Validation of a Demography-Based Adaptive QTc Formula using Pediatric and Adult Datasets Acquired from Humans and Guinea Pigs.

Introduction: A variety of QT rate-correction (QTc) formulae have been utilized for both clinical and research purposes. However, these formulae are not universally effective, likely due to significant influences of demographic diversity on the QT-HR relationship. To address this limitation, we proposed an adaptive QTc (QTcAd) formula that adjusts to subject demographics (i.e., age). Further, we compared the efficacy and accuracy of the QTcAd formula to other widely used alternatives. Method: Using age as a demographic parameter, we tested the QTcAd formula across diverse age groups with different heart rates (HR) in both humans and guinea pigs. Utilizing retrospective human (n=1360) and guinea pig electrocardiogram (ECG) data from in-vivo (n=55) and ex-vivo (n=66) settings, we evaluated the formula's effectiveness. Linear regression fit parameters of HR-QTc (slope and R²) were utilized for performance assessment. To evaluate the accuracy of the predicted QTc, we acquired epicardial electrical and optical voltage data from Langendorff-perfused guinea pig hearts. Results: In both human subjects and guinea pigs, the QTcAd formula consistently outperformed other formulae across all age groups. For instance, in a 20-year-old human group (n=300), the QTcAd formula successfully nullified the inverse HR-QT relationship (R²=5.1E-09, slope=-3.5E-05), while the Bazett formula (QTcB) failed to achieve comparable effectiveness (R²= 0.20, slope=0.91). Moreover, the QTcAd formula exhibited better accuracy than the age-specific Benatar formula (QTcBe), which overcorrected QTc (1-week human QT: 263.8±14.8 ms, QTcAd: 263.8±7.3 ms, p=0.62; QTcBe: 422.5±7.3 ms, p<0.0001). The optically measured pseudo-QT interval (143±22.5 ms, n=44) was better approximated by QTcAd (180.6±17.0 ms) compared to all other formulae. Furthermore, we demonstrated that the QTcAd formula was not inferior to individual-specific QTc formulae. Conclusion: The demography-based QTcAd formula showed superior performance across human and guinea pig age groups, which may enhance the efficacy of QTc for cardiovascular disease diagnosis, risk stratification, and drug safety testing. What is known: Corrected QT (QTc) is a well-known ECG biomarker for cardiovascular disease risk stratification and drug safety testing. Various QT rate-correction formulae have been developed, but these formulae do not perform consistently across diverse datasets (e.g., sex, age, disease, species). What the study adds: We introduce a novel QTc formula (QTcAd) that adapts to demographic variability, as the parameters can be modified based on the characteristics of the study population. The formula (QTcAd = QT + (|m|*(HR-HR mean )) - includes the absolute slope (m) of the linear regression of QT and heart rate (HR) and the mean HR of the population (HR mean ) as population characteristics parametersˍUsing datasets from both pediatric and adult human subjects and an animal model, we demonstrate that the QTcAd formula is more effective at eliminating the QT-HR inverse relationship, as compared to other commonly used correction formulae.

Disrupting Na+ ion homeostasis and Na+/K+ ATPase activity in breast cancer cells directly modulates glycolysis in vitro and in vivo.

BACKGROUND: Glycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na+/K+-ATPase (NKA) due to altered sodium ion homeostasis in cancer cells. METHODS: Live whole-cell measurements of intracellular sodium [Na+]i were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered 23Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by 2H NMR to monitor conversion of [6,6-2H2]D-glucose to [2H]-labelled L-lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na+]i was titrated using isotonic buffers with varying [Na+] and [K+] and introducing an artificial Na+ plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, 1H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[18F]fluoroglucose (18F-FDG) positron emission tomography (PET). RESULTS: Intracellular [Na+]i was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na+]i and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na+]i in MDA-MB-231 and 4T1 cells and a Na+-dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. 18FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower 18FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected. CONCLUSIONS: Glycolytic flux correlates with Na+-driven NKA activity in breast cancer cells, providing evidence for the 'centrality of the [Na+]i-NKA nexus' in the mechanistic basis of the Warburg effect.

Elevated Na is a dynamic and reversible modulator of mitochondrial metabolism in the heart.

