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

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 ␣3 subunit-containing isoforms of the Na,K-ATPase in the mammalian brain. Both agrin and the ␣3 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 ␣3 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-␣3 Na,K-ATPase interaction and suggest that agrin modulation of the ␣3 Na,KATPase is important in regulating heart function. Na,K-ATPases, or sodium pumps, are integral membrane enzymes found in all animal cells. Using energy from the hydrolysis of ATP they transport three Na ϩ ions out of the cell for every two K ϩ ions into the cell, resulting in a transmembrane chemical gradient that is reflected in the resting membrane potential and used to drive a variety of secondary transport processes. Each Na,K-ATPase is a heterodimer consisting of an ␣-and ␤-subunit. The ␣-subunit is the catalytic subunit and contains the binding sites for Na ϩ and K ϩ . The ␤-subunit is required for pump function and targeting of the ␣-subunit to the plasma membrane. Four ␣-and three ␤-subunit genes have been identified. All combinations of ␣-and ␤-subunits form functional pumps, but developmental, cellular, and subcellular differences in expression suggest functional adaptation of the different isoforms (1).Na,K-ATPases play a central role in regulating the contractile activity of cardiac muscle (2). They are directly responsible for the Na ϩ gradient required for propagation of action potentials that initiate myocyte contraction. Moreover, because of the dependence of the Na ϩ /Ca 2ϩ exchanger (NCX) 3 on the Na ϩ gradient as the source of counterions for transport of Ca 2ϩ out of the cell, they play a critical role in Ca 2ϩ homeostasis and excitation-contraction coupling. For example, inhibition of Na,K-ATPases by digoxin, ouabain, or other cardiac glycoside results in a decline of the Na ϩ gradient, reducing NCX activity and Ca 2ϩ efflux. The inotropic effects of cardiac glycosides result from uptake of this "excess" cytoplasmic Ca 2ϩ into the sarcoplasmic reticulum, raising the level of Ca ...

A marked animal–vegetal polarity in the localization of Na+,K+‐ATPase activity and its down‐regulation following progesterone‐induced maturation

“…Moreover, we know that only the animal hemisphere of the stage‐VI oocyte is responsive to progesterone. Similarly, Hilgenberg et al (2006, 2009) have shown that the α3‐subunit of Na + ,K + ‐ATPase acts as a receptor for Agrin (both in neuronal and cardiac tissues), which down‐regulates Na + ,K + ‐ATPase activity and modulates membrane potential and action potential frequencies of cortical neurons and cardiomyocytes contraction.…”

“…During early development of Xenopus, Na + ,K + ‐ATPase activity has been implicated in blastocoel formation (Slack et al, 1973), for the maintenance of neural plate potentials (Blackshaw and Warner, 1976), and in neuronal differentiation (Messenger and Warner, 2000). Na + ,K + ‐ATPase has also recently been found to have roles in cell adhesion (Shoshani et al, 2005), cell polarity (Laprise et al, 2009), phosphotidylinositol 3‐kinase (Barwe et al, 2005), and Src kinase signaling (Xie and Askari, 2002), endocytosis (Cai et al, 2008), tubulogenesis (Krupinski and Beitel, 2009), tight junction formation (Rajasekaran et al, 2005), and as receptor for progesterone (Morrill et al, 2005) and Agrin (Hilgenberg et al, 2006, 2009).…”

Profiling of the muscle-specific dystroglycan interactome reveals the role of Hippo signaling in muscular dystrophy and age-dependent muscle atrophy

“…Another ion channel proteins that we identified to be putatively associated with Dg are -ATPα and Nrv1 (Nervana1), which are αand β-subunits of the Na + , K + ATPase ion pump important for maintaining cellular membrane potential in the muscles [97]. Previous studies report that the activity of α3 Na + , K + ATPase can be modulated by the ECM protein Agrin in mouse cardiac myocytes, influencing their contraction [98]. The Nterminal region of Agrin binds laminin, while the Cterminal end binds to several transmembrane ECM receptors, including Dg; however, whether Agrin-linked regulation of Na + , K + ion pump depends on Dg remains unrevealed.…”

The Expanding Spectrum of Neurological Phenotypes in Children With ATP1A3 Mutations, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia-Parkinsonism, CAPOS and Beyond

“…44 In a study of ATP1A3 mutant mice, “restraint stress” was found to induce significant deficits in motor coordination and balance, as well as decreased thermal sensitivity and alterations in monoamine metabolism. 38 An impact on function or expression of the cardiac-specific alpha3 isomer 45 could provide an alternative explanation for autonomic dysfunction.…”