A Secretion Trap Screen in Yeast Identifies Protease Inhibitor 16 as a Novel Antihypertrophic Protein Secreted From the Heart
Background— Cardiomyocyte hypertrophy is of central importance in the development of congestive heart failure. Whether proteins secreted from the myocardium itself contribute to myocardial hypertrophy is largely unknown.
Methods and Results— We performed a genetic yeast secretion trap screen using a murine cardiac cDNA library and identified 54 cardiac proteins that contained a secretion signal. When determining their mRNA expression in the myocardium of failing hearts, we found protease inhibitor 16 (PI16) to be strongly upregulated in hypertrophic and failing myocardium. PI16, a 489–amino acid protein with an unknown function, also displayed enhanced expression on the protein level after serum stimulation of primary cardiomyocytes and in failing myocardium. We found PI16 to be secreted rapidly by primary cardiomyocytes into the culture medium, where it inhibited cardiomyocyte growth. RNA interference–mediated suppression of endogenous PI16 in primary cardiomyocytes significantly enhanced cardiomyocyte size. Transgenic mice overexpressing PI16 in a cardiomyocyte-specific manner showed normal cardiac function but had smaller hearts with hypotrophic cardiomyocytes.
Conclusions— Taken together, we identified 54 putatively secreted cardiac proteins. PI16, a novel protein secreted from the heart, is strongly upregulated early in heart failure and inhibits growth of cardiomyocytes both in vitro and in vivo. PI16 might represent a novel therapeutic target in heart failure.
Received February 16, 2007; accepted August 10, 2007.
Heart failure remains one of the most frequent causes of death in industrialized countries despite significant progress in pharmacological treatment.1 Remodeling of the myocardium, characterized by hypertrophy of cardiomyocytes and interstitial fibrosis, is of central importance for the development and progressive clinical course of heart failure. Several lines of evidence indicate that communication between cardiac cells via secreted factors may contribute to myocardial remodeling. For example, conditioned medium from cultured fibroblasts induces hypertrophy of cardiomyocytes, and conditioned medium from cardiomyocytes promotes proliferation of fibroblasts.2 Furthermore, evidence is growing that stem cell therapy after myocardial infarction prevents cardiac remodeling through paracrine factors.3,4
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Secreted and transmembrane proteins show promise as therapeutic targets or possibly even as therapeutic agents. They are accessible to various drug-delivery mechanisms, as they are present on the cell surface or within the extracellular space.
Paracrine factors that are known to contribute to cardiomyocyte hypertrophy and myocardial remodeling include angiotensin II, transforming growth factor-β, endothelin, catecholamines, and insulin-like growth factor-1.5,6 The number of identified proteins secreted from the heart is small, possibly owing to their low expression level. In contrast, bioinformatic analyses imply very high numbers of secreted proteins, comprising up to 2000 proteins for the mouse secretome.7,8 These approaches assume that the presence of a secretion signal sequence per se is sufficient to mediate effective secretion of a particular protein; however, no strictly defined consensus sequence exists for a functionally active signal sequence. Signal sequences are typically characterized by hydrophobic amino acids followed by a signal peptidase cleavage site; but not all sequences with these characteristics function as signal sequences, for example, because the signal sequence is not accessible after folding of the tertiary structure. In addition, identification of the genes themselves in genomic data is still difficult, and estimates of the total number of human genes vary widely.9 Expressed sequence tags, on the other hand, are often truncated and miss the N-terminal signal sequence. Furthermore, tissue-specific expression data for the majority of the identified proteins are insufficient, which makes the identification of tissue-specific secretomes difficult. Lastly, many of the putative secreted genes were identified by homology screening, but homologs do not need to have the same subcellular localization. Thus, computational screens may miss secreted proteins and may yield a high rate of false-positive results.
In the present study, we performed a genetic yeast secretion trap screen to systematically search for genes encoding proteins that are secreted by the heart. Using a murine cardiac cDNA library, we identified 54 cardiac proteins that contained a secretion signal. Many of these proteins have not been studied previously or even discovered in the heart, and the function of several is completely unknown. Among the latter is protease inhibitor 16 (PI16), a protein we found to be secreted by cardiomyocytes and that inhibits cardiomyocyte hypertrophy both in vitro and in vivo.
