Biodesign of a renal-protective peptide based onalternative splicing of B-type natriuretic peptideShuchong Pana,1, Horng H. Chena,1, Deborah M. Dickeyb, Guido Boerrigtera, Candace Leea, Laurel S. Kleppea,Jennifer L. Hallc, Amir Lermana; Margaret M. Redfielda, Lincoln R. Potterb, John C. Burnett, Jra, and Robert D. Simaria,2

aDivision of Cardiovascular Diseases and Internal Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905; bDepartment of Biochemistry, MolecularBiology, and Biophysics, University of Minnesota, 321 Church Street SE, Minneapolis, MN 55455; and cDivision of Cardiology, University of Minnesota, 420Delaware Street SE, Minneapolis, MN 55455

Edited by Jonathan G. Seidman, Harvard Medical School, Boston, MA, and approved May 11, 2009 (received for review November 21, 2008)

Alternative RNA splicing may provide unique opportunities toidentify drug targets and therapeutics. We identified an alternativespliced transcript for B-type natriuretic peptide (BNP) resultingfrom intronic retention. This transcript is present in failing humanhearts and is reduced following mechanical unloading. The intron-retained transcript would generate a unique 34 amino acid (aa)carboxyl terminus while maintaining the remaining structure ofnative BNP. We generated antisera to this carboxyl terminus andidentified immunoreactivity in failing human heart tissue. Thealternatively spliced peptide (ASBNP) was synthesized and unlikeBNP, failed to stimulate cGMP in vascular cells or vasorelax pre-constricted arterial rings. This suggests that ASBNP may lack thedose-limiting effects of recombinant BNP. Given structural consid-erations, a carboxyl-terminal truncated form of ASBNP was gen-erated (ASBNP.1) and was determined to retain the ability of BNPto stimulate cGMP in canine glomerular isolates and culturedhuman mesangial cells but lacked similar effects in vascular cells. Ina canine-pacing model of heart failure, systemic infusion ofASBNP.1 did not alter mean arterial pressure but increased theglomerular filtration rate (GFR), suppressed plasma renin andangiotensin, while inducing natriuresis and diuresis. Consistentwith its distinct in vivo effects, the activity of ASBNP.1 may not beexplained through binding and activation of NPR-A or NPR-B. Thus,the biodesigner peptide ASBNP.1 enhances GFR associated withheart failure while lacking the vasoactive properties of BNP. Thesefindings demonstrate that peptides with unique properties may bedesigned based on products of alternatively splicing.

vasoactive � myocardial � kidney

Genome-wide analyses have revealed the prevalence of al-ternative splicing of multiexonic genes (1, 2). In fact, much

of the complexity of the human proteome is accounted for byalternative splicing of messenger RNA. Identification of thesealtered forms may allow for unique opportunities to diagnose,understand, and treat human disease. Therefore, we hypothesizedthat it might be possible to identify splice variants and to designtherapeutics based on their unique structure and function.

As an example of the wide potential of this technology to alterdisease states, we focused on a peptide with broad mechanistic,diagnostic, and therapeutic importance in cardiovascular dis-ease, B-type natriuretic peptide (BNP). BNP is encoded by asmall multiexonic gene, and although discovered in brain (3), isexpressed primarily in the heart (4). BNP, like atrial natriureticpeptide and C-type natriuretic peptide, is expressed as aprepro-hormone that is processed to a mature [32-amino acid(aa)] form by extracellular proteases (5). Mature BNP containsshort carboxyl and amino termini and a central 17-aa ring. BNPhas important autocrine, paracrine, and endocrine actions thatare mediated through the NPRA receptor and activation ofcGMP in target cells (6). Infusion of a recombinant form ofmature BNP (nesiritide) has been used clinically in heart failurefor its vasodilatory properties (7), but its use has been limited

by hypotension and concerns regarding worsening of renalfunction (8).

We identified an alternatively spliced transcript of B-typenatriuretic peptide resulting from intron 2 retention in failing leftventricular tissue. We further used the unique sequence of thededuced peptide to design a peptide with unique renal protectiveactions without attendant hypotension.

