Effect of Carbenoxolone on Arrhythmogenesis in Rat Ventricular Muscle.

BACKGROUND: Connexin43 (Cx43) is a major connexin that forms gap junction (GJ) channels in the heart and is also present in the cell membrane as unopposed/non-junctional hemichannels and in the inner mitochondrial membrane. By using carbenoxolone (CBX), a blocker of Cx43, the effect of the blockade of Cx43 on Ca(2+)waves and triggered arrhythmias in the myocardium with non-uniform contraction was examined. METHODS AND RESULTS: Trabeculae were obtained from rat hearts. Force, [Ca(2+)]i, and the diffusion coefficient were measured. Non-uniform contraction was produced with a 2,3-butanedione monoxime jet. Ca(2+)waves were induced by electrical stimulation. Inducibility of arrhythmias was estimated based on the minimal [Ca(2+)]oat which arrhythmias were induced. The Ca(2+)spark rate was measured in isolated single rat ventricular myocytes. CBX reduced the GJ permeability, whereas it did not change force and [Ca(2+)]itransients. CBX increased the Ca(2+)leak from the sarcoplasmic reticulum in trabeculae and increased the Ca(2+)spark rate in isolated single myocytes. CBX increased the velocity of Ca(2+)waves and further increased the inducibility of arrhythmias. Modulation of mitochondrial KATPchannels by diazoxide, cromakalim and 5-hydroxydecanoic acid affected the inducibility of arrhythmias increased by CBX. CONCLUSIONS: These results suggest that in diseased hearts, Cx43 plays an important role in the occurrence of triggered arrhythmias, probably under the modulation of mitochondrial KATPchannels.

Role of reactive oxygen species and Ca(2+) dissociation from the myofilaments in determination of Ca(2+) wave propagation in rat cardiac muscle.

Ca(2+) waves are initiated not only by Ca(2+) leak from the sarcoplasmic reticulum (SR), but also by Ca(2+) dissociation from the myofilaments in the myocardium with nonuniform contraction. We investigated whether contractile properties and the production of reactive oxygen species (ROS) affect Ca(2+) wave propagation. Trabeculae were obtained from 76 rat hearts. Force was measured with a strain gauge, sarcomere length with a laser diffraction technique, and [Ca(2+)](i) with fura-2 and a CCD camera (24°C, 2.0mmol/L [Ca(2+)](o)). ROS production was estimated from 2',7'-dichlorofluorescein (DCF) fluorescence. Trabeculae were regionally exposed to a jet of solution containing 1) 10mmol/L Ca(2+) to initiate Ca(2+) waves by SR Ca(2+) leak due to Ca(2+) overload within the jet-exposed region, and 2) 0.2mmol/L Ca(2+) or 5mmol/L caffeine to initiate such waves by Ca(2+) dissociation from the myofilaments due to nonuniform contraction. Ca(2+) waves were induced by stimulus trains for 7.5s. Ten-percent muscle stretch increased DCF fluorescence and accelerated Ca(2+) waves initiated due to both Ca(2+) overload and nonuniform contraction. Preincubation with 3µmol/L diphenyleneiodonium or 10µmol/L colchicine suppressed the increase in DCF fluorescence but suppressed acceleration of Ca(2+) waves initiated only due to Ca(2+) overload. Irrespective of preincubation with colchicine, reduction of force after the addition of 10µmol/L blebbistatin did not decelerate Ca(2+) waves initiated due to Ca(2+) overload, while it did decelerate waves initiated due to nonuniform contraction. These results suggest that Ca(2+) wave propagation is modulated by ROS production through an intact microtubule network only during stretch and may be additionally modulated by Ca(2+) dissociated from the myofilaments in the case of nonuniform contraction.

Inhalation of budesonide/formoterol increases diaphragm muscle contractility.

