Isolated Guinea Pig Hearts Research Articles

The endocannabinoid ARA-S acts as an endogenous rescuing factor of Kv7.1/KCNE1 carrying Long QT syndrome mutations and paves the way for drug development strategies

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union’s Horizon 2020 research and innovation program Long QT Syndrome (LQTS) is a cardiac arrhythmia characterized by delayed ventricular repolarization. LQTS occurs when there are loss-of-function mutations in genes encoding for voltage-gated channels and their beta subunits that contribute to the cardiac action potential. Among them, the most mutated channel is Kv7.1/KCNE1, which encodes for the slowly activating component of the delayed rectifier K+ current (IKs). Families with inherited mutations in the Kv7.1/KCNE1 exhibit inconsistent correlations between genotypes and phenotypes making the clinical management of LQTS challenging. A possible strategy to help guide clinical approaches involves exploring endogenous compounds capable of modulating the Kv7.1/KCNE1 channel function, acting as potential disease modifiers. Using the two-electrode voltage clamp technique, we previously found that the endogenous endocannabinoid-like compound ARA-S, which has an arachidonic acid tail and a serine head group, facilitates activation of the wild-type Kv7.1/KCNE1 channel expressed in Xenopus oocytes. This is seen as a left shift in the voltage dependence of channel opening and an increase in conductance. Moreover, we showed that ARA-S restores a physiological QT interval in isolated guinea pig hearts suffering from drug-induced LQTS. Hence, ARA-S is an interesting endogenous Kv7.1/KCNE1 channel modulator that may influence disease severity. However, it is uncertain whether ARA-S also activates the Kv7.1/KCNE1 when it carries LQTS-associated mutations. Therefore, the current study involves studying the effect of ARA-S on mutated Kv7.1/KCNE1 channels using two-electrode voltage clamp electrophysiology. We find that ARA-S facilitates the activation of all tested LQTS mutants. However, the magnitude of the effect varies among them, suggesting that the compound´s effect is dependent on the genetic background. To understand why ARA-S has different effects, we use computational simulation approaches to study how mutations in the putative ARA-S binding sites change ARA-S efficacy. This understanding aids in developing new substances that can activate different mutations, by, for example, modifying the ARA-S tail to enhance its efficacy. We also try to understand the concentration of ARA-S required to restore a wild-type-like function of each mutant, considering the mutant's intrinsic behavior and the apparent affinity of ARA-S for the specific mutant. Altogether, this work finds ARA-S to be an efficient activator of LQTS-associated mutant Kv7.1/KCNE1 channels and is a first step towards understanding how the genetic background determines the effect of this endogenous modulator.

Further Quinolizidine Derivatives as Antiarrhythmic Agents- 3.

Fourteen quinolizidine derivatives, structurally related to the alkaloids lupinine and cytisine and previously studied for other pharmacological purposes, were presently tested for antiarrhythmic, and other cardiovascular effects on isolated guinea pig heart tissues in comparison to well-established reference drugs. According to their structures, the tested compounds are assembled into three subsets: (a) N-(quinolizidinyl-alkyl)-benzamides; (b) 2-(benzotriazol-2-yl)methyl-1-(quinolizidinyl)alkyl-benzimidazoles; (c) N-substituted cytisines. All compounds but two displayed antiarrhythmic activity that was potent for compounds 4, 1, 6, and 5 (in ascending order). The last compound (N-(3,4,5-trimethoxybenzoyl)aminohomolupinane) was outstanding, exhibiting a nanomolar potency (EC50 = 0.017 µM) for the increase in the threshold of ac-arrhythmia. The tested compounds shared strong negative inotropic activity; however, this does not compromise the value of their antiarrhythmic action. On the other hand, only moderate or modest negative chronotropic and vasorelaxant activities were commonly observed. Compound 5, which has high antiarrhythmic potency, a favorable cardiovascular profile, and is devoid of antihypertensive activity in spontaneously hypertensive rats, represents a lead worthy of further investigation.

Acute osimertinib exposure induces electrocardiac changes by synchronously inhibiting the currents of cardiac ion channels.

