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Am J Physiol Heart Circ Physiol 295: H509-H521, 2008. First published June 6, 2008; doi:10.1152/ajpheart.00368.2008
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Myocardial expression, signaling, and function of GPR22: a protective role for an orphan G protein-coupled receptor

John W. Adams,1,* Jianming Wang,2,* James R. Davis,1 Chen Liaw,1 Ibragim Gaidarov,1 Joel Gatlin,1 Nancy D. Dalton,2 Yusu Gu,2 John Ross, Jr.,2 Dominic Behan,1 Ken Chien,3 and Daniel Connolly1

1Arena Pharmaceuticals Incorportated, San Diego; 2University of California-San Diego, La Jolla, California; and 3Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts

Submitted 8 April 2008 ; accepted in final form 28 May 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
G protein-coupled receptors (GPCRs) play an essential role in the regulation of cardiovascular function. Therapeutic modulation of GPCRs has proven to be beneficial in the treatment of human heart disease. Myocardial "orphan" GPCRs, for which the natural ligand is unknown, represent potential novel therapeutic targets for the treatment of heart disease. Here, we describe the expression pattern, signaling pathways, and possible physiological role of the orphan GPR22. GPR22 mRNA analysis revealed a highly restricted expression pattern, with remarkably abundant and selective expression in the brain and heart of humans and rodents. In the heart, GPR22 mRNA was determined to be expressed in all chambers and was comparable with transcript levels of the β1-adrenergic receptor as assessed by Taqman PCR. GPR22 protein expression in cardiac myocytes and coronary arteries was demonstrated in the rat heart by immunohistochemistry. When transfected into HEK-293 cells, GPR22 coupled constitutively to Gi/Go, resulting in the inhibition of adenyl cyclase. No constitutive coupling to Gs or Gq was observed. Myocardial mRNA expression of GPR22 was dramatically reduced following aortic banding in mice, suggesting a possible role in response to the stress associated with increased afterload. The absence of detectable GPR22 mRNA expression in the hearts of GPR22–/– mice had no apparent effect on normal heart structure or function; however, these mice displayed increased susceptibility to functional decompensation following aortic banding. Thus, we described, for the first time, the expression pattern and signaling for GPR22 and identified a protective role for GPR22 in response to hemodynamic stress resulting from increased afterload.

heart failure; hypertrophy; orphan receptor

G PROTEIN-COUPLED RECEPTORS (GPCRs) represent the richest single class of proteins targeted by drug therapy (27). A wide variety of extracellular ligands, such as hormones, neurotransmitters, nucleotides, amino acids, peptides, lipids, and fatty acid derivatives, recognize and modulate the function of GPCRs. The four primary families of G proteins (Gs, Gi/Go, Gq, and G11/12) diverge with respect to downstream signaling molecules. For example, the modulation of intracellular cAMP via adenylyl cyclase is primarily influenced by Gi (inhibitory) and Gs (stimulatory) activities, whereas the generation of inositol triphosphate and diacylglycerol is due to Gq activity (17, 20).

Within the cardiovascular system, several GPCRs are well characterized and widely appreciated for their therapeutic value; drugs targeting GPCRs account for the majority of prescriptions for cardiovascular diseases (10). For example, seminal cardiovascular pharmacology studies have established the role of the Gs-coupled β-adrenergic receptor in the regulation of heart rate and contractility in response to catecholamines (29). GPCRs coupled to Gi oppose Gs signaling by inhibiting adenylyl cyclase. Accordingly, the activation of cardiac Gi-coupled receptors, such as the M2 muscarinic receptor, results in bradycardia and decreased cardiac contractility (14, 21, 36). Stimulation of myocardial Gi/Go-coupled receptors can also activate survival pathways in cardiomyocytes, resulting in cardioprotection in the setting of ischemia and reperfusion injury. In fact, the activation of the Gi/Go-coupled adenosine A1 receptor is thought to mediate the cardioprotective effect of adenosine observed in recent clinical studies [such as the Acute Myocardial Infarction Study of Adenosine (AMISTAD) I and AMISTAD II trials] (16, 19, 31). Through these and other mechanisms, it is clear that GPCR-mediated signaling is essential to normal heart function. Conversely, abnormalities in cardiac GPCR activity can result in pathological sequalae, leading to a variety of cardiovascular diseases, including heart failure.

GPCRs for which the natural ligands have not been identified are referred to as "orphan" receptors. It is estimated that the heart may express in the neighborhood of 100 GPCRs, of which ~40% are orphans (8). Despite the relative success of targeting GPCRs for the treatment of heart disease, heart failure remains a leading cause of global morbidity and mortality, and a greater understanding of the repertoire of cardiac GPCRs and their role in heart disease could lead to a meaningful improvement in heart failure treatment.

GPR22 is an orphan GPCR discovered using a customized search of a database of expressed sequence tags (23). While GPR22 mRNA expression has been reported in the brain (23) and more recently in the heart (15), little is known about regional or cellular cardiac expression, signaling mechanisms, or function of this receptor in the context of cardiovascular physiology. The present study demonstrates, for the first time, that GPR22 mRNA and protein are highly expressed in cardiac myocytes and coronary arteries. In addition, we generated GPR22 knockout mice that showed increased susceptibility to heart failure under conditions of hemodynamic stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
cDNA constructs. Wild-type GPR22 was obtained by PCR using genomic DNA as a template and recombinant Thermus thermophilus polymerase (Perkin-Elmer) with the buffer system provided by the manufacturer. The sense primer (S1) contained an added SmaI site, and the antisense primer (AS1) contained an added BamHI site (see Table 1). PCR was performed with 30 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1.5 min after an initial denaturation at 94°C for 5 min. After cycling, the reaction was followed by an extension time of 10 min at 72°C. The amplified 1.38-kb fragment was cloned into the EcoRV and BamHI sites of an expression vector under the control of the cytomegalovirus (CMV) promoter.


