Masayoshi Kuwahara

Power Spectral Analysis of Heart Rate Variability as a New Method for Assessing Autonomic Activity in the Rat

The authors studied power spectral analysis of heart rate variability in the rat, hypothesizing that the quantitative information provided by this analysis reflects the interaction between sympathetic and parasympathetic regulatory activities. For this purpose, an electrocardiogram was recorded from conscious and unrestrained Wistar rats (Nippon, Shizuoka) (12-16 weeks old) by a telemetry system and analyzed by a power spectrum. Because it was thought that the electrocardiogram recorded by the telemetry system could provide more reliable data to assess autonomic nervous activity than the tethering system, the telemetry recording system was used. There were two major spectral components in the power spectrum at low frequency (LF) (0.6 Hz) and high frequency (HF) (approximately 1.4 Hz). On the basis of these data, the authors defined two frequency bands of interest: LF (0.04-1.0 Hz) and HF (1.0-3.0 Hz). The power of LF was higher than that of HF in the normal rat. Atropine (2 mg/kg intraperitoneally) significantly reduced both HF and LF power. Propranolol (4 mg/kg intraperitoneally) also significantly reduced LF power; however, it had no significant effect on HF power. Thus, this study in the rat confirmed earlier observations in the conscious dog and human. Furthermore, the decrease in the parasympathetic mechanism produced by atropine was reflected by a slight increase in the LF/HF ratio. The LF/HF ratio appeared to follow the reductions of sympathetic activity produced by propranolol. From these results, the LF/HF ratio seemed to be a convenient index of parasympathetic and sympathetic interactions in the rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Statin Protects Endothelial Nitric Oxide Synthase Activity in Hypoxia-Induced Pulmonary Hypertension

Objective—We investigated the effects of fluvastatin on hypoxia-induced (1 to 3 weeks, 10% O2) pulmonary hypertension with focus on endothelial nitric oxide synthase (eNOS) activity. Methods and Results—Oral fluvastatin treatment (1 mg/kg daily) prevented the causing and progression of pulmonary hypertension as determined by the right ventricular pressure, right ventricular hypertrophy, and muscularization of pulmonary artery. We also revealed that fluvastatin treatments prevented the hypoxia-induced decrease in cGMP production in the rat lung and restored the endothelium-dependent relaxation in the pulmonary artery. We revealed that this beneficial effect was not dependent on the increase in eNOS mRNA or protein expression, but was dependent on the inhibition of the eNOS-tight coupling with caveolin-1, the eNOS dissociation from heat shock protein 90, and the decrease in eNOS Ser1177–phosphorylation induced by hypoxia. Furthermore, in a whole-mount immunostaining the hypoxia-induced eNOS protein condensation with caveolin-1 of pulmonary endothelial cells was restored by the fluvastatin-treatment. Conclusion—These results suggest that the fluvastatin exerts beneficial effects on chronic hypoxia-induced pulmonary hypertension by protecting against the eNOS activity at the post-transcriptional level.

Assessment of autonomic nervous function by power spectral analysis of heart rate variability in the horse

We studied power spectral analysis of heart rate (HR) variability in the horse, with the hypothesis that the quantitative information provided by the spectral analysis of HR variability reflects the interaction between sympathetic and parasympathetic regulatory activities. For this purpose, electrocardiogram, blood pressure (BP) and respiratory (Resp) waveform were simultaneously recorded from Thoroughbred horses (3-5 years old) and analyzed by power spectrum. There were two major spectral components at low-frequency (LF) and high-frequency (HF) bands for HR variability. The peak of Resp variability clearly occurred at the HF range. In contrast to Resp variability, the power spectra of BP variability occurred at lower frequencies. The maximum coherence between HR and Resp variabilities and HR and BP variabilities occurred at approximately 0.15 and approximately 0.03 Hz, respectively. These relationships were similar to the ensemble spectra. On the basis of these data, we have defined two frequency bands of interest: LF (0.01-0.07 Hz) and HF (0.07-0.6 Hz). Therefore, we believe that power spectral analysis of HR variability provides a very powerful technique for assessing autonomic nervous activity in the horse.

