Search

Aim: Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial KATP channels affect the occurrence of triggered arrhythmias. Methods and Results: To generate cardiac-specific Cx43-deficient (cCx43-/-) mice, Cx43flox/flox mice were crossed with α-MHC (Myh6)-cre+/- mice. The resulting offspring, Cx43flox/flox/ Myh6-cre+/- mice (cCx43-/- mice) and their littermates (cCx43+/+ mice), were used. Trabeculae were dissected from right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from ventricles of mouse hearts. Force was measured with a strain gauge in trabeculae (22°C). To assess arrhythmia susceptibility, the minimal extracellular Ca2+ concentration ([Ca2+]o,min), at which arrhythmias were induced by electrical stimulation, was determined in trabeculae. ROS production was estimated with 2’,7’-dichlorofluorescein (DCF), mitochondrial membrane potential with tetramethylrhodamine methylester(TMRM), and Ca2+ spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. Mitochondrial Ca2+ was estimated with rhod-2 in trabeculae. Diazoxide was used to open mitochondrial KATP. Most of cCx43-/-mice died suddenly within 8 weeks. Cx43 was present in the inner mitochondrial membrane in cCx43+/+mice, but not in cCx43-/-mice. In cCx43-/- mice, the [Ca2+]o,min was lower, Ca2+ spark frequency and DCF oxidation rate were more increased, and TMRM fluorescence was more decreased. These changes in cCx43-/-mice were suppressed by diazoxide. Besides, in cCx43-/- mice, antioxidant peptide SS-31 increased the [Ca2+]o,min. Rhod-2 fluorescence was increased at high [Ca2+]o only in Cx43-/- mice. Conclusions: These results suggest that the deficiency of Cx43 activates Ca2+ leak from the SR probably due to an increase in ROS production, depolarization of mitochondrial membrane potential, and an increase in mitochondrial Ca2+, thereby causing triggered arrhythmias and that the deficiency of Cx43 hemichannels may be compensated by activation of mitochondrial KATP channels in mouse hearts.

In non-diabetic patients with severe disease, such as acute myocardial infarction or acute heart failure, admission blood glucose level is associated with their short-term and long-term mortality. We examined whether transient elevation of glucose affects contractile properties in non-diabetic hearts. Force, intracellular Ca2+ ([Ca2+]i), and sarcomere length were measured in trabeculae from rat hearts. To assess contractile properties, maximum velocity of contraction (Max dF/dt) and minimum velocity of relaxation (Min dF/dt) were calculated. The ratio of phosphorylated troponin I (P-TnI) to troponin I (TnI) was measured. One hour after elevation of glucose from 150 to 400 mg/dL, developed force, Max dF/dt, and Min dF/dt were reduced without changes in [Ca2+]i transients at 2.5 Hz stimulation and 2.0 mM [Ca2+]o, while developed force and [Ca2+]i transients showed no changes at 0.5 Hz stimulation and 0.7 mM [Ca2+]o. In the presence of 1 μM KN-93, a Ca2+/calmodulin-dependent protein kinaseII (CaMKII) inhibitor, or 50 μM diazo-5-oxonorleucine, a l-glutamine-d-fructose-6-phosphate amidotransferase inhibitor, the reduction of contractile properties after elevation of glucose was suppressed. Furthermore, 1 h after elevation of glucose to 400 mg/dL at 2.0 mM [Ca2+]o, the ratio of P-TnI to TnI was increased. These results suggest that in non-diabetic hearts under higher Ca2+-load, transient elevation of glucose for 1 h reduces contractile properties probably by activating CaMKII through O-GlcNAcylation. Thus, in the patients with severe disease, transient elevation of blood glucose, such as due to stress, may worsen cardiac function and thereby affect their mortality without known diabetes.

Background In non-diabetic patients with acute coronary syndrome, stress hyperglycemia occasionally occurs and is related to their mortality. Whether transient elevation of glucose affects arrhythmia susceptibility in non-diabetic hearts with non-uniform contraction was examined.Methods and Results:Force, intracellular Ca2+([Ca2+]i), and membrane potential were measured in trabeculae from rat hearts. Non-uniform contraction was produced by a jet of paralyzing solution. Ca2+waves and arrhythmias were induced by electrical stimulation (2.0 mmol/L [Ca2+]o). The activity of Ca2+/calmodulin-dependent protein kinaseII (CaMKII) was measured. An elevation of glucose from 150 to 400 mg/dL increased the velocity of Ca2+waves and the number of spontaneous action potentials triggered by electrical stimulation. Besides, the elevation of glucose increased the CaMKII activity. In the presence of 1 μmol/L KN-93, the elevation of glucose did not increase the velocity of Ca2+waves and the number of triggered action potentials. In addition, in the presence of 1 μmol/L autocamtide-2 related inhibitory peptide or 50 μmol/L diazo-5-oxonorleucine, the elevation of glucose did not increase the number of triggered action potentials. Furthermore, the elevation of glucose by adding L-glucose did not increase their number. Conclusions In non-diabetic hearts with non-uniform contraction, transient elevation of glucose increases the velocity of Ca2+waves by activating CaMKII,probably through glycosylation with O-linked β-N-acetylglucosamine, thereby increasing arrhythmia susceptibility.

Introduction: Patient survival with pulmonary arterial hypertension (PAH) is determined from right ventricular (RV) function. CMR feature tracking (CMR-FT) is used to assess RV function. It is not determined, however, whether CMR-FT can assess RV functional reserve in patients with PAH. Hypothesis: We assessed the hypothesis that CMR-FT can estimate RV functional reserve in rats with PAH. Methods: Rats were received injections with monocrotaline (MCT-rats, n=19) or solvent (Ctr-rats, n=5). After four weeks, retrospective ECG-gated cine MR (16 phases/beat) was imaged using a 7-T MRI scanner, and RVEF, RV global longitudinal strain (RVGLS) and RV global circumferential strain (RVGCS) were measured before and after addition of 0.5~3 nmol endothelin-1 (ET-1). After the measurements, trabeculae were dissected from the RVs and electrically stimulated with 2-s intervals ([Ca 2+ ] o 2.0 mM, 24°C). Force and maximum dF/dt (dF/dt max ) were obtained using a silicon strain gauge in the absence and presence of 0.1 μM ET-1. Results: MCT-rats showed higher RV pressure (RVP), lower RVEF, lower RVGLS, lower developed force and lower dF/dt max . RVGLS showed a liner correlation with dF/dt max (r=0.53, p50%), suggesting that RVGLS reflects RV contractile properties and is reduced earlier than RVEF. Addition of ET-1 increased RVP in MCT-rats (49.0±19.3 to 59.7±16.8 mmHg, n=6) and Ctr-rats (17.3±7.5 to 20.4±7.8 mmHg, n=2) and increased developed force and dF/dt max in MCT-rats (12.2±5.7 to 17.4±3.1 mN/mm 2 , 0.08±0.03 to 0.14±0.06 mN/mm 2 /sec, n=6). When MCT-rats with preserved-RVEF (>50%) were divided into 2 groups depending on RVGCS, addition of ET-1 further reduced RVGCS in reduced-RVGCS (>-20%) group but not in increased-RVGCS ( Conclusions: CMR-FT can estimate RV functional reserve earlier and more accurately with RVGLS and RVGCS in rats with PAH. Therefore, RV strain with CMR may become an important parameter to assess RV functional reserve in patients with PAH.

