Whole-heart multiparametric optical imaging reveals sex-dependent heterogeneity in cAMP signaling and repolarization kinetics.

Cyclic adenosine 3',5'-monophosphate (cAMP) is a key second messenger in cardiomyocytes responsible for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. However, whether cAMP signaling occurs heterogeneously throughout the intact heart and how this drives sex-dependent functional responses are unknown. Here, we developed and validated a novel cardiac-specific fluorescence resonance energy transfer-based cAMP reporter mouse and a combined voltage-cAMP whole-heart imaging system. We showed that in male hearts, cAMP was uniformly activated in response to pharmacological ß-adrenergic stimulation. In contrast, female hearts showed that cAMP levels decayed faster in apical versus basal regions, which was associated with nonuniform action potential changes and notable changes in the direction of repolarization. Apical phosphodiesterase (PDE) activity was higher in female versus male hearts, and PDE inhibition prevented repolarization changes in female hearts. Thus, our imaging approach revealed sex-dependent regional breakdown of cAMP and associated electrophysiological differences.

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

CaMKII Serine 280 O-GlcNAcylation Links Diabetic Hyperglycemia to Proarrhythmia.

[Figure: see text].

CaMKII and PKA-dependent phosphorylation co-regulate nuclear localization of HDAC4 in adult cardiomyocytes.

Nuclear histone deacetylase 4 (HDAC4) represses MEF2-mediated transcription, implicated in the development of heart failure. CaMKII-dependent phosphorylation drives nucleus-to-cytoplasm HDAC4 shuttling, but protein kinase A (PKA) is also linked to HDAC4 translocation. However, the interplay of CaMKII and PKA in regulating adult cardiomyocyte HDAC4 translocation is unclear. Here we sought to determine the interplay of PKA- and CaMKII-dependent HDAC4 phosphorylation and translocation in adult mouse, rabbit and human ventricular myocytes. Confocal imaging and protein analyses revealed that inhibition of CaMKII-but not PKA, PKC or PKD-raised nucleo-to-cytoplasmic HDAC4 fluorescence ratio (FNuc/FCyto) by ~ 50%, indicating baseline CaMKII activity that limits HDAC4 nuclear localization. Further CaMKII activation (via increased extracellular [Ca2+], high pacing frequencies, angiotensin II or overexpression of CaM or CaMKIIδC) led to significant HDAC4 nuclear export. In contrast, PKA activation by isoproterenol or forskolin drove HDAC4 into the nucleus (raising FNuc/FCyto by > 60%). These PKA-mediated effects were abolished in cells pretreated with PKA inhibitors and in cells expressing mutant HDAC4 in S265/266A mutant. In physiological conditions where both kinases are active, PKA-dependent nuclear accumulation of HDAC4 was predominant in the very early response, while CaMKII-dependent HDAC4 export prevailed upon prolonged stimuli. This orchestrated co-regulation was shifted in failing cardiomyocytes, where CaMKII-dependent effects predominated over PKA-dependent response. Importantly, human cardiomyocytes showed similar CaMKII- and PKA-dependent HDAC4 shifts. Collectively, CaMKII limits nuclear localization of HDAC4, while PKA favors HDAC4 nuclear retention and S265/266 is essential for PKA-mediated regulation. These pathways thus compete in HDAC4 nuclear localization and transcriptional regulation in cardiac signaling.

Intracellular ß1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility.

RATIONALE: ß1ARs (ß1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular ß1AR in cardiac contractility remains to be elucidated. OBJECTIVE: Test localization and function of intracellular ß1AR on cardiac contractility. METHODS AND RESULTS: Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of ß1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant ßAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant ßAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility. CONCLUSIONS: Functional ß1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular ß1ARs requires catecholamine transport via OCT3.

Hyperglycemia regulates cardiac K+ channels via O-GlcNAc-CaMKII and NOX2-ROS-PKC pathways.

