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Alpha-1-adrenergic receptors (α1-ARs) play adaptive roles in the heart and protect against the development of heart failure (HF). The three α1-AR subtypes,α1A, α1B, and α1D, have distinct physiological roles in mouse heart, but very little is known about α1-subtypes in human heart. Here we test the hypothesis that the α1A and α1B subtypes are present in human myocardium, similar to the mouse, and are not down-regulated in heart failure.
Hearts from transplant recipients and unused donors were failing (n = 12; mean EF 24%) or non-failing (n = 9; mean EF 59%), and similar in age (~44 years) and sex (~70% male). We measured the α1-AR subtypes in multiple regions of both ventricles by quantitative real-time reverse transcription PCR and radioligand binding. All three α1-AR subtype mRNAs were present, and α1A mRNA was most abundant (~65% of total α1-AR mRNA). However, only α1A and α1B binding were present, and the α1B was most abundant (60% of total). In failing hearts, α1A and α1B binding were not down-regulated, in contrast with β1-ARs.
Our data show for the first time that the α1A and α1B subtypes are both present in human myocardium, but α1D binding is not, and that the α1-subtypes are not down-regulated in HF. Since α1-subtypes in the human heart are similar to mouse, where adaptive and protective effects of α1-subtypes are most convincing, it might become feasible to treat HF with a drug targeting the α1A and/or α1B.
The myocardium contains adrenergic receptors (ARs) of two main classes, β and α1. Myocardial β-ARs are studied extensively, and blocking β-ARs in heart failure (HF), when the AR agonist norepinephrine (NE) is elevated, is now a cornerstone of therapy. Much less is known about myocardial α1-ARs. However, animal and human data suggest that activation of myocardial α1-ARs in HF is adaptive and protective, in contrast with the toxic effects of chronic β-AR stimulation.
α1-ARs exist as three molecular subtypes, α1A, α1B, and α1D. Knockout (KO) mouse models have provided the most convincing evidence for the beneficial effects of α1-AR stimulation, and have begun to reveal the distinct physiological roles of the cardiac α1-subtypes.1 KO of the two main myocardial α1-AR subtypes, the α1A and α1B, impairs normal post-natal cardiac growth, and causes severe dilated cardiomyopathy and death after pressure overload.2, 3 In KO models, the role of the α1B subtype appears to be physiological cardiac hypertrophy,2, 4, 5 whereas the α1A is cardio-protective.3, 6 Although all three subtypes can constrict peripheral arteries,4, 7, 8 the cardiac α1D stimulates coronary vasoconstriction.9, 10
α1-AR gain-of-function in cardiac transgenic models generally supports the KO results, although phenotypes vary greatly with receptor level, promoter, and absence or presence of an activating mutation. Thus, cardiac α1B overexpression can cause hypertrophy, but this can be associated with β-AR down-regulation and late cardiomyopathy.11–14 Cardiac α1A overexpression can stimulate contractility and cause cardio-protection,15–18 but there can be late fibrosis and sudden death.19 Recently, we found that modest augmentation of cardiac α1A signaling with a subpressor dose of an α1A-selective agonist can prevent doxorubicin-induced cardiomyopathy in mice.20
In the human heart, α1-subtype roles have not been studied. However, in human clinical trials, non-selective blockade of all three α1-subtypes caused a two-fold increase in HF in the ALLHAT trial,21 and a trend toward increased mortality in the V-HeFT trial.22 Non-selective activation of all α1-subtypes in vitro has a robust positive inotropic effect in failing human myocardium.23, 24 Non-selective α1-activation in vitro also protects against ischemia.25–27 These beneficial effects of α1-AR activation suggest a novel interpretation of the harmful results of excessive NE reduction in clinical trials (MOXSE, MOXCON, and BEST),28–30 specifically, that some degree of α1-activation is essential in HF. Further consistent with a beneficial or compensatory role for human myocardial α1-ARs, chronic therapy with the cardioprotective β-blocker carvedilol potentiates α1-AR effects,31 and total α1-ARs are not down-regulated in HF, in contrast with β-ARs that are down-regulated.32–34
The distinct and important roles of the α1A, α1B, and α1D in mouse heart, and, in human heart, the contrasting harmful effects of non-selective α1-blockade and beneficial effects of non-selective α1-stimulation, emphasize the need to define expression of the α1-subtypes in the human heart. Recently, we found that the α1D is the predominant subtype in human coronary arteries.35 However, little is known about the α1-subtypes in human myocardium. In fact, prior limited mRNA studies have concluded that the human heart expresses only the α1A,36–39 and that mouse models of α1-AR biology might not be relevant to human heart disease.40, 41 The α1-subtype proteins in human myocardium have never been measured.
