Abstract-Cardiac sympathetic stimulation activates ␤-adrenergic (␤-AR) receptors and protein kinase A (PKA) phosphorylation of proteins involved in myocyte Ca regulation. The Na/K-ATPase (NKA) is essential in regulating intracellular [Na] ([Na] i ), which in turn affects [Ca] i via Na/Ca exchange. However, how PKA modifies NKA function is unknown. Phospholemman (PLM), a member of the FXYD family of proteins that interact with NKA in various tissues, is a major PKA substrate in heart. Here we tested the hypothesis that PLM phosphorylation is responsible for the PKA effects on cardiac NKA function using wild-type (WT) and PLM knockout (PLM-KO) mice. We measured NKA-mediated [Na] i decline and current (I Pump ) to assess ␤-AR effects on NKA function in isolated myocytes. In WT myocytes, 1 mol/L isoproterenol (ISO) increased PLM phosphorylation and stimulated NKA activity mainly by increasing its affinity for internal Na (K m decreased from 18.8Ϯ1.4 to 13.6Ϯ1.5 mmol/L), with no significant effect on the maximum pump rate. This led to a significant decrease in resting [Na] i (from 12.5Ϯ1.8 to 10.5Ϯ1.4 mmol/L). In PLM-KO mice under control conditions K m (14.2Ϯ1.5 mmol/L) was lower than in WT, but comparable to that for WT in the presence of ISO. Furthermore, ISO had no significant effect on NKA function in PLM-KO mice. ATPase activity in sarcolemmal vesicles also showed a lower K m (Na) in PLM-KO versus WT (12.9Ϯ0.9 versus 16.2Ϯ1.5). Thus, PLM inhibits NKA activity by decreasing its [Na] i affinity, and this inhibitory effect is relieved by PKA activation. We conclude that PLM modulates the NKA function in a manner similar to the way phospholamban affects the related SR Ca-ATPase (inhibition of transport substrate affinity, that is relieved by phosphorylation). Key Words: Na pump Ⅲ phospholemman Ⅲ signal transduction Ⅲ ion channels A ctivation of the sympathetic nervous system and cardiac ␤-adrenergic (␤-AR) receptors causes cAMP formation and activation of protein kinase A (PKA). In cardiac myocytes, PKA phosphorylates several targets with key roles in the control of excitation-contraction coupling (ECC), including L-type Ca 2ϩ channels, phospholamban (PLB) and troponin-I, as well as other sarcolemmal proteins such as voltage-gated Na and K channels and phospholemman (PLM).During sympathetic activation, the larger Ca influx via more frequent and larger Ca current must be balanced by enhanced Ca extrusion via the Na/Ca exchange (NCX) that is driven by larger Ca transients. This increases Na influx at each beat, along with more frequent and larger Na current, which increases intracellular [Na] ([Na] i ). To limit the rise in [Na] i , the greater Na influx must be compensated for by an enhanced Na extrusion via the Na/K pump (NKA). Indeed, many early studies indicated stimulation of the Na-pump by ␤-AR activation. 1-3 However, there is controversy at present because some recent studies in single myocytes using NKA pump current (I Pump ) found either stimulation, 4 -6 inhibition, 7,8 or no change 9 in I Pump on ␤-...

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.

Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB and yellow fluorescent protein-PLB in AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 ؎ 1.3%. The observed FRET corresponds to a probe separation distance of 58.7 ؎ 0.5 Å , according to a computational model of intrapentameric FRET. This is consistent with models of the PLB pentamer in which cytoplasmic domains fan out from the central bundle of transmembrane helices. An I40A mutation of PLB did not alter pentamer conformation but increased the concentration of half-maximal FRET (K D ) by >4-fold. This is consistent with the previous observation that this putatively monomeric mutant still oligomerizes in intact membranes but forms more dynamic pentamers than wild type PLB. PLB association with SERCA, measured by FRET between cyan fluorescent protein-SERCA and yellow fluorescent protein-PLB, was increased by the I40A mutation without any detectable change in probe separation distance. The data indicate that the regulatory complex conformation is not altered by the I40A mutation. A naturally occurring human mutation (L39Stop) greatly reduced PLB oligomerization and SERCA binding and caused mislocalization of PLB to the cytoplasm and nucleus. Overall, the data suggest that the PLB pentamer adopts a "pinwheel" shape in cell membranes, as opposed to a more compact "bellflower" conformation. I40A mutation decreases oligomerization and increases PLB binding to SERCA. Truncation of the transmembrane domain by L39Stop mutation prevents anchoring of the protein in the membrane, greatly reducing PLB binding to itself or its regulatory target, SERCA.The 52-amino acid protein phospholamban (PLB) 2 is an important regulator of cardiac calcium handling (1). PLB binds avidly (2) but reversibly (3) to the sarco(endo)plasmic reticulum calcium ATPase (SERCA), reducing this calcium pump's affinity for calcium (4). PLB also oligomerizes into pentamers through leucine zipper interactions in its transmembrane domain (5, 6). NMR studies indicate that PLB tertiary structure consists of an N-terminal (cytosolic) ␣-helix (domain IA) connected by a flexible loop (domain IB) to a second ␣-helix (domain II) that is anchored in the membrane of the sarcoplasmic reticulum (7). Several possible configurations of the cytoplasmic domains of pentameric PLB have been described. Some of these portray the domain IA ␣-helix as being nearly normal to the surface of the membrane (8, 9), whereas others indicate axial declination of domain IA (7, 10 -14), permitting contact with the surface of the membrane (15, 16). These previous studies were performed using in vitro preparations of PLB in defined lipids or detergent. To investigate the quaternary structure of PLB in the membranes of living cells and distinguish between these structural mo...

Ca(2+) in cardiac myocytes regulates contractility and relaxation, and Ca(2+) and Na (+)regulation are linked via Na(+)/Ca(2+) exchange (NCX). Heart failure (HF) is accompanied by contractile dysfunction and arrhythmias, both of which may be due to altered cellular Ca(2+) handling. Smaller Ca(2+) transient and sarcoplasmic reticulum (SR) Ca(2+) content cause systolic dysfunction in HF. The reduced SR Ca(2+) content is due to: (a) reduced SR Ca(2+)-ATPase function (which also contributes to diastolic dysfunction), (b) increased expression and function of NCX (which competes with SR Ca(2+)-ATPase during relaxation, but preserves diastolic function), and (c) enhanced diastolic SR Ca(2+) leak. Relative contributions of these may vary with HF etiology and stage. Triggered arrhythmias (e.g., delayed afterdepolarizations [DADs]) are prominent in HF. DADs are due to spontaneous SR Ca(2+) release and consequent activation of transient inward NCX current, which in HF allows DADs to more readily trigger arrhythmogenic action potentials. Thus NCX and Na(+) are critical in systolic and diastolic function and arrhythmias. [Na(+)](i) is elevated in HF, which may limit SR unloading and provide some Ca(2+) influx during the HF action potential, thus limiting the depression of systolic function. High [Na(+)](i) in HF is due to enhanced Na(+) influx. Cellular Na(+)/K(+)-ATPase (NKA) function appears unaltered, despite reduced NKA expression. This dichotomy led us to test NKA regulation by phospholemman (PLM). We find that PLM regulates NKA in a manner analogous to phospholamban regulation of SR Ca(2+)-ATPase (i.e., inhibition that is relieved by PLM phosphorylation). We measured intermolecular FRET between PLM and NKA, which is reduced upon PLM phosphorylation. The lower expression level of more phosphorylated PLM in HF may explain the above dichotomy. Thus, altered Ca(2+) and Na(+) handling contributes to altered contractile function and arrhythmogenesis in HF.

