Phospholemman: a novel cardiac stress protein

Abstract

Phospholemman (PLM), a member of the FXYD family of regulators of ion transport, is a major sarcolemmal substrate for protein kinases A and C in cardiac and skeletal muscle. In the heart, PLM co-localizes and co-immunoprecipitates with Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and L-type Ca(2+) channel. Functionally, when phosphorylated at serine(68), PLM stimulates Na(+)-K(+)-ATPase but inhibits Na(+)/Ca(2+) exchanger in cardiac myocytes. In heterologous expression systems, PLM modulates the gating of cardiac L-type Ca(2+) channel. Therefore, PLM occupies a key modulatory role in intracellular Na(+) and Ca(2+) homeostasis and is intimately involved in regulation of excitation-contraction (EC) coupling. Genetic ablation of PLM results in a slight increase in baseline cardiac contractility and prolongation of action potential duration. When hearts are subjected to catecholamine stress, PLM minimizes the risks of arrhythmogenesis by reducing Na(+) overload and simultaneously preserves inotropy by inhibiting Na(+)/Ca(2+) exchanger. In heart failure, both expression and phosphorylation state of PLM are altered and may partly account for abnormalities in EC coupling. The unique role of PLM in regulation of Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and potentially L-type Ca(2+) channel in the heart, together with the changes in its expression and phosphorylation in heart failure, make PLM a rational and novel target for development of drugs in our armamentarium against heart failure. Clin Trans Sci 2010; Volume 3: 189-196.

Figure 1

Molecular model of phospholemman. Nuclear magnetic resonance studies of highly purified phospholemman in micelles revealed four helices of the protein with a single transmembrane domain (after Francesca Marassi). ⁸ , ¹⁰ The FXYD motif is in the extracellular domain and the important serine⁶³ and serine⁶⁸ are in the cytoplasm.

Figure 2

Phospholemman co‐localizes with α1‐subunit of Na⁺‐K⁺‐ATPase. Indirect immunofluorescence of adult rat ventricular myocytes doubly labeled with mouse monoclonal antibody against α1‐subunit of Na⁺‐K⁺‐ATPase (A) and rabbit polyclonal anti‐PLM antibody (B) are shown. Primary antibodies are visualized with Alexa Fluor 488‐labeled goat anti‐mouse IgG (A) and Alexa Fluor 594‐ labeled goat anti‐rabbit IgG (B). Note the orange color in the merged image (C), indicating co‐localization of PLM and Na⁺‐K⁺‐ATPase. Bar = 5 μm.

Figure 3

Cardiac excitation–contraction coupling. Membrane depolarization is initiated by opening of the Na⁺ channel (not shown) with Na⁺ entry. Extracellular Ca²⁺ enters via L‐type Ca²⁺ channel (I_Ca) and Na⁺/Ca²⁺ exchanger (NCX1), causing Ca²⁺ release from the ryanodine receptor (RyR) in the sarcoplasmic reticulum (SR). Ca²⁺ binds to troponin and initiates myofilament contraction. During diastole, Ca²⁺ is pumped back to the SR by SR Ca²⁺‐ATPase (SERCA) under the control of phospholamban (PLB). A small amount of Ca²⁺ is also taken up by the mitochondrial Ca²⁺ uniporter. The amount of Ca²⁺ that has entered during systole is extruded by Na⁺/Ca²⁺ exchanger and to a lesser extent by sarcolemmal Ca²⁺‐ATPase. Na⁺ that has entered via Na⁺ channel and Na⁺/Ca²⁺ exchanger is pumped out by Na⁺‐K⁺‐ATPase. Repolarization is mediated by opening of K⁺ channels (only the transient outward K⁺ current I_to responsible for early repolarization is shown). Phospholemman (PLM) associates with and is an endogenous regulator of Na⁺/Ca²⁺ exchanger, Na⁺‐K⁺‐ATPase, and possibly L‐type Ca²⁺ channel. Na⁺/Ca²⁺ exchanger is depicted as operating in the forward mode (Ca²⁺ efflux) in the sarcolemma and reverse mode (Ca²⁺ influx) in the t‐tubules. Broken arrows point to ion transporters, ion channels, and myofilaments that are altered after myocardial infarction.

Figure 4

Effects of activation of Na⁺‐K⁺‐ATPase by phosphorylated phos‐pholemman on β‐adrenergic response in vivo. Shown are normalized in vivo hemodynamics (+dP/dt) of anesthetized wild type (•; n= 9) and phospholemman‐null (€n= 14) mice after stimulation with 25 ng of isoproterenol. Note time‐dependent decline of +dP/dt in wild type but not phospholemman‐null hearts. Phospholemman phosphorylated at serine⁶⁸ activates Na⁺‐K⁺‐ATPase, ²⁵ , ³² leading to decreases in [Na⁺]_i in wild type but not phospholemman‐null cardiac myocytes. ³² , ⁷¹

Figure 5

Effects of inhibition of Na⁺/Ca²⁺ exchanger by phosphorylated phospholemman on β‐adrenergic response in vivo. Left ventricles of phospholemman‐null (KO) mice are injected with recombinant adeno‐associated virus, serotype 9, expressing either green fluorescent protein (GFP) (€; n= 5) or the phosphomimetic phospholemman S68E mutant (•; n= 7). S68E mutant inhibits Na⁺/Ca²⁺ exchanger but has no effect on Na⁺‐K⁺‐ATPase. ¹² , ³¹ , ⁵⁷ Five weeks after virus injection, in vivo hemodynamics (+dP/dt) are measured in anesthetized mice. ³² Note that with increasing doses of isoproterenol, KO‐S68E hearts contract significantly better than KO‐GFP hearts. Since isoproterenol has no effect on Na⁺‐K⁺‐ATPase in phospholemman‐null myocytes, ²⁵ , ³² [Na⁺]_i is similar between KO‐S68E and KO‐GFP hearts (data not shown). Enhanced contractility in KO‐S68E hearts is therefore due to inhibition of Na⁺/Ca²⁺ exchanger by the S68E mutant.