GSK3β Phosphorylates Newly Identified Site in the Proline-Alanine–Rich Region of Cardiac Myosin–Binding Protein C and Alters Cross-Bridge Cycling Kinetics in HumanNovelty and Significance
Rationale: Cardiac myosin–binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C.
Objective: To identify and characterize additional phosphorylation sites in human cMyBP-C.
Methods and Results: Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine–rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3β (GSK3β) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3β showed phosphorylation on Ser133. In addition, GSK3β phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A–induced phosphorylation at Ser304. GSK3β treatment of single membrane–permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment.
Conclusions: GSK3β phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine–rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3β at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.
- cardiac myosin–binding protein C
- contractile proteins
- heart failure
- myocardial contractility
Cardiac myosin–binding protein C (cMyBP-C) is increasingly being recognized as an important regulator of sarcomere function with consequences for in vivo cardiac performance. The main regulatory role of cMyBP-C seems to be its effect on cross-bridge cycling kinetics of sarcomere contraction.1,2 Cardiac MyBP-C itself is regulated by phosphorylation.3 It has been proposed that cMyBP-C acts as a structural constraint limiting cross-bridge formation and that phosphorylation of cMyBP-C accelerates cross-bridge kinetics, which is required for enhanced rates of relaxation and force development in diastole and systole, respectively.2
Classically, protein kinase A (PKA), which is activated on β-adrenergic receptor stimulation, was described as the main kinase responsible for cMyBP-C phosphorylation.4 At least 3 sites on cMyBP-C can be phosphorylated in vivo by PKA,4,5 that is, Ser275, Ser284, and Ser304 (numbering based on human sequence), whereas Ser311 phosphorylation was shown to be phosphorylated by PKA in vitro.6 All these phosphorylation sites are located in the cardiac isoform-specific M-domain (Figure 1A). More recent work has shown that the phosphorylation sites in the M-domain can be targeted by many other kinases (reviewed by Bardswell et al7). Moreover, additional sites have been described on the basis of animal experiments,8 and studies on human cardiac tissue revealed that at least 1 additional phosphorylation site should exist in humans, because up to 4 phosphate groups are present in isolated human cMyBP-C.5
To identify phosphorylation sites on cMyBP-C, we performed tandem mass spectrometry on semipurified cMyBP-C from human heart tissue. A novel phosphorylation site (Ser133) was identified in the linker domain of cMyBP-C, which was not a target of PKA, but was phosphorylated by the hypertrophy kinase, glycogen synthase kinase 3β (GSK3β). Interestingly, GSK3β treatment of single membrane–permeabilized human cardiomyocytes significantly enhanced kinetics of force redevelopment, suggesting a novel functional role for GSK3β at the sarcomere level.
An expanded Methods section is available in the Online Data Supplement.
In Vitro Kinase Assays
Recombinant C0C2 fragment was incubated for 2 hours at 37°C in relax solution (pH 7.0; in mmol/L: free Mg2+ 1, KCl 145, EGTA 2, ATP 4, imidazole 10) with 750 U PKA (Calbiochem) or 10 μL GSK3β (Sigma Aldrich). This reaction was stopped using 2-dimensional clean-up kit (GE Healthcare).
Novel Phosphorylation Site in Human cMyBP-C
Previous data showed that at least 1 additional phosphorylation site is present in human cMyBP-C.5 To investigate the presence of novel phosphorylation sites, cMyBP-C was semipurified from donor and failing (idiopathic dilated cardiomyopathy) heart tissue and was analyzed for phosphorylated peptides by tandem mass spectrometry. Peptide coverage was 58% of the total amino acid sequence (Figure 1B). In donor cMyBP-C, 2 phosphorylation sites were identified. One corresponded to the known Ser284 site4 (Figure 1C), whereas a novel phosphorylation site was identified at serine 133 (Figure 1D). Analysis of cMyBP-C from failing heart did not reveal any phosphorylated peptides (data not shown).
