Electropharmacological effects of intracellular Ca2+ handling modulator caldaret on the heart assessed in the halothane-anesthetized dogs.

We analyzed how the enhancement of net sarcoplasmic reticulum (SR) Ca2+ uptake may affect cardiac electrophysiological properties in vivo by using caldaret which can decrease SR diastolic Ca2+ leak, enhance SR Ca2+ reuptake and inhibit reverse-mode Na+/Ca2+ exchanger. Caldaret in doses of 0.5, 5 and 50 µg/kg was intravenously administered over 10 min to the halothane-anesthetized beagle dogs (n = 5), attaining pharmacologically active plasma concentration. The low and middle doses of caldaret increased the ventricular contraction, which could be explained by its on-target pharmacological activities. The high dose enhanced the sinus automaticity followed by its suppression in addition to the increase of the total peripheral resistance, which may be unfavorable for treating diastolic heart failure. The low and middle doses enhanced the atrioventricular conduction, which may have some potential for predisposing the atria to the onset of atrial fibrillation via an induction of mitral and/or tricuspid regurgitation. The middle and high doses of caldaret prolonged the ventricular effective refractory period without altering the intraventricular conduction or repolarization period, which may prevent the onset of ventricular arrhythmias. Thus, modulation of intracellular Ca2+ handling by caldaret can induce not only inotropic effect, but also various electrophysiological actions on the in situ heart.

Analysis of torsadogenic and pharmacokinetic profile of E-4031 in dogs bridging the gap of information between in vitro proarrhythmia assay and clinical observation in human subjects.

We analyzed torsadogenic and pharmacokinetic profile of E-4031 using chronic atrioventricular block dogs. E-4031 in intravenous doses of 0.03, 0.1 and 0.3 mg/kg over 10 min prolonged QT/QTc, and increased short-term variability of QT in a dose-related manner (n = 4), resulting in onset of torsade de pointes in 1 animal after the middle dose and 4 animals after the high dose, while it attained peak plasma concentrations of 16.5, 60.5 and 182.5 ng/mL at 10 min after their start of administration, respectively (n = 2). These results bridge the gap of information between in vitro proarrhythmia assay and clinical observation in human subjects.

Use of microminipigs for unveiling unknown mechanisms of azithromycin-induced cardiovascular death.

Although azithromycin can suppress cardiac INa, IKr, IKs, ICa,L and IK1, its onset mechanisms for cardiovascular death have not been fully investigated. We examined electropharmacological effects of azithromycin in intravenous doses of 0.3, 3 and 30 mg/kg using microminipigs under the halothane anesthesia (n = 4), which provided plasma concentrations of 3.1, 11.2 and 120.4 µg/mL, respectively. The low dose did not alter any of the cardiohemodynamic or electrocardiographic variables. The middle dose significantly shortened QT interval for 10-20 min and QTc for 10-30 min. The high dose significantly decreased mean blood pressure for 5-60 min, prolonged QRS width at 20 min, but shortened QT interval for 15-20 min and QTc for 15-30 min (n = 3). Cardiohemodynamic collapse occurred in 1 animal after the start of the high dose infusion, which might be associated with the cardiovascular death in patients with vasomotor dysfunction. Prolongation of QRS width indicates that azithromycin may suppress ventricular INa in vivo, which may unmask latent type of Brugada electrocardiographic genotype. Meanwhile, abbreviation of the QTc might cause potentially lethal, short QT-related, cardiac arrhythmia syndrome. These findings with microminipigs suggest the possible entry point for analyzing the mechanisms of cardiovascular death clinically seen with this antibiotic.

Changes of electrocardiogram and hemodynamics in response to dipyridamole: In vivo comparative analyses using anesthetized beagle dogs and microminipigs.