Introduction: As the third generation of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), osimertinib has demonstrated more significant cardiotoxicity than previous generations of EGFR-TKIs. Investigating the mechanism of osimertinib cardiotoxicity can provide a reference for a comprehensive understanding of osimertinib-induced cardiotoxicity and the safety of the usage of this drug in clinical practice. Methods: Multichannel electrical mapping with synchronous ECG recording was used to investigate the effects of varying osimertinib concentrations on electrophysiological indicators in isolated Langendorff-perfused hearts of guinea pigs. Additionally, a whole-cell patch clamp was used to detect the impact of osimertinib on the currents of hERG channels transfected into HEK293 cells and the Nav1.5 channel transfected into Chinese hamster ovary cells and acute isolated ventricular myocytes from SD rats. Results: Acute exposure to varying osimertinib concentrations produced prolongation in the PR interval, QT interval, and QRS complex in isolated hearts of guinea pigs. Meanwhile, this exposure could concentration-dependently increase the conduction time in the left atrium, left ventricle, and atrioventricular without affecting the left ventricle conduction velocity. Osimertinib inhibited the hERG channel in a concentration-dependent manner, with an IC50 of 2.21 ± 1.29μM. Osimertinib also inhibited the Nav1.5 channel in a concentration-dependent manner, with IC50 values in the absence of inactivation, 20% inactivation, and 50% inactivation of 15.58 ± 0.83μM, 3.24 ± 0.09μM, and 2.03 ± 0.57μM, respectively. Osimertinib slightly inhibited the currents of L-type Ca2+ channels in a concentration-dependent manner in acutely isolated rat ventricular myocytes. Discussion: Osimertinib could prolong the QT interval; PR interval; QRS complex; left atrium, left ventricle, and atrioventricular conduction time in isolated guinea pig hearts. Furthermore, osimertinib could block the hERG, Nav1.5, and L-type Ca2+ channels in concentration-dependent manners. Therefore, these findings might be the leading cause of the cardiotoxicity effects, such as QT prolongation and decreased left ventricular ejection fraction.

The endocannabinoid ARA-S displays variably activating effects on Kv7.1/KCNE1 carrying long QT syndrome mutations.

The cardiac arrhythmia long QT syndrome (LQTS) is characterized by a delayed ventricular repolarization. The most common cause of inherited LQTS is loss-of-function mutations in the KV7.1/KCNE1 channel. Therefore, the KV7.1/KCNE1 channel is an interesting target for developing new antiarrhythmic drugs, which ideally would restore the function of mutated KV7.1/KCNE1 channels. In this study, we evaluate the endogenous endocannabinoid-like compound ARA-S as an activator of the human KV7.1/KCNE1 channel carrying diverse loss-of-function mutations linked to LQTS. Using the two-electrode voltage clamp technique, we have previously found ARA-S to facilitate activation of the wild type KV7.1/KCNE1 channel expressed in Xenopus oocytes, seen as a left-shift in voltage dependence of channel opening and a conductance increase. Moreover, we have previously shown that ARA-S restores a physiological QT interval in isolated guinea pig hearts suffering from drug-induced LQTS. Hence, ARA-S is an interesting endogenous KV7.1/KCNE1 channel modulator which may inspire drug development. Now, using the same technique on LQTS-mutated KV7.1/KCNE1 channels, we find that ARA-S facilitates activation of all tested mutants, but the magnitude of the effect varies among them. For instance, the effect of ARA-S is reduced on the mutant I204F but increased on the mutant A370V, compared to the effect on the wild-type channel. This suggests that the effect of the compound is dependent on the genetic background. To better understand its impact, we group the tested mutants based on their response to ARA-S and factors such as whether they are localized to putative ARA-S binding sites or other interesting sites, such as PIP2 sites. Altogether, this work finds ARA-S as an efficient activator of LQTS-associated mutant KV7.1/KCNE1 channels and is a first step towards understanding how the genetic background determines the effect of this modulator.

Abstract 11526: Voltage Gated Sodium Channel β1 Subunit Mimetic Peptide PS2L Increases Regulated Intramembrane Proteolysis of SCN1B/β1

Clinical treatment for cardiac arrhythmias based on targeting ion channels is limited and can have deadly side effects. We previously reported that acute administration of SCN1B mimetic peptide βadp1 resulted in decreased action potential conduction velocity and increased arrhythmias in isolated guinea pig hearts. These changes were associated with disruption of trans-homophilic adhesion of the β1 subunit within gap junction-adjacent perinexal domains in intercalated discs.Following a strategy reported by two other independent groups working on intercellular adhesion molecules containing Ig domains, N-cadherin and desmoglein-2, we dimerized a c-terminal βadp1 sequence resulting in the peptide PS2L.Using Electric Cell-Substrate Impedance Sensing (ECIS) with Chinese Hamster 1610 cells stably expressing β1, we found that PS2L increases β1 mediated adhesion not only acutely, but unexpectedly for over 48 hours post-treatment. Regulated Intramembrane Proteolysis (RIP) of β1 sequentially by BACE-1 and γ-secretase results in a cleaved intracellular fragment that translocates to the nucleus and alters transcription of various genes, including VGSC subunits. Therefore, we hypothesized the long-term gain-of-adhesion-function with PS2L treatment resulted from increased RIP of β1, with downstream VGSC gene expression effects. Inhibition of γ-secretase with DAPT accumulates a C-terminal fragment (CTF) of β1 during RIP, enabling quantification of changes to RIP. Thus, 1610β1 cells were treated with 50μM PS2L, 1μM DAPT, 50μM PS2L+1μM DAPT or 0.1% DMSO control. Cells were fixed for β1 immunofluorescence (IF) or lysate was collected at 6-, 24-, and 48-hours post treatments for Western blotting.IF showed an increase in abundance of β1 48 hours after βadp1 treatment compared to DMSO, especially at cell borders. DAPT treatment significantly increased CTF levels compared to control at all timepoints (p<.0001). However, PS2L+DAPT treatment resulted in greater accumulation of β1 CTF levels over DAPT alone at the 6-hour timepoint but not later. (n=3, p<.05) In sum, our data indicates that PS2L may upregulate RIP of β1 with potential downstream effects on gene transcription of VGSC subunits, suggesting a path to a novel class of antiarrhythmics.

