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.

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.

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.

Real-time ex vivo perfusion of human lymph nodes invaded by cancer (REPLICANT): a feasibility study.

Understanding how breast cancer (BC) grows in axillary lymph nodes (ALNs), and refining how therapies might halt that process, is clinically important. However, modelling the complex ALN microenvironment is difficult, and no human models exist at present. We harvested ALNs from ten BC patients, and perfused them at 37 °C ex vivo for up to 24 h. Controlled autologous testing showed that ALNs remain viable after 24 h of ex vivo perfusion: haematoxylin and eosin-stained histological appearance and proliferation (by Ki67 immunohistochemistry) did not change significantly over time for any perfused ALN compared with a control from time-point zero. Furthermore, targeted gene expression analysis (NanoString PanCancer IO360 panel) showed that only 21/750 genes were differentially expressed between control and perfused ALNs (|log2 FC| > 1 and q < 0.1): none were involved in apoptosis and metabolism, but rather all 21 genes were involved in immune function and angiogenesis. During perfusion, tissue acid-base balance remained stable. Interestingly, the flow rate increased (p < 0.001) in cancer-replaced (i.e. metastasis occupied more than 90% of the surface area on multiple levels) compared to cancer-free nodes (i.e. nodes with no metastasis on multiple sections). CXCL11 transcripts were significantly more abundant in cancer-replaced nodes, while CXCL12 transcripts were significantly more abundant in cancer-free nodes. These cytokines were also detected in the circulating perfusate. Monoclonal antibodies (nivolumab and trastuzumab) were administered into a further three ALNs to confirm perfusion efficacy. These drugs saturated the nodes; nivolumab even induced cancer cell death. Normothermic ALN perfusion is not only feasible but sustains the tumour microenvironment ex vivo for scientific investigation. This model could facilitate the identification of actionable immuno-oncology targets. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.

Ion-Channel modulator TH1177 reduces glomerular injury and serum creatinine in chronic mesangial proliferative disease in rats.

BACKGROUND: T-type calcium channels (TTCC) are involved in mesangial cell proliferation. In acute thy-1 nephritis in the rat TTCC inhibition reduces glomerular damage and cell proliferation. This work is extended here by a study of the non-selective TTCC inhibitor TH1177 in a chronic model of proliferative glomerulonephritis (GN) including late treatment starting after the initial inflammation has resolved. The objective was to determine the effects of TH1177 in a model of chronic mesangioproliferative renal disease. METHODS: Chronic GN was induced in WKY rats by unilateral nephrectomy (day - 7) followed by day 0 injection of Ox7 thy-1 mAb. Treatment with TH1177 (10-20 mg/Kg daily IP) was started on day 2 (early treatment) or on day 14 (late treatment) and compared to vehicle-treated controls until sacrifice at day 42. Glomerular disease was assessed with a damage score, fibrosis assay, cellular counts and renal function measured by serum creatinine. RESULTS: Treatment with TH11777 was associated with reduced serum creatinine, less glomerular damage, reduced fibrosis and reduced glomerular cellularity. The results for early and late TH1177 treatments were essentially the same and differed significantly from vehicle. CONCLUSIONS: The ion-channel modulator TH1177 is capable of improving glomerular outcome in chronic rat GN even when treatment starts 14 days after initiation of the disease. These data are discussed in the context of the possible targets of TH1177 including TTCC, TRP family, Stim/Orai group and other cation channels. The work supports the use of genetic models to examine the roles of individual cation channels in progressive glomerulonephritis to further define the targets of TH1177.

Ablation of Toll-like receptor 9 attenuates myocardial ischemia/reperfusion injury in mice.

