Background-In ␣1-AR knockout (␣1ABKO) mice that lacked cardiac myocyte ␣1-adrenergic receptor (␣1-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 ␣1-ARs and whether the ␣1A, ␣1B, or both subtypes mediated protection. Therefore, we investigated ␣1A and ␣1B subtype-specific survival signaling in cultured cardiac myocytes to test for a direct protective effect of ␣1-ARs in cardiac myocytes. Methods and Results-We cultured ␣1ABKO myocytes and reconstituted ␣1-AR signaling with adenoviruses expressing ␣1-GFP fusion proteins. Myocyte death was induced by norepinephrine, doxorubicin, or H 2 O 2 and was measured by annexin V/propidium iodide staining. In ␣1ABKO myocytes, all 3 stimuli significantly increased apoptosis and necrosis. Reconstitution of the ␣1A subtype, but not the ␣1B, rescued ␣1ABKO myocytes from cell death induced by each stimulus. To address the mechanism, we examined ␣1-AR activation of extracellular signal-regulated kinase (ERK). In ␣1ABKO hearts, aortic constriction failed to activate ERK, and in ␣1ABKO myocytes, expression of a constitutively active MEK1 rescued ␣1ABKO myocytes from norepinephrine-induced death. In addition, only the ␣1A-AR activated ERK in ␣1ABKO myocytes, and expression of a dominant-negative MEK1 completely blocked ␣1A survival signaling in ␣1ABKO myocytes. Conclusions-Our results demonstrate a direct protective effect of the ␣1A subtype in cardiac myocytes and define an ␣1A-ERK signaling pathway that is required for myocyte survival. Absence of the ␣1A-ERK pathway can explain the failure to activate ERK after aortic constriction in ␣1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.

Abstract-We previously identified an ␣1-AR-ERK (␣1A-adrenergic receptor-extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of ␣1-AR subtypes (␣1A and ␣1B) and how their localization influences ␣1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent ␣1-AR antagonist, we localized endogenous ␣1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify ␣1 subtype localization, we reconstituted ␣1 signaling in cultured ␣1A-and ␣1B-AR double knockout cardiac myocytes using ␣1-AR-green fluorescent protein (GFP) fusion proteins. Similar to endogenous ␣1-ARs and ␣1A-and ␣1B-GFP colocalized with LAP2 at the nuclear membrane. ␣1-AR nuclear localization was confirmed in vivo using ␣1-AR-GFP transgenic mice. The ␣1-signaling partners G␣q and phospholipase C␤1 also colocalized with ␣1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that ␣1-mediated activation of ERK was not inhibited by a membrane impermeant ␣1-blocker, suggesting ␣1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe ␣1-AR localization to caveolae, but we found that ␣1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear ␣1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes. (Circ Res. 2008;103:992-1000.)Key Words: ␣1-adrenergic receptors Ⅲ cardiac myocytes Ⅲ ERK C ardiovascular disease is the leading killer in the United States, accounting for 1.4 million deaths a year. Five million Americans experience heart failure, leading to 970 000 hospitalizations annually, a number that has tripled in the last 25 years. 1 In heart failure, increased activation of the sympathetic nervous system is correlated with pathophysiologic remodeling of the heart, 2 which has led to the therapeutic use of ␤-adrenergic receptor (AR) antagonists in heart failure. However, the general conclusion that inhibition of catecholamine activation of ARs is beneficial in heart failure is disputed by clinical trials with ␣1-AR antagonists. The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) demonstrated that the ␣1-antagonist doxazosin increased the risk of heart failure by 80% and stroke by 26% leading to termination of the trial. 3,4 Similar detrimental effects were seen in the VasodilatorHeart Failure Trials (V-HeFT), in which prazosin was compared with other vasodilators for the prevention of death in heart failure. 5 All 3 ␣1-AR subtypes (A, B, and D) are expressed in the heart 6 -9 ; however, cardiac myocytes only express the ␣1A and ␣1B. 9 Using ␣1A-and ␣1B-AR double knockout mice (␣1ABKO), we demonstrated previously that ␣1-ARs are required for postnatal hypertrophy and adaptation to pathological stress. 9,10 In ␣1ABKO mice, we found that aortic constriction induced dilated cardiomyopathy that le...