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Impaired binding of ZASP/Cypher with phosphoglucomutase 1 is associated with dilated cardiomyopathy

Takuro Arimura , Natsuko Inagaki , Takeharu Hayashi , Daisuke Shichi , Akinori Sato , Kunihiko Hinohara , Matteo Vatta , Jeffrey A. Towbin , Taishiro Chikamori , Akira Yamashina , Akinori Kimura
DOI: http://dx.doi.org/10.1093/cvr/cvp119 80-88 First published online: 18 April 2009

Abstract

Aims Z-band alternatively spliced PDZ-motif protein (ZASP)/Cypher is a Z-disc component of which several dilated cardiomyopathy (DCM)-associated mutations have been reported. Most of the mutations were found in exons 4 and 10 of ZASP/Cypher gene LDB3 and both exons were expressed preferentially in the heart. The aim of this study was to investigate the functional alteration of ZASP/Cypher caused by the DCM-associated mutations.

Methods and results The yeast-two-hybrid method was used to identify the protein bound to a domain encoded by exon 4 of LDB3. Interaction of ZASP/Cypher with the binding protein was investigated in relation to the functional alterations caused by LDB3 mutations. Localization of the ZASP/Cypher-binding protein was examined at the cellular level in rat cardiomyocytes. Phosphoglucomutase 1 (PGM1), a metabolic enzyme involved in glycolysis and gluconeogenesis, was identified as a protein interacting with ZASP/Cypher. PGM1 bound to ZASP/Cypher at the domains encoded by exons 4 and 10. Two LDB3 mutations in exon 4 (Ser189Leu and Thr206Ile) and another mutation in exon 10 (Ile345Met) reduced the binding to PGM1. PGM1 showed diffuse localization in the cytoplasm of rat cardiomyocytes under standard culture conditions, and distribution at the Z-discs was observed under stressed culture conditions. Binding of endogenous PGM1 and ZASP/Cypher was found to be enhanced by stress in rat cardiomyocytes.

Conclusion ZASP/Cypher anchors PGM1 to Z-disc under conditions of stress. The impaired binding of PGM1 to ZASP/Cypher might be involved in the pathogenesis of DCM.

KEYWORDS
  • Cardiomyopathy
  • Energy metabolism
  • Mutation
  • ZASP/Cypher
  • Z-disc
  • Phosphoglucomutase 1

1. Introduction

The sarcomere is a contractile unit of striated muscle composed of highly organized proteins.1 The structure of the striated muscle, i.e. the cardiac and skeletal muscles, represents the Z-disc, I-band, A-band, and M-line; the M-line is surrounded by A-bands which in turn are surrounded by I-bands and the I-bands are linked to the Z-discs. The structure of the M-line and the A-band is represented by thick and thin filaments, mainly composed of myosin and actin, respectively, and the thin filaments are inserted into the Z-disc.2 Because the force generated by the contraction of sarcomere is transmitted through a complex network of proteins in the Z-disc, the Z-disc plays a pivotal role in the cardiomyocytes, i.e. in sarcomeric organization and force transduction.3 The Z-disc also mediates as a functional link between the sarcolemma and the nuclear membrane.4 Since the Z-disc is important in establishing the mechanical coupling of the sarcomere, functional defects in the Z-disc proteins lead to cardiac dysfunction.5

Dilated cardiomyopathy (DCM) is a cardiac disease characterized by cardiac enlargement associated with systolic dysfunction and it often manifests with congestive heart failure.6 Genetic abnormalities cause DCM in some of patients7 and we have previously reported mutations in the gene for Z-band alternatively spliced PDZ-motif protein (ZASP)/Cypher (LDB3) associated with DCM.8,9 ZASP/Cypher is a Z-disc protein containing PDZ and LIM domains expressed in the striated muscles,10,11 which is also called Oracle.12 Human LDB3 consists of 16 exons encoded for six different isoforms due to the alternative splicing of several exons.8 Although the expression pattern of the human ZASP isoform has not been completely elucidated as yet, there are six known Cypher isoforms that demonstrate specific expression profiles in murine striated muscle. The cardiac-specific isoforms 1c, 2c, and 3c utilize exon 4, whereas the skeletal muscle isoforms 1s, 2s, and 3s use exons 5 and 6, i.e. exon 4 and exons 5/6 are mutually exclusive. Both human and mouse LDB3 share a similar structural genomic organization, although the mouse gene has been reported to consist of 17 exons due to the presence of the additional exon 7.8,13 The PDZ domain coded by exons 1–3 is expressed in all isoforms and is required for binding to α-actinin-2 in the Z-disc,11 whereas the LIM domains coded by exons 12–16 in ZASP are expressed by exons 13–17 in murine 1c, 2c, 1s, and 2s, and are involved in binding to protein kinase C.8,10 Another domain containing a ZASP-like motif composed of 26 amino acid (aa) residues necessary for interaction with α-actinin-2 was found in the internal region coded by exon 4 (cardiac isoforms) or exon 6 (skeletal isoforms).14 Alternative splicing of LDB3 in rat has not been investigated in detail, but it was possible to generate several isoforms from the genomic gene (http://genome.ucsc.edu/cgi-bin/hgTracks?position=chr16:10221362-10247798&hgsid=123741090&refGene=pack&hgFind.matches=NM_001110490).