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  		Table 1. PCR primer sequences

 
Transient transfection of wild-type GPR22 resulted in weak mRNA expression. Under these conditions, it was relatively difficult to measure the functional activity of the receptor. To improve expression, we created a mutant GPR22 receptor with enhanced mRNA stability. Following the design of a full-length nucleic acid sequence encoding expression-enhanced GPR22 (see Supplemental Fig. 1),1 the entire wild-type GPR22 sequence was dissected into a series of contiguous segments of ~80–150 bp in length. Restriction sites were engineered in between two neighboring segments. Each segment consisted of one or two sets of complementary oligonucleotides that, upon annealing, would generate double-stranded DNA fragments with ends compatible with specific restriction sites. The annealed fragments were cloned into a standard bacterial cloning vector, and the sequence was confirmed by sequencing before the neighboring segment, generated similarly as described above, was inserted into the restriction sites. This process was repeated until a full-length GPR22-encoding cDNA clone was generated. The "synthetic" GPR22 (sGPR22) cDNA so generated had 59% G/C content versus 36% G/C content for native GPR22 cDNA.

Northern blots and dot blots. Total RNA was prepared from mouse ventricular tissue using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Agarose gel electrophoresis and Northern blot hybridization were performed as previously described (9). RNA was stained after transfer with methylene blue and then scanned to obtain an image to demonstrate RNA quality and loading and transfer equality between samples. Rat and mouse multitissue Northern blots were performed following the protocol recommended by the manufacturer (Clontech). For Northern blot experiments, all probes were labeled with 32P using the High Prime Random Primer DNA Labeling kit according to the manufacturer's instructions (Roche). The labeled rat or mouse GPR22 probe was hybridized to mRNA at 65°C for 18 h. Nonspecific hybridization was removed by washing at high stringency (0.1x SSC and 0.1% SDS at 55°C). Washed blots were exposed to X-ray film for 8 h at –80°C. Human and rat cDNA probes were generated by PCR from respective genomic DNA samples (Clontech) using primers S2 and AS2 (see Table 1), which were designed to anneal to GPR22 sequences conserved across the two species. PCR fragments were directionally ligated into TOPO II plasmids following the manufacturer's instructions. For Northern blots, probes were retrieved from TOPO II by restriction digestion and separated by gel electrophoresis. Similarly, the rat atrial natriuretic factor (ANF) probe was generated by PCR from rat genomic DNA using primers S3/AS3 (see Table 1) and was also ligated into the TOPO II plasmid following the manufacturer's instructions (Invitrogen). The mouse GPR22 cDNA probe was obtained by PCR amplification of the entire coding region of the mouse DNA sequence and verified by sequencing. The cDNA probe for the human sGPR22 sequence was obtained by PCR amplification of nucleotides 400–935 of sGPR22.

Human multitissue dot blots were performed according to the protocol recommended by the manufacturer (Clontech). Briefly, the full-length human GPR22 cDNA sequence corresponding to the coding region was labeled with 32P using the High Prime Random Primer DNA Labeling kit according to the manufacturer's instructions (Roche). The labeled human GPR22 probe was hybridized to mRNA on the dot blot at 65°C for 18 h. Nonspecific hybridization was removed by washing at high stringency (0.1x SSC and 0.5% SDS at 55°C). After being washed in PBS, the blot was exposed to X-ray film for 8 h at –80°C. Densitometry was performed on the hybridization signal generated from each tissue sample, which was then normalized to the signal for whole brain expression to generate the relative expression histogram. The tissue map grid for the human dot blot is provided in the online supplemental data (Table 1).

Real-time PCR. Regional expression of GPR22 mRNA in the human heart was analyzed by Taqman real-time PCR analysis of a human cardiovascular cDNA panel (Clontech). The expression of the human β1-adrenergic receptor was used as a reference GPCR. Taqman primers and probes (Applied Biosystems) for real-time PCR of human GPR22 and human β1-adrenergic receptor are described in Table 2. The primer/probe set for β-actin was obtained commercially (human ACTB endogenous control, Applied Biosystems). For each assay, a standard curve was obtained by analyzing a dilution series of plasmid cDNA standards for human GRP22 or β1-adrendergic receptor. The concentration of the plasmid cDNA was estimated by measuring the absorbance at 260 nm using a spectrophotometer, and the absolute weight of the cDNA encoding the receptor was determined. Quantitation of the amount of target in unknown samples (grams obtained) was accomplished by Sequence Detection System software (version 2.0, Applied Biosystems). Quantities of GPR22 and β1-adrenergic receptor products obtained were normalized to the β-actin PCR product obtained from the same cDNA samples measured on the same day.


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  		Table 2. Taqman primer/probe sequences

 
In situ hybridization. In situ hybidization probes were generated from the TOPO II-GPR22 (mouse) plasmid using the RiboProbe In Vitro Transcription Kit (Promega) following the manufacturer's instructions. Sense and antisense 33P-radiolabeled probes were generated by in vitro transcription by incubating linearized plasmids in transcription buffer containing RNasin (40 units), DTT (2 mM), ATP, CTP, and GTP (0.33 mM), [{alpha}-33P]UTP (Perkin-Elmer, 50 µCi, NEG307 H001MC), and the appropriate polymerase [T7 (50 units) or T3 (20 units)]. Probes were treated with DNase, purified by ethanol precipitation, and resuspended in 2x hybridization mix (8x SET, 2x Denhardt's solution, 0.4% SDS, 200 mM DTT, 500 µg/ml tRNA, 50 µg/ml polyA, and 50 µg/ml polyC). Frozen mouse heart sections (8 µm thickness) were thawed, fixed with 10% formalin, and hybridized with mouse GPR22 riboprobe as previously described (11). Labeled slides were exposed to BioMax MR film for 3 days at room temperature.

Immunohistochemistry. A polyclonal antibody directed against a peptide located in the intracellular COOH terminus of GPR22 (NH2-VEADPLPNNAVIHNSWIDPKRN-COOH, Synpep, Dublin, CA) was raised in the rabbit and affinity purified by Covance (Denver, PA) using a two-rabbit protocol and combining serum. Based on transfection experiments in COS7 cells, we determined that anti-GPR22-C1 antibody selectively recognized human and rat GPR22 (data not shown). Specificity was assessed using cells transfected with the pCMV vector alone, other nonrelated GPCRs, or preincubation of anti-GPR22-C1 antibody for 30 min with a 10-fold molar excess of the corresponding immunogenic peptide. Myosin light chain (MLC)-2v antibody was used as a marker to label cardiomyocytes in heart sections. In some experiments, rabbit universal IgG control primary antibody (Dako) was used as a control to determine nonspecific staining.