Secretion Trap Screen
The secretion trap screen was performed as described previously10,11 with some modifications. Briefly, the host yeast strain, O66-2 lacks invertase, an enzyme that must be secreted for the strain to grow on sucrose. A cDNA library was prepared (SuperScript Plasmid System, Invitrogen, Carlsbad, Calif) from left heart ventricles of 4- to 5-month-old wild-type FVB mice by use of random hexamer primers. The cDNA library was then cloned into a yeast expression vector encoding a mutant invertase (pSuc2dMSP), which lacked the start codon for methionine and the signal sequence necessary for secretion. After transformation of the host strain with the library, colonies could form on sucrose only in those cases in which a clone from the cDNA library provided both a start codon and a functional signal sequence in frame with the invertase. cDNA inserts of positive clones were amplified by polymerase chain reaction (PCR) with flanking primers, sequenced, and analyzed by a BLAST search (Basic Local Alignment and Search Tool). To avoid sequencing of redundant clones, we performed an iterative cross-hybridization of the PCR products of newly identified clones with 32P-labeled probes directed against repeatedly found cDNAs.
RNA Isolation and Real-Time Quantitative PCR
Total RNA was extracted from frozen tissue with the RNeasy Mini Kit (Qiagen, Hilden, Germany), including DNase digestion according to the manufacturer’s instructions. Real-time PCR was performed with SYBR Green as fluorescent dye, and data were calculated by the 2-ΔΔCT method.12 Please refer to the online-only Data Supplement for details.
Human Heart Tissue
Please refer to the online-only Data Supplement for a detailed description. The present study was approved by the Ethics Committee of the Medical Faculty of the University of Wuerzburg.
Generation of a PI16-Specific Antibody and Western Blot Analysis
Polyclonal antibodies directed against PI16 were generated and affinity purified as detailed in the online-only Data Supplement.
Isolation of Primary Cardiac Cells
Neonatal rat cardiomyocytes were isolated as described previously.13,14
Generation of PI16 Adenovirus, Immunofluorescent Staining, Isoleucine Incorporation, RNA Interference Transfection, Histological Analyses, and In Vivo Hemodynamic Analysis
Please refer to the online-only Data Supplement for a description.
Generation of Transgenic Mice
A detailed description is presented in the online-only Data Supplement. All animal experiments were approved by the responsible authorities.
For comparison of 2 individual groups, unpaired 2-tailed t tests were performed with the Prism software package (GraphPad, San Diego, Calif). For comparison of 1 factor in >2 groups, ANOVA followed by Bonferroni multiple-comparison posttest was performed. To test for differences in means of 2 factors, 2-factor ANOVA was performed. If the interaction was significant, the t test was used instead. Data are presented as mean±SEM. Differences were considered significant when P<0.05.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Identification of Proteins Secreted in the Heart by a Secretion Trap Screen
We generated a murine cardiac cDNA library with a complexity of 2×107 independent clones, which means an average gene transcript was represented >100 times. The cDNA library also contained transcripts from weakly expressed genes, as tested by PCR amplification of a variety of genes (data not shown). We then successively plated 1.7×107 yeast transformants (approximately one the cDNA library) and isolated 1900 clones that grew on selection media containing 2% sucrose. Only yeast clones carrying cDNA fragments encoding for secreted proteins grew under selection conditions (Figure 1A). Using an iterative cross-hybridization strategy, we could reduce the number of clones that were sequenced to 347 and thereby identified 54 nonredundant cardiac cDNAs encoding for putatively secreted proteins (online-only Data Supplement Table II). Currently, the function of approximately one third of these genes is unknown. We then determined the mRNA expression of 25 of the identified genes in a well-characterized murine heart failure model (β1-adrenergic receptor transgenic mice15; Figure 1B). Compared with healthy wild-type littermates, we found upregulation of PI16 (420±130%, P<0.001) and atrial natriuretic peptide (620±170%, P<0.001). We therefore aimed to further characterize PI16.