Elevated intracellular sodium Nai adversely affects mitochondrial metabolism and is a common feature of heart failure. The reversibility of acute Na induced metabolic changes is evaluated in Langendorff perfused rat hearts using the Na/K ATPase inhibitor ouabain and the myosin-uncoupler para-aminoblebbistatin to maintain constant energetic demand. Elevated Nai decreases Gibb's free energy of ATP hydrolysis, increases the TCA cycle intermediates succinate and fumarate, decreases ETC activity at Complexes I, II and III, and causes a redox shift of CoQ to CoQH2, which are all reversed on lowering Nai to baseline levels. Pseudo hypoxia and stabilization of HIF-1α is observed despite normal tissue oxygenation. Inhibition of mitochondrial Na/Ca-exchange with CGP-37517 or treatment with the mitochondrial ROS scavenger MitoQ prevents the metabolic alterations during Nai elevation. Elevated Nai plays a reversible role in the metabolic and functional changes and is a novel therapeutic target to correct metabolic dysfunction in heart failure.

Targeted degradation of zDHHC-PATs decreases substrate S-palmitoylation.

Reversible S-palmitoylation of protein cysteines, catalysed by a family of integral membrane zDHHC-motif containing palmitoyl acyl transferases (zDHHC-PATs), controls the localisation, activity, and interactions of numerous integral and peripheral membrane proteins. There are compelling reasons to want to inhibit the activity of individual zDHHC-PATs in both the laboratory and the clinic, but the specificity of existing tools is poor. Given the extensive conservation of the zDHHC-PAT active site, development of isoform-specific competitive inhibitors is highly challenging. We therefore hypothesised that proteolysis-targeting chimaeras (PROTACs) may offer greater specificity to target this class of enzymes. In proof-of-principle experiments we engineered cell lines expressing tetracycline-inducible Halo-tagged zDHHC5 or zDHHC20, and evaluated the impact of Halo-PROTACs on zDHHC-PAT expression and substrate palmitoylation. In HEK-derived FT-293 cells, Halo-zDHHC5 degradation significantly decreased palmitoylation of its substrate phospholemman, and Halo-zDHHC20 degradation significantly diminished palmitoylation of its substrate IFITM3, but not of the SARS-CoV-2 spike protein. In contrast, in a second kidney derived cell line, Vero E6, Halo-zDHHC20 degradation did not alter palmitoylation of either IFITM3 or SARS-CoV-2 spike. We conclude from these experiments that PROTAC-mediated targeting of zDHHC-PATs to decrease substrate palmitoylation is feasible. However, given the well-established degeneracy in the zDHHC-PAT family, in some settings the activity of non-targeted zDHHC-PATs may substitute and preserve substrate palmitoylation.

Glutathione-dependent depalmitoylation of phospholemman by peroxiredoxin 6.

Phospholemman (PLM) regulates the cardiac sodium pump: PLM phosphorylation activates the pump whereas PLM palmitoylation inhibits its activity. Here, we show that the anti-oxidant protein peroxiredoxin 6 (Prdx6) interacts with and depalmitoylates PLM in a glutathione-dependent manner. Glutathione loading cells acutely reduce PLM palmitoylation; glutathione depletion significantly increases PLM palmitoylation. Prdx6 silencing abolishes these effects, suggesting that PLM can be depalmitoylated by reduced Prdx6. In vitro, only recombinant Prdx6, among several peroxiredoxin isoforms tested, removes palmitic acid from recombinant palmitoylated PLM. The broad-spectrum depalmitoylase inhibitor palmostatin B prevents Prdx6-dependent PLM depalmitoylation in cells and in vitro. Our data suggest that Prdx6 is a thioesterase that can depalmitoylate proteins by nucleophilic attack via its reactive thiol, linking PLM palmitoylation and hence sodium pump activity to cellular glutathione status. We show that protein depalmitoylation can occur via a catalytic cysteine in which substrate specificity is determined by a protein-protein interaction.

Discovery of a novel cardiac-specific myosin modulator using artificial intelligence-based virtual screening.

