more selectively and avoid off-target effects. Traditional experimental approaches, however, may not detect dysregulation of small, localized pools of a kinase.

GSK-3β (Glycogen synthase kinase 3β) is a prolific cytosolic serine/threonine kinase. In the heart, GSK-3β acts as a negative regulator of hypertrophic signaling, preventing translocation of proteins to the nucleus that activate hypertrophic gene expression. [6][7][8][9] There is a lack of consensus on whether GSK-3β is protective or harmful in heart failure (HF), 6,[10][11][12] possibly because there exists a subcellular pool of GSK-3β with unexplored actions. Indeed, we previously found that GSK-3β activity was depressed in a dog model of HF concurrent with cardiac dyssynchrony, 13 which typically arises from conduction abnormalities and results in premature activation of one region of the ventricular wall. 13 The observed decrease in GSK-3β activity correlated with depressed myofilament calcium sensitivity, which could be rescued in vitro with exposure to exogenous GSK-3β. Furthermore, GSK-3β was uncoupled from Akt, its canonical upstream de-activator, suggesting an independently regulated pool of GSK-3β. 13 However, there is no direct evidence that GSK-3β localizes to the sarcomere or regulates sarcomere function in vivo.

Changes in myofilament calcium sensitivity are frequently observed in HF due to altered phosphorylation of thin filament proteins. 14 However, dependent on the specific HF cause, [15][16][17] comorbidities, 18 and treatments, 19 myofilament calcium desensitization and oversensitization are both observed. Unfortunately, both situations are detrimental, with desensitization resulting in hypocontractility and worsening of the weakened heart while oversensitization can cause arrhythmias and slowed relaxation. 15 In addition, calcium sensitivity is dynamically regulated in response to acute changes, such as stretch. Length-dependent activation (LDA) is the mechanism by which stretch increases calcium sensitivity in cardiomyocytes and underlies the organ level Frank-Starling Law 20,21 that allows the heart to respond to changes in blood volume on a beat-to-beat basis. Animal models of HF exhibit a depressed Frank-Starling response, 5,22 however, in humans this is less clear, as Frank-Starling/LDA has been found to be both diminished [23][24][25] and unaffected 26,27 by HF.

It is unknown whether GSK-3β regulates myofilament function in vivo, whether it does so through a localized pool at the myofilament, the mechanisms involved, and

What Is Known?

• GSK-3β (Glycogen synthase kinase 3β) can target myofilament proteins and alter calcium sensitivity in vitro. • Stretch increases myofilament calcium sensitivity through a process known as length-dependent activation that is not well understood but involves titin-based strain. • Length-dependent activation is reduced in heart failure.

• GSK-3β phosphorylates myofilament proteins at the z-disc and contributes to length-dependent activation in vivo. • GSK-3β's effect on length-dependent activation occurs through interaction of its myofilament target, abLIM-1 (actin-binding LIM protein 1), with the z-disc region of titin. • Loss of myofilament GSK-3β in human heart failure correlates with decreased length-dependent activation.

Length-dependent activation (LDA) refers to the process by which increased sarcomere length results in increased myofilament calcium sensitivity. While much work has been done to elucidate the mechanism by which LDA occurs, a clear picture remains elusive. Our in vivo work shows that the primarily cytosolic kinase GSK-3β localizes to the myofilament and alters LDA via titin-based strain. We provide evidence that GSK-3β's z-disc substrate, abLIM-1 is a negative regulator of LDA. Additionally, abLIM-1 is a novel binding partner to titin's z-disc bound Z1Z2 domains. In human heart failure myofilament GSK-3β is diminished in samples that also have reduced LDA. These findings suggest that LDA can be regulated by protein interactions at the z-disc, which could be beneficial for targeting the diminished LDA response that can occur in heart failure.

whether this occurs in humans. In this study, we directly address these questions using inducible cardiomyocytespecific GSK-3β knockout mice, engineered heart tissue (EHT), and human left ventricular (LV) tissue from nonfailing rejected donor hearts and patients with HF. Confirming our hypothesis, ablation of GSK-3β in vivo results in reduction in LDA and interestingly, passive tension, a property of titin which has been established as a facilitator of LDA. 28 Exogenous treatment of skinned myocytes with recombinant GSK-3β can increase calcium sensitivity at long sarcomere lengths (SLs) and increase passive tension. We also found that GSK-3β's z-disc phosphorylation target, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc region of titin (Z1Z2 domains) and modulates LDA. Importantly, in human HF GSK-3β mislocalizes away from the z-disc, which correlates with an absent LDA. GSK-3β localizes to the sarcomeric z-disc via its own phosphorylation at Y216, which reveals a novel mechanism for targeting this kinase to specific myofilament targets. Overall, this study uncovers a novel subcellular pool of GSK-3β that is critical for maintaining normal contractile function.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