Cloning of PI16 and Identification of a Splice Variant
The murine PI16 gene comprises 7 exons (exon 7 encodes for the 3′-UTR) spanning ≈10 200 bp and resides on chromosome 17.16 We cloned murine PI16 using primers at the 5′ and 3′ ends of the putative coding sequence and confirmed the predicted sequence by sequencing (1470 bp). In the heart, we thereby identified a more weakly expressed splice variant comprising 684 bp that lacks exon 5 (online-only Data Supplement, Figure IA). The coding sequence of the human full-length homolog is shorter (1392 bp), but due to a smaller exon 5, the putative splice variant is slightly longer (714 bp). Bioinformatic analysis with SMART (Simple Modular Architecture Research Tool)17 revealed that PI16 contains a sperm-coating glycoprotein domain (online-only Data Supplement, Figure IB). There are homologs to PI16 in other mammals, such as opossum (ENSMODP00000016841; aa identity 55.9%) and chicken (ENSGALP00000000778; aa identity 47.0%; online-only Data Supplement, Figure IC). The evolutionarily most distant homolog that we could identify was in fish (ENSDARP00000067664; aa identity 32.9%), which suggests that PI16 evolved in vertebrates.
Protein Expression of PI16
The open reading frame of full-length PI16 encodes a protein of 489 amino acids. We generated a polyclonal antibody directed specifically against the full-length PI16 protein (PI16-FL antibody). To examine the organ expression pattern of PI16, we isolated diverse organs from 3-month-old wild-type FVB mice and determined expression of PI16 by Western blotting. PI16 protein expression was strongest in aorta and skin and weaker in adipose tissue (online-only Data Supplement, Figure IIA). In healthy hearts, PI16 protein expression was low. Surprisingly, the polyclonal PI16-FL antibody detected 3 specific PI16 bands (74, 100, and 108 kDa) in tissue lysates under both reducing and nonreducing conditions. We generated 2 additional antibodies directed against exon 5 (excluding the sperm-coating glycoprotein domain, which is localized in exons 1 to 4) and a small peptide in exon 2 of PI16, respectively. Both our original antibody directed against full-length PI16 and the 2 new antibodies detected a virtually identical pattern of PI16 bands when used for Western blotting analysis in tissue lysates (online-only Data Supplement, Figure IIB). Bioinformatic analysis indicated potential glycosylation sites. Therefore, we deglycosylated myocardial lysates from PI16 transgenic mice using PNGaseF. Enzymatic deglycosylation led to a significant shift of the PI16 bands to lower molecular weights, which suggests glycosylation of the PI16 protein (online-only Data Supplement, Figure IIC). However, a different glycosylation pattern cannot explain the existence of more than 1 PI16 band. The theoretical molecular weight of PI16 is 52.7 kDa; however, recombinant PI16 from Escherichia coli fused to a GST tag (27 kDa) and, lacking its signal sequence (2 kDa), ran at ≈95 kDa, which suggests a molecular weight of unmodified PI16 of ≈70 kDa (data not shown). This is in line with the deglycosylation experiment. The higher-molecular-weight bands of PI16 may represent a further modification or a covalent linkage to another protein.
PI16 Is a Secreted Protein
The identification of PI16 in the secretion trap screen and the presence of a bioinformatically predicted N-terminal signal peptide (online-only Data Supplement, Table II) both suggest secretion of PI16. To determine whether PI16 is secreted from mammalian cells, we transfected neonatal rat cardiomyocytes with a recombinant adenovirus expressing murine PI16 (Adv-PI16). Interestingly, we detected predominantly the 74- and 108-kDa bands in lysates from cells transfected with PI16 (Figure 2A). In contrast, the 100-kDa band predominated in the culture medium. This indicates that the N-terminal signal sequence of PI16 is functional and that PI16 is secreted from cardiomyocytes. The 2 cellular PI16 bands and the soluble extracellular PI16 together represent the 3 bands detected in tissue lysates that are a mixture of cells and extracellular material (online-only Data Supplement, Figure IIA).
Classically secreted proteins pass the endoplasmic reticulum and the Golgi apparatus and are ultimately transported to the plasma membrane in secretory vesicles. To elucidate the intracellular localization of PI16, we transfected neonatal rat cardiomyocytes with Adv-PI16 (multiplicity of infection [MOI] 0.1). Immunofluorescent staining with the affinity-purified antibody directed against PI16 (PI16-FL antibody) revealed a distribution of PI16 in small cytoplasmatic vesicles, as is typical for secreted proteins (Figure 2B). Endogenous PI16 protein in nontransfected cardiomyocytes was only scarcely detected. This could be due to the low PI16 expression in nonstimulated cardiomyocytes or to a rapid and effective secretion of PI16.