Levels of Phosphorylated Ser133 in Human Cardiac Tissue
To assess Ser133 phosphorylation levels in nonfailing and failing human hearts, a phospho-specific antibody was generated. Phosphorylation levels in failing heart samples from dilated and ischemic heart disease patients were significantly lower than in donors (Figure 2), which was in agreement with the mass spectrometry (MS)/MS data. Phosphorylation at the in vivo–established sites on cMyBP-C (Ser275, Ser284, and Ser304) was significantly lower in idiopathic dilated cardiomyopathy and ischemic heart disease compared with donor hearts (Figure 2).
Ser133 Is a Target of GSK3β
PKA is the archetypical kinase that phosphorylates cMyBP-C at all previously identified sites.4,6,9 To study whether PKA can also phosphorylate Ser133, the N-terminal human recombinant peptide spanning the C0C2 domain (amino acids 1–451) of cMyBP-C was incubated with PKA. Robust phosphorylation of Ser275, Ser284, and Ser304 sites was detected, whereas Ser133 was not phosphorylated by PKA (Figure 3A). To identify the kinase responsible for Ser133 phosphorylation, in silico kinase prediction was performed. This yielded GSK3β as the most likely candidate (score 0.52). In vitro incubation of the C0C2 peptide with GSK3β revealed marked phosphorylation at Ser133 and Ser304, whereas the other sites were not phosphorylated (Figure 3B). Analysis of C0C2 treated with GSK3β or PKA loaded on the same immunoblot and stained with the antibodies against phosphorylated Ser133 and Ser304 (Figure 3C) confirmed that Ser133 was phosphorylated by GSK3β, but not by PKA. Interestingly, no phosphorylation signal was obtained at Ser304 for GSK3β-treated C0C2, whereas phosphorylation signals for the PKA-treated C0C2 were extremely intense even though PKA activity was lower compared with GSK3β activity (respectively, 10 versus 168 pmol/min per microgram). Overall, this suggests that Ser133 may be the preferred target of GSK3β on cMyBP-C.
To show that endogenous GSK3β targets Ser133, the recombinant human 40-kDa fragment (amino acids, 1–271, also known as the 29-kDa fragment10) was incubated with a rough cytosolic fraction from donor heart tissue with and without 2 μM GSK3β antagonist CT99021. At this dose, CT99021 almost completely prevented the ability of exogenous GSK3β to phosphorylate Ser133 (Online Figure I). Phosphorylation of Ser133 was significantly lower in the presence of CT99021 (Figure 3D), suggesting that GSK3β present in the cytosolic fraction is able to phosphorylate cMyBP-C at Ser133.
GSK3β protein levels were similar in donor and failing samples (Figure 3E), whereas phosphorylation of β-catenin (determined by Phos-tag analysis),11 another cellular substrate of GSK3β, was lower in idiopathic dilated cardiomyopathy and ischemic heart disease compared with donor (Figure 3F), consistent with low Ser133 cMyBP-C phosphorylation in failing myocardium (Figure 2).
Effect of GSK3β on Sarcomere Function
To study the effects of GSK3β on sarcomere function, force measurements in membrane-permeabilized cardiomyocytes were performed. Cardiomyocytes from failing human tissue (low basal Ser133 phosphorylation; Figure 2) were used and sarcomere function was measured before and after incubation with GSK3β. Incubations in kinase buffer without enzyme served as control. Idiopathic dilated cardiomyopathy cardiac tissue incubated with GSK3β showed increased phosphorylation at Ser133, whereas no increase was found at Ser304 (Figure 4A). GSK3β incubation had no effect on maximal and passive force (data not shown). However, GSK3β significantly increased the maximal rate of tension redevelopment, whereas no effect was seen in the absence of the kinase (Figure 4B).
Tandem MS identified a novel phosphorylation site at Ser133 in human cMyBP-C. We also revealed that GSK3β, but not PKA, could phosphorylate Ser133. Functional studies in cardiomyocytes from failing hearts showed that GSK3β also has a modulating effect on sarcomere function by increasing the rate of tension redevelopment.