Abstract P1092: Voltage Gated Sodium Channel Subunit Mimetic Peptide βadp1 Increases Regulated Intramembrane Proteolysis Of Scn1b/β1

Clinical treatment for cardiac arrhythmias based on targeting ion channels is limited and can have deadly side effects. We previously reported that acute administration of βadp1 resulted in decreased action potential conduction velocity and increased arrhythmias in isolated guinea pig hearts. These changes were associated with disruption of trans-homophilic adhesion of the β1 subunit within gap junction-adjacent perinexal domains in intercalated discs. Using Electric Cell-Substrate Impedance Sensing (ECIS), we found that βadp1 shows opposing acute and long-term effects on β1 mediated adhesion. βadp1 decreases β1 mediated adhesion in 1610 cells stably expressing SCN1B/β1 (1610β1) over the first 24 hours, but after 48 hours is associated with increased adhesion. Regulated Intramembrane Proteolysis (RIP) of β1 sequentially by BACE-1 and γ-secretase results in a cleaved intracellular fragment that translocates to the nucleus and alters transcription of various genes, including VGSC subunits. Therefore, we hypothesized the long-term gain-of-adhesion-function with βadp1 treatment resulted from increased RIP of β1, with downstream VGSC gene expression effects. Inhibition of γ-secretase with DAPT accumulates a C-terminal fragment (CTF) of β1 during RIP, enabling quantification of changes to RIP. Thus, 1610β1 cells were treated with 50μM βadp1, 1μM DAPT, 50μM βadp1+1μM DAPT or 0.1% DMSO control. Cells were fixed for β1 immunofluorescence (IF) or lysate was collected at 6-, 24-, and 48-hours post treatments for Western blotting. DAPT treatment significantly increased CTF levels compared to control at all timepoints (p<.0001). However, βadp1+DAPT treatment resulted in greater accumulation of β1 CTF levels over DAPT alone at each timepoint. (n=6, 6 hr: p<.01, 24 hr: p<.01, 48 hr: p<.05). βadp1+DAPT treatment caused a significant increase in full-length β1 at 6 hours, but no difference at 24 or 48 hours. The data suggest depletion of β1 by increased RIP of β1 after βadp1 treatment. IF showed an increase in abundance of β1 48 hours after βadp1 treatment compared to DMSO. In sum, our data indicates that βadp1 may upregulate RIP of β1 with potential downstream effects on gene transcription of VGSC subunits, suggesting a path to a novel class of antiarrhythmics.

The Cardiotoxic Effect of Roundup® is not Induced by Glyphosate: A Non-specific Blockade of Human CaV1.2 Channels.

In Roundup®, the active principle glyphosate is formulated with adjuvants that help it to penetrate the plants' cell membranes. Several reports and reviews report cardiovascular effects of Roundup®, pointing the presence of arrhythmias as a potential consequence of Roundup® toxicity and death cause. However, it still remains debatable whether these cardiac events are related to glyphosate per se or to the Roundup® adjuvants. The present study aims to compare the pro-arrhythmogenic properties of Roundup® and glyphosate in an animal model and in human cardiomyocytes. In isolated guinea pig heart, the cardiotoxicity of Roundup® (significant effect on heart rate and depressive effect on ventricular contractility) was demonstrated with the highest concentrations (100µM). In human cardiomyocytes, the cardiotoxicity is confirmed by a marked effect on contractility and a strong effect on cell viability. Finally, this Roundup® depressive effect on heart contractility is due to a concentration-dependent blocking effect on cardiac calcium channel CaV1.2 with an IC50 value of 3.76µM. Surprisingly, no significant effect on each parameter has been shown with glyphosate. Glyphosate was devoid of major effect on cardiac calcium channel with a maximal effect at 100µM (- 27.2 ± 1.7%, p < 0.01). In conclusion, Roundup® could induce severe cardiac toxicity by a blockade of CaV1.2 channel, leading to a worsening of heart contractility and genesis of arrhythmias. This toxicity could not be attributed to glyphosate.

