Characterization of the electrocardiogram of microminipig in comparison with that of Clawn miniature swine: impacts of miniaturization of body size on electrocardiographic indices.

Effects of body size reduction on electrocardiographic indices were examined using microminipigs in comparison with Clawn miniature swine (Clawn). Electrocardiogram was recorded using Holter electrocardiograph in conscious state for 24 hr for microminipigs (male: 11.6 ± 0.1 kg, 12-17 months, n=5; and female: 9.9 ± 0.4 kg, 6 months, n=5) and Clawn (female: 20.3 ± 0.4 kg, 8-9 months, n=8). Microminipig had shorter PR interval and QRS width than Clawn, whereas no significant difference was detected in JTcF/QTcF between them. Ratios of PR interval, QRS width, and body weight cubic root for microminipigs to Clawn ranged between 0.713 and 0.830. These findings indicate that PR interval and QRS width will depend on distance for excitatory current propagation, whereas JTcF/QTcF may be governed by local electrical activities.

In vivo analysis of concentration-dependent effects of halothane or isoflurane inhalation on the electrocardiographic and hemodynamic variables in dogs.

We assessed concentration-dependent effects of halothane or isoflurane inhalation on the electrocardiographic and hemodynamic variables using a cross-over design in intact beagle dogs (n = 4). Elevation of inhaled halothane from 1.0% to 2.0% or isoflurane from 1.5% to 2.5% decreased the mean blood pressure and prolonged the QRS width without significantly altering the heart rate, PR interval or QT interval. However, the observed changes disappeared after regressions of both anesthetic conditions to their initial settings. These results indicate that hypotension-induced, reflex-mediated increase of sympathetic tone may have counterbalanced the direct negative chronotropic, dromotropic and repolarization slowing effects of the anesthetics.

In vivo comparison of dl-sotalol-induced electrocardiographic responses among halothane anesthesia, isoflurane anesthesia with nitrous oxide, and conscious state.

We compared dl-sotalol-induced electrocardiographic responses in intact dogs using a repeated-measures design among 1% halothane anesthesia, 1.5% isoflurane anesthesia with nitrous oxide (N2O), and conscious state to clarify influences of the anesthetics (n = 4). Basal PR interval was longer in halothane than either in isoflurane with N2O or in conscious state, reflecting sympathetic nerve suppression for the atrioventricular node by halothane. Both anesthetics exhibited longer basal QRS width than conscious state, suggesting their ventricular INa inhibition. Also, both anesthetics showed longer basal QT interval, QTcF and Tpeak-Tend than conscious state, indicating their ventricular IKr inhibition. Meanwhile, dl-sotalol prolonged PR interval similarly in isoflurane with N2O and in conscious state, which was less great in halothane, suggesting further sympathetic nerve suppression for the atrioventricular node might be limited in halothane. dl-Sotalol prolonged QT interval and QTcF >3 times greater in either of the anesthetics than in conscious state; moreover, dl-sotalol prolonged Tpeak-Tend similarly in both anesthetics, but hardly altered it in conscious state; indicating isoflurane with N2O as well as halothane may have reduced the repolarization reserve to increase the sensitivity of ventricle toward IKr suppression. Thus, isoflurane with nitrous oxide could be useful for in vivo IKr assay like halothane.

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.

Electropharmacological characterization of microminipigs as a laboratory animal using anti-influenza virus drug oseltamivir.

We analyzed electropharmacological characteristics of microminipigs under halothane-anesthesia using anti-influenza virus drug oseltamivir, which has been known to possess multi-channel blocking properties, including Na+, Ca2+ and K+ channels (n = 4). Oseltamivir in doses of 0.3, 3 and 30 mg/kg was intravenously infused over 10 min with an interval of 20 min, which provided peak plasma concentrations 1.4, 7.4 and 125.5 µg/mL, respectively. The low dose did not alter any of the cardiovascular variables. The middle dose decreased the heart rate at 30 min after the start of the infusion. The high dose transiently returned the heart rate toward the baseline for 10-15 min, but decreased it for 20-60 min; decreased the mean blood pressure for 5-60 min; prolonged the PR interval for 10-60 min, and the QRS width for 10-20 min; but shortened the QT interval for 10-30 min, and the QTc for 5-60 min. Thus, oseltamivir can suppress the sinus automaticity, and atrioventricular nodal and intraventricular conduction; and decrease the mean blood pressure, extents of which were greater in microminipigs than in beagle dogs in our previous observation in spite of similar plasma concentrations, reflecting higher sensitivity of microminipigs for Na+ and Ca2+ channel inhibition than that of beagle dogs. In contrast to beagle dogs, oseltamivir shortened the repolarization period in microminipigs, indicating that oseltamivir can more potently inhibit the inward currents than the outward ones in the hearts of microminipigs. This information may help improve utilizatione of microminipigs as a laboratory animal.