Snap-frozen heart tissues from male Sprague-Dawley rats were cryosectioned at a thickness of 8 µm and stored at –80°C. Sections were removed from the freezer and allowed to come to room temperature. Sections were fixed with formalin, and heat-induced epitope retrieval was performed through microwave irradiation (13). Immunohistochemical detection was performed using the Vectastain ABC System (Vector Laboratories) according to the manufacturer's instructions. Briefly, slides were incubated for 5 min in 0.3% H2O2-PBS and then washed in PBS. Sections were incubated with the primary antibody diluted 1:200 in PBS containing 5% normal goat serum and 0.1% Triton X-100 for 1 h at room temperature and washed two times with PBS. Sections were then incubated at room temperature for 30 min with secondary antibody solution composed of biotinylated goat anti-rabbit antibody diluted 1:200 in PBS containing 5% normal goat serum and 0.1% Triton X-100. After being washed with PBS, sections were incubated with Vectastain ABC reagents for 30 min at room temperature. Slides were rinsed and stained with Dako DAB Chromagen (K3465, Dako) and counterstained with hematoxylin QS (H3404, Vector Laboratories) for 20 s. Sections were then washed with PBS and dehydrated with ethanol before mounting media and a coverslip were applied. Pictures were taken using an upright Leica DMRA2 microscope and Open Lab software (Improvision).

Generation of the sGPR22 stable cell line. To generate a sGPR22-expressing stable cell line, HEK-293 cells were transfected with linearized pcDNA-neo-Hygro plasmids containing sGPR22 cDNA tagged at the NH2 terminus with a sequence encoding the hemaglutanin (HA) epitope. Forty-eight hours after transfection, the growth medium was supplemented with 200 µg/ml G418. Antibiotic-resistant cells were dilution cloned, and the resulting clonal cell lines were tested for receptor expression by flow cytometry using Alexa 488-conjugated anti-HA antibody 16B12 (Invitrogen).

Flow cytometry. Cells (1 x 106) were resuspended in PBS supplemented with 1% FBS and incubated with 5 µg/ml Alexa 488-conjugated anti-HA antibody 16B12 (Invitrogen) for 10 min on ice. Cells were washed twice and analyzed on a FACSScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Analysis was done on cells from the live gate using forward/sidescatter plots.

Immunocytochemistry. Transiently transfected cells or stable cell lines were plated on 12-mm glass coverslips in 24-well tissue culture plates (100,000 cells/well). Cells were fixed with 3.7% formaldehyde, washed with PBS, and blocked with 4% normal goat serum in PBS. Cells were incubated with 5 µg/ml Alexa 488-conjugated anti-HA antibody 16B12 in PBS plus 4% normal goat serum, washed with PBS, fixed, and mounted on glass slides using antifade reagent supplemented with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen). Immunofluorescence analysis was performed on a Leica DMRA2 microscope equipped with a Retiga EX charge-coupled device camera.

Inositol phosphate assay. HEK-293 cells were grown in DMEM, 10% fetal BSA, 2 mM glutamine, and 1 mM sodium pyruvate at 37°C. Cells were detached from culture dishes with 0.05% trypsin-EDTA. Cells were then seeded at 80% confluence in 200-mm culture dishes and transfected with 60 µl Lipofactamine 2000 with 16 mg plasmid/dish. Cells were collected 24 h after transfection. Transfected cells were plated at 20,000 and 40,000 cells/well in 384-well plates and incubated for 1 h at 37°C. Cells were stimulated with PBS-0.5% DMSO and reference compounds (ghrelin and substance P, Bachem) for 1 h. Lysis buffer containing LiCl was added to cells, and total inositol phosphate accumulation levels were determined using the IP-1 kit according to the manufacturer's instructions (62IP1 PEC Cisbio).

cAMP assay. cAMP accumulation in stably transfected sGPR22 cells was analyzed in 96-well microplates using an Adenylyl Cyclase Activation Flashplate Assay kit (Perkin-Elmer) following the manufacturer's protocol. Briefly, cells were treated overnight with either vehicle or 100 ng/ml pertussis toxin. Cells incubated with stimulation buffer (supplied with the assay kit) containing 100 µM IBMX were stimulated in a test plate (100,000 cells/well) for 1 h at room temperature with either vehicle or 10 µM isoproterenol. 125I-labeled cAMP diluted in detection buffer (supplied in the kit) was added, and the assay reaction was incubated further for 2 h. Plates were counted on a Wallac Microbeta counter 1450.

[35S]GTP{gamma}S binding assay. Untransfected HEK-293 cells or cells stably expressing sGPR22 were treated overnight with either vehicle or 100 ng/ml pertussis toxin. Membranes were prepared and diluted in assay buffer [20 mM HEPES (pH 7.4), 100 mM NaCl, and 1 mM MgCl2] in Wallac Scintistrip plates (15 µg/assay point) and incubated with 10 µM GDP for 10 min before the addition of [35S]GTP{gamma}S to 0.3 nM. The binding reaction was incubated for 1 h before plates were centrifugued at 4,000 rpm for 15 min at room temperature. Supernatants were aspirated, and plates were counted on a Wallac Microbeta counter 1450.

Transverse aortic constriction surgery and transthoracic echocardiography. Transverse aortic constriction (TAC) was performed on mice for two independent experiments. First, normal 6- to 8-wk-old C57BL/6 mice were subjected to TAC or sham surgery as previously described (30). After 7 days, animals from the TAC (n = 6) and sham-operated (n = 6) groups were killed, and the hearts were removed. Left ventricles (LVs) were weighed and quickly frozen in liquid nitrogen. Total RNA was isolated from each sample for Northern blot analysis of GPR22 mRNA expression. Second, homozygous knockout animals (GPR22–/–, n = 13) and their wild-type littermate controls (n = 13) were subjected to (TAC) surgery, and transthoracic echocardiography was performed as previously described (34) 24 h before TAC or sham surgery and once more 8 days after surgery. Echocardiograph evaluation and measurements were obtained by an examiner blinded to the genotype of the animals.

