Abstract 15890: Reduced Na/K Pump Current, Enhanced Contractility and Negative Force-frequency Relationship in Cardiomyocytes and Hearts Isolated from Mice Expressing Unphosphorylatable Phospholemman
Phospholemman (PLM) is a member of the FXYD family of Na/K ATPase accessory proteins expressed in cardiac tissue. It has been suggested that PLM phosphorylation provides the primary mechanism linking kinase activation to Na/K pump stimulation. PLM has previously been shown to be basally phosphorylated (by around 40%) and this can be further increased by PKC and/or PKA activation and by rapid pacing. Na/K pump current in myocytes isolated from PLM knock-out mice (PLMKO) is increased and, in isolated hearts, deletion of PLM causes a reduction in left ventricular pressure (LVDP) and a switch to a positive force-frequency gradient (FFRG) (cf a negative FFRG in WT hearts). To investigate the role of PLM phosphorylation a novel knock-in mouse (PLM-KI) was generated in which the endogenous allele was globally replaced with one expressing unphosphorylatable PLM (serines 63, 68 and 69 substituted with alanines). Na/K pump current was recorded using the perforated-patch technique in voltage-clamped myocytes isolated from PLM-KI and wild-type (WT) littermates. Maximum K-activated Na/K pump current (pipet Na=50mM) was decreased in PLM-KI myocytes from 3.7±0.2 to 2.9±0.1pA/pF with no change in K affinity (PLM-KI Km=1.1±0.2mM vs WT Km=1.5±0.1). The voltage-dependence of Na/K pump current showed normal rectification at positive potentials and was similar in both genotypes. There was no difference between the two genotypes at a saturating pipet [Na] of 140mM suggesting a change in Na/K pump Na affinity rather than a change in Vmax. In isolated Langendorff-perfused hearts (paced at 550bpm) basal LVDP was substantially increased in PLM-KI hearts (112.2±7.2mmHg) cf WT (86.8±4.9mmHg) (p<0.0001). Unpaced heart rate, time-to-peak and half-relaxation times were unaffected by genotype. Unlike the PLMKO, hearts isolated from PLM-KI mice showed a negative FFRG (PLM-KI FFRG=-0.08±0.01, WT FFRG=-0.10±0.02mmHg.bpm-1). These results are consistent with the hypothesis that basal phosphorylation of PLM in WT hearts maintains Na/K pump activation and limits Na accumulation (particularly at higher hearts rates) thus influencing cardiac contractility.
- © 2010 by American Heart Association, Inc.