ResultsA BLAST search identified 2 ESTs (accession numbersBQ130258 and CN267260), consistent with intron 2 retention ofthe BNP transcript that would result in a unique carboxylterminus while maintaining the amino terminus and ring struc-ture of mature BNP (Fig. 1). Using primers specific for thistranscript, we identified polyadenylated mRNA containing thefirst and second exons and second intron (lacking the first intronof the BNP gene) in failing human heart tissue (Fig. 2 A and B).Sequencing of the PCR product confirmed the identity of thistranscript. Using left ventricular tissue obtained pre- andpost-LVAD placement, we demonstrated that the level ofexpression of the alternatively spliced transcript is 2 log-foldless than the wild-type transcript pre-LVAD (Fig. 2C). Addi-tionally, expression of the alternatively spliced form wasdecreased with the mechanical unloading associated withLVAD placement (Fig. 2D).

To determine whether this alternatively spliced transcriptresulted in expression of the proposed peptide (referred to asASBNP), we generated a rabbit polyclonal antiserum to theunique carboxyl terminus of the proposed peptide sequencesfrom the alternatively spliced form. This antiserum specificallydetected recombinant ASBNP expressed from baculovirus in Sf9

Author contributions: S.P., H.H.C., D.M.D., G.B., C.L., A.L., L.R.P., J.C.B., and R.D.S. designedresearch; S.P., H.H.C., D.M.D., G.B., C.L., L.S.K., J.L.H., and R.D.S. performed research; S.P.,H.H.C., C.L., L.S.K., J.L.H., M.M.R., J.C.B., and R.D.S. contributed new reagents/analytic tools;S.P., D.M.D., A.L., L.R.P., J.C.B., and R.D.S. analyzed data; and S.P., H.H.C., A.L., J.C.B., andR.D.S. wrote the paper.

Conflict of interest statement: Drs. Pan, Chen, Burnett, and Simari are named inventors ona Mayo Clinic owned patent, which has been licensed to Anexon, Inc.

This article is a PNAS Direct Submission.

1S.P. and H.H.C. contributed equally to this work.

2To whom correspondence should be addressed. E-mail: simari.robert@mayo.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0811851106/DCSupplemental.

BNP: SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRHBNP: SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH

ASBNP: SPKMVQGSGCFGRKMDRISSSSGLGCKGKH PLPPRPPSPIPVCDTVRVTLGFVVSGNHTLASBNP: SPKMVQGSGCFGRKMDRISSSSGLGCKGKH PLPPRPPSPIPVCDTVRVTLGFVVSGNHTL

ASBNP.1: SPKMVQGSGCFGRKMDRISSSSGLGCKGKH PLPPRPPSPIPVASBNP.1: SPKMVQGSGCFGRKMDRISSSSGLGCKGKH PLPPRPPSPIPV

ANP: SLRRSSCFGGRMDRIGAQSGLGCNSFR YANP: SLRRSSCFGGRMDRIGAQSGLGCNSFR Y

CNP: YKGANKKGLSKGCFGLKLDRIGSMSGLGCCNP: YKGANKKGLSKGCFGLKLDRIGSMSGLGC

DNP: EVKYDPCFGHKIDRINHVSNLGCPSLRDPR PNAPSTSADNP: EVKYDPCFGHKIDRINHVSNLGCPSLRDPR PNAPSTSA

Fig. 1. Amino acid sequence of mature ANP, BNP, CNP, DNP, ASBNP, andASBNP.1. Residues resulting from alternative splicing are highlighted in bold.

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cells compared with uninfected cell supernatants (Fig. 3) as wellas ASBNP but not BNP produced by synthesis. Most impor-tantly, we identified immunoreactivity for ASBNP from cardi-omyocytes in failing human hearts (Fig. 3).

Since BNP is known to activate cGMP in an NPRA-dependentmanner in endothelium, we compared the ability of ASBNP(generated synthetically) to that of BNP in vitro. ASBNP (unlike

BNP) lacked the ability to stimulate cGMP in HUVECs (Fig.4A). Furthermore, we also demonstrated that ASBNP lacked theability of BNP to vasorelax preconstricted arterial rings (Fig. 4B).Thus, in these in vitro studies, ASBNP lacked the vascular effectsof BNP. The potential reasons for the lack of activity in vascularcells and arterial rings would include the length or sequence ofthe carboxyl terminus which might interfere with receptorbinding or activation.

