Philip M. Swigart

Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific

Commercial antibodies are used widely to quantify and localize the α1-adrenergic receptor (AR) subtypes, α1A, α1B, and α1D. We tested ten antibodies, from abcam and Santa Cruz, using western blot with heart and brain tissue from wild-type (WT) mice and mice with systemic knockout (KO) of one or all three subtypes. We found that none of the antibodies detected a band in WT that was absent in the appropriate KO or in the KO that was null for all α1-ARs (ABDKO). We conclude that the antibodies we tested are not specific for α1-ARs. These results raise caution with prior studies using these reagents. For now, competition radioligand binding is the only reliable approach to quantify the α1-AR subtype proteins. Receptor protein localization remains a challenge.

α1-Adrenergic receptors prevent a maladaptive cardiac response to pressure overload

An alpha1-adrenergic receptor (alpha1-AR) antagonist increased heart failure in the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT), but it is unknown whether this adverse result was due to alpha1-AR inhibition or a nonspecific drug effect. We studied cardiac pressure overload in mice with double KO of the 2 main alpha1-AR subtypes in the heart, alpha 1A (Adra1a) and alpha 1B (Adra1b). At 2 weeks after transverse aortic constriction (TAC), KO mouse survival was only 60% of WT, and surviving KO mice had lower ejection fractions and larger end-diastolic volumes than WT mice. Mechanistically, final heart weight and myocyte cross-sectional area were the same after TAC in KO and WT mice. However, KO hearts after TAC had increased interstitial fibrosis, increased apoptosis, and failed induction of the fetal hypertrophic genes. Before TAC, isolated KO myocytes were more susceptible to apoptosis after oxidative and beta-AR stimulation, and beta-ARs were desensitized. Thus, alpha1-AR deletion worsens dilated cardiomyopathy after pressure overload, by multiple mechanisms, indicating that alpha1-signaling is required for cardiac adaptation. These results suggest that the adverse cardiac effects of alpha1-antagonists in clinical trials are due to loss of alpha1-signaling in myocytes, emphasizing concern about clinical use of alpha1-antagonists, and point to a revised perspective on sympathetic activation in heart failure.

β-Myosin Heavy Chain Is Induced by Pressure Overload in a Minor Subpopulation of Smaller Mouse Cardiac Myocytes

Rationale: Induction of the fetal hypertrophic marker gene &bgr;-myosin heavy chain (&bgr;-MyHC) is a signature feature of pressure overload hypertrophy in rodents. &bgr;-MyHC is assumed present in all or most enlarged myocytes. Objective: To quantify the number and size of myocytes expressing endogenous &bgr;-MyHC by a flow cytometry approach. Methods and Results: Myocytes were isolated from the left ventricle of male C57BL/6J mice after transverse aortic constriction (TAC), and the fraction of cells expressing endogenous &bgr;-MyHC was quantified by flow cytometry on 10 000 to 20 000 myocytes with use of a validated &bgr;-MyHC antibody. Side scatter by flow cytometry in the same cells was validated as an index of myocyte size. &bgr;-MyHC-positive myocytes constituted 3±1% of myocytes in control hearts (n=12), increasing to 25±10% at 3 days to 6 weeks after TAC (n=24, P<0.01). &bgr;-MyHC-positive myocytes did not enlarge with TAC and were smaller at all times than myocytes without &bgr;-MyHC (≈70% as large, P<0.001). &bgr;-MyHC-positive myocytes arose by addition of &bgr;-MyHC to &agr;-MyHC and had more total MyHC after TAC than did the hypertrophied myocytes that had &agr;-MyHC only. Myocytes positive for &bgr;-MyHC were found in discrete regions of the left ventricle in 3 patterns: perivascular, in areas with fibrosis, and in apparently normal myocardium. Conclusions: &bgr;-MyHC protein is induced by pressure overload in a minor subpopulation of smaller cardiac myocytes. The hypertrophied myocytes after TAC have &agr;-MyHC only. These data challenge the current paradigm of the fetal hypertrophic gene program and identify a new subpopulation of smaller working ventricular myocytes with more myosin.