LV tissue from control (n=4) and GSK-3β knockout (n=5) mice were enriched for the myofilament, digested in trypsin, desalted, and phospho-enriched using titanium oxide beads. Samples were analyzed by liquid chromatography mass spectrometry (LC-MS/MS) using Data-Independent Acquisition (Orbitrap Fusion Lumos Tribrid Mass Spectrometer). Data were processed using Data-Independent Acquisition-Umpire and then underwent Median-based normalization.

X-ray diffraction patterns were collected from freshly skinned mouse muscle strips using the small-angle instrument at BioCAT beamline 18ID at the Advanced Photon Source, Argonne National Laboratory. 32 SL was adjusted by laser diffraction using a 4-mW HeNe laser. The data were analyzed using data reduction programs belonging to the MuscleX software package developed at BioCAT, 33 as described previously. 34

EHTs were made by seeding induced pluripotent stem cell cardiomyocytes and human adult cardiac fibroblasts onto decellularized porcine ventricular scaffolds as previously reported. 35 Two weeks after seeding, EHTs were treated with scrambled or abLIM-1 siRNA for 4 hours. Seventy-two hours after treatment, passive and active mechanics of EHTs were measured as previously described. 36

Variables S1). Correlations between categorical variables were performed with linear regression. P<0.05 were considered statistically significant. A priori power analyses were performed based on data from published studies 13,37,38 and pilot experiments. Further details are provided in the Supplemental Material.

We used a cardiomyocyte-specific inducible GSK-3β knockout mouse to assess whether GSK-3β affects myofilament function in vivo. To evaluate knockdown efficiency, we prepared 2 sets of protein samples from the LV, those processed from whole tissue and those enriched for the myofilament (see Figure S1 for specificity of this enrichment). 39 We detected significant GSK-3β in the myofilament-enriched samples from GSK-3β fl/fl / Cre-tamoxifen-treated control mice (Figure 1A through 1C) suggesting there is a pool of GSK-3β which localizes to the myofilament. In the GSK-3β fl/fl /Cre+ tamoxifentreated knockout mice, there was an ≈70% reduction in GSK-3β in both the whole tissue and myofilamentenriched samples (Figure 1A through 1C). At 12 weeks of age (used for all studies), there were no in vivo functional or structural differences between control and knockout mice as assessed by echocardiography (Table S2), except a mild hypertrophy in the anterior wall, consistent with GSK-3β's role as an inhibitor of hypertrophy.

We previously found decreased GSK-3β activity in a dog model of ventricular mechanical dyssynchrony 13 that has out-of-phase stress-strain relationships. Thus, we hypothesized GSK-3β may be involved in a mechanotransduction pathway at the myofilament, one of the most critical being LDA. 40 Thus, we performed force-calcium measurements at a SL of 1.9 µm (short) then stretched the myocyte to SL=2.3 µm (long) in skinned myocytes isolated from LV myocardium from control and knockout mice (n=4-5 mice/group, 2-4 cells/mouse; Figure 1D and1E). Stretching control myocytes increased both F max and calcium sensitivity (P=6.4×10 -4 , P=4.97×10 -4 , respectively, via 2-way repeated measures ANOVA, Figure 1F and 1G, Table S3), the expected effect of LDA. Conversely, myocytes from GSK-3β knockout mice exhibited no significant change in either F max or EC 50 with stretch. Additionally, there was no statistical difference in calcium sensitivity between the 2 groups at the short SL (P=0.33). Thus, GSK-3β is critical for the ability of the myofilament to adequately respond to stretch, and its genetic removal ablates LDA.