Here we test the hypothesis that the α1A and α1B subtypes are both present in human myocardium, similar to the mouse, and are not down-regulated in HF. This is the first report of α1-AR subtype proteins in human heart, and the first characterization of α1-subtypes in non-failing and failing heart.
With the approval of the University of California, San Francisco (UCSF) Committee for Human Research, we obtained tissue from hearts removed at the time of transplant at UCSF, or from organ donors whose hearts were not transplanted for technical reasons. Full informed consent was obtained from all UCSF transplant recipients prior to surgery. The California Transplant Donor Network (CTDN) provided the unused donor hearts and obtained informed consent from the donor’s next of kin.
Cold cardioplegia was perfused antegrade prior to cardiectomy, and the explanted heart was placed immediately in ice-cold physiologic solution. Full-thickness samples from multiple regions of the LV and RV were cleaned rapidly of all epicardial fat, flash frozen in liquid nitrogen, and stored at −80°C.
Tissue was homogenized in TRIzol reagent (Invitrogen, Gibco BRL), using a rotor-stator homogenizer (Polytron) at speed 7 out of 10. RNA was extracted in chloroform and isopropyl alcohol, purified on Qiagen Mini-Prep columns, treated with DNase (Turbo DNAfree, Ambion), and quantified using spectrophotometry (BioRad SmartSpec 3000). Selected RNA samples were analyzed to confirm the absence of significant degradation (Agilent 2100 BioAnalyzer).
Primer3 (v0.4.0) and BLAST were used to design multiple potential primer pairs for each target and reference gene. α1-AR subtype primers spanned the 25 kb intron at the end of the 6th transmembrane domain, and final primer pairs (Online Figure 1) were chosen for comparable reaction efficiencies as measured by serial dilution. Specificity of amplification was confirmed by (1) sequencing, (2) PCR with human α1-AR cDNAs, and (3) a dissociation step in all qRT-PCR reactions. Amplification of genomic DNA was excluded by (1) use of intron-spanning primers, (2) DNase treatment of RNA, and (3) PCR run on agarose gels using no-RT templates as negative controls.
For qRT-PCR, one μg of RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen) with both random hexamers (Invitrogen) and oligo-dT (Roche). qRT-PCR reactions contained 5% of the cDNA product, primers at 125 nM per reaction, and SYBR Green Master (Roche) with ROX reference dye. All reactions were performed in triplicate in an ABI PRISM 7900HT Sequence Detection System. Data were analyzed with SDS software version 2.3 (Applied Biosystems).
Relative quantitation of PCR products used the ΔΔCt method.42 Values for each mRNA are arbitrary units (AU) relative to two reference genes, β-actin and TATA-binding protein (TBP), for improved accuracy,43 as AU = 2−Δ−ΔCT × 1000, where ΔΔCT=[(mean target gene CT) − (mean reference genes CT)].
Approximately 120 mg wet weight of tissue was homogenized (5 mM Tris-HCl, 5 mM EDTA, 250 M Sucrose pH 7.4 plus 0.1 mM PMSF), and centrifuged at 100,000 × g for 1 h. The pellet was resuspended in homogenization buffer and centrifuged as before. The resulting final membrane pellet was resuspended in assay buffer (α1 binding: 50 mM Tris pH 7.4, 1 mM EDTA; β-AR binding: 154 mM NaCl, 5 mM MgCl, 20 mM Tris pH 7.4), and used for saturation and competition radioligand binding.
α1-AR saturation binding was at 30°C for 60 min with 200 μg membrane protein per tube (~2.5 mg tissue), 6 concentrations (0.04–1.2 nM) in triplicate of 3H-prazosin (Perkin Elmer), and phentolamine (10 μM) to define non-specific binding.44 β-AR binding was at 25°C for 90 min with 50 μg membrane protein per tube, 6 concentrations (0.04–1.0 nM) in triplicate of 125I-cyanopindolol (CYP, NEN Life Sciences), and L-propranolol (1 μM) to define non-specific binding.
The subtype proteins were quantified by competition binding. For α1-ARs, 3H-prazosin binding (0.5 nM) was competed with 22 concentrations (0.05 nM–500 μM) in duplicate of BMY-7378, an α1D-selective antagonist,8, 45 or 5-methylurapidil (5-MU) an α1A-selective antagonist.7 For β-ARs, 125I-CYP binding (50 pM) was competed with 22 concentrations (50 pM–500 μM) in duplicate of the β1-selective antagonist, CGP 20712A, or the β2-selective antagonist, ICI-118,551. Binding data were analyzed using GraphPad Prism 4.0b (GraphPad Software Inc., San Diego, CA). Subtype percents were calculated from fitting competition curves, and subtype levels in fmol/mg were calculated from total binding in saturation analysis, multiplied by percent from competition in the same preparation.