BACKGROUND: Although budesonide/formoterol (BUD/FORM) is used clinically as a steroid/ß(2)-agonist single inhaler, it has not yet been clarified whether BUD/FORM has inotropic effects on diaphragm muscles after inhalation. METHODS: We examined the effects of BUD/FORM inhalation, endotoxin injection, and BUD/FORM inhalation plus endotoxin injection on diaphragm contractile properties and nitric oxide (NO) production. After these three treatments, the diaphragm muscle was dissected, and its contractile properties were measured. Histochemistry for the reduced form of nicotinamide adenine dinucleotide phosphate diaphorase was performed for each muscle to assess NO production. RESULTS: The force-frequency curves showed an upward shift 1 h after inhalation (p < 0.05) in the BUD/FORM inhalation only group. The force-frequency curves showed a downward shift 4 h after injection (p < 0.001) in the endotoxin injection groups. In the BUD/FORM inhalation plus endotoxin injection groups, a downward shift in the force-frequency curves at 4 h after endotoxin injection was prevented. NO production was inhibited in the BUD/FORM inhalation plus endotoxin injection group compared with that of the endotoxin injection only groups. CONCLUSIONS: BUD/FORM inhalation has an inotropic effect on diaphragm muscle, protects diaphragm muscle deterioration after endotoxin injection, and inhibits NO production. Increments in muscle contractility with BUD/FORM inhalation are induced through a synergistic effect of an anti-inflammatory agent and ß(2)-agonist.

Regional increase in extracellular potassium can be arrhythmogenic due to nonuniform muscle contraction in rat ventricular muscle.

In the ischemic myocardium, extracellular potassium ([K(+)](o)) increases to ≥20 mmol/l. To determine how lethal arrhythmias occur during ischemia, we investigated whether the increased spatial pattern of [K(+)](o), i.e., a regional or a global increase, affects the incidence of arrhythmias. Force, sarcomere length, membrane potential, and nonuniform intracellular Ca(2+) ([Ca(2+)](i)) were measured in rat ventricular trabeculae. A "regional" or "global" increase in [K(+)](o) was produced by exposing a restricted region of muscle to a jet of 30 mmol/l KCl or by superfusing trabeculae with a solution containing 30 mmol/l KCl, respectively. The increase in [Ca(2+)](i) (Ca(CW)) during Ca(2+) waves was measured (24°C, 3.0 mmol/l [Ca(2+)](o)). A regional increase in [K(+)](o) caused nonuniform [Ca(2+)](i) and contraction. In the presence of isoproterenol, the regional increase in [K(+)](o) induced sustained arrhythmias in 10 of 14 trabeculae, whereas the global increase did not induce such arrhythmias. During sustained arrhythmias, Ca(2+) surged within the jet-exposed region. In the absence of isoproterenol, the regional increase in [K(+)](o) increased Ca(CW), whereas the global increase decreased it. This increase in Ca(CW) with the regional increase in [K(+)](o) was not suppressed by 100 µmol/l streptomycin, whereas it was suppressed by 1) a combination of 10 µmol/l cilnidipine and 3 µmol/l SEA0400; 2) 20 mmol/l 2,3-butanedione monoxime; and 3) 10 µmol/l blebbistatin. A regional but not a global increase in [K(+)](o) induces sustained arrhythmias, probably due to nonuniform excitation-contraction coupling. The same mechanism may underlie arrhythmias during ischemia.

Effect of nonuniform muscle contraction on sustainability and frequency of triggered arrhythmias in rat cardiac muscle.