Microminipigs are expected as a novel animal model for cardiovascular pharmacological experiments. Since inherent vulnerability of coronary circulation of microminipigs has not been characterized, we performed dipyridamole-stress test to both microminipigs and beagle dogs, and compared the results. Dipyridamole in doses of 0.056 and 0.56 mg/kg were intravenously infused over 10 min (n = 4 for each animal). Dipyridamole decreased the systolic/diastolic blood pressures and double product in dogs as well as in microminipigs; but it did not significantly alter the heart rate or the global balance between the myocardial oxygen demand and supply in either animal. While organic coronary arterial stenosis was not detected in either animal, dogs have well-developed epicardial intracoronary networks unlike microminipigs. Like in humans, dipyridamole did not affect the ST segment of microminipigs, whereas it substantially depressed that in dogs. The results indicate the onset of subendocardial ischemia by dipyridamole in dogs may be partly associated with their well-developed native coronary collateral channels. Microminipigs would be more useful to evaluate the drugs which may affect the coronary circulation in the pre-clinical study than dogs.

Effects of moxifloxacin on the proarrhythmic surrogate markers in healthy Filipino subjects: Exposure-response modeling using ECG data of thorough QT/QTc study.

Effects of moxifloxacin on QTc as well as proarrhythmic surrogate markers including J-Tpeakc, Tpeak-Tend and short-term variability (STV) of repolarization were examined by using both standard E14 time-based evaluation and exposure-response modeling. The study was conducted with a single-blind, randomized, single-dose, placebo-controlled and two-period cross-over design in healthy Filipino subjects. QT interval was corrected by Fridericia's formula (QTcF). In the E14 time-based evaluation of ECG data, the largest ΔΔQTcF with 90% confidence interval was 14.1 ms (11.2-16.9) with Cmax of 3.39 µg/mL at 3 h post-dose (n = 69; male: 35, female: 34), indicating a positive effect on the QTcF. Moxifloxacin significantly increased the ΔΔJ-Tpeakc and ΔΔTpeak-Tend, whereas the ΔΔSTV was not altered. Meanwhile in the exposure-response modeling of the same ECG data, the slope of moxifloxacin plasma concentration-ΔΔQTcF relationship was 4.84 ms per µg/mL and the predicted ΔΔQTcF with 90% confidence interval was 13.8 ms (13.1-15.1) at Cmax, also indicating a positive effect on the QTcF. Importantly, results in each proarrhythmic surrogate marker obtained by the exposure-response modeling also showed high similarity to those obtained by the E14 statistical evaluation. Thus, these results of moxifloxacin may become a guide to estimate proarrhythmic potential of new chemical entities.

Effects of Red Wine Vinegar Beverage on the Colonic Tissue of Rodents: Biochemical, Functional and Pharmacological Analyses.

A beverage made of red wine vinegar and grape juice (Yamanashi-no-megumi™) was developed as a supplemental fluid containing polyphenols, which has been clinically shown to enhance the colonic transit. In this study, we assessed the mechanism of its prokinetic action by analyzing the effects on both the colonic phosphodiesterase activity of rats (n=4) and the isolated colonic strip preparation of guinea pigs (n=4). The 7% (v/v) solution of the beverage significantly decreased the phosphodiesterase activity by 9% (n=4). The beverage in concentrations of 0.7, 2.1 and 7% (v/v) relaxed the colonic strips pre-contracted by 1 µmol/L of carbachol in a concentration-related manner with 50, 58 and 79%, each response of which was diminished to 11, 19 and 46%, respectively in the presence of 100 µmol/L of L-nitro-arginine methyl ester. These results obtained by biochemical, functional and pharmacological analyses suggest that the beverage could relax the colon through both cAMP-associated and nitric oxide-dependent pathways, which may partly explain clinically observed prokinetic effect of the beverage.

Efficacy of Precordial Percussion Pacing Assessed in a Cardiac Standstill Microminipig Model.

