Katsuya Onishi

Activation of RhoA and Inhibition of Myosin Phosphatase as Important Components in Hypertension in Vascular Smooth Muscle

Abstract— Two mechanisms are proposed to account for the inhibition of myosin phosphatase (MP) involved in Ca2+ sensitization of vascular muscle, ie, phosphorylation of either MYPT1, a target subunit of MP or CPI-17, an inhibitory phosphoprotein. In cultured vascular aorta smooth muscle cells (VSMCs), stimulation with angiotensin II activated RhoA, and this was blocked by pretreatment with 8-bromo-cGMP. VSMCs stimulated by angiotensin II, endothelin-1, or U-46619 significantly increased the phosphorylation levels of both MYPT1 (at Thr696) and CPI-17 (at Thr38). The angiotensin II-induced phosphorylation of MYPT1 was completely blocked by 8-bromo-cGMP or Y-27632 (a Rho-kinase inhibitor), but not by GF109203X (a PKC inhibitor). In contrast, phosphorylation of CPI-17 was inhibited only by GF109203X. Y-27632 dramatically corrected the hypertension in N&ohgr;-nitro-l-arginine methyl ester (L-NAME)-treated rats, and this hypertension also was sensitive to isosorbide mononitrate. The level of the active form of RhoA was significantly higher in aortas from L-NAME-treated rats. Expression of RhoA, Rho-kinase, MYPT1, CPI-17, and myosin light chain kinase were not significantly different in aortas from L-NAME-treated and control rats. Activation of RhoA without changes in levels of other signaling molecules were observed in three other rat models of hypertension, ie, stroke-prone spontaneously hypertensive rats, renal hypertensive rats, and DOCA-salt rats. These results suggest that independent of the cause of hypertension, a common point in downstream signaling and a critical component of hypertension is activation of RhoA and subsequent activation of Rho-kinase.

Tenascin-C may aggravate left ventricular remodeling and function after myocardial infarction in mice

Tenascin-C (TN-C) is an extracellular matrix glycoprotein with high bioactivity. It is expressed at low levels in normal adult heart, but upregulated under pathological conditions, such as myocardial infarction (MI). Recently, we (Ref. 34) reported that MI patients with high serum levels of TN-C have a greater incidence of maladaptive cardiac remodeling and a worse prognosis. We hypothesized that TN-C may aggravate left ventricular remodeling. To examine the effects of TN-C, MI was induced by ligating coronary arteries of TN-C knockout (KO) mice under anesthesia and comparing them with sibling wild-type (WT) mice. In WT+MI mice, TN-C expression was upregulated at day 1, peaked at day 5, downregulated and disappeared by day 28, and the molecule was localized in the border zone between intact myocardium and infarct lesions. The morphometrically determined infarct size and survival rate on day 28 were comparable between the WT+MI and KO+MI groups. Echocardiography and hemodynamic analyses demonstrated left ventricular end-diastolic diameter, myocardial stiffness, and left ventricular end-diastolic pressure to be significantly increased in both WT+MI and KO+MI mice compared with sham-operated mice. However, end-diastolic pressure and dimension and myocardial stiffness of KO+MI were lower than those of the WT+MI mice. Histological examination revealed normal tissue healing, but interstitial fibrosis in the residual myocardium in peri-infarcted areas was significantly less pronounced in KO+MI mice than in WT+MI mice. TN-C may thus accelerate adverse ventricular remodeling, cardiac failure, and fibrosis in the residual myocardium after MI.

Serum intact parathyroid hormone levels predict hospitalisation for heart failure

Objective: To assess whether circulating levels of intact parathyroid hormone (intact PTH) in outpatients predict hospitalisation for heart failure (HF). Methods: Eighty-eight consecutive outpatients with HF were enrolled in the study. The independent association between intact PTH and hospitalisation for HF was assessed using Cox regression analysis. Results: Mean (SD) serum intact PTH levels significantly increased as New York Heart Association classes increased (I: 40 (21), II: 55 (24), III: 76 (46), IV: 131 (45) pg/ml). The receiver operating characteristic (ROC) curves showed intact PTH levels ⩾47 pg/ml to be the optimal cut-off points for hospitalisation for HF, with sensitivity 87%, specificity 71% and area under the ROC curve 0.82 (95% CI 0.72 to 0.91). After adjustment for variables accepted to be predictors for hospitalisation due to HF (age, gender, hypertension, diabetes mellitus, atrial fibrillation, ischaemic heart disease, left ventricular ejection fraction, B-type natriuretic peptide, estimated glomerular filtration rate and cardiac drugs), intact PTH levels ⩾47 pg/ml were associated with a hazard ratio of 7.13 for hospitalisation for HF (95% CI 1.79 to 28.4). Conclusion: Serum intact PTH levels obtained in outpatients with HF were shown to be an independent predictor of hospitalisation for HF.