In addition to the DCM-associated LDB3 mutations,9 there were other mutations associated with isolated non-compaction of the left ventricular myocardium9 or skeletal muscle myopathy, such as myofibrillar myopathy (MFM) and distal myopathy.15,16 Pathological changes, including functional alterations due to the LDB3 mutations, have not been fully elucidated, except that we were able to reveal an increased binding to PKCs by a DCM-associated mutation, Asp626Asn in the LIM domain.8 Further, the aetiological link between hereditary cardiomyopathy and skeletal muscle myopathy has raised the question: how do the LDB3 mutations cause heart-specific disease phenotypes in the isolated DCM? Because three other DCM-associated mutations were found in exon 4 (Ser189Leu and Thr206Ile) and exon 10 (Ile345Met),9 while these exons were preferentially expressed in the cardiac muscle,8 the ZASP/Cypher domains encoded by these exons may play a crucial role.

We hypothesized that there might be a molecule interacting with the ZASP/Cypher domains encoded by exons 4 and/or 10, and the DCM-associated mutations would impair the interaction. In this paper, we have demonstrated that phosphoglucomutase 1 (PGM1) was the molecule of issue. This is the first report suggesting a role for metabolic enzymes in the Z-disc function and pathogenesis of DCM.

2. Methods

2.1 Plasmid constructions

We obtained cDNA fragments of human LDB3 and PGM1 (GenBank accession nos M_007078 and NM_002633, respectively) by reverse transcription (RT)–PCR from human heart cDNA. A plasmid pGBKT7-ZASP/Cypher-Ex4 containing an exon 4 region corresponding to aa 100–238 of ZASP/Cypher in pGBKT7 (Matchmaker GAL4 two-hybrid system 3, Clontech, CA, USA) was obtained to be used in the yeast-two-hybrid (Y2H) screening. An equivalent region of LDB3 exon 4 was also cloned into pBIND containing GAL4 as bait (CheckMate mammalian two-hybrid system, Promega, WI, USA). Two mutants carrying C to T substitutions (DCM-associated Ser189Leu and Thr206Ile) were obtained by the primer-directed mutagenesis method. Other LDB3 cDNAs, corresponding to exons 6 and 10 (LDB3 Ex6-WT in NM_007078 or LDB3 Ex10-WT in NM_001080114 corresponding aa 300–362 or aa 114–183, respectively), were cloned into pGBKT7. Four exon 6 mutants carrying substitutions of G to A (INLVH-associated Asp117Asn and MFM-associated Ala147Thr), A to T (DCM-associated Lys136Met), or C to T (MFM-associated Ala165Val) and one exon 10 mutant carrying C to G substitution (DCM-associated Ile345Met) were created by the primer-directed mutagenesis method. Wild-type (WT) PGM1 cDNA fragments PGM1-WT, PGM1-WTs1, PGM1-WTs2, and PGM1-WTs3, corresponding to aa 406–562, aa 483–562, aa 483–510, and aa 510–562, respectively, were cloned into pACT containing VP16 as a prey (CheckMate mammalian two-hybrid system, Promega). Full-length WT PGM1 cDNA corresponding to aa 1–562 was also cloned into pACT. To create a green fluorescence protein (GFP)-fused PGM1, three PGM1cDNAs corresponding to aa 1–562, aa 1–426, and aa 427–562 were cloned into pEGFP-C1 (Clontech). All constructs were sequenced to ensure that no errors were introduced. Sequences of all primers used in this study are available upon request.

2.2 Y2H analysis

All the procedures were performed according to the manufacturer's instructions for Matchmaker GAL4 Two-Hybrid System 3 (Clontech). For the library screening, yeast strain AH109 was transformed with the bait plasmid pGBKT7-ZASP/Cypher-Ex4 and maintained on SD/-Trp. A human heart cDNA prey library was introduced into the transformant and plated onto SD/-Trp/-Leu/-His supplemented with 3-aminotriazole (3-AT). Positive clones were picked-up and re-plated onto SD/-Trp/-Leu/-His/-Ade with 3-AT. Colonies surviving the quadruple selection were assayed on filters for β-galactosidase activity, and plasmids from β-galactosidase-positive colonies were isolated, transferred to Escherichia coli, and sequenced.

