An α1A-Adrenergic–Extracellular Signal-Regulated Kinase Survival Signaling Pathway in Cardiac Myocytes
Background— In α1-AR knockout (α1ABKO) mice that lacked cardiac myocyte α1-adrenergic receptor (α1-AR) binding, aortic constriction induced apoptosis, dilated cardiomyopathy, and death. However, it was unclear whether these effects were attributable to a lack of cardiac myocyte α1-ARs and whether the α1A, α1B, or both subtypes mediated protection. Therefore, we investigated α1A and α1B subtype–specific survival signaling in cultured cardiac myocytes to test for a direct protective effect of α1-ARs in cardiac myocytes.
Methods and Results— We cultured α1ABKO myocytes and reconstituted α1-AR signaling with adenoviruses expressing α1-GFP fusion proteins. Myocyte death was induced by norepinephrine, doxorubicin, or H2O2 and was measured by annexin V/propidium iodide staining. In α1ABKO myocytes, all 3 stimuli significantly increased apoptosis and necrosis. Reconstitution of the α1A subtype, but not the α1B, rescued α1ABKO myocytes from cell death induced by each stimulus. To address the mechanism, we examined α1-AR activation of extracellular signal-regulated kinase (ERK). In α1ABKO hearts, aortic constriction failed to activate ERK, and in α1ABKO myocytes, expression of a constitutively active MEK1 rescued α1ABKO myocytes from norepinephrine-induced death. In addition, only the α1A-AR activated ERK in α1ABKO myocytes, and expression of a dominant-negative MEK1 completely blocked α1A survival signaling in α1ABKO myocytes.
Conclusions— Our results demonstrate a direct protective effect of the α1A subtype in cardiac myocytes and define an α1A-ERK signaling pathway that is required for myocyte survival. Absence of the α1A-ERK pathway can explain the failure to activate ERK after aortic constriction in α1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.
Received September 15, 2006; accepted December 11, 2006.
Alpha-1-adrenergic receptors (α1-ARs) are classically associated with the regulation of vascular smooth-muscle contraction,1 but recent studies suggest important α1-AR functions in the heart. Clinically, α1-AR antagonists are used to treat hypertension and prostate enlargement with urinary symptoms. However, in the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT)2 and the Veterans Administration Heart Failure Trial (V-HeFT),3 the use of α1-AR antagonists worsened heart failure and increased mortality in hypertensive and heart failure patients. Because of the recent recommendation for increased use of α1-AR antagonist therapy in prostate hyperplasia,4 determining the exact role of α1-ARs in the heart has important clinical implications.
Clinical Perspective p 772
Using α1-AR knockout mice (α1ABKO) that lacked the α1A- and α1B-AR subtypes, we demonstrated previously that α1-ARs are required for postnatal physiological hypertrophy of cardiac myocytes and for adaptation to myocardial stress.5,6 In α1ABKO mice, which lack cardiac myocyte α1-AR binding, aortic constriction induces apoptosis, dilated cardiomyopathy, and death. These results suggest that α1-ARs mediate survival signaling in cardiac myocytes, which could explain the adverse outcomes after aortic constriction in α1ABKO mice. In addition, the adverse outcomes in α1ABKO mice after aortic constriction correlate with results observed in ALLHAT and V-HeFT,2,3 cautioning against the use of α1-AR antagonists.
Despite these findings, several questions remain regarding α1-AR–mediated survival signaling. Because the α1ABKO was a systemic knockout, it is unclear whether the adverse outcomes after aortic constriction were caused directly by the absence of α1-ARs in cardiac myocytes. Furthermore, it is unclear whether the α1A, α1B, or both subtypes are required for α1-AR–mediated survival signaling and adaptation to myocardial stress.
