吴茱萸次碱对肺动脉高压大鼠右心室重构的影响

目的：观察吴茱萸次碱（Rut）对野百合碱（MCT）诱导的肺动脉高压（PH）大鼠右心室重构的作用及机制。方法：SD大鼠48只适应性喂养一周,随机分为正常对照组、Mcr组、MCT＋Rut（20mg／kg）及Mcr＋Rut（40mg／kg）剂量组（n=12）。MCT（60mg／kg）皮下注射诱导PH大鼠模型。连续给药4周后,右颈外静脉插管测定大鼠右心室收缩压（RVSP）、平均肺动脉压（MPAP）。分离大鼠右心室（RV）、左心室＋室间隔（LV＋s）并称重,剥离大鼠胫骨并测量其长度,计算av／（LV＋s）gRV／胫骨长度的比值。HE染色观察右心室病理学变化,Masson染色观察右心室胶原沉积的变化。比色法测定右心室总抗氧化能力（T-AOC）、丙二醛（MDA）含量。Real time PCR、Western blot及免疫组化检测右心室NADPH氧化酶4（NOX4）mRNA和蛋白表达。结果：Rut连续给药4周后能明显降低MCT诱导的PH大鼠RVSP及mPAP,减轻RV／（Lv＋s）及RV重量／胫骨长度的比值,改善右心室病理变化,降低右心室胶原的沉积及collagenI、collagenHI的表达,提高右心室T-AOC水平,降低右心室NOX4的表达及MDA含量。结论：Rut能够缓解野百合碱诱导的PH大鼠右心室重构,其机制可能与抑制NOX4的表达,进而降低氧化应激损伤有关。     

Muscle RING Finger-1 Promotes a Maladaptive Phenotype in Chronic Hypoxia-Induced Right Ventricular Remodeling

Exposure to chronic hypoxia (CH) induces elevated pulmonary artery pressure/resistance, leading to an eventual maladaptive right ventricular hypertrophy (RVH). Muscle RING finger-1 (MuRF1) is a muscle-specific ubiquitin ligase that mediates myocyte atrophy and has been shown to play a role in left ventricular hypertrophy and altered cardiac bioenergetics in pressure overloaded hearts. However, little is known about the contribution of MuRF1 impacting RVH in the setting of CH. Therefore, we hypothesized that MuRF1 deletion would enhance RVH compared to their wild-type littermates, while cardiac-specific overexpression would reduce hypertrophy following CH-induced pulmonary hypertension. We assessed right ventricular systolic pressure (RVSP), right ventricle to left ventricle plus septal weight ratio (RV/LV+S) and hematocrit (Hct) following a 3-wk isobaric CH exposure. Additionally, we conducted dual-isotope SPECT/CT imaging with cardiac function agent ²⁰¹Tl-chloride and cell death agent ^99mTc-annexin V. Predictably, CH induced pulmonary hypertension, measured by increased RVSP, RV/LV+S and Hct in WT mice compared to normoxic WT mice. Normoxic WT and MuRF1-null mice exhibited no significant differences in RVSP, RV/LV+S or Hct. CH-induced increases in RVSP were also similar between WT and MuRF1-null mice; however, RV/LV+S and Hct were significantly elevated in CH-exposed MuRF1-null mice compared to WT. In cardiac-specific MuRF1 overexpressing mice, RV/LV+S increased significantly due to CH exposure, even greater than in WT mice. This remodeling appeared eccentric, maladaptive and led to reduced systemic perfusion. In conclusion, these results are consistent with an atrophic role for MuRF1 regulating the magnitude of right ventricular hypertrophy following CH-induction of pulmonary hypertension.     

Nicorandil prevents right ventricular remodeling by inhibiting apoptosis and lowering pressure overload in rats with pulmonary arterial hypertension

Background

Most of the deaths among patients with severe pulmonary arterial hypertension (PAH) are caused by progressive right ventricular (RV) pathological remodeling, dysfunction, and failure. Nicorandil can inhibit the development of PAH by reducing pulmonary artery pressure and RV hypertrophy. However, whether nicorandil can inhibit apoptosis in RV cardiomyocytes and prevent RV remodeling has been unclear.