Not all Long-QTs Are The Same, Proarrhytmic Quantification with Action Potential Triangulation and Alternans.

Long-QT is commonly associated with an increased risk of polymorphic ventricular tachycardia from drug therapy. However, not all drugs prolonging QT interval are proarrhythmic. This study aimed to characterize cellular and tissue mechanisms under which QT-interval prolonging drugs and their combination are proarrhythmic, examining arrhythmia susceptibility due to action potential (AP) triangulation and spatial dispersion of action potential duration (APD). Additionally, we aimed to elucidate that Torsades de Pointe (TdP) associated with long-QT are not necessarily caused by early-after-depolarization (EADs) but are related to the presence of AP alternans in both time and space. Isolated Guinea Pig hearts were Langendorff perfused, and optical mapping was done with a voltage dye-sensitive dye. Two commonly used drugs at the beginning of the COVID-19 pandemic, hydroxychloroquine (HCQ) and Azithromycin (AZM), were added to study the effects of QT interval prolongation. Alternans in time and space were characterized by performing restitution pacing protocols. Comparing APs, HCQ prolongs APD during phase-III repolarization, resulting in a higher triangulation ratio than AZM alone or AZM combined with HCQ. Lower triangulation ratios with AZM are associated with phase-II prolongation, lower arrhythmia, and lower incidence of spatially discordant alternans.

The Investigation of Combined Na+/Ca2+ Exchanger and the L-type Ca2+- Channel Inhibition in Langendorff-Perfused Isolated Guinea Pig Hearts

Abstract Objective The sodium/calcium exchanger (NCX) and the L-type Ca2+-channel (LTCC) are nowadays considered the major transmembrane transport mechanisms that control Ca2+ homeostasis. In pathophysiological conditions the altered function of these currents may influence the Ca2+ homeostasis and cardiac contractility and thereby, may enhance the development of severe tachyarrhythmias. The blockade of NCX current has been proposed as possible approach in the prevention and/or suppression of arrhythmias; however, this mechanism is not always favourable because the inhibition of both modes of NCX may induce Ca2+-overload. The decrease of the Ca2+ level by partial LTCC inhibition may be beneficial in increasing the antiarrhythmic efficacy. Therefore, the aim of our study was to investigate the antiarrhythmic effects of combined NCX and LTCC blockade in the ex vivo guinea pig arrhythmia model. Methods We have performed Langendorff experiments in isolated guinea pig hearts. We have recorded electrocardiograms (ECG) and left ventricle pressure. We have applied 1 μM ORM-10962 (ORM), a compound that block NCX current and 30 nM nisoldipine for the inhibition of LTCC. Arrhythmias have been provoked by decreasing the activity of the sodium/potassium pump with 5 μM ouabain. Results We found that neither LTCC nor NCX blockade alone increased, while the combined inhibition of the two currents significantly delayed (p&lt;0.05) the mean time of appearance of ouabain-induced ventricular fibrillation. The heart frequency was affected by none of the drugs, only the left ventricular pressure (end-systolic and diastolic difference) was significantly elevated by ORM (p&lt;0.001). Conclusion In the Langendorff-perfused guinea pig heart, specific, combined NCX and LTCC blockade may be favourable than the inhibition of NCX or LTCC alone. However, further investigations are necessary to identify the pathological settings in which this combined cardiac drug therapy may be a potential new approach.

Reduction of Carnitine Content by Inhibition of Its Biosynthesis Results in Protection of Isolated Guinea Pig Hearts Against Hypoxic Damage.

BACKGROUND: 3-(2,2,2-trimethylhydrazinium) propionate (THP or mildronate) is an inhibitor of carnitine biosynthesis. This study was carried out to determine whether feeding of guinea pigs with THP results in decreased myocardial-free carnitine content and, as a result, attenuates hypoxic damage in isolated and paced work-performing hearts. METHODS AND RESULTS: Guinea pigs were administered either distilled water of 100 mg THP/kg/day orally for 10 days. The treatment resulted in about a 50% decline in myocardial-free carnitine content, from 11.1 +/- 0.2 (n = 5) to 5.6 +/- 0.2 (n = 5) µM/g dry weight of the heart. The left ventricular contractile function of the hearts was measured during normoxic perfusion (PO(2) = 590 mmHg), hypoxic perfusion (PO(2) = 149 mmHg), and reperfusion (PO(2) = 590 mmHg). In both untreated and THP-treated groups, the rate of development of intraventricular pressure (+dP/dt) under normoxic perfusion was similar; however, +dP/dt declined to about 10% of the initial rate within 20 minutes of hypoxic perfusion. In the THP-treated group of hearts, the initial decline was slower than that of the untreated animal hearts. After 20 minutes of normoxic reperfusion following 60 minutes of hypoxic perfusion, the recovery of +dP/dt and -dP/dt was greater in the THP-treated group than in the untreated group. The elevation of end-diastolic pressure during hypoxia was completely reversed by normoxic reperfusion of the THP-treated group but not in the untreated group. Mitochondria isolated from hearts from the THP-treated group after normoxic reperfusion following hypoxic perfusion exhibited better respiratory function than those from untreated hearts. CONCLUSIONS: The data suggest that feeding guinea pigs with THP results in reduced myocardial-free carnitine content and attenuation of hypoxic and reperfusion injury in isolated hearts.