Targeted disruption of murine GPR22. GPR22 genomic clones were isolated by screening a mouse 129/sv genomic library (ResGen's RPCI-22 Mouse BAC Library HD Membranes, Invitrogen). The targeting vector was constructed from a plasmid containing Neo cassettes flanked by two Frt sites. To generate a floxed allele targeting construct, a 2-kb BamHI-BamHI genomic DNA fragment containing the second exon was cloned into a site flanked by two LoxP sites. A 4.7-kb XhoI-SalI upstream fragment containing exon 1 and intron 1 and a 4.6-kb ClaI-NotI downstream fragment were cloned into the vector as the 5'-arm or 3'-arm, respectively. The targeting vector was linearized with NotI and electroporated into ES cells derived from 129/sv mice. After G418 selection, homologous recombinants were identified by digesting genomic DNA with XbaI and hybridizing with a 990-bp 5'-probe and verified by hybridizing to a 930-bp 3'-probe. All fragments were generated by PCR with high-fidelity Takara LA DNA polymerase (Fisher) cloned into a PCR2.1 TA vector (Invitrogen), and the sequence was verified. The 5'-probe (990 bp) and 3'-probe (930 bp) were generated with oligonucleotides S4/AS4 and S5/AS5, respectively (see Table 1). A diphtheria toxin A (DTA) expression cassette was placed at the 5' end of the targeting construct to facilitate the rate of homologous recombination.

ES cells with a floxed GPR22 allele were injected into C57BL/6 blastocysts. Chimeras were mated with C57BL/6 females, and offspring were genotyped by PCR and Southern blot analysis of tail DNA. After germline transmission of the targeted allele, targeted mice (GPR22loxP/+) were crossed with FLPe mice, a FLPe recombinase variant under the control of the human ACTB promoter (Jackson laboratory), to delete the neomycin resistant gene. Mice carrying the GPR22loxP/+ neo(–) allele were bred into homozygosity. GPR22loxP/loxP neo(–) mice were viable and fertile, with grossly no differences from wild-type mice. Heterozygous knockout (GPR22+/–) mice were obtained by crossing GPR22loxP/+ neo(–) mice with protamine-Cre mice (24). Homozygous knockout (GPR22–/–) mice were generated by crossing two heterozygous mice. Genotypes were determined by PCR with primer triplet S6/AS6a/AS6b (see Table 1). The wild-type allele band was 485 bp (by S6/AS6a), the floxed allele gave a product of 565 bp (by S6/AS6a), and the knockout allele was 754 bp (by S6/AS6b). The FLPe transgene was determined by primer pair S7/AS7. Protamine-Cre transgene was identified by primers S8/AS8.

Statistical analysis. All results are expressed as means ± SE. We performed statistical analysis using one-way ANOVA with Tukey's or Newman-Keuls Multiple-Comparison post hoc analysis or t-tests as indicated. P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
GPR22 mRNA expression in human and rodent tissues. To examine the expression profile of the orphan receptor GPR22 in rodent and human tissues, we performed a series of dot blot, Northern blot, and in situ hybridization experiments. Consistent with previous reports, we observed abundant GPR22 mRNA expression in discreet regions of the human brain, with enrichment in the accumbens, amygdala, cerebellum, cortex, and hippocampus [Fig. 1, A and B; and online supplemental data (Table 1)] (15, 23). The highest level of GPR22 mRNA was detected in adult and fetal heart tissue. A similar expression pattern was observed by Northern blot analysis in rat and mouse tissues, with abundant mRNA levels detected in the brain and heart and no detectable expression in other peripheral tissues (Fig. 1C). Northern blot analysis revealed hybridization of the rat GPR22 probe to three distinct mRNA transcripts of different sizes (3.5, 4.5, and 6 Kb), likely reflecting mRNA transcript processing.


Figure 1
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  		Fig. 1. GPR22 mRNA expression in human and rat tissues. A: RNA dot blot analysis was performed on mRNA samples from multiple human tissues. High-level expression was detected in the heart and multiple brain regions. A complete tissue map of the dot blot map is provided in the online Supplemental Data. B: densitometry was performed on hybridized 32P-labeled human GPR22 probe signals from each sample. The densitometric signal from each tissue sample was normalized to the control signal (A1, whole brain) and is represented in the histogram as a ratio where the dashed line at 1 represents equal expression in the tissue sample relative to whole brain expression. C: multitissue rat and mouse Northern blots were analyzed for GPR22 mRNA expression by hybridization with 32P-labeled rat (left) and mouse (right) GPR22 probes, respectively.

 
We used real-time PCR to quantify transcript copy numbers in mRNA samples taken from discreet regions of the normal adult human heart to assess regional levels of cardiac GPR22 mRNA expression (Fig. 2A). GPR22 expression in the heart was dramatically higher compared with control pooled mRNA from mixed human tissues. For reference to a therapeutically relevant GPCR, we also measured the expression of the β1-adrenergic receptor. Since differences in transcript levels measured by PCR can reflect primer efficiency as well as mRNA numbers, it is not possible to accurately compare the abundance of two different receptors using these methods. Nonetheless, when normalized to endogenous β-actin expression, we determined that the level of mRNA expression was severalfold higher for GPR22 than for the β1-adrenergic receptor in the normal human heart.


Figure 2
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  		Fig. 2. GPR22 mRNA expression in human and rodent hearts. A: real-time PCR analysis was performed on cDNA samples obtained from different regions of adult human hearts. Pooled cDNA samples from multiple human tissues were used as a control and confirmed the relative enrichment of myocardial GPR22 expression (top). For comparison, expression of the β1-adrenergic receptor (β1-AdrR) in the same cDNA samples is shown (bottom). The quantity of the PCR product for each G protein-coupled receptor (GPCR) was normalized to β-actin expression and is expressed as a ratio. PCR primers and probes are listed in MATERIALS AND METHODS. B: in situ hybridization of the antisense mouse GPR22 riboprobe demonstrated mRNA expression across all chambers of the mouse heart. Sense GPR22 riboprobes were used as a control to demonstrate nonspecific hybridization.

 
High-level expression of GPR22 across all chambers in the mouse heart was confirmed by in situ hybridization (Fig. 2B). Antisense GPR22 riboprobes hybridized to target mRNA throughout all chambers in the mouse heart, whereas control "sense" riboprobes showed a low level of nonspecific hybridization. A similar expression pattern in the rat heart was also demonstrated by in situ hybridization (data not shown).