Methodology/Principal Findings

RV remodeling was induced in rats by intraperitoneal injection of monocrotaline (MCT). RV systolic pressure (RVSP) was measured at the end of each week after MCT injection. Blood samples were drawn for brain natriuretic peptide (BNP) ELISA analysis. The hearts were excised for histopathological, ultrastructural, immunohistochemical, and Western blotting analyses. The MCT-injected rats exhibited greater mortality and less weight gain and showed significantly increased RVSP and RV hypertrophy during the second week. These worsened during the third week. MCT injection for three weeks caused pathological RV remodeling, characterized by hypertrophy, fibrosis, dysfunction, and RV mitochondrial impairment, as indicated by increased levels of apoptosis. Nicorandil improved survival, weight gain, and RV function, ameliorated RV pressure overload, and prevented maladaptive RV remodeling in PAH rats. Nicorandil also reduced the number of apoptotic cardiomyocytes, with a concomitant increase in Bcl-2/Bax ratio. 5-hydroxydecanoate (5-HD) reversed these beneficial effects of nicorandil in MCT-injected rats.

Conclusions/Significance

Nicorandil inhibits PAH-induced RV remodeling in rats not only by reducing RV pressure overload but also by inhibiting apoptosis in cardiomyocytes through the activation of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels. The use of a mitoK_ATP channel opener such as nicorandil for PAH-associated RV remodeling and dysfunction may represent a new therapeutic strategy for the amelioration of RV remodeling during the early stages of PAH.     

Synergistic effects of ANP and sildenafil on cGMP levels and amelioration of acute hypoxic pulmonary hypertension

We hypothesized that the phosphodiesterase 5 inhibitor, sildenafil, and the guanosine cyclase stimulator, atrial natriuretic peptide (ANP), would act synergistically to increase cGMP levels and blunt hypoxic pulmonary hypertension in rats, because these compounds act via different mechanisms to increase the intracellular second messenger. Acute hypoxia: Adult Sprague-Dawley rats were gavaged with sildenafil (1 mg/ kg) or vehicle and exposed to acute hypoxia with and without ANP (10(-8)-10(-5) M ). Sildenafil decreased systemic blood pressure (103 +/- 10 vs. 87 +/- 6 mm Hg, P < 0.001) and blunted the hypoxia-induced increase in right ventricular systolic pressure (RVSP; percent increase 73.7% +/- 9.4% in sildenafil-treated rats vs. 117.2% +/- 21.1% in vehicle-treated rats, P = 0.03). Also, ANP and sildenafil had synergistic effects on blunting the hypoxia-induced increase in RVSP (P < 0.001) and on rising plasma cGMP levels (P < 0.05). Chronic hypoxia: Other rats were exposed to prolonged hypoxia (3 weeks, 0.5 atm) after subcutaneous implantation of a sustained-release pellet containing lower (2.5 mg), or higher (25 mg) doses of sildenafil, or placebo. Higher-dose, but not lower-dose sildenafil blunted the chronic hypoxia-induced increase in RVSP (P = 0.006). RVSP and plasma sildenafil levels were inversely correlated in hypoxic rats (r(2) = 0.68, P = 0.044). Lung cGMP levels were increased by both chronic hypoxia and sildenafil, with the greatest increase achieved by the combination. Plasma and right ventricular (RV) cGMP levels were increased by hypoxia, but sildenafil had no effect. RV hypertrophy and pulmonary artery muscularization were also unaffected by sildenafil. In conclusion, sildenafil and ANP have synergistic effects on the blunting of hypoxia-induced pulmonary vasoconstriction. During chronic hypoxia, sildenafil normalizes RVSP, but in the doses used, sildenafil has no effect on RV hypertrophy or pulmonary vascular remodeling.     

