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Am J Physiol Heart Circ Physiol 293: H1581-H1589, 2007. First published May 25, 2007; doi:10.1152/ajpheart.00278.2007
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Histidine-rich Ca-binding protein interacts with sarcoplasmic reticulum Ca-ATPase

Demetrios A. Arvanitis,1 Elizabeth Vafiadaki,1 Guo-Chang Fan,2 Bryan A. Mitton,2 Kimberly N. Gregory,2 Federica Del Monte,3 Aikaterini Kontrogianni-Konstantopoulos,4 Despina Sanoudou,1,* and Evangelia G. Kranias1,2,*

1Molecular Biology Division, Center for Basic Research, Foundation for Biomedical Research of the Academy of Athens, Athens, Greece; 2Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati, Ohio; 3Department of Cardiology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts; and 4Department of Physiology, School of Medicine, University of Maryland Baltimore, Baltimore, Maryland

Submitted 6 March 2007 ; accepted in final form 21 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Depressed cardiac Ca cycling by the sarcoplasmic reticulum (SR) has been associated with attenuated contractility, which can progress to heart failure. The histidine-rich Ca-binding protein (HRC) is an SR component that binds to triadin and may affect Ca release through the ryanodine receptor. HRC overexpression in transgenic mouse hearts was associated with decreased rates of SR Ca uptake and delayed relaxation, which progressed to hypertrophy with aging. The present study shows that HRC may mediate part of its regulatory effects by binding directly to sarco(endo)plasmic reticulum Ca-ATPase type 2 (SERCA2) in cardiac muscle, which is confirmed by coimmunostaining observed under confocal microscopy. This interaction involves the histidine- and glutamic acid-rich domain of HRC (320–460 aa) and the part of the NH2-terminal cation transporter domain of SERCA2 (74–90 aa) that projects into the SR lumen. The SERCA2-binding domain is upstream from the triadin-binding region in human HRC (609–699 aa). Specific binding between HRC and SERCA was verified by coimmunoprecipitation and pull-down assays using human and mouse cardiac homogenates and by blot overlays using glutathione S-transferase and maltose-binding protein recombinant proteins. Importantly, increases in Ca concentration were associated with a significant reduction of HRC binding to SERCA2, whereas they had opposite effects on the HRC-triadin interaction in cardiac homogenates. Collectively, our data suggest that HRC may play a key role in the regulation of SR Ca cycling through its direct interactions with SERCA2 and triadin, mediating a fine cross talk between SR Ca uptake and release in the heart.

triadin; calcium cycling

DIASTOLIC DYSFUNCTION, associated with impaired cardiomyocyte relaxation, is a common hallmark of all forms of heart failure. The depressed relaxation reflects abnormal Ca sequestration by the sarcoplasmic reticulum (SR) because of decreased sarco(endo)plasmic reticulum Ca-ATPase (SERCA) type 2 (SERCA2) protein levels and/or increased inhibition by its regulatory phosphoprotein phospholamban (5, 14, 24). Indeed, SERCA2a overexpression and phospholamban inhibition remarkably improved myocyte dysfunction and/or remodeling in experimental heart failure (7, 8, 11, 30), as well as in human failing cardiomyocytes (6). The attenuated SR Ca uptake during relaxation, coupled with aberrant ryanodine receptor Ca release in heart failure (28, 29, 42), results in decreased SR Ca load and deteriorated cardiac function.

Ca storage in the SR is mediated by calsequestrin and the 170-kDa histidine-rich Ca-binding protein (HRC) (15, 25). Both proteins exhibit high capacity but low affinity for Ca (3, 15, 32). Biochemical studies in skeletal muscle have shown that HRC binds directly to triadin in a Ca-dependent manner. Increases in Ca concentrations disrupt the HRC-triadin interaction, suggesting a regulatory role for HRC in SR Ca release (25, 33, 34). There is a single isoform of HRC (13, 26, 28, 31), and its deduced amino acid sequence reveals structural similarities to calsequestrin, which indicates a Ca storage role for this protein during excitation-contraction coupling (25). The function of HRC is further supported by the increased SR Ca storage capacity in rat neonatal or adult cardiomyocytes upon acute overexpression of HRC (10, 19). Furthermore, in vivo overexpression of HRC in mouse hearts resulted in delayed cytoplasmic Ca decline and depressed cardiomyocyte SR Ca uptake, which progressed to cardiac hypertrophy with aging (12). These findings suggested that HRC may also regulate SR Ca sequestration and cardiac relaxation (12). However, it is not clear whether the inhibitory effects of HRC involve a direct physical binding of this protein to the SR Ca transport proteins or indirect mechanisms.