Background: Connexin43 (Cx43) forms gap junctions in cardiac ventricles and exits as hemichannels in the inner mitochondrial membrane (mCx43). Objective: To examine whether mCx43 is involved in the occurrence of arrhythmias with modulation of mitochondrial K ATP channels (mK ATP ). Methods: To generate cardiac-specific Cx43-deficient ( Cx43 -/- ) mice, Cx43 flox/flox mice were crossed with α-myosin heavy chain -cre +/- mice. The resulting offspring, Cx43 -/- -mice and their littermates ( Cx43 +/+ -mice) were used. Trabeculae were obtained from right ventricles. Force was measured with a strain gauge, Ca 2+ within the mitochondria (Ca m ) with rhod-2, intracellular Ca 2+ (Ca i ) with microinjected fura-2 (22°C). The minimal extracellular Ca 2+ concentration (Cao min ), at which arrhythmias were induced by electrical stimulation, was determined. Using isolated single ventricular myocytes, mitochondrial membrane potential (ΔΨ m ) was estimated with tetramethylrhodamine methylester (TMRM), ROS production with 2’,7’-dichlorofluorescein (DCF), and Ca 2+ spark frequency with fluo-4 and confocal microscopy. Results: Cx43 -/- -mice showed sudden death within 8 weeks (p Cx43 +/+ -mice but not in Cx43 -/- -mice. Developed force and Ca i transients by 0.5 Hz electrical stimulation showed no difference between Cx43 -/- - and Cx43 +/+ -mice (n=7). The Cao min in Cx43 -/- -mice was lower than that in Cx43 +/+ -mice (n=5, pCx43 -/- -mice and is suppressed by DZX. Ca 2+ spark frequency and DCF oxidation rate in Cx43 -/- -mice were higher (pCx43 -/- -mice, and it was increased by DZX (p2+ in Cx43 -/- -mice but not in Cx43 +/+ -mice. Conclusions: Cx43 deficiency depolarizes ΔΨ m and increases Ca m at higher Ca o , ROS production, Ca 2+ spark frequency, and arrhythmia susceptibility. These changes in Cx43 -/- -mice are suppressed by DZX. Thus, mCx43 can be involved in the occurrence of arrhythmias with modulation of mK ATP .

Background: Ca 2+ within the sarcoplasmic reticulum affects the propagation of intracellular Ca 2+ (Ca i ) waves, and the velocity of Ca i waves play important roles in the occurrence of triggered arrhythmias. It is still unknown, however, whether Ca 2+ within the mitochondria (Ca m ) affects the velocity of Ca i wave and the occurrence of arrhythmias in right ventricular (RV) hypertrophy. Objective: To know the roles of Ca m in the velocity of Ca i waves and the occurrence of triggered arrhythmias in rats with RV hypertrophy. Methods: Rats were subcutaneously injected with 60 mg/kg monocrotaline (MCT-rats) or solvent (Ctr-rats). Four weeks after the injection, trabeculae were dissected from the RVs. The force was measured using a silicon strain gauge, Ca m using rhod-2, Ca i using microinjected fura-2 and a CCD camera, and production of reactive oxygen species (ROS) from the slope DCF fluorescence during electrical stimulation. Ca 2+ waves and arrhythmias were induced by electrical stimulation (24°C). Results: MCT-rats showed a higher RV pressure, a larger weight ratio of RV free wall to left ventricle (RV/LV), and faster and larger Ca i waves than Ctr-rats (pi transients, suggesting that inhibition of MCU decreases Ca m in MCT-rats with more severe RV hypertrophy. In addition, Ru360 decreased the velocity of Ca i waves depending on systolic RV pressure (r=-0.60, n=6) and the RV/LV (r=-0.64, n= 7), suggesting that inhibition of MCU decreased the velocity of Ca i waves in MCT-rats with higher RV pressure and more severe RV hypertrophy. Ru360 decreased the slope of DCF fluorescence (p Conclusions: In the hearts with severe RV hypertrophy, inhibition of MCU can cause larger reduction of Ca m , decrease ROS production, and decrease the velocity of Ca i waves and arrhythmias. Thus, in the hearts with severe RV hypertrophy, more Ca 2+ may enter the mitochondria through the MCU, and higher Ca m may be involved in the occurrence of arrhythmias.

Mechano-electric feedback means that muscle stretching causes depolarization of membrane potential. We investigated whether muscle stretching induces action potential and twitch contraction with a threshold of sarcomere length (SL) and what roles stretch-activated channels (SACs) and stretch-activated NADPH oxidase (X-ROS signaling) play in the induction. Trabeculae were obtained from the right ventricles of rat hearts. Force, SL, and [Ca

