Langendorff-perfused Mouse Hearts Research Articles

Efficient termination of cardiac arrhythmias using optogenetic resonant feedback pacing.

Malignant cardiac tachyarrhythmias are associated with complex spatiotemporal excitation of the heart. The termination of these life-threatening arrhythmias requires high-energy electrical shocks that have significant side effects, including tissue damage, excruciating pain, and worsening prognosis. This significant medical need has motivated the search for alternative approaches that mitigate the side effects, based on a comprehensive understanding of the nonlinear dynamics of the heart. Cardiac optogenetics enables the manipulation of cellular function using light, enhancing our understanding of nonlinear cardiac function and control. Here, we investigate the efficacy of optically resonant feedback pacing (ORFP) to terminate ventricular tachyarrhythmias using numerical simulations and experiments in transgenic Langendorff-perfused mouse hearts. We show that ORFP outperforms the termination efficacy of the optical single-pulse (OSP) approach. When using ORFP, the total energy required for arrhythmia termination, i.e., the energy summed over all pulses in the sequence, is 1 mJ. With a success rate of 50%, the energy per pulse is 40 times lower than with OSP with a pulse duration of 10 ms. We demonstrate that even at light intensities below the excitation threshold, ORFP enables the termination of arrhythmias by spatiotemporal modulation of excitability inducing spiral wave drift.

Dual-Dye Optical Mapping of Hearts from RyR2 R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia.

The pro-arrhythmic cardiac disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests as polymorphic ventricular tachycardia episodes following physical activity, stress, or catecholamine challenge, which can deteriorate into potentially fatal ventricular fibrillation. The mouse heart is a widespread species for modeling inherited cardiac arrhythmic diseases, including CPVT. Simultaneous optical mapping of transmembrane potential (Vm) and calcium transients (CaT) from Langendorff-perfused mouse hearts has the potential to elucidate mechanisms underlying arrhythmogenesis. Compared with the cellular level investigation, the optical mapping technique can test some electrophysiological parameters, such as the determination of activation, conduction velocity, action potential duration, and CaT duration. This paper presents the instrumentation setup and experimental procedure for high-throughput optical mapping of CaT and Vm in murine wild-type and heterozygous RyR2-R2474S/+ hearts, combined with programmed electrical pacing before and during the isoproterenol challenge. This approach has demonstrated a feasible and reliable method for mechanistically studying CPVT disease in an ex vivo mouse heart preparation.

Paracrine cardioprotective signaling by red blood cells through hypoxia-mediated activation of soluble guanylate cyclase and release of cyclic guanosine monophosphate

Abstract Background Red blood cells (RBCs) are known to regulate cardiovascular function under hypoxic conditions and to mediate cardioprotection via nitric oxide (NO)-like bioactivity. However, the molecular signalling behind this effect and the identity of any mediator released by the RBCs remain unknown. Previous studies revealed that NO activates soluble guanylate cyclase (sGC) and increases formation of the second messenger cyclic guanosine monophosphate (cGMP) in RBCs. The functional role of this signalling in RBCs during hypoxia/ischemia is still unclear. Aim To determine the functional role of the NO-sGC-cGMP signalling pathway in RBCs during hypoxia and myocardial ischemia. Method RBCs collected from wild-type and sGC knockout mice were exposed to 1% hypoxia or normoxia for 1 h. The RBCs or the extracellular supernatant were then administered to isolated Langendorff-perfused mouse hearts subjected to 40 min global ischemia and 60 min reperfusion. Afterwards, left ventricular developed pressure and infarct size were determined. Additional groups of mice and patients with mild hypertension were given oral nitrate (10mM in their drinking water) before the collection of RBCs to further increase NO signalling. Result Administration of RBCs or the extracellular supernatant collected from wild-type mouse RBCs exposed to hypoxia to isolated hearts subjected to ischemia-reperfusion improved post-ischemic cardiac function and reduced infarct size (Fig. 1A and B). By contrast, supernatant collected from hypoxic RBCs of sGC knockout mice failed to induce cardioprotection (Fig. 1C). The cardioprotection induced by hypoxic RBCs of wild-type mice was blocked by MK571, an inhibitor of cyclic nucleotide transport (Fig. 1D). Hypoxia increased extracellular export of hate cGMP from mouse RBCs (Fig. 1E), and exogenous cGMP resulted in similar cardioprotection induced by the supernatant. The cardioprotective effect of RBCs was blocked by an inhibitor of cardiac cGMP-dependent protein kinase G (PKG) (Fig. 1F) and was associated with increased cardiac PKG-dependent phosphorylation of vasodilator-stimulated phosphoprotein. Oral administration of nitrate to mice for 4 weeks to increase NO bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. RBCs collected from patients randomized to a 5-week nitrate-rich diet induced cardioprotection in the isolated rat heart via an effect dependent on sGC activation in the RBCs (Fig 2). Conclusion RBCs generate and export cGMP as a physiological response to hypoxia mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention with nitrate suggesting preventive and therapeutic opportunities in ischemic heart disease.Figure 1Figure 2