Direct modulation of cardiac myosin function has emerged as a therapeutic target for both heart disease and heart failure. However, the development of myosin-based therapeutics has been hampered by the lack of targeted in vitro screening assays. In this study we use Artificial Intelligence-based virtual high throughput screening (vHTS) to identify novel small molecule effectors of human ß-cardiac myosin. We test the top scoring compounds from vHTS in biochemical counter-screens and identify a novel chemical scaffold called 'F10' as a cardiac-specific low-micromolar myosin inhibitor. Biochemical and biophysical characterization in both isolated proteins and muscle fibers show that F10 stabilizes both the biochemical (i.e. super-relaxed state) and structural (i.e. interacting heads motif) OFF state of cardiac myosin, and reduces force and left ventricular pressure development in isolated myofilaments and Langendorff-perfused hearts, respectively. F10 is a tunable scaffold for the further development of a novel class of myosin modulators.

Empagliflozin improves cardiac energetics during ischaemia/reperfusion by directly increasing cardiac ketone utilization.

AIMS: Empagliflozin (EMPA), a potent inhibitor of the renal sodium-glucose cotransporter 2 and an effective treatment for Type 2 diabetes, has been shown to have cardioprotective effects, independent of improved glycaemic control. Several non-canonical mechanisms have been proposed to explain these cardiac effects, including increasing circulating ketone supply to the heart. This study aims to test whether EMPA directly alters cardiac ketone metabolism independent of supply. METHODS AND RESULTS: The direct effects of EMPA on cardiac function and metabolomics were investigated in Langendorff rat heart perfused with buffer containing 5 mM glucose, 4 mM ß-hydroxybutyrate (ßHb) and 0.4 mM intralipid, subject to low flow ischaemia/reperfusion. Cardiac energetics were monitored in situ using 31P NMR spectroscopy. Steady-state 13C labelling was performed by switching 12C substrates for 13C1 glucose or 13C4 ßHb and 13C incorporation into metabolites determined using 2D 1H-13C HSQC NMR spectroscopy. EMPA treatment improved left ventricular-developed pressure during ischaemia and reperfusion compared to vehicle-treated hearts. In EMPA-treated hearts, total adenosine triphosphate (ATP) and phosphocreatine (PCr) levels, and Gibbs free energy for ATP hydrolysis were significantly higher during ischaemia and reperfusion. EMPA treatment did not alter the incorporation of 13C from glucose into glycolytic products lactate or alanine neither during ischaemia nor reperfusion. In ischaemia, EMPA led to a decrease in 13C1 glucose incorporation and a concurrent increase in 13C4 ßHb incorporation into tricarboxylic acid (TCA) cycle intermediates succinate, citrate, and glutamate. During reperfusion, the concentration of metabolites originating from 13C1 glucose was similar to vehicle but those originating from 13C4 ßHb remained elevated in EMPA-treated hearts. CONCLUSION: Our findings indicate that EMPA causes a switch in metabolism away from glucose oxidation towards increased ketone utilization in the rat heart, thereby improving function and energetics both during ischaemia and recovery during reperfusion. This preference of ketone utilization over glucose was observed under conditions of constant supply of substrate, suggesting that EMPA acts directly by modulating cardiac substrate preference, independent of substrate availability. The mechanisms underlying our findings are currently unknown, warranting further study.

Sensing Cytosolic DNA Lowers Blood Pressure by Direct cGAMP-Dependent PKGI Activation.