GPR22 protein expression in the rat heart. We developed a polyclonal antibody that specifically recognized rat GPR22 to evaluate GPR22 protein expression in the heart. Immunostaining of rat heart sections revealed abundant GPR22 protein expression in all chambers (data not shown). In the ventricle, we observed the expression of GPR22 protein in cardiomyocytes and coronary arteries (Fig. 3, bottom). MLC-2v is the ventricle-specific isoform of the MLC sarcomeric protein, and its expression was used as a control to identify cardiomyocytes in the ventricular sections (Fig. 3, middle). Cardiomyocyte GPR22 cell surface expression was detectable when samples were viewed at a higher magnification (Fig. 4, middle). The expression pattern on cardiomyocytes was nonuniform and formed clusters in distinct regions on the cell surface (Fig. 4, red arrows). To verify the antibody selectivity for GPR22 in these sections, the GPR22 antibody solution was preabsorbed with a 10-fold molar excess of the corresponding immunogenic peptide (Fig. 4, bottom).


Figure 3
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  		Fig. 3. GPR22 protein expression in the rat heart. Formalin-fixed longitudinal cryosections of the normal rat whole heart were stained with either control [rabbit (Rb) universal IgG], anti-myosin light chain (MLC)-2v antibody, or GPR22-c1 antisera. Microscopic analysis of the left ventricular (LV) free wall is shown, where the brown color indicates positive staining for MLC-2v in cardiomyocytes (middle) and GPR22 in cardiomyocytes (bottom left) and coronary arteries (red arrow, bottom right). The dark blue color indicates hematoxylin counterstaining of cell nuclei. Scale bar = 200 µm.

 

Figure 4
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  		Fig. 4. GPR22 cell surface expression in cardiomyocytes. Sections of the LV were stained with control (Rb universay IgG), GPR22-c1, or GPR22-c1 antisera preabsorbed with blocking peptide. Brown staining indicates GPR22 expression on the surface of cardiomyocytes (middle, red arrows) where the receptor is clustered to specific areas of some cells. Preincubation of GPR22-c1 antibody with the blocking peptide resulted in the absence of cardiomyocyte GPR22 staining (bottom). Scale bar = 100 µm.

 
GPR22 signaling. Analysis of GPCR signaling can be difficult in the absence of a known ligand. However, many GPCRs are capable of coupling constitutively to their preferred G protein in the absence of ligand (3). Thus, we sought to determine if GPR22 harbored constitutive G protein coupling when overexpressed either transiently or stably in heterologous cells. Transient transfection of wild-type GPR22 into HEK-293 or COS7 cells resulted in weak expression of GPR22 mRNA, requiring 48–72 h of exposure for detection (Fig. 5A). Under the conditions used in this experiment, HA-tagged GPR22 protein expression was not readily detectable in transfected cells by immunocytochemistry (Fig. 5B, left). Not surprisingly, measurement of G protein coupling to Gs, Gi/Go, or Gq in wild-type GPR22 transfected cells was suboptimal under these conditions (data not shown).


Figure 5
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  		Fig. 5. Heterologous expression of wild-type GPR22 and "synthetic" GPR22 (sGPR22). A: Northern blot analysis of total RNA from COS7 cells tranfected with control (pCMV), wild-type GPR22 (wtGPR22), and sGPR22 expression plasmids. Top left, 30-min autoradiographic exposure following binding of 32P-labeled sGPR22 probes to RNA derived from COS7 cells transfected with either wtGPR22 or sGPR22. Top right, 72-h autoradiographic exposure following binding of human wtGPR22 probes to RNA derived from COS7 cells transfected with wtGPR22. Note that a much shorter exposure time was required for sGPR22 signals relative to wtGPR22 signals. Bottom, methylene blue stain of 28S rRNA on the Northern blot membrane demonstrating equal loading and transfer and integrity of RNA. B: immunocytochemical staining of COS7 cells transfected with hemmaglutinin (HA)-tagged wtGPR22 and HA-tagged sGPR22. Anti-HA antibody staining is seen clearly in sGPR22 transfected cells (b) but was not detectable under these assay conditions in cells transfected with wtGPR22 (a). Higher magnification images show cell surface expression of HA-tagged wtGPR22 (c). In d–f, the presence of similar cell densities was confirmed by 4',6-diamidino-2-phenylindole (DAPI) staining of the same cells shown in a–c. Anti-HA antibody staining of HEK-293 cells stably expressing HA-tagged sGPR22 (sGPR22st) demonstrated high-level protein expression in the majority of cells (h). Anti-HA antibody staining of control HEK-293 cells showed levels of nonspecific staining (g), and a higher magnification image is shown in i. DAPI staining showed similar cell densities (j–l). Scale bar = 25 µm. C: flow cytometry of HEK-293 clonal cells stably expressing HA-sGPR22 (red tracing) and control HEK-293 cells (blue tracing) demonstrated prominent cell surface HA-sGPR22 expression.

 
Analysis of the GPR22 nucleotide sequence revealed that it is unusually A/T rich (64%), whereas most human genes have only ~40% A/T content. Since A/T-rich sequences are known to affect mRNA stability (4, 12, 38), we hypothesized that one or more of those A/T-rich regions was responsible for the rapid degradation of GPR22 mRNA in heterologous transfected cell systems. To mitigate the possible role of A/T-rich content on decreased mRNA stability, we used a "shotgun" approach that introduced nucleotide changes that converted the A/T-rich regions to G/C-rich regions, thus reversing the A/T-to-G/C ratio without modifying the wild-type amino acid sequence. Using this approach, we generated a synthetic version of GPR22 (sGPR22) having an amino acid sequence identical to the wild type yet having an A/T content of 41%. A nucleotide comparison between wild-type GPR22 and sGPR22 is available in the online supplemental data (Fig. 1).

Transient transfection of COS7 cells using sGPR22 resulted in a dramatic increase in mRNA levels, as demonstrated by Northern blot analysis (Fig. 5A, left). In contrast to the low mRNA levels observed when the natural GPR22 sequence was used for transfection, sGPR22 mRNA levels were easily detected after hybridization using a 32P-labeled sGPR22 probe and exposing the blot to film for 30 min. Relatively abundant expression of cell surface receptor protein was observed by immunocytochemical staining of sGPR22 compared with wild-type GPR22-transfected cells with antibodies to the HA epitope tag (Fig. 5B, right). Given the dramatic increase in GPR22 receptor expression with the synthetic construct, a HEK-293 cell line stably expressing sGPR22 was generated. High-level cell surface GPR22 expression was confirmed in sGPR22 stable cells by flow cytometry (Fig. 5C).