2.3 Gene expression analysis

Expression of PGM1 and a control gene GAPDH was investigated by RT–PCR analysis using mRNAs from various human tissues (Clontech) as described previously.17 Conditions for PCR was 25 consecutive cycles of 95°C 30 s, 55°C 30 s, and 72°C 30 s.

2.4 Mammalian two-hybrid and co-immunoprecipitation assays

Mammalian two-hybrid (M2H) and co-immunoprecipitation (co-IP) assays using HeLa and COS-7 cells, respectively, were performed as described previously.18,19 Cells were co-transfected with a combination of pBIND-LDB3 constructs (4 µg) and pACT- or pEGFP-PGM1constructs (4 µg) in 16 µL of TransFectin (for HeLa) or 9.6 µL COSFectin (for COS-7) Lipid Reagent (Bio-Rad, CA, USA), according to the manufacturer's instructions, to analyse the binding of ZASP/Cypher with PGM1.

The care and treatment of animals were in accordance with the guidelines for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH Publication 85-23, revised 1996) and were subject to prior approval from the local animal protection authority at Tokyo Medical and Dental University. Neonatal cardiomyocytes were isolated from 1-day-old Sprague–Dawley rats. Briefly, the ventricles placed in cooled CBFHH buffer (137 mM NaCl, 5.36 mM KCl, 0.81 mM MgSO4, 5.55 mM dextrose, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, and 20 mM HEPES, pH 7.5) were mechanically dissociated and treated by repetitive digestion with 0.07 mg/mL Liberase blendzyme 3 (Roche, IN, USA) and 0.02 mg/mL DNaseII, and then purified by the discontinuous gradient method with Percoll.20 Cells (5 × 106) were seeded on the collagen-I coated 100 mm dishes (Corning, NY, USA) in low glucose (1 g/L) Dulbecco's modified Eagle's medium supplemented with 0.01 mg/mL insulin (Sigma-Aldrich, MO, USA), 10% foetal bovine serum (FBS), and 1% penicillin/streptomycin at 37°C with 5% CO2. Cardiomyocytes were then cultured in standard (10% FBS and 1 g/L glucose medium) and stress (FBS-free and 1 g/L glucose medium or 10% FBS and glucose-free medium) conditions for 72 h. Cells were harvested, permeabilized, and subjected to brief sonication in 250 µL of TNE buffer (1% NP-40, 1 mM EDTA, 150 mM NaCl, and 10 mM Tris–HCl, pH 7.8) with a protease inhibitor cocktail (Sigma-Aldrich). Cell debris was removed by centrifugation. After measuring protein concentration by using a BCA protein assay regent (Pierce Biotechnology), aliquots were removed for use in direct immunoblotting and co-IP assays, using the remaining supernatants containing equal amount of proteins and 6 µg of goat anti-ZASP/Cypher polyclonal antibody (Ab) (Novus Biologicals, CO, USA), or goat IgG (negative control), and were performed using the Catch and Release v2.0 Reversible Immunoprecipitation System, according to the manufacturer's instructions. Immunoprecipitates were separated on SDS–PAGE gels and transferred to a nitrocellulose membrane. After a pre-incubation with 3% skim milk in PBS, the membrane was incubated with primary mouse anti-PGM1 monoclonal Ab (1:200, Abnova, Taipei, Taiwan) or goat anti-ZASP/Cypher polyclonal Ab (1:200), and with secondary rabbit anti-mouse (for monoclonal Ab) or goat anti-rabbit (for polyclonal Ab) IgG HRP-conjugated Ab (1:2000, Dako A/S, Grostrup, Denmark). Signals were visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA) and Luminescent Image Analyzer LAS-3000mini (Fujifilm, Tokyo, Japan), and their densities were quantified by using Multi Gauge ver3.0 (Fujifilm).

Numerical data were expressed as mean ± SE. Statistical differences were analysed using one-way ANOVA and Student's t-test for paired values. P-values <0.05 were considered to be statistically significant.