Introduction Mechano-electric coupling (MEC) means that muscle stretching can induce action potentials. Stretch-activated channels (SACs) have been believed to play important roles in their induction. Purpose To investigate what degree of muscle stretching can induce MEC-mediated action potentials and what roles SACs play in their induction. Methods Trabeculae were obtained from right ventricles of rat hearts. Force was measured with a strain gauge, sarcomere length (SL) with a laser diffraction technique, and [Ca2+]i with fura-2 (24°C). The SL was set at 2.0 μm at the resting condition. Trabeculae were stimulated electrically at 400-ms intervals for 7.5 s. Various degrees of muscle stretching were applied at 500 ms after the last stimulus of the electrical train to determine the minimal SL (SL-AP) at which an action potential or a twitch contraction was induced by the stretching (0.7 mM [Ca2+]o). Results The SL-AP was 2.34±0.02 μm (n=8) when trabeculae were stretched rapidly from a SL of 2.0 μm (400-ms stimulation intervals, 0.7 mM [Ca2+]o). The SL-AP was not changed by increasing the stimulation intervals from 400 to 2000 ms (n=7), by increasing [Ca2+]o from 0.7 to 2 mM (n=8), and by adding 1 μM isoproterenol (n=8), suggesting that Ca2+ loading within the myocardium has no effect on the SL-AP. Surprisingly, the SL-AP was not changed by adding 5 μM GsMTx4 (n=8), 10 mM Gd3+ (n=9), 100 μM (n=8) and 200 μM streptomycin (n=11), revealing that SACs play no roles in the determination of SL-AP. The SL-AP was not changed by adding 1 μM ryanodine (n=5) and 30 μM cyclopiazonic acid and was not changed by adding 3 μM diphenyleneiodonium chloride (n=8) and 10 μM colchicine, suggesting that Ca2+ leak from the SR and activation of NADPH oxidase has no effect on the SL-AP. In contrast, elevation of temperature from 23 to 36°C decreased the SL-AP from 2.35±0.01 to 2.34±0.02 μm (p Conclusions These results suggest that muscle stretching causes membrane excitation, which potentially induces arrhythmias and that activation of SACs, Ca2+ release from the SR, and activation of NADPH oxidase by muscle stretching are not involved in the excitation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): JSPS KAKENHI, Grant-in-Aid for Early-Career Scientists. Background Pulmonary arterial hypertension (PAH) remains a fatal disorder characterized by elevated pulmonary arterial pressure. Survival of the patients with PAH is determined from right ventricular (RV) function. CMR has become an attractive modality for following up and providing prognosis in such patients, and CMR feature tracking has been used as a newer useful parameter to assess RV function. However, it has not yet been determined whether CMR feature tracking can assess RV functional reserve in patients with PAH. Purpose We investigated whether CMR feature tracking can estimate RV functional reserve using a rat model with PAH. Methods Rats were received injections with monocrotaline (MCT-rats, n = 19) or solvent (Ctr-rats, n = 5). Four weeks after the injections, we performed CMR on 7-T MRI scanner and imaged retrospective ECG-gated cine MR (16 phases/beat). RV ejection fraction (RVEF) and RV strain were analyzed before and after addition of 0.5∼3 nmol endothelin-1 (ET-1). After the measurements, we dissected trabeculae (length = 1.45 ± 0.07 mm, width = 334 ± 27 µm, thickness = 114 ± 6 µm) from the RVs of rat hearts. Trabeculae were electrically stimulated with 2-s intervals at extracellular Ca2+ of 0.7 and 2.0 mmol/L (24°C). Force and maximum dF/dt (dF/dtmax) were then measured using a silicon strain gauge in the absence and presence of 0.1 µM ET-1. Results MCT-rats showed higher systolic RV pressure (RVP), lower RVEF, and lower RV global longitudinal strain (RVGLS) in CMR imaging and showed lower developed force and lower dF/dtmax in their trabeculae. Correlation between RVGLS and dF/dtmax was higher (r = 0.53, p 50%), RVGLS had already been reduced, suggesting that RVGLS is reduced earlier than RVEF. ET-1 increased developed force and dF/dtmax in trabeculae from MCT-rats (12.2 ± 5.7 to 17.4 ± 3.1 mN/mm2 and 0.08 ± 0.03 to 0.14 ± 0.06 mN/mm2/sec, respectively, n = 6), and ET-1 also increased RVP in MCT-rats and Ctr-rats (49.0 ± 19.3 to 59.7 ± 16.8 mmHg in MCT-rats, n = 6, 17.3 ± 7.5 to 20.4 ± 7.8 mmHg in Ctr-rats, n = 2). According to RV global circumferential strain (RVGCS) and RVEF, we could divide MCT-rats into three groups as follows: MCT-rats with reduced-RVGCS (> -20%)/preserved-RVEF (> 50%), MCT-rats with increased-RVGCS (< -30%)/preserved-RVEF and MCT-rats with reduced-RVGCS/reduced-RVEF. ET-1 reduced RVGCS in MCT-rats with reduced-RVGCS/preserved-RVEF, while ET-1 did not change RVGCS in MCT-rats with increased-RVGCS/preserved-RVEF. MCT-rats with reduced-RVGCS/reduced-RVEF died after injection of ET-1. In Ctr-rats, ET-1 did not change RVGCS and RVEF. These results suggest that RVGCS can be useful to assess RV functional reserve. Conclusion CMR feature tracking can estimate RV functional reserve earlier and more accurately than RVEF in rats with PAH. RV strain may become an important parameter to assess RV functional reserve in patients with PAH.

Background Right ventricular (RV) function is an important prognostic factor in patients with pulmonary arterial hypertension (PAH). Recently, CMR has become an attractive modality for follow up and providing prognosis in the patients, and strain has been used as a newer parameter to assess contractile properties of ventricle. It has not yet been established, however, whether RV strain with CMR can estimate RV functional reserve in the patients with PAH. Purpose We focused on CMR imaging of RV, investigating whether RV longitudinal strain can estimate RV functional reserve using a rat model with PAH. Method Rats were given a subcutaneous injection of 60 mg/kg monocrotaline (MCT-rats) or solvent (Ctr-rats). Four weeks after the injection, 25% of MCT-rats died due to RV failure. In the survivors of MCT- (n=19) and Ctr-rats (n=5), retrospective ECG-gated cine MR (16 phases/beat) was imaged with a 7T scanner. Subsequently, we measured RV pressure (RVP) via right internal jugular vein and dissected trabeculae (length = 1.45±0.07 mm, width = 334±27 μm, thickness = 114±6 μm) from RVs. We calculated weight ratio of RV free wall to left ventricle (LV) by RV/(LV+septum). Trabeculae were electrically stimulated with 2-s stimulus intervals, and force was measured using a silicon strain gauge (0.7 mM extracellular Ca2+, 24°C). To determine contractile properties of RV muscle, dF/dt was calculated. Using CMR imaging, we measured RV ejection fraction (RVEF) and RV longitudinal strain (RVLS). To modulate RVP, we intravenously injected 5 nmol endothelin-1 (ET-1) and again measured RVEF and RVLS. Results MCT-rats showed higher systolic RVP (62.5±16.6 vs. 25.9±1.86 mmHg, p50%), suggesting that RVLS decreases earlier than RVEF in MCT-rats. Ten minutes after the injection of ET-1, RVP was increased from 49.4±7.9 to 57.9±6.4 mmHg in MCT-rats (n=6). In MCT-rats with preserved RVEF and decreased RVLS, the increase in RVP chiefly decreased RVEF while it did not change RVLS and RVEF in Ctr-rats, meaning that RV functional reserve had been decreased in MCT-rats. In trabeculae, developed force and dF/dt were increased after the addition of 0.1 μM ET-1 in MCT- (n=11, p Conclusion These results suggest that in rats with PAH, RVLS obtained from CMR can estimate RV functional reserve earlier and more accurately than RVEF. Therefore, RV strain with CMR may become an important parameter to assess RV functional reserve in patients with PAH. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Young Scientists