To demonstrate a direct protective effect of α1-ARs in cardiac myocytes and to define the mechanism of protection, we examined α1A- and α1B-AR subtype–specific signaling in cultured cardiac myocytes. We also studied the role of extracellular signal-regulated kinase (ERK), a known regulator of myocyte survival,7–9 in mediating α1-AR survival signaling in cardiac myocytes. In cultured α1ABKO cardiac myocytes, which are susceptible to death from β-AR stimulation and oxidative stress,6 we reconstituted α1A and α1B subtype signaling in an attempt to reverse the susceptibility to death and, thereby, demonstrate a direct protective effect of α1-ARs. Our results show that reconstitution of α1A, but not α1B, subtype signaling rescued α1ABKO myocytes from cell death induced by norepinephrine (NE), doxorubicin, and H2O2. We also found that activation of ERK was sufficient to protect α1ABKO myocytes from cell death and was required for α1A-mediated survival signaling. Therefore, our results demonstrate a direct protective effect of the α1A subtype in cardiac myocytes and define an α1A-ERK signaling pathway that is required for myocyte survival. Absence of the α1A-ERK pathway can explain the failure to activate ERK after aortic constriction in α1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.
Generation of Adenoviral Constructs
To generate α1A-GFP and α1B-GFP fusion proteins, cDNAs for the human α1A-AR (NM000680) and α1B-AR (NM000679) were amplified by polymerase chain reaction with primers designed to remove the stop codon and insert Bgl II and Mlu I restriction sites 5′ and 3′, respectively. The amplified α1A and α1B products were cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif) and then subcloned into the Bgl II–Mlu I restriction sites in the multicloning site of the humanized pGFP2-N3 vector (BioSignal Packard, Montreal, Quebec, Canada), with GFP at the C-terminus.10–13
To generate adenoviruses expressing the α1A-GFP and α1B-GFP fusion proteins under control of the cytomegalovirus promoter, the α1A-AR-GFP2 and α1B-AR-GFP2 were amplified by polymerase chain reaction with primers designed to insert Pme I and Xba I restriction sites at the 5′ and 3′ ends, respectively. The amplified α1A-GFP and α1B-GFP products were cloned into the pCR2.1-TOPO vector (Invitrogen) and then subcloned into the Pme I–Xba I restriction sites in the Ad5CMV K-NpA vector (ViraQuest, North Liberty, Ia) under control of the cytomegalovirus promoter. The Ad5 plasmids with α1-AR inserts were then recombined with an adenoviral cell line. Clones positive for recombination were transfected into HEK293 cells. Viral products evident 7 to 10 days after transfection were amplified, purified through 2 rounds of CsCl gradients, and dialyzed against a 3% sucrose/phosphate buffer solution. Viral titer was determined by observing plaque formation in agarose overlay assays.
The constitutively active and dominant-negative MEK1 adenoviruses were generated as described previously.8,14
Culture of Adult Mouse Cardiac Myocytes
Ventricular cardiac myocytes from adult male mice were cultured as previously described6,15,16 (see the Methods section of the online-only Data Supplement for more detail).
Measurement of α1-AR Expression
α1-AR levels and binding affinity in cultured wild type (WT) and α1ABKO myocytes expressing α1-ARs were measured by saturation binding as previously described5 (see the Methods section of the online-only Data Supplement for more detail).
Localization of α1-ARs in Adult Mouse Cardiac Myocytes by Confocal Microscopy
WT or α1ABKO myocytes were cultured on glass coverslips. α1ABKO myocytes were infected with adenovirus expressing α1-GFPs or untagged α1-ARs. For uninfected WT myocytes and α1ABKO myocytes infected with untagged α1-ARs, 50 nmol/L BODIPY prazosin (Molecular Probes, Eugene, Ore) was added to the culture after 24 hours. After an additional 16 hours, myocytes were fixed with 4% paraformaldehyde and mounted on slides with fluoromount G (Electron Microscopy Sciences, Hatfield, Pa). Fluorescent images were captured by confocal microscopy using Fluoview software (Olympus BX50 confocal microscope; Olympus America Inc., Melville, NY). Images were processed for publication using Imaris software (Bitplane Scientific Solutions, St. Paul, Minn).