Allicin in garlic protects against coronary endothelial dysfunction and right heart hypertrophy in pulmonary hypertensive rats

We recently reported that coronary endothelial cell (CEC) dysfunction may contribute to the development of right ventricular (RV) hypertrophy (RVH) in monocrotaline (MCT)-induced pulmonary hypertensive rats. This present study investigated whether preservation of CEC function with garlic and its active metabolite allicin could abrogate RVH. Rats were fed with 1% raw garlic (RG)-supplemented diet 1 day or 3 wk before and 1 day after MCT injection, and changes in RV pressure (RVP), RVH, and CEC function were assessed 3 wk after MCT administration. In all cases, RG feeding significantly inhibited the development of RVP and RVH in these MCT rats. However, similar treatments with either boiled garlic (BG) or aged garlic (AG), which do not contain the active allicin metabolite, were ineffective. CEC function, assessed with acetylcholine-induced dilation as well as N(omega)-nitro-l-arginine methyl ester-induced constriction, revealed marked attenuation in right, but not left, coronary arteries of the MCT rats. This is consistent with our earlier report. Feeding of RG, but not BG or AG, preserved the CEC function and prevented the exaggerated vasoconstrictory responses of the MCT coronary arteries. There was no change in the coronary dilatory responses to a nitric oxide donor sodium nitroprusside. Further testings of vasoactivity to garlic extracts showed that only RG, but not BG or AG, elicited a potent, dose-dependent dilation on the isolated coronaries. Taken together, these findings show that the protective effect of garlic against the development of RVP and RVH in MCT-treated rats is probably mediated via its active metabolite allicin action on coronary endothelial function and vasoreactivity.     

Human immunodeficiency virus transgenic rats exhibit pulmonary hypertension

Human immunodeficiency virus (HIV)-associated pulmonary arterial hypertension (PAH) is a serious noninfectious disease involving an aberrant increase in pressure in the blood vessels of the lung, which leads to right ventricular (RV) heart failure and can eventually result in death. A lack of viable animal models of HIV-PAH has limited the identification of signaling pathways involved in HIV-mediated onset and progression of PAH. To determine whether the HIV-1 transgenic (HIV Tg) rat displays pathophysiological end points associated with PAH, we evaluated peak RV systolic pressure (RVSP), RV hypertrophy, pulmonary vessel remodeling, and alterations in gene expression by real-time PCR and microarray. RVSP was measured by RV catheterization via the right jugular vein in 3- and 9-mo-old HIV Tg and age-matched Fischer 344 (control) male rats while under 2% isoflurane anesthesia. RVSP was elevated in the HIV Tg rats (34.2 ± 2.5 mmHg) compared with the F344 controls (21.2 ± 2.5 mmHg), with more significant elevations in the 9-mo-old HIV Tg rats (42.5 ± 3.7 mmHg). We observed significant increases in RV wall thickness in HIV Tg rats compared with controls, both histologically and by echocardiograph measurement. HIV Tg rats also show increased thickening of the pulmonary artery and remodeling of small pulmonary arteries, as well as altered expression of gene pathways associated with PAH. These data represent the first analysis of PAH in HIV Tg rats and suggest that this model will be useful for investigating pathways and identifying potential therapies for HIV-PAH.     

Characterization of right ventricular function after monocrotaline-induced pulmonary hypertension in the intact rat

We characterized hemodynamics and systolic and diastolic right ventricular (RV) function in relation to structural changes in the rat model of monocrotaline (MCT)-induced pulmonary hypertension. Rats were treated with MCT at 30 mg/kg body wt (MCT30, n = 15) and 80 mg/kg body wt (MCT80, n = 16) to induce compensated RV hypertrophy and RV failure, respectively. Saline-treated rats served as control (Cont, n = 13). After 4 wk, a pressure-conductance catheter was introduced into the RV to assess pressure-volume relations. Subsequently, rats were killed, hearts and lungs were rapidly dissected, and RV, left ventricle (LV), and interventricular septum (IVS) were weighed and analyzed histochemically. RV-to-(LV + IVS) weight ratio was 0.29 +/- 0.05 in Cont, 0.35 +/- 0.05 in MCT30, and 0.49 +/- 0.10 in MCT80 (P < 0.001 vs. Cont and MCT30) rats, confirming MCT-induced RV hypertrophy. RV ejection fraction was 49 +/- 6% in Cont, 40 +/- 12% in MCT30 (P < 0.05 vs. Cont), and 26 +/- 6% in MCT80 (P < 0.05 vs. Cont and MCT30) rats. In MCT30 rats, cardiac output was maintained, but RV volumes and filling pressures were significantly increased compared with Cont (all P < 0.05), indicating RV remodeling. In MCT80 rats, RV systolic pressure, volumes, and peak wall stress were further increased, and cardiac output was significantly decreased (all P < 0.05). However, RV end-systolic and end-diastolic stiffness were unchanged, consistent with the absence of interstitial fibrosis. MCT-induced pressure overload was associated with a dose-dependent development of RV hypertrophy. The most pronounced response to MCT was an overload-dependent increase of RV end-systolic and end-diastolic volumes, even under nonfailing conditions.     