To ensure this was a direct effect of GSK-3β, we next tested whether exogenous GSK-3β would similarly enhance calcium sensitivity at long SLs while having no impact at short SLs. To avoid potential rundown from 4 subsequent activations, we performed paired pre-GSK-3β and post-GSK-3β treatments separately at short and long SLs in control and knockout mice. Exogenous GSK-3β treatment (0.1 µg for 15 minutes) had no effect in myocytes at a short SL (n=8 cells from 3 mice/group), but increased calcium sensitivity when the myocytes were stretched to a long SL (n=11 myocytes from 4 mice; P=0.037 by paired t test; Figure S2). Interestingly, when analyzed by 2-way ANOVA, there was no interaction between genotype and treatment, hence the data were analyzed by a paired t test. This result indicates exogenous GSK-3β can enhance LDA in both control and knockout myocytes and suggests the functional impact of GSK-3β is not saturated in normal hearts.

We next sought to explore the mechanism of GSK-3β's impact on LDA. We first showed by Western blot that phosphorylation sites on cardiac troponin I (S23/24) and cMyBP-C (S273, S282, and S302) that can impact LDA 41 are unchanged in the GSK-3β knockout mice (Figure S3). There is evidence LDA is impacted by changes in interfilament spacing, which describes the distance between thick and thin filaments. When the sarcomere is stretched, interfilament lattice spacing decreases, promoting force generation. 42 Using synchrotron small-angle x-ray diffraction, we measured interfilament lattice spacing (d10) in isolated, skinned papillary muscles in relaxing solution at both short and long SLs. Lattice spacing significantly decreased with stretch, but there were no statistical differences between GSK-3β control and knockout fibers (Figure 2A), eliminating it as a plausible mechanism.

As the molecular spring responsible for passive tension 43 and resting SL, titin acts as a stretch sensor for LDA. For example, transgenic expression of a more compliant titin isoform depressed LDA. 28 To assess whether titin may be contributing to GSK-3β's modulation of LDA, we assessed passive tension in control and knockout myocytes. Myocytes from knockout mice had significantly decreased passive tension at long SLs (2.4 and 2.6 µm, P=0.0083 and P=0.016 by unpaired t test, n=4 mice/group, 3-4 cells/mouse, Figure 2B, Table S4). If GSK-3β indeed modulates LDA via passive tension, recombinant GSK-3β should be able to increase passive tension similar to its effect on calcium sensitivity at long SLs. We performed passive tension experiments in control and GSK-3β knockout myocytes before and after treatment with exogenous GSK-3β (0.1 µg GSK-3β for 5 minutes). GSK-3β treatment significantly increased passive tension in both control and knockout myocytes (calculated by paired t test, n=3 mice/group, 4-5 cells/ mouse, Figure 2C and2D).

In addition to passive tension and consistent with a more compliant titin, resting SL was also increased in GSK-3β knockout myocytes compared with control myocytes (n=3 mice/group, 30 cells/mouse, P=0.024 by Mann-Whitney test, Figure 2E and2F). Together, these results indicate that loss of GSK-3β from the myofilament results in a more compliant titin, which is known to depress LDA.

There are 2 isoforms of titin, the stiff N2B and more compliant N2BA, 44 and it is possible the observed change in passive tension was due to a switch in the relative expression of these 2 isoforms. We prepared 45 LV samples from control and knockout mice and quantified the ratio of N2BA/N2B (n=5-6/group, Figure 2G) and found the isoform composition to be unchanged. Thus, the decrease in passive tension in knockout cells was likely due to reduced phosphorylation of GSK-3β's myofilament targets.

We next determined whether genetic removal of a protein in the LDA pathway, specifically, cMyBP-C, would block the ability of exogenous GSK-3β to rescue calcium sensitivity at a long SL. LDA requires MyBP-C, 46 whose C terminus binds titin 47 and N terminus binds the thin filament. 48,49 Importantly, we observed no evidence of a direct interaction of GSK-3β and cMyBP-C (ie, no change in cMyBP-C phosphorylation in the knockout mice), so here we are testing whether GSK-3β's functional impact lies upstream of cMyBP-C.