Increased cell surface expression in cells transfected with sGPR22 enabled us to measure constitutive G protein coupling in both transiently transfected cells and HEK-293 cells stably overexpressing sGPR22. As a positive control for Gq coupling, inositol phosphate generation was shown to be constitutively increased in cells transfected with the ghrelin receptor. This activity could be upmodulated by an agonist (ghrelin) or downmodulated by an inverse agonist (substance P) (Fig. 6A). However, no detectable inositol phosphate accumulation was observed in cells transfected with sGPR22, indicating a lack of constitutive coupling of sGPR22 to Gq. Similarly, sGPR22 was unable to constitutively couple to Gs (Fig. 6B). In contrast, isoproterenol-stimulated cAMP accumulation was significantly reduced in cells stably overexpressing sGPR22, indicating constitutive Gi/Go coupling (Fig. 6B). Gi/Go coupling was confirmed by reversing the inhibition with pertussis toxin treatment. In addition, we observed increased [35S]GTP{gamma}S binding to membranes derived from sGPR22 stable cells, another indicator of Gi/Go coupling (Fig. 6C).


Figure 6
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  		Fig. 6. Constitutive Gi signaling by sGPR22. A: inositol phosphate (IP) accumulation was measured in control untransfected HEK-293 cells (vehicle) and HEK-293 cells transiently transfected with GPR22 or the ghrelin receptor (GHSR), which was used as a positive control. The GHSR agonist ghrelin and the inverse agonist substance P (SubP) demonstrated the expected modulation of the GHSR IP response. *P < 0.001 compared with vehicle; **P < 0.001 compared with GHSR. B: cAMP accumulation was measured in untransfected (control) HEK-293 cells and in HEK-293 cells stably expressing sGPR22 (sGPR22st). The increase in cAMP stimulated by 10 µM isoproterenol (Iso) in sGPR22st cells was significantly lower than that seen in control HEK-293 cells with Iso, suggesting constitutive Gi coupling. *P < 0.001 compared with HEK-293 + Iso. Pretreatment with 100 ng/ml pertussis toxin (PTX) confirmed sGPR22 Gi coupling by blocking the sGPR22-mediated inhibition of the Iso cAMP response. C: GTP{gamma}S binding was significantly increased in HEK-293 cells stably expressing sGPR22 compared with control untransfected HEK-293 cells. Preincubation with 100 ng/ml PTX completely blocked sGPR22st GTP{gamma}S activity, indicating constitutive Gi coupling. *P < 0.001 compared with untransfected HEK-293 controls.

 
Regulation of GPR22 expression in vivo. To determine if GPR22 mRNA expression was regulated in animal models of heart disease, we measured mRNA levels in mice subjected to chronically increased afterload induced by surgical banding of the aorta (TAC). Abundant mRNA expression of GPR22 in normal mouse ventricles was confirmed in sham-operated mice, whereas 7 days of aortic banding resulted in a striking decrease in mRNA levels (Fig. 7A). ANF mRNA expression was also monitored in this experiment, since it has previously been shown that ANF is upregulated in response to pressure overload in the murine heart (30). The substantial upregulation of ANF in the hearts of TAC animals but not control animals verified the myocardial stress response to the increase hemodynamic load associated with aortic banding. When GPR22 mRNA levels in ventricles of seven sham and nine TAC mice were normalized to 28S rRNA levels using densitometry and averaged, the decrease in GPR22 mRNA in the TAC group was found to be approximately threefold lower and highly statistically significant (P < 0.0001; Fig. 7B).


Figure 7
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  		Fig. 7. Decreased GPR22 mRNA expression in mouse hearts following aortic banding. Total RNA was isolated from the ventricles of wild-type mice 7 days following aortic banding by transverse aortic constriction (TAC) or sham-operated nonbanded controls (Sham). A: Northern blot analysis using a 32P-labeled mouse GPR22 probe demonstrated decreased GPR22 mRNA expression following banding (top) in 3 mice subjected to TAC relative to 2 mice subjected to sham surgery. To confirm that TAC elicited a stress response in the LV, all RNA samples were also tested for the induction of atrial natriuretic factor (ANF) expression (middle). Methylene blue staining of 28S rRNA confirmed equal loading and transfer and RNA integrity (bottom). B: densitometric analysis of GPR22 hybridization signals normalized to the intensity of 28S rRNA staining. Numbers in parentheses indicate numbers of animals. *P < 0.0001 compared with sham controls.

 
Disruption of GPR22 expression in mice. As GPR22 is abundantly expressed in the heart and brain, we anticipated that disruption of expression might result in a dramatic phenotype. Thus, to avoid possible embryonic lethality, we employed a Cre-loxP gene-targeting strategy, where disruption of the gene could be controlled in a tissue and developmentally specific manner (see Supplemental Fig. 2). Heterozygous mice carrying the targeted allele GPR22loxP/+ neo(–) were bred into homozygosity. We verified that homozygous GPR22loxP/loxP neo(–) mice were viable, fertile, and grossly not different from wild-type mice. These mice were crossed to protamine-Cre transgenic mice expressing Cre DNA recombinase under the control of protamine 1 promoter (24) to generate a GPR22-null allele in male germ cells. Germ line knockout mice (GPR22+/–) were obtained by germline transmission of the GPR22-null allele. Homozygous knockouts (GPR22–/–) were generated by crossing two heterozygous (GPR22+/–) mice. Genetic deletion of the floxed GPR22 alleles was determined, and genotypes were confirmed by Southern blot analysis (Fig. 8B) using the restriction enzymes and probes shown in Fig. 8A.


