Beneficial Effects of Low-Intensity Pulsed Ultrasound Therapy on Right Ventricular Dysfunction in Animal Models.

Right ventricular failure (RVF) is a leading cause of death in patients with pulmonary hypertension; however, effective treatment remains to be developed. We have developed low-intensity pulsed ultrasound therapy for cardiovascular diseases. In this study, we demonstrated that the expression of endothelial nitric oxide synthase (eNOS) in RVF patients was downregulated and that eNOS expression and its downstream pathway were ameliorated through eNOS activation in 2 animal models of RVF. These results indicate that eNOS is an important therapeutic target of RVF, for which low-intensity pulsed ultrasound therapy is a promising therapy for patients with RVF.

Mitochondrial connexin43 and mitochondrial KATP channels modulate triggered arrhythmias in mouse ventricular muscle.

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. 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 the right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from the 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 methyl ester (TMRM), and Ca2+ spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Ca2+ with rhod-2 in trabeculae. Diazoxide was used to activate 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, and Ca2+ spark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased in cCx43-/- mice. Most of these changes were suppressed by diazoxide. In addition, in cCx43-/- mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca2+]o,min. These results suggest that Cx43 deficiency activates Ca2+ leak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca2+, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial KATP channels in mouse hearts.

Roles of stretch-activated channels and NADPH oxidase 2 in the induction of twitch contraction by muscle stretching in rat ventricular muscle.

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 [Ca2+]i were measured. Various degrees of stretching from the SL of 2.0 µm were applied 0.5 s after the last stimulus of the electrical train with 0.4-s intervals for 7.5 s. The SLtwitch was defined as the minimal SL at which twitch contraction was induced by the stretching. Muscle stretching induced twitch contraction with a threshold of SL at 0.4-s stimulus intervals ([Ca2+]o = 0.7 mmol/L). The SLtwitch was not changed by increasing the stimulus intervals and [Ca2+]o and by adding 1 µmol/L isoproterenol. The SLtwitch was not changed by adding 10 µmol/L Gd3+, 100 µmol/L or 200 µmol/L streptomycin, and 5 µmol/L GsMTx4. The SLtwitch was not changed by adding 1 µmol/L ryanodine and 3 µmol/L diphenyleneiodonium chloride. In contrast, the SLtwitch was increased by elevating extracellular K+ from 5 to 10 mmol/L and by adding the stretching during the refractory period of membrane potential. The addition of the stretching-induced twitch contraction more frequently induced arrhythmias. These results suggest that muscle stretching can induce twitch contraction with a threshold of SL and concern the occurrence of arrhythmias and that SACs and X-ROS signaling play no roles in the induction.

Effect of transient elevation of glucose on contractile properties in non-diabetic rat cardiac muscle.

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.

Enumeration of Legionella CFU by colony hybridization using specific DNA probes.

Oligonucleotide probes and colony hybridization (CH) were applied to enumerate organisms of the genus Legionella in cooling tower water. The CH counts indicated almost the same results as CFU counts in cultivated samples derived from the water. It was concluded that it is possible to substitute the CH procedure for the conventional one.