In myocardial ischemia/reperfusion injury, the innate immune and subsequent inflammatory responses play a crucial role in the extension of myocardial damage. Toll-like receptor 9 (TLR9) is a critical receptor for recognizing unmethylated CpG motifs that mitochondria contain in their DNA, and induces inflammatory responses. The aim of this study was to elucidate the role of TLR9 in myocardial ischemia/reperfusion injury. Isolated hearts from TLR9-deficient and control wild-type mice were subjected to 35 min of global ischemia, followed by 60 min of reperfusion with Langendorff apparatus. Furthermore, wild-type mouse hearts were infused with DNase I and subjected to ischemia/reperfusion. Ablation of TLR9-mediated signaling pathway attenuates myocardial ischemia/reperfusion injury and inflammatory responses, and digestion of extracellular mitochondrial DNA released from the infarct heart partially improved myocardial ischemia/reperfusion injury with no effect on inflammatory responses. TLR9 could be a therapeutic target to reduce myocardial ischemia/reperfusion injury.

Autonomic conflict exacerbates long QT associated ventricular arrhythmias.

This study tested the hypothesis that concomitant sympathetic and parasympathetic stimulation ("autonomic conflict") may act as a trigger for arrhythmias in long QT syndrome (LQTS). Studies were performed in isolated innervated rabbit hearts treated with clofilium (100 nmol/L); a potassium channel blocker. The influence of vagus nerve stimulation (VNS) on spontaneous ventricular arrhythmias was assessed in the absence/presence of sustained noradrenaline perfusion (100 nmol/L) and with sudden adrenergic stress (injections of noradrenaline into the perfusion line). Hearts were instrumented for a pseudo-electrocardiogram and monophasic action potential recordings. VNS, which slows heart rate, was associated with a stimulation frequency-dependent incidence of spontaneous early after-depolarisations (EADs) and ventricular tachycardia (VT), best predicted by the duration of the electrocardiographic T-wave and by triangulation of the ventricular action potential. In the presence of sustained (steady-state) noradrenaline perfusion, the incidence of EADs and VT with VNS was decreased from 73/55% to 45/27%, respectively. However, sudden adrenergic stress, imposed during periods of sustained VNS, was associated with a transient increase in the incidence of severity of observed arrhythmias, as indicated by an increase in the average arrhythmias score (1.6 ±â€¯0.4 vs. 2.1 ±â€¯0.7, p = .01). Analysis of electrophysiological parameters suggests that sudden adrenergic stress is associated with a transient prolongation, and increased triangulation, of the ventricular action potential, which may predispose to triggered activity. This study demonstrates that autonomic conflict is a pro-arrhythmic stimulus in LQTS. However, combined adrenergic and parasympathetic stimulation has a complex relationship with arrhythmogenicity, with differences in the effects of steady-state adrenergic activation vs. sudden adrenergic stress.

Is there a causal link between intracellular Na elevation and metabolic remodelling in cardiac hypertrophy?

Alterations in excitation-contraction coupling and elevated intracellular sodium (Nai) are hallmarks of pathological cardiac remodelling that underline contractile dysfunction. In addition, changes in cardiac metabolism are observed in cardiac hypertrophy and heart failure (HF) that lead to a mismatch in ATP supply and demand, contributing to poor prognosis. A link between Nai and altered metabolism has been proposed but is not well understood. Many mitochondrial enzymes are stimulated by mitochondrial calcium (Camito) during contraction, thereby sustaining production of reducing equivalents to maintain ATP supply. This stimulation is thought to be perturbed when cytosolic Nai is high due to increased Camito efflux, potentially compromising ATPmito production and leading to metabolic dysregulation. Increased Nai has been previously shown to affect Camito; however, whether Nai elevation plays a causative role in energetic mismatching in the hypertrophied and failing heart remains unknown. In this review, we discuss the relationship between elevated Nai, NaK ATPase dysregulation and the metabolic phenotype in the contexts of pathological hypertrophy and HF and their link to metabolic flexibility, capacity (reserve) and efficiency that are governed by intracellular ion homeostasis. The development of non-invasive analytical techniques using nuclear magnetic resonance able to probe metabolism in situ in the functioning heart will enable a better understanding of the underlying mechanisms of Nai overload in cardiac pathophysiology. They will lead to novel insights that help to explain the metabolic contribution towards these diseases, the incomplete rescue observed with current therapies and a rationale for future energy-targeted therapies.

Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy.

Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACß > PP2ACα > PP4C > PP6C), NRVM (PP2ACß > PP2ACα = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACα > PP2ACß > PP6C > PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACα, PP2ACß, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (γH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of γH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium.

Substrate recognition by the cell surface palmitoyl transferase DHHC5.

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/Discs-large/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.

Increased oxidative metabolism following hypoxia in the type 2 diabetic heart, despite normal hypoxia signalling and metabolic adaptation.

KEY POINTS: Adaptation to hypoxia makes the heart more oxygen efficient, by metabolising more glucose. In contrast, type 2 diabetes makes the heart metabolise more fatty acids. Diabetes increases the chances of the heart being exposed to hypoxia, but whether the diabetic heart can adapt and respond is unknown. In this study we show that diabetic hearts retain the ability to adapt their metabolism in response to hypoxia, with functional hypoxia signalling pathways. However, the hypoxia-induced changes in metabolism are additive to abnormal baseline metabolism, resulting in hypoxic diabetic hearts metabolising more fat and less glucose than controls. This stops the diabetic heart being able to recover its function when stressed. These results demonstrate that the diabetic heart retains metabolic flexibility to adapt to hypoxia, but is hindered by the baseline effects of the disease. This increases our understanding of how the diabetic heart is affected by hypoxia-associated complications of the disease. ABSTRACT: Hypoxia activates the hypoxia-inducible factor (HIF), promoting glycolysis and suppressing mitochondrial respiration. In the type 2 diabetic heart, glycolysis is suppressed whereas fatty acid metabolism is promoted. The diabetic heart experiences chronic hypoxia as a consequence of increased obstructive sleep apnoea and cardiovascular disease. Given the opposing metabolic effects of hypoxia and diabetes, we questioned whether diabetes affects cardiac metabolic adaptation to hypoxia. Control and type 2 diabetic rats were housed for 3 weeks in normoxia or 11% oxygen. Metabolism and function were measured in the isolated perfused heart using radiolabelled substrates. Following chronic hypoxia, both control and diabetic hearts upregulated glycolysis, lactate efflux and glycogen content and decreased fatty acid oxidation rates, with similar activation of HIF signalling pathways. However, hypoxia-induced changes were superimposed on diabetic hearts that were metabolically abnormal in normoxia, resulting in glycolytic rates 30% lower, and fatty acid oxidation 36% higher, in hypoxic diabetic hearts than hypoxic controls. Peroxisome proliferator-activated receptor α target proteins were suppressed by hypoxia, but activated by diabetes. Mitochondrial respiration in diabetic hearts was divergently activated following hypoxia compared with controls. These differences in metabolism were associated with decreased contractile recovery of the hypoxic diabetic heart following an acute hypoxic insult. In conclusion, type 2 diabetic hearts retain metabolic flexibility to adapt to hypoxia, with normal HIF signalling pathways. However, they are more dependent on oxidative metabolism following hypoxia due to abnormal normoxic metabolism, which was associated with a functional deficit in response to stress.

Intensive training and reduced volume increases muscle FXYD1 expression and phosphorylation at rest and during exercise in athletes.

The present study examined the effect of intensive training in combination with marked reduction in training volume on phospholemman (FXYD1) expression and phosphorylation at rest and during exercise. Eight well-trained cyclists replaced their regular training with speed-endurance training (10-12 × âˆ¼30-s sprints) two or three times per week and aerobic high-intensity training (4-5 × 3-4 min at 90-95% of peak aerobic power output) 1-2 times per week for 7 wk and reduced the training volume by 70%. Muscle biopsies were obtained before and during a repeated high-intensity exercise protocol, and protein expression and phosphorylation were determined by Western blot analysis. Expression of FXYD1 (30%), actin (40%), mammalian target of rapamycin (mTOR) (12%), phospholamban (PLN) (16%), and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) γ/δ (25%) was higher (P < 0.05) than before the training intervention. In addition, after the intervention, nonspecific FXYD1 phosphorylation was higher (P < 0.05) at rest and during exercise, mainly achieved by an increased FXYD1 Ser-68 phosphorylation, compared with before the intervention. CaMKII, Thr-287, and eukaryotic elongation factor 2 Thr-56 phosphorylation at rest and during exercise, overall PKCα/ß, Thr-638/641, and mTOR Ser-2448 phosphorylation during repeated intense exercise as well as resting PLN Thr-17 phosphorylation were also higher (P < 0.05) compared with before the intervention period. Thus, a period of high-intensity training with reduced training volume increases expression and phosphorylation levels of FXYD1, which may affect Na(+)/K(+) pump activity and muscle K(+) homeostasis during intense exercise. Furthermore, higher expression of CaMKII and PLN, as well as increased phosphorylation of CaMKII Thr-287 may have improved intracellular Ca(2+) handling.