BACKGROUND: Potential cardiovascular benefits of precordial percussion pacing (PPP) during cardiac standstill are unknown.Methods and Results:A cardiac standstill model in amicrominipigwas created by inducing complete atrioventricular block with a catheter ablation technique (n=7). Next, the efficacy of cardiopulmonary resuscitation by standard chest compressions (S-CPR), PPP and ventricular electrical pacing in this model were analyzed in series (n=4). To assess the mechanism of PPP, a non-selective, stretch-activated channel blocker, amiloride, was administered during PPP (n=3). Peak systolic and diastolic arterial pressures during S-CPR, PPP and ventricular electrical pacing were statistically similar. However, the duration of developed arterial pressure with PPP was comparable to that with ventricular electrical pacing, and significantly greater than that with S-CPR. Amiloride decreased the induction rate of ventricular electrical activity by PPP in a dose-related manner. Each animal survived without any neurological deficit at 24, 48 h and 1 week, even with up to 2 h of continuous PPP. CONCLUSIONS: In amicrominipigmodel of cardiac standstill, PPP can become a novel means to significantly improve physiological outcomes after cardiac standstill or symptomatic bradyarrhythmias in the absence of cardiac pacing. Activation of the non-selective stretch-activated channels may mediate some of the mechanophysiological effects of PPP. Further study of PPP by itself and together with S-CPR is warranted using cardiac arrest models of atrioventricular block and asystole.

Characterization of microminipigs as an in vivo experimental model for cardiac safety pharmacology.

We pharmacologically characterized microminipigs as an in vivo experimental model by assessing cardiovascular effects of pilsicainide, verapamil and E-4031, which can preferentially inhibit cardiac Na+, Ca2+ and K+ channels, respectively. Intravenous infusion of 1 mg/kg of pilsicainide (n = 4), 0.1 mg/kg of verapamil (n = 4) and 0.01 followed by 0.1 mg/kg of E-4031 (n = 5) over 10 min decreased the heart rate, mean blood pressure and ventricular contractility. Moreover, pilsicainide prolonged the PR interval, QRS width and QTc; verapamil prolonged the PR interval, but shortened the QRS width and QTc; and E-4031 prolonged the QTc, whereas no substantial change was detected in the PR interval or QRS width. Peak plasma concentrations of pilsicainide, verapamil and E-4031 in microminipigs were 1.7-4.8 times higher than those expected in humans and dogs, possibly due to smaller effective volume of drug distribution. The extent of the drug-induced cardiovascular responses was generally greater in microminipigs than in humans and dogs, which could be explained by the following possibilities; namely unique pharmacokinetic profile, less great reflex-mediated increase of sympathetic tone and/or smaller repolarization reserve in microminipigs. These information may make it feasible to apply this new-type animal to a tool for assessing cardiac safety profiles of new chemical entities.

Development of correction formula for field potential duration of human induced pluripotent stem cell-derived cardiomyocytes sheets.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used in many studies to assess proarrhythmic risks of chemical compounds. In those studies, field potential durations (FPD) of hiPSC-CMs have been corrected by clinically used Fridericia's and/or Bazett's formulae, however, the rationale for the use of these formulae has not been well established. In the present study, we developed a correction formula for experiments using hiPSC-CMs. First, we analyzed the effect of beating rate on FPD in the hiPSC-CMs sheets with electrical stimuli and a HCN channel inhibitor zatebradine. Next, we examined the relationship between the electrophysiological properties and the expression levels of ion channel genes in the cell sheets. Zatebradine slowed the beating rate and allowed to analyze FPD changes at various pacing cycle lengths. Rate-dependent change in the repolarization period was smaller in the cell sheets than that reported on the human hearts, which can be partly explained by lower gene expression level of hKCNJ2 and hKCNE1. Thus, non-linear equation for correcting FPD in the cell sheet; FPDc = FPD/RR0.22 with RR given in second was obtained, which may make it feasible to assess net repolarization delay by various chemical compounds with a chronotropic action.

Analysis of proarrhythmic potential of an atypical antipsychotic drug paliperidone in the halothane-anesthetized dogs.