2.5 Immunofluorescence microscopy

Preparation of neonatal rat cardiomyocytes was performed as above, and cell culture and the transfection procedure were performed as described previously.18,21 For transient transfection into the neonatal cardiomyocytes, GFP- or VP16-tagged PGM1 constructs (0.4 µg) was added with 0.8 µL of TransFectin Lipid Reagent (Bio-Rad) according to the manufacturer's instructions. After the transfection, cardiomyocytes were cultured in standard (10% FBS and 1 g/L glucose medium) and stress (FBS-free and 1 g/L glucose, or 10% FBS and glucose-free medium) conditions for 24 or 72 h. Non-transfected cardiomyocytes were also cultured under the same conditions for 72 h. The cells were washed with PBS, fixed for 15 min in 100% ethanol at −20°C, incubated in a blocking solution, and stained by primary mouse anti-ZASP/Cypher monoclonal Ab (1:150, Abnova), followed by secondary Alexa fluor 568 goat anti-mouse IgG (1:500, Molecular Probes, OR, USA), or primary mouse anti-ZASP/Cypher monoclonal Ab and goat anti-PGM1 polyclonal Ab (1:40, Santa Cruz Biotechnology Inc., CA, USA), followed by secondary Alexa fluor 488 rabbit anti-mouse IgG and Alexa fluor 568 rabbit anti-goat IgG (1:500, Molecular Probes). Images of at least 200 cells were collected and analysed with an LSM510 laser-scanning microscope (Carl Zeiss Microscopy, Jena, Germany).

3. Results

3.1 Identification of ZASP/Cypher-binding protein

A cDNA fragment corresponding to exon 4 of human LDB3 was cloned into the Y2H bait vector pGBKT7 to obtain pGBKT7-ZASP/Cypher-Ex4. By screening a human heart cDNA library with pGBKT7-ZASP/Cypher-Ex4, over 1000 potentially interacting clones were observed and 192 clones were randomly selected and sequenced. It was found that the most frequent (20 clones) cDNA was that for PGM1, a catalytic protein transferring a phosphate between glucose-1-phiospate and glucose-6-phosphate. They were classified into 13 independent cDNAs, corresponding to the C-terminal part of PGM1. All clones contained a conserved domain named phosphoglucomutase/phosphomannomutase α/β/α (PGM/PMM) domain IV (Figure 1A). We next analysed expression of PGM1 in various human tissues by RT–PCR. As shown in Figure 1B, PGM1 expressed ubiquitously but high-level expression was observed in the heart, skeletal muscle, kidney, liver, and lungs.

Figure 1

Structure and expression of PGM1. (A) Upper panel shows the domain structure of PGM1. Numbers indicate the amino acid positions. Dotted boxes represent PGM/PMM domains I–IV. Lower panel indicates human PGM1 clones isolated by the Y2H screening from the human heart cDNA library. Numbers indicate the start positions. (B) RT reactions were performed using total RNA from human tissues followed by PCR to detect the expression of PGM1 and GAPDH. GAPDH was an external control showing that similar amounts of cDNAs were analysed.

3.2 Analysis of interaction between PGM1 and the heart-specific domain of ZASP/Cypher

To investigate the binding of PGM1 to the heart-specific domain of ZASP/Cypher, we performed M2H assays. A bait construct containing WT LDB3 exon 4 (pBIND-LDB3 Ex4-WT) was co-transfected with a prey plasmid containing WT-PGM1 corresponding to the PGM/PMM domain IV (pACT-PGM1-WT), and luciferase activities of the co-transformants were measured. Negative controls containing either LDB3 Ex4-WT or PGM1-WT construct alone showed negligible luciferase activity, indicating no self-activation in these constructs (Figure 2A). The luciferase activity in the transfectants of PGM1-WT and LDB3 Ex4 was 0.94 ± 0.25 AU, indicating the binding between the PGM/PMM domain of PGM1 and heart-specific domain of ZASP/Cypher (Figure 2A). Next, we analysed the effect of LDB3 mutations in exon 4. It was found that the luciferase activities in the transfectants containing the PGM1 construct and DCM-associated Ser189Leu or Thr206Ile LDB3 constructs (0.30 ± 0.05 or 0.23 ± 0.01 AU, respectively) were significantly lower than those of PGM1-WT and LDB3 Ex4-WT (Figure 2A).

Figure 2

Binding of PGM1 with heart-specific domain of ZASP/Cypher encoded by exon 4 of LDB3. (A) Luciferase activities obtained in the M2H assay. Co-transfected pairs were PGM/PMM domain IV of PGM1 (PGM1) with WT-, S189L-, and T206I-ZASP/Cypher exon 4. Dashes indicate no VP16- or GAL4-tagged proteins (transfection with pACT or pBIND vectors, respectively). Data are expressed as mean ± SE (n = 4 for each). *P < 0.001 vs. WT; **P < 0.01 vs. WT. (B) Binding of PGM1 with ZASP/Cypher in (A) was analysed by co-IP using anti-VP16 antibody. GAL4-ZASP/Cypher proteins co-precipitated with VP16-PGM1 were detected by using anti-GAL4 antibody (top panel). Equal expression of GAL4-ZASP/Cypher (middle panel) and VP16-PGM1 (lower panel) was confirmed by immunoblotting of whole-cell supernatants. (C) Densitometric analysis of GAL4-blotting data obtained in the co-IP assays shown in (B). Data for WT-PGM1 with WT-ZASP/Cypher exon 4 were arbitrarily defined as 1.00 AU. Data are represented as the mean ± SE (n = 4 for each case). *P < 0.001 vs. WT. (D) GAL4-ZASP/Cyphers co-precipitated with VP16-PGM1 WTs1 (aa 483–562), WTs2 (aa 483–510), or WTs3 (aa 510–562) were detected by using anti-GAL4 antibody (top panel). Expressed amounts of GAL4-ZASP/Cypher (middle panel) and VP16-PGM1 (lower panel) were confirmed by immunoblotting of whole-cell supernatants. Dashes indicate no GAL4- or VP16-tagged proteins.