The present study demonstrates for the first time that HRC binds directly to SERCA2 in human and mouse cardiac homogenates. Furthermore, the region of human HRC that binds SERCA is different from the HRC-binding domain to triadin. Importantly, increasing concentrations of Ca attenuated the HRC-SERCA interaction, whereas it enhanced the HRC-triadin binding interaction in a dose-dependent manner. Thus HRC appears to play an important role in the fine tuning of SR Ca uptake and release, linking the two processes in the lumen of the SR membrane through its binding partners.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue specimens and protein preparation. Cardiac homogenates from wild-type mice or postmortem human specimens were prepared using an Ultra-Turrax Tissuemizer (IKA-Werke, Staufen, Germany) in ice-cold lysis buffer (10 mM NaPO4, pH 7.2, 2 mM EDTA, 10 mM NaN3, 120 mM NaCl, and 1% Nonidet P-40) supplemented with a mixture of protease inhibitors (Sigma-Aldrich Chemie, Munich, Germany) (22). The Quant-iT protein assay kit (Molecular Probes/Invitrogen Labeling and Detection, Eugene, OR) was used to determine protein concentration on a Qubit fluorometer (Invitrogen) according to the manufacturer's instructions. The homogenates were aliquoted and stored at –80°C. The research protocol was approved by the institutional ethical committee and conformed to the principles outlined in the Declaration of Helsinki (1).

Coimmunostaining. Single cardiomyocytes were isolated from adult mouse hearts as described previously (12) and then attached to Poly-Prep slides (Sigma-Aldrich Chemie) for 30 min. After they were rinsed briefly in PBS, these cardiomyocytes were fixed by immersion in 3.7% paraformaldehyde solution (containing 0.5% Triton X-100) for 30 min. Staining was performed with polyclonal rabbit anti-HRC [1:1,000 dilution; a generous gift from Dr. Woo Jin Park (25)] followed by Alexa Fluor 488 goat anti-rabbit IgG (1:500 dilution; Invitrogen, Carlsbad, CA) and with mouse anti-SERCA2 (1:100 dilution; Affinity Bioreagents, Golden, CO) followed by Alexa Fluor 594 goat anti-mouse IgG (1:500 dilution; Invitrogen) antibodies. These slides were analyzed under a laser scanning confocal microscope (model LSM 510, Carl Zeiss MicroImaging, Thornwood, NY).

Coimmunoprecipitation experiments. For the coimmunoprecipitation experiments, protein lysates, extracted from mouse cardiac homogenates, were precleared with prewashed protein G-agarose beads (Roche, Indianapolis, IN) overnight on a rotary wheel at 4°C. The anti-HRC and anti-SERCA2 (polyclonal rabbit anti-SERCA) (10) antibodies were separately conjugated to prewashed protein G-agarose beads overnight (1 µl antibody/10 µl agarose beads). Subsequently, antibody-bound beads were incubated with 0.5 mg of precleared protein lysates overnight on a rotary wheel at 4°C. Immunoprecipitates were collected, washed three times in 0.2% Triton X-100, 20 mM Tris-Cl (pH 7.4), and 0.15 M NaCl solution, solubilized in 2x SDS sample buffer, boiled at 90°C for 5 min, analyzed by SDS-PAGE, and processed for immunoblotting.

Immunoblotting. SDS-PAGE and immunoblotting analysis were performed as previously described (13). Briefly, samples analyzed by SDS-PAGE were transferred to nitrocellulose membranes (Schleicher & Schuell Bioscience, Dassel, Germany). The membranes were incubated with one of the following primary antibodies: polyclonal rabbit anti-HRC (1:5,000 dilution) (25), polyclonal rabbit anti-SERCA2 (1:5,000 dilution) (10), or monoclonal mouse anti-SERCA2 (1:1,000 dilution; Affinity Bioreagents), monoclonal mouse anti-triadin (1:1,000 dilution; Abcam, Cambridge, UK), rabbit anti-glutathione S-transferase (GST, 1:2,500 dilution; Amersham Biosciences Europe, Uppsala, Sweden), or rabbit anti-maltose binding protein (MBP, 1:10,000 dilution; New England Biolabs, Beverly MA). The nitrocellulose membranes were subsequently washed in 50 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20 and then incubated with a peroxidase-conjugated anti-mouse (1:14,000 dilution; Sigma-Aldrich Chemie) or anti-rabbit (1:33,000 dilution; Amersham Biosciences Europe) secondary antibody. Protein signals were detected using electrogenerated chemiluminescence (ECL) reagents according to the manufacturer's protocol (Amersham Biosciences Europe). The intensities of the bands of interest from at least three different experiments were quantified using Image J software (version 1.33a).