BACKGROUND: The major cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) has emerged as a key mediator of inflammation that underlies cardiovascular disease. On interaction with double-stranded DNA, cGAS generates the second messenger 2',3'-cyclic GMP-AMP (cGAMP) that directly binds to and activates the stimulator of interferon genes, which in turn leads to enhanced expression of genes encoding interferons and proinflammatory cytokines. Here, we show that cGAMP generated by cGAS also directly activates PKGI (cGMP-dependent protein kinase 1), a mechanism that underlies crosstalk between inflammation and blood pressure regulation. METHODS: The ability of cGAS and cGAMP to activate PKGI was assessed using molecular, cellular, and biochemical analyses, and in myography experiments, as well. The release of cGAMP from the endothelium was measured using an ELISA, and its uptake into the vascular smooth muscle was assessed using molecular and biochemical approaches, including the identification and targeting of specific cGAMP transporters. The blood pressure of wild-type and cGAS-/- mice was assessed using implanted telemetry probes. cGAS was activated by in vivo transfection with G3-YSD or mice were made septic by administration of lipopolysaccharide. RESULTS: The detection of cytosolic DNA by cGAS within the vascular endothelium leads to formation of cGAMP that was found to be actively extruded by MRP1 (multidrug resistance protein 1). Once exported, this cGAMP is then imported into neighboring vascular smooth muscle cells through the volume-regulated anion channel, where it can directly activate PKGI. The activation of PKGI by cGAMP mediates vasorelaxation that is dependent on the activity of MRP1 and volume-regulated anion channel, but independent of the canonical nitric oxide pathway. This mechanism of PKGI activation mediates lowering of blood pressure and contributes to hypotension and tissue hypoperfusion during sepsis. CONCLUSIONS: The activation of PKGI by cGAMP enables the coupling of blood pressure to cytosolic DNA sensing by cGAS, which plays a key role during sepsis by mediating hypotension and tissue hypoperfusion.

Fasting increases susceptibility to acute myocardial ischaemia/reperfusion injury through a sirtuin-3 mediated increase in fatty acid oxidation.

Fasting increases susceptibility to acute myocardial ischaemia/reperfusion injury (IRI) but the mechanisms are unknown. Here, we investigate the role of the mitochondrial NAD+-dependent deacetylase, Sirtuin-3 (SIRT3), which has been shown to influence fatty acid oxidation and cardiac outcomes, as a potential mediator of this effect. Fasting was shown to shift metabolism from glucose towards fatty acid oxidation. This change in metabolic fuel substrate utilisation increased myocardial infarct size in wild-type (WT), but not SIRT3 heterozygous knock-out (KO) mice. Further analysis revealed SIRT3 KO mice were better adapted to starvation through an improved cardiac efficiency, thus protecting them from acute myocardial IRI. Mitochondria from SIRT3 KO mice were hyperacetylated compared to WT mice which may regulate key metabolic processes controlling glucose and fatty acid utilisation in the heart. Fasting and the associated metabolic switch to fatty acid respiration worsens outcomes in WT hearts, whilst hearts from SIRT3 KO mice are better adapted to oxidising fatty acids, thereby protecting them from acute myocardial IRI.

Dynamic but discordant alterations in zDHHC5 expression and palmitoylation of its substrates in cardiac pathologies.

S-palmitoylation is an essential lipid modification catalysed by zDHHC-palmitoyl acyltransferases that regulates the localisation and activity of substrates in every class of protein and tissue investigated to date. In the heart, S-palmitoylation regulates sodium-calcium exchanger (NCX1) inactivation, phospholemman (PLM) inhibition of the Na+/K+ ATPase, Nav1.5 influence on membrane excitability and membrane localisation of heterotrimeric G-proteins. The cell surface localised enzyme zDHHC5 palmitoylates NCX1 and PLM and is implicated in injury during anoxia/reperfusion. Little is known about how palmitoylation remodels in cardiac diseases. We investigated expression of zDHHC5 in animal models of left ventricular hypertrophy (LVH) and heart failure (HF), along with HF tissue from humans. zDHHC5 expression increased rapidly during onset of LVH, whilst HF was associated with decreased zDHHC5 expression. Paradoxically, palmitoylation of the zDHHC5 substrate NCX1 was significantly reduced in LVH but increased in human HF, while palmitoylation of the zDHHC5 substrate PLM was unchanged in all settings. Overexpression of zDHHC5 in rabbit ventricular cardiomyocytes did not alter palmitoylation of its substrates or overall cardiomyocyte contractility, suggesting changes in zDHHC5 expression in disease may not be a primary driver of pathology. zDHHC5 itself is regulated by post-translational modifications, including palmitoylation in its C-terminal tail. We found that in HF palmitoylation of zDHHC5 changed in the same manner as palmitoylation of NCX1, suggesting additional regulatory mechanisms may be involved. This study provides novel evidence that palmitoylation of cardiac substrates is altered in the setting of HF, and that expression of zDHHC5 is dysregulated in both hypertrophy and HF.

Stimulated phosphorylation of ERK in mouse kidney mesangial cells is dependent upon expression of Cav3.1.