Caveolae-associated cAMP/Ca2+-mediated mechano-chemical signal transduction in mouse atrial myocytes

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca2+ transients and caused a rise in Ca2+ spark activity. Similar changes in Ca2+ signaling were observed after chemical (methyl-β-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser2030 and Ser2808). Caveolae-mediated stimulatory effects on Ca2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes.

Naringin exerts antiarrhythmic effects by inhibiting channel currents in mouse cardiomyocytes.

Naringin, a flavonoid extracted from citrus plants, has a variety of biological effects. Studies have shown that increasing the consumption of flavonoid-rich foods can reduce the incidence of cardiac arrhythmia. Naringin has been reported to have beneficial cardiovascular effects and thus can be used to prevent cardiovascular diseases, but the electrophysiological mechanism through which it prevents arrhythmias has not been elucidated. This study was conducted to investigate the effect of naringin on the transmembrane ion channel currents in mouse ventricular myocytes and the antiarrhythmic effect of this compound on Langendorff-perfused mouse hearts. Action potentials (APs) and ionic currents were recorded in isolated ventricular myocytes using the whole-cell patch-clamp technique. Anemone toxin II (ATX II) and CaCl2 were used to induce early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs), respectively. Electrocardiogram (ECG) recordings were conducted in Langendorff-perfused mouse hearts with a BL-420F biological signal acquisition and analysis system. At the cellular level, naringin shortened the action potential duration (APD) of ventricular myocytes and decreased the maximum depolarization velocity (Vmax) of APs.Naringin inhibited the L-type calcium current (ICa.L) and ATX II enhanced the late sodium current (INa.L) in a concentration-dependent manner with IC50 values of 508.5μmol/L (n=9) and 311.6μmol/L (n=10), respectively. In addition, naringin also inhibited the peak sodium current (INa·P) and delayed the rectifier potassium current (IK) and the transient outward potassium current (Ito). Moreover, naringin reduced ATX II-induced APD prolongation and EADs and had a significant inhibitory effect on CaCl2-induced DADs as well. At the organ level, naringin reduced the incidence of ventricular tachycardia (VT) and ventricular fibrillation (VF) induced by ATX II and shortened the duration of both in isolated hearts. Naringin can inhibit the occurrence of EADs and DADs at the cellular level; furthermore, it can inhibit INa.L, ICa.L, INa·P, IK, and Ito in ventricular myocytes. Naringin also inhibits arrhythmias induced by ATX II in hearts. By investigating naringin with this electrophysiological method for the first time, we determined that this flavonoid may be a multichannel blocker with antiarrhythmic effects.

Assessment of Tissue Viability by Functional Imaging of Membrane Potential.

