Characterization of the phospholemman knockout mouse heart: depressed left ventricular function with increased Na-K-ATPase activity

Abstract

Dunn MJ, Shattock MJ. Characterization of the phospholemman knockout mouse heart: depressed left ventricular function with increased Na-K-ATPase activity. Am J Physiol Heart Circ Physiol 294: H613-H621, 2008. First published December 7, 2007 doi:10.1152/ajpheart.01332.2007, abundantly expressed in the heart, is the primary cardiac sarcolemmal substrate for PKA and PKC. Evidence supports the hypothesis that PLM is part of the cardiac Na-K pump complex and provides the link between kinase activity and pump modulation. PLM has also been proposed to modulate Na/Ca exchanger activity and may be involved in cell volume regulation. This study characterized the phenotype of the PLM knockout (KO) mouse heart to further our understanding of PLM function in the heart. PLM KO mice were bred on a congenic C57/BL6 background. In vivo conductance catheter measurements exhibited a mildly depressed cardiac contractile function in PLM KO mice, which was exacerbated when hearts were isolated and Langendorff perfused. There were no significant differences in action potential morphology in paced Langendorff-perfused hearts. Depressed contractile function was associated with a mild cardiac hypertrophy in PLM KO mice. Biochemical analysis of crude ventricular homogenates showed a significant increase in Na-K-ATPase activity in PLM KO hearts compared with wild-type controls. SDS-PAGE and Western blot analysis of ventricular homogenates revealed small, nonsignificant changes in Na-K-ATPase subunit expression, with two-dimensional gel (isoelectric focusing, SDS-PAGE) analysis revealing minimal changes in ventricular protein expression, indicating that deletion of PLM was the primary reason for the observed PLM KO phenotype. These studies demonstrate that PLM plays an important role in the contractile function of the normoxic mouse heart. Data are consistent with the hypothesis that PLM modulates Na-K-ATPase activity, indirectly affecting intracellular Ca and hence contractile function. FXYD1; contractile function; intracellular sodium regulation IN EXCITABLE TISSUES, the activity of the plasma membrane Na-K-ATPase is vital for the maintenance of normal electrical activity, ionic homeostasis, cell volume control, and substrate and amino acid transport and for setting cellular Ca load and hence contractility. Interventions that influence Na-K-ATPase activity and/or the transmembrane Na gradient can therefore profoundly affect myocardial function. In essence, the Na-KATPase not only influences a wide range of transmembrane transport processes but also indirectly controls myocardial contractility.It has recently been recognized that the FXYD family of small single transmembrane-spanning proteins are tissue-specific regulators of the Na-K-ATPase (3-5, 28). Phospholemman (PLM, FXYD1) is expressed in excitable tissues and is unique among the FXYD proteins in that it contains a cytoplasmic region with consensus phosphorylation sites for kinases that include PKC and PKA (21, 30). In fact, PLM was originally identified as the primary sarcolemmal ...

Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+-K+-ATPase

“…This V max effect of PLM on I pump is in agreement with the observations in adult rat myocytes overexpressing PLM (26) and in freshly isolated guinea pig myocytes stimulated with forskolin (16). A comparison of Na ϩ -K ϩ -ATPase activities measured under saturating Na ϩ conditions in heart homogenates obtained from WT and PLM KO mice also indicated a V max effect of PLM (2). In contrast, when pump activity was measured as the Na (11).…”

“…3 and Table 1), in agreement with our previous results (22) and those of others (2). After 48 h of culture, PLM KO myocytes retained similar amounts of NCX1 and SERCA2 but suffered an ϳ18% loss in the ␣ 1 -subunit of Na ϩ -K ϩ -ATPase compared with freshly isolated PLM KO myocytes ( Fig.…”

“…A good example of this is that despite evidence of hypertrophy in PLM KO hearts (2, 12), neither we (22) nor Despa et al (8) could detect hypertrophy in myocytes isolated from PLM KO hearts. Indeed, whether contractile performance in PLM KO hearts was superior (12) or depressed (2) in the intact animal remains controversial at the present time. The second limitation is that contractility of cultured adult mouse myocytes, although preserved, was not quite "normal," as evidenced by the high incidence of contractile failure at 0.6 mM [Ca 2ϩ ] o , and contraction amplitudes did decline slightly with increasing days in Values are means Ϯ SE; n, no.…”

Regulation of in vivo cardiac contractility by phospholemman: role of Na+/Ca2+exchange

“…Under basal conditions, PLM-knockout (KO) hearts contract just as well, if not better, than wild-type (WT) hearts (3,10,26). This is inconsistent with the hypothesis that with loss of inhibition of Na (3,8,21,26), PLM-KO hearts should exhibit lower contractility compared with WT hearts. On the other hand, when subjected to catecholamine stress resulting in high intracellular Na ϩ concentration ([Na ϩ ] i ), WT but not PLM-KO hearts suffer a time-dependent decline in contractility (26).…”

“…Sarkosyl (1%) was added, and the disrupted pellet was incubated with gentle shaking for 1 h. Insoluble material was removed by centrifugation (2,500 g, 20 min, 4°C). The supernatant was transferred to a 14 ϫ 89-mm ultracentrifuge tube underlaid with 5 ml of 1.3 g/cm 3 CsCl and 2 ml of 1.5 g/cm 3 CsCl (both in 10 mM sodium phosphate, pH 7.4). Gradients were spun at 36,000 rpm and 18°C with a SW41 rotor (Beckman) for 20 h with no brake.…”