Nuclear localization of a1A-adrenergic receptors is required for signaling in cardiac myocytes: an “inside-out” a1-AR signaling pathway.
BACKGROUND
Recent studies indicate that a1-adrenergic receptors (a1-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 a1-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 a1-ARs with their physiologic function by examining a1-AR-induced contractility in adult cardiac myocytes.


RESULTS
By measuring shortening in unloaded, cultured adult cardiac myocytes, we found that the a1A-subtype regulated contractility through phosphorylation of cardiac troponin I (cTnI) at the protein kinase C (PKC) site, threonine 144. Reconstitution of an a1A-subtype nuclear localization mutant in cardiac myocytes lacking a1-ARs failed to rescue nuclear a1A-mediated phosphorylation of cTnI and myocyte contractility. Leptomycin B, the nuclear export inhibitor, also blocked a1A-mediated phosphorylation of cTnI. These data indicate that a1-AR signaling originates in the nucleus. Consistent with these observations, we localized the a1A-subtype to the inner nuclear membrane, identified PKCa, d, and e in the nucleus, and found that a1-ARs activate PKCd in nuclei isolated from adult cardiac myocytes. Finally, we found that a PKCd nuclear localization mutant blunted a1-induced phosphorylation of cTnI.


CONCLUSIONS
Together, our data identify a novel, “inside-out” nuclear a1A-subtype/PKCd/cTnI-signaling pathway that regulates contractile function in adult cardiac myocytes. Importantly, these data help resolve the discrepancy between nuclear localization of a1-ARs and a1-AR-mediated physiologic function.
Steven C
SC
Wu
Erika F
EF
Dahl
Casey D
CD
Wright
Andrew L
AL
Cypher
Chastity L
CL
Healy
Timothy D
TD
O'Connell
D000818:Animals; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism*; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism; D018340:Receptors, Adrenergic, alpha-1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D019210:Troponin I / Q000378:metabolism
C547714:Adra1a protein, mouse; D018340:Receptors, Adrenergic, alpha-1; D019210:Troponin I; C497258:PRKCD protein, human; D051745:Protein Kinase C-delta