Electrical activity plays a key role in physiology, in particular for signaling and coordination. Cellular electrophysiology is often studied with micropipette-based techniques such as patch clamp and sharp electrodes, but for measurements at the tissue or organ scale, more integrated approaches are needed. Epifluorescence imaging of voltage-sensitive dyes ("optical mapping") is a tissue non-destructive approach to obtain insight into electrophysiology with high spatiotemporal resolution. Optical mapping has primarily been applied to excitable organs, especially the heart and brain. Action potential durations, conduction patterns, and conduction velocities can be determined from the recordings, providing information about electrophysiological mechanisms, including factors such as effects of pharmacological interventions, ion channel mutations, or tissue remodeling. Here, we describe the process for optical mapping of Langendorff-perfused mouse hearts, highlighting potential issues and key considerations.

Heterogeneities in Ventricular Conduction Following Treatment with Heptanol: A Multi-Electrode Array Study in Langendorff-Perfused Mouse Hearts.

Background: Previous studies have associated slowed ventricular conduction with the arrhythmogenesis mediated by the gap junction and sodium channel inhibitor heptanol in mouse hearts. However, they did not study the propagation patterns that might contribute to the arrhythmic substrate. This study used a multi-electrode array mapping technique to further investigate different conduction abnormalities in Langendorff-perfused mouse hearts exposed to 0.1 or 2 mM heptanol. Methods: Recordings were made from the left ventricular epicardium using multi-electrode arrays in spontaneously beating hearts during right ventricular 8 Hz pacing or S1S2 pacing. Results: In spontaneously beating hearts, heptanol at 0.1 and 2 mM significantly reduced the heart rate from 314 ± 25 to 189 ± 24 and 157 ± 7 bpm, respectively (ANOVA, p < 0.05 and p < 0.001). During regular 8 Hz pacing, the mean LATs were increased by 0.1 and 2 mM heptanol from 7.1 ± 2.2 ms to 19.9 ± 5.0 ms (p < 0.05) and 18.4 ± 5.7 ms (p < 0.05). The standard deviation of the mean LATs was increased from 2.5 ± 0.8 ms to 10.3 ± 4.0 ms and 8.0 ± 2.5 ms (p < 0.05), and the median of phase differences was increased from 1.7 ± 1.1 ms to 13.9 ± 7.8 ms and 12.1 ± 5.0 ms by 0.1 and 2 mM heptanol (p < 0.05). P5 took a value of 0.2 ± 0.1 ms and was not significantly altered by heptanol at 0.1 or 2 mM (1.1 ± 0.9 ms and 0.9 ± 0.5 ms, p > 0.05). P50 was increased from 7.3 ± 2.7 ms to 24.0 ± 12.0 ms by 0.1 mM heptanol and then to 22.5 ± 7.5 ms by 2 mM heptanol (p < 0.05). P95 was increased from 1.7 ± 1.1 ms to 13.9 ± 7.8 ms by 0.1 mM heptanol and to 12.1 ± 5.0 ms by 2 mM heptanol (p < 0.05). These changes led to increases in the absolute inhomogeneity in conduction (P5–95) from 7.1 ± 2.6 ms to 31.4 ± 11.3 ms, 2 mM: 21.6 ± 7.2 ms, respectively (p < 0.05). The inhomogeneity index (P5–95/P50) was significantly reduced from 3.7 ± 1.2 to 3.1 ± 0.8 by 0.1 mM and then to 3.3 ± 0.9 by 2 mM heptanol (p < 0.05). Conclusion: Increased activation latencies, reduced CVs, and the increased inhomogeneity index of conduction were associated with both spontaneous and induced ventricular arrhythmias.

Ageing Increases Cardiac Electrical Remodelling in Rats and Mice via NOX4/ROS/CaMKII-Mediated Calcium Signalling.