We used 2 genetically engineered mouse lines, one with complete ablation of cMyBP-C (cMyBP-C knockout) 30 and one transgenic expressing a truncated cMyBP-C lacking the N-terminal C0 and C1 domains that interact with the thin filament (ΔC0-C1f). 31 Both strains have been previously studied, although we confirmed their cMyBP-C expression profiles here (Figure S4A). We measured force-calcium relationships before and after exogenous GSK-3β treatment (n=3 mice/group, 3 cells/mouse). Although GSK-3β increased calcium sensitivity at long SLs in Con myocytes, it failed to affect calcium sensitivity in the cMyBP-C knockout or cMyBP-C ΔC0C1f mice (Figure S4B through S4G). Together, these findings indicate cMyBP-C, and particularly the N terminus, is downstream of GSK-3β's titin-based modulation of calcium sensitivity. However, the precise role of cMyBP-C in LDA has not

To further understand the mechanism by which GSK-3β modulates LDA, we next sought to identify the myofilament S5). Using cutoffs of P<0.05 and log 2 FC<-0.5, we found decreased phosphorylation at 9 S/T residues on 8 proteins in the knockout mice, including the structural z-disc protein abLIM-1, 50 and z-disc affiliated proteins Supervillin, 51 Synaptopodin, 52 and SPEG (striated muscle preferentially expressed protein kinase) (Figure 3A and 3B), suggesting they are GSK-3β targets. Two phosphorylation sites increased (log 2 FC>0.5, HSPB6 (heat shock protein beta-6) and B3AT [band 3 anion transport protein]). We did not detect changes in phosphorylation of any thin filament proteins known to impact calcium sensitivity as well as any changes in titin phosphorylation that could be linked to the decreased passive tension. The mass spectrometry results indicated GSK-3β's myofilament targets are primarily at the z-disc in vivo, which we verified with immunofluorescence on LV skinned myocytes from control and knockout mice (n=3), using antibodies against phosphorylated serine/threonine residues, and α-actinin. As a positive control, a subset of control myocytes was incubated with alkaline phosphatase. We used the α-actinin channel to delineate the z-disc and create a region of interest to quantify the pSer/Thr signal. The area of z-disc pSer/Thr staining significantly decreased in GSK-3β knockout mice compared with control animals (P=0.0091, Figure 3C and3D). Decreased phosphorylated z-disc area was also observed in cells incubated with alkaline phosphatase (P=1.0×10 -6 ).

Consistent with its targets, we also found GSK-3β localizes to the myofilament z-disc. Myocytes isolated from human nonfailing LV myocardium were skinned before plating/fixing to remove the confounding presence of the cytosolic pool of GSK-3β. While total GSK-3β displayed a mild colocalization with α-actinin, pY216 GSK-3β displayed a much stronger localization to the z-disc. Comparatively, pS9 GSK-was not at the z-disc and instead appeared to localize to the intercalated disc (Figure 4A). This intercalated disc localization was not unique to the pS9 antibody, however, as all GSK-3β antibodies localized to the intercalated disc (Figure S5). Immunofluorescence images for IgG and secondary antibody only controls to test for nonspecific binding in human myocytes are shown in Figure S6.

That phosphorylation at Y216 on GSK-3β increased its affinity for the myofilament was further shown by immunoprecipitation experiments in human nonfailing LV myocardium enriched for the myofilament. Using total GSK-3β, pS9 GSK-3β, or pY216 GSK-3β as bait, total and pS9 GSK-3β did not pull-down myofilament proteins, while pY216 GSK-3β showed high levels of the primary myofilament proteins (Figure 4B). Because of the strong association of myofilament proteins for each other, this result does not indicate a specific binding partner of pY216 GSK-3β. As total GSK-3β should include pY216 GSK-3β, it was surprising there was little interaction between total GSK-3β and the myofilament. One explanation is that Y216 phosphorylation makes up a small percentage of total GSK-3β, however, it is also possible this is due to differences between the primary antibodies.

To confirm whether phosphorylation at Y216 is necessary and sufficient for GSK-3β's association with the myofilament, we generated myc-tagged adenoviruses (so the same primary antibody could be used in each) for wildtype GSK-3β, phospho-null (Y216F) GSK-3β, and phospho-mimetic (Y216E) GSK-3β and transduced them into neonatal rat ventricular myocytes. Probing for the myctag revealed equal expression in the transduced neonatal rat ventricular myocytes (Figure 4C and4D). Coimmunoprecipitation in myofilament-enriched samples showed Y216E GSK-3β strongly bound to the myofilament, while almost no binding was observed with the phospho-null Y216F GSK-3β (Figure 4E and4F). A modest amount of myc-wild-type GSK-3β also associated with the myofilament, which we attributed to endogenous Y216 phosphorylation, as shown by Western blot (Figure 4G).