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.

Pharmacological characterization of microminipig as a model to assess the drug-induced cardiovascular responses for non-clinical toxicity and/or safety pharmacology studies.

We tried to establish the halothane-anesthetized microminipigs as an alternative animal model for non-clinical toxicity and/or safety pharmacology studies. In order to characterize the halothane-anesthetized microminipigs, we firstly clarified the effects of halothane anesthesia on their cardiovascular system (n = 5). Then, we examined the cardiovascular effects of dl-sotalol in doses of 0.1, 0.3 and 1 mg/kg, i.v. on the halothane-anesthetized microminipigs (n = 6). Induction of the halothane anesthesia by itself prolonged the QT interval as well as QTcF, suggesting that the halothane anesthesia can reduce the cardiac repolarization reserve in microminipigs like in dogs. dl-Sotalol showed more potent negative chronotropic, dromotropic and hypotensive effects together with repolarization delay in microminipigs than in dogs, although each cardiovascular response to dl-sotalol was directionally similar between them, suggesting greater basal sympathetic tone and/or smaller volume of distribution of the drug in microminipigs than in dogs. Analyses of proarrhythmic surrogate markers indicate that Tpeak-Tend and short-term variability of QT interval may be more sensitive to detect the dl-sotalol-induced direct electrophysiological changes in microminipigs than in dogs, but its reverse will be true for J-Tpeakc. Thus, these results may help better understand the drug-induced cardiovascular responses in microminipigs.

Twenty-six-week oral toxicity of diheptyl phthalate with special emphasis on its induction of liver proliferative lesions in male F344 rats.

The 26-week oral toxicity of diheptyl phthalate (DHP), a peroxisome proliferator-activated receptor α (PPARα) agonist, with special emphasis on the potential induction of hepatocellular proliferative lesions was investigated in this study. DHP was administered to male F344 rats via gavage at 0 (control), 1,000 or 2,000 mg/kg/day for 26 weeks. Body weight gain was significantly lower, whereas food and water consumption was significantly higher in DHP-treated rats compared with controls. DHP-treated rats exhibited decreases in blood triglyceride, total cholesterol, phospholipid and glucose levels, which were likely related to biological effects of the PPARα agonist. Absolute and relative organ weights of the livers with pale brown discoloration and dark brown spots significantly increased in DHP-treated rats. Histopathological examinations revealed remarkable diffuse hypertrophy of hepatocytes with ground-glass appearance, intracytoplasmic inclusion bodies and/or vacuolation in the DHP-treated groups. These findings were associated with increases in serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and γ-glutamyltranspeptidase. The number and area of glutathione S-transferase placental form positive foci, a marker of hepatocellular preneoplastic lesions in rats, significantly increased in DHP-treated groups. Additionally, proliferating cell nuclear antigen positive liver cell counts in DHP-treated groups were significantly higher than those of the controls. Testicular alterations were not detected histopathologically, whereas absolute and relative prostate weights significantly decreased at both doses. These results indicate that DHP induces liver pre-neoplastic foci, and suggest the possibility that DHP is a possible genotoxic carcinogen in the liver of rats.

Measurements of cardiac ion channel subunits in the chronic atrioventricular block dog.

The chronic atrioventricular block (CAVB) dog has been widely used as an in vivo proarrhythmia model. mRNA levels of K(+) and Ca(2+) channels in the isolated ventricular tissues from normal and CAVB dogs were assayed using a real-time PCR. The mRNA levels of KvLQT1 and MiRP1 were significantly less in the CAVB heart compared with those in the intact heart, whereas no significant difference was detected in the mRNA levels of other K(+)- or Ca(2+)-channel subunits. Adaptation against chronic bradycardia-related pathophysiology may have decreased the mRNA levels of cardiac K(+) channels, which may partly explain the arrhythmogenic property of this model.

Microminipig, a non-rodent experimental animal optimized for life science research: in vivo proarrhythmia models of drug-induced long QT syndrome: development of chronic atrioventricular block model of microminipig.

A new in vivo proarrhythmia model of drug-induced long QT syndrome was developed using the Microminipig, an incredibly small minipig established by Fuji Micra Inc. (Shizuoka). The atrioventricular (AV) node of the Microminipig of either sex weighing approximately 6 - 7 kg was ablated under halothane anesthesia, and proper care was taken for them. Proarrhythmic effects of drugs were assessed at >2 months after the onset of AV block using a Holter recording system. Oral administration of dl-sotalol (10 mg/kg) to the AV-block Microminipig prolonged the QT interval; moreover, it frequently induced dangerous ventricular premature beats, whereas no arrhythmia was detected after the vehicle administration (n = 4). Such dl-sotalol-induced ventricular arrhythmias were not detected in the intact Microminipig with sinus rhythm, although significant QT prolongation was observed (n = 4). Thus, the sensitivity and specificity of the AV-block Microminipig for detecting the drug-induced long QT syndrome can be considered to be comparable to previously established AV-block animal models of dogs and monkeys.