Chronic hyperglycemia and diabetes lead to impaired cardiac repolarization, K+ channel remodeling and increased arrhythmia risk. However, the exact signaling mechanism by which diabetic hyperglycemia regulates cardiac K+ channels remains elusive. Here, we show that acute hyperglycemia increases inward rectifier K+ current (IK1), but reduces the amplitude and inactivation recovery time of the transient outward K+ current (Ito) in mouse, rat, and rabbit myocytes. These changes were all critically dependent on intracellular O-GlcNAcylation. Additionally, IK1 amplitude and Ito recovery effects (but not Ito amplitude) were prevented by the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor autocamtide-2-related inhibitory peptide, CaMKIIδ-knockout, and O-GlcNAc-resistant CaMKIIδ-S280A knock-in. Ito reduction was prevented by inhibition of protein kinase C (PKC) and NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS). In mouse models of chronic diabetes (streptozotocin, db/db, and high-fat diet), heart failure, and CaMKIIδ overexpression, both Ito and IK1 were reduced in line with the downregulated K+ channel expression. However, IK1 downregulation in diabetes was markedly attenuated in CaMKIIδ-S280A. We conclude that acute hyperglycemia enhances IK1 and Ito recovery via CaMKIIδ-S280 O-GlcNAcylation, but reduces Ito amplitude via a NOX2-ROS-PKC pathway. Moreover, chronic hyperglycemia during diabetes and CaMKII activation downregulate K+ channel expression and function, which may further increase arrhythmia susceptibility.

Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes.

RATIONALE: Diabetes mellitus is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII (Ca2+/calmodulin-dependent kinase II) activation may occur in diabetes mellitus and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality. OBJECTIVE: To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII. METHODS AND RESULTS: We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using DCF (2',7'-dichlorodihydrofluorescein diacetate) and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation-an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac-specific CaMKIIδ KO (knockout) mice. CaMKII KO or inhibition also prevented Hi-Glu-induced sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks). Thus, CaMKII activation is required for Hi-Glu-induced ROS generation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes. To test the involvement of O-GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (ThmG), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1 [(αR)-α-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide]) prevented ROS induction in response to either Hi-Glu or ThmG. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIδ was ablated (S280A), neither Hi-Glu nor ThmG induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX (NADPH oxidase) 2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (MitoTempo), and NOS (NO synthase) pathway inhibitors (L-NAME, L-NIO, and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/ThmG-induced ROS generation. CONCLUSIONS: Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires O-GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.

Emergency Spatiotemporal Shift: The Response of Protein Kinase D to Stress Signals in the Cardiovascular System.

Protein Kinase D isoforms (PKD 1-3) are key mediators of neurohormonal, oxidative, and metabolic stress signals. PKDs impact a wide variety of signaling pathways and cellular functions including actin dynamics, vesicle trafficking, cell motility, survival, contractility, energy substrate utilization, and gene transcription. PKD activity is also increasingly linked to cancer, immune regulation, pain modulation, memory, angiogenesis, and cardiovascular disease. This increasing complexity and diversity of PKD function, highlights the importance of tight spatiotemporal control of the kinase via protein-protein interactions, post-translational modifications or targeting via scaffolding proteins. In this review, we focus on the spatiotemporal regulation and effects of PKD signaling in response to neurohormonal, oxidant and metabolic signals that have implications for myocardial disease. Precise targeting of these mechanisms will be crucial in the design of PKD-based therapeutic strategies.

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

RATIONALE: In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. OBJECTIVE: This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. METHODS AND RESULTS: We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced ß2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of ß2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. CONCLUSIONS: In hypertrophic rabbit myocytes, selectively enhanced ß2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine ß2 adrenergic receptor signaling and restore myocyte contractility in response to ß adrenergic stimulation.

Novel Epac fluorescent ligand reveals distinct Epac1 vs. Epac2 distribution and function in cardiomyocytes.