Introduction Connexin43 (Cx43) forms gap junction channels in ventricular muscle and also exits in the inner mitochondrial membrane (mCx43). We have previously reported that carbenoxolone, a blocker of Cx43, increased the occurrence of arrhythmias with modulation of mitochondrial KATP channels (mKATP). Purpose To examine how mCx43 and mKATP affect arrhythmogenesis, using cardiac-specific Cx43-deficient (Cx43−/−) mice. Methods To generate cardiac-specific Cx43−/− mice, Cx43flox/flox mice were crossed with α-myosin heavy chain (Myh6)-cre+/− mice. The resulting offspring, Cx43flox/flox/ Myh6-cre+/− mice (Cx43−/− mice) and their littermates (Cx43+/+ mice), were used. Trabeculae were dissected from right ventricles of mouse hearts (length ∼0.9 mm, width ∼200 μm, thickness ∼80 μm). Force was measured with a strain gauge, and intracellular Ca2+ (Cai) with fura-2 and a CCD camera (22°C). To assess arrhythmogenesis, the minimal extracellular Ca2+ concentration (Caomin), at which arrhythmias were induced by electrical stimulation (0.3-s stimulus intervals, 100 nM isoproterenol), was determined in mouse trabeculae. Using isolated single ventricular myocytes, mitochondrial membrane potential (ΔΨm) was estimated with tetramethylrhodamine methylester (TMRM) fluorescence, ROS production was estimated with 2',7'-dichlorofluorescein (DCF) fluorescence, and Ca2+ spark frequency was measured with fluo-4 and confocal microscopy. To investigate the roles of mKATP, 0.1 mM diazoxide, a mKATP channel opener, and 0.2 mM 5-hydroxydecanoic acid (5-HD), a mKATP channel inhibitor, were used. Results Most of Cx43−/− mice suddenly died within 8 weeks (p Conclusions These results suggest that with modulation of mKATP, mCx43 plays important roles in ΔΨm determination, ROS production, and Ca2+ leak from the sarcoplasmic reticulum, thereby affecting arrhythmogenesis. Therefore, it is likely that mCx43 and mKATP could become a therapeutic target for controlling arrythmias. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science

Introduction Delayed afterdepolarizations (DADs) causes triggered arrhythmias, and intracellular Ca2+ (Cai) waves play important roles in the formation of DADs. It remains unclear, however, whether Ca2+ loading within mitochondria affects arrhythmogenesis in right ventricular (RV) hypertrophy. Purpose We focused on the properties of Cai wave propagation, investigating whether changes in Ca2+ loading within mitochondria affects arrhythmogenesis in a rat model of RV hypertrophy. Methods Rats were given a subcutaneous injection of 60 mg/kg monocrotaline (MCT-rats) or solvent (Ctr-rats). Four weeks after the injection, trabeculae were dissected from RVs. Force was measured using a silicon strain gauge, membrane potential using a microelectrode, Cai using microinjected fura-2 and a CCD camera, and ROS production using 2',7'-dichlorofluorescein (DCF) fluorescence. Cai waves and arrhythmias were induced by electrical stimulation (24°C). To change Ca2+ loading within mitochondria, Ru360 (10 μM), a mitochondrial calcium uptake inhibitor, was added. Results MCT-rats showed lower survival rate (p=0.075), higher RV systolic pressure (48.8±4.6 vs 21.3±6.5 mmHg), and heavier weight ratio of RV free wall to left ventricle (LV) (0.44±0.03 vs 0.25±0.04). MCT-rats showed a wider action potential (APD90, 320±20 vs 205±11 ms), a higher diastolic Cai (177±20 vs 110±8 nM), and faster and larger Cai waves (p Conclusions In RV hypertrophy, acceleration of Cai waves occurs due to an increase in diastolic Cai and an increase in Ca2+ sensitivity of the SR Ca2+ release channels, thereby causing arrhythmias. In more severe RV hypertrophy, Ca2+ within mitochondria may also be involved in arrhythmogenesis probably through an increase in ROS production. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science

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.

In diseased hearts, impaired muscle within the hearts is passively stretched by contractions of the more viable neighboring muscle during the contraction phase. We investigated whether in the myocardium with nonuniform contraction such passive stretch regionally generates ROS within the stretched region and exacerbates arrhythmias. In trabeculae from rat hearts, force, intracellular Ca2+, and membrane potential were measured. To assess regional ROS generation, the slope of the change in the 2′,7′-dichlorofluorescein fluorescence (DCFslope) was calculated at the each pixel position along the long axis of trabeculae using DCF fluorescence images. Ca2+ waves and arrhythmias were induced by electrical stimulation. A H2O2 (1 mmol/L) jet regionally increased the DCFslope within the jet-exposed region. A blebbistatin (10 μmol/L) jet caused passive stretch of the muscle within the jet-exposed region during the contraction phase and increased the DCFslope within the stretched region, the velocity of Ca2+ waves, and the number of beats after electrical stimulation (0.2 μmol/L isoproterenol), while 3 μmol/L diphenyleneiodonium (DPI), NADPH oxidase inhibitor, decreased them. A jet of a solution containing 0.2 mmol/L H2O2 in addition to 10 µmol/L blebbistatin also increased them. A H2O2 jet within the region where Ca2+ waves propagated increased their velocity. In the myocardium with nonuniform contraction, passive stretch of the muscle by contractions of the neighboring muscle regionally increases ROS within the stretched region, and the regional ROS exacerbates arrhythmias by activating the propagation of Ca2+ waves.

Objective: We investigated diaphragm contractile and inflammatory properties of mice with OVA sensitization and challenge. Methods: BALB/c mice were sensitized to OVA by intraperitoneal (i.p.) injection at 0 and 7 days, and challenged with aerosolized OVA on 21, 22, and 23 days (O/O group). Budesonide/Formoterol combination was inhaled on days 21, 22, and 23 before OVA challenge on those same days (O/OC group). Control mice were sensitized and challenged with an aerosolized saline (O-group). The diaphragm contractile and inflammatory properties were measured on day 24. NOS activity in the diaphragm muscle was evaluated by NADPH diaphorase staining. IL-4 and IL-13 levels of BALF, as well as lung tissue and diaphragm muscle homogenates were measured by ELISA. Results: Force-frequency (F/f) curves of O/O and O/OC shifted downward in comparison with O- (p

Background: Although budesonide/formoterol (BUD/FORM) is used clinically as a steroid/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

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 μmol/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°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 μmol/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 2+ sensitizer of myofilaments (n=10). Conclusion— 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.