We therefore made cryosections of murine wild-type hearts and stained PI16 by immunofluorescence (Figure 2C). Again, only small amounts of PI16 were visible intracellularly; however, PI16 was readily detectable in the intercellular space, which suggests extracellular accumulation and possibly membrane association of PI16 in the heart after rapid secretion.
Expression of PI16 Is Upregulated in Heart Failure
In β1-adrenergic receptor transgenic mice, a well-characterized heart failure model that uses cardiac-specific overexpression of the β1-adrenergic receptor, we found profound upregulation (up to 470±55%, P<0.001) of PI16 full-length mRNA (Figure 3A). PI16 upregulation in this model starts in very young animals (2 months old) and precedes the macroscopically visible myocardial remodeling, which suggests a possible role in the pathogenesis of heart failure. Messenger RNA expression of PI16 was also strongly upregulated (200±70%, P<0.05) in mice after 5 weeks of pressure overload of the left ventricle by thoracic aortic banding. Furthermore, PI16 mRNA appeared to be upregulated (323±160%, P=0.11) in human heart failure. In accordance with these findings, we also found PI16 protein expression to be markedly upregulated in heart failure (Figure 3B). Similar to the adult murine heart, only weak PI16 expression occurred in isolated neonatal rat cardiomyocytes under basal conditions; however, PI16 expression was strongly induced in cardiomyocytes after serum stimulation (Figure 3C).
PI16 Inhibits Hypertrophy of Cardiomyocytes
To determine the effect of PI16 on cardiomyocyte growth, we infected neonatal rat cardiomyocytes with Adv-PI16. Although PI16 overexpression had no effect on cardiomyocyte size under normal conditions (data not shown), it inhibited isoproterenol/phenylephrine-induced cardiomyocyte hypertrophy as assessed by morphometric analysis of cell size and by isoleucine incorporation (Figure 4A and 4B). Interestingly, immunofluorescent staining demonstrated that the average cardiomyocyte size was already strongly reduced by overexpression of PI16 in only ≈10% of the cells (MOI 0.1, immunofluorescent detection of PI16 expression; data not shown), consistent with secretion of PI16 (Adv-lacZ versus Adv-PI16, P<0.01; Figure 4B). At MOI 0.4, ≈30% of the cells expressed PI16, and at MOI 1.6, ≈90% expressed PI16. Use of higher MOIs resulted in accumulation of PI16 in the endoplasmic reticulum and Golgi apparatus, probably due to time-consuming posttranslational modifications. We next analyzed the effect of PI16 expression on changes in the gene expression program typically associated with cardiomyocyte hypertrophy. Treatment of neonatal rat cardiomyocytes with isoproterenol/phenylephrine led to a marked induction of atrial natriuretic peptide and brain natriuretic peptide mRNA expression, which was blunted in Adv-PI16–expressing cells (Figure 4C). We then sought to determine whether endogenous PI16 is required for cardiomyocyte growth control. We transfected synthetic interfering RNA directed against PI16 into neonatal rat cardiomyocytes. This led to a marked suppression of PI16 protein levels (Figure 4D). Subsequent analysis of neonatal rat cardiomyocyte cell size by immunofluorescent staining (Figure 4D) revealed a marked increase in cardiomyocyte cell size and reorganization of the sarcomeres. These characteristics of cardiomyocyte hypertrophy were accompanied by a significant increase in cardiomyocyte protein synthesis through RNA interference–mediated suppression of PI16 (Figure 4E).
To determine the role of PI16 in the intact mammalian heart, we generated transgenic mice that overexpress PI16 in a cardiomyocyte-specific manner (Figure 5A). To assess expression of the transgenic protein relative to endogenous PI16 levels, we performed stepwise dilutions of myocardial lysates from PI16 transgenic mice with lysates from wild-type littermates (Figure 5A). We thereby determined the level of transgene expression to be ≈20-fold higher than endogenous PI16 expression in nonfailing myocardium.