As our interest has been focused on developing therapeuticpeptides, consideration was given to the length of ASBNP (60 aa)that might limit the ability for large-scale synthesis and thepresence of 3 cysteines that might lead to dimerization and riskof aberrant ring formation. Based on these structural consider-ations, we designed a carboxyl-terminal truncated form ofASBNP referred to as ASBNP.1 (Fig. 1). In vitro, compared withBNP, ASBNP.1 like ASBNP fails to stimulate cGMP in humanendothelial or vascular smooth muscle cells at equimolar dosesor relax preconstricted human arterial rings (Fig. 5 A–C).However, in freshly isolated canine glomeruli, ASBNP.1 hadsimilar ability to stimulate cGMP at equimolar doses to that ofBNP (Fig. 5D). Studies in primary human kidney mesangial cellsconfirm that ASBNP.1 retained the ability to stimulate cGMP toa similar degree as BNP (Fig. 5E). Additional studies in mes-angial cells demonstrate that the cGMP response to ASBNP.1 isinhibited by HS-142-1 (Tokyo Research Laboratories, KyowaHakko Kogyo Co.), suggesting that the effect is mediatedthrough a particulate guanylyl cyclase linked receptor (Fig. 5F).These data suggest that ASBNP.1 retained the effects of BNP onrenal cells but lacked the effects on vascular cells and intactvascular rings.

To determine whether ASBNP.1 activates NPR-A or NPR-B,in vitro studies were performed using HEK cells that stablyexpress human NPR-A or human NPR-B (9) (Fig. 6 A and B).

DC

+RT-RTBA

Fig. 2. ASBNP mRNA is present in failing human hearts. (A) Schematic ofgenetic structure of BNP gene and primer localization for RT-PCR. (B) PCRproduct ASBNP with and without reverse transcriptase (RT). (C) Comparison ofBNP and ASBNP transcripts in failing left ventricles. (D) Comparison of ASBNPtranscripts pre- and post-LVAD placement. *, P � 0.05.

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Fig. 3. ASBNP carboxyterminus is present in failing human hearts. (A) Sectionof human left atrium stained with rabbit antiserum generated to peptideencoded by intron 2 (specific for ASBNP) or (B) section stained with preimmuneserum. (C) Immunoblot of recombinant full-length peptide (ASBNP) or controlstained with specific rabbit antisera.

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For NPR-A, the EC50 for BNP was 33.5 nM, and for ASBNP.1,the EC50 was 3.86 �M. For NPR-B, the EC50 for CNP was 48.4nM, and for ASBNP.1, the EC50 was �10 �M. Since NPR-C doesnot activate cGMP, formal binding studies were performed. TheIC50 for BNP was 4.52 nM and was 38.8 nM for ASBNP.1. Thesedata suggest that ASBNP.1 is a poor activator of NPR-A and hasreduced binding to NPR-C compared with BNP. These data hintat a receptor or receptor subtype in the kidney to whichASBNP.1 is a ligand.

Based on the distinct in vitro properties of ASBNP.1 includingthe ability to stimulate cGMP in isolated canine glomeruli andin cultured human mesangial cells without similar effects onvascular cells, we hypothesized that ASBNP.1 might have ther-apeutic properties in a disease state such as heart failureindependent of vasoactivity in vivo. A dose escalation study (2,10, and 100 pmol/kg/min) of i.v. infusion of ASBNP.1 in a caninerapid-pacing model of overt heart failure (10) demonstratedincreased aquaresis (from 0.19 � 0.04 to 0.32 � 0.07, 0.46 � 0.11,and 0.40 � 0.09 mL/min, P � 0.05) with a trend for urinarysodium excretion to increase (Table S1). Importantly, ASBNP.1enhanced the glomerular filtration rate (GFR) (from 31 � 4 to47 � 8, 69 � 10, and 57 � 9 mL/min, P � 0.05). These renalactions were associated with increases in urinary BNP, ANP, andcGMP (P � 0.05) excretion. ASBNP.1 did not have any systemicor renal vasodilatory action demonstrated by a lack of change inmean arterial blood pressure, renal blood flow, or cardiac-fillingpressures even at the highest dose.

These data led to a further study of longer infusion at thehighest dose (n � 5). A sustained IV infusion of ASBNP.1 at 100

pmol/kg/min had no effect on mean arterial pressure (Fig. 7).Importantly, ASBNP.1 markedly enhanced GFR while inducingnatriuresis and diuresis. These specific renal actions were asso-ciated with increases in plasma and urinary cGMP excretion.ASBNP.1 also significantly suppressed both plasma renin andangiotensin II during the infusion. No change in renal blood flowwas noted at any time during the study. Thus, in vitro and in vivowe demonstrated that ASBNP.1 has renal-enhancing actionswhile lacking any dose-limiting hypotensive effects. Taken to-gether, ASBNP.1 has significant potential as a unique anddistinct renal protective and GFR-enhancing therapeutic.

