This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation–contraction coupling and arrhythmias: Na+ channel and Na+ transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na+/Ca2+ exchange (NCX) and Na+/K+-ATPase (NKA). While the relevance of Ca2+ homeostasis in cardiac function has been extensively investigated, the role of Na+ regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na+ content have multiple effects on the heart by influencing intracellular Ca2+ and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na+ homeostasis. Among the proteins that accomplish this task are the Na+/Ca2+ exchanger (NCX) and the Na+/K+ pump (NKA). By transporting three Na+ ions into the cytoplasm in exchange for one Ca2+ moved out, NCX is one of the main Na+ influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na+ ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na+ and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na+ homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na+/Ca2+ exchanger (NCX1) and Na+/K+ pump and the controversies that still persist in the field.

The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discslarge/ZO-1 homology (PDZ) binding motif, but requires a ∼120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.phospholemman | sodium pump | palmitoylation | DHHC | ion transport P rotein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol via a thioester bond, is catalyzed by Asp-His-His-Cys motif-containing palmitoyl acyltransferases (DHHC-PATs); there are 23 human isoforms (1). These zinc-finger-containing enzymes typically have four transmembrane (TM) domains, with a conserved ∼50 amino acid cysteine-rich cytosolic core located between TM2 and -3, which contains a conserved DHHC motif, the active site. In contrast, the intracellular amino and carboxyl termini are poorly conserved, and likely contribute to DHHC isoform substrate selectivity (1). DHHC-PATs are expressed throughout the secretory pathway, but DHHC5 is widely recognized as one of very few cell-surfacelocalized PATs (2, 3). The final four amino acids of DHHC5 form a canonical class II PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, which interacts with postsynaptic density protein 95 (PSD-95) (2), although PSD-95 is not itself a DHHC5 substrate.An appreciation is now growing that protein palmitoylation turns over rapidly (in minutes) for certain proteins (4-8). For example, dynamic surface membrane protein palmitoylation by DHHC5 underlies a novel form of endocytosis, massive endocytosis (MEND), in which up to 70% of the cell surface membrane is internalized (7, 8). Calci...

AimsElevation of intracellular Na in the failing myocardium contributes to contractile dysfunction, the negative force–frequency relationship, and arrhythmias. Although phospholemman (PLM) is recognized to form the link between signalling pathways and Na/K pump activity, the possibility that defects in its regulation contribute to elevation of intracellular Na has not been investigated. Our aim was to test the hypothesis that the prevention of PLM phosphorylation in a PLM3SA knock-in mouse (in which PLM has been rendered unphosphorylatable) will exacerbate cardiac hypertrophy and cellular Na overload. Testing this hypothesis should determine whether changes in PLM phosphorylation are simply bystander effects or are causally involved in disease progression.Methods and resultsIn wild-type (WT) mice, aortic constriction resulted in hypophosphorylation of PLM with no change in Na/K pump expression. This under-phosphorylation of PLM occurred at 3 days post-banding and was associated with a progressive decline in Na/K pump current and elevation of [Na]i. Echocardiography, morphometry, and pressure-volume (PV) catheterization confirmed remodelling, dilation, and contractile dysfunction, respectively. In PLM3SA mice, expression of Na/K ATPase was increased and PLM decreased such that net Na/K pump current under quiescent conditions was unchanged (cf. WT myocytes); [Na+]i was increased and forward-mode Na/Ca exchanger was reduced in paced PLM3SA myocytes. Cardiac hypertrophy and Na/K pump inhibition were significantly exacerbated in banded PLM3SA mice compared with banded WT.ConclusionsDecreased phosphorylation of PLM reduces Na/K pump activity and exacerbates Na overload, contractile dysfunction, and adverse remodelling following aortic constriction in mice. This suggests a novel therapeutic target for the treatment of heart failure.