Measurement of α1-Mediated Inositol Phosphate Generation
To quantify α1-mediated inositol phosphate generation in both HeLa cells and α1ABKO myocytes expressing α1-ARs, we measured total inositol phosphate in response to phenylephrine treatment, using a slight modification to our previously described protocol17 (see the Methods section of the online-only Data Supplement for more detail).
Measurement of Cell Death
Myocyte death was measured using annexin V (AnnV)/propidium iodide (PI) staining as described previously.6,16 For cell death assays, myocytes were infected with adenovirus and cultured for 40 hours. At 40 hours, myocytes were treated for 2 hours with NE (1 μmol/L), H2O2 (10 μmol/L), doxorubicin (1 μmol/L), or vehicle (100 μmol/L ascorbic acid for NE; saline for doxorubicin). After 2 hours, AnnV-Fluos (Roche Diagnostics Corp, Indianapolis, Ind) and PI (Roche Diagnostics Corp) were added directly to the culture medium. After 10 minutes, myocytes were photographed under both phase contrast and fluorescent microscopy. For each condition, 300 to 400 myocytes were counted in randomly selected fields, and each condition was measured in duplicate. Apoptotic myocytes were defined as AnnV positive and PI negative, and necrotic myocytes were defined as AnnV and PI positive.
Measurement of MEK/ERK Signaling
ERK activity, the effects of the constitutively active MEK1 (MEK1 CA) and dominant-negative MEK1 (MEK1 D/N) mutants on ERK activity, and MEK1 levels were all measured by Western blot (phospho- and total ERK and MEK1 antibodies, Cell Signaling Technology, Beverly, Mass), as described previously.5,8
Transverse Aortic Constriction
Transverse aortic constriction surgery was performed without intubation under anesthesia with isoflurane, as described previously.5,6
The α1ABKO mice used in this study have been described previously.5,6 In all experiments, we used congenic C57Bl/6 male WT or knockout mice, ages 10 to 15 weeks. All protocols involving animal use were reviewed and approved by the internal animal care and use committee at the University of South Dakota.
In all experiments, values were compared by 1-way ANOVA with Bonferonni posttest, and P<0.05 was considered significant. The number of experiments (n), given in each figure legend, refers to independent cultures from different hearts.
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.
Expression and Localization of α1A-GFP and α1B-GFP Fluorescent Fusion Proteins in Cardiac Myocytes
To study α1-AR–mediated survival signaling, we reconstituted α1-AR signaling in α1ABKO myocytes, which lack α1-AR binding, using adenoviruses expressing α1A- or α1B-GFP fluorescent fusion proteins. The GFP tags allow for visualization of receptor expression and immunodetection by Western blot using a GFP antibody, because no reliable α1 subtype–specific antibodies are available. As a control, we also made adenoviruses expressing untagged α1-ARs. We measured expression levels of the α1-GFP and untagged α1 constructs in binding assays with 3H-prazosin, and we defined a 2.5- to 4-fold level of overexpression for both the GFP-tagged and untagged α1 constructs used in all subsequent experiments (binding assays not shown).
On expressing the α1A- and α1B-GFP fusion proteins in α1ABKO cardiac myocytes, we observed that the α1A and α1B-GFP both localized to the nucleus and perinucleus (Figures 1a and 1c). To verify that this localization was not an artifact caused by the GFP tag, we also examined the localization of the untagged α1A and α1B subtypes in α1ABKO cardiac myocytes using BODIPY prazosin, a prazosin analog that fluoresces when bound to a receptor. As with the α1A- and α1B-GFP, we observed that the untagged α1A and -α1B subtypes localized to the nucleus and perinucleus (Figures 1b and 1d). Using BODIPY prazosin, we also found that α1-ARs in WT myocytes localized to the nucleus and perinucleus (Figure 1e), validating our expression system.
