Nuclear alpha1-adrenergic receptors signal activated ERK localization to caveolae in adult cardiac myocytes.

We previously identified an alpha1-AR-ERK (alpha1A-adrenergic receptor-extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of alpha1-AR subtypes (alpha1A and alpha1B) and how their localization influences alpha1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent alpha1-AR antagonist, we localized endogenous alpha1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify alpha1 subtype localization, we reconstituted alpha1 signaling in cultured alpha1A- and alpha1B-AR double knockout cardiac myocytes using alpha1-AR-green fluorescent protein (GFP) fusion proteins. Similar to endogenous alpha1-ARs and alpha1A- and alpha1B-GFP colocalized with LAP2 at the nuclear membrane. alpha1-AR nuclear localization was confirmed in vivo using alpha1-AR-GFP transgenic mice. The alpha1-signaling partners Galphaq and phospholipase Cbeta1 also colocalized with alpha1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that alpha1-mediated activation of ERK was not inhibited by a membrane impermeant alpha1-blocker, suggesting alpha1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe alpha1-AR localization to caveolae, but we found that alpha1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear alpha1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes.

An alpha1A-adrenergic-extracellular signal-regulated kinase survival signaling pathway in cardiac myocytes.

BACKGROUND: In alpha1-AR knockout (alpha1ABKO) mice that lacked cardiac myocyte alpha1-adrenergic receptor (alpha1-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 alpha1-ARs and whether the alpha1A, alpha1B, or both subtypes mediated protection. Therefore, we investigated alpha1A and alpha1B subtype-specific survival signaling in cultured cardiac myocytes to test for a direct protective effect of alpha1-ARs in cardiac myocytes. METHODS AND RESULTS: We cultured alpha1ABKO myocytes and reconstituted alpha1-AR signaling with adenoviruses expressing alpha1-GFP fusion proteins. Myocyte death was induced by norepinephrine, doxorubicin, or H2O2 and was measured by annexin V/propidium iodide staining. In alpha1ABKO myocytes, all 3 stimuli significantly increased apoptosis and necrosis. Reconstitution of the alpha1A subtype, but not the alpha1B, rescued alpha1ABKO myocytes from cell death induced by each stimulus. To address the mechanism, we examined alpha1-AR activation of extracellular signal-regulated kinase (ERK). In alpha1ABKO hearts, aortic constriction failed to activate ERK, and in alpha1ABKO myocytes, expression of a constitutively active MEK1 rescued alpha1ABKO myocytes from norepinephrine-induced death. In addition, only the alpha1A-AR activated ERK in alpha1ABKO myocytes, and expression of a dominant-negative MEK1 completely blocked alpha1A survival signaling in alpha1ABKO myocytes. CONCLUSIONS: Our results demonstrate a direct protective effect of the alpha1A subtype in cardiac myocytes and define an alpha1A-ERK signaling pathway that is required for myocyte survival. Absence of the alpha1A-ERK pathway can explain the failure to activate ERK after aortic constriction in alpha1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.