DiscussionThe complexity of the human proteome provides a diverse arrayof peptides that might be used as therapeutic targets or biologictherapeutics. As alternative splicing usually affects coding re-gions (11), altered functions of resulting proteins would bepredicted. We identified an alternatively spliced transcript ofBNP resulting from retention of intron 2 in failing human leftventricular tissue. This alternatively spliced transcript is ex-pressed at levels 2 log-fold less than BNP and is down regulatedin the setting of ventricular unloading. As BNP expression isup-regulated in heart failure, it is not surprising that alternativeforms might exist in this setting where regulated and stochasticmechanisms might exist (12). We further identified proteinexpression of the unique carboxyl terminus from cardiac myo-cytes in humans using a specific antiserum, which supports thatthe alternative transcript is translated in humans. Determining

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Fig. 5. The biodesigner peptide ASBNP.1 retains renal effects but lacks vascular effects of BNP in vitro. (A) ASBNP.1 differs from BNP in its inability to stimulatecGMP in endothelial cells and (B) vascular smooth muscle cells and (C) in its inability to dilate preconstricted human left internal mammary arteries. (D) ASNP.1retains the ability to stimulate cGMP in canine glomerular isolates (*, P � 0.001 vs. control; †P � 0.0001 vs. control, BNP vs. ASBNP.1 not significant) and (E) inprimary human mesangial cells. (F) The cGMP effect in mesangial cells was inhibited by HS-142–1.

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the extent and degree of expression in health and disease willrequire further study.

The deduced peptide ASBNP was synthesized and shown tolack equipotency of BNP in stimulating cGMP in vascular cellsand lacked the ability to vasorelax preconstricted arterial rings.Based on structural considerations regarding the alternativetranscript, we then generated a carboxyl terminus-deleted formof the peptide (ASBNP.1) that also lacked vascular effects butimportantly possessed renal properties. ASBNP.1 stimulatedcGMP in a dose-dependent manner in isolated canine glomeruliand human mesangial cells, although these effects requiredhigher concentrations of either BNP or ASBNP.1. The cGMPeffects in mesangial cells were inhibited in a dose-dependentmanner by HS-142-1, an inhibitor of particulate guanylyl cyclasereceptors. Interestingly, ASBNP.1 is a poor activator of NPR-Aand does not activate NPR-B in cells which specifically expresshuman forms. Additionally, ASBNP.1 binds NPR-C less avidlythan BNP. These data suggest a mechanism for the effects ofASBNP.1 including through an undefined particulate guanylylcyclase linked receptor (13). The functional effects of ASBNP.1should provide the opportunity to define this mechanism.

Furthermore, in vivo studies in experimental heart failurecorroborated these effects. ASBNP.1 has distinct renal effectsbut lacked the dose-limiting hypotensive effects of BNP. As such,ASBNP.1 may have therapeutic potential in patients with thecardiorenal syndrome in whom the GFR enhancement might beachieved without risk of hypotension. These effects would bedistinct from BNP and are of great interest given the contrain-dication of nesiritide in hypotensive patients. In future studies,

it would be of interest to determine whether the renal actions ofASBNP.1 may be potentiated by inhibition of neutral endopep-tidase which degrades the endogenous natriuretic peptides as itis highly expressed in the kidney.

While the precise mechanism for selective renal actions ofASBNP.1 will require further study, this designer peptide rep-resents a natriuretic peptide analogue totally devoid of systemichemodynamic actions but with GFR-enhancing properties. Sucha biological profile is highly attractive in human disease syn-dromes of reduced GFR in which most GFR-enhancing thera-peutic interventions are associated with reductions in arterialpressure and compromised renal perfusion. Thus, alternativelyspliced transcripts may not only provide drug targets and mech-anisms of disease but may also provide opportunities to designtherapeutics with unique features.

MethodsCell Culture. Human umbilical vein endothelial cells (HUVECs) were purchasedfrom American Type Culture Collection and cultured in endothelial growthmedium (EGM) (Cambrex) containing FBS, bovine brain extract, human epi-dermal growth factor, hydrocortisone, gentamicin, and amphotericin B at37 °C in humidified 5% CO2 in air. Primary human glomerular mesangial cells(Cell Systems Inc.) were grown in endothelial growth media. Cells frompassages 2–5 were used in this study. Sf9 cells (BD Biosciences Pharmingen)were cultured in Hank’s TNM-FH medium supplemented with 10% FBS(Mediatech).