To determine whether the GFP tag altered α1-mediated signaling, we measured α1-mediated inositol phosphate generation in HeLa cells and cardiac myocytes. In HeLa cells, the α1-GFP fusion proteins and untagged α1-ARs all increased inositol phosphate levels, and there was no difference between the GFP-tagged or untagged receptors (see Figure I in the online-only Data Supplement). Maximum total inositol phosphate production was greater with the α1A subtype than with the α1B, as it was in previous studies with GFP-tagged α1-ARs.13 However, we were not able to detect α1-mediated inositol phosphate generation in α1ABKO cardiac myocytes expressing our α1-AR constructs (endothilin-1 did increase inositol phosphate levels, data not shown), similar to previous reports in cultured WT adult mouse cardiac myocytes.18
In summary, these results suggest that the GFP fluorescent moiety did not alter receptor localization or function, thus defining a reconstitution system using α1A- and α1B-GFP fluorescent fusion proteins.
The α1A Subtype, but Not the α1B Subtype, Rescues α1ABKO Cardiac Myocytes From NE-Induced Cell Death
Previously, we demonstrated that α1ABKO cardiac myocytes were susceptible to death induced by NE through β-ARs and by oxidative stress (H2O2).6 Here, we reconstituted α1-AR signaling in α1ABKO cardiac myocytes to determine whether an α1-AR subtype—the α1A, α1B, or both—could reverse the susceptibility of α1ABKO myocytes to death, thus demonstrating a direct protective effect of α1-AR signaling in cardiac myocytes. To reconstitute α1-AR signaling, cultured α1ABKO myocytes were infected with the α1-AR adenoviruses (α1A-GFP, MOI 1000; untagged α1A, MOI 50; α1B-GFP, MOI 3000; untagged α1B 1500) to obtain roughly equal levels of expression of each receptor (2.5- to 4-fold over basal). Myocyte death was induced with NE, which is known to cause myocyte apoptosis by activating β1-AR signaling.19,20 Myocyte death was quantified by AnnV/PI staining, where AnnV-positive and PI-negative cells were considered apoptotic, and AnnV- and PI-double-positive cells were considered necrotic (Figure 2).
As we observed previously,6 NE (1 μmol/L) did not induce cell death in WT myocytes, but in α1ABKO myocytes, NE significantly increased both apoptosis (P<0.05 versus WT NE) and necrosis (P<0.05 versus WT NE), which was correlated with a significant loss in rod-shaped myocyte morphology (P<0.05 versus WT NE) (Figure 2a through c). Reconstitution of α1A subtype signaling (α1A-GFP or untagged α1A) rescued α1ABKO myocytes from NE-induced cell death (apoptosis, necrosis, and rod shape: all P<0.05 versus α1ABKO NE), but reconstitution of α1B signaling (α1B-GFP or untagged α1B) did not (apoptosis, necrosis, and rod shape: all P=NS versus α1ABKO NE) (Figure 2a through c). We verified this result by examining NE-induced cell death in cardiac myocytes from single α1AKO and α1BKO mice, which, again, showed that the α1A subtype protected against NE-induced cell death (seen as increased cell death in α1AKO myocytes; Figure 3). To ensure that the higher levels of adenovirus used to express the α1B constructs did not induce cell death, we tested a β-galactosidase control virus in the same experimental protocol and found no death at the highest level of virus (Figure II in the online-only Data Supplement). In summary, our results demonstrate that the α1A subtype mediates survival signaling in cardiac myocytes.
The α1A Subtype Rescues α1ABKO Cardiac Myocytes From Doxorubicin- and H2O2-Induced Cell Death
To determine whether the α1A subtype could protect cardiac myocytes from other known mediators of cell death, we reconstituted α1A subtype signaling in α1ABKO cardiac myocytes and measured cell death in response to doxorubicin, a chemotherapeutic agent with known cardiotoxicity, and H2O2, an inducer of oxidative stress (Figure 4). In α1ABKO myocytes, both doxorubicin (1 μmol/L) and H2O2 (10 μmol/L) induced cell death (apoptosis: P=NS versus α1ABKO control; necrosis and rod shape: P<0.05 versus α1ABKO control). Once again, reconstitution of α1A subtype signaling rescued α1ABKO myocytes from both doxorubicin- and H2O2-induced cell death (apoptosis: P=NS; necrosis and rod shape: P<0.05 versus α1ABKO doxorubicin or H2O2) (Figure 4a through b). In summary, the α1A subtype can prevent myocyte death induced by several different stimuli.