Anti-Anaphylactic Activity of Isoquercitrin (Quercetin-3-O-β-d-Glucose) in the Cardiovascular System of Animals.

Effects of isoquercitrin (IQ) on anaphylactic responses were examined in cardiovascular systems of experimental animals. In pithed rats, IQ at 30 and 100 mg/kg (intravenous) significantly blunted both the initial hypertensive and the ensuing hypotensive responses during anaphylaxis. Death rate and tachycardia were also significantly inhibited after the same IQ doses in these rats. In isolated guinea pig hearts, IQ infusion at 30–100 μg/mL markedly reduced anaphylaxis-related coronary flow decrease, contractile force change, and heart rate responses (both tachycardia and arrhythmia). Cardiac histamine and creatine kinase releases were similarly diminished by IQ during anaphylaxis in the isolated guinea pig hearts. In two different isolated guinea pig vasculatures, the pulmonary artery and mesenteric arterial bed, anaphylactic vasoconstriction was reduced by IQ 30 and 100 μg/mL. It was observed that IQ had a marked inhibitory effect on histamine release from rat mast cells, and this mechanism was suggested as the major anti-anaphylactic mechanism. Direct inhibition of histamine-induced muscle contraction did not seem to be relevant, but IQ treatment successfully repressed intracellular calcium influx/depletion in mast cells. Overall, this study provided evidence for the beneficial effect of IQ on cardiac anaphylaxis, thus suggesting its potential applications in the treatment and prevention of related diseases.

Mechanisms of Action of the KCa2-Negative Modulator AP30663, a Novel Compound in Development for Treatment of Atrial Fibrillation in Man.

AimsSmall conductance Ca2+-activated K+ channels (SK channels, KCa2) are a new target for treatment of atrial fibrillation (AF). AP30663 is a small molecule inhibitor of KCa2 channels that is currently in clinical development for treatment of AF. The aim of this study is to present the electrophysiological profile and mechanism of action of AP30663 and its efficacy in prolonging atrial refractoriness in rodents, and by bioinformatic analysis investigate if genetic variants in KCNN2 or KCNN3 influence the expression level of these in human heart tissue.Methods and ResultsWhole-cell and inside-out patch-clamp recordings of heterologously expressed KCa2 channels revealed that AP30663 inhibits KCa2 channels with minor effects on other relevant cardiac ion channels. AP30663 modulates the KCa2.3 channel by right-shifting the Ca2+-activation curve. In isolated guinea pig hearts AP30663 significantly prolonged the atrial effective refractory period (AERP) with minor effects on the QT-interval corrected for heart rate. Similarly, in anaesthetized rats 5 and 10 mg/kg of AP30663 changed the AERP to 130.7±5.4% and 189.9±18.6 of baseline values. The expression quantitative trait loci analyses revealed that the genome wide association studies for AF SNP rs13376333 in KCNN3 is associated with increased mRNA expression of KCNN3 in human atrial appendage tissue.ConclusionsAP30663 is a novel negative allosteric modulator of KCa2 channels that concentration-dependently prolonged rodent atrial refractoriness with minor effects on the QT-interval. Moreover, AF associated SNPs in KCNN3 influence KCNN3 mRNA expression in human atrial tissue. These properties support continued development of AP30663 for treatment of AF in man.

P699Mechanisms of action of the small conductance Ca2+-activated K+-channel modulator AP30663, a novel compound being developed for treatment of atrial fibrillation in man