PI16 transgenic mice developed normally, had normal cardiac function (Figure 5B), and showed a normal myocardial structure without any signs of interstitial fibrosis (Sirius red staining, data not shown); however, PI16 transgenic mice had significantly smaller hearts than wild-type mice (Figure 5C). In line with these findings, individual cardiomyocytes from PI16 transgenic mice were significantly smaller (−24%, P<0.01; Figure 5C).
To the best of our knowledge, the present study describes the first systematic search for secreted proteins in the heart using a biological screen. We identified 54 cardiac cDNAs that contain a secretion signal by a secretion trap screen in yeast. Among them are well-known genes such as atrial natriuretic peptide, but they also include genes with unknown cardiac expression and genes with unknown function and expression. Thirty of the identified proteins are putatively secreted. Another 15 proteins have secretion signals and 1 membrane domain but might be secreted after being shed from the plasma membrane.18
Secreted proteins play a major role in intercellular communication. Several secreted peptides and proteins have been described as being involved in cardiac remodeling.6 However, many proteins known to be secreted in the heart, such as extracellular matrix proteins, proteases, and autocrine/paracrine factors such as transforming growth factor-β, tumor necrosis factor-α, fibroblast growth factor, platelet-derived growth factor, and interleukin-6, have been shown to be secreted by activated cardiac fibroblasts.6 Little is known about the secretome of cardiomyocytes, although proteins secreted from cardiomyocytes might play an important role in myocardial remodeling. Cardiomyocyte-specific overexpression of the β1-adrenergic receptor, for example, is accompanied by activation of fibroblasts and interstitial fibrosis, although the initial stimulus for remodeling was set in cardiomyocytes.15 To avoid enrichment of mRNAs encoding for secreted proteins from noncardiomyocytes, we generated a cardiac cDNA library from young, healthy wild-type mice that showed no signs of fibroblast activation.
We then studied regulation of the identified genes during the development of heart failure. The mRNA expression of PI16, a thus far largely uncharacterized protein, was strongly upregulated early in heart failure. Here, we demonstrate that PI16 is expressed and secreted in the mammalian heart and subsequently accumulates extracellularly. Although there are putative PI16 homologs in several species, the function of PI16 is unknown. Bioinformatic analysis revealed that PI16 contains a sperm-coating glycoprotein domain. This evolutionary highly conserved protein domain can be found in >600 mainly extracellular eukaryotic proteins of many different species, including bacteria.19 It was recently shown that PSP94 binding protein, the human homolog of PI16, may be partially bound in the serum to the prostate secretory protein of 94 amino acids (PSP94).20 PSP94 is a protein that is upregulated in prostate cancer and may have growth-regulating properties in this disease,21 but the signaling pathway and the receptor are unknown. However, we could not detect any PSP94 mRNA or protein expression in either the healthy or the failing heart, which suggests a different signaling pathway in this setting. In the gene ontology classification, PI16 was inferred as protease by electronic annotation. Our own sequence analysis (ProtFun 2.222) supports an enzymatic function (probability 0.48; odds 1.67) and negates a function as a structural protein (probability 0.008; odds 0.28). Proteases have several important functions in cardiac disease; for example, matrix metalloproteases and their inhibitors (tissue inhibitors of matrix metalloproteinase) play a crucial role in extracellular matrix modification in heart failure.23
Expression of PI16 in primary cardiomyocytes revealed a potent function of PI16 as an endogenous repressor of cardiomyocyte growth. Interestingly, PI16 exerted its antihypertrophic effect even when only a small fraction of cardiomyocytes were infected with Adv-PI16, which supports a paracrine function of PI16. When we expressed PI16 in the hearts of transgenic mice, we found a profound inhibition of cardiac growth in the absence of any impairment of cardiac structure or function. In line with the experiments conducted in vitro, we found cardiomyocytes from PI16 transgenic hearts to be significantly smaller than wild-type cardiomyocytes. In vitro, PI16 did not influence cardiomyocyte cell size under resting conditions, which indicates that the ability of PI16 to control cardiomyocyte size critically depends on the cellular environment that accompanies cell growth.
The rapid and strong induction of PI16 and its growth-inhibitory effect under conditions of cardiac stress are reminiscent of atrial natriuretic peptide and brain natriuretic peptide24,25 and the cytokine GDF15 (growth-differentiation factor 15).26 All 3 secreted proteins are induced in cardiac disease and serve as endogenous feedback mechanisms to control excessive growth-promoting stimuli.