Microminipig, a Non-rodent Experimental Animal Optimized for Life Science Research: In Vivo Proarrhythmia Models of Drug-Induced Long QT Syndrome: Development of Chronic Atrioventricular Block Model of Microminipig.

A new in vivo proarrhythmia model of drug-induced long QT syndrome was developed using the Microminipig, an incredibly small minipig established by Fuji Micra Inc. (Shizuoka). The atrioventricular (AV) node of the Microminipig of either sex weighing approximately 6 - 7 kg was ablated under halothane anesthesia, and proper care was taken for them. Proarrhythmic effects of drugs were assessed at >2 months after the onset of AV block using a Holter recording system. Oral administration of dl-sotalol (10 mg/kg) to the AV-block Microminipig prolonged the QT interval; moreover, it frequently induced dangerous ventricular premature beats, whereas no arrhythmia was detected after the vehicle administration (n = 4). Such dl-sotalol-induced ventricular arrhythmias were not detected in the intact Microminipig with sinus rhythm, although significant QT prolongation was observed (n = 4). Thus, the sensitivity and specificity of the AV-block Microminipig for detecting the drug-induced long QT syndrome can be considered to be comparable to previously established AV-block animal models of dogs and monkeys.

Spontaneous Basal cell carcinoma of the submandibular gland in a rat.

At necropsy, a white nodule (about 5 × 3 mm in size) was observed in the right submandibular gland of a 10-week-old female GALAS rat. Histopathologically, oval to spindle-shaped and pale basophilic tumor cells proliferated closely, and formed variably sized foci. The nodule partially spread into or invaded the surrounding normal tissue, and necrotic foci were recognized in the tumor. Immunohistochemically, the nuclei of the tumor cells showed a diffusely positive reaction for p63, and the cytoplasm showed a diffusely positive reaction for cytokeratin and negative reaction for αSMA, vimentin, desmin and S-100. Many tumor cells were positive for PCNA. Ultrastructurally, the tumor cells contained many tonofilaments in the cytoplasm and a few desmosomes at the intercellular portion. Based on these findings, the tumor was diagnosed as a basal cell carcinoma originating from the duct in the rat submandibular gland.

Halothane-anaesthetized, closed-chest, guinea-pig model for assessment of drug-induced QT-interval prolongation.

For the halothane-anaesthetized, closed-chest, guinea-pig model, corrected QT interval (QTc) has been empirically used to estimate the extent of drug-induced QT-interval prolongation. In the present study, we employed an atrial pacing method to clarify a net effect of a drug on the QT interval in this model. The atrial pacing catheter was inserted via the jugular vein with a minimal surgical invasion, and the effects of d-sotalol (0.3 and 3 mg/kg, intravenously) and verapamil (0.01 and 0.1 mg/kg, intravenously) on electrocardiogram parameters were assessed under the sinus rhythm and during the atrial pacing of 200 and 240 beats/min. d-Sotalol significantly prolonged the QT interval in a reverse use-dependent manner and decreased the heart rate, while verapamil prolonged the PR interval without affecting the heart rate or QT interval, indicating the sensitivity and specificity of this model in assessing the pharmacodynamics of the drug-induced QT-interval prolongation. Using the QT/RR relationship under the sinus rhythm, we obtained the following two types of QT-interval correcting formulae; namely, QTc = QT - 0.207(RR - 300) by a linear regression method; and QTc = QT/(RR/300)0.332 by a non-linear regression method, the latter of which is equal to 0.67 times of Fridericia's formula, providing rationale for the use of mathematical correction in this model. Thus, the halothane-anaesthetized, closed-chest, guinea-pig model may be highly useful for assessing the drug-induced QT-interval prolongation, which may become an alternative to current models for the in vivo QT assay.

Halothane sensitizes the guinea-pig heart to pharmacological IKr blockade: comparison with urethane anesthesia.