Results are presented as mean ± SEM. Significant differences (p < 0.05) were tested using one-way ANOVA and Tukey’s multiple comparison for more than two groups, or Student’s unpaired t-test for two groups, and a normal distribution was assumed for all continuous variables. Linear regression tested for association between mRNA abundance and clinical variables. The F test compared goodness-of-fit to one-or two-site models for competition binding analyses (GraphPad Prism v4.0).
The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.
We tested α1-AR subtype expression in human myocardium from 21 explanted hearts of 14 transplant recipients and 7 unused donors. As shown in Table 1, there were 9 non-failing (NF) and 12 failing (F) patients, one-third of which had ischemic cardiomyopathy. NF and F were similar in age and sex, but the mean ejection fraction (EF) was 59 ± 3% in the NF group, and 24 ± 2% in the F group (p<0.0001).
To characterize NF and F LV myocardium, we assayed select myocyte and non-myocyte mRNAs by qRT-PCR, and correlated these with EF. As EF decreased, LV β-MyHC levels did not change (p = 0.71), but α-MyHC decreased (p = 0.01), and Type I collagen increased (p = 0.02) (Figure 1A–C). α-MyHC mRNA in NF was 19% of total MyHC, but only 1% of total in F. Importantly, the levels of the reference genes, β-actin and TBP, were comparable in the NF and F groups (Figure 1D).
Competition binding for β-AR subtypes using ICI-118,551, an antagonist selective for the β2-subtype, showed that β1-subtype levels decreased markedly in F, whereas the β2-subtype was not changed (Figure 1E). Competition binding with CGP 20712A, a β1-selective antagonist, confirmed these values (data not shown).
In summary, we found in F LV the expected repression of α-MyHC, induction of collagen indicative of fibrosis, and down-regulation of the β1-AR subtype, showing a molecular phenotype similar to prior studies in human HF.33, 46–48
To begin to test if all three α1-subtypes were present in human myocardium, we did qRT-PCR with validated primers spanning the long intron in each α1-subtype gene. We made RNA from transmural samples of myocardium from three regions, LV free wall (LVFW), LV septum (LVS, the LV side of the interventricular septum), and RVFW, all taken at the level of the papillary muscles. Figure 2 (left) shows that the α1A was the predominant α1-subtype mRNA in all NF myocardial regions. The α1B and α1D were present at much lower levels (p < 0.05), and also did not differ among the regions (Figure 2). In NF myocardial LVFW and LVS, the α1A was ~63% of total α1-AR mRNA, the α1B was 22%, and the α1D was 15% (Table 2).
We quantified α1-AR subtype protein levels using radioligand binding. Recently, we found that commercial α1-AR antibodies are not specific for α1-ARs,49 and thus we could not use immunoblot or immunohistochemistry to detect or quantify the α1-subtype proteins.
Saturation radioligand binding in myocardial membranes with 3H-prazosin, a non-selective α1-AR antagonist, produced typical curves (Figure 3A), with Bmax ~ 4 fmol/mg protein. Specific binding at the 3H-prazosin Kd (0.2 nM) averaged 40% of total 3H-prazosin bound. Specific total α1-binding reflecting all α1-subtypes in NF myocardium was about 10% of β-AR binding (Table 2), similar to prior studies.32–34 Total α1-binding was similar in NF LVFW, LVS, and RVFW (Figure 3B left).
To detect and quantify the α1-subtype proteins, we used competition for 3H-prazosin binding with subtype-selective antagonists. Competition with 5-MU, an α1A-selective antagonist,7 produced classic two-site curves (Figure 4A left). In NF LVFW myocardium, there was a high affinity component (Ki 11 ± 4 nM, n = 6), representing the α1A subtype, and a low affinity component (Ki 99 ± 53 μM, n = 6), which could have been the α1B and/or α1D. To distinguish these, we did competition binding in the same preparations with BMY-7378, an α1D-selective antagonist.8, 45 Competition binding with BMY-7378 yielded one-site binding curves (Figure 4A right), with only a low-affinity component (Ki 11 ± 6 μM, n = 6), indicating no detectable α1D binding. Taken together, these results suggested that the α1A and α1B subtype proteins were both present in human NF LV myocardium, but the α1D was not. NF LV had 40 ± 2% α1A and 60 ± 2%α1B (p < 0.001, n = 6) (Figure 4B left, Table 2). Total α1-binding in NF RV myocardium was similar to LV (Figure 3B), but the NF RV contained relatively less α1A (25 ± 5%, n = 3, p = 0.02) (Figure 4B left).