Rationale Mitochondrial [Ca2+] ([Ca2+]mito) regulates mitochondrial energy production, provides transient Ca2+buffering under stress and can be involved in cell death. Mitochondria are near the sarcoplasmic reticulum (SR) in cardiac myocytes and evidence for crosstalk exists. However, quantitative measurements of [Ca2+]mito are limited and spatial [Ca2+]mito gradients have not been directly measured. Objective To directly measure local [Ca2+]mito during normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial [Ca2+]mito gradients. Methods and Results We used in-situ calibration of the mitochondrially targeted inverse pericam indicator Mitycam and directly measured [Ca2+]mito during SR Ca2+ release in intact rabbit ventricular myocytes by confocal microscopy. During steady state pacing Δ[Ca2+]mito amplitude was 29 ± 3 nM, rising rapidly (similar to cytosolic [Ca2+]i) but declining much more slowly. Taking advantage of the structural periodicity of cardiac sarcomeres, we found that [Ca2+]mito near SR Ca2+ release sites (Z-lines) vs. mid sarcomere (M-line) reached a higher peak amplitude (37 ± 4 vs. 26 ± 4 nM, respectively P < 0.05) which occurred earlier in time. This difference was attributed to ends of mitochondria being physically closer to SR Ca2+ release sites, because the mitochondrial Ca2+ uniporter was homogeneously distributed and elevated [Ca2+] applied laterally did not produce longitudinal [Ca2+]mito gradients. Conclusions We developed methods to measure spatiotemporal [Ca2+]mito gradients quantitatively during excitation-contraction coupling. The amplitude and kinetics of [Ca2+]mito transients differ significantly from those in the cytosol and are higher and faster near the Z- vs. M-line. This approach will help clarify SR-mitochondrial Ca2+ signaling.

Background A hallmark of heart failure is impaired cytoplasmic Ca2+ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca2+ handling – via altered excitation-transcription coupling – contribute to the development and progression of heart failure. Methods and Results Using tissue and isolated cardiomyocytes from non-failing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca2+ handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment there was also reduced expression of Ca2+ pumps and ryanodine receptors, and increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca2+ transporters. These changes were associated with altered nucleoplasmic Ca2+ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca2+ levels with diminished and prolonged nuclear Ca2+ transients and slowed intranuclear Ca2+ diffusion. Altered nucleoplasmic Ca2+ levels were translated to higher activation of nuclear Ca2+/calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca2+ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca2+ changes that are typical of heart failure. Conclusions During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca2+ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca2+ regulation may, therefore, be a novel therapeutic approach for preventing adverse cardiac remodeling.

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Abstract-Cardiac hypertrophy and heart failure (HF) are associated with reactivation of fetal cardiac genes, and class II histone deacetylases (HDACs) (eg, HDAC5) have been strongly implicated in this process. We have shown previously that inositol trisphosphate, Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII), and protein kinase (PK)D are involved in HDAC5 phosphorylation and nuclear export in normal adult ventricular myocytes and also that CaMKII␦ and inositol trisphosphate receptors are upregulated in HF. Here we tested whether, in our rabbit HF model, nucleocytoplasmic shuttling of HDAC5 was altered either at baseline or in response to endothelin-1, which would indicate HDAC5 phosphorylation and transcription effects. The fusion protein HDAC5-green fluorescent protein (HDAC5-GFP) was more cytosolic in HF myocytes (F nuc /F cyto 3.3Ϯ0.3 vs 7.2Ϯ0.4 in control), and HDAC5 was more phosphorylated. Despite this baseline cytosolic HDAC5 shift, endothelin-1 produced more rapid HDAC5-GFP nuclear export in HF versus control myocytes. We also find that PKD and CaMKII␦ C expression and activation state are increased in both rabbit and human HF. Inhibition of either CaMKII or PKD in HF myocytes partially restored the HDAC5-GFP F nuc /F cyto toward control, and simultaneous inhibition restored F nuc /F cyto to that in control myocytes. Moreover, adenovirus-mediated overexpression of PKD, CaMKII␦ B , or CaMKII␦ C reduced baseline HDAC5 F nuc /F cyto in control myocytes (3.4Ϯ0.5, 3.8Ϯ0.5, and 5.2Ϯ0.5, respectively), approaching that seen in HF. We conclude that chronic upregulation and activation of inositol trisphosphate receptors, CaMKII, and PKD in HF shifts HDAC5 out of the nucleus, derepressing transcription of hypertrophic genes. This may directly contribute to the development and/or maintenance of HF.