Objective Ageing is one of the risk factors associated with cardiovascular diseases including cardiac arrhythmias and heart failure. Ageing-related cardiac dysfunction involves a complicated pathophysiological progress. Abnormal membrane voltage and Ca2+ dynamics in aged cardiomyocytes contribute to ageing-related arrhythmias. However, its underlying mechanisms have not been well clarified. Methods Young and old rats or mice were included in this study. Cardiac electrophysiological properties and functions were assessed by ECG, echocardiography, and ex vivo heart voltage and Ca2+ optical mapping. Proteomics, phosphor-proteomics, Western blotting, Masson staining, and ROS measurement were used to investigate the underlying mechanisms. Results Ageing increased the incidence of cardiac hypertrophy and fibrosis in rats. Moreover, ageing increased the occurrence of ventricular tachycardia or ventricular fibrillation induced by rapid pacing and during isoprenaline (ISO) (1 mg/kg i.p.) challenge in mice in vivo. Optical mapping with dual dyes (membrane voltage (Vm) dye and intracellular Ca2+ dye) simultaneously recording revealed that ageing increased the action potential duration (APD) and Ca2+ transient duration (CaTD) and slowed the ventricular conduction with the Langendorff-perfused mouse heart. More importantly, ageing increased the ISO-induced (1 μM) changes of APD (ΔAPD80) and CaTD (ΔCaTD50). Ageing also delayed the decay of Ca2+ transient by extending the decay time constant from 30% to 90% (τ30−90). In addition, ageing decreased the Vm/Ca2+ latency which represented the coupling of Vm/Ca2+ including between the midpoint of AP depolarization and Ca2+ upstroke, peak transmembrane voltage and peak cytosolic calcium, and time to 50% voltage repolarization and extrusion of cytosolic calcium. Optical mapping also revealed that ageing increased the ISO-induced arrhythmia incidence and occurrence of the excitation rotor. Proteomics and phosphor-proteomics assays from rat hearts demonstrated ageing-induced protein and phosphor-protein changes, suggesting that CaMKII was involved in ageing-induced change. Ageing increased the level of ROS and the expression of NOX4, oxidative CaMKII (ox-CaMKII), phosphorated CaMKII (p-CaMKII), and periostin. Conclusion Ageing accelerates cardiac remodelling and increases the susceptibility to ventricular arrhythmias through NOX4/ROS/CaMKII pathway-mediated abnormal membrane voltage and intracellular Ca2+ handling and Vm/Ca2+ coupling.

Abstract 11137: Caveolin 1 Ameliorated Coronary Flow Responsible for Cardiac Function Improvement After Cardiac Ischemia Reperfusion

Introduction: Caveolin-1 (Cav-1) is the main structural protein of caveolae, which is dominantly expressed in vascular tissue. It associates with vascular homeostasis by regulating vascular signals. However, Cav-1 KO aggravated the cardiac dysfunction of systolic and diastolic function after cardiac ischemia reperfusion. Injection of Cav-1 peptides successfully preserved left ventricular function post-ischemic reperfusion through the increase in nitric oxide (NO). Hypothesis and Methods: To evaluate the role of Cav-1 in cardiac function, we assessed cardiac function, heart rate and coronary artery blood flow through global ischemia and reperfusion using Langendorff-perfused mouse heart model. Results: In the final analysis of 15 mice, 7 were wild type (WT) and 8 were Cav-1 KO at the age of 12-23 weeks. There are significantly differences in baseline of Left ventricular pressure (LVP), heart rate (HR), max and minimum dp/dp before and after ischemia in each mouse group. We further concluded that the markedly decrease of Coronary artery flow (CF) is the reason for the inhibition of cardiac diastolic function, while heart rate (HR) did not show obvious difference in WT and Cav-1 KO mice. Cav-1 deficiency inhibited the coronary artery flow after ischemia reperfusion and led to aggravation of cardiac dysfunction. Conclusions: As further experiments are needed, both cardiac functions and coronary flow in Cav-1 KO could be impaired. More experiments will be performed and results will be updated.