Construction of recombinant proteins. For the generation of human HRC, SERCA2a, and triadin constructs, total RNA was extracted from human postmortem nonfailing left ventricular heart tissue using TRIzol (Invitrogen) and reverse transcribed by Superscript II RNase H reverse transcriptase (Invitrogen) to cDNA, which was used as the template for PCR amplification. PCR products were digested with EcoR I (Takara Bio, Otsu, Shiga, Japan) and Sal I (Takara Bio) and subcloned into EcoR I/Sal I cloning sites of pGEX-5x-1 (Amersham Biosciences Europe) or pMAL-c2X vector (New England Biolabs). The following primer sets were used for amplification: 5'-GAA TTC CAC CAC CTC CAC AGC CCT-3' and 5'-GTC GAC CTC ATC CTC TTC ACT C-3' for HRC-A (59–225 aa residues), 5'-GAA TTC GGG AGT GAA GAG GAT GA and 5'-GTC GAC GTG GGC CTG GTG TCC A-3' for HRC-B (220–320 aa), 5'-GAA TTC GTC TCC ACT GAG TAT GGA-3' and 5'-GTC GAC GTG TCC TGG GGG GTG A-3' for HRC-C (310–468 aa), 5'-GAA TTC GAG ATG AGC CAT CAC C-3' and 5'-GTC GAC CTC ACC GCT TTC CTC CT-3' for HRC-D (460–608 aa), 5'-GAA TTC GCC TCC AGC GAG GAG GA-3' and 5'-GTC GAC GGG TTC CGG CGT TTC CA-3' for HRC-E (600–699 aa), 5'-GAA TTC GTT TTG GCT TGG TTT G-3' and 5'-GTC GAC TTC TAC AAA GGC TGT A-3' for SERCA2a-A (74–90 aa), 5'-GAA TTC AAT ATT GGG CAC TTC A-3' and 5'-GTC GAC GTA GTA AAT AGC ACC T-3' for SERCA2a-B (275–295 aa), 5'-AA TTC GCC CTT GGA TTT CCC GAG GCT TTG-3' and 5'-TC GAC CAA AGC CTC GGG AAA TCC AAG GGC-3' for SERCA2a-C (779–786 aa), 5'-GAA TTC TGG TGG TTC ATT GCT GCT GAC-3' and 5'-GTC GAC GGA TTC AAA GAT TGC ACA-3' for SERCA2a-D (853–892 aa), 5'-AA TTC CCC TTG CCA CTC ATC TTC CAG ATC ACA CCG-3' and 5'-TC GAC CGG TGT GAT CTG GAA GAT GAG TGG CAA GGG-3' for SERCA2a-E (951–960 aa), 5'-GAA TTC GAT TTA GTG GAT TAC A-3' and 5'-GTC GAC TGT AAC TTT AGT TTG T-3' for triadin-A (69–163 aa), 5'-GAA TTC GAT GAA GAA GAT GAT GA-3' and 5'-GTC GAC TTT TTC CTT GTG AGT TGC T-3' for triadin-B (129–189 aa), and 5'-GAA TTC GTT ACA CAC AAA GAA-3' and 5'-GTC GAC ATC TTT CTG TTC ATG CT-3' for triadin-C (164–265 aa). For the generation of SERCA2a-C and SERCA2a-E, sense and antisense primer pairs were allowed to anneal at 70°C for 10 min in 500 mM Tris·HCl (pH 8), 100 mM MgCl2, and 500 mM NaCl. After these DNA fragments were prepared and digested with EcoR I and Sal I, they were subcloned in the pGEX-5x-1 and pMAL-c2X vectors, and these clones were verified by direct sequencing (Macrogen, Seoul, Korea).

GST and MBP recombinant polypeptides were generated after induction with 0.5 mM isopropyl-beta-D-thiogalactopyranoside for 3 h in BL21 Star (DE3, Invitrogen) and library efficient DH5a (Invitrogen) Escherichia coli strains, respectively, according to the manufacturers' instructions. Recombinant polypeptides were purified by affinity chromatography with glutathione-Sepharose 4B for GST fusion proteins (Amersham Biosciences Europe) or amylose resin for MBP fusion proteins (New England Biolabs).