RNA Isolation and Reverse Transcription. Tissue from explanted failing humanhearts was obtained using a human protocol approved by the InstitutionalReview Boards of the Mayo Foundation and the University of Minnesota. TotalRNA was prepared by homogenization of heart tissues using TRIzol (Invitro-gen) method and reverse transcription was performed using standard tech-niques and reagents. (RT-PCR kit, Invitrogen).

Generation of ASBNP cDNA. The forward primer 5�-AGACATGGATCCCCAGA-CAG-3� (start codon with first 4 nucleotides from 5� ULT region) and reverseprimer 5�-GGCTGCCAAATGATAAACAG-3� (selected from second intron) wereused for cloning ASBNP gene. A 2-�L aliquot of the reverse transcriptionreaction product was used as DNA template in PCR and was carried out inMinicycler (MJ Research). The PCR amplified cDNA fragments were subclonedinto the pCR2.1-TOPO vector using TOPO TA Cloning Kit (Invitrogen). Theplasmid containing PCR fragments were purified using Miniprep kit (QIAGEN),and the sequence of the insert was conformed at Mayo Sequencing CoreFacility.

Quantitative Real-time PCR. The mRNA levels of BNP and ASBNP were com-pared by real-time quantitative RT-PCR (PCR) analysis using the Light Cyclerthermocycler (Roche Diagnostics Corp.). Details are provided in SI Methods.Toquantify the amount of mRNA for ASBNP in failing human hearts, left ven-tricular tissues (n � 6) from patients were obtained (14). The mRNA levels ofBNP and ASBNP in left ventricular tissue were examined in specimens usingquantitative real-time PCR.

Antisera Generation and Purification. The deduced unique 33-aa ASBNP car-boxyl terminus (GKHPLPPRPPSPI PVCDTVRVTLGFVVSGNHTL) was synthesizedin Mayo Proteomic Research Center. A polyclonal anti-ASBNP c-terminusantiserum was generated using standard techniques (Cocalico Biologicals).The anti-ASBNP rabbit serum was collected, and the IgG was purified usingaffi-Gel Protein A MAPS II Kit (BIO-RAD) according to the manufacturer’sinstructions. The antisera detects ASBNP (and ASBNP.1) but does not detectANP, CNP, DNP, urodilatin, or VNP) (Fig. S1).

Immunohistochemistry. Failing human heart samples were sectioned into5-�m thick sections and deparaffinized and incubated 30 min in 10 mM citratebuffer, pH 6.0, at 100 °C for 30 min. The sections were blocked 30 min with10% normal donkey serum followed with an incubation time for 60 min withrabbit anti-ASBNP antibody at 32 �g/mL. Normal rabbit IgG replaced theprimary antibody to service as the negative control. The sections were thenincubated with biotinylated donkey anti-rabbit (GE Healthcare Bio-SciencesCorp.) at 1:200 for 30 min followed by alkaline phosphatase streptavidin(Vector Laboratories) at 1:300 for 45 min. The reaction was visualized usingVector Blue (Vector Laboratories).

Fig. 6. ASBNP.1 is a poor activator of human natriuretic peptide receptors.(A) ASBNP is a poor activator of HEK293 cells stably expressing human NPR-A(P value � 0.0007). (B) ASBNP is a poor activator of human NPR-B stablyexpressed in HEK293 cells (BNP vs. ASBNP.1 not significant).

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Construction of pAcGP67B-ASBNP. The secretory pAcGP67B transfer vector (BDBiosciences) was chosen to match the codon sequence. A forward primer5�-CGGGATCCAGCCCCAAGATGGTGCAA-3� containing 18 bases from 2nd exonwith a BamHI site and a reverse primer 5�-GAAGATCTTTAAAGAGTGTGGT-TCCC-3� containing 18 nucleotides from 2nd intron with a BglII site were usedas PCR primers to amplify cDNA fragments that encoded mature ASBNP. Theobtained PCR fragments were purified and subcloned into transfer vectorpAcGP67B between the BamHI and BglII sites. The sequence of the insert wasconfirmed by double-stranded sequencing (Mayo Molecular Core Facility).