Wide Area Uniform Illumination Scheme Using LED Matrix for Optogenetic Cardiac Pacing

Control of heart rhythm is vital in the case of arrhythmia. Cardiac optogenetics is a promising technique to replace electrical stimulation in a next generation of pacemakers and defibrillators. Therefore, further research towards optimizing light delivery methods is essential. A major requirement is the uniform stimulation of all cells in the area of interest while reducing side effects such as photodamage. Here, a 2 × 2 blue (470 nm, InGaN-based) light-emitting diode (LED) optoelectronic module for uniform ex vivo cardiac muscle illumination is demonstrated. It satisfies two important requirements in optogenetics, which are high illumination homogeneity and high irradiance. CCD camera images show an average 90% homogeneity over the central illumination area of close to 38 mm2 at 1 cm distance from the light source. The module is used to perform physiological experiments on channelrhodopsin 2-expressing Langendorff-perfused mouse hearts. Successful ventricular pacing is obtained for an optical power density threshold below 2 mW/mm2 with light pulses as short as 1 ms. For 10 ms long pulses, the threshold was below 0.2 mW/mm2. The large homogeneous illumination area enabled optogenetic pacing with less than half the optical power of previous attempts with smaller areas of 2 mm2 and thus, presumably, will result in less phototoxicity.

Only the NAD precursor nicotinamide riboside maintains cardioprotection under clinically relevant conditions, possibly through activation of glycolysis

Abstract Purpose Cardioprotective strategies against ischaemia-reperfusion injury (IRI) that remain effective in the clinical arena need to be developed. Here, we examined the efficacy of the cardioprotective compounds fingolimod (Fingo), empagliflozin (Empa), melatonin (Mela) and nicotinamide riboside (NR) in the presence of drugs routinely used clinically in IRI conditions such as cardiac by-pass surgery and PCI procedures (opiates, benzodiazepines, P2Y12 antagonist, propofol). Furthermore, we examine the mechanism of protection of effective compound(s). Methods Following pilot dose-response curves of each compound, cardioprotective efficacy of Fingo, Empa, Mela and NR in an in vivo rat model employing a clinically relevant anaesthetic background therapy (fentanyl-midazolam) were examined alone or in combination. Drugs were administered 30 min before 25 min left ascending coronary (LAD) ischaemia. Infarct size (IS) was determined following 2 h of reperfusion. Effective treatments were then tested in the presence of a P2Y12 antagonist (cangrelor) or propofol anaesthesia. Finally, the underlying metabolic mechanism of the efficient compound(s) was explored in an ex vivo Langendorff-perfused mouse heart model of cardiac IRI. Results We found that among these four compounds, only singular NR was able to reduce IS (30±14% vs 60±16%, P=0.009 vs control, Fig.1A) in vivo in the presence of clinically relevant background anaesthesia. NR still reduced IS in the presence of cangrelor (51±18% vs 71±4%, P=0.016 vs control, Fig.1B), but lost protection in the presence of propofol anaesthesia (62±16% vs 60±14%, P=0.839 vs control, Fig.1C). Furthermore, NR showed protection in the ex vivo model of IRI, where isolated hearts were perfused with glucose (G) and free fatty acid (FFA) palmitate (G+FFA): (IS%: 40±14% vs 64±12%, P=0.003 vs control; LDH release: 28.3±7.2 U/min/GWW vs 37.8±3.7 U/min/GWW, P=0.005 vs control; Rate-Pressure-Product (RPP) recovery (43±12% vs 31±8%, P=0.031 vs control, Fig.2A-C). However, NR's protection was lost when 1) glycolysis was by-passed, by replacing glucose in the perfusate with lactate (L) and pyruvate (P) (LP+FFA) or 2) glycolysis was overly activated by adding insulin to the perfusate (G+FFA+insulin) (Fig.2). Conclusion Our data suggests that NR is a promising cardioprotective agent to target cardiac ischaemia-reperfusion injury in clinical conditions employing opioid agonists, benzodiazepines and platelet P2Y12 inhibitors. This protection may, at least partly, be due to NR stimulation of cardiac glycolysis. Funding Acknowledgement Type of funding sources: None.