BACKGROUND: T-type calcium channels (TTCC) are low voltage activated channels that are widely expressed in the heart, smooth muscle and neurons. They are known to impact on cell cycle progression in cancer and smooth muscle cells and more recently, have been implicated in rat and human mesangial cell proliferation. The aim of this study was to investigate the roles of the different isoforms of TTCC in mouse mesangial cells to establish which may be the best therapeutic target for treating mesangioproliferative kidney diseases.  METHODS: In this study, we generated single and double knockout (SKO and DKO) clones of the TTCC isoforms CaV3.1 and CaV3.2 in mouse mesangial cells using CRISPR-cas9 gene editing. The downstream signals linked to this channel activity were studied by ERK1/2 phosphorylation assays in serum, PDGF and TGF-ß1 stimulated cells. We also examined their proliferative responses in the presence of the TTCC inhibitors mibefradil and TH1177. RESULTS: We demonstrate a complete loss of ERK1/2 phosphorylation in response to multiple stimuli (serum, PDGF, TGF-ß1) in CaV3.1 SKO clone, whereas the CaV3.2 SKO clone retained these phospho-ERK1/2 responses. Stimulated cell proliferation was not profoundly impacted in either SKO clone and both clones remained sensitive to non-selective TTCC blockers, suggesting a role for more than one TTCC isoform in cell cycle progression. Deletion of both the isoforms resulted in cell death. CONCLUSION: This study confirms that TTCC are expressed in mouse mesangial cells and that they play a role in cell proliferation. Whereas the CaV3.1 isoform is required for stimulated phosphorylation of ERK1/2, the Ca V3.2 isoform is not. Our data also suggest that neither isoform is necessary for cell proliferation and that the anti-proliferative effects of mibefradil and TH1177 are not isoform-specific. These findings are consistent with data from in vivo rat mesangial proliferation Thy1 models and support the future use of genetic mouse models to test the therapeutic actions of TTCC inhibitors.

With a grain of salt: Sodium elevation and metabolic remodelling in heart failure.

Elevated intracellular Na (Nai) and metabolic impairment are interrelated pathophysiological features of the failing heart (HF). There have been a number of studies showing that myocardial sodium elevation subtly affects mitochondrial function. During contraction, mitochondrial calcium (Camito) stimulates a variety of TCA cycle enzymes, thereby providing reducing equivalents to maintain ATP supply. Nai elevation has been shown to impact Camito; however, whether metabolic remodelling in HF is caused by increased Nai has only been recently demonstrated. This novel insight may help to elucidate the contribution of metabolic remodelling in the pathophysiology of HF, the lack of efficacy of current HF therapies and a rationale for the development of future metabolism-targeting treatments. Here we review the relationship between Na pump inhibition, elevated Nai, and altered metabolic profile in the context of HF and their link to metabolic (in)flexibility and mitochondrial reprogramming.

Off-target effects of sodium-glucose co-transporter 2 blockers: empagliflozin does not inhibit Na+/H+ exchanger-1 or lower [Na+]i in the heart.

AIMS: Emipagliflozin (EMPA) is a potent inhibitor of the renal sodium-glucose co-transporter 2 (SGLT2) and an effective treatment for type-2 diabetes. In patients with diabetes and heart failure, EMPA has cardioprotective effects independent of improved glycaemic control, despite SGLT2 not being expressed in the heart. A number of non-canonical mechanisms have been proposed to explain these cardiac effects, most notably an inhibitory action on cardiac Na+/H+ exchanger 1 (NHE1), causing a reduction in intracellular [Na+] ([Na+]i). However, at resting intracellular pH (pHi), NHE1 activity is very low and its pharmacological inhibition is not expected to meaningfully alter steady-state [Na+]i. We re-evaluate this putative EMPA target by measuring cardiac NHE1 activity. METHODS AND RESULTS: The effect of EMPA on NHE1 activity was tested in isolated rat ventricular cardiomyocytes from measurements of pHi recovery following an ammonium pre-pulse manoeuvre, using cSNARF1 fluorescence imaging. Whereas 10 µM cariporide produced near-complete inhibition, there was no evidence for NHE1 inhibition with EMPA treatment (1, 3, 10, or 30 µM). Intracellular acidification by acetate-superfusion evoked NHE1 activity and raised [Na+]i, reported by sodium binding benzofuran isophthalate (SBFI) fluorescence, but EMPA did not ablate this rise. EMPA (10 µM) also had no significant effect on the rate of cytoplasmic [Na+]i rise upon superfusion of Na+-depleted cells with Na+-containing buffers. In Langendorff-perfused mouse, rat and guinea pig hearts, EMPA did not affect [Na+]i at baseline nor pHi recovery following acute acidosis, as measured by 23Na triple quantum filtered NMR and 31P NMR, respectively. CONCLUSIONS: Our findings indicate that cardiac NHE1 activity is not inhibited by EMPA (or other SGLT2i's) and EMPA has no effect on [Na+]i over a wide range of concentrations, including the therapeutic dose. Thus, the beneficial effects of SGLT2i's in failing hearts should not be interpreted in terms of actions on myocardial NHE1 or intracellular [Na+].