Eplerenone attenuates myocardial fibrosis in the angiotensin II-induced hypertensive mouse: involvement of tenascin-C induced by aldosterone-mediated inflammation.

Tenascin-C is an extracellular matrix glycoprotein that is supposed to be a profibrotic molecule in various fibrogenic processes. To elucidate its significance for myocardial fibrosis in the hypertensive heart, we used a mouse model with infusion of angiotensin II and examined results by histology, immunohistochemistry, in situ hybridization, and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Angiotensin II treatment elevated blood pressure and expression of tenascin-C by interstitial fibroblasts in perivascular fibrotic lesions, and angiotensin II infusion caused accumulation of macrophages. It also upregulated expression of collagen Iα2; IIIα1; and proinflammatory/profibrotic mediators including transforming growth factor beta (TGFβ), platelet-derived growth factor alpha (PDGF-A), PDGF-B, and PDGF-receptor α, but not IL-1β and PDGF-receptor β, in the myocardium. Treatment with an aldosterone receptor antagonist, eplerenone, significantly attenuated angiotensin II-induced fibrosis, expression of tenascin-C, and inflammatory changes without affecting the blood pressure level. In vitro, neither eplerenone nor aldosterone exerted any influence on tenascin-C expression of cardiac fibroblasts, whereas angiotensin II, TGF-β1, and PDGF significantly upregulated expression of tenascin-C. These results suggest that, in the angiotensin II-induced hypertensive mouse heart: (1) tenascin-C may be involved in the progression of cardiac fibrosis and (2) aldosterone may elicit inflammatory reactions in myocardium, which might, in turn, induce tenascin-C synthesis of fibroblasts through at least 2 pathways mediated by TGF-β and PDGF-A-B/PDGF-receptor α.

The Negative Inotropic Effect of β3-Adrenoceptor Stimulation in the Beating Guinea Pig Heart

Although beta3-adrenoceptors (ARs) have been extensively characterized in brown and white adipocytes, their actions in the beating heart are unclear. We examined the effects of a beta3-AR agonist, BRL37344, on cardiac function and calcium transients in Langendorff-perfused guinea pig hearts by simultaneously measuring left ventricular (LV) pressure and Ca2+-dependent indo-1 fluorescence. BRL37344 induced a dose-dependent negative inotropic effect at concentrations from 10(-11) to 10(-8) M. Maximally, LV developed pressure decreased to 80+/-2%, +dP/dt to 81+/-2%. and -dP/dt to 81+/-3% of their respective control values (p < 0.01). The amplitude of the Ca2+ transient also decreased (to 92+/-3% of the control level; p < 0.01). The BRL37344 dose-response curve was not altered by nadolol (10(-5) M), a potent beta1- and beta2-AR antagonist, but completely suppressed by bupranolol (10(-6) M), a potent beta1-, beta2- and beta3-AR antagonist. To assess the potential role of a nitric oxide synthase (NOS) pathway, we determined whether the NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), modified the contractile response to BRL37344. L-NAME (10(-7) and 10(-4) M) attenuated the negative inotropic effects on LV developed pressure by 35 and 50%, suggesting that beta3-AR stimulation induces a negative inotropic effect on guinea pig hearts partly through a decrease in the Ca2+ transient and partly by the NOS pathway.

Regional myocardial perfusion reserve determined using myocardial perfusion magnetic resonance imaging showed a direct correlation with coronary flow velocity reserve by Doppler flow wire