In summary, human NF myocardium expressed all three α1-subtype mRNAs, with a striking predominance of the α1A. However, binding assays detected only the α1A and the α1B, and the α1B was predominant.
The relative pattern of α1-subtype mRNA expression within the F myocardium was identical to NF, with a predominance of α1A mRNA, and no differences between α1B and α1D in any region (Figure 2 right). Total α1-subtype mRNA levels were also similar in F and NF myocardium, although α1A mRNA was increased in F LV (p < 0.05, Table 2), and tended to increase in F RV (Figure 2).
Total α1-binding was not reduced in any region of F myocardium (Figure 3B right). Similarly, binding levels of the α1A and α1B subtypes were not reduced in F myocardium (Figure 4B), and the α1D remained undetectable (data not shown). The relative levels of α1A and α1B binding also were unchanged in F LV (Table 2), but in RV there was a relative increase in α1A binding in F versus NF (44% versus 27%, p = 0.04) (Figure 4B).
In summary, the α1A and α1B were not repressed or down-regulated in the F myocardium. The α1A tended to increase.
We measured β-AR subtype mRNAs and binding, to test if the two AR families had distinct regulation in HF. Myocardium from F LV had a significant decrease versus NF in β2-subtype mRNA and β1-subtype binding (Table 2 and Figure 1E). Stable binding levels in HF of the α1A- and α1B-subtypes and down-regulation of the β1-subtype caused a marked increase in the ratio of binding of α1-ARs to β-ARs, from about ~10% in NF LV to ~20–40% in F LV (Table 2; the 41% α1/β ratio in the F LV in Table 2 is from four samples with α1- and β-binding on the same membranes).
The qRT-PCR results were analyzed to determine whether demographic or clinical factors affected the expression of α1-subtype mRNAs. We found that age, EF, sex, β-blocker use, and CAD had no effect on myocardial total or α1-subtype mRNAs (Figure 5A–D and data not shown). On the other hand, β-agonist use was associated with a decrease in both α1B (p = 0.04) and α1D (p < 0.01), but did not affect α1A levels (Figure 5E). We compared β-AR mRNAs in a similar analysis, and found that β1- and β2-subtype mRNA levels in myocardium did not change with age, EF, sex, β-blocker use, β-agonist use, or CAD (Online Figure 2 and data not shown).
We present here the first data on α1-AR subtype proteins in human heart, and the first comparison of α1-AR subtypes in NF and F human hearts. The main findings are that the α1A is the predominant α1-subtype mRNA, the α1A and α1B are both present by binding, with the α1B predominant, and the α1D is undetectable by binding. In HF, α1-subtype mRNAs are not repressed, and α1-binding is not down-regulated, in contrast with β-AR subtypes.
Prior studies of human myocardial α1-AR subtypes used semi-quantitative mRNA assays with a very limited number of undefined patients, and never measured α1-subtype proteins.36–39 These studies identified the α1A as the most abundant or only α1-subtype mRNA in myocardium, and a separate study concluded that the α1A was the only α1-subtype in human coronary arteries.40 These results prompted the conclusion that the human heart is exclusively α1A, and thus that mouse models are irrelevant to human cardiac α1-AR biology.40, 41 On the contrary, the present and our recent studies show that α1-subtype expression is the same in the human and mouse heart, with the α1A and α1B subtypes in myocardium, and the α1D in coronary arteries. This is important, because it implies that findings in mouse genetic models, where α1-subtype functions can be studied with precision, are relevant to human cardiac α1-AR biology, in particular, the adaptive and protective effects of the α1A- and α1B-subtypes.
Technical aspects of this study warrant emphasis. Although our patient population was modest in size, it was significantly larger and more thoroughly characterized than in prior studies. We quantified α1-subtype mRNAs in DNase-treated RNA, using qRT-PCR with carefully evaluated primer pairs that cross the large intron in all α1-AR genes, to eliminate contamination from genomic DNA. We quantified α1-subtype proteins by radioligand binding. Prior studies have used commercial antibodies to measure α1-subtypes in human non-cardiac tissues, but we find that ten different antibodies are not specific for α1-ARs,49 indicating that binding is currently the only valid method for detecting and quantifying α1-AR proteins. Our membrane preparation for binding was not “purified”, i.e. we did not discard any low speed pellets. This meant that we did not discard the large number of receptors that are found in low speed pellets,1 and also that our denominator of mg protein was higher than in purified membranes.