Fatal cases with the use of atypical antipsychotic drug paliperidone have been reported; however, there was no clinical report describing paliperidone-induced torsade de pointes. In this study we assessed its electropharmacological effects together with its proarrhythmic potential in intravenous doses of 0.03, 0.3 and 3 mg/kg using the halothane-anesthetized dogs (n = 5), which could provide approximately 2, 20 and 200 times higher peak plasma drug concentrations than its therapeutic level, respectively. Paliperidone exerted potent vasodilator effect resulting in hypotension, which may be largely explained by its α1-adrenoceptor blocking action. In vivo electrophysiological results suggest that paliperidone may inhibit human ether-à-go-go-related gene K+ channel in a dose-related manner and modestly suppress Na+ channel in the in situ heart. The high dose of paliperidone may have some potential to induce early afterdepolarization that can trigger lethal ventricular arrhythmias, whereas the low and middle doses lack such proarrhythmic possibility, indicating that at least 20 times higher plasma concentration may be considered to be safe.

L/N-type Ca2+ channels blocker cilnidipine ameliorated the repolarization abnormality in a chronic hemodialysis patient.

A 53-year-old woman with end-stage renal disease and hypertension, who had received regular artificial hemodialysis for 10 years, has been treated with candesartan in a dose of 8 mg/day against her hypertension, but premature ventricular contractions were often observed during the hemodialysis. QT interval was 445 ms before hemodialysis, which was prolonged to 515 ms immediately after it, possibly reflecting the presence of reduced repolarization reserve in her heart. Since the blood pressure was often elevated to >160 mmHg before the hemodialysis, a daily dose of an L/N-type Ca2+ channels blocker cilnidipine of 5 mg/day was added. Three months later, the electrocardiogram was obtained before hemodialysis, revealing the basal QT interval was shortened to 416 ms. More importantly, in the electrocardiogram recorded immediately after the hemodialysis, the QT interval was 429 ms, indicating that 3 months administration of cilnidipine may restore the reduced repolarization reserve. As well, we observed that premature ventricular contractions during the hemodialysis had disappeared. Thus, cilnidipine may become a new upstream therapy to reduce the risk of lethal arrhythmias.

Impacts of Surgically Performed Renal Denervation on the Cardiovascular and Electrophysiological Variables in the Chronic Atrioventricular Block Dogs　- Comparison With Those of Amiodarone Treatment.

BACKGROUND: In order to begin to precisely clarify the impact of renal denervation on the blood pressure, atrial fibrillation and ventricular tachyarrhythmias, in addition to proarrhythmic potential, its cardiovascular effects were assessed by using the chronic complete atrioventricular block dogs. METHODS AND RESULTS: Cardiohemodynamic and electrophysiological effects, together with neurohumoral factors and/or electrolytes, were assessed before and 4 weeks after either renal denervation (n=5) or amiodarone treatment (n=6). Amiodarone hydrochloride was given orally to the animals every day in a dose of 200 mg/day for the first 7 days followed by 100 mg/day for the following 21 days. The renal denervation decreased the systolic pressure, idioventricular rate, prolonged ventricular effective refractory period, and slightly suppressed the adrenergic tone and the renin-angiotensin-aldosterone system, but hardly affected the atrial effective refractory period and terminal repolarization period. Amiodarone prolonged the atrial effective refractory period, whereas no significant change was detected in the other variables. CONCLUSIONS: Surgically performed renal denervation may possess the anti-ventricular tachyarrhythmic rather than anti-atrial fibrillatory potentials, and it also modestly decreased the blood pressure. Thus, currently obtained information may be used as guidance for better understanding the utility and limitation of renal denervation against various types of cardiovascular diseases. (Circ J 2016; 80: 1556-1563).

Possibility as an anti-cancer drug of astemizole: Evaluation of arrhythmogenicity by the chronic atrioventricular block canine model.