To further investigate the binding between PGM1 and ZASP/Cypher, we performed a co-IP assay. Western blot analysis of immunoprecipitates from the transfectants of pBIND-LDB3 Ex4-WT in combination with pACT-PGM1-WT revealed that the heart-specific domain of ZASP/Cypher encoded by exon 4 of LDB3 bound to the PGM/PMM domain IV of PGM1 (Figure 2B). Consistent with the weaker interaction between the PGM1 and mutant ZASP/Cypher observed in the M2H assays, despite equal expression of proteins, ZASP/Cypher in the presence of Ser189Leu or Thr206Ile mutation bound to PGM1-WT significantly less (0.59 ± 0.11 or 0.46 ± 0.17 AU, respectively) than WT (expressed as 1.00 AU) (Figure 2B and C, P < 0.001 for each cases).

To map the ZASP/Cypher-binding site in the PGM/PMM domain IV of PGM1, deletion constructs of PGM1, WTs1 (aa 483–562), WTs2 (aa 483–510), or WTs3 (aa 510–562) were tested for co-IP with ZASP/Cypher. As shown in Figure 2D, all deletion mutants were co-immunoprecipitated by anti-VP16 antibody, demonstrating that both the N- and the C-terminus of PGM/PMM domain IV of PGM1 were capable of interacting with ZASP/Cypher.

3.3 Analysis of the interaction between PGM1 and other regions of ZASP/Cypher

The heart-specific domain encoded by exon 4 of LDB3 contained proline-rich motifs and a ZASP-like motif, implying that these domains might be involved in the binding to PGM1. Quite interestingly, a region of ZASP/Cypher encoded by exon 10 of LDB3 also contained a proline-rich motif, whereas another region encoded by exon 6 carried a ZASP-like motif, suggesting that one or both of these regions might be binding targets of PGM1.

Western blot analysis of immunoprecipitates from the transfectant of pBIND-LDB3 Ex6- or Ex10-WT in combination with pEGFP-PGM1-WT C-terminus revealed that both regions were capable of binding to the PGM/PMM domain IV of PGM1 (Figure 3). The binding between mutant LDB3 Ex6 (INLVH-associated Asp117Asn, DCM-associated Lys136Met, and MFM-associated Ala147Thr and Ala165Val) and PGM1-WT was not significantly different from that of LDB3 Ex6-WT to PGM1-WT (Figure 3A). In clear contrast, despite the equal expression of proteins, mutant LDB3 Ex10 (DCM-associated Ile345Met) bound to PGM1-WT significantly less (0.35 ± 0.14 AU) than LDB3 Ex10-WT (expressed as 1.00 AU) (Figure 3B and C, P < 0.001). These data demonstrated that the regions encoded by exons 4, 6, and 10 of LDB3 could serve as binding motifs to the C-terminal domain of PGM1 and the binding of ZASP/Cypher with PGM1 was specifically impaired by the LDB3 mutations in exons 4 and 10.

Figure 3

Binding of PGM1 with the region of ZASP/Cypher encoded by exon 6 or exon 10 of LDB3. (A and B) Binding of PGM/PMM domain IV of PGM1 to regions of ZASP/Cypher encoded by exon 6 (A) or 10 (B) of LDB3 was analysed by co-IP. GAL4-ZASP/Cyphers co-precipitated with GFP-PGM1 C-terminus were detected by using anti-GAL4 antibody (top panel). Expressed amounts of GAL4-ZASP/Cypher (middle panel) and GFP-PGM1 C-terminus (lower panel) were confirmed by immunoblotting of whole-cell supernatants. Dashes indicate no GAL4- or GFP-tagged proteins. Binding pairs were WT-PGM1 with WT-, D117N-, K136M-, A147T-, and A165V-ZASP/Cypher exon 6 in (A), or with WT- and I345M-ZASP/Cypher exon 10 in (B). (C) Densitometric analysis of GAL4-blotting data obtained in (B). Data for WT-PGM1 with WT- ZASP/Cypher were arbitrarily defined as 1.00 AU and data were represented as the mean ± SE (n = 4 for each case). *P < 0.001 vs. WT.