Exchange proteins directly activated by cAMP (Epac1 and Epac2) have been recently recognized as key players in ß-adrenergic-dependent cardiac arrhythmias. Whereas Epac1 overexpression can lead to cardiac hypertrophy and Epac2 activation can be arrhythmogenic, it is unknown whether distinct subcellular distribution of Epac1 vs. Epac2 contributes to differential functional effects. Here, we characterized and used a novel fluorescent cAMP derivate Epac ligand 8-[Pharos-575]-2'-O-methyladenosine-3',5'-cyclic monophosphate (Φ-O-Me-cAMP) in mice lacking either one or both isoforms (Epac1-KO, Epac2-KO, or double knockout, DKO) to assess isoform localization and function. Fluorescence of Φ-O-Me-cAMP was enhanced by binding to Epac. Unlike several Epac-specific antibodies tested, Φ-O-Me-cAMP exhibited dramatically reduced signals in DKO myocytes. In WT, the apparent binding affinity (Kd = 10.2 ± 0.8 µM) is comparable to that of cAMP and nonfluorescent Epac-selective agonist 8-(4-chlorophenylthio)-2-O-methyladenosine-3'-,5'-cyclicmonophosphate (OMe-CPT). Φ-O-Me-cAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. Φ-O-Me-cAMP is a weak partial agonist for purified Epac, but functioned as an antagonist for four Epac signaling pathways in myocytes. Epac2 and Epac1 were differentially concentrated along T tubules and around the nucleus, respectively. Epac1-KO abolished OMe-CPT-induced nuclear CaMKII activation and export of transcriptional regulator histone deacetylase 5. In conclusion, Epac1 is localized and functionally involved in nuclear signaling, whereas Epac2 is located at the T tubules and regulates arrhythmogenic sarcoplasmic reticulum Ca leak.

Na+/Ca2+ exchange and Na+/K+-ATPase in the heart.

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(+)/Ca(2+) exchange (NCX) and Na(+)/K(+)-ATPase (NKA). While the relevance of Ca(2+) 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 Ca(2+) 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(+)/Ca(2+) exchanger (NCX) and the Na(+)/K(+) pump (NKA). By transporting three Na(+) ions into the cytoplasm in exchange for one Ca(2+) 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(+)/Ca(2+) exchanger (NCX1) and Na(+)/K(+) pump and the controversies that still persist in the field.

Junctional cleft [Ca²âº]i measurements using novel cleft-targeted Ca²âº sensors.

RATIONALE: Intracellular Ca(2+) concentration ([Ca(2+)]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca(2+) release and signaling are controlled by local [Ca(2+)]i in the junctional cleft ([Ca(2+)]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [Ca(2+)]Cleft directly are needed. OBJECTIVE: To construct novel sensors that allow direct measurement of [Ca(2+)]Cleft. METHODS AND RESULTS: We constructed cleft-targeted [Ca(2+)] sensors by fusing Ca(2+)-sensor GCaMP2.2 and a new lower Ca(2+)-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [Ca(2+)]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [Ca(2+)]Cleft reaches higher levels with faster kinetics than global [Ca(2+)]i during excitation-contraction coupling. Diastolic sarcoplasmic reticulum Ca(2+) leak or sarcolemmal Ca(2+) entry may raise local [Ca(2+)]Cleft above bulk cytosolic [Ca(2+)]i ([Ca(2+)]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [Ca(2+)]Cleft-[Ca(2+)]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [Ca(2+)] measured without gradients as a reference point. This diastolic difference ([Ca(2+)]Cleft=194 nmol/L versus [Ca(2+)]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca(2+) leak rather than sarcolemmal Ca(2+) flux. CONCLUSIONS: We have developed junctional cleft-targeted sensors to measure [Ca(2+)]Cleft versus [Ca(2+)]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [Ca(2+)]i gradient, which could influence local Ca(2+)-dependent signaling within the junctional cleft.