Abstract P311: Blocking Succinate Release Enhances Mitochondrial Reactive Oxygen Species Generation And Worsens Ischemia-reperfusion Injury

Succinate is a metabolite that plays a central role in ischemia-reperfusion (IR) injury,which is relevant to myocardial infarction (heart attack) and stroke. Succinateaccumulates during ischemia and is rapidly consumed at reperfusion driving reactiveoxygen species (ROS) generation at complex-I (Cx-I) and III of the mitochondrial electrontransport chain. This ROS production triggers cell-death, leading to tissue necrosis.Although succinate oxidation has been extensively studied and exploited as a noveltherapeutic target, only 1/3 of the succinate accumulated in ischemia is oxidized atreperfusion, with the remaining 2/3 being released from the cell via monocarboxylatetransporter 1 (MCT1). Extracellular succinate is thought to be pro-inflammatory, and ithas been proposed that preventing succinate release may be therapeutically beneficial.To determine the impact of preventing succinate release on IR injury, we comparedfunctional recovery (i.e. rate x pressure product, RPP) and infarction (i.e. tissue necrosis)of Langendorff perfused mouse hearts treated with an MCT1 inhibitor, AR-C155858,versus vehicle control. This revealed that succinate retention worsens IR injury (i.e.increased infarction and decreased functional recovery) likely due to increased ROS. Totest this hypothesis, we utilized a Langendorff apparatus positioned within aspectrofluorimeter, which permits real-time fluorescence measurements in beatingmouse hearts. Using the mitochondria targeted superoxide probe, MitoSOX red tomeasure ROS production at reperfusion + AR-C155858, demonstrated that succinateretention leads to enhanced mitochondrial ROS generation at the onset of reperfusion.Overall, these results suggest that inhibiting succinate release in the context of IR injurymay not be a viable therapeutic approach, regardless of any downstream anti-inflammatory effects.

The Cx43 Carboxyl-Terminal Mimetic Peptide αCT1 Protects Endothelial Barrier Function in a ZO1 Binding-Competent Manner.

The Cx43 carboxyl-terminus (CT) mimetic peptide, αCT1, originally designed to bind to Zonula Occludens 1 (ZO1) and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing (ECIS), a TRITC-dextran Transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, αCT1 was found to protect the endothelium from thrombin-induced breakdown in cell–cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular chiral orientation distribution. In line with observations of increased cortical F-actin, αCT1 upregulated cell–cell border localization of endothelial VE-cadherin, the tight junction protein Zonula Occludens 1 (ZO1), and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1 binding-incompetent variant of αCT1, αCT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of αCT1 in the treatment of vascular edema.

B-AB01-04 INTERACTION BETWEEN ISCHEMIA AND SYMPATHETIC ACTIVITY ON CARDIAC ELECTROPHYSIOLOGY AND ARRHYTHMIA IN THE INNERVATED LANGENDORFF-PERFUSED MOUSE HEART

Sympathetic activation of the heart plays a key role in the modulation of cardiac electrophysiology and arrhythmogenesis. It is generally believed that sympathetic activation is proarrhythmic in the setting of cardiac ischemia. However, few studies have looked at the dynamic effects of physiological sympathetic nerve activation on cardiac electrophysiological activities during ischemia. To investigate the effects of sympathetic activation in the innervated Langendorff-perfused mouse heart subjected to acute ischemia. The heart and posterior thoracic cavity from mice (C57Bl6, N=4) were dissected and perfused through the descending aorta for dual optical mapping of transmembrane potential (Vm) and intracellular Ca2+ to study the effects of sympathetic nerve stimulation (SNS). SNS, achieved through spinal cord stimulation at T1-T3, was performed before, during and immediately after 5 min of low-flow ischemia (10% of pre-ischemic flow rate). Early afterdepolarizations (EADs) and ventricular tachycardia (VT) occurred in all 4 animals within 5 min of ischemia and immediately upon reperfusion. Both SNS and ventricular pacing, during either ischemia or reperfusion, completely abolished EADs and VT induced by global low-flow ischemia and reperfusion (Fig). In the isolated innervated mouse heart, we showed that sympathetic stimulation protects against ischemia-induced cardiac arrhythmias potentially by over-driving ventricular ectopic activity.