Mechanism of succinate efflux upon reperfusion of the ischaemic heart.

AIMS: Succinate accumulates several-fold in the ischaemic heart and is then rapidly oxidized upon reperfusion, contributing to reactive oxygen species production by mitochondria. In addition, a significant amount of the accumulated succinate is released from the heart into the circulation at reperfusion, potentially activating the G-protein-coupled succinate receptor (SUCNR1). However, the factors that determine the proportion of succinate oxidation or release, and the mechanism of this release, are not known. METHODS AND RESULTS: To address these questions, we assessed the fate of accumulated succinate upon reperfusion of anoxic cardiomyocytes, and of the ischaemic heart both ex vivo and in vivo. The release of accumulated succinate was selective and was enhanced by acidification of the intracellular milieu. Furthermore, pharmacological inhibition, or haploinsufficiency of the monocarboxylate transporter 1 (MCT1) significantly decreased succinate efflux from the reperfused heart. CONCLUSION: Succinate release upon reperfusion of the ischaemic heart is mediated by MCT1 and is facilitated by the acidification of the myocardium during ischaemia. These findings will allow the signalling interaction between succinate released from reperfused ischaemic myocardium and SUCNR1 to be explored.

Phospholemman Phosphorylation Regulates Vascular Tone, Blood Pressure, and Hypertension in Mice and Humans.

BACKGROUND: Although it has long been recognized that smooth muscle Na/K ATPase modulates vascular tone and blood pressure (BP), the role of its accessory protein phospholemman has not been characterized. The aim of this study was to test the hypothesis that phospholemman phosphorylation regulates vascular tone in vitro and that this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in humans. METHODS: In mouse studies, phospholemman knock-in mice (PLM3SA; phospholemman [FXYD1] in which the 3 phosphorylation sites on serines 63, 68, and 69 are mutated to alanines), in which phospholemman is rendered unphosphorylatable, were used to assess the role of phospholemman phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements. In vivo BP and regional blood flow were assessed using Doppler flow and telemetry in young (14-16 weeks) and old (57-60 weeks) wild-type and transgenic mice. In human studies, we searched human genomic databases for mutations in phospholemman in the region of the phosphorylation sites and performed analyses within 2 human data cohorts (UK Biobank and GoDARTS [Genetics of Diabetes Audit and Research in Tayside]) to assess the impact of an identified single nucleotide polymorphism on BP. This single nucleotide polymorphism was expressed in human embryonic kidney cells, and its effect on phospholemman phosphorylation was determined using Western blotting. RESULTS: Phospholemman phosphorylation at Ser63 and Ser68 limited vascular constriction in response to phenylephrine. This effect was blocked by ouabain. Prevention of phospholemman phosphorylation in the PLM3SA mouse profoundly enhanced vascular responses to phenylephrine both in vitro and in vivo. In aging wild-type mice, phospholemman was hypophosphorylated, and this correlated with the development of aging-induced essential hypertension. In humans, we identified a nonsynonymous coding variant, single nucleotide polymorphism rs61753924, which causes the substitution R70C in phospholemman. In human embryonic kidney cells, the R70C mutation prevented phospholemman phosphorylation at Ser68. This variant's rare allele is significantly associated with increased BP in middle-aged men. CONCLUSIONS: These studies demonstrate the importance of phospholemman phosphorylation in the regulation of vascular tone and BP and suggest a novel mechanism, and therapeutic target, for aging-induced essential hypertension in humans.