Figure 8
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  		Fig. 8. Southern and Northern blot analysis of GPR22–/– mice. A: the 3 different alleles described in this study. The boxed areas indicate the part of the genomic region included in the targeting vector. Filled boxes indicate targeted genomic regions encoding the GPR22 seven-transmembrane domain (7TM exon). The targeted allele (GPR22loxP/+) is the result of the gene-targeting event. The knockout (GPR22+/–) is the null allele derived from Cre-mediated recombination between the two loxP sites. Only a single loxP site remains in the modified locus. loxP and Frt sites are indicated as large and small arrows, respectively. The genomic region is not drawn to scale. B: genomic DNA samples from wild-type mice (+/+), heterozygous and homozygous floxed mice (loxP/+ and loxP/loxP), and heterozygous and homozygous knockout mice (+/– and –/–) were isolated from mouse tails, digested with XbaI, transferred by Southern blot, and hybridized using the 5'-probe. The XbaI-XbaI fragment detected by the 5'-probe was 18.8 kb in the wild-type allele, 10.9 kb in the floxed allele, and 8.9 kb in the knockout allele, as indicated in the restriction map in A. Genotypes are indicated, as are the positions of the different bands. C: total RNA samples were isolated from heart and brain tissues of wild type (+/+), heterozygous (+/–), and homozygous (–/–) mice. The Northern blot was hybridized using a mGPR22 cDNA probe. A cDNA fragment encoding 18S rRNA was used as a control probe for RNA quality. Genotypes are indicated.

 
To examine the mRNA expression of GPR22 in heterozygous and homozygous knockout mice, RNA samples from heart and brain tissues of wild-type, heterozygous knockout, and homozygous knockout mice were isolated. Northern blot analysis verified that the GPR22 transcript was undetectable in GPR22-null (GPR22–/–) mice (Fig. 8C). In contrast, the transcript level was not altered in heterozygous knockout mice (GPR22+/–) compared with wild-type littermate controls. Thus, we had generated mice with disruption of GPR22 mRNA expression.

Cardiac function in GPR22 KO mice subjected to aortic banding. GPR22–/– mice were viable, fertile, and grossly indistinguishable from wild-type littermates. Upon dissection, the hearts from mutant animals were of normal weight and appeared normal in morphology compared with wild-type littermates (data not shown). Echocardiograph analysis indicated that cardiac dimensions and function did not differ between wild-type and GPR22–/– mice (Table 3, pre-TAC).


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  		Table 3. Echocardiographic measurements of cardiac structure and function in GPR22–/– mice and WT littermates before and after aortic banding

 
Since normal cardiac function did not appear to be affected by genetic disruption of GPR22, we next examined cardiac function under conditions of chronic hemodynamic stress. A sustained increase in afterload, mimicking chronic hypertension, is achieved experimentally in mice by surgical narrowing of the transverse aorta (30). We therefore performed TAC surgery to generate a sustained increase in cardiac afterload. Echocardiography was performed before and after 7 days of aortic banding. TAC resulted in a surgical and postoperative mortality of ~30% in both wild-type and knockout mice.

In wild-type mice, the expected compensatory cardiac hypertrophy was observed after 7 days of aortic banding, as indicated by significant increases in interventricular septum thickness at end diastole, LV posterior wall thickness at end diastole, and LV mass (Table 3). Compensatory hypertrophy was also evident in GPR22–/– mice subjected to TAC, with significant increases in interventricular septum thickness, LV posterior wall thickness, and LV mass. Cardiac function was not significantly impaired under these conditions in wild-type mice, as indicated by the percent fractional shortening (44.1% before TAC vs. 41.0% after TAC, P > 0.05) and mean velocity of circumferential fiber shortening (VCF; 7.9 circumferences/s before TAC vs. 6.8 circumferences/s after TAC, P > 0.05) measurements. However, in contrast to wild-type mice, GPR22–/– mice did show impaired cardiac function, as indicated by significant decreases in %FS (44.4% before TAC vs. 34.8% after TAC, P < 0.01) and mean VCF (8.1 circumferences/s before TAC vs. 5.8 circumferences/s after TAC, P < 0.01). Consistent with the onset of decompensated heart failure in GPR22–/– mice, chamber dilation was also observed, with LV end-systolic diameter significantly increased and LV end-diastolic diameter showing a trend toward dilation, albeit not statistically significant. Thus, while TAC resulted in compensated hypertrophy in normal mice, a transition from hypertrophy to decompensated heart failure occurred in GPR22–/– mice.


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Our findings indicate a unique expression pattern for GPR22. Especially interesting was the dramatic enrichment of expression in the heart and brain relative to other tissues. While it is difficult to directly compare mRNA expression levels for different gene transcripts, our real-time PCR data suggested that cardiac GPR22 mRNA levels were substantially higher than the functionally important and therapeutically relevant β1-adrenergic receptor. Demonstration of GPR22 protein expression in the heart by immunohistochemistry confirmed abundant myocardial expression. In addition, we identified cardiomyocytes and coronary vascular cells as sites of enriched GPR22 protein expression in the heart. Antibody staining indicated a unique expression pattern characterized by focused clustering to specific areas of the cardiomyocyte cell surface. The reason for the clustering pattern of protein expression is unknown but is likely to be related to its role in the regulation of heart function. It was recently shown that the β1-adrenergic receptor is enriched at sites of sympathetic nerve synapses in cardiomyocytes (33). Interestingly, we also observed enriched GPR22 protein expression in the highly innervated cells of the sinoatrial node and atrioventricular node in rat hearts (data not shown). The enriched expression in coronary vascular cells suggests a possible role for this receptor in the regulation of coronary blood flow.

GPCR signaling in the heart has profound biological and therapeutic implications. With the discovery of a novel, highly expressed myocardial GPCR, we attempted to identify G protein coupling. Initially, weak expression of wild-type GPR22 was detected when it was transiently transfected into mammalian cells. We hypothesized that mRNA instability might explain the weak expression. In an effort to improve GPR22 mRNA stability, we converted portions of the DNA coding sequence into a synthetic version of GPR22 (sGPR22) with a lower percentage of A/T pairings without altering the amino acid sequence. The resulting sGPR22 showed a dramatic increase in mRNA and protein expression upon transfection in HEK-293 and COS7 cells. This, in turn, resulted in a robust functional response associated with constitutive Gi/Go coupling.