Chronic kidney disease (CKD) is a global public health issue, and strategies for its early detection and intervention are imperative. The latest Japanese CKD guideline recommends that patients without diabetes should be classified using the urine protein-to-creatinine ratio (PCR) instead of the urine albumin-to-creatinine ratio (ACR); however, no validation studies are available. This study aimed to validate the PCR-based CKD risk classification compared with the ACR-based classification and to explore more accurate classification methods. We analyzed two previously reported datasets that included diabetic and/or cardiovascular patients who were classified into early CKD stages. In total, 860 patients (131 diabetic patients and 729 cardiovascular patients, including 193 diabetic patients) were enrolled. We assessed the CKD risk classification of each patient according to the estimated glomerular filtration rate and the ACR-based or PCR-based classification. The use of the cut-off value recommended in the current guideline (PCR 0.15 g/g creatinine) resulted in risk misclassification rates of 26.0% and 16.6% for the two datasets. The misclassification was primarily caused by underestimation. Moderate to substantial agreement between each classification was achieved: Cohen's kappa, 0.56 (95% confidence interval, 0.45-0.69) and 0.72 (0.67-0.76) in each dataset, respectively. To improve the accuracy, we tested various candidate PCR cut-off values, showing that a PCR cut-off value of 0.08-0.10 g/g creatinine resulted in improvement in the misclassification rates and kappa values. Modification of the PCR cut-off value would improve its efficacy to identify high-risk populations who will benefit from early intervention.

Background The propagation velocity of Ca 2 + waves determines delayed afterdepolarization and affects the occurrence of triggered arrhythmias in cardiac muscle. We focused on myofilament Ca 2 + sensitivity, investigating how the velocity of Ca 2 + waves responds to its increased sensitivity resulting from muscle stretch or the addition of a myofilament Ca 2 + sensitizer, SCH00013. We further investigated whether production of reactive oxygen species (ROS) may be involved in the change in velocity. Methods Trabeculae were obtained from rat hearts. Force, sarcomere length, and [Ca 2 + ] i were measured. ROS production was estimated from 2′,7′-dichlorofluorescein (DCF) fluorescence. Trabeculae were exposed to a 10 mM Ca 2 + jet for the induction of Ca 2 + leak from the sarcoplasmic reticulum in its exposed region. Ca 2 + waves were induced by 2.5-Hz stimulus trains for 7.5 s (24 °C, 2.0 mM [Ca 2 + ] o ). Muscle stretch of 5, 10, and 15% was applied 300 ms after the last stimulus of the train. Results Muscle stretch increased the DCF fluorescence, the amplitude of aftercontractions, and the velocity of Ca 2 + waves depending on the degree of stretch. After preincubation with 3 μM diphenyleneiodonium (DPI), muscle stretch increased only the amplitude of aftercontractions but not the DCF fluorescence nor the velocity of Ca 2 + waves. SCH00013 (30 μM) increased the DCF fluorescence, the amplitude of aftercontractions, and the velocity of Ca 2 + waves. DPI suppressed these increases. Conclusions Muscle stretch increases the velocity of Ca 2 + waves by increasing ROS production, not by increasing myofilament Ca 2 + sensitivity. In the case of SCH00013, ROS production increases myofilament Ca 2 + sensitivity and the velocity of Ca 2 + waves. These results suggest that ROS rather than myofilament Ca 2 + sensitivity plays an important role in the determination of the velocity of Ca 2 + waves, that is, arrhythmogenesis.

Background and Objective: Although a combination of inhaled corticosteroid (ICS) and inhaled long-acting β2- agonist (LABA) reduces exacerbation of asthma, whether these affect diaphragm muscle contraction is still unclear. Methods: We investigated the effects of ICS and LABA inhalation separately, endotoxin injection-only, and ICS or LABA inhalation plus endotoxin injection on diaphragm contractile properties and nitric oxide (NO) production during 4 h, using BALB/c mice (n=84). In this study, budesonide was used as the ICS, and formoterol fumarate dehydrate was used as the LABA. After administrations of these drugs, the diaphragm muscles were dissected, and their contractile properties, including force/frequency (F/f) curves and twitch contraction, were measured by electrical pulse stimulation in an isometric condition. A reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry was performed to assess NO production, which induces cellular damages in diaphragm muscle fibers. Results: The ICS inhalation did not significantly shift the F/f curves; but the LABA inhalation significantly shifted them upward compared with shams at 1 h (p < 0.01), 2 h (p < 0.01), and 4 h (p < 0.05) after inhalation, that is, it showed an inotropic effect. Although endotoxin injection resulted in a downward shift in F/f curves at 4 h (p < 0.01) and induced NO production, the endotoxin plus either ICS or LABA inhalation prevented downward shifts of the F/f curves and inhibited NO production. Conclusions: These results indicate that the inhalation of LABA potentiates diaphragm muscle contractility more than ICS inhalation and that both ICS and LABA inhalation inhibit NO production induced by endotoxin injection.

Background Patients with severe pulmonary emphysema have a greatly increased oxygen cost of breathing (O 2 cost), and this is the cause of serious malnutrition, or respiratory cachexia, in such patients. Study objectives To clarify the effect of lung volume reduction surgery (LVRS) on respiratory function and the nutritional state of these patients through a reduction in the O 2 cost of the respiratory muscles. Design Prospective cohort study. Setting, patients, and interventions Twenty-three patients who underwent LVRS in Tohoku University Hospital. Measurements Pulmonary function and O 2 cost were measured perioperatively by utilizing a method of continuous dead space. In addition, we calculated the proportion of oxygen consumption ( V ˙ o 2 ) of respiratory muscles to total V ˙ o 2 (% V ˙ o 2 resp) from the measured energy expenditure and the predicted values. Results FEV 1 and arterial oxygen pressure increased after surgery while lung volume and dyspnea decreased (p 2 cost was also reduced from 0.044 to 0.026 log(mL/min)/(L/min) [p 2 cost had a strong negative correlation with that of FEV 1 ( r = − 0.70, p r = 0.54, p < 0.01). % V ˙ o 2 resp was 23.1% at rest and 55.5% at maximal ventilation. LVRS reduced % V ˙ o 2 resp at maximal ventilation to 49.0% (p < 0.05), but % V ˙ o 2 resp at rest did not decrease after surgery. Conclusions LVRS reduces energy expenditure of respiratory muscles especially during exercise by decreasing small airway obstruction and hyperinflated lung volume. This may reverse the malnourished state in end-stage emphysema.