AIMS Quantitative analysis of rest-stress myocardial perfusion magnetic resonance imaging (MRI) can provide assessments of regional myocardial perfusion reserve (MPR). The purpose of this study was to compare regional MPR determined by myocardial perfusion MRI with coronary flow reserve (CFR) by intracoronary Doppler flow wire. METHODS AND RESULTS Twenty patients with suspected coronary artery disease (CAD) were studied. Average peak velocity was measured by Doppler flow wire in the resting state and during adenosine triphosphate (ATP) stress in 36 coronary arteries. CFR measurements for each patient were performed in the culprit and one non-culprit non-stenotic artery. First-pass, contrast-enhanced myocardial perfusion MR images were obtained in the resting state and during ATP stress within the week before the Doppler wire procedure. Regional myocardial blood flow (MBF) was quantified in 16 myocardial segments by analysing arterial input and myocardial output using a Patlak plot method. MPR was calculated as stress MBF divided by rest MBF. CFR measured by Doppler flow wire was compared with MPR in the myocardial segments corresponding to vessel territories. The average MPR measured by perfusion MRI was 1.77 +/- 0.62 for the culprit arteries and 3.45 +/- 0.78 for the non-culprit arteries, respectively (P < 0.001). The averaged CFR by Doppler flow wire was 1.72 +/- 0.44 in the culprit arteries and 3.14 +/- 0.74 in the non-culprit arteries, respectively (P < 0.001). For both culprit and non-culprit vessel groups, significant direct correlations were observed between MR assessments of MPR and Doppler assessments of CFR (culprit artery: R = 0.87, Non-culprit artery: R = 0.86) On Bland-Altman analysis, the mean differences between MPR determined by myocardial perfusion MRI and CFR measured by Doppler wire were 0.05 in culprit arteries (95% limit of agreement; -0.65 to 0.56) and 0.36 in non-culprit arteries (95% limit of agreement; -1.24 to 0.44). The sensitivity and specificity of MR measurement of MPR for predicting physiologically significant reduction of Doppler CFR (<2) was 88% (95% CI 61.7-98.5) and 90% (95% CI 68.3-98.8), respectively. CONCLUSION The current results using Doppler flow wire as a reference method demonstrated that quantitative analysis of stress-rest myocardial perfusion MRI can provide a non-invasive assessment of reduced MPR in patients with CAD.

Reversible Right Ventricular Regional Non-Uniformity Quantified by Speckle-Tracking Strain Imaging in Patients With Acute Pulmonary Thromboembolism

BACKGROUND The aim of this study was to evaluate the effects of acute right ventricular (RV) pressure overload (RVPO) on RV systolic function and its regional uniformity using speckle-tracking strain analysis in patients with acute pulmonary thromboembolism (APTE). METHODS Twenty-three patients with APTE (mean age, 59 +/- 16 years) and 23 age-matched and gender-matched normal subjects (the control group) were examined using echocardiography. Global and segmental longitudinal RV peak systolic strain (PSS) was analyzed using speckle-tracking strain echocardiography. The heterogeneity of RV regional function was assessed by calculating the standard deviation from 6-segmental PSS divided by the absolute value of global PSS. The standard deviation of the heart rate-corrected intervals from QRS onset to PSS for the 6 segments was used to quantify RV dyssynchrony. RESULTS Patients with APTE had reduced regional PSS, resulting in reduced global PSS and augmented regional heterogeneity, and had delayed myocardial contraction in the basal and mid RV lateral walls, resulting in large dyssynchrony (global PSS, -14 +/- vs -25 +/- 3%; heterogeneity, 0.54 +/- 0.26 vs 0.24 +/- 0.09; dyssynchrony, 91 +/- 38 vs 25 +/- 10 ms; P < .05 vs controls for all comparisons). After the amelioration of acute RVPO by primary treatment, both RV heterogeneity and dyssynchrony returned to normal values. CONCLUSION Speckle-tracking strain echocardiography can effectively quantify reversible RV regional nonuniformity caused by acute RVPO and can characterize the pattern of RV regional impairment in patients with APTE.

Role of Radial Strain and Displacement Imaging to Quantify Wall Motion Dyssynchrony in Patients With Left Ventricular Mechanical Dyssynchrony and Chronic Right Ventricular Pressure Overload

Left ventricular (LV) deformation with ventricular septal shift is one of the most distinctive echocardiographic observations in patients with chronic right ventricular (RV) pressure overload (PO). However, little is known about the effects of RVPO on LV performance and regional synchrony. Accordingly, our objective was to test the hypothesis that chronic RVPO affects regional wall motion, synchronicity, and global LV function using a novel speckle-tracking approach to quantify and characterize regional LV wall motion dyssynchrony. Displacement and strain imaging echocardiographic studies were performed in 20 patients with RVPO from pulmonary arterial hypertension or pulmonic stenosis (mean age 53 +/- 16 years, New York Heart Association class 2.6 +/- 0.7, and peak RV systolic pressure 73 +/- 28 mm Hg) and 20 age-matched normal subjects (mean age 47 +/- 16 years). Segmental signals from 6 segments around the mid-LV short axis were defined as dyssynchronous if their changes were opposite to that of the global LV signal at each time frame, and overall LV dyssynchrony was calculated as the percentage of dyssynchrony in all 6 segments within the specified time interval from onset of QRS to the end of isovolumic relaxation. RVPO was associated with a large degree of regional dyssynchrony with paradoxical ventricular septal motion observed by displacement imaging (21 +/- 6%, p <0.05 vs control group), which was closely associated with LV eccentricity index (r = 0.79, p <0.05) and LV myocardial performance index with linear regression (r = 0.76, p <0.05). In contrast, strain imaging showed uniform segmental radial thickening in the RVPO group, which was similar to the control group, and suggests that there was no intrinsic LV contractile dyssynchrony. In conclusion, LV wall motion dyssynchrony assessed by displacement imaging, not intrinsic contractile dyssynchrony by strain imaging, coexists with LV chamber deformation with ventricular septal shift and is closely associated with impairment of LV performance.