Activation of ERK Is Sufficient to Rescue α1ABKO Myocytes From NE-Induced Cell Death
Previously, we demonstrated that aortic constriction induced apoptosis, dilated cardiomyopathy, and death in α1ABKO mice, indicating that α1-AR signaling is required for myocardial adaptation to stress.5,6 α1-AR–mediated activation of ERK in cardiac myocytes is a well-characterized signaling pathway, and others suggest that α1-AR survival signaling is mediated by ERK in neonatal rat cardiac myocytes.21 To determine what role ERK plays in the α1-mediated response to myocardial stress, we measured ERK activation in α1ABKO mice after aortic constriction. In α1ABKO hearts, basal ERK activity was reduced versus WT, as demonstrated previously (Figure 5a).5 Furthermore, aortic constriction activated ERK in WT mice but failed to activate ERK in α1ABKO mice (Figure 5a). The failure to activate ERK after aortic constriction correlates with our previous finding that aortic constriction induces apoptosis, dilated cardiomyopathy, and death in α1ABKO mice.5,6
In agreement with the reduced level of ERK activation in the α1ABKO heart (Figure 5a), there is no α1-AR–mediated activation of ERK in α1ABKO myocytes.5 To determine whether the activation of ERK could prevent NE-induced cell death in α1ABKO myocytes, we infected α1ABKO myocytes with an adenovirus expressing a constitutively active mutant of MEK1 (MEK1 CA), a kinase that directly activates ERK, and we measured NE-induced cell death (Figure 5). Expression of the MEK1 CA rescued α1ABKO myocytes from NE-induced cell death (apoptosis, necrosis, and rod shape: all P<0.05 versus α1ABKO NE) (Figure 5b). In these experiments, the level of MEK1 CA used (MOI 20) produced a modest level of ERK activation (Figure 5c). In summary, moderate activation of ERK is sufficient to protect α1ABKO myocytes from NE-induced cell death.
Activation of ERK Is Required for α1A Subtype–Mediated Survival Signaling in Cardiac Myocytes
To determine whether α1A-mediated survival signaling was linked with the activation of ERK, we measured α1 subtype–specific activation of ERK in α1ABKO myocytes. Myocytes were infected with the α1A-GFP or α1B-GFP, and phenylephrine-induced activation of ERK was measured by Western blot (Figure 6). Reconstitution of α1A, but not α1B, subtype-specific signaling restored α1-AR–mediated activation of ERK (Figure 6a). To ensure that the α1B-GFP construct was not defective, we measured phenylephrine-induced activation of ERK in HeLa cells as well, and we found that α1A-GFP and α1B-GFP both activated ERK (Figure 6a). Further, we found that phorbol 12-myristate, 13-acetate activated ERK in myocytes expressing either the α1A or α1B subtype, suggesting that failure to activate ERK in myocytes expressing the α1B subtype was not attributable to a defect in ERK signaling (Figure 6a). In summary, the α1A subtype activates ERK in cardiac myocytes, but the α1B does not.