Abstract Background Small conductance Ca2+-activated K+-channels (SK-channels) are a promising new atrial selective target for treatment of atrial fibrillation (AF). AP30663 is a small molecule inhibitor of SK-channels that is effective in converting vernakalant-resistant AF in tachy-paced pigs. Detailed understanding of the molecular mechanism of AP30663 is important for the development of SK channel inhibition for use in man. Purpose To establish the electrophysiological profile, mechanism of action and efficacy in prolonging atrial refractoriness ex vivo of AP30663. Methods AP30663 potency and mechanism of action were established by whole cell and inside-out patch clamp recordings of expressed SK channels. The ion channel selectivity profile of AP30663 was investigated on heterologous expressed channels. Effects of AP30663 or vehicle (DMSO) on atrial refractoriness (AERP) and ventricular repolarization (QTcB) were investigated on isolated perfused guinea pig hearts. Results AP30663 was found to be a selective negative allosteric modulator of SK channels (IC50=0.77±x0.13 μM) with no or minor effects on a panel of other cardiac ion channels, including hERG/KV11.1, (IKr), KV7.1/KCNE1 (IKs), KV4.3/KChiP2 (Ito), Kir2.1 (IK1), Kir3.1/Kir3.4 (IKACh), KV1.5 (IKur), NaV1.5 (INa) and CaV1.2 (ICa). AP30663 inhibited the SK-channel by right-shifting the calcium activation curve of the SK-channel (the EC50 of Ca2+ increased from 0.43±0.02 μM (control, n=6) to 1.37±0.05 μM (in the presence of 7μM AP30663, n=6). In isolated guinea pig hearts, administration of vehicle had no effect on AERP or QTcB. AP30663 significantly prolonged the AERP in 3 μM (to 131±6% of baseline) and 10 μM (to 165±3% of baseline) without any effects on the QTcB. Conclusion AP30663 is a selective negative allosteric modulator of SK channels, acting by means of shifting the calcium dependence of SK-channel activation. AP30663 prolonged atrial refractoriness without affecting the QT-interval in isolated perfused heart preparations. These properties support continued development of AP30663 for treatment of AF in man. Acknowledgement/Funding Innovation Fund Denmark, Wellcome Trust

Prevention of mitochondrial pH gradient dissipation: a novel role for cyclosporin A on inhibiting calcium‐hydrogen exchange activity in cardiac isolated mitochondria

Calcium‐hydrogen exchange (CHE) can be induced in cardiac isolated mitochondria (m) either indirectly, by dinitrophenol (DNP)‐induced proton leakage into the mitochondrial matrix when proton pumping by complex V ATPase is inhibited by oligomycin, or directly by acidifying the extra‐mitochondrial buffer after adding excess CaCl2. Because the matrix efflux of Ca2+ by mCHE is partially matched by its reuptake via the mitochondrial Ca2+ uniporter (MCU), adding the MCU blocker ruthenium red after adding CaCl2 exposes the total release of Ca2+ via the mCHE. In addition to mCHE two other Ca2+ efflux pathways are the well‐known, the sodium‐hydrogen exchanger (mNCE) and the purported transient, low conductance, mitochondrial permeability transition pore (mPTP), through which ions and small molecules might exit the mitochondria in a “flickering” manner. We questioned if the effects of cyclosporine A (CsA) to block Ca2+ efflux resulted from transient mPTP opening rather than Ca2+ efflux via mCHE. We isolated Guinea pig heart mitochondria and tested if 500 nM CsA, a known inhibitor of mPTP opening, inhibits mCa2+ efflux induced after adding CaCl2 to an acidic (pH 6.9) or alkaline (pH 7.6) medium. We used a Na+ free mitochondrial buffer that contained ≈40 μM EGTA, carried over from the isolation buffer, to block mNCE. A single bolus of 40 μM CaCl2 was added to the buffer. Ca2+ uptake by mitochondria was assessed by the disappearance of Ca2+ from the buffer using the fluorescent dye Fura‐4 penta‐K+ salt. We also examined for buffer pH‐induced differences in Ca2+ retention capacity (CRC), identified by adding consecutive boluses of 10 μM CaCl2 every 90 s until external Ca2+ was no longer taken up by mitochondria. We found that CsA, in all pH conditions, delayed mPTP opening as shown by the greater CRCs. However, compared to without CsA, external (buffer) [Ca2+]e did not rise, and matrix pHm and membrane potential (ΔΨm) did not fall over time in acidic buffer when CsA was added before adding CaCl2. In addition, with or without CsA, CRC was greater at external pHe 7.6, and least at pHe 6.9. Although we found that CsA blocked mCa2+ efflux, we hypothesize that this was not mediated via mPTP flickering. CsA did not directly prevent the ΔΨm depolarization that occurs during matrix acidification or after addition of 30 μM DNP alone. In the absence of CsA, the mCHE‐mediated changes in pHm, external [Ca2+]e, and ΔΨm, induced by adding CaCl2 at extra‐matrix pH 6.9, occurred very slowly over 25‐30 min; this time course is indicative of slow cation exchange activity. Moreover, full ΔΨm depolarization was incomplete. Our results suggest a novel role of CsA in which, directly or indirectly, it prevents dissipation of the mitochondrial pH gradient induced when the extra‐matrix pH is low. Potentially, this may be due to inhibition of mCHE activity via mitochondrial Ca2+ buffering.Support or Funding InformationVA Merit BX‐002539‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BPA disrupts the cardioprotection by 17β-oestradiol against ischemia/reperfusion injury in isolated guinea pig hearts