To date several phosphorylation sites have been identified in the M-domain of human cMyBP-C (Figure 1A),4,6,9 and our recent study suggested that at least 1 more site exists.5 Starting from this, we performed tandem MS experiments to identify the missing site in cMyBP-C semipurified from donor myocardium. This resulted in the identification of 1 of the known sites (Ser284) and a novel site Ser133. The other PKA sites (Ser275, Ser304, and Ser311) were not found in our analysis, because they were not present in the identified peptides (Figure 1B). Complete sequence coverage of a protein is typically not achieved in MS/MS experiments, because of a variety of reasons, such as the generation of peptides that are either too large or too small to be adequately detected.12
To our knowledge, we are the first to link phosphorylation of cMyBP-C to GSK3β. GSK3β is a key signaling molecule in the insulin/phosphoinositide 3-kinase/Akt signaling cascade13 and is active under resting conditions, and inactivated by phosphorylation.14 GSK3β is considered to be anti-hypertrophic,15 through the inhibition of pro-hypertrophic transcription factors.13,15 Reduced GSK3β activity has been reported in human heart failure, whereas protein levels were similar (Haq et al16; Figure 3E). In line with reduced GSK3β activity, the level of phosphorylated Ser133 was significantly lower in end-stage failing than in donor myocardium (Figure 2). Ser133 is positioned within the proline-alanine–rich linker domain between C0 and C1 (Figure 1A), which was thought to interact with the thin filament actin,17 although recently it has been shown that N-terminal fragments of cMyBP-C lacking the M-domain bind actin only weakly in vitro.18 Other proposed interaction partners for the N-terminal part of cMyBP-C are myosin S2 domain and possibly the regulatory light chain (RLC).19 The true worth of actin, myosin S2, and regulatory light chain interactions with cMyBP-C on sarcomere function is yet to be established. Phosphorylation of Ser133 likely affects these protein interactions and might explain the observed change in cross-bridge kinetics. To directly link cMyBP-C Ser133 phosphorylation to the observed increase in cross-bridge kinetics, additional experiments using transgenic mice or exchange with recombinant protein are needed. Previous studies showed that PKA-mediated cMyBP-C phosphorylation is centrally involved in enhancement of cardiac kinetics during β-adrenergic receptor stimulation. Likewise, we showed that GSK3β increases the rate of force redevelopment in human cardiomyocytes and may represent another kinase, which is able to modify myocardial kinetics, possibly via phosphorylation of a newly identified site in the linker domain of cMyBP-C.
Sources of Funding
We acknowledge support from the 7th Framework Program of the European Union (BIG-HEART, grant agreement 241577), The Netherlands Organization for Scientific Research (NWO; VIDI grant), and National Institutes of Health grants R01HL105826 and K02HL114749.
In November 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.8 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.275602/-/DC1.
- cardiac myosin-binding protein C
- glycogen synthase kinase 3β
- Mass spectrometry
- protein kinase A
- Received June 14, 2012.
- Revision received December 23, 2012.
- Accepted December 28, 2012.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Cardiac myosin–binding protein C (cMyBP-C, or MYBPC3), a thick filament associated protein, is an important regulator of cardiac contractility.
Phosphorylation cMyBP-C regulates cardiac function.
Phosphorylation of cMyBP-C is reduced in end-stage heart failure.
What New Information Does This Article Contribute?
A new phosphorylation site (Ser133) was identified in the proline-alanine–rich linker sequence between the C0 and C1 domains of cMyBP-C.
Ser133 is phosphorylated by glycogen synthase kinase 3β (GSK3β). Level of Ser133 phosphorylated cMyBP-C protein is lower in heart failure.
Incubation of the skinned ventricular myocytes isolated from failing human hearts with GSK3β leads to an increase in the kinetics of cardiac contraction.
Phosphorylation of cMyBP-C is required for normal cardiac function. We have identified a novel phosphorylation site on cMyBP-C, that is, Ser133 in the proline-alanine–rich linker sequence between C0 and C1 domains. Hearts from end-stage heart failure patients have reduced level of Ser133 phosphorylated cMyBP-C compared with donor hearts. Ser133 is phosphorylated by the antihypertrophy kinase GSK3β. Incubation of skinned ventricular myocytes isolated from failing human hearts with GSK3β increased kinetics of contraction. These findings imply that GSK3β is involved in direct regulation of cardiac contractility through phosphorylation of a myofilament protein. Regulating GSK3β activity may represent a new mechanism for regulating the kinetics of cardiac contraction in human myocardium.