Aspirin Attenuates Pulmonary Arterial Hypertension in Rats by Reducing Plasma 5-Hydroxytryptamine Levels

Pulmonary arterial hypertension (PAH) is characterized by increasing pulmonary pressure, right ventricular failure, and death. The typical pathological changes include medial hypertrophy, intimal fibrosis and in situ thrombosis. Serotonin (5-HT) and other factors contribute to the development of pathologic lesions. Aspirin (ASA), a platelet aggregation inhibitor, inhibits 5-HT release from platelets. The aim of this study was to determine the efficacy of ASA in preventing or attenuating PAH. Sprague–Dawley rats injected with monocrotaline (MCT) developed severe PAH within 31 days. One hundred forty rats were randomized to receive either vehicle or ASA (0.5, 1, 2, or 4 mg/kg/day). The pre-ASA group was treated with ASA (1 mg/kg/day) for 30 days before the MCT injection. Thirty-one days after the injection (day 61 for the pre-ASA group), pulmonary arterial pressure (PAP), right ventricular hypertrophy and pulmonary arteriole thickness were measured. Plasma 5-HT was measured by high-performance liquid chromatography. Aspirin suppressed PAH and increased the survival rate compared with the control group (84 vs. 60%, P < 0.05). Aspirin treatment also reduced right ventricular hypertrophy and pulmonary arteriole proliferation in ASA-treated PAH model. In addition, plasma 5-HT was decreased in our ASA-treated PAH model. The degree of 5-HT reduction was associated with systolic PAP, right ventricular hypertrophy and wall thickness of pulmonary arterioles in rats. These results showed that ASA treatment effectively attenuated MCT-induced pulmonary hypertension, right ventricular hypertrophy, and occlusion of the pulmonary arteries. The effects of ASA was associated with a reduction of 5-HT.     

Arrhythmogenic substrate in hearts of rats with monocrotaline-induced pulmonary hypertension and right ventricular hypertrophy

Mechanisms associated with right ventricular (RV) hypertension and arrhythmias are less understood than those in the left ventricle (LV). The aim of our study was to investigate whether and by what mechanisms a proarrhythmic substrate exists in a rat model of RV hypertension and hypertrophy. Rats were injected with monocrotaline (MCT; 60 mg/kg) to induce pulmonary artery hypertension or with saline (CON). Myocardial levels of mRNA for genes expressing ion channels were measured by real-time RT-PCR. Monophasic action potential duration (MAPD) was recorded in isolated Langendorff-perfused hearts. MAPD restitution was measured, and arrhythmias were induced by burst stimulation. Twenty-two to twenty-six days after treatment, MCT animals had RV hypertension, hypertrophy, and decreased ejection fractions compared with CON. A greater proportion of MCT hearts developed sustained ventricular tachycardias/fibrillation (0.83 MCT vs. 0.14 CON). MAPD was prolonged in RV and less so in the LV of MCT hearts. There were decreased levels of mRNA for K(+) channels. Restitution curves of MCT RV were steeper than CON RV or either LV. Dispersion of MAPD was greater in MCT hearts and was dependent on stimulation frequency. Computer simulations based on ion channel gene expression closely predicted experimental changes in MAPD and restitution. We have identified a proarrhythmic substrate in the hearts of MCT-treated rats. We conclude that steeper RV electrical restitution and rate-dependant RV-LV action potential duration dispersion may be contributing mechanisms and be implicated in the generation of arrhythmias associated with in RV hypertension and hypertrophy.     

Increased in vivo mitochondrial oxygenation with right ventricular failure induced by pulmonary arterial hypertension: mitochondrial inhibition as driver of cardiac failure?