Positive Inotropic Effects of ATP Released via the Maxi-Anion Channel in Langendorff-Perfused Mouse Hearts Subjected to Ischemia-Reperfusion.

The organic anion transporter SLCO2A1 constitutes an essential core component of the ATP-conductive large-conductance anion (Maxi-Cl) channel. Our previous experiments using Langendorff-perfused mouse hearts showed that the Maxi-Cl channel contributes largely to the release of ATP into the coronary effluent observed during 10-min reperfusion following a short period (6 min) of oxygen-glucose deprivation. The present study examined the effect of endogenous ATP released via Maxi-Cl channels on the left ventricular contractile function of Langendorff-perfused mouse hearts, using a fluid-filled balloon connected to a pressure transducer. After the initial 30-min stabilization period, the heart was then perfused with oxygen-glucose-deprived Tyrode solution for 6 min, which was followed by a 10-min perfusion with oxygenated normal Tyrode solution in the absence and presence of an ATP-hydrolyzing enzyme, apyrase, and/or an adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). In the absence of apyrase and DPCPX, the left ventricular developed pressure (LVDP) decreased from a baseline value of 72.3 ± 7.1 to 57.5 ± 5.5 mmHg (n = 4) at the end of 6-min perfusion with oxygen-glucose-deprived Tyrode solution, which was followed by a transient increase to 108.5 ± 16.5 mmHg during subsequent perfusion with oxygenated normal Tyrode solution. However, in the presence of apyrase and DPCPX, the LVDP decreased to the same degree during 6-min perfusion with oxygen-glucose-deprived Tyrode solution, but failed to exhibit a transient increase during a subsequent perfusion with oxygenated normal Tyrode solution. These results strongly suggest that endogenous ATP released through Maxi-Cl channels contributes to the development of transient positive inotropy observed during reperfusion after short-period hypoxia/ischemia in the heart.

Measures of repolarization variability predict ventricular arrhythmogenesis in heptanol-treated Langendorff-perfused mouse hearts.

BackgroundTime-domain and non-linear methods can be used to quantify beat-to-beat repolarization variability but whether measures of repolarization variability can predict ventricular arrhythmogenesis in mice have never been explored.MethodsLeft ventricular monophasic action potentials (MAPs) were recorded during constant right ventricular 8 ​Hz pacing in Langendorff-perfused mouse hearts, in the presence or absence of the gap junction and sodium channel inhibitor heptanol (0.1, 0.5, 1 or 2 ​mM).ResultsUnder control conditions, mean action potential duration (APD) was 39.4 ​± ​8.1 ​ms. Standard deviation (SD) of APDs was 0.3 ​± ​0.2 ​ms, coefficient of variation was 0.9 ​± ​0.8% and the root mean square (RMS) of successive differences in APDs was 0.15 ​± ​0.14 ​ms. Poincaré plots of APDn+1 against APDn revealed ellipsoid morphologies with a SD along the line-of-identity (SD2) to SD perpendicular to the line-of-identity (SD1) ratio of 4.6 ​± ​2.1. Approximate and sample entropy were 0.61 ​± ​0.12 and 0.76 ​± ​0.26, respectively. Detrended fluctuation analysis revealed short- and long-term fluctuation slopes of 1.49 ​± ​0.27 and 0.81 ​± ​0.36, respectively. Heptanol at 2 ​mM induced ventricular tachycardia in five out of six hearts. None of the above parameters were altered by heptanol during which reproducible electrical activity was observed (KW-ANOVA, P ​> ​0.05). Contrastingly, SD2/SD1 decreased to 2.03 ​± ​0.41, approximate and sample entropy increased to 0.82 ​± ​0.12 and 1.45 ​± ​0.34, and short-term fluctuation slope decreased to 0.82 ​± ​0.19 during the 20-s period preceding spontaneous ventricular tachy-arrhythmias (n ​= ​6, KW-ANOVA, P ​< ​0.05).ConclusionMeasures of repolarization variability, such as SD2/SD1, entropy, and fluctuation slope are altered preceding the occurrence of ventricular arrhythmogenesis in mouse hearts. Changes in these variables may allow detection of impending arrhythmias for early intervention.

