLIM-1 siRNA treated EHT.

Figure 6.. Myofilament-localized GSK-3β and LDA is reduced in human heart failure

(A) Example blots and quantification of myofilament-enriched (n=17, p=0.0019 by unpaired t-test) and (B) Whole tissue preparation (n values: NF=15, HF=19) of LV from human NF and HF samples. (C) Mean force as a function of calcium concentration and fitted curves for skinned myocytes from the LV of NF and (D) HF patients from which measurements were taken at short (1.9 μm) and long (2.3 μm) SL. (E) F_max and (F) EC₅₀ depicted as mean± SEM (n = 9 myocytes from 3 patients). P-values were calculated from two-way repeated measures ANOVA with pair-wise comparisons.