Bisphenol A (BPA) is an environmental oestrogen or xenoestrogen (XEs). XEs represent a health risk due to their potential for endocrine disruption and ability to mimic estrogenic activity. The effects of BPA on isolated hearts under normal and ischemia/reperfusion (I/R) conditions were investigated for the first time, with a focus on the effects of BPA and 17β-oestradiol (E2) co-administration on I/R injury. Our results indicated that BPA at 10−7 M and 10−5 M did not significantly affect heart rate (HR), coronary flow (CF), lactate dehydrogenase (LDH) or creatine kinase (CK) release in normal or I/R isolated hearts within the 90 min. However, E2 exerted a protective effect against I/R injury, whereas, BPA inhibited the cardio-protective effects of E2 on HR, CF, and LDH and CK release. Furthermore, BPA in combination with E2 aggravated I/R injury by increasing infarct size and causing a more severe ultrastructural disruption as compared to treatment with E2 alone. Based on our results, we conclude that BPA inhibits the cardio-protective effects of E2 on I/R-injured hearts, despite not significantly affecting normal or I/R isolated hearts.

The cardiac electrophysiology effects of higenamine in guinea pig heart

BackgroundHigenamine (HG) is an active compound derived from Aconiti root with a cardiotonic effect. It has been approved by the Chinese SFDA for clinical trials due to its effect as a potent inotropic and chronotropic agent in the heart. However, the direct mode of action of HG on cardiac electrophysiology is unclear. MethodsThe experiments were performed at both cell levels and the isolated organ. The major cardiac ion currents and the action potential duration (APD) were measured using patch-clamps in single guinea-pig left ventricular myocytes. ECG was recorded in isolated guinea pig hearts. ResultsIn the left ventricular myocytes, HG increased ICa-L and IKs in concentration- and voltage-dependent manners in the left ventricular myocytes. It potentiated the ICa-L and IKs simultaneously for synchronization. The EC50 values were 0.27 μM and 0.64 μM for the ICa-L and IKs, respectively. HG (0.1 μM, 0.5 μM and 1 μM) had no effect on the IKr and INa. HG slightly prolonged APD at lower concentrations, and shortened the APD at higher concentrations. HG can induce the delayed after depolarization (DAD), which showed some pro-arrhythmic effect. In the isolated perfused heart, HG increased the heart rate via an action on the sinoatrial node cells, but did not induce cardiac arrhythmias, even at high concentrations. The EC50 value for the sinoatrial node that controls the heart rate was 0.13 μM. The sinoatrial node cells appeared to be more sensitive than ventricular myocytes to HG. The effects of HG on ventricular cells and sinoatrial node cells were both mediated through stimulation of β1-AR. ConclusionWe show for the first time that HG produced a predominant action on the sinoatrial node. HG appears to control the cardiac electrophysiology through its predominant effect on the sinoarial node cells, without induction of the ectopic activity that causes cardiac arrhythmias. Thus, HG might be useful for the treatment of bradycardia.

The efficiency of heart protection with HTK or HTK-N depending on the type of ischemia

We investigated isolated guinea pig hearts (n = 121) in an ischemia/ reperfusion model with the aim to compare the efficiency of the cardioplegic solution HTK with its novel replacement HTKN.Following consolidation with Tyrode's solution, ischemia started either immediately or after preceding cardioplegia with HTK, HTKN, or modified HTK enriched with Ca. Ischemia lasted either 80 min at 30 °C, or 360 min at 5 °C, or 81 min at 30 °C with intermittent cardioplegic perfusion. During ischemia we measured intracellular calcium (iCa++) and the time of gap junction uncoupling (t-in). During reperfusion we measured the reestablishment of cell coupling (t-ret), left ventricular developed pressure (LVDP), and heart rhythm (VC-RR).In 5 °C groups, iCa++ at t-in was significantly higher than before ischemia, and longest t-in, shortest t-ret, and best VC-RR were observed after HTK-protection. Of all 30 °C groups, the intermittent group with modified HTK showed shortest t-ret, best VC-RR, and the highest LVDP. At 5 °C, HTK groups had higher LVDP than HTK-N groups, but not at 30 °C.The data suggest that the higher calcium level in the HTK-N solution improves reperfusion after short ischemia at 30 °C but for long lasting ischemia at 5 °C it is beneficial to use the HTK solution.