Of the GSK-3β phospho-targets we identified by mass spectrometry, abLIM-1 was of specific interest because (1) multiple phosphorylation sites on the protein were decreased in the knockout mice indicating strong regulation by GSK-3β (Figure 5A), (2) we previously identified abLIM-1 as a target of GSK-3β in a dog model of HF, 13 and (3) LIM domains are involved in stress sensing. 53 No statistical differences in protein (n=5; Figure 5B and5C) or transcript levels (n=4; Figure S7A) of abLIM-1 were detected in knockout mice. Immunofluorescence in control and knockout myocytes showed abLIM-1 localizes to the z-disc (Figure 5D) as previously shown 50 and is solely a sarcomeric protein, as abLIM-1 was only detected in the myofilament fraction and absent from the soluble, primarily cytosolic, fraction (n=3 control, 4 knockout; Figure S7B andS7C). Immunofluorescence images for IgG and secondary antibody only controls to test for nonspecific binding in mouse myocytes are shown in Figure S8.

The N terminus of titin, specifically the Z1Z2 domains and z-repeats, localizes to the z-disc. 54 The Z1Z2 domains bind to TCAP (telethonin), a complex that is important for stress sensing at the z-disc 55 and involved in maintaining passive tension. 56 As passive tension was decreased in the GSK-3β knockout mice, we hypothesized abLIM-1 interacts with the Z1Z2 domains of titin at the z-disc. We Using the GST-tag on abLIM-1 as bait, we were able to identify Z1Z2 in the elutants via mass spectrometry (Figure 5E), indicating that abLIM-1 can bind the Z1Z2 domains on titin. To determine whether this interaction can be regulated by GSK-3β, we treated abLIM-1 with GSK-3β and found this ablated its interaction with Z1Z2 (n=4-5 samples/group; P=0.0188 by ANOVA as previous studies have indicated IP-MS/MS (immunoprecipitation-mass spectrometry) experiments in the myofilament can be analyzed by parametric statistical tests 57 ).

We next sought to determine mechanistically whether abLIM-1 is involved in LDA signaling in the myocyte. We generated engineered human heart tissues (EHT) 35 by seeding decellularized myocardium with human induced pluripotent stem cell cardiomyocytes and allowed them to mature for 2 weeks. The EHTs were then treated with either scrambled siRNA or abLIM-1 siRNA, which resulted in a 30% knockdown of abLIM-1 (P=0.032, Figure 5F and5G). The abLIM-1 treatment did not result in alteration of cross-sectional area of the EHTs (Figure 5H). EHTs were paced at 1 Hz in Tyrodes solution while simultaneously measuring isometric twitch force. To measure LDA, the EHT was slacked to -10% of the tissue culture length and then stretched to +10%, all while continuing 1 Hz pacing (n=6 scrambled, 7 abLIM-1). Both scrambled and abLIM-1 siRNA treated EHTs exhibited increased twitch force with stretch, however, the EHTs with reduced abLIM-1 showed enhanced length sensitivity compared with the scrambled group, with a divergence occurring around 2% stretch (Figure 5I). The greatest difference in force occurred at 10% stretch (P=0.0043; (Figure 5J). These experiments indicate that abLIM-1 binds titin at the z-disc to depress passive tension and thus LDA, and that its phosphorylation by GSK-3β relieves this inhibition and results in normal function.

Myofilament GSK-3β Is Decreased in HF Samples With Dampened LDA

We next determined whether myofilament-localized GSK-3β plays a role in human HF. We used LV myocardium from human nonfailing rejected donor hearts (n=19) and explanted hearts from patients with HF (n=22, demographics in the Table ). These 41 samples were collected from 2 biobanks (Loyola University Chicago and Cleveland Clinic). Myofilament GSK-3β was significantly reduced in myocardium from patients with HF compared with nonfailing hearts, while whole tissue GSK-3β remained unchanged when normalized to either the total protein stain (Figure 6A and 6B) or the actin band of the total protein stain (Figure S9). No statistical differences were detected in phosphorylated GSK-3β (pS9 and pY216 normalized to total GSK-3β) in either whole tissue or the myofilament (Figure S10A through S10E).

