Nuclear Localization of α1A‐Adrenergic Receptors Is Required for Signaling in Cardiac Myocytes: An “Inside‐Out” α1‐AR Signaling Pathway

Abstract

BackgroundRecent studies indicate that α1‐adrenergic receptors (α1‐ARs) are cardioprotective by preventing cardiac myocyte death and augmenting contractility in heart failure. Although G‐protein‐coupled receptors are assumed to localize to and signal at the plasma membrane, we previously demonstrated that endogenous α1‐ARs localize to the nuclei in adult cardiac myocytes. However, the functional consequence of this nuclear localization remains unclear. Here, we attempted to reconcile nuclear localization of α1‐ARs with their physiologic function by examining α1‐AR‐induced contractility in adult cardiac myocytes.Methods and ResultsBy measuring shortening in unloaded, cultured adult cardiac myocytes, we found that the α1A‐subtype regulated contractility through phosphorylation of cardiac troponin I (cTnI) at the protein kinase C (PKC) site, threonine 144. Reconstitution of an α1A‐subtype nuclear localization mutant in cardiac myocytes lacking α1‐ARs failed to rescue nuclear α1A‐mediated phosphorylation of cTnI and myocyte contractility. Leptomycin B, the nuclear export inhibitor, also blocked α1A‐mediated phosphorylation of cTnI. These data indicate that α1‐AR signaling originates in the nucleus. Consistent with these observations, we localized the α1A‐subtype to the inner nuclear membrane, identified PKCα, δ, and ε in the nucleus, and found that α1‐ARs activate PKCδ in nuclei isolated from adult cardiac myocytes. Finally, we found that a PKCδ nuclear localization mutant blunted α1‐induced phosphorylation of cTnI.ConclusionsTogether, our data identify a novel, “inside‐out” nuclear α1A‐subtype/PKCδ/cTnI‐signaling pathway that regulates contractile function in adult cardiac myocytes. Importantly, these data help resolve the discrepancy between nuclear localization of α1‐ARs and α1‐AR‐mediated physiologic function.

Nuclear Calcium in Cardiac Myocytes

“…42, 43 When two different subtypes of functional α-ARs (α1A and α1B) were expressed as GFP fusion proteins in α1AB-KO cardiomyocytes, both could be observed at nuclear and perinuclear locations. 44 Although evidence for colocalization of both α1A and α1B subtypes with G-protein and PLC in the nuclear membrane is available, the involvement of α1-AR in the regulation of [Ca 2+ ] nuc remains unknown.…”

G Protein–Coupled Receptor Signaling in Cardiac Nuclear Membranes

“…6,60,61 In both neonatal and adult cardiomyocytes, the effects of extracellular phenylephrine are mediated, in part, by intracellular a 1 AR. 7,61,62 Although the responsiveness of nuclear bARs to extracellular ligands requires further study, extracellularly applied [ 3 H]norepinephrine was previously shown to accumulate in the nuclei of neonatal ventricular cardiomyocytes. 63 Potential NLS sequences identified in the rat sequences for the indicated GPCRs using cNLS Mapper (http://nls-mapper.iab.keio.ac.jp).…”

“…6 In adult cardiomyocytes, the role of nuclear a 1 AR has been examined by expressing a 1 AR subtypes bearing mutations within the NLS sequence in a a 1 AR knockout background. 7,62 In addition, as mentioned above, several genetic approaches have been used to study intracrine Ang II signaling, including adenovirusmediated overexpression of Ang II in neonatal cardiomyocytes, 102 injection of mice with a plasmid encoding Ang II under control of the a-myosin heavy chain promoter to achieve cardiomyocyte-specific overexpression, 102 and generating a line of a-myosin heavy chain-Ang II transgenic mice. 103 Experiments using caged ligands have allowed us to examine the role of intracellular GPCRs in live cells isolated fresh from any source.…”

Regulation of Phosphatidylinositol-specific Phospholipase C at the Nuclear Envelope in Cardiac Myocytes

“…Arguing in favor of GPCR localization at the NE are studies with α1-adrenergic receptor (α1-AR) showing that mutation of a nuclear localization sequence in the C terminal tail (α1A-AR-NLSmut) leads to localization away from the nucleus [90]. In myocytes isolated from α1AB-AR knockout mice, wild type α1A-AR rescues phenylephrine induced contraction while α1A-AR-NLS does not.…”