3.4 Distribution of PGM1 in cardiomyocytes cultured under stress conditions

To investigate the localization of PGM1 in cardiomyocyte, we analysed the distribution of GFP signals in neonatal rat cardiomyocytes transfected with GFP-fused full-length PGM1 WT under standard (10% FBS/glucose) and stress (FBS-free/glucose or 10% FBS/glucose-free) culture conditions (Figure 4A and B). GFP-PGM1 WT showed diffuse localization in the cytoplasm and did not co-localize with ZASP/Cypher under standard culture conditions for 24 h (Figure 4A, a–c) and 72 h (Figure 4B, a–c). Under stressed culture conditions (FBS-free or glucose-free) for 24 h, GFP-PGM1 WT also showed diffuse localization in the cytoplasm, whereas ZASP/Cypher was assembled in the striated pattern at the Z-discs (Figure 4A, d–f and g–i). In addition, it was observed in ∼60% of transfected cardiomyocytes cultured in FBS-free/glucose medium for 72 h that ZASP/Cypher showed a dot-like pattern and GFP-PGM1 WT was still diffusely distributed in the cytoplasm (data not shown). In contrast, ∼40% of the transfected cardiomyocytes showed well-organized Z-discs (as represented by localization of ZASP/Cypher) under serum-free condition during the 72 h and GFP-PGM1 WT was assembled in the striated pattern at the Z-discs in about half of the transfected cardiomyocytes with well-organized Z-discs (Figure 4B, d–f). After 72 h, under 10% FBS/glucose-free condition, ZASP/Cypher localized at Z-discs as a striated staining pattern in >95% of transfected cardiomyocytes, albeit that atrophic features were observed in 10% of all cardiomyocytes, and GFP-PGM1 WT signals were observed in the cytoplasm with a few striated pattern in ∼40% of transfected cardiomyocytes (Figure 4B, g–i). Similar distribution of PGM1 was observed in neonatal rat cardiomyocytes transfected with VP16-tagged full-length PGM1 WT under stressed culture conditions (data not shown). Control cells expressing only GFP and VP16 showed weak intensity and diffuse localization of GFP and VP16, respectively, in the cytoplasm with striated staining pattern of ZASP/Cypher at the Z-disc under standard and stressed culture conditions (data not shown).

Figure 4

Fluorescence images of transiently expressed GFP-tagged PGM1. Neonatal rat cardiomyocytes transfected with GFP-PGM1 full-length WT (a, d, and g) were cultured under 10% FBS/glucose (a–c), FBS-free/glucose (d–f), or 10% FBS/glucose-free (g–i) conditions for 24 h (A) or 72 h (B), fixed and stained with anti-ZASP/Cypher antibody followed by secondary antibody (b, e, and h). Merged images were shown in c, f, and i. (A) Diffuse localization of GFP-PGM1 protein in the cytoplasm was observed and no strict co-localization with ZASP/Cypher was observed after 24 h culture. (B) After 72 h under FBS-free/glucose condition, GFP-PGM1 WT protein was assembled in a striated pattern at the Z-discs and co-localized with ZASP/Cypher (d–f, white arrows), whereas diffuse localization in the cytoplasm was observed under 10% FBS/glucose condition (a–c). Under 10% FBS/glucose-free condition, GFP-PGM1 WT protein showed diffuse localization with a few striated pattern (co-localization with ZASP/Cypher, white arrows) in the cytoplasm (g–i); scale bars = 10 µm.

Increased recruitment and binding of endogenous PGM1 to endogenous ZASP/Cypher in the Z-disc under stressed culture conditions was investigated in neonatal rat cardiomyocytes by using immunofluorescence analysis and co-IP assay. The immunofluorescence analysis showed co-localization of endogenous PGM1 and ZASP/Cypher in the striated pattern at the Z-discs in about half of the rat cardiomyocytes with well-organized Z-discs under serum-free culture condition for 72 h, whereas endogenous PGM1 was diffusely distributed in the cytoplasm under standard culture conditions (Figure 5A). Under glucose-free culture conditions, endogenous PGM1 was observed in the cytoplasm and weakly at the Z-discs in ∼40% of the cardiomyocytes (Figure 5A, g–i). Western blot analyses of immunoprecipitates, using whole-cell lysates extracted by NP-40 from the cardiomyocytes, demonstrated that ZASP/Cypher bound PGM1 in standard culture conditions, and a significantly higher amount of PGM1 was co-immunoprecipitated under the FBS-free/glucose (0.97 ± 0.08 AU, P < 0.001) and 10% FBS/glucose-free (0.43 ± 0.07 AU, P < 0.05) conditions than under the 10% FBS/glucose condition (0.23 ± 0.02 AU) (Figure 5B and C). Similar amounts of PGM1 in the NP-40 un-extracted fraction (debris fraction) in stressed and standard culture conditions were observed (data not shown). These data demonstrated that the amounts of endogenous PGM1 in the cytoplasm were large and a part of them was tethered to ZASP/Cypher more efficiently under stress.