Is rate-pressure product of any use in the isolated rat heart? Assessing cardiac 'effort' and oxygen consumption in the Langendorff-perfused heart.

NEW FINDINGS: What is the central question of this study? Rate-pressure product (RPP) is commonly used as an index of cardiac 'effort'. In canine and human hearts (which have a positive force-frequency relationship), RPP is linearly correlated with oxygen consumption and has therefore been widely adopted as a species-independent index of cardiac work. However, given that isolated rodent hearts demonstrate a negative force-frequency relationship, its use in this model requires validation. What is the main finding and its importance? Despite its widespread use, RPP is not correlated with oxygen consumption (or cardiac 'effort') in the Langendorff-perfused isolated rat heart. This lack of correlation was also evident when perfusions included a range of metabolic substrates, insulin or ß-adrenoceptor stimulation. Langendorff perfusion of hearts isolated from rats and mice has been used extensively for physiological, pharmacological and biochemical studies. The ability to phenotype these hearts reliably is, therefore, essential. One of the commonly used indices of function is rate-pressure product (RPP); a rather ill-defined index of 'work' or, more correctly, 'effort'. Rate-pressure product, as originally described in dog or human hearts, was shown to be correlated with myocardial oxygen consumption (MV̇O2). Despite its widespread use, the application of this index to rat or mouse hearts (which, unlike the dog or human, have a negative force-frequency relationship) has not been characterized. The aim of this study was to examine the relationship between RPP and MV̇O2 in Langendorff-perfused rat hearts. Paced hearts (300-750 beats min(-1)) were perfused either with Krebs-Henseleit (KH) buffer (11 mm glucose) or with buffer supplemented with metabolic substrates and insulin. The arteriovenous oxygen consumption (MV̇O2) was recorded. Metabolic status was assessed using (31) P magnetic resonance spectroscopy and lactate efflux. Experiments were repeated in the presence of isoprenaline and in unpaced hearts where heart rate was increased by cumulative isoprenaline challenge. In KH buffer-perfused hearts, MV̇O2 increased with increasing heart rate, but given that left ventricular developed pressure decreased with increases in rate, RPP was not correlated with MV̇O2, lactate production or phosphocreatine/ATP ratio. Although the provision of substrates or ß-adrenoceptor stimulation changed the shape of the RPP-MV̇O2 relationship, neither intervention resulted in a positive correlation between RPP and oxygen consumption. Rate-pressure product is therefore an unreliable index of oxygen consumption or 'cardiac effort' in the isolated rat heart.

Geometrical considerations in cardiac electrophysiology and arrhythmogenesis.

The rate of repolarization (RRepol) and so the duration of the cardiac action potential are determined by the balance of inward and outward currents across the cardiac membrane (net ionic current). Plotting action potential duration (APD) as a function of the RRepol reveals an inverse non-linear relationship, arising from the geometric association between these two factors. From the RRepol-APD relationship, it can be observed that a longer action potential will exhibit a greater propensity to shorten, or prolong, for a given change in the RRepol (i.e. net ionic current), when compared with one that is initially shorter. This observation has recently been used to explain why so many interventions that prolong the action potential exert a greater effect at slow rates (reverse rate-dependence). In this article, we will discuss the broader implications of this simple principle and examine how common experimental observations on the electrical behaviour of the myocardium may be explained in terms of the RRepol-APD relationship. An argument is made, with supporting published evidence, that the non-linear relationship between the RRepol and APD is a fundamental, and largely overlooked, property of the myocardium. The RRepol-APD relationship appears to explain why interventions and disease with seemingly disparate mechanisms of action have similar electrophysiological consequences. Furthermore, the RRepol-APD relationship predicts that prolongation of the action potential, by slowing repolarization, will promote conditions of dynamic electrical instability, exacerbating several electrophysiological phenomena associated with arrhythmogenesis, namely, the rate dependence of dispersion of repolarization, APD restitution, and electrical alternans.