Background

The leading cause of mortality due to pulmonary arterial hypertension (PAH) is failure of the cardiac right ventricle. It has long been hypothesized that during the development of chronic cardiac failure the heart becomes energy deprived, possibly due to shortage of oxygen at the level of cardiomyocyte mitochondria. However, direct evaluation of oxygen tension levels within the in vivo right ventricle during PAH is currently lacking. Here we directly evaluated this hypothesis by using a recently reported technique of oxygen-dependent quenching of delayed fluorescence of mitochondrial protoprophyrin IX, to determine the distribution of mitochondrial oxygen tension (mitoPO₂) within the right ventricle (RV) subjected to progressive PAH.

Methods

PAH was induced through a single injection of monocrotaline (MCT). Control (saline-injected), compensated RV hypertrophy (30 mg/kg MCT; MCT30), and RV failure (60 mg/kg MCT; MCT60) rats were compared 4 wk after treatment. The distribution of mitoPO₂ within the RV was determined in mechanically-ventilated, anaesthetized animals, applying different inspired oxygen (FiO₂) levels and two increment dosages of dobutamine.

Results

MCT60 resulted in RV failure (increased mortality, weight loss, increased lung weight), MCT30 resulted in compensated RV hypertrophy. At 30% or 40% FiO₂, necessary to obtain physiological arterial PO₂ in the diseased animals, RV failure rats had significantly less mitochondria (15% of total mitochondria) in the 0-20 mmHg mitoPO₂ range than hypertrophied RV rats (48%) or control rats (54%). Only when oxygen supply was reduced to 21% FiO_2, resulting in low arterial PO₂ for the MCT60 animals, or when oxygen demand increased with high dose dobutamine, the number of failing RV mitochondria with low oxygen became similar to control RV. In addition, metabolic enzyme analysis revealed similar mitochondrial mass, increased glycolytic hexokinase activity following MCT, with increased lactate dehydrogenase activity only in compensated hypertrophied RV.

Conclusions

Our novel observation of increased mitochondrial oxygenation suggests down-regulation of in vivo mitochondrial oxygen consumption, in the absence of hypoxia, with transition towards right ventricular failure induced by pulmonary arterial hypertension.     

PAF antagonists inhibit monocrotaline-induced lung injury and pulmonary hypertension.

Lung platelet-activating factor (PAF) levels increased in some rats at 1-3 wk after subcutaneous injection of monocrotaline (MCT). We tested the effect of specific PAF antagonists, WEB 2086 and WEB 2170, on MCT-induced lung injury and subsequent pulmonary hypertension and right ventricular hypertrophy. Treatment with either agent decreased MCT-induced pulmonary hypertension and right ventricular hypertrophy at 3 wk after injection. Treatment with WEB 2170 reduced MCT-induced pulmonary vascular leak at 1 wk after injection, and WEB 2086-treatment exclusively during the early leak phase also decreased MCT-induced right ventricular hypertrophy at 3 wk. Treatment with WEB 2170 between the 3rd and 4th wk after MCT injection inhibited the progression of right ventricular hypertrophy at 4 wk. These results suggest that PAF contributes to the early pulmonary vascular leak, and this leak phase is important for the development of pulmonary hypertension and right ventricular hypertrophy in MCT-treated rats. Furthermore, it appears that PAF action contributes to the maintenance of a chronic inflammatory process that involves the synthesis of other lipid mediators (prostaglandins and leukotrienes) and leads to pulmonary hypertension. We conclude that PAF has a role in the MCT-induced inflammatory lung injury and pulmonary hypertension.     