Short-term effects of low-dose tolvaptan on hemodynamic parameters in patients with chronic heart failure

BACKGROUND We evaluated the short-term effects of low-dose tolvaptan treatment on hemodynamic parameters in patients with chronic heart failure (HF). METHODS We studied 22 patients (69 ± 10 years) with chronic HF and excess fluid retention despite receiving appropriate medical therapy, including loop and/or thiazide diuretics. The therapeutic effects of low-dose (7.5mg) once-daily tolvaptan on hemodynamics associated with changes in fluid balance and neurohumoral activations were investigated after a seven day treatment period. RESULTS After the treatment period, body weight decreased (-2.7 ± 2.3 kg) associated with increases in daily urine output. Whereas plasma arginine-vasopressin levels, serum aldosterone concentration, and plasma renin activity mildly increased, plasma levels of B-type natriuretic peptide and atrial natriuretic peptide significantly decreased after tolvaptan treatment. Serum electrolytes were not adversely affected by tolvaptan treatment. Although cardiac index and systemic vascular resistance index remained unchanged, mean pulmonary artery wedge pressure (22 ± 7 mmHg vs. 17 ± 7 mmHg, p<0.05), mean right atrial pressure (12 ± 5 mmHg vs. 9 ± 5 mmHg, p<0.05), mean pulmonary artery pressure (32 ± 9 mmHg vs. 25 ± 7 mmHg, p<0.05), and pulmonary vascular resistance index (332 ± 207 dynes/cm(-5)/m(2) vs. 245 ± 110 dynes/cm(-5)/m(2), p<0.05) significantly decreased after tolvaptan treatment. The extent of the reduction in pulmonary vascular resistance index after tolvaptan treatment strongly correlated with baseline values. CONCLUSIONS Short-term treatment with low-dose tolvaptan improved hemodynamic parameters and correlated with significant fluid removal in patients with chronic HF.

Patients with a Hypertensive Response to Exercise Have Impaired Left Ventricular Diastolic Function

An exaggerated increase in systolic blood pressure prolongs myocardial relaxation and increases left ventricular (LV) chamber stiffness, resulting in an increase in LV filling pressure. We hypothesize that patients with a marked hypertensive response to exercise (HRE) have LV diastolic dysfunction leading to exercise intolerance, even in the absence of resting hypertension. We recruited 129 subjects (age 63±9 years, 64% male) with a preserved ejection fraction and a negative stress test. HRE was evaluated at the end of a 6-min exercise test using the modified Bruce protocol. Patients were categorized into three groups: a group without HRE and without resting hypertension (control group; n=30), a group with HRE but without resting hypertension (HRE group; n=25), and a group with both HRE and resting hypertension (HTN group; n=74). Conventional Doppler and tissue Doppler imaging were performed at rest. After 6-min exercise tests, systolic blood pressure increased in the HRE and HTN groups, compared with the control group (226±17 mmHg, 226±17 mmHg, and 180±15 mmHg, respectively, p<0.001). There were no significant differences in LV ejection fraction, LV end-diastolic diameter, and early mitral inflow velocity among the three groups. However, early diastolic mitral annular velocity (E′) was significantly lower and the ratio of early diastolic mitral inflow velocity (E) to E′ (E/E′) was significantly higher in patients of the HRE and HTN groups compared to controls (E′: 5.9±1.6 cm/s, 5.9±1.7 cm/s, 8.0±1.9 cm/s, respectively, p<0.05). In conclusion, irrespective of the presence of resting hypertension, patients with hypertensive response to exercise had impaired LV longitudinal diastolic function and exercise intolerance.