Pull-down assays. Pull-down assays were performed as previously described (22). Briefly, equal amounts of recombinant GST, GST-HRC-A (59–225 aa), GST-HRC-B (220–320 aa), GST-HRC-C (310–468 aa), GST-HRC-D (460–608 aa), and GST-HRC-E (600–699 aa) fusion proteins bound to glutathione-Sepharose 4B (Amersham Biosciences Europe) were incubated at 4°C for 16 h with 0.5 mg of whole left ventricular cardiac extracts. Samples were washed twice with 10 mM NaPO4, pH 7.2, 120 mM NaCl, 1 mM NaN3, and 0.1% Tween 20 at 4°C and analyzed by 10% SDS-PAGE and immunoblotting.

Titration with Ca. The effect of alterations in free Ca concentration on the HRC-SERCA2 or HRC-triadin interaction was studied in protein homogenates incubated with 10–8–10–3 M CaCl2 at room temperature for 5 min in 150 µl of reaction buffer (20 mM Tris·HCl, pH 6.8, 100 mM KCl, 5 mM MgCl2, 5 mM ATP, 1 mM EGTA, and 5 mM potassium oxalate), as described elsewhere (2). Coimmunoprecipitation and GST pull-down assays were then performed (see above).

Blot-overlay assays. Blot-overlay assays were performed as previously reported with minor modifications (23). Briefly, ~2.5 µg of bacterially expressed, affinity-purified MBP, MBP-SERCA2a-A (74–90 aa), MBP-SERCA2a-B (275–295 aa), MBP-SERCA2a-C (779–786 aa), MBP-SERCA2a-D (853–892 aa), and MBP-SERCA2a-E (951–960 aa), as well as MBP-triadin-A (69–163 aa), MBP-triadin-B (129–189 aa), and MBP-triadin-C (164–265 aa), fusion proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. Nonspecific sites on the nitrocellulose membranes were blocked in buffer A (50 mM Tris, pH 7.2, 140 mM NaCl, 0.1% Tween 20, 5% nonfat milk, 2 mM DTT, and 0.5% Nonidet P-40) plus protease inhibitors for 3 h at 25°C and then incubated with 3 µg/ml of GST-HRC-C (310–468 aa) or GST-HRC-E (600–699 aa) fusion proteins in the same buffer in the presence of 1 mM ATP for 16 h at 4°C. Blots were washed five times (15 min each) with buffer A and once with buffer B (1x PBS, pH 7.2, 10 mM NaN3, and 0.1% Tween 20). After nonspecific reactive sites were blocked in buffer C (1x PBS, pH 7.2, 10 mM NaN3, 0.1% Tween 20, and 3% nonfat milk), membranes were probed with rabbit anti-GST antibody (Amersham Biosciences Europe) diluted in buffer C. Immunoreactive bands were visualized using ECL reagents (Amersham Biosciences).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
HRC binds to SERCA2. HRC overexpression in transgenic mouse hearts has been reported to result in a 35% reduction of the maximal velocity of SR Ca uptake (12). To investigate whether this inhibitory effect may be mediated by a physical interaction between HRC and SERCA2, we used antibodies to HRC or SERCA2 to generate immunoprecipitates from homogenates of wild-type adult mouse hearts (Fig. 1A) and human failing hearts (Fig. 1B). Both immunoprecipitates contained bands at ~165 and ~115 kDa that reacted with anti-HRC (Fig. 1, A and B, top) and anti-SERCA2 (Fig. 1, A and B, bottom) antibodies, respectively. For negative controls, protein samples were precleared and incubated with fresh beads without specific antibodies (data not shown) or empty protein G-agarose beads (Fig. 1A, lane 3). The coimmunoprecipitation of SERCA2 and HRC is consistent with their presence in a complex within the myoplasm of mouse and human cardiac myofibers. Furthermore, coimmunostaining of adult wild-type mouse cardiomyocytes with HRC and SERCA2 antibodies clearly demonstrated their colocalization (Fig. 1C).


Figure 1
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  		Fig. 1. In vivo binding of histidine-rich Ca-binding protein (HRC) and sarco(endo)plasmic reticulum Ca-ATPase type 2 (SERCA2). A: immunoprecipitations (IP) with polyclonal rabbit anti-HRC antibodies coupled to protein G-agarose beads and precleared mouse cardiac homogenates. Lane 1, mouse cardiac homogenates precleared with protein G-agarose beads; lane 2, immunoprecipitates generated with HRC antibodies; lane 3, immunoprecipitates generated with empty protein G-agarose beads (negative control). B: coimmunoprecipitations with anti-SERCA2 or anti-HRC antibodies in human failing heart homogenates obtained from 4 different individuals (lanes 1–4). C: coimmunostaining with anti-HRC (green) and anti-SERCA2 (red) antibodies in adult mouse cardiomyocytes.