To determine whether the activation of ERK was required for α1A-AR–mediated survival signaling, we coinfected α1ABKO myocytes with adenoviruses expressing the α1A-GFP and a dominant-negative mutant of MEK1 (MEK1 D/N), and we measured NE-induced cell death. Expression of the MEK1 D/N alone had no effect on NE-induced cell death (apoptosis, necrosis, and rod shape: all P=NS versus α1ABKO NE), but it completely reversed α1A-AR survival signaling (apoptosis, necrosis, and rod shape: P=NS versus α1ABKO NE, but P<0.05 versus α1ABKO + α1A-GFP NE) (Figure 6b). The coexpression of the α1A-GFP and MEK1 D/N adenoviruses did result in lower expression levels of the MEK1 D/N (Figure 6c). However, the MEK1 D/N completely inhibited α1A-mediated activation of ERK (Figure 6d). The MEK1 D/N also inhibited α1A-AR–mediated survival signaling in α1BKO myocytes, which express only the endogenous α1A subtype, confirming these results (see Figure III in the online-only Data Supplement). In summary, activation of ERK is required for α1A-mediated survival signaling in cardiac myocytes.
The development of dilated cardiomyopathy and death after aortic constriction in α1ABKO mice demonstrates that α1-ARs are required for myocardial adaptation to stress.5,6 However, on the basis of our previous results, we could not determine whether the adverse outcomes after aortic constriction in α1ABKO mice were caused directly by the loss of α1-ARs in cardiac myocytes, nor could we determine which α1-AR subtype(s) was required for the adaptive response to stress. To address the limitations of our previous study, we investigated α1A and α1B subtype–specific survival signaling in cultured cardiac myocytes to test for a direct protective effect of α1-ARs in cardiac myocytes. Here, we found that reconstitution of α1A, but not α1B, subtype signaling rescued α1ABKO myocytes from cell death induced by NE, doxorubicin, and H2O2. Therefore, these results demonstrate a direct protective effect of the α1A subtype in cardiac myocytes.
In addition to defining a direct protective role for the α1A subtype in cardiac myocytes, we also identified an α1A-ERK signaling pathway that is critical for α1-AR–mediated survival signaling. In previous studies, inhibition of ERK increased apoptosis in both cultured cardiac myocytes and isolated perfused hearts subjected to ischemia.7 Further, cardiac-specific transgenic overexpression of MEK1 inhibited apoptosis induced by ischemia/reperfusion.8 In ERK2 knockout mice, ischemia/reperfusion increased apoptosis and myocardial injury.9 These findings clearly demonstrate that ERK signaling is protective in the heart. Here, we found that aortic constriction failed to activate ERK in α1ABKO mice and that activation of ERK, using a constitutively active mutant of MEK1, was sufficient to protect cultured α1ABKO cardiac myocytes from NE-induced death. We also found that the α1A, but not the α1B, mediated ERK activation in α1ABKO cardiac myocytes, and inhibition of ERK, with a dominant-negative mutant of MEK1, completely blocked α1A survival signaling. Therefore, our findings are consistent with a protective role for an α1A-ERK pathway. Further, the lack of α1A-ERK survival signaling could explain the failure to activate ERK after aortic constriction in α1ABKO mice, and this might ultimately explain the development of apoptosis, dilated cardiomyopathy, and death.
An unexpected result was that α1-ARs localized to the nucleus and perinucleus in WT cardiac myocytes and α1ABKO myocytes expressing α1-GFP fluorescent fusion proteins. However, we cannot exclude the possibility that inactive, unoccupied receptor resides at the plasma membrane. Classical models of GPCR function suggest that GPCRs are expressed on the membrane and only internalize after desensitization. However, a previous report found that α1-ARs are expressed in myocyte nuclei.22 Moreover, both β1-ARs and endothelin receptors localize to the nuclear membrane and activate nuclear signaling in the cardiac myocytes.23,24 In cultured adult mouse myocytes, we failed to observe α1-mediated inositol phosphate generation; this was consistent with previous studies.18 However, we are currently testing whether we can detect inositol phosphate generation in nuclei isolated from cultured adult mouse myocytes.