To evaluate the role of interleukin (IL)-18 in endotoxin-induced diaphragm muscle deterioration, we studied three treatment groups, namely, a saline+endotoxin group, an IL-18+endotoxin group and an IL-18 only group using Wistar rats. Five minutes after saline or IL-18 (0.25 microg) injection, E. coli endotoxin (30 mg/kg) was injected intraperitoneally. In the saline+endotoxin group, the force-frequency curves by ANOVA (p0.01), twitch tension (TT) and slope during contraction time (TT/CT) were significantly lower at 4 hours than those at 0 hours due to endotoxin (p0.01 and p0.05, respectively) and nitric oxide (NO) production had increased at 4 hours as shown by NADPH diaphorase staining. In the IL-18+endotoxin group, the decrement of the force-frequency curves in a higher frequency range (50-120 Hz), and the decrements of TT and TT/CT observed at 4 hours in the saline+endotoxin group were significantly prevented, and NO production was blocked at 4 hours. In the IL-18 only group, the force-frequency curves did not change except for fatigability, and NO production did not occur. It is suggested that IL-18 itself naturally blocks NO production, even when endotoxin coexists, resulting in the prevention of deterioration of diaphragm muscle contraction by endotoxin.

Background The Kidney Disease: Improving Global Outcomes chronic kidney disease (CKD) guidelines recommend that CKD be classified based on the etiology, glomerular filtration rate (GFR) and degree of albuminuria. The present study aimed to establish a method that predicts the presence of microalbuminuria by measuring the total urine protein-to-creatinine ratio (TPCR) in patients with cardiovascular disease (CVD) risk factors. Methods and Results We obtained urine samples from 1,033 patients who visited the cardiovascular clinic at St. Luke's International Hospital from February 2012 to August 2012. We measured the TPCR and the urine albumin-to-creatinine ratio (ACR) from random spot urine samples. We performed correlation, receiver operating characteristic (ROC) curve, sensitivity, and subgroup analyses. There was a strong positive correlation between the TPCR and ACR (R2 = 0.861, p

The effects of an intravenous injection of Interleukin-13 (IL-13) after endotoxin administration on diaphragm muscle were studied using Wistar rats. Two treatment groups, a control (saline+endotoxin) group and an IL-13 (IL-13+endotoxin) group were studied. E. coli endotoxin (10 mg/kg) was injected intraperitoneally 5 minutes after saline or IL-13 (0.25 microg) injection. The force-frequency curves, twitch kinetics and fatigability were measured at 0 and 4 hours after endotoxin injection. The force-frequency curves and twitch tension in the control group were significantly lower at 4 hours than those at 0 hour due to endotoxin. On the other hand, IL-13 prevented the decrement of the force-frequency curves and twitch tension induced by endotoxin. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry showed positive staining at 4 hours due to endotoxin in the control group; however, IL-13 also blocked NADPH diaphorase staining at 4 hours. Furthermore, the positive muscle fibers detected by the NADPH diaphorase staining were classified as type I (slow twitch) muscle fibers by ATPase staining. We conclude that IL-13 prevents the deterioration of contraction induced by endotoxin by inhibiting nitric oxide production in the diaphragm muscle, mainly the type I muscle fibers.

The effects of Interleukin (IL)-10 intravenous injection after endotoxin administration on diaphragm muscle were studied using Wistar rats. The animals were divided into two treatment groups: A saline + endotoxin group as control and an IL-10 + endotoxin group. E. coli endotoxin (10 mg/kg) was injected intraperitoneally 5 minutes after saline or IL-10 (1250 U, 0.25 mg) injection. The force-frequency curves, twitch kinetics and fatigability were measured at 0 and 4 hours after endotoxin injection. In the saline + endotoxin group, the force-frequency curves and half relaxation time were significantly decreased at 4 hours (p0.001 and p0.05, respectively) compared to those at 0 hour. In the IL-10 + endotoxin group, the decrement in the force-frequency curves by endotoxin was prevented at 4 hours compared to that at 0 hour. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry showed positive staining in the saline + endotoxin at 4 hours, but there was no significant staining at 0 or 4 hours in the IL-10 + endotoxin group. These data suggest that IL-10 prevents the deterioration of contraction induced by endotoxin by inhibiting nitric oxide production in the diaphragm muscle.

We examined whether streptozotocin (STZ)-induced diabetic rats have an impairment in diaphragm contractility, and if so, whether N-acetylcysteine (NAC), a nonspecific antioxidant, prevents this impairment. First, diaphragm contractility, assessed by tension-frequency relationships and twitch kinetics in in vitro diaphragm strip preparations of Wistar rats, was obtained on Days 3 and 7 after administration of STZ of 30 or 60 mg/kg body weight, and compared with that of the control group. Second, NAC at 500 mg/kg body weight or vehicle solution was administered orally every day in rats treated with STZ at 60 mg/kg body weight, and diaphragm function on Day 7 after starting NAC treatment was compared between vehicle control and STZ-treated groups. We found that diaphragm function in STZ-treated rats, which had hyperglycemia, decreased in a dose- and time-dependent manner. NAC inhibited the decrease in diaphragm contractility in STZ-treated rats without reducing blood glucose. These findings suggest that the loss of diaphragm function in STZ-induced diabetic rats is not directly related to hyperglycemia. The data are consistent with secondary alterations of normal cytokine signaling or changes in the redox state of the cell, both of which could be affected by NAC treatment.

We studied gene expression and production of TNF-alpha in the diaphragm tissue and changes of muscle contractile properties after endotoxin injection (Escherichia coli, 20 mg/kg) in 88 rats. We assessed the muscle contractile properties by force-frequency curves and twitch kinetics using dissected diaphragm muscle strips. The peak tensions of force-frequency curves decreased from control values (2.15 +/- 0.2 kg/cm2) up to 4 h (0.81 +/- 0.17, p < 0.001), and then increased at 6 h (1.36 +/- 0.19, p < 0.05) after endotoxin injection. The cytotoxic activity on L929 cells in arterial blood samples maximally increased at 2 h (p < 0.001), then decreased to 6 h (p < 0.05). TNF-alpha mRNA in diaphragm tissue was detected by Northern blot method at 1 and 1.5 h, and the immunolocalization of TNF-alpha was evaluated at 2 and 4 h by immunohistochemistry in the muscle tissues. Furthermore, preinjection with anti-m TNF-alpha antibody prevented the decrement of force-frequency curves after endotoxin injection of 10 microliters/kg. From this evidence that TNF-alpha gene expression and production occurred in the diaphragm tissue, but anti-m TNF-alpha antibody preinjection prevented the deterioration of diaphragm muscle contractile properties, we suggest that TNF-alpha may act on muscle cells extracellularly.