Potential utility of halothane-anesthetized guinea pigs for detecting drug-induced repolarization delay was analyzed in comparison with urethane-anesthesia (n = 4 for both groups). Basal QT interval was significantly greater under halothane-anesthesia than urethane-anesthesia (192 +/- 7 vs 132 +/- 5 ms, respectively), whereas the reverse was true for the heart rate (190 +/- 7 vs 248 +/- 11 beats/min, respectively). The typical I(Kr)-blocker dl-sotalol (0.1 to 3 mg/kg, i.v.) induced dose-related bradycardia and QT interval prolongation under each anesthesia. The extent of maximum prolongation in the QT interval was greater under halothane-anesthesia than urethane-anesthesia (+101 +/- 15 vs +49 +/- 3 ms, respectively), whereas that of peak change in the heart rate was smaller under the former than the latter (-49 +/- 8 vs -63 +/- 5 beats/min, respectively). Pretreatment of the animals under urethane-anesthesia with the selective I(Ks) blocker chromanol 293B (n = 6) increased the extent of the dl-sotalol-induced QT interval prolongation to +57 +/- 8 ms, which was only 0.56 times of that under the halothane-anesthesia, whereas the pretreatment increased the peak change in the heart rate to -76 +/- 12 ms. These results indicate that the halothane-anesthesia may effectively sensitize the guinea-pig heart to pharmacological I(Kr) blockade.

The endothelium-dependent vasodilator action of a new beverage made of red wine vinegar and grape juice.

A new non-alcoholic beverage made of red wine vinegar and grape juice (Budo-no-megumi) has been recently demonstrated to lower the blood pressure of human as well as rats. In this study, we pharmacologically analyzed the mechanism of its hypotensive action. The thoracic aorta with intact endothelium was isolated from Sprague-Dawley rats, and incubated with a Tyrode's solution. The beverage in concentrations of 0.25 to 2% relaxed the pre-contracted aorta with an alpha-adrenoceptor agonist phenylephrine in a concentration-dependent manner, 2% of which induced 59% relaxation. In contrast, the vasodilator response disappeared in the aorta without endothelium. L-Nitro-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, significantly reduced the vasodilator effect of the beverage, whereas indomethacin, an inhibitor of cyclooxygenase, hardly affected it. These results suggest that the beverage can activate the nitric oxide synthase activity to exert vasodilation, which may partly explain its previously reported hypotensive action.

QT PRODACT: in vivo QT assay in anesthetized dog for detecting the potential for QT interval prolongation by human pharmaceuticals.

The purpose of this study was to assess the utility of the isoflurane-anesthetized dog model for detecting the potential for QT interval prolongation by human pharmaceuticals. The effects of 10 positive compounds with torsadogenic potential, 8 negative compounds with little torsadogenic potential, and dl-sotalol as a common positive compound were evaluated in 5 facilities in accordance with the common protocol approved by QT PRODACT. Each test compound was cumulatively infused into male beagle dogs anesthetized with isoflurane. Surface lead II ECG, blood pressure, and plasma concentrations for the positive compounds were measured. Repeated administration of the vehicle examined in each facility before the start of the experiments resulted in a slight, but not significant, change in corrected QT (QTc) interval, indicating that this model only shows slight experimental variation. Although an inter-facility variability in the extent of dl-sotalol-induced QT interval prolongation was observed, dl-sotalol significantly prolonged QTc interval in all facilities. All positive compounds significantly prolonged QTc interval at plasma levels up to 10 times those in patients who developed prolonged QTc interval or TdP, whereas no negative compounds did so. These data suggest that the in vivo QT assay using the anesthetized dog is a useful model for detecting the potential for QT interval prolongation by human pharmaceuticals.

Electrophysiological, anatomical and histological remodeling of the heart to AV block enhances susceptibility to arrhythmogenic effects of QT-prolonging drugs.

The present study was designed to investigate what kinds of adaptation occurred in the canine chronic AV block model, which has been used to study torsade de pointes (TdP). Dogs at 7-10 days (acute phase) and 28-56 days (chronic phase) after AV block were assessed. Ventricular effective refractory period and monophasic action potential duration were prolonged in chronic animals compared with acute animals; moreover the electrically vulnerable period was prolonged in chronic animals. Non-specific IKr channel blocker cisapride (1 and 10 mg/kg, p.o.) was administered without anesthesia to estimate the feasibility of QT prolongation. In chronic animals, QT prolongation followed by TdP was induced in one dog by the low dose and in all by the high dose, which was not observed in acute animals. MR images indicated increases of diameter and wall thickness of both ventricles in chronic animals. The degree of hypertrophy was prominent in the right ventricular wall and septal wall. Heart weight of the chronic animals was 1.7 times greater than that of normal control subjects. Photo- and electron-micrograph analyses showed myocardial cell hypertrophy with parallel increases of collagen fiber and extracellular space in chronic animals. These electrophysiological, anatomical and histological adaptations may predispose the chronic AV block heart to drug-induced QT prolongation with enhanced risk of re-entry and early after depolarization, leading to the onset of TdP.