When PI16 was overexpressed to an extent that is comparable to conditions of cardiac disease, we observed a significant suppression of physiological cardiac growth. This finding sets PI16 apart from other antihypertrophic molecules such as GDF1526 and NAB1,27 which exert their antihypertrophic properties only in cardiac disease. This pivotal difference suggests alternative downstream growth control pathways that are affected by PI16. Secretion of PI16 may serve a similar role as an endogenous cardioprotective signaling pathway and might be used as a therapeutic strategy in disease states such as cardiac failure and hypertrophic cardiomyopathy.
In summary, using a genetic yeast screen of a heart cDNA library, we identified 54 cardiac cDNAs encoding for proteins that contain a secretion signal. Among them are many genes with unknown cardiac expression and function. PI16, a newly identified protein secreted by the heart, is strongly upregulated early in the development of heart failure and inhibits cardiomyocyte hypertrophy in vivo and in vitro.
We would like to thank M.J. Lohse (Institute of Pharmacology, University of Wuerzburg) for many helpful discussions during this study. We thank A. Spang (Friedrich Miescher laboratory of the Max-Planck Institute (MPI), Tuebingen, Germany) for advice on yeast work; C. Weitz (Department of Neurobiology, Harvard Medical School, Boston, Mass) for kindly providing the host yeast strain 066-2 and the mutant invertase plasmid pSuc2dMSP; A. Kramer (Humboldt University, Berlin, Germany) for support with the yeast screen; and A. Ahles for help with real-time PCR analysis, E. Schmitteckert for pronuclear injection of the transgene construct, and M. Babl for support with ventricular catheterization (Rudolf Virchow Center, University of Wuerzburg). We gratefully acknowledge the help of A. Kopp-Schneider (Department of Biostatistics of the German Cancer Research Center, Heidelberg, Germany) with statistical analysis, J. Schultz and coworkers (Department of Bioinformatics, University of Wuerzburg) with bioinformatic analysis, and A. Sickmann (Rudolf Virchow Center, University of Wuerzburg) with protein biochemistry.
Sources of Funding
These studies were supported by grants from the Deutsche Forschungsgemeinschaft (DFG), the German Cardiac Society (Hengstberger award), the Rudolf Virchow Center/DFG Research Center for Experimental Biomedicine, the Bavarian Ministry of Technology, Trigen, and Sanofi-Aventis. Dr Frost is a fellow of the MD/PhD program of the University of Wuerzburg funded by the Interdisciplinary Center for Clinical Research.
Drs Frost and Engelhardt have filed a patent application for the use of PI16 in cardiovascular disease.
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Left ventricular hypertrophy is a strong and independent predictor of outcome in cardiac failure. Because the heart is a largely postmitotic organ, growth of the heart in response to mechanical or neurohumoral stimulation primarily occurs through an increase in cardiomyocyte size. Cardiomyocyte hypertrophy may initially serve a compensatory role; a classic example of this is the partial normalization of an increase in wall stress after pressure overload of the left ventricle. With the chronic presence of a detrimental stimulus, such as enhanced catecholamine concentrations in heart failure, cardiomyocyte hypertrophy is believed to ultimately become maladaptive and contribute to the progression of heart failure. Therefore, a more detailed understanding of the molecular events that mediate and regulate cardiomyocyte growth may lead to novel therapeutic strategies in cardiac hypertrophy and heart failure. In this respect, proteins that are secreted from the heart, such as the cardioprotective atrial natriuretic peptides, are of particular interest, but only a limited set of such proteins are currently known. The present work sought to identify additional proteins that are secreted from the heart and that may act as regulators of cardiomyocyte growth. To this end, a genetic yeast-based screening approach was applied to the heart to systematically identify novel secreted proteins. Among the discovered proteins, proteinase inhibitor 16 was identified as being strongly upregulated in diseased myocardium and as acting as a potent repressor of cardiomyocyte growth. Although its effects are speculative at this early point, proteinase inhibitor 16 might eventually be used as a therapeutic means to treat cardiac hypertrophy and heart failure.
The online-only Data Supplement, consisting of an expanded Methods section, figures, and tables, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.696468/DC1.