14-3-3 binding creates a memory of kinase action by stabilizing the modified state of phospholamban.

The cardiac membrane protein phospholamban (PLN) is targeted by protein kinase A (PKA) at Ser16 and by Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr17 ß-Adrenergic stimulation and PKA-dependent phosphorylation of Ser16 acutely stimulate the sarcoplasmic reticulum calcium pump (SERCA) by relieving its inhibition by PLN. CaMKII-dependent phosphorylation may lead to longer-lasting SERCA stimulation and may sustain maladaptive Ca2+ handling. Here, we demonstrated that phosphorylation at either Ser16 or Thr17 converted PLN into a target for the phosphoadaptor protein 14-3-3 with different affinities. 14-3-3 proteins were localized within nanometers of PLN and endogenous 14-3-3 coimmunoprecipitated with pentameric PLN from cardiac membranes. Molecular dynamics simulations predicted different molecular contacts for peptides phosphorylated at Ser16 or Thr17 with the binding groove of 14-3-3, resulting in varied binding affinities. 14-3-3 binding protected either PLN phosphosite from dephosphorylation. ß-Adrenergic stimulation of isolated adult cardiomyocytes resulted in the membrane recruitment of endogenous 14-3-3. The exogenous addition of 14-3-3 to ß-adrenergic-stimulated cardiomyocytes led to prolonged SERCA activation, presumably because 14-3-3 protected PLN pentamers from dephosphorylation. Phosphorylation of Ser16 was disrupted by the cardiomyopathy-associated ∆Arg14 mutation, implying that phosphorylation of Thr17 by CaMKII may become crucial for 14-3-3 recruitment to ∆Arg14 PLN. Consistent with PLN acting as a dynamic hub in the control of Ca2+ handling, our results identify 14-3-3 binding to PLN as a contractility-augmenting mechanism.

Intracellular sodium elevation reprograms cardiac metabolism.

Intracellular Na elevation in the heart is a hallmark of pathologies where both acute and chronic metabolic remodelling occurs. Here, we assess whether acute (75 µM ouabain 100 nM blebbistatin) or chronic myocardial Nai load (PLM3SA mouse) are causally linked to metabolic remodelling and whether the failing heart shares a common Na-mediated metabolic 'fingerprint'. Control (PLMWT), transgenic (PLM3SA), ouabain-treated and hypertrophied Langendorff-perfused mouse hearts are studied by 23Na, 31P, 13C NMR followed by 1H-NMR metabolomic profiling. Elevated Nai leads to common adaptive metabolic alterations preceding energetic impairment: a switch from fatty acid to carbohydrate metabolism and changes in steady-state metabolite concentrations (glycolytic, anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 ameliorates the metabolic changes. In silico modelling indicates altered metabolic fluxes (Krebs cycle, fatty acid, carbohydrate, amino acid metabolism). Prevention of Nai overload or inhibition of Na/Camito may be a new approach to ameliorate metabolic dysregulation in heart failure.

Control of protein palmitoylation by regulating substrate recruitment to a zDHHC-protein acyltransferase.

Although palmitoylation regulates numerous cellular processes, as yet efforts to manipulate this post-translational modification for therapeutic gain have proved unsuccessful. The Na-pump accessory sub-unit phospholemman (PLM) is palmitoylated by zDHHC5. Here, we show that PLM palmitoylation is facilitated by recruitment of the Na-pump α sub-unit to a specific site on zDHHC5 that contains a juxtamembrane amphipathic helix. Site-specific palmitoylation and GlcNAcylation of this helix increased binding between the Na-pump and zDHHC5, promoting PLM palmitoylation. In contrast, disruption of the zDHHC5-Na-pump interaction with a cell penetrating peptide reduced PLM palmitoylation. Our results suggest that by manipulating the recruitment of specific substrates to particular zDHHC-palmitoyl acyl transferases, the palmitoylation status of individual proteins can be selectively altered, thus opening the door to the development of molecular modulators of protein palmitoylation for the treatment of disease.