 
Determination of the interacting domains of HRC with SERCA2a and triadin. To identify the minimal domain of human HRC required for binding to human SERCA2a, we performed a series of pull-down assays. We generated a series of overlapping HRC constructs fused to GST (Fig. 2, A and B) : 1) the NH2-terminal histidine-rich and histidine/glutamic acid-rich region (HRC-A, 59–225 aa), 2) the histidine/glutamic acid/aspartic acid-rich domain (HRC-B, 220–320 aa), 3) the histidine/glutamic acid-rich region (HRC-C, 310–468 aa), 4) the glutamic acid-rich region (HRC-D, 460–608 aa), and 5) the COOH-terminal cysteine-rich domain (HRC-E 600–699 aa), with estimated molecular sizes of ~45, ~37.5, ~45, ~43, and ~37 kDa, respectively (Fig. 2B). Equivalent amounts of GST-HRC fusion peptides, along with control GST protein (~25 kDa) bound to glutathione matrices, were allowed to interact with human cardiac homogenates and subsequently tested for their ability to adsorb native SERCA2a by Western blotting using the appropriate antibodies. These experiments demonstrated that only HRC-C (310–468 aa) specifically retained native SERCA2a (Fig. 2C). None of the other GST-HRC constructs or the control GST protein adsorbed SERCA2a from adult cardiac homogenates (Fig. 2C). It therefore appears that the 320- to 460-amino acid region of HRC contains the binding site for SERCA2a.


Figure 2
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  		Fig. 2. Different HRC domains bind SERCA2 and triadin. A: schematic representation of human HRC constructs (A–E) that were produced as GST-fusion peptides. B: GST-HRC fusion proteins A–E, with estimated molecular sizes of 45.0, 37.5, 45.0, 43.0, and 37.0 kDa, respectively, and control GST protein were analyzed by SDS-PAGE and visualized by staining with Coomassie blue. Equivalent amounts of GST-HRC recombinant proteins bound to glutathione matrices were incubated with homogenates of adult human cardiac muscle. C: binding of native SERCA2. D: triadin (TRDN) examined by immunoblot analysis. GST-HRC-C specifically retained endogenous SERCA2, whereas GST-HRC-E specifically precipitated native triadin.

 
The presence of triadin, a known binding partner of HRC in rabbit cardiac muscle fibers (25, 34), was also examined in precipitates generated by GST-HRC fusion peptides after incubation with human cardiac homogenates, since the rabbit and human HRC homologs are substantially diverse (16). Notably, HRC-E (600–699 aa), but not GST or any of the other GST-HRC polypeptides, was able to adsorb native triadin from human cardiac homogenates (Fig. 2D). The multiple bands detected by the triadin antibody may represent different triadin isoforms (21, 36). Thus it appears that distinct domains within HRC mediate binding to SERCA2a and triadin in the human heart; specifically, the histidine/glutamic acid-rich motif (310–468 aa) supports binding to SERCA2a, whereas the cysteine-rich domain (600–699 aa) mediates binding to triadin.

Identification of the minimal interacting domains between HRC, SERCA2a, and triadin. To determine whether HRC binds directly to SERCA2a and to characterize the minimal interacting domains of human SERCA2a or triadin to HRC, we generated a series of successive constructs for both proteins, which were fused to MBP.

SERCA2a consists of 10 SR membrane-spanning helices, 6 stretches composed of 48, 132, 437, 48, 17, and 12 amino acids that are projected toward the cytosol, and 5 stretches containing 16, 20, 7, 39, and 9 amino acids that are projected in the SR lumen (9, 37, 39). Since HRC is located in the SR lumen (15, 25), we focused our studies on the five SERCA2a amino acid stretches that are projected in the SR lumen. These were expressed as MBP fusion peptides in E. coli. In particular, SERCA2a-A (74–90 aa) contains part of the NH2-terminal cation transporter motif and SERCA2a-B (275–295 aa) contains part of the E1-E2 enzyme form, whereas SERCA2a-C (779–786 aa), SERCA2a-D (853–892 aa), and SERCA2a-E (951–960 aa) contain portions of the COOH-terminal domain of the cation transporter ATPase (Fig. 3A). Equal amounts of the aforementioned MBP-SERCA2a peptides, with molecular sizes of ~44.5, ~45.0, ~43.3, ~47.1, and ~43.5 kDa, respectively (Fig. 3B), along with control MBP, were analyzed by SDS-PAGE and transferred to nitrocellulose. The membranes were overlaid with affinity-purified GST-HRC-C (310–468 aa) and immunoprobed with anti-GST. GST-HRC-C bound specifically and efficiently to MBP-SERCA2a-A (74–90 aa), but not to any of the other MBP-SERCA2a fusion peptides or control MBP (Fig. 3C). These findings indicate that the COOH-terminal histidine- and glutamic acid-rich domain of HRC (310–468 aa) binds to the NH2-terminal cation transporter domain of SERCA2a (74–90 aa).


