Protein Kinase A Hyperphosphorylation Increases Basal Current but Decreases -Adrenergic Responsiveness of the Sarcolemmal Na -Ca Exchanger in Failing Pig Myocytes

The sodium-calcium exchanger (NCX) protein is the major cardiac calcium extrusion mechanism and is upregulated in heart failure (HF). NCX expression level and functional activity as regulated by -adrenergic receptor ( -AR) stimulation in swine with and without tachycardia-induced heart failure were studied. The Ni -sensitive NCX current was measured in myocytes from HF and control animals in the basal state or in the presence of isoproterenol, forskolin, 8-Br-cAMP, okadaic acid, or protein phosphatase type 1. Western blot analysis revealed a significant increase in both the 120-kDa (29%) and 80-kDa (69%) fragments in HF (P 0.05 versus control). Despite this modest increase in protein, the basal peak outward NCX current was increased almost 5-fold in HF (P 0.05 versus control). Stimulation with isoproterenol, however, increased the control currents to a significantly greater extent than HF (500% increase in control versus 100% increase in HF, P 0.01); peak stimulated current was not different in HF and control. This reduction in responsiveness to -AR stimulation was refractory to forskolin, 8-Br-cAMP, or okadaic acid stimulation. In vitro protein kinase A back-phosphorylation revealed higher phosphorylation capacity of NCX protein in control versus HF, consistent with increased phosphorylation in vivo (hyperphosphorylation) in HF. Protein phosphatase type 1 exposure resulted in a significant reduction (73%) in peak basal current in HF (compared with no significant difference in controls), confirming that the increased basal NCX current in HF is predominately attributable to hyperphosphorylation. NCX expression and activity are thus increased in HF, although -AR responsiveness is decreased because of NCX hyperphosphorylation. (Circ Res. 2003;92:897-903.)