A potential technical concern was the discordance between α1-subtype mRNA levels and binding levels. The α1A mRNA in human myocardium was by far most abundant, but α1A binding was less than α1B. The myocardial α1D mRNA was as abundant as α1B, but α1D binding was undetectable. Importantly, the same discordance between the levels of α1A and α1B mRNAs and binding is seen also in the mouse and rat heart,1, 44 and mouse myocardium has α1D mRNA without α1D binding.2 Furthermore, we are able to detect α1D binding in human coronary arteries, where the α1D is 75% of total binding,35 ruling out a technical problem. In summary, these results together indicate that the α1-AR subtypes in myocardium have substantial post-transcriptional regulation in man and rodent.
A major finding was that α1-subtype mRNAs and α1-subtype binding were unchanged or increased in HF, in contrast with β-AR subtypes, where the β2 mRNA was repressed, and β1-binding, as shown before,33 was down-regulated. To simplify characterization of the AR phenotype in F versus NF myocardium, we normalized all subtype binding, given in Table 2, to binding of the α1A in NF LV. Normalization provides the following binding ratios in human LV myocardium of α1A:α1B:β1:β2: in NF LV, 1:2:17:7, and in F LV, 1:2:6:7. Thus, the failing human myocardium has potentially much greater relative signaling through the α1A, α1B, and β2 subtypes. The F RV was especially notable, with a significant increase in relative α1A binding (Figure 4).
Loss and gain of function studies in the mouse heart show adaptive and protective effects of stimulation of the α1A and/or α1B subtypes (Introduction), raising the possibility of using α1-subtype-selective agonists as therapy for HF. We demonstrate here that the α1A and α1B are both present in the human myocardium and are not down-regulated in HF, indicating that this idea might be feasible. Furthermore, the fact that the α1-subtype in human coronary arteries is the α1D suggests that an agonist for the α1A or α1B would not cause coronary constriction.35 On the other hand, an α1D-selective antagonist might be safe and efficacious in prostate disease,50 without the potential harmful side effects of non-selective blockade of myocardial α1A and α1B subtypes.21, 22
In this study, we characterize the α1-AR subtypes in the non-failing and failing human heart. As in the mouse, the α1A and α1B are the predominant binding subtypes in human myocardium, and the α1D is absent. In contrast to β1-ARs, the α1A and α1B are not down-regulated in HF. Thus, α1-subtypes in the human heart appear to be similar to mouse heart, where adaptive and protective effects of α1-subtypes are most convincing. Further studies will help determine whether it might be feasible to treat HF with a drug targeting the α1A and/or α1B.
For assistance with obtaining heart tissue we thank the CTDN, Celia Rifkin and the staff in UCSF operating rooms 9 and 10, and J. Eduardo Rame, MD MPhil.
FUNDING SOURCES: Support was from the Department of Veterans Affairs and the NIH (PCS), a Young Investigators Award from the GlaxoSmithKline Research and Education Foundation for Cardiovascular Disease (BCJ), and the UCSF Foundation for Cardiac Research (BCJ).
SUMMARY and CLINICAL RELEVANCE: Elevated levels of catecholamines bind two classes of adrenergic receptors (ARs) in the failing heart: α1-ARs and β-ARs. The toxic effect of excessive β-AR stimulation is well-studied. Less is known about α1-ARs in the human heart, although evidence ranging from genetically-altered mice to large clinical trials suggests that they play adaptive and protective roles. α1-ARs exist as three distinct molecular subtypes: A, B, and D. In the rodent heart, the A and B subtypes are on cardiomyocytes, where they mediate beneficial processes including positive inotropy, physiological hypertrophy, and protection from cell death. The α1D protein is absent in rodent cardiomyocytes, but regulates coronary vasoconstriction. In a separate study, we found that the α1D is also the most abundant subtype in human coronary arteries. However, very little is known about the α1-AR subtypes in human myocardium. Here we used tissue from failing and non-failing hearts to show that the α1A and α1B are the predominant α1-subtypes in human myocardium, and that their abundance is maintained in HF. Our findings demonstrate that the distribution of α1-AR subtypes in the human heart is similar to that in the mouse heart, indicating that findings from mouse models can be applicable to human α1-biology. Our findings also suggest the possibility of therapeutically activating a beneficial α1-subtype in the myocardium without causing α1D-mediated coronary vasoconstriction. Likewise, it is possible that the benefits of α1-blockade in the treatment of prostate disease might best be achieved by selective antagonism of the α1D.