Figure 3
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  		Fig. 3. Amino acids 74–90 of the SERCA2 luminal domain interact with HRC. A: generation of 5 recombinant maltose-binding protein (MBP)-SERCA2a fusion constructs (A–E) corresponding to human SERCA2a domains that project into the sarcoplasmic reticulum (SR) lumen. B: MBP-SERCA2a fusion proteins A–E (predicted molecular sizes 44.5, 45.0, 43.3, 47.1, and 43.6 kDa, respectively) and control MBP protein were analyzed by SDS-PAGE and visualized by Coomassie blue staining. C: equivalent amounts of control MBP protein and MBP-SERCA2a fusion peptides were subjected to SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and incubated with recombinant GST-HRC-C (310–468 aa). Bound GST-HRC-C was detected by Western blotting with antibodies to GST. Binding of GST-HRC-C was specific to MBP-SERCA2a-A (74–90 aa).

 
To determine the minimal binding region of triadin to HRC, we followed a similar approach. The first NH2-terminal 69 amino acids of triadin, containing the cytoplasmic domain and the signal peptide, were excluded from the analysis because the triadin-HRC interaction within the SR lumen. Three overlapping constructs spanning amino acids 69–265 were generated: triadin-A contained residues 69–163, triadin-B included residues 129–189, and triadin-C contained residues 164–265 (Fig. 4A), with estimated molecular sizes of ~53.5, ~51.0, and ~54.5 kDa, respectively (Fig. 4B). Blot-overlay assays for these recombinant proteins were performed as described above using GST-HRC-E (600–699 aa). Immunostaining with anti-GST antibody revealed that GST-HRC-E bound specifically to MBP-triadin-C (164–265 aa), but not to the other two MBP-triadin fusion constructs or to control MBP protein (Fig. 4C). Thus the binding site for the cysteine-rich domain of human HRC is confined to amino acids 189–265 of the consensus sequence of human triadin.


Figure 4
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  		Fig. 4. KEKE motif of triadin supports binding to HRC. A: generation of 3 overlapping recombinant MBP-triadin fusion constructs (A–C) corresponding to human triadin domains that project into the SR lumen. B: MBP-triadin fusion proteins (A–C) with respective molecular sizes of 53.5, 51.1, and 54.5 kDa and control MBP were analyzed by SDS-PAGE and stained with Coomassie blue. C: equivalent amounts of recombinant MBP-triadin peptides and control MBP were analyzed by SDS-PAGE, electrotransferred to nitrocellulose membranes, and overlaid with GST-HRC-E (600–699 aa). Bound GST-HRC-E was detected by Western blotting with antibodies to GST. GST-HRC-E bound specifically and directly to MBP-triadin-C (164–265 aa), which contains the previously characterized KEKE motif.

 
Effect of Ca concentration on the HRC-SERCA2a or HRC-triadin interaction. Previous cross-linking and coimmunoprecipitation studies demonstrated that elevation of Ca from pCa 8 to pCa 5 causes a concentration-dependent decrease in the amount of phospholamban interacting with SERCA (2, 18), suggesting a critical role for Ca concentration in the regulation of interactions between these SR proteins. Moreover, a recent study reported that the affinity of HRC for triadin increases as Ca concentration rises from 10–8 to 10–3 M (33).

To determine whether Ca levels may also influence the binding of HRC to SERCA2a or triadin, we performed a series of Ca titration experiments and determined the amount of native SERCA2a or triadin that interacted with GST-HRC-C (310–468 aa) or GST-HRC-E (600–699 aa), respectively (Fig. 5A). Quantification studies indicated opposite effects of increasing Ca concentration on the HRC-C-SERCA2a and the HRC-E-triadin interactions. The peak amount of precipitated human SERCA2a by GST-HRC-C was achieved at pCa 7, whereas the peak amount of precipitated human triadin by GST-HRC-E was reached at pCa 4. The recombinant HRC-C-SERCA2a interaction was reduced by 75%, whereas the recombinant HRC-E-triadin interaction was increased by 65%, when the Ca concentration was raised from 0.1 to 100 µM (Fig. 5B; n = 3, P < 0.05).