Since astemizole in an oral dose of 50 mg/kg/day was recently reported to exert anti-cancer effect in mice, we evaluated its proarrhythmic potential using the atrioventricular block dogs in order to clarify its cardiac safety profile. An oral dose of 3 mg/kg prolonged the QT interval without affecting the QTc (n = 4), whereas that of 30 mg/kg increased the short-term variability of repolarization and induced premature ventricular contractions in each animal, resulting in the onset of torsade de pointes in 1 animal (n = 4). Thus, proarrhythmic dose of astemizole would be lower than anti-cancer one, limiting its re-profiling as an anti-cancer drug.

Intravenous anti-influenza drug oseltamivir will not induce torsade de pointes: Evidences from proarrhythmia model and action-potential assay.

We evaluated proarrhythmic risk of intravenous oseltamivir with chronic atrioventricular block canine model (n = 4) and action-potential assay on guinea-pig right ventricle (n = 5). Oseltamivir in doses of 3-30 mg/kg, i.v. did not induce torsade de pointes in the canine model, whereas that in concentrations of 30-300 µM decreased maximum rate of phase 0 depolarization, shortened action potential duration at 30%, 60% and 90% repolarization levels, but prolonged difference in action-potential duration between 30% and 90% repolarization levels in a concentration-related manner. These results indicate that oseltamivir will not induce torsade de pointes clinically, since it inhibits both inward and outward currents.

Assessment of the Anti-anginal Effect of Tetramethylpyrazine Using Vasopressin-Induced Angina Model Rats.

Intravenous tetramethylpyrazine has been widely used in China as a complementary and/or alternative medicine to treat patients with ischemic heart disease. We assessed the anti-anginal effect of tetramethylpyrazine (10 mg/kg, intravenously (i.v.), n=6) in comparison with that of its vehicle, saline (1 mL/kg, i.v., n=6), using vasopressin-induced angina model rats. First, Donryu rats were anesthetized with pentobarbital sodium (60 mg/kg, intraperitoneally (i.p.)), and the surface lead I electrocardiogram was continuously monitored. Administration of vasopressin (0.5 IU/kg, i.v.) to the rats depressed the S-wave level of the electrocardiogram, indicating the onset of subendocardial ischemia. However, pretreatment with tetramethylpyrazine suppressed the vasopressin-induced depression of the S-wave level, which was not observed following pretreatment with its vehicle alone (saline), suggesting that tetramethylpyrazine ameliorated the vasopressin-induced subendocardial ischemia in vivo. These results may provide experimental evidence for the empirically known clinical efficacy of tetramethylpyrazine against ischemic heart disease, and could provide clues to better understanding its in vivo mechanism of action.

Comparison of anti-anginal effect of cilnidipine with those of nicardipine and nifedipine in the vasopressin-induced angina model of rats.

We assessed the anti-anginal effects of cilnidipine in comparison with those of nicardipine and nifedipine (1 and 10 µg/kg, n = 6 for each drug) or vehicle (n = 6) by using the vasopressin-induced angina model of rats. The administration of vasopressin (0.5 IU/kg, i.v.) to the rats depressed the S-wave level of the electrocardiogram reflecting the presence of subendocardial ischemia, whereas it significantly increased the mean blood pressure, resulting in the decrease of the heart rate and the prolongation of the PR interval possibly through a reflex-mediated increase in vagal tone. Cilnidipine suppressed the vasopressin-induced depression of the S-wave level in a dose-related manner, which was not observed by nicardipine or nifedipine. In addition, the low dose of cilnidipine hardly affected the vasopressin-induced pressor response, but it attenuated the negative dromotropic effect, suggesting N-type Ca2+ channel inhibition by cilnidipine might have suppressed the parasympathetic nerve activity in vivo like those reported in the sympathetic nerve. Thus, cilnidipine may become a useful strategy for inhibiting coronary vasospasm-induced anginal attack.

Comparison of direct effects of clinically available vasodilators; nitroglycerin, nifedipine, cilnidipine and diltiazem, on human skeletonized internal mammary harvested with ultrasonic scalpel.