BACKGROUND: Arrhythmias are benign or lethal, depending on their sustainability and frequency. To determine why lethal arrhythmias are prone to occur in diseased hearts, usually characterized by nonuniform muscle contraction, we investigated the effect of nonuniformity on sustainability and frequency of triggered arrhythmias. METHODS AND RESULTS: Force, membrane potential, and intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured in 51 rat ventricular trabeculae. Nonuniform contraction was produced by exposing a restricted region of muscle to a jet of 20 mmol/L 2,3-butanedione monoxime (BDM) or 20 mumol/L blebbistatin. Sustained arrhythmias (>10 seconds) could be induced by stimulus trains for 7.5 seconds only with the BDM or blebbistatin jet (100 nmol/L isoproterenol, 1.0 mmol/L [Ca(2+)](o), 24 degrees C). During sustained arrhythmias, Ca(2+) surges preceded synchronous increases in [Ca(2+)](i), whereas the stoppage of the BDM jet made the Ca(2+) surges unclear and arrested sustained arrhythmias (n=6). With 200 nmol/L isoproterenol, 2.5 mmol/L [Ca(2+)](o), and the BDM jet, lengthening or shortening of the muscle during sustained arrhythmias accelerated or decelerated their cycle in both the absence (n=10) and presence (n=10) of 100 mumol/L streptomycin, a stretch-activated channel blocker, respectively. The maximum rate of force relaxation correlated inversely with the change in cycle lengths (n=14; P<0.01). Sustained arrhythmias with the BDM jet were significantly accelerated by 30 mumol/L SCH00013, a Ca(2+) sensitizer of myofilaments (n=10). CONCLUSIONS: These results suggest that nonuniformity of muscle contraction is an important determinant of the sustainability and frequency of triggered arrhythmias caused by the surge of Ca(2+) dissociated from myofilaments in cardiac muscle.

A neuropeptide Y Y5 antagonist selectively ameliorates body weight gain and associated parameters in diet-induced obese mice.

Neuropeptide Y (NPY) is thought to have a major role in the physiological control of energy homeostasis. Among five NPY receptors described, the NPY Y5 receptor (Y5R) is a prime candidate to mediate some of the effects of NPY on energy homeostasis, although its role in physiologically relevant rodent obesity models remains poorly defined. We examined the effect of a potent and highly selective Y5R antagonist in rodent obesity and dietary models. The Y5R antagonist selectively ameliorated diet-induced obesity (DIO) in rodents by suppressing body weight gain and adiposity while improving the DIO-associated hyperinsulinemia. The compound did not affect the body weight of lean mice fed a regular diet or genetically obese leptin receptor-deficient mice or rats, despite similarly high brain Y5R receptor occupancy. The Y5R antagonist acts in a mechanism-based manner, as the compound did not affect DIO of Y5R-deficient mice. These results indicate that Y5R is involved in the regulation and development of DIO and suggest utility for Y5R antagonists in the treatment of obesity.

Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin.

Mutations in the glycogen synthase kinase 3beta (GSK3beta) phosphorylation sites of the beta-catenin gene exon 3 are found in 20-30% of human primary hepatocellular carcinoma (HCC), whereas mutations in the APC or AXIN genes are found in other HCC populations. These data strongly suggest that the Wnt signaling pathway is involved in hepatocarcinogenesis. To determine the role of beta-catenin in intestinal tumorigenesis, we earlier constructed a mutant mouse strain Catnb(lox(ex3)), in which exon 3 of the beta-catenin gene was sandwiched by loxP sequences. By genetic crosses of these mice with the Fabpl-cre transgenic mice that express the cre gene controlled by the fatty acid binding protein gene promoter, we introduced the beta-catenin stabilizing mutation into the small intestine and liver. Although numerous polyps were formed in the small intestine, we did not find any neoplastic (i.e., dysplastic) foci in the liver, and the mice died in 5 weeks after birth because of acute liver damage accompanying mitochondrial swelling. When a recombinant adenovirus that expresses the cre gene from a human cytomegalovirus early gene promoter was constructed and inoculated at a high multiplicity (10(9) plaque-forming units/mouse), the Catnb(lox(ex3)) mice showed marked hepatomegaly, with similar mitochondrial swelling in the hepatocytes, and died within 3 weeks after infection. On the other hand, when inoculated at lower multiplicities of infection (10(7) and 10(8) plaque-forming units/mouse, respectively), the Catnb(lox(ex3)) mice survived >6 months without any neoplastic foci in the liver, although the nuclear localization of beta-catenin was found in some hepatocytes even after 6 months. These results suggest that, in contrast to intestinal polyposis, the Wnt pathway activation by stabilized beta-catenin is not sufficient for hepatocarcinogenesis, but additional mutations or epigenetic changes may be required.