Figure 5
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  		Fig. 5. Differential effects of Ca concentration on HRC-SERCA2 and HRC-triadin interactions in vitro. A: GST pull-down assay with GST-HRC-C (310–468 aa; top) and GST-HRC-E (600–699 aa; bottom) at different free Ca concentrations. B: quantitative assessment of HRC-SERCA2a (solid line) or HRC-triadin (dashed line) interaction. Data are expressed as percentage of maximal binding. Peak binding of HRC to SERCA2a was obtained at pCa 7, whereas peak binding of HRC to triadin was detected at pCa 4 (n = 3).

 
The findings on the HRC-SERCA2 interaction were further confirmed by a series of coimmunoprecipitation experiments in which mouse cardiac homogenates and increasing concentrations of Ca were used. The amount of HRC that was in complex with SERCA2 was dramatically reduced as Ca concentration was increased (Fig. 6A). Similar to the results we obtained from our pull-down experiments, quantitative analysis of coimmunoprecipitation experiments indicated a ~80% reduction in the amount of native HRC interacting with native SERCA at 10–3 M Ca compared with the amount obtained at 10–7 M Ca (Fig. 6B; n = 5, P < 0.05).


Figure 6
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  		Fig. 6. Coimmunoprecipitation of HRC with SERCA2 is reduced at high Ca. A: anti-SERCA2 antibody, coupled to protein G-agarose beads, was used for coimmunoprecipitation of HRC at different Ca concentrations. Top: immunoblotting with SERCA2 antibodies. Bottom: immunoblotting with HRC antibodies. B: quantitative analysis of endogenous HRC-SERCA2 interaction at different Ca concentrations. Peak binding between native HRC and SERCA2 was detected at pCa 7 (n = 5).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have identified the HRC protein as a novel binding partner of SERCA2a in mouse and human hearts. This HRC-SERCA2 interaction may contribute to the reduced rates of SR Ca uptake and impaired Ca signal decay observed in cardiomyocytes upon acute or chronic HRC overexpression (10, 12). Importantly, the binding of HRC to SERCA2 was Ca dependent, and it was attenuated upon increased Ca levels. Thus, in addition to the previously reported involvement of HRC in Ca release (10), our findings suggest that HRC may also play a regulatory role in Ca sequestration by the cardiac SR.

The domain of human HRC that binds to SERCA2 encompasses amino acid residues 320–468. This region contains 87 of the 385 charged amino acids and several of the potential Ca-binding sites of HRC. Furthermore, similar to calsequestrin, this domain may be involved in Ca-mediated HRC polymerization (29, 39). Interestingly, the minimal domain of SERCA2 required for binding to HRC contains part of its NH2-terminal region, composed of amino acid residues 74–90, which projects into the SR lumen. These 16 amino acids are identical in all SERCA1 and SERCA2 isoforms (4, 20, 27, 41). The binding of HRC to this SERCA domain, which is in close proximity to the cation transporter domain (38, 40), may regulate SR Ca sequestration, as observed in HRC-overexpressing hearts (12). Thus increases in the apparent stoichiometry of HRC-SERCA2 may impair SR Ca cycling. Indeed, the depressed Ca handling in human failing hearts may reflect, at least partially, an increase in the relative HRC-SERCA2 levels (5, 10) due to less reduction of HRC (17%) than SERCA2 (40%). This would lead to a significant (1.4-fold) increase in the relative HRC-to-SERCA2 protein ratio, which may contribute to the depressed SR Ca uptake and impaired Ca cycling in heart failure.

