Agrin Regulation of α3 Sodium-Potassium ATPase Activity Modulates Cardiac Myocyte Contraction

doi:10.1074/jbc.M806855200

Agrin Regulation of α3 Sodium-Potassium ATPase Activity Modulates Cardiac Myocyte Contraction*

From the Departments of ^‡Anatomy and Neurobiology and
^§Developmental and Cell Biology, University of California, Irvine, California 92697

² To whom correspondence should be addressed:
Dept. of Anatomy and Neurobiology, Joan Irvine-Smith Hall, Rm. 110, University of California, Irvine, CA 92697-1280.
Fax: 949-824-0043; E-mail: masmith{at}uci.edu.

Abstract

Drugs that inhibit Na,K-ATPases, such as digoxin and ouabain, alter cardiac myocyte contractility. We recently demonstrated that agrin, a protein first identified at the vertebrate neuromuscular junction, binds to and regulates the activity of α3 subunit-containing isoforms of the Na,K-ATPase in the mammalian brain. Both agrin and the α3 Na,K-ATPase are expressed in heart, but their potential for interaction and effect on cardiac myocyte function was unknown. Here we show that agrin binds to the α3 subunit of the Na,K-ATPase in cardiac myocyte membranes, inducing tyrosine phosphorylation and inhibiting activity of the pump. Agrin also triggers a rapid increase in cytoplasmic Na⁺ in cardiac myocytes, suggesting a role in cardiac myocyte function. Consistent with this hypothesis, spontaneous contraction frequencies of cultured cardiac myocytes prepared from mice in which agrin expression is blocked by mutation of the Agrn gene are significantly higher than in the wild type. The Agrn mutant phenotype is rescued by acute treatment with recombinant agrin. Furthermore, exposure of wild type myocytes to an agrin antagonist phenocopies the Agrn mutation. These data demonstrate that the basal frequency of myocyte contraction depends on endogenous agrin-α3 Na,K-ATPase interaction and suggest that agrin modulation of the α3 Na,K-ATPase is important in regulating heart function.

Footnotes

↵¹ Undergraduate research supported in part by an HHMI professor's grant (to D. K. O'D.).
↵* This work was supported, in whole or in part, by National Institutes of Health Grants NS33213 (to M. A. S.) and NS27501 (to D. K. O'D.). This work was also supported by a grant-in-aid from the American Heart Association Western States Affiliate (to M. A. S.) and a Howard Hughes Medical Institute (HHMI) professor's grant (to D. K. O'D.).
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–4.
↵3 The abbreviations used are:

NCX

Na⁺/Ca²⁺ exchanger

E18

embryonic day 18

P0

postnatal day 0

ANOVA

analysis of variance

PBS

phosphate-buffered saline

BS³

bis(sulfosuccinimidyl) suberate

MOPS

4-morpholinepropanesulfonic acid

DS

dissecting solution

FCS

fetal calf serum

SBFI

sodium-binding benzofuran isophthalate

MuSK

muscle-specific kinase.
- Received September 4, 2008.
- Revision received March 25, 2009.