Abstract 16046: Rapid Pacing Stimulates Na/K ATPase in Rat Ventricular Myocytes via a Nitric Oxide and Phospholemman-dependent Mechanism
Nitric oxide (NO) is a potent signaling molecule that has diverse effects throughout the cardiovascular system. NO has been reported to both inhibit and stimulate the Na/K ATPase. The effects of NO appear to correlate with whether the tissue expresses phospholemman (PLM) as its Na/K ATPase accessory protein. Since rapid pacing has been reported to phosphorylate the analogous ATPase accessory protein phospholamban (with some studies suggesting a role for NO), we examined whether rapid pacing stimulates intracellular NO production and investigated its effects on PLM and Na/K ATPase activity. Adult rat ventricular myocytes (ARVM) stained with DAF-2FM, were paced for 20 min at 3 Hz to monitor NO production. Rapid pacing increased [NO]i (from 13±2 to 40±4µM; n=6, p<0.05) and this was abolished by 1mM L-NAME (16±2µM, n=6). Rapid pacing had no effect on total PLM expression, however, there was a significant increase in PLM phosphorylation at Ser 63 and 68, 10 min after the initiation of pacing. Phosphorylation at Thr 69 was transient and peaked at 5 min. Preincubation of ARVM with a PKG inhibitor (KT-5832; 1µM), or a PKC inhibitor (bisindolylmaleimide; 2µM), abolished rapid pacing-induced phosphorylation at Ser 63 and Ser 68. Preincubation of ARVM with a PKA inhibitor (H89; 2µM) had no effect on phosphorylation. Na/K ATPase pump current (Ip) was measured by perforated patch clamp technique. In ARVM, 20 min of exposure to spermine NONOate (SpNO; 10µM) significantly increased Ip (1.48±0.12 pA/pF, n=6) compared to control (1.05±0.13 pA/pF, n=6). In contrast, ‘depleted’ SpNO (10µM) had no effect on pump current. The stimulatory effect of SpNO was absent in ventricular myocytes isolated from PLM knockout mice (Ip=1.68±0.18pA/pF, n=6) compared to control (1.51±0.17 pA/pF, n=6) while a significant stimulation was seen in wild-type littermates (1.88±0.12 pA/pF, n=6; p<0.05 cf control). We conclude that in adult cardiac myocytes, rapid pacing induces NO synthesis, which causes PLM phosphorylation at Ser 63 and Ser 68 through a PKG/PKC dependent mechanism and thereby stimulates Na/K ATPase. This may provide an intrinsic mechanism for the beat-to-beat functional modulation of cardiac Na/K ATPase.
- © 2010 by American Heart Association, Inc.