ß-adrenergic signaling inhibits Gq-dependent protein kinase D activation by preventing protein kinase D translocation.

RATIONALE: Both ß-adrenergic receptor (ß-AR) and Gq-coupled receptor (GqR) agonist-driven signaling play key roles in the events, leading up to and during cardiac dysfunction. How these stimuli interact at the level of protein kinase D (PKD), a nodal point in cardiac hypertrophic signaling, remains unclear. OBJECTIVE: To assess the spatiotemporal dynamics of PKD activation in response to ß-AR signaling alone and on coactivation with GqR-agonists. This will test our hypothesis that compartmentalized PKD signaling reconciles disparate findings of PKA facilitation and inhibition of PKD activation. METHODS AND RESULTS: We report on the spatial and temporal profiles of PKD activation using green fluorescent protein-tagged PKD (wildtype or mutant S427E) and targeted fluorescence resonance energy transfer-based biosensors (D-kinase activity reporters) in adult cardiomyocytes. We find that ß-AR/PKA signaling drives local nuclear activation of PKD, without preceding sarcolemmal translocation. We also discover pronounced interference of ß-AR/cAMP/PKA signaling on GqR-induced translocation and activation of PKD throughout the cardiomyocyte. We attribute these effects to direct, PKA-dependent phosphorylation of PKD-S427. We also show that phosphomimetic substitution of S427 likewise impedes GqR-induced PKD translocation and activation. In neonatal myocytes, S427E inhibits GqR-evoked cell growth and expression of hypertrophic markers. Finally, we show altered S427 phosphorylation in transverse aortic constriction-induced hypertrophy. CONCLUSIONS: ß-AR signaling triggers local nuclear signaling and inhibits GqR-mediated PKD activation by preventing its intracellular translocation. PKA-dependent phosphorylation of PKD-S427 fine-tunes the PKD responsiveness to GqR-agonists, serving as a key integration point for ß-adrenergic and Gq-coupled stimuli.

Nuclear translocation of cardiac G protein-Coupled Receptor kinase 5 downstream of select Gq-activating hypertrophic ligands is a calmodulin-dependent process.

G protein-Coupled Receptors (GPCRs) kinases (GRKs) play a crucial role in regulating cardiac hypertrophy. Recent data from our lab has shown that, following ventricular pressure overload, GRK5, a primary cardiac GRK, facilitates maladaptive myocyte growth via novel nuclear localization. In the nucleus, GRK5's newly discovered kinase activity on histone deacetylase 5 induces hypertrophic gene transcription. The mechanisms governing the nuclear targeting of GRK5 are unknown. We report here that GRK5 nuclear accumulation is dependent on Ca(2+)/calmodulin (CaM) binding to a specific site within the amino terminus of GRK5 and this interaction occurs after selective activation of hypertrophic Gq-coupled receptors. Stimulation of myocytes with phenylephrine or angiotensinII causes GRK5 to leave the sarcolemmal membrane and accumulate in the nucleus, while the endothelin-1 does not cause nuclear GRK5 localization. A mutation within the amino-terminus of GRK5 negating CaM binding attenuates GRK5 movement from the sarcolemma to the nucleus and, importantly, overexpression of this mutant does not facilitate cardiac hypertrophy and related gene transcription in vitro and in vivo. Our data reveal that CaM binding to GRK5 is a physiologically relevant event that is absolutely required for nuclear GRK5 localization downstream of hypertrophic stimuli, thus facilitating GRK5-dependent regulation of maladaptive hypertrophy.

Acute ß-adrenergic activation triggers nuclear import of histone deacetylase 5 and delays G(q)-induced transcriptional activation.