Despite observing no statistical difference in whole tissue GSK-3β between the groups, we selected the samples with the highest and lowest levels of whole tissue GSK-3β (n=5-7 cells/ 3 hearts per group) and measured force-calcium relationships at long SL. There were no statistical differences between these groups (Figure S11), supporting that whole tissue (primarily cytosolic) GSK-3β is not responsible for modulating sarcomeric function.

Last, we tested whether HF patients with diminished sarcomere-localized GSK-3β had depressed LDA as the mouse model would predict. We measured force-calcium relationships at both short and long SLs in nonfailing and HF LV (n=3 hearts/group, 3 cells/heart). Both nonfailing and HF groups experienced an increase in F max with stretch; however, while we observed increased calcium sensitivity in nonfailing myocytes when increasing SL, this effect was entirely absent in the HF group (Figure 6C through 6F, Table S6). These data suggest z-disc localized GSK-3β is lost in human HF, which correlates with a loss of LDA.

GSK-3β is a central kinase in multiple critical signaling pathways in the cardiomyocyte, and recent evidence 13,58 suggests it also regulates sarcomere contractile function, although direct evidence is lacking. To address this gap, we used an inducible cardiomyocyte-specific GSK-3β knockout mouse model. We discovered GSK-3β is essential for normal LDA, the ability of the myocyte to respond to stretch and a critical component of the Frank-Starling mechanism, resulting in calcium desensitization at longer SLs. Mechanistically, this is via decreased titinbased passive tension observed in the knockout mice, which is a critical length sensor for LDA. GSK-3β localizes to the sarcomere z-disc via its own phosphorylation at Y216 and phosphorylates several z-disc proteins, including abLIM-1. Almost nothing is known about abLIM-1 in the heart, but we have found that it localizes to the z-disc where it can bind to the Z1Z2 domains of titin, and this binding is blocked by GSK-3β phosphorylation. Furthermore, abLIM-1 acts as an inhibitor of passive tension and LDA, which is relieved by GSK-3β. Importantly, in LV myocardium from human patients with HF there was reduced sarcomeric GSK-3β (but not whole tissue GSK-3β) and diminished LDA compared with nonfailing samples. As reduced LDA in HF is detrimental, altering Y216 phosphorylation to restore sarcomeric GSK-3β localization is a potential therapeutic strategy for restoring the Frank-Starling mechanism in patients with HF.

The Frank-Starling law of the heart states that the stroke volume of the LV increases as LV volume increases, 59 allowing the heart to respond to changes in volume on a beat-to-beat basis. This behavior stems from the response of cardiomyocytes to stretch, which results in an increase in force production, termed LDA. LDA, and thus the Frank-Starling mechanism, can become depressed in HF. [23][24][25][26][27] While most studies are conducted in patients with end-stage HF, animal models have shown that this mechanism may be lost in the earlier stages, before hypertrophy and fibrosis. 5 Despite the fact it was initially discovered over a century ago-the molecular mechanisms of LDA are still unclear, which may explain why some studies have found the Frank-Starling mechanism to be unaltered in HF. 26,27 There are several hypotheses supported by the existing work, and these may not be exclusive, since such a critical behavior could warrant redundant pathways to operate. There are currently 3 primary mechanisms for explaining LDA 20 : (1) decreased interfilament spacing from sarcomeric stretch brings myosin heads closer to actin to increase the likelihood of crossbridge formation, (2) phosphorylation/ regulation of thin filament proteins with stretch increases calcium sensitivity, and (3) stretch is sensed by titin We were able to rule out the first 2 mechanisms by which GSK-3β regulates LDA, as lattice spacing was not altered in GSK-3β knockout mice and phospho-proteomics did not identify any phosphorylation sites connected to LDA or calcium sensitivity. 20 Instead, our data implicate the titin-based mechanism. Titin's ability to create passive tension directly correlates with an enhanced LDA response 28,60,61 ; and myocytes with a loss of titin compliance have a blunted LDA just as we observed in the GSK-3β knockout mice. Additionally, incubation with recombinant GSK-3β increased passive tension. How, then, does GSK-3β modify titin compliance? GSK-3β did not affect titin itself, as MS revealed GSK-3β did not phosphorylate any titin residues, and there was no change in titin isoforms. Thus, GSK-3β must regulate the interaction between titin and z-disc proteins.