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.

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 Ca2+([Ca2+]i) were measured in rat ventricular trabeculae. A “regional” or “global” increase in [K+]owas 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 [Ca2+]i(CaCW) during Ca2+waves was measured (24°C, 3.0 mmol/l [Ca2+]o). A regional increase in [K+]ocaused nonuniform [Ca2+]iand contraction. In the presence of isoproterenol, the regional increase in [K+]oinduced sustained arrhythmias in 10 of 14 trabeculae, whereas the global increase did not induce such arrhythmias. During sustained arrhythmias, Ca2+surged within the jet-exposed region. In the absence of isoproterenol, the regional increase in [K+]oincreased CaCW, whereas the global increase decreased it. This increase in CaCWwith the regional increase in [K+]owas 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+]oinduces sustained arrhythmias, probably due to nonuniform excitation-contraction coupling. The same mechanism may underlie arrhythmias during ischemia.

We measured the oxygen consumption ( V ˙ o 2 ) of respiratory muscles in 8 COPD patients and 12 age-matched healthy subjects using a closed circuit device which allows a continuous increase in external dead space and is equipped with a 9-L Collins spirometer. Furthermore, we measured simultaneously mouth occlusion pressure at 0.1 s of inspiration (P0.1), minute ventilation (VE), and other ventilatory parameters during the measurement of total V ˙ o 2 ( V ˙ o 2 tot). We found that the logarithm of V ˙ o 2 tot (log V ˙ o 2 tot) had a good correlation with V ˙ E in both groups. The mean slope of the regression line of log V ˙ o 2 tot and V ˙ E (Δlog V ˙ o 2 tot/Δ V ˙ E) of COPD patients was significantly higher than that of normal subjects (p < 0.001). However, the mean Y-intercept (metabolic V ˙ o 2 [ V ˙ o 2 met]) of the regression lines did not differ between the two groups. The P0.1 in COPD patients was higher than that in normal subjects at the corresponding dead space loading. However, the VE did not differ between the two groups except for at rest and the first 1 min after dead space loading. These results suggest that the V ˙ o 2 of respiratory muscles in patients with COPD is higher at given ventilation compared with that in age-matched normal subjects and that this increased V ˙ o 2 partly may be due to an augmented ventilatory drive.

SHINDOH, C., HIDA, W., KUROSAWA, H., EBIHARA, S., KIKUCHI, Y., TAKISHIMA, T. and SHIRATO, K. Effects of Unilateral Phrenic Nerve Denervation on Diaphragm Contractility in Rat. Tohoku J. Exp. Med., 1994, 173 (3), 291-302- We examined the early effects of phrenic nerve denervation on the diaphragm muscle 1, 3, 7 and 14 days after unilateral denervation in rats. In the denervated hemidiaphragms, force frequency curves at 3, 7 and 14 days decreased significantly by 51%, 50% and 38% respectively of the peak tension of the force frequency curves of the diaphragms of rats with sham operation. Twitch tensions increased significantly at 14 days, and contraction times and half relaxation times slowed significantly at 3, 7 and 14 days. The tensions of denervated diaphragms at 5min during the fatigue runs was significantly increased at 14 days. As determined by histological staining, the mean cross sectional area of fast-twitch fibers (type II) decreased significantly from 2, 742 (sham) to 1, 599μm2 (14 days), but that of the slow-twitch fibers (type I) did not change significantly during the same period. These findings suggest that, during the first two weeks of denervation, fast twitch fibers (type II) atrophy more rapidly than slow twitch fatigue resistant fibers (type I), as confirmed by the contractile properties and histological findings.

A 52-year-old man was given a diagnosis of ulcerative colitis and treated with mesalazine for 7.5 years. However, the unusually long administration of mesalazine induced lung injury and the patient complained of a dry cough and dyspnea on limited exertion. Infiltrative shadows were observed in bilateral lung fields on a chest radiograph and computed tomographic images. The histological findings obtained by transbronchial biopsy and bronchoalveolar lavage showed organizing pneumonia. His drug-induced lymphocyte stimulation test (DLST) for mesalazine was positive. Improvements in his clinical symptoms and radiographic abnormalities occurred spontaneously after the discontinuation of mesalazine. This case indicates that the long-term administration of mesalazine may lead to an adverse pulmonary reaction.

Background: Triggered arrhythmias arise from delayed afterdepolarizations (DADs), with Ca2+ waves playing an important role in their formation. In ventricular hypertrophy, however, it remains unclear how Ca2+ waves change their propagation features and affect arrhythmogenesis. We addressed this important issue in a rat model of hypertrophy. Methods and Results: Rats were given a subcutaneous injection of 60mg/kg monocrotaline (MCT-rats) or solvent (Ctr-rats). After 4 weeks, MCT-rats showed high right ventricular (RV) pressure and RV hypertrophy. Trabeculae were dissected from 36 right ventricles. The force was measured using a silicon strain gauge and regional intracellular Ca2+ ([Ca2+]i) was determined using microinjected fura-2. Reproducible Ca2+ waves were induced by stimulus trains (2Hz, 7.5s). MCT-rats showed a higher diastolic [Ca2+]i and faster and larger Ca2+ waves (P

Background: Although oxitropium bromide is used clinically as an anticholinergic drug (i.e., parasympathetic antagonist) to relax airway smooth muscle, we examined whether it has or does not have any effects on diaphragm muscle. Methods: Three treatment sets, an oxitropium bromide inhalation only group, an oxitropium bromide inhalation plus endotoxin injection group (in vivo) and an oxitropium bromide incubation group (in vitro) were studied as to diaphragm muscle contractile properties. Results: Oxitropium bromide inhalation shifted force-frequency curves upward at 2 h after inhalation (p < 0.05) and inhibited the decrease of force-frequency curves due to endotoxin injection in vivo. Incubation with oxitropium bromide of untreated diaphragm muscle and diaphragm muscle injected with endotoxin did not increase the force-frequency curves dose-dependently in vitro; however, it caused both types of muscle to be fatigue resistant. Conclusions: We speculate that the increment of muscle contractility with the inhalation of oxitropium bromide was induced by the antagonization of musucarinic acetylcholine receptors (mAChR). In addition, the changes of fatigue resistance provoked by oxitropium bromide, which also is speculated to antagonize mAChR, may be beneficial in the treatment of patients with COPD.