Measurements of Total Cellular cGMP from Cells in Culture. The intracellularcGMP were measured by using BIOTRAK cGMP enzyme immunoassay systemKit (GE Healthcare Bio-Sciences Corp.) as previously described (15). HUVECswere plated in 96-well Falcon plates and incubated 24 h to reach 80%confluence. Then cells were stimulated with various diluted conditionedmedia from transfected cells and different concentrations of synthetic BNPpeptides dissolved in endothelial growth medium containing 0.5 mM IBMX for30 min at 37 °C with 5% CO2, and cells were subjected to lysis following the

instruction of BIOTRAK Kit. The amount of cGMP was calculated according to thestandard curve that was generated from parallel reactions within the sameexperiment. HS-142–1 was used to investigate whether the effect of ASBNP.1 onmesangial cells was dependent upon a particulate guanylyl cyclase receptor.

Studies in Isolated Canine Glomeruli. Normal canine kidneys were harvestedand glomeruli were isolated (16, 17). After 10 min of preincubation (37 °C),aliquots of glomeruli suspended in Krebs buffer were incubated with humanBNP (hBNP, Phoenix Pharmaceuticals, Inc.) or ASBNP.1 (final concentration10�5 M for both hBNP and ASBNP.1) for 10 min in the presence of isobutyl-methylxanthine (0.3 mM). The controls consist of the same composition withthe exception that Krebs buffer alone was used instead of glomeruli sus-pended in Krebs buffer. The final assay volume was 500 �L. The reaction wasterminated by addition of ice-cold trichloroacetic acid (final concentration6.6%) (16). Following centrifugation, a supernatant aliquot (800 �L) wasextracted with ether for cGMP assay (15, 16), and the remaining supernatantwas assayed for protein levels (BCA protein assay, Pierce Biotechnology).Results were corrected for protein levels (mean � SEM).

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Fig. 7. The biodesigner peptide ASBNP.1 retains renal effects but lacks vascular effects of BNP in a canine model of congestive heart failure. Infusion of i.v.ASBNP.1 increases GFR (A), urinary sodium excretion (C), urine flow (D), without affecting mean arterial blood pressure (B). Plasma (E) and urine (F) cGMP wereincreased whereas plasma renin (G) and angiotensin II (H) were decreased. Open bars, baseline; black bars, infusion of 100 pmol/kg/min; gray bars, postinfusionperiod. *, P � 0.05.

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Expression of Recombinant ASBNP Mature Peptide and Western Blot Analysis.Recombinant mature ASBNP peptides were expressed by using BD BaculoGoldBright system (BD Biosciences). Details are provided in SI Methods.

Arterial Vasoactivity Studies. To determine the arterial and venous vasodilatoreffects of ASBNP, ASBNP.1 in comparison with BNP, canine carotid artery, anddiscarded human left internal mammary artery (LIMA) were used as previouslydescribed (18, 19). Details are provided in SI Methods.

Whole-cell Binding Assays. Human embryonic kidney 293 cells stably expressinghuman NPR-A or NPR-B or NPR-C were previously described (9). Binding of125I-ANP to HEK 293 cells stably expressing various natriuretic peptide recep-tors was performed as previously described (9).

In Vivo Animal Studies. Studies were conducted in anesthetized male mongreldogs (18–23 kg). Specifically, 2 groups of dogs with overt chronic heart failure

produced by rapid ventricular pacing at 240 beats per minute (bpm) for 10days were used: (i) a dose escalation study (2, 10, and 100 pmol/kg/min) of i.v.infusion of ASBNP.1 (n � 10) and (ii) a sustained IV infusion of ASBNP.1 at 100pmol/kg/min (n � 5). Studies were performed in accordance with the AnimalWelfare Act and with approval of the Mayo Clinic Institutional Animal Careand Use Committee. Experimental details are provided in SI Methods.

Statistical Analysis. Results are reported as mean � 1 SEM. For the cell culturestudies, each experiment was performed in triplicate in 2 or 3 (human NPR-Cbinding) separate assays. Differences between groups were made using un-paired t-tests. Statistical analysis was performed with the SAS software pack-age. Physiologic parameters in study groups were compared with 1-wayANOVA or repeated measures ANOVA. The normal distribution was testedwith univariate analysis. A P value �0.05 was considered significant.

ACKNOWLEDGMENTS. This work was funded in part by grants from Anexon,Inc., the Mayo Foundation, and the National Institutes of Health (HL 76611).

1. Matlin AJ, Clark F, Smith CW (2005) Understanding alternative splicing: Towards acellular code. Nat Rev Mol Cell Biol 6:386–398.

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