α1-Adrenergic Receptor Subtypes in Nonfailing and Failing Human Myocardium

Background—&agr;1-adrenergic receptors (&agr;1-ARs) play adaptive roles in the heart and protect against the development of heart failure. The 3 &agr;1-AR subtypes, &agr;1A, &agr;1B, and &agr;1D, have distinct physiological roles in mouse heart, but very little is known about &agr;1 subtypes in human heart. Here, we test the hypothesis that the &agr;1A and &agr;1B subtypes are present in human myocardium, similar to the mouse, and are not downregulated in heart failure. Methods and Results—Hearts from transplant recipients and unused donors were failing (n=12; mean ejection fraction, 24%) or nonfailing (n=9; mean ejection fraction, 59%) and similar in age (≈44 years) and sex (≈70% male). We measured the &agr;1-AR subtypes in multiple regions of both ventricles by quantitative real-time reverse-transcription polymerase chain reaction and radioligand binding. All 3 &agr;1-AR subtype mRNAs were present, and &agr;1A mRNA was most abundant (≈65% of total &agr;1-AR mRNA). However, only &agr;1A and &agr;1B binding were present, and the &agr;1B was most abundant (60% of total). In failing hearts, &agr;1A and &agr;1B binding was not downregulated, in contrast with &bgr;1-ARs. Conclusions—Our data show for the first time that the &agr;1A and &agr;1B subtypes are both present in human myocardium, but &agr;1D binding is not, and the &agr;1 subtypes are not downregulated in heart failure. Because &agr;1 subtypes in the human heart are similar to those in the mouse, where adaptive and protective effects of &agr;1 subtypes are most convincing, it might become feasible to treat heart failure with a drug targeting the &agr;1A and/or &agr;1B.

The Alpha-1D Is the Predominant Alpha-1-Adrenergic Receptor Subtype in Human Epicardial Coronary Arteries

OBJECTIVES The goal was to identify alpha-1-adrenergic receptor (AR) subtypes in human coronary arteries. BACKGROUND The alpha1-ARs regulate human coronary blood flow. The alpha1-ARs exist as 3 molecular subtypes, alpha1A, alpha1B, and alpha1D, and the alpha1D subtype mediates coronary vasoconstriction in the mouse. However, the alpha1A is thought to be the only subtype in human coronary arteries. METHODS We obtained human epicardial coronary arteries and left ventricular (LV) myocardium from 19 transplant recipients and 6 unused donors (age 19 to 70 years; 68% male; 32% with coronary artery disease). We cultured coronary rings and human coronary smooth muscle cells. We assayed alpha1- and beta-AR subtype messenger ribonucleic acid (mRNA) by quantitative real-time reverse transcription polymerase chain reaction and subtype proteins by radioligand binding and extracellular signal-regulated kinase (ERK) activation. RESULTS The alpha1D subtype was 85% of total coronary alpha1-AR mRNA and 75% of total alpha1-AR protein, and alpha1D stimulation activated ERK. In contrast, the alpha1D was low in LV myocardium. Total coronary alpha1-AR levels were one-third of beta-ARs, which were 99% the beta2 subtype. CONCLUSIONS The alpha1D subtype is predominant and functional in human epicardial coronary arteries, whereas the alpha1A and alpha1B are present at very low levels. This distribution is similar to the mouse, where myocardial alpha1A- and alpha1B-ARs mediate beneficial functional responses and coronary alpha1Ds mediate vasoconstriction. Thus, alpha1D-selective antagonists might mediate coronary vasodilation, without the negative cardiac effects of nonselective alpha1-AR antagonists in current use. Furthermore, it could be possible to selectively activate beneficial myocardial alpha1A- and/or alpha1B-AR signaling without causing coronary vasoconstriction.