Direct vasodilator effects of nitroglycerin, nifedipine, cilnidipine and diltiazem on human skeletonized internal mammary artery graft harvested with ultrasonic scalpel were assessed in the presence of 0.1 or 0.2 µM of noradrenaline. Ring preparations were made of distal end section of the bypass grafts, and those dilated by acetylcholine were used for assessment. Each drug dilated the artery in a concentration-related manner (0.01-10 µM, n = 6 for each drug) with a potency of nitroglycerin > nifedipine = cilnidipine > diltiazem. These results indicate that nitroglycerin can be useful for treating internal mammary artery spasm, that clinical utility of diltiazem may not depend on its vasodilator effect on the bypass graft, and that cilnidipine as well as nifedipine will have anti-spastic action which is in the middle between those of nitroglycerine and diltiazem.

Possible effects of inhibition of IKr and IKs on field-potential waveforms in the human iPS cell-derived cardiomyocytes sheet.

In order to investigate how IKr and IKs inhibitions affect waveforms of the field potential in the human iPS cell-derived cardiomyocytes sheet, we analyzed the effects of E-4031 and chromanol 293B on the maximum upslope and peak amplitude of its second wave (n = 7 for each drug). E-4031 in 10-100 nM as well as chromanol 293B in 3-30 µM prolonged the field-potential duration, whereas E-4031 decreased the upslope in 10-100 nM and amplitude at 100 nM, which was not observed by chromanol 293B. Thus, the decrease of the upslope can be used as a supplemental marker of drug-induced IKr inhibition.

Beneficial and Adverse Effects of Electro-acupuncture Assessed in the Canine Chronic Atrio-ventricular Block Model Having Severe Hypertension and Chronic Heart Failure.

Regarding the effects of electro-acupuncture for severe hypertension, we assessed its acute cardiovascular consequences with 4 subjects of the chronic atrioventricular block dogs having severe hypertension and chronic heart failure. The electro-acupuncture consisting of 2 mA at 2 Hz frequency was carried out for 30 min at Renying (ST-9) and Taichong (LR-3) every other day. Seven sessions were performed within 2 weeks. In the 1st and 7th sessions, the animals were anesthetized with pentobarbital to analyze the effects of the electro-acupuncture on cardiovascular variables. No significant change was detected in any of the basal control values of the cardiohemodynamic or electrophysiological variables between the 1st and 7th sessions. During the 1st session, electo-acupuncture produced a peak increase in mean blood pressure by 8.7% at 35 min (p < 0.05), whereas during the 7th session the peak increase was 6.5% at 35 min (p = 0.06). There was no significant change in the cardiac output, total peripheral resistance, a product of the heart rate and systolic blood pressure (= double product) reflecting myocardial oxygen consumption, QRS width or QT interval during the electrical stimulation in the 1st or 7th session. The results suggest that electroacupuncture may not exert lethal adverse effect except the vasopressor response, but that it can decrease the treatment-induced sympathetic response including vasopressor reaction and tachycardia. Since electro-acupuncture may have some potential to induce hypertensive crisis at the beginning, clinicians have to pay attention on its use for patients with hypertension.

Common parameters of acupuncture for the treatment of hypertension used in animal models.

Hypertension is associated with at least 7.6 million annual deaths worldwide. While pharmacotherapy may provide good control for blood pressure, it sometimes induces adverse effects. Meanwhile, acupuncture has been used for the treatment of cardiovascular diseases, such as hypertension, coronary artery disease, and stroke, but its mechanisms of actions remain poorly understood. The efficacy of acupuncture depends on multiple constituent elements including acupoints, manipulation skills, and implementation programs, which are termed as acupuncture prescription. This review summarized the previous information of experimental use of acupuncture on animals including species, hypertension models, acupoints selection, acupoint location, stimulation protocols, and evaluation of effectiveness to provide useful guidance for researchers when performing acupuncture in animal experiments.