The observation that GPR22 was highly expressed on cardiomyocytes, and that the receptor coupled to Gi/Go, suggested a possible role in the regulation of cardiac contractile function. For example, increased Gi protein expression in the setting of heart failure has been implicated in the decreased contractile response to activation of the β-adrenergic Gs pathway (2). In addition to its role in regulating contractility, Gi/Go signaling has been shown to promote cardiomyocyte survival by opposing the proapoptotic action of β1-adrenergic stimulation (5, 6, 39). Cardiomyocyte Gi/Go signaling in response to adenosine and opioid receptor activation has also been shown to activate prosurvival pathways in the setting of ischemia-reperfusion injury, implicating a role for Gi/Go-coupled receptors in cardioprotection (26, 32). Furthermore, we cannot rule out a operative role for Go signaling by GPR22, especially in light of recent findings suggesting modulation of cardiac calcium signaling and contractile function by Go in transgenically altered mice (40).

In the absence of a known ligand, analysis of the acute or chronic effects of GPR22 signaling activity on myocyte function is difficult. Thus, we turned to a genetically altered mouse model to study the role of GPR22 in the heart. We generated mice with undetectable myocardial GPR22 expression and used these receptor-deficient mice to evaluate its role in cardiac physiology and pathophysiology. To mitigate possible early embryonic lethality in GPR22-null mice, we chose a multiple step knockout strategy. Generation of floxed GPR22 mice (GPR22loxP/loxP) allows spatial and temporal disruption of the gene simply by crossing floxed mice to Cre transgenic mice driven by various promoters. In this study, germline knockouts of GPR22 were obtained by crossing GPR22loxP/loxP mice with protamine 1-Cre transgenics, mice expressing Cre under the protamine 1 promotor, which expresses the Cre transgene in male germ cells (24). We did not observe any negative effects of GPR22 deletion on embryonic survival or postnatal heart development or on normal cardiac morphology or function. Thus, there was no immediate need for our anticipated secondary crossbreeding strategy to generate temporal- and spatial-specific knockouts.

Clinically, in response to stress such as hypertension, myocardial injury, or neurohumoral activation, the heart initially compensates with an adaptive hypertrophic increase in cardiac mass. Under prolonged stress, the heart undergoes further irreversible changes, resulting in chamber dilation and diminished performance, characteristics of heart failure. TAC creates an experimental condition mimicking clinical hypertension and is well established as an animal model to study cardiac hypertrophy and heart failure. Both GPR22–/– mice and wild-type littermates were able to respond to the increased afterload in the TAC model with similar degrees of hypertrophy, as indicated by increased ventricular wall thickness and LV mass in echocardiograph measurements. In contrast, GPR22–/– mice were more susceptible to heart failure than wild-type littermates, as indicated by the significant decrease in both %FS and VCF. The mechanisms governing the transition from stable cardiac hypertrophy to decompensated heart failure are poorly understood but are thought to involve a shift in the balance of survival and apoptotic signaling pathway activity in cardiomyocytes (1, 35). Our results suggest a role for GPR22 in the transition process from hypertrophy to heart failure, which we postulate would involve the regulation of survival pathways in cardiomyocytes.

Changes in GPCR expression are known to accompany the pathogenesis of cardiovascular disease (18, 22, 25, 28). In some cases, alterations in myocardial GPCR expression have been suggested to be key pathogenic factors contributing to the severity of the disease. Does the dramatic reduction in myocardial GPR22 mRNA levels we observed in response to TAC suggest a role for this receptor in the pathophysiological responses that follow? Cardiac hypertrophy in response to TAC, which is known to occur primarily through the activation of Gq/11 signaling pathways (37), was unaffected by loss of GPR22 expression. In contrast, contractile function was significantly worsened following TAC in mice lacking GPR22. It is of interest to consider that the decrease in GPR22 mRNA expression observed following TAC might occur as an adaptive response related to the sustained demand for increased myocardial contractile function. Our data describe a highly expressed, constitutively active Gi/Go-coupled receptor that could act to balance cardiomyocyte β-adrenergic Gs signaling to cAMP. In the short term, reduced GRP22 expression could result in less Gi/Go activity in cardiac myocytes, thus allowing unimpeded β-adrenergic Gs signaling and consequently increased contractility. However, the long-term effect of decreased GPR22-Gi/Go activity and chronically elevated or unbalanced Gs pathway activation would be expected to result in cardiomyocyte apoptosis. In addition, reduced or ablated GPR22 expression in this setting could result in the reduced activation of well-known Gi-coupled survival pathways in heart cells, including Gβ{gamma}/phosphoinositide-3-kinase/Akt. Further support for this hypothesis comes from a recently published study (7) demonstrating that sustained inhibition of myocardial Gi signaling in the setting of ischemic stress resulted in increased apoptosis and decreased contractile performance. Although cardiomyocyte apoptosis in our model was unconfirmed, this scenario is consistent with our observation of decreased contractile function in GPR22–/– mice subjected to chronic TAC. Thus, it is possible that the regulation of GPR22 expression could play a role in the transition from compensated hypertrophy to decompensated heart failure.

Based on the data presented here, GPR22 represents a possible new target for the treatment of heart failure. However, the natural ligand for this receptor remains undiscovered. Comparing the GPR22 amino acid sequence with other GPCRs revealed the highest homology with the human cholecystokinin B receptor, with 34% identity in transmembrane domains. The lack of closely related receptors increases the difficulty of identifying the natural ligand. An alternative strategy would be to screen large chemical libraries for small molecule modulators of GPR22. In either case, a greater understanding of the nature of GPR22 function in the heart will come with the discovery of its natural ligand or identification of small molecules that modulate GPR22 function.


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This work was funded by Arena Pharmaceuticals Incorporated.


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Some of the GRP22 expression data included in this article also appears in patent WO2004/013285 A2. The synthesis and functional analysis of the sGPR22 construct have been previously described in patent form (WO 2007/047520 A1).


   ACKNOWLEDGMENTS
 
We thank Carolyn Ferrell, Tong Zhang, and Thomas Herman for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. W. Adams, Cardiovascular Biology, Arena Pharmaceuticals Incorporated, 6166 Nancy Ridge Dr., San Diego, CA 92121 (e-mail: jadams{at}arenapharm.com)

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.

* J. W. Adams and J. Wang contributed equally to this work. Back

1 Supplemental data for this article is available online at the American Journal of Physiology-Heart and Circulatory Physiology website. Back


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H. H. Patel and R. S. Ostrom
An orphan GPCR finds a home in the heart
Am J Physiol Heart Circ Physiol, August 1, 2008; 295(2): H479 - H481.
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