Abstract 615: Laminin Acts via FAK/PI-(3)K/Akt Signaling to Down-Regulate β1-Adrenergic Receptor Stimulation of L-Type Calcium Current in Cat Atrial Myocytes
We previously reported that binding of laminin (LMN) to β1-integrin receptors decreases β1-adrenergic receptor (β1-AR) stimulation of L-type Ca2+ current (ICa,L) in cat atrial myocytes. This study investigated the underlying signaling mechanisms. β1-AR stimulation was achieved by 0.01 μM isoproterenol (ISO) plus 0.1 μM ICI 118551, a selective β2-AR antagonist. Atrial myocytes were plated on glass coverslips (2 hr) either not coated with LMN (-LMN) or coated with LMN (+LMN). Western blots showed that compared with control (-LMN) +LMN atrial myocytes exhibited increased Akt phosphorylation at Ser473, which was prevented by 10 μM LY294002 (LY), an inhibitor of phosphatidylinositol-3′ kinase (PI-(3)K). In -LMN atrial myocytes LY had no effect on β1-AR stimulation of ICa,L. However, in +LMN atrial myocytes LY significantly increased β1-AR stimulation of ICa,L (LMN, 48±5% vs LMN + LY, 120±21%; P<0.05, n=6). Using another approach, +LMN atrial myocytes were infected (100 moi, 24 h) over night with replication-defective adenoviruses (Adv) expressing dominant-negative (dn) inhibitors of focal adhesion kinase (FAK)(Adv-FRNK or Adv-Y397F) or Akt (Adv-dnAkt). Control cells were infected with Adv-βgal. Compared with controls, cells infected with FRNK (cont, 38±6% vs FRNK, 67±5%; P<0.05, n=8); Y397F (cont, 41±5% vs Y397F, 60±4%; P<0.05, n=8) or dnAkt (cont, 42±4% vs dn-Akt, 70±6%; P<0.05, n=10) each showed significantly greater β1-AR stimulation of ICa,L. In -LMN atrial myocytes forskolin (FSK)-induced stimulation of ICa,L was unaffected by LY. However, in +LMN atrial myocytes LY significantly increased FSK-induced stimulation of ICa,L (cont, 42±2% vs LY, 62±6%; P<0.05, n=8). Similar results were obtained in +LMN atrial myocytes infected with Adv-FRNK compared to control. We conclude that in atrial myocytes LMN binding to β1-integrin receptors acts via FAK/PI-(3)K/Akt signaling to inhibit adenylate cyclase and thereby down-regulate β1-AR signaling. Furthermore, in isolated atrial myocytes not plated on LMN, β1-AR stimulation of ICa,L does not involve PI-(3)K/Akt signaling. These findings provide insight into the cellular mechanisms by which the extracellular matrix may participate in remodeling of β1-AR signaling in the failing heart.