慢性低氧对大鼠左右心室的功能及TRPC亚家族表达的影响

目的：探讨慢性低氧3周对大鼠左右心室的影响以及规范性瞬时感受器电位亚家族（TRPC）在慢性低氧诱导的右心室心肌肥厚中的表达。方法：将SD雄性大鼠48只随机分为对照组（CON组）和慢性低氧肺动脉高压模型组（CH组）（n=24）,CH组将大鼠置于连续的慢性低氧（10％±0．2％）环境饲养三周以诱导大鼠发生心肌肥厚。通过左、右心室插管法测定右心室内压（RVSP）、左心室内压（LVSP）、心率（HR）、平均体循环动脉压（mSAP）、左、右心室内压力最大上升速率（＋dp／dtmax）、最大下降速率（-dp／dkmax）、右心肥大指数（RVMI）、左心肥大指数（LVMI）;HE染色观察左、右心室心肌组织切片;通过SYBR Green荧光定量PCR法检测CON组、CH组大鼠的肥厚侧心室心肌组织编码TRPC 1／3／4／5／6／7的rnRNA表达;结合real-time RT-PCR结果对mRNA表达有显著变化的TRPC亚型通过免疫印迹法检测相应蛋白的表达。结果：与CON组相比：CH组的RVSP、RVMI、右心室±dp／dtmax显著增高（P〈0．01）,LVSP、左心室±dp／dmax无显著变化,LVMI显著降低（P〈0．01）;CH组右心室心肌细胞显著增粗（P〈0．01）,细胞内肌原纤维数量增多,心肌纤维排列紊乱,细胞核深染,形状不整;左心室心肌纤维无明显改变;CH组编码TRPCI的mRNA和蛋白显著增高（P〈0．05）,而编码其余TRPC亚型的mRNA无显著变化。结论：慢性低氧3周可特异性诱导sD大鼠产生右心室心肌肥厚,上调了编码右心室心肌细胞TRPCI通道蛋白的mRNA和蛋白的表达,TRPCI可能参与了心肌肥厚的发生发展。     

Endogenous production of ghrelin and beneficial effects of its exogenous administration in monocrotaline-induced pulmonary hypertension

We investigated the endogenous production of ghrelin as well as cardiac and pulmonary vascular effects of its administration in a rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH). Adult Wistar rats randomly received a subcutaneous injection of MCT (60 mg/kg) or an equal volume of vehicle. One week later, animals were randomly assigned to receive a subcutaneous injection of ghrelin (100 mug/kg bid for 2 wk) or saline. Four groups were analyzed: normal rats treated with ghrelin (n=7), normal rats injected with saline (n=7), MCT rats treated with ghrelin (n=9), and MCT rats injected with saline (n=9). At 22-25 days, right (RV) and left ventricular (LV) pressures were measured, heart and lungs were weighted, and samples were collected for histological and molecular analysis. Endogenous production of ghrelin was almost abolished in normal rats treated with ghrelin. In MCT-treated animals, pulmonary expression of ghrelin was preserved, and RV myocardial expression was increased more than 20 times. In these animals, exogenous administration of ghrelin attenuated PH, RV hypertrophy, wall thickening of peripheral pulmonary arteries, and RV diastolic disturbances and ameliorated LV dysfunction, without affecting its endogenous production. In conclusion, decreased tissular expression of ghrelin in healthy animals but not in PH animals suggests a negative feedback in the former that is lost in the latter. A selective increase of ghrelin mRNA levels in the RV of animals with PH might indicate distinct regulation of its cardiac expression. Finally, ghrelin administration attenuated MCT-induced PH, pulmonary vascular remodeling, and RV hypertrophy, indicating that it may modulate PH.     

Progressive development of pulmonary hypertension leading to right ventricular hypertrophy assessed by echocardiography in rats.

The present study aimed to evaluate the development of pulmonary hypertension by serial echocardiography, including measurements of pulmonary artery (PA) flow velocities, and correlate echocardiographic indices with pathological findings in rats administered monocrotaline (MCT). MCT (60 mg/kg body weight) or physiologic saline was administered to a total of 9 male Wistar rats at the age of 4 weeks (MCT group: n = 4, control group: n = 5, respectively). Echocardiography was performed serially until the age of 8 weeks. The ratio of right ventricular (RV) outflow tract dimensions to aortic dimensions increased progressively in the MCT group and became significantly greater than that of the control group after the age of 6 weeks. Peak PA velocity (Peak V) in the MCT group was significantly less than that of the control group at the ages of 7 and 8 weeks. The ratio of acceleration time to ejection time (AT/ET) in PA flow waveforms declined progressively and was significantly less than that of the control group after the age of 6 weeks. The ratio of RV weight to body weight (RVW/BW) in the MCT group was significantly greater than that of the control group. Both AT/ET ratio and Peak V were significantly inversely correlated with RVW/BW ratio. Furthermore, these echocardiographic findings were also significantly inversely correlated with the mean cross-sectional RV myocyte area. In conclusion, the progressive development of pulmonary hypertension leading to RV hypertrophy can be evaluated appropriately by echocardiography including PA flow Doppler indices in rats.     