Autonomic nervous function in mice and voles (Microtus arvalis): investigation by power spectral analysis of heart rate variability

We have studied the autonomic nervous function in voles (Microtus arvalis) and mice. For this purpose, ECGs were recorded from conscious and unrestrained voles and mice using radiotelemetry and the autonomic nervous function was investigated by the power spectral analysis of heart rate variability. Heart rate in voles was lower than mice and the coefficient of variance was larger in voles. In the power spectra of voles and mice, there were two major spectral components with the high frequency (HF) peak generally appearing between 2.0 and 4.0 Hz, and the low frequency (LF) peak appearing below 0.6 Hz. On the basis of this data, we set the two frequency bands as LF (0.1-1.0 Hz) and HF (1.0-5.0 Hz) to evaluate autonomic nervous function. The LF and HF powers were larger in voles than mice. The LF/HF ratio was thought to provide a convenient index of autonomic nervous balance and was smaller in voles than mice. The LF powers in both species were reduced by atropine, but propranolol reduced the LF power only in mice. The HF power was reduced by atropine only in voles. The intrinsic heart rate produced by a double blockade with atropine and propranolol in voles was almost the same as control levels, but in mice was lower than controls. The ratio of the LF and HF powers by a double blockade were almost the same as those of the administration of atropine in voles, but nearer to propranolol in mice. These results suggested that the parasympathetic nervous function was predominant in voles, but the sympathetic one was predominant in mice.

Diurnal Variation of Autonomic Nervous Activity in the Rat Investigation by Power Spectral Analysis of Heart Rate Variability

We studied the diurnal variations of autonomic nervous function in rats. For this purpose, a long-term electrocardiogram (ECG) was recorded from conscious and unrestrained rats using a telemetry system, and the autonomic nervous function was investigated by the power spectral analysis of heart rate variability. No arrhythmias were observed in the ECG of any of the rats. Nocturnal patterns, in which the values of heart rate in the dark phase (2000-0600) were higher than those in the light phase (0600-2000), were observed. All normal rats shared a characteristic pattern in their power spectrum analysis. Both low-frequency and high-frequency power in the light phase were higher than those in the dark phase. However, these differences were not statistically significant (P > .05). The low frequency to high frequency ratio also showed a nocturnal pattern. The value in the dark phase was significantly higher (P < .05) than that in the light phase. These results suggest that the sympathetic nervous activity is predominant in the dark phase in rats. Therefore, we believe that this information may be useful for future biobehavioral studies.

Role of Reactive Oxygen Species on Diesel Exhaust Particle-Induced Cytotoxicity in Rat Cardiac Myocytes

Exposure to air pollution containing diesel exhaust particles (DEP) is associated with an increase in mortality rate attributed to cardiovascular diseases, but the mechanisms by which DEP produces adverse cardiovascular effects at the cellular level are not elucidated. This study investigated the cytotoxic mechanisms underlying DEP-induced neonatal rat cardiac myocytes effects in vitro, focusing on the role of reactive oxygen species (ROS). DEP extracts (DEPE) damaged cells in a concentration- and a time-dependent manner. Lactate dehydrogenase activity leaked to medium was also increased in a concentration-dependent manner after 24 h of DEPE exposure. DEPE-induced cytotoxicity was markedly reduced by treatment with superoxide dismutase, catalase, and N-(2-mercaptopropionyl)-glycine. Furthermore, superoxide was produced from both DEPE and myocardial cells. These results suggest that ROS such as superoxide, hydrogen peroxide, and hydroxyl radical are involved in DEPE-induced cardiac cell damage.

Opioid mediated suppressive effect of milk-derived lactoferrin on distress induced by maternal separation in rat pups

The present study assessed the effects of bovine milk-derived lactoferrin (bLf) on distress activities induced by maternal separation in 5- to 18-day-old rat pups. The rat pups were injected with BSA (100 mg/kg, i.p.; control) or bLf (100 mg/kg, i.p.) 30 min before the behavioral test. Distress activity was estimated by means of recording body movements or ultrasonic vocalizations (USVs). After 5 min of maternal separation, bLf significantly (P<0.01) suppressed body movements, particularly in the 10-day-old pups. This suppressive effect of bLf was reversed by pretreatment with naloxone, CTOP, and norBNI at doses of 0.1-1 mg/kg. Additionally, USVs were also suppressed by bLf, which was reversed by pretreatment with naloxone. Inhibition of nitric oxide synthase (NOS) with nitro-L-arginine methyl ester (L-NAME) dose dependently (3-10 mg/kg) suppressed separation-induced USV production in 10-day-old pups. Interestingly, the suppressive effect of bLf was completely reversed by pretreatment with a low dose (1 mg/kg) of L-NAME, which did not affect the USVs with single application. These findings demonstrate that milk-derived bLf suppresses distress induced by maternal separation via an opioid-mediated mechanism. Furthermore, bLf possibly activates NOS, and an elevated nitric oxide may cause some modification of the opioid system.