α1-adrenergic stimulation increases ventricular action potential duration in the intact mouse heart

The role of α1-adrenergic receptors (α-ARs) in the regulation of myocardial function is less well-understood than that of β-ARs. Previous reports in the mouse heart have described that α1-adrenergic stimulation shortens action potential duration in isolated cells or tissues, in contrast to prolongation of the action potential reported in most other mammalian hearts. It has since become appreciated, however, that the mouse heart exhibits marked variation in inotropic response to α1-adrenergic stimulation between ventricles and even individual cardiomyocytes. We investigated the effects of α1-adrenergic stimulation on action potential duration at 80% of repolarization in the right and left ventricles of Langendorff-perfused mouse hearts using optical mapping. In hearts under β-adrenergic blockade (propranolol), phenylephrine or noradrenaline perfusion both increased action potential duration in both ventricles. The increased action potential duration was partially reversed by subsequent perfusion with the α-adrenergic antagonist phentolamine (1 μmol L−1). These data show that α1-receptor stimulation may lead to a prolonging of action potential in the mouse heart and thereby refine our understanding of how action potential duration adjusts during sympathetic stimulation.

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+].

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.

Identifying Regulators of O‐GlcNAcylation in the Broken Heart

The post‐translational modification of intracellular proteins by monosaccharides of O‐linked b‐N‐acetylglucosamine (O‐GlcNAc) has emerged as a critical regulator of cellular function. Previous research has demonstrated that enhanced O‐GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia reperfusion (I/R) injury. The cycling of O‐GlcNAc is regulated by the collective efforts of two enzymes: O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA), which catalyze the addition and hydrolysis of O‐GlcNAc respectively. The goal of these studies is to understand the mechanisms by which the heart regulates O‐GlcNAcylation in response to injury. Sex and gender have been linked to the pathophysiology of cardiovascular disease; however, the impact of sex on O‐GlcNAc cycling has not been studied. Thus, to address our goal, we harvested male and female hearts from mice and extracted the total heart proteome and assessed the levels of O‐GlcNAc, OGT, and OGA as well as key enzymes within the hexosamine biosynthetic pathway (HBP). We observed a modest elevation in O‐GlcNAc levels in female hearts. Counter‐intuitively, OGA levels appeared to be elevated in the female hearts. To assess the impact of sex on O‐GlcNAc cycling in response to injury, we used the Langendorff perfused mouse heart model to collect male and female hearts exposed to either control, ischemic or I/R conditions. Hearts were partitioned into a soluble fraction enriched in cytosolic and mitochondrial proteins, as well as a myofibrillar fraction enriched in contractile proteins. Our data demonstrate that the majority of the O‐GlcNAc‐modified proteins are detected in the soluble fraction. Ischemia results in a suppression of O‐GlcNAcylation; however, O‐GlcNAc levels were modestly elevated in hearts subjected to I/R injury. Future work will assess the impact of sex on the activity of OGT and OGA, as well as the nucleotide sugar UDP‐GlcNAc. Collectively, these findings will provide a foundation for understanding whether sex is an important factor to consider when assessing molecular mechanisms regulating O‐GlcNAc cycling in the heart.Support or Funding InformationK12 HL141952, U01 CA2300978, R01 HL129640, P01 HL107153