Vagal modulation of dispersion of repolarisation in the rabbit heart.

Bradycardia is a risk factor for arrhythmia in several disorders, including acquired long QT syndrome, whereby slowing of heart rate facilitates ectopic activity and torsade de pointes. Slowing of rate is associated with an increase in the spatiotemporal dispersion of ventricular repolarisation (DOR) in electrically paced hearts. However, there have been conflicting reports on the effect of the vagus nerve, which mediates the physiological slowing of heart rate, on DOR. The aim of this study was to investigate the effect of vagus nerve stimulation (VNS) on the heterogeneity of ventricular repolarisation, as assessed using the T-wave peak-to-end interval (TpTe) and monophasic action potentials (MAPs), in normal hearts and in hearts with acquired long QT syndrome. Experiments were conducted in an isolated innervated rabbit heart preparation. The effect of VNS on cardiac electrograms, MAPs and ventricular function was investigated in control and following perfusion of E4031 (50nmol/L); an inhibitor of the rapid delayed rectifying potassium current. VNS was associated with a stimulation frequency-dependent bradycardia (-74±6 [10Hz] vs. -25±4bpm [2Hz], P<0.05). VNS prolonged the TpTe interval (29±1 vs. 20±2ms, P<0.05) and increased T-wave amplitude (1.7±0.3 vs. 0.7±0.2mV, P<0.05) in association with increased apicobasal DOR. The effects of VNS were exacerbated by E4031, with a greater prolongation of TpTe (ΔTpTe 42±6 vs. 8±1ms, P<0.05) and max-min apicobasal time of repolarisation (TRepol; 45±11 vs. 5±2ms, P<0.05). ΔTpTe was strongly correlated with the Δmax-minTRepol (r(2)=0.87, P<0.05) and TpTe was prolonged to a greater degree in hearts exhibiting spontaneous ventricular tachyarrhythmia. Rate dependent differences in regional action potential prolongation were replicated using computational models. These data demonstrate that VNS increases ventricular DOR and that the effects of the vagus nerve on ventricular electrophysiology are exacerbated in pharmacologically acquired long QT syndrome.

Multiple quantum filtered (23)Na NMR in the Langendorff perfused mouse heart: Ratio of triple/double quantum filtered signals correlates with [Na]i.

We investigate the potential of multiple quantum filtered (MQF) (23)Na NMR to probe intracellular [Na]i in the Langendorff perfused mouse heart. In the presence of Tm(DOTP) shift reagent the triple quantum filtered (TQF) signal originated largely from the intracellular sodium pool with a 32±6% contribution of the total TQF signal arising from extracellular sodium, whilst the rank 2 double-quantum filtered signal (DQF), acquired with a 54.7° flip-angle pulse, originated exclusively from the extracellular sodium pool. Given the different cellular origins of the (23)Na MQF signals we propose that the TQF/DQF ratio can be used as a semi-quantitative measure of [Na]i in the mouse heart. We demonstrate a good correlation of this ratio with [Na]i measured with shift reagent at baseline and under conditions of elevated [Na]i. We compare the measurements of [Na]i using both shift reagent and TQF/DQF ratio in a cohort of wild type mouse hearts and in a transgenic PLM(3SA) mouse expressing a non-phosphorylatable form of phospholemman, showing a modest but measurable elevation of baseline [Na]i. MQF filtered (23)Na NMR is a potentially useful tool for studying normal and pathophysiological changes in [Na]i, particularly in transgenic mouse models with altered Na regulation.

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.