Figure 5

Localization and co-IP of endogenous PGM1 and ZASP/Cypher from neonatal rat cardiomyocytes. (A) Neonatal rat cardiomyocytes were cultured in 10% FBS/glucose (a–c), FBS-free/glucose (d–f), or 10% FBS/glucose-free (g–i) conditions for 72 h, fixed and stained with anti-PGM1 (a, d, and g) and anti-ZASP/Cypher antibody (b, e, and h) followed by secondary antibody. Merged images are shown in c, f, and i. Under FBS-free/glucose condition, endogenous PGM1 protein was assembled into striated patterns at the Z-discs and co-localized with ZASP/Cypher (d–f, white arrows), whereas diffuse localization in the cytoplasm was observed under standard 10% FBS/glucose condition (a–c). Under 10% FBS/glucose-free conditions, endogenous PGM1 showed a few striated pattern (co-localization with ZASP/Cypher, white arrows) in the cytoplasm (g–i); scale bars = 10 µm. (B) Neonatal rat cardiomyocytes were cultured in 10% FBS/glucose, FBS-free/glucose, or 10% FBS/glucose-free conditions for 72 h. Endogenous PGM1 could be co-immunoprecipitated with endogenous ZASP/Cypher (top panel). Expressions of endogenous PGM1 (middle panel) and ZASP/Cypher (lower panel) were confirmed by immunoblotting of whole-cell lysates extracted by NP-40. (C) Densitometric analysis of PGM1-blotting data in (A). Data are expressed as mean ± SE. (n = 4–6 for each case). *P < 0.001 vs. WT. **P < 0.05 vs. WT.

To map the domain of PGM1 involved in the Z-disc localization, we examined the distribution of N- or C-terminus of PGM1 (aa 1–426 or aa 427–562 containing PGM/PMM domains I–III or IV, respectively) and endogenous ZASP/Cypher under standard or stressed (during 72 h with FBS-free/glucose) culture conditions. Under both conditions, similar diffuse localization of GFP-PGM1 N-terminus in the cytoplasm was observed (Figure 6, a–c and d–f). On the other hand, co-localization of GFP-tagged PGM1 C-terminal protein with ZASP/Cypher was found in ∼60% of cardiomyocytes (Figure 6, g–i and j–l), although a part of the GFP signals fused to PGM1 C-terminus was not co-localized with ZASP/Cypher (Figure 6, g and j). These data indicate that the PGM/PMM domain IV of PGM1 was indispensable for recruitment to the Z-disc under stress conditions and the N-terminal domains of PGM1 inhibited recruitment under standard conditions.

Figure 6

Fluorescence images of transiently expressed N- or C-terminus of PGM1 tagged by GFP. Neonatal rat cardiomyocytes transfected with GFP-PGM1 N-terminus (aa 1–426, a–c and d–f) or C-terminus (aa 427–562, g–i and j–l) were cultured under 10% FBS/glucose (a–c and g–i) or FBS-free/glucose (d–f and j–l) conditions for 72 h, fixed and stained with anti-ZASP/Cypher antibody followed by secondary antibody (b, e, h, and k). Merged images are shown in c, f, i, and l. Under both 10% FBS/glucose and FBS-free/glucose conditions, diffuse localization of GFP-PGM1 N-terminus protein in the cytoplasm was observed (a–c and d–f), whereas GFP-PGM1 C-terminus protein was assembled in a striated pattern at the Z-discs and co-localized with ZASP/Cypher (g–i and j–l, white arrows). GFP-PGM1 C-terminus protein that was not co-localized with ZASP/Cypher was also detected in the cytoplasm of cardiomyocytes (white arrowheads); scale bars = 10 µm.

4. Discussion

In this study, we identified PGM1 as a protein that binds to ZASP/Cypher. The most important finding was that PGM1 bound to different domains encoded by exons 4, 6, and 10 of LDB3; DCM-associated mutations found in exons 4 and 10 impaired the binding, whereas other mutations found in exon 6 did not. We have previously demonstrated that exons 4 and 10 are preferentially expressed in the heart, whereas the expression of exon 6 was not confined to the heart.8 Therefore, it was speculated that mutations in the heart-specific domains of ZASP/Cypher were associated with DCM via impaired binding to PGM1. The region encoded by exon 4 consisted of 122 aa residues with a high percentage of proline (15.6%), serine (13.9%), and alanine (12.3%), and there were several proline-rich motifs similar to the core-binding target (consensus was P) of the Src homology-3 (SH3) domain. Although the SH3 domain is known to be involved in protein–protein interaction, it is unlikely to participate in the binding of ZASP/Cypher and PGM1, because PGM1 has no SH3 domain. We also revealed the binding of PGM1 with another heart-specific region encoded by exon 10 of LDB3. The region was composed of 62 aa residues that are rich in proline (19.4%), serine (16.1%), threonine (12.9%), and alanine (30.6%). The region also contains the proline-rich motif, which could be the target of the PGM/PMM domain IV. However, such a proline-rich motif was not found in the region encoded by exon 6.