Distinctive ERK and p38 signaling in remote and infarcted myocardium during post-MI remodeling in the mouse.

Global activation of MAP kinases has been reported in both human and experimental heart failure. Chronic remodeling of the surviving ventricular wall after myocardial infarction (MI) involves both myocyte loss and fibrosis; we hypothesized that this cardiomyopathy involves differential shifts in pro‐ and anti‐apoptotic MAP kinase signaling in cardiac myocyte (CM) and non‐myocyte. Cardiomyopathy after coronary artery ligation in mice was characterized by echocardiography, ex vivo Langendorff preparation, histologic analysis and measurements of apoptosis. Phosphorylation (activation) of signaling molecules was analyzed by Western blot, ELISA and immunohistochemistry. Post‐MI remodeling involved dramatic changes in the phosphorylation of both stress‐activated MAP (SAP) kinase p38 as well as ERK, a known mediator of cell survival, but not of SAP kinase JNK or the anti‐apoptotic mediator of PI3K, Akt. Phosphorylation of p38 rose early after MI in the infarct, whereas a more gradual rise in the remote myocardium accompanied a rise in apoptosis in that region. In both areas, ERK phosphorylation was lowest early after MI and rose steadily thereafter, though infarct phosphorylation was consistently higher. Immunostaining of p‐ERK localized to fibrotic areas populated primarily by non‐myocytes, whereas staining of p38 phosphorylation was stronger in areas of progressive CM apoptosis. Relative segregation of CMs and non‐myocytes in different regions of the post‐MI myocardium revealed signaling patterns that imply cell type‐specific changes in pro‐ and anti‐apoptotic MAP kinase signaling. Prevention of myocyte loss and of LV remodeling after MI may therefore require cell type‐specific manipulation of p38 and ERK activation. J. Cell. Biochem. 109: 1185–1191, 2010.

α1-Adrenergic Receptor Subtypes in Nonfailing and Failing Human MyocardiumCLINICAL PERSPECTIVE

Background—&agr;1-adrenergic receptors (&agr;1-ARs) play adaptive roles in the heart and protect against the development of heart failure. The 3 &agr;1-AR subtypes, &agr;1A, &agr;1B, and &agr;1D, have distinct physiological roles in mouse heart, but very little is known about &agr;1 subtypes in human heart. Here, we test the hypothesis that the &agr;1A and &agr;1B subtypes are present in human myocardium, similar to the mouse, and are not downregulated in heart failure. Methods and Results—Hearts from transplant recipients and unused donors were failing (n=12; mean ejection fraction, 24%) or nonfailing (n=9; mean ejection fraction, 59%) and similar in age (≈44 years) and sex (≈70% male). We measured the &agr;1-AR subtypes in multiple regions of both ventricles by quantitative real-time reverse-transcription polymerase chain reaction and radioligand binding. All 3 &agr;1-AR subtype mRNAs were present, and &agr;1A mRNA was most abundant (≈65% of total &agr;1-AR mRNA). However, only &agr;1A and &agr;1B binding were present, and the &agr;1B was most abundant (60% of total). In failing hearts, &agr;1A and &agr;1B binding was not downregulated, in contrast with &bgr;1-ARs. Conclusions—Our data show for the first time that the &agr;1A and &agr;1B subtypes are both present in human myocardium, but &agr;1D binding is not, and the &agr;1 subtypes are not downregulated in heart failure. Because &agr;1 subtypes in the human heart are similar to those in the mouse, where adaptive and protective effects of &agr;1 subtypes are most convincing, it might become feasible to treat heart failure with a drug targeting the &agr;1A and/or &agr;1B.