Tulobuterol, a sympathomimetic drug used as a transdermal patch, increases normal diaphragm muscle strength. Because diaphragm muscle weakness (i.e. decrease of contraction) is a feature of bronchial asthma and sepsis, we examined the in vitro and in vivo effects of tulobuterol on the contractility of diaphragm muscles prepared from mice treated with endotoxin. We measured contractile parameters of force-frequency curves and twitch kinetics using untreated or treated diaphragm muscles at 0 (E0) and 4 (E4) hours after E. coli endotoxin (20 mg/kg) administration. The force-frequency curve of E4 diaphragm muscle was decreased from that of E0 diaphragm muscle (p < 0.001). E4 diaphragm muscle was incubated in an organ buffer containing 10(-7) or 10(-5) M concentrations of tulobuterol for 1 h (in vitro). The force-frequency curves of both 10(-7) (p < 0.01) or 10(-5) M (p < 0.001) tulobuterol concentrations shifted significantly upward from those of no tulobuterol, indicating that tulobuterol can recover the diaphragm muscle contractility that was decreased by endotoxin. In the in vivo treatment, E0 and E4 diaphragm muscles were analyzed at 0, 12, and 24 h after transdermal tulobuterol treatment. The force-frequency curves of E0 and E4 diaphragm muscles at three time points were not significantly changed each other, indicating that tulobuterol patch restores the muscle contractility. Thus, diaphragm muscle contractility was maintained during 4 h of endotoxin administration with tulobuterol patch for over 24 h. We suggest that this treatment of bronchial asthma may protect against endotoxin contained in inhaled house dust.

The purposes of the present study were to determine the changes in functional residual capacity (FRC) during inspiratory loading and to examine their mechanisms. We studied seven normal subjects seated in a body plethysmograph. In both graded inspiratory elastic (35, 48, and 68 cmH2O/l) and resistive (21, 86, and 192 cmH2O.l-1.s) loading, FRC invariably decreased from control FRC and phasic expiratory activity increased. The reduction in FRC was greater with greater loads. A single inspiratory effort against an inspiratory occlusion at three different target mouth pressures (-25, -50, and -75 cmH2O) and durations (1, 2, and 5 s) also resulted in a decrease in FRC with an increase in expiratory electromyogram activity in the following expiration. The decrease in FRC was greater with greater target pressure and duration. This decrease in FRC is qualitatively similar to that during inspiratory loaded breathing, and we suspect that the same mechanisms are at work. Because neither vagal nor chemoreceptor reflex can account for these responses, we suspect conscious awareness of breathing or behavioral control to be responsible. In an additional study, the sensation of discomfort of breathing during elastic loading decreased with a decrease in FRC. These results suggest that the reduced FRC may be due to behavioral control of breathing to reduce the sensation of dyspnea during inspiratory loading.

Clinically, patients suffering from bronchial asthma are often treated transdermally with tulobuterol patches to dilate the bronchi. Tulobuterol, a synthetic beta(2) agonist, is also thought to act as a diaphragm muscle contractor, like other beta(2) sympathomimetic drugs. However, it has not been clarified that transdermal treatment with tulobuterol influences diaphragm muscle contractility. We therefore examined its effects on contractile properties of such muscles obtained from BALB/c mice. Two systems, a tulobuterol incubation group (in vitro) and a tulobuterol transdermal treatment group (in vivo), were employed. In both groups, the contractile properties of the dissected diaphragm muscles were measured by field stimulation in an organ bath. In the incubation group, the diaphragm muscle of untreated mice was incubated in an organ buffer at 10(-7), 10(-6), or 10(-5) M tulobuterol for 1 hr and then measured for contractility. Tulobuterol significantly increased force-frequency curves at a concentration of 10(-5) M at 1 (p0.01), 30, 50, 70, 100, and 120 Hz (p0.05, each) compared with the values at 0 M. In the transdermal treatment group, the diaphragm muscle was dissected from animals at 1, 4, 8, 12, or 24 hrs after treatment and measured for contractility, showing that the force-frequency curves were significantly increased and maintained from 4 to 24 hrs (each p0.01 as compared with the sham-treated group). We suggest that transdermal tulobuterol treatment in case of bronchial asthma is useful not only for bronchial dilatation, but also for increasing diaphragm muscle contractility.

To find predicted values of respiratory function tests in young persons in Japan, we measured slow vital capacity and forced vital capacity in 1,141 subjects aged from 10 to 20 years. The values obtained from 636 persons (363 males and 273 females) who were not smokers or had no suspected rhinitis or asthma were analyzed. Although FEV1% by the Gaensler and Tiffeneau methods were almost constant regardless of age, all other values of respiratory function tests increased with age and then reached a plateau level in late teens. Excluding the Gaensler and Tiffeneau FEV1%, in a single regression analysis using gender, age, and height, both the contribution ratio and regression coefficient were the highest using height, followed by age. In addition, the result was similar with multiple regression analysis. Therefore, for a young person aged from 10 to 20 years, body height is the most important predictor variable of respiratory function tests, compared to gender and age. For each respiratory function test, we showed the prediction relation according to gender obtained from multiple linear regression analysis using 2 variables of height and age.

We performed a transsubxiphoidal LV puncture (TSLVP) to evaluate left ventricular function in 21 patients with both mechanical prosthetic aortic and mitral valves and successfully obtained hemodynamic information on each patient. Analyzing cardiac hemodynamic information and ventriculographic findings obtained with TSLVP, we concluded that seven of the patients required repair of their prosthetic valves. Five of these seven patients agreed to replacement of their valves, whereas two did not. TSLVP was performed adequately and safely without severe complications, suggesting that this maneuver is easier than that of transapical LV puncture and should be recommended for the assessment of left ventricular hemodynamic functions instead of the transapical LV puncture, especially in patients requiring replacement of two valves.

The effects of nitric oxide on diaphragm contraction after endotoxin administration were studied in Wistar rats. The animals were divided into seven treatments: a saline-injected group as control, three groups injected with l-NAME (0.01, 0.1, 1 mg/kg) and three groups injected with l-arginine (1, 10, 100 mg/kg). Escherichia coli endotoxin was injected into the peritoneal cavity 15 min later. Twitch kinetics and force-frequency curves were measured 0, 2, and 4 hr after endotoxin injection. In the control group, the force-frequency curves significantly decreased from 0 hr to 4 hr. In the l-NAME group, the force-frequency curves at 4 hr showed significant increases in a dose-dependent manner. In the l-arginine group, the force-frequency curve with 100 mg/kg at 4 hr showed a significant increase. There was no consistent change in the contraction time, half relaxation time, or fatiguability. NADPH diaphorase histochemistry performed on diaphragm muscle samples 4 hr after endotoxin injection showed positive in the control and l-arginine group, but was only weakly observed in l-NAME group. These data suggest that nitric oxide contributes to the endotoxin induced diaphragm contractile deterioration.