It has been reported that HRC binds to triadin in rabbit striated muscle (25, 33, 34). However, results regarding the minimal HRC interaction domain are contradictory. One report suggested that the 9.5 histidine and acidic-rich repeats, including amino acids 199–470 of rabbit HRC, interact with the luminal domain of triadin (25). Other studies indicated that the COOH-terminal domain, containing amino acids 569–852 of rabbit HRC, binds to the cytoplasmic region of triadin, and the 13-amino acid polyglutamic stretch of HRC is responsible for this interaction (33, 34). Although triadin is conserved among human and rabbit (87.5% identity), the sequence of HRC in these species is substantially different, with only 47.1% identity. Therefore, one could hypothesize that the interacting domains between HRC and triadin proteins in human may be different from those in rabbit. To address this issue, we used human cardiac homogenates and pull-down assays with recombinant human HRC peptides fused to GST to identify the HRC domain that is responsible for the HRC-triadin interaction. We found that the COOH-terminal cysteine-rich domain of HRC, which contains amino acids 600–699 of the human sequence, binds to native human triadin. These results are consistent with those of Sacchetto et al. (33, 34) but different from those of Lee et al. (25), suggesting that there may be multiple domains of HRC interacting with triadin. This domain is different from the previously reported 199- to 470-amino acid region (25) but is located within the 569- to 852-amino acid domain of rabbit HRC (33), which has been suggested to interact with triadin. In contrast to the rest of the HRC sequence, the cysteine-rich domain is highly conserved between human and rabbit, with 87.0% identity. The HRC-triadin interaction was also found to be Ca sensitive, with the highest binding at moderate Ca concentrations, similar to previous studies in rabbit (25, 33). Indeed, the amino acids that support binding of human triadin to HRC are located within the COOH terminus of the protein (189–265 aa), which is extensively charged and rich in acidic and basic residues. In particular, it contains 64 of the 119 (54%) charged and 23 of the 55 (42%) acidic amino acid residues of the human triadin consensus sequence. This human triadin domain also contains an EQKKAKTAEKSEEKTKKE sequence; this sequence is similar to the EEKKARTKEKIEEKTKKE (KEKE) motif of rabbit triadin, which was shown to interact with rabbit HRC (25). The KEKE motif of triadin, located in the SR lumen, has been proposed to facilitate protein-protein interactions with calsequestrin (21) and ryanodine receptor (42). Although different domains of HRC have been shown to be responsible for its binding to triadin in rabbit (25, 33, 42) and human, they appear to target a common triadin (KEKE) motif (25). This implies the possible existence of multiple HRC sites for binding to triadin. Binding of HRC to triadin may affect ryanodine receptor function through the triadin-ryanodine receptor interaction and, thus, delay the release of SR Ca, as observed in isolated HRC-overexpressing cardiomyocytes (10).

Our findings suggest that HRC may be an important modifier of SERCA2 and ryanodine receptor function through direct and indirect binding, respectively. However, the HRC interactions with SERCA2 or triadin exhibited different Ca sensitivities in vitro. Thus it may be hypothesized that, under local reduction of SR luminal Ca concentration (35) during relaxation, HRC may bind to SERCA2 and, thereby, control the rate of SR Ca uptake. As the concentration of SR Ca is rapidly restored, the HRC-SERCA2 interaction may be attenuated, resulting in dissociation of SERCA2 from HRC. HRC is released and binds to triadin, regulating Ca release through the ryanodine receptor (Fig. 7). The effects of HRC on SERCA2 and the ryanodine receptor may not alter SR Ca load, as recently shown in HRC-deficient cardiac homogenates (17). However, it remains to be determined whether the HRC-SERCA2 or HRC-triadin interactions play a role in excitation-contraction coupling of living intact cardiomyocytes.


Figure 7
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  		Fig. 7. HRC interactions in the SR lumen of cardiomyocytes. HRC may interact with SERCA2 or triadin. Focal alterations of Ca concentration in the SR lumen may alter the ability of HRC to bind SERCA2 or triadin. Consequently, HRC may play a critical role in coordinating sarcoplasmic reticulum Ca uptake and release. PLN, phospholamban.

 
In conclusion, HRC binds directly to SERCA2 and triadin through different domains, and this may regulate SR Ca sequestration and Ca release. Thus HRC may serve as a nodal point bridging these two key Ca processes in the heart. Future studies may be designed to elucidate the functional significance of these interactions under physiological and pathophysiological conditions.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported by research funds from the Foundation of Biomedical Research of the Academy of Athens, the John F. Kostopoulos Foundation, National Heart, Lung, and Blood Institute Grants HL-26057, HL-64018, and HL-77101, and the Leducq Foundation Trans-Atlantic Alliance. E. Vafiadaki and D. Sanoudou are supported by the European Union 6th Framework Program for Research and Technological Development "Life sciences, genomics and biotechnology for health" VALAPODYN Contract LSHG-CT-2006-037277.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. G. Kranias, Dept. of Pharmacology and Cell Biophysics, College of Medicine, Univ. of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0575 (e-mail: Litsa.Kranias{at}uc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* D. Sanoudou and E. G. Kranias contributed equally to this work. Back


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