While a great deal remains unknown about titin's structure, function, and interacting partners at the z-disc, there are studies that support the hypothesis that altering titin's interaction with z-disc proteins can alter passive tension. For example, the Z1Z2 domains of titin, which are located in its z-disc region, are highly compliant in isolation, but are stabilized via z-disc binding partners. 54 Of the GSK-3β phosphorylated proteins identified in our phospho-proteomics screen, we were most interested in abLIM-1. abLIM-1 has been the focus of very few studies, only one of which identified it in the heart. 50 However, several proteins of the same family, which contain LIM domains, such as MLP (muscle LIM protein) and FHL1 and FHL2 (Four and a half LIM domain protein) have been shown to be stress/strain sensors. 62 Indeed, this work establishes abLIM-1 as a critical z-disc protein in maintaining normal function. First, abLIM-1 interacts with the Z1Z2 domains of titin in vitro which also interact with TCAP, an interaction important for stress sensing, maintaining passive tension, 55,56 and likely LDA although this has not yet been shown.

Similar to TCAP, abLIM-1 is also required for normal stress sensing at the z-disc, as siRNA-induced reduction in abLIM-1 significantly increased LDA. The direction of this impact was somewhat surprising, as we anticipated facilitation or enhancement of LDA by abLIM-1, and that its reduction would lead to depressed LDA. However, these results show that abLIM-1 is not simply required for the LDA mechanism to proceed but modulates the response of the cell to stretch by acting as a brake on stretch sensing and LDA. This regulatory paradigm is not unprecedented, as there are other proteins in cardiac excitation-contraction coupling that inhibit activity in a phosphorylation-dependent manner, such as cMyBP-C and phospholamban. In fact, our experiments with recombinant Z1Z2 and abLIM-1

show that GSK-3β ablates their interaction and relieves abLIM-1's inhibition of passive tension and LDA. These results introduce a new player in sarcomere mechanosensing, and future studies are warranted to understand the structural basis for abLIM-1's novel functional role and how its phosphorylation alters this function.

It is likely that declining myofilament GSK-3β contributes to decreased LDA and Frank-Starling mechanism observed in the failing human heart. 5,24,26 As GSK-3β can directly enhance calcium sensitivity at long SLs, it is an intriguing candidate for a HF therapeutic. Indeed, a therapeutic that could increase calcium sensitivity at long SLs while not affecting it at short or resting SLs would be highly beneficial, especially since LDA takes place within 5 ms 21 so it can regulate calcium sensitivity during a single beat. A straight-forward calcium sensitizer would increase contractility but would also slow relaxation. However, a therapeutic that increased calcium sensitivity at long SLs when the heart is filled with blood just before systole would enhance contractility, and as the LV chamber volume decreases during systole and myocyte SL shortens calcium sensitivity would decrease, aiding relaxation. Indeed, compounds that aim to enhance sarcomere-based contractility have frequently resulted in depressed relaxation, 63 creating a benefit-cost tradeoff.

However, GSK-3β is such a promiscuous signaling kinase 64 that broadly elevating GSK-3β would likely result in catastrophic off-target effects. Thus, the ability to manipulate strictly myofilament-localized GSK-3β is imperative, which we found is regulated by its phosphorylation at Y216. Several kinases have been reported to phosphorylate Y216, including Fyn (tyroine-protein kinaes fyn), 65 PYK2 (protein-tyrosine kinase 2-beta), 66 MEK1 (dual specificity mitogen-activated protein kinase 1), 67 and as an auto-phosphorylation event. 68 Generally, tyrosine kinases phosphorylate fewer targets than serine/threonine kinases, 69 thus the large number of kinases targeting Y216 suggests dynamic regulation. Whether targeting the Y216 phosphorylation site directly or by modulating the activity of an upstream kinase would be a useful therapeutic approach to restore myofilament GSK-3β and thus LDA in HF will need to be established in future studies.

We have identified a novel mechanism by which GSK-3β regulates LDA, arising from its z-disc localization and phosphorylation targets, most likely abLIM-1, a new player in sarcomere function. While LDA and the Frank-Starling mechanism were described well over a century ago, many aspects of the mechanism remain unresolved. This study reveals new kinase regulation of LDA and suggests myofilament GSK-3β is a strong therapeutic