During hemodynamic stress, catecholamines and neurohumoral stimuli may induce co-activation of G(q)-coupled receptors and ß-adrenergic receptors (ß-AR), leading to cardiac remodeling. Dynamic regulation of histone deacetylase 5 (HDAC5), a transcriptional repressor, is crucial during stress signaling due to its role in epigenetic control of fetal gene markers. Little is known about its regulation during acute and chronic ß-AR stimulation and its cross-interaction with G(q) signaling in adult cardiac myocytes. Here, we evaluate the potential cross-talk between G(q)-driven and ß-AR mediated signaling at the level of nucleocytoplasmic shuttling of HDAC5. We show the translocation of GFP-tagged wild type HDAC5 or mutants (S279A and S279D) in response to ß-AR or G(q) agonists. Isoproterenol (ISO) or PKA activation results in strong nuclear accumulation of HDAC5 in contrast to nuclear export driven by Ca(2+)-calmodulin protein kinase II and protein kinase D. Moreover, nuclear accumulation of HDAC5 under acute ISO/PKA signaling is dependent on phosphorylation of Ser-279 and can block subsequent G(q)-mediated nuclear HDAC5 export. Intriguingly, the attenuation of G(q)-induced export is abolished after chronic PKA activation, yet nuclear HDAC5 remains elevated. Last, the effect of chronic ß-AR signaling on HDAC5 translocation was examined in adult myocytes from a rabbit model of heart failure, where ISO-induced nuclear import is ablated, but G(q)-agonist mediated export is preserved. Acute ß-AR/PKA activation protects against hypertrophic signaling by delaying G(q)-mediated transcriptional activation. This serves as a key physiological control switch before allowing genetic reprogramming via HDAC5 nuclear export during more severe stress, such as heart failure.

Na⁺/K⁺-ATPase E960 and phospholemman F28 are critical for their functional interaction.

Na(+)-K(+)-ATPase (NKA) establishes the transmembrane [Na(+)] gradient in cells. In heart, phospholemman (PLM) inhibits NKA activity by reducing its apparent Na(+) affinity, an effect that is relieved by PLM phosphorylation. The NKA crystal structure suggests regions of PLM-NKA interaction, but the sites important for functional effects in live cells are not known. We tested wild type (WT) and CFP-NKA-α1 point mutants (alanine substitution at F956, E960, L964, and F967) for fluorescence resonance energy transfer (FRET) with WT-PLM-YFP in HEK293 cells. NKA-PLM FRET was unaltered with F956A or F967A, reduced with L964A, and nearly abolished with E960A. Mutating the PLM site (F28A) identified by structural analysis to interact with E960-NKA also nearly abolished NKA-PLM FRET. In contrast, NKA-PLM coimmunoprecipitation was only slightly reduced by E960A-NKA or F28A-PLM mutants, consistent with an additional interaction site. FRET titrations indicate that the additional site has higher affinity than that between E960-NKA and F28-PLM. To test whether the FRET-preventing mutations also prevent PLM functional effects, we measured NKA-mediated Na(+)-transport in intact cells. For WT-NKA, PLM reduced apparent Na(+)-affinity of NKA and PLM phosphorylation reversed the effect. In contrast, for E960A-NKA the apparent Na(+)-affinity was unaltered by either PLM or forskolin-induced PLM phosphorylation. We conclude that E960 on NKA and F28 on PLM are critical for PLM effects on both NKA function and NKA-PLM FRET, but also there is at least one additional site that is critical for tethering PLM to NKA.

The human phospholamban Arg14-deletion mutant localizes to plasma membrane and interacts with the Na/K-ATPase.

Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired sarcoplasmic reticulum (SR) function in human and experimental heart failure. Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN mutations in humans have been associated with dilated cardiomyopathy (DCM). We previously reported the deletion of the highly conserved amino acid residue arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid is important in maintaining the upstream consensus sequence for PKA phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic lines expressing mutant PLN-R14Del at similar protein levels to wild types exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics also remained highly stimulated in PLN-R14Del cardiomyocytes, similar to PLN-KO, and isoproterenol did not further stimulate these hyper-contractile basal parameters. Consistent with the lack of inhibition on SR Ca-transport and contractility, confocal microscopy indicated that the PLN-R14Del failed to co-localize with SERCA2a. Moreover, PLN-R14Del did not co-immunoprecipitate with SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells indicated significant fluorescence resonance energy transfer between PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman. Despite the enhanced cardiac function in PLN-R14Del hearts (as in PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO) coupled with activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to the sarcolemma, in the absence of endogenous PLN, and alters NKA activity, leading to cardiac remodeling.

Fluorescence resonance energy transfer-based sensor Camui provides new insight into mechanisms of calcium/calmodulin-dependent protein kinase II activation in intact cardiomyocytes.

RATIONALE: Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of intracellular signaling in the heart. However, the tools currently available for assessing dynamic changes in CaMKII localization and activation in living myocytes are limited. OBJECTIVE: We use Camui, a novel FRET-based biosensor in which full-length CaMKII is flanked by CFP and YFP, to measure CaMKII activation state in living rabbit myocytes. METHODS AND RESULTS: We show that Camui and mutant variants that lack the sites of CaMKII autophosphorylation (T286A) and oxidative regulation (CM280/1VV) serve as useful biosensors for CaMKIIδ activation state. Camui (wild-type or mutant) was expressed in isolated adult cardiac myocytes, and localization and CaMKII activation state were determined using confocal microscopy. Camui, like CaMKIIδ, is concentrated at the z-lines, with low baseline activation state. Camui activation increased directly with pacing frequency, but the maximal effect was blunted with the T286A, consistent with frequency-dependent phosphorylation of CaMKII at T286 mainly at high-frequency and high-amplitude Ca transients. Camui was also activated by 4 neurohormonal agonists. Angiotensin II and endothelin-1 activated Camui, largely through an oxidation-dependent mechanism, whereas isoproterenol- and phenylephrine-mediated mechanisms had a significant autophosphorylation-dependent component. CONCLUSIONS: Camui is a novel, nondestructive tool that allows spatiotemporally resolved measurement of CaMKII activation state in physiologically functioning myocytes. This represents a first step in using Camui to elucidate key mechanistic details of CaMKII signaling in live hearts and myocytes.

Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase.

Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.

Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na+-K+-ATPase.

Phospholemman (PLM) is a major target for phosphorylation mediated by both PKA (at Ser68) and PKC (at both Ser63 and Ser68) in the heart. In intact cardiac myocytes, PLM associates with and inhibits Na(+)-K(+)-ATPase (NKA), mainly by reducing its affinity for internal Na(+). The inhibition is relieved upon PLM phosphorylation by PKA or PKC. The aim here was to distinguish the role of the Ser63 and Ser68 PLM phosphorylation sites in mediating kinase-induced modulation of NKA function. We expressed wild-type (WT) PLM and S63A, S68A, and AA (Ser63 and Ser68 to alanine double mutant) PLM mutants in HeLa cells that stably express rat NKA-α(1) and we measured the effect of PKA and PKC activation on NKA-mediated intracellular Na(+) concentration decline. PLM expression (WT or mutant) significantly decreased the apparent NKA affinity for internal Na(+) and had no significant effect on the maximum pump rate (V(max)). PKA activation with forskolin (20 µM) restored NKA Na(+) affinity in cells expressing WT but not AA PLM and did not affect V(max) in either case. Similarly, PKC activation with 300 nM phorbol 12,13-dibutyrate increased NKA Na(+) affinity in cells expressing WT, S63A, and S68A PLM and had no effect in cells expressing AA PLM. Neither forskolin nor phorbol 12,13-dibutyrate affected NKA function in the absence of PLM. We conclude that PLM phosphorylation at either Ser63 or Ser68 is both necessary and sufficient for completely relieving the PLM-induced NKA inhibition.