Nuclear accumulation of androgen receptor in gender difference of dilated cardiomyopathy due to lamin A/C mutations

AIMS Dilated cardiomyopathy (DCM) is characterized by ventricular dilation associated with systolic dysfunction, which could be caused by mutations in lamina/C gene (LMNA). LMNA-linked DCM is severe in males in both human patients and a knock-in mouse model carrying a homozygous p.H222P mutation (LmnaH222P/H222P). The aim of this study was to investigate the molecular mechanisms underlying the gender difference of LMNA-linked DCM. METHODS AND RESULTS A whole-exome analysis of a multiplex family with DCM exhibiting the gender difference revealed a DCM-linked LMNA mutation, p.R225X. Immunohistochemical analyses of neonatal rat cardiomyocytes expressing mutant LMNA constructs and heart samples from the LMNA-linked DCM patients and LmnaH222P/H222P mice demonstrated a nuclear accumulation of androgen receptor (AR) and its co-activators, serum response factor, and four-and-a-half LIM protein-2. Role of sex hormones in the gender difference was investigated in vivo using the LmnaH222P/H222P mice, where male and female mice were castrated and ovariectomized, respectively, or treated with testosterone or an antagonist of AR. Examination of the mice by echocardiography, followed by the analyses of histological changes and gene/protein expression profiles in the hearts, confirmed the involvement of testicular hormone in the disease progression and enhanced cardiac remodelling in the LmnaH222P/H222P mice. CONCLUSION These observations indicated that nuclear accumulation of AR was associated with the gender difference in LMNA-linked DCM.

Antihypertensive effect of angiotensin I-converting enzyme inhibitory peptides derived from hemoglobin

From proteolytic digest of swine hemoglobin, we isolated four peptide, E-1 (Phe-Gln-Lys-Val-Val-Ala), E-2 (Phe-Gln-Lys-Val-Val-Ala-Gly), peptide 30-3 (Phe-Gln-Lys-Val-Val-Ala-Lys) and H-1 (Gly-Lys-Lys-Val-Leu-Gln). These peptides inhibited angiotensin I-converting enzyme activity with an IC50 of 5.8, 7.4, 2.1 and 1.9 microM, respectively. Oral administration of 50 mg/kg E-1 and 50 mg/kg H-1 decreased blood pressure in spontaneously hypertensive rats. In normotensive rats, oral administration of 500 mg/kg E-1 and 500 mg/kg H-1 inhibited the pressor effect of i.v. administrated 300 ng/kg angiotensin I, possibly by inhibiting its conversion to angiotensin II. These results suggest that these peptides are orally effective inhibitors of angiotensin I-converting enzyme that have a hypotensive effect.

ECG changes during furosemide-induced hypokalemia in the rat

Electrolyte abnormalities have become an increasingly important cause of arrhythmias owing to the widespread use of high-potency diuretics. Hypokalemia is one of the common complications of diuretic use. Although some studies of hypokalemia induced by furosemide as well as of potassium-deficient diets in the rat have been reported, the electrocardiographic (ECG) changes during hypokalemia in the rat are poorly understood. This study was designed to examine such changes. For this purpose, hypokalemia was induced by furosemide administration, and the diagnostic criteria for ECG manifestations of hypokalemia were determined. During hypokalemia, conduction in most parts of the heart was suppressed to an extent depending on plasma potassium concentration. Prolongation of the QT interval was also observed, which agrees with findings in humans and dogs. Furthermore, prolonged durations of the P wave and QRS complex were observed during hypokalemia in the rat. The extent of alteration of the PR interval induced by hypokalemia was less significant than that of P wave and QRS complex durations. These results suggest that the excitabilities of the myocardium in the atria and ventricles may be affected by extracellular potassium level rather than by the atrioventricular conduction system in the rat. Wave amplitude, except that of the P wave, was decreased by severe hypokalemia. These changes were not dependent on the plasma potassium concentration. Typical T wave changes observed with hypokalemia in humans and dogs did not occur in the rat. The ECG manifestations of acute hypokalemia in the rat did not include the typical T wave changes seen in species with ST-segment type ECGs; however, other ECG parameter changes occurring with hypokalemia were qualitatively similar to those in other species. These results may be useful for testing the toxicity of potassium-depleting drugs in the rat.