Functional alpha-1B adrenergic receptors on human epicardial coronary artery endothelial cells

Alpha-1-adrenergic receptors (α1-ARs) regulate coronary arterial blood flow by binding catecholamines, norepinephrine (NE), and epinephrine (EPI), causing vasoconstriction when the endothelium is disrupted. Among the three α1-AR subtypes (α1A, α1B, and α1D), the α1D subtype predominates in human epicardial coronary arteries and is functional in human coronary smooth muscle cells (SMCs). However, the presence or function of α1-ARs on human coronary endothelial cells (ECs) is unknown. Here we tested the hypothesis that human epicardial coronary ECs express functional α1-ARs. Cultured human epicardial coronary artery ECs were studied using quantitative real-time reverse transcription polymerase chain reaction, radioligand binding, immunoblot, and 3H-thymidine incorporation. The α1B-subtype messenger ribonucleic acid (mRNA) was predominant in cultured human epicardial coronary ECs (90–95% of total α1-AR mRNA), and total α1-AR binding density in ECs was twice that in coronary SMCs. Functionally, NE and EPI through the α1B subtype activated extracellular signal-regulated kinase (ERK) in ECs, stimulated phosphorylation of EC endothelial nitric oxide synthase (eNOS), and increased deoxyribonucleic acid (DNA) synthesis. These results are the first to demonstrate α1-ARs on human coronary ECs and indicate that the α1B subtype is predominant. Our findings provide another potential mechanism for adverse cardiac effects of drug antagonists that nonselectively inhibit all three α1-AR subtypes.

Adrenergic Receptors in Individual Ventricular Myocytes

Rationale: It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), &bgr;1, &bgr;2, &bgr;3, &agr;1A, and &agr;1B. The &bgr;1 and &bgr;2 are thought to be the dominant myocyte ARs. Objective: Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results: We studied ventricular myocytes from wild-type mice, mice with &agr;1A and &agr;1B knockin reporters, and &bgr;1 and &bgr;2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal–regulated kinase and phospholamban), and contraction. We found that the &bgr;1 and &agr;1B were present in all myocytes. The &agr;1A was present in 60%, with high levels in 20%. The &bgr;2 and &bgr;3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total &bgr;-ARs were &bgr;2 and 20% were &bgr;3, both mainly in nonmyocytes. Conclusion: The dominant ventricular myocyte ARs present in all cells are the &bgr;1 and &agr;1B. The &bgr;2 and &bgr;3 are mostly absent in myocytes but are abundant in nonmyocytes. The &agr;1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with &bgr;1 and &agr;1B only; 60% that also have the &agr;1A; and 5% each that also have the &bgr;2 or &bgr;3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms.Rationale: It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), β1, β2, β3, α1A, and α1B. The β1 and β2 are thought to be the dominant myocyte ARs. Objective: Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results: We studied ventricular myocytes from wild-type mice, mice with α1A and α1B knockin reporters, and β1 and β2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal–regulated kinase and phospholamban), and contraction. We found that the β1 and α1B were present in all myocytes. The α1A was present in 60%, with high levels in 20%. The β2 and β3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total β-ARs were β2 and 20% were β3, both mainly in nonmyocytes. Conclusion: The dominant ventricular myocyte ARs present in all cells are the β1 and α1B. The β2 and β3 are mostly absent in myocytes but are abundant in nonmyocytes. The α1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with β1 and α1B only; 60% that also have the α1A; and 5% each that also have the β2 or β3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms. # Novelty and Significance {#article-title-74}

A Myocardial Slice Culture Model Reveals Alpha-1A-Adrenergic Receptor Signaling in the Human Heart

Summary The authors used 52 nonfailing and failing human hearts to develop a simple, high throughput left ventricular myocardial slice model that is stable by ATP and viability assays for at least 3 days. The model supports studies of signaling, contraction, and viral transduction. They use the model to show for the first time that the alpha-1A-adrenergic receptor, which is present at very low abundance in the human myocardium, activates cardioprotective ERK with nanomolar EC50 in failing heart slices and stimulates a positive inotropic effect. This model should be useful for translational studies, to test whether molecules discovered in basic experiments are functional in the human heart.