PGM is a key enzyme in cellular glucose utilization and energy homeostasis. There are several PGM isoforms encoded by different, but structurally related, genes: PGM1 to PGM5, and PGM1 predominate, representing ∼90% of total PGM activity, in most cell types, except in red blood cells where PGM2 is a major enzyme and in milk where PGM4 is the sole isoenzyme.22,23 In this study, we found abundant expression of PGM1 in the heart at a similar level as in the skeletal muscles and liver, two major tissues for glycogen storage and metabolism. This finding is in agreement with the view that the working heart requires a continuous supply of energy to support its contractile activity and the pathway of glucose metabolism (glucose uptake and phosphorylation, glycolysis, and glucose oxidation) is important to produce an essential fuel under both normal and stress conditions in the heart.24,25 Quite interestingly, PGM1 showed translocation from cytoplasm to the Z-discs in cardiomyocytes under cultured conditions of stress.

We found that PGM1 bound to ZASP/Cypher in the Z-discs and the binding was enhanced under conditions of stress. The mechanism for the enhanced binding was not clear, but there are several possibilities: phosphorylation of PGM1 and/or ZASP/Cypher, isoform change of ZASP/Cypher or alterations in another protein that binds or blocks the potential binding site on one of the proteins. Because the function of PGM1 at the Z-discs needs to be elucidated and it is unknown how the depletion of serum or glucose from the culture medium could mimic the stress conditions purportedly related to heart failure, our study had a limitation, in that the translocation of PGM1 into Z-discs and its binding to ZASP/Cypher would have a functional significance in heart failure. However, one could hypothesize that PGM1 at the Z-discs might relieve damage to cardiomyocytes under conditions of stress, and hence, the impaired binding might predispose early progression to heart failure. In this regard, it is worth noting that a missense mutation of FHL2, encoding FHL2 protein, found in DCM altered the binding of FHL2 to the N2B region of titin/connectin.19 Because FHL2 tethers metabolic enzymes, including phosphofructokinase which is another glycolytic enzyme, it is suggested that mutations in LDB3 and FHL2 lead to impaired recruitment of the glycolytic enzymes to the Z-disc and I-band, respectively, and result in cardiac dysfunction. Because it is not clarified how PGM1 was recruited into the Z-discs under stressed culture conditions and whether the whole glycolysis complex or other glycolytic enzymes would be distributed to the Z-discs, further studies are needed to reveal the functional role of ‘Z-disc PGM1’ and other glycolysis-related proteins, if any, at the Z-discs under stress conditions.

It was reported that PGM1 deficiency was associated with an infantile muscle glycogen storage disease accompanied by systemic carnitine deficiency and the disease phonotype was improved by oral carnitine administration.26 Because systemic carnitine deficiency is well known to cause cardiomyopathy in addition to skeletal myopathy in children,27 it is of interest to investigate the PGM1 gene mutations in DCM. Our preliminary study revealed that there are several DCM patients carrying mutations in PGM1. However, most of the mutations were also found in healthy controls, demonstrating that they were polymorphisms (data not shown). Some PGM1 mutations found in the patients were not observed in the 200 healthy controls, but none of them were mapped within the domain required for binding to ZASP/Cypher. Further functional studies are required to investigate the role of PGM1 mutations, if any, in DCM.

In summary, we identified PGM1 as a binding partner of ZASP/Cypher and showed that DCM-associated mutations impaired the interaction. Although the molecular function of PGM1 at the Z-discs remains to be elucidated, our findings imply that DCM may be associated with the abnormal recruitment of PGM1 at the Z-disc under stress.

Funding

This work was supported in part by Grant-in-Aids from the Ministry of Education, Culture, Sports, Science and Technology, Japan, a grant for Japan–Korea collaboration research from the Japan Society for the Promotion of Science (JSPS), a research grant from the Ministry of Health, Labour and Welfare, Japan, a research grant from the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), and research grants from the Japan Heart Foundation and the ‘Association Francaise contre les Myopathies’ (AFM) [Grant No. 11737].

Acknowledgements

We are grateful to H. Shibata and M. Takahashi for their contribution in the initial stages of this work. We also thank Ms M. Emura and Ms A. Nishimura for their technical assistance.

Conflict of interest: none declared.

Footnotes

  • These authors contributed equally to this work.

References

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