The “M” Conformation of Adenine Nucleotide Translocase Enhances Cyclosporine A-Induced Delay of Mitochondrial Ca2+ Uptake after Cardiac Ischemia/Reperfusion Injury

Mitochondria can take up large amounts of Ca2+ during IR injury. How excess mitochondrial Ca2+ leads to formation/opening of the mitochondrial permeability transition pore (mPTP) is poorly understood. Adenine nucleotide translocase (ANT), while not a mPTP component, may regulate mPTP opening. Our aim was to assess if ANT has a role in regulating mPTP after IR injury of isolated guinea pig hearts. We measured Ca2+ retention capacity (CRC), a marker of bioenergetic activity, in mitochondria isolated after no IR (time control, TC) or after IR injury. Cyclophilin D (CypD) interacts with mitochondrial proteins, including ANT, to initiate mPTP opening. Cyclosporine A (CSA) blocks CypD, thus delaying mPTP opening induced by Ca2+ overload if CSA is given during initiation of Ca2+ efflux. Therefore, we examined if ANT inhibitors carboxyatractyloside (cATR) or bonkrekic acid (BKA) alter CSA-induced delay of mPTP opening. We found that CSA-induced delay of mPTP opening (assessed by Ca2+ release) was greater for IR+BKA vs. IR only; all data vs. TC (100%): For substrates NaPyr and NaSucc: IR+BKA, 88±10%, 97±10% (p<.05) vs. IR, 61±7%, 64±7%, respectively. In contrast IR+cATR exhibited no difference from IR (p>0.5) with NaPyr or NaSucc, 55±7%, 71±6%. BKA induces the “m” conformation of ANT (only ADP/ATP transport), while cATR induces the “c” conformation of ANT (ADP/ATP+nonspecific transport), so the differential effect of BKA on CRC suggests at least a partial role for modulation by ANT in the “m” conformation when CSA was given to delay mPTP opening. These results indicate enhanced efficiency of CSA to stall mPTP opening in IR injured mitochondria based on the contention that BKA promotes ANT to “m” being favorable over the “c” conformation induced by cATR or by IR injury.

Sympathetic Nervous Regulation of Calcium and Action Potential Alternans in the Intact Heart.

Rationale: Arrhythmogenic cardiac alternans are thought to be an important determinant for the initiation of ventricular fibrillation. There is limited information on the effects of sympathetic nerve stimulation (SNS) on alternans in the intact heart and the conclusions of existing studies, focused on investigating electrical alternans, are conflicted. Meanwhile, several lines of evidence implicate instabilities in Ca handling, not electrical restitution, as the primary mechanism underpinning alternans. Despite this, there have been no studies on Ca alternans and SNS in the intact heart. The present study sought to address this, by application of voltage and Ca optical mapping for the simultaneous study of APD and Ca alternans in the intact guinea pig heart during direct SNS.Objective: To determine the effects of SNS on APD and Ca alternans in the intact guinea pig heart and to examine the mechanism(s) by which the effects of SNS are mediated.Methods and Results: Studies utilized simultaneous voltage and Ca optical mapping in isolated guinea pig hearts with intact innervation. Alternans were induced using a rapid dynamic pacing protocol. SNS was associated with rate-independent shortening of action potential duration (APD) and the suppression of APD and Ca alternans, as indicated by a shift in the alternans threshold to faster pacing rates. Qualitatively similar results were observed with exogenous noradrenaline perfusion. In contrast with previous reports, both SNS and noradrenaline acted to flatten the slope of the electrical restitution curve. Pharmacological block of the slow delayed rectifying potassium current (IKs), sufficient to abolish IKs-mediated APD-adaptation, partially reversed the effects of SNS on pacing-induced alternans. Treatment with cyclopiazonic acid, an inhibitor of the sarco(endo)plasmic reticulum ATPase, had opposite effects to that of SNS, acting to increase susceptibility to alternans, and suggesting that accelerated Ca reuptake into the sarcoplasmic reticulum is a major mechanism by which SNS suppresses alternans in the guinea pig heart.Conclusions: SNS suppresses calcium and action potential alternans in the intact guinea pig heart by an action mediated through accelerated Ca handling and via increased IKs.

Protective role of STVNa in myocardial ischemia reperfusion injury by inhibiting mitochondrial fission.

It has been reported that isosteviol, a widely known sweeteners, can protect against myocardial ischemia-reperfusion (IR) injury in isolated guinea pig heart. Here, we aim to confirm the cardioprotective effect of its sodium salt, isosteviol sodium (STVNa), against IR injury and its potential molecular mechanism in H9c2 cardiomyocytes. STVNa significantly improved cell viability, restored mitochondrial membrane potential, decreased cellular reactive oxygen species generation, and inhibited cell apoptosis. Furthermore, STVNa treatment changed the morphology of mitochondria from fragmented, discontinuous forms to normal elongated, tubular forms. Cyto-immunofluorescence and western blot analysis revealed that STVNa inhibited mitochondrial fission proteins dynamin-related protein 1 (Drp1), and mitochondrial fission 1 (Fis1), thus plays a key role in cardioprotection. These findings, for the first time, suggest that STVNa can protect against myocardial IR injury through reverse mitochondrial fission.