The neurohormonal hypothesis of heart failure states that the basis for pathological ventricular remodeling in heart failure is increased sympathetic activity and NE release. Clinically, this provided the basis for the successful use of β-blockers to treat heart failure.25 Interestingly, β1-ARs induce myocyte apoptosis,19,20 and transgenic overexpression of β1-ARs induces progressive heart failure with increased cardiac myocyte apoptosis.26,27 Our current and previous results demonstrate that the α1A subtype prevents cell death and seems to offset β1-AR proapoptotic signaling.5,6 Because low levels of apoptosis are sufficient to induce heart failure in mice28 (and quite possibly also in humans29,30), our results demonstrate a direct protective function of the α1A subtype in cardiac myocytes that might prevent or delay the onset of heart failure. Our results might also explain the adverse effects of α1-antagonists in ALLHAT and V-HeFT.2,3 In addition, these findings support a reconsideration of the neurohormonal hypothesis of heart failure and the idea that blocking all adrenergic receptor activation is beneficial in the treatment of heart failure.
In summary, our results demonstrate a direct protective effect of the α1A subtype in cardiac myocytes and define an α1A-ERK signaling pathway that is required for myocyte survival. Our results also imply that systemic factors do not explain the maladaptive phenotype of the α1ABKO after aortic constriction. Instead, absence of the α1A-ERK pathway in cardiac myocytes can explain the failure to activate ERK after aortic constriction in α1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.6 Finally, these data suggest a plausible mechanism for the adverse effects of α1-antagonists in ALLHAT and V-HeFT.2,3 These data also raise the possibility that α1A subtype–selective agonist therapy might be cardioprotective, as also suggested by some recent studies with α1A-transgenic mice.31,32
Sources of Funding
This work was supported by grants from the Pharmaceutical Research and Manufacturers of America Foundation (post-doctoral fellowship to Dr Wright), the American Heart Association (Scientist Development Grant 0435338Z, Dr O’Connell), the South Dakota State Legislature (2010 Grant, Dr O’Connell), the Veterans Administration (Dr Simpson), and the National Institute of Health (HL31113, Dr Simpson; P20 RR-017662, Dr O’Connell).
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The neurohormonal hypothesis suggests that pathological ventricular remodeling in heart failure is attributable to increased sympathetic activity and catecholamine release (norepinephrine and epinephrine). Moreover, increased norepinephrine levels are a prominent finding in patients with heart failure, predicting disease severity and mortality and providing the foundation for the successful use of β-AR antagonists, or β-blockers, to treat heart failure (Metoprolol CR/XL Randomized Intervention Trial, Cardiac Insufficiency Bisoprolol Study II, Carvedilol or Metoprolol European Trial). However, catecholamines also activate cardiac α1-ARs, and in 2 clinical trials, the Veterans Administration Heart Failure Trial (prazosin), which examined vasodilator therapy in heart failure, and the Antihypertensive and Lipid Lowering Treatment to Prevent Heart Attack Trial (doxazosin), which included 25 000 hypertensive patients, α1-blockers adversely affected mortality or heart failure. Recently, we used knockout mice with systemic deletion of the 2 main α1-subtypes, α1A and α1B, to show that α1-ARs are required for postnatal physiological growth of the heart and adaptation to the pathological stress of aortic constriction. These results provide direct evidence that the adverse effects of α1-blockers in the Veterans Administration Heart Failure Trial and the Antihypertensive and Lipid Lowering Treatment to Prevent Heart Attack Trial may have been caused by blockade of myocyte α1-ARs and not by vascular or nonspecific effects. The current study advances the prior results in 2 ways. First, it demonstrates a direct protective effect of the α1A subtype to rescue α1ABKO myocytes from death induced by β-AR stimulation, doxorubicin, or oxidative stress. Second, it identifies an α1A-ERK signaling pathway required for survival signaling. In summary, our studies call for a reassessment of the neurohormonal hypothesis, indicating that myocyte α1A-ARs are essential for cardiac adaptation. Another important consideration is the potential cardiac effects of α1-blockers (tamsulosin) used to treat prostate hyperplasia.
The online-only Data Supplement, consisting of expanded Methods and figures, can be found at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.664862/DC1.