Chronic hypoxia induces right heart failure in caveolin-1-/- mice

Caveolin-1 (Cav-1)-/- mice develop mild pulmonary hypertension as they age. In this study, we sought to determine the effect of chronic hypoxia, an established model of pulmonary hypertension, on young Cav-1-/- mice with no measurable signs of pulmonary hypertension. Exposure of Cav-1-/- mice to chronic hypoxia resulted in an initial rise in right ventricular (RV) systolic pressure (RVSP) similar to wild-type (WT) mice. By three weeks RVSP decreased in the Cav-1-/- mice, whereas it was maintained in WT mice. The drop in RVSP in Cav-1-/- mice was accompanied by decreased cardiac output, increased RV hypertrophy, RV interstitial fibrosis, decreased RV sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a mRNA and decreased RV function compared with WT mice. Importantly, minimal differences were noted in pulmonary vascular remodeling between WT and Cav-1-/- mice, and left ventricular function was normal in hypoxic Cav-1-/- mice. Mechanistically, increased endothelial nitric oxide synthase uncoupling and increased tyrosine nitration of protein kinase G were detected in the RV of Cav-1-/- mice. These hemodynamic, histological, and molecular changes were prevented in Cav-1-/- mice expressing an endothelial-specific Cav-1 transgene or by nitric oxide synthase inhibition. These data suggest that, in Cav-1-/- mice, increased oxidative/nitrosative stress due to endothelial nitric oxide synthase uncoupling modifies the response of the RV to pressure overload, accelerating the deterioration of RV function.     

Early changes in rat hearts with developing pulmonary arterial hypertension can be detected with three-dimensional electrocardiography

The study aim was to assess three-dimensional electrocardiogram (ECG) changes during development of pulmonary arterial hypertension (PAH). PAH was induced in male Wistar rats (n = 23) using monocrotaline (MCT; 40 mg/kg sc). Untreated healthy rats served as controls (n = 5). ECGs were recorded with an orthogonal three-lead system on days 0, 14, and 25 and analyzed with dedicated computer software. In addition, left ventricular (LV)-to-right ventricular (RV) fractional shortening ratio was determined using echocardiography. Invasively measured RV systolic pressure was 49 (SD 10) mmHg on day 14 and 64 (SD 10) mmHg on day 25 vs. 25 (SD 2) mmHg in controls (both P < 0.001). Baseline ECGs of controls and MCT rats were similar, and ECGs of controls did not change over time. In MCT rats, ECG changes were already present on day 14 but more explicit on day 25: increased RV electromotive forces decreased mean QRS-vector magnitude and changed QRS-axis orientation. Important changes in action potential duration distribution and repolarization sequence were reflected by a decreased spatial ventricular gradient magnitude and increased QRS-T spatial angle. On day 25, LV-to-RV fractional shortening ratio was increased, and RV hypertrophy was found, but not on day 14. In conclusion, developing PAH is characterized by early ECG changes preceding RV hypertrophy, whereas severe PAH is marked by profound ECG changes associated with anatomical and functional changes in the RV. Three-dimensional ECG analysis appears to be very sensitive to early changes in RV afterload.     

Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats

Hill, Nicholas S., Rod R. Warburton, Linda Pietras, andJames R. Klinger. Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats.J. Appl. Physiol. 83(4):1209-1215, 1997.Endothelin-1 (ET-1), a potent vasoactive andmitogenic peptide, has been implicated in the pathogenesis ofseveral forms of pulmonary hypertension. We hypothesized thatnonspecific blockade of ET receptors would blunt the development ofmonocrotaline (MCT)-induced pulmonary hypertension in rats. Asingle dose of the nonspecific ET blocker bosentan (100 mg/kg) given tointact rats by gavage completely blocked the pulmonary vasoconstrictoractions of Big ET-1 and partially blunted hypoxic pulmonaryvasoconstriction. After 3 wk, MCT-injected (105 mg/kg sc) rats gavagedonce daily with bosentan (200 mg/kg) had lower right ventricular (RV)systolic pressure (RVSP), RV-to-body weight (RV/BW) andRV-to-left ventricular (LV) plus septal (S) weight [RV/(LV+S)] ratiosand less percent medial thickness of small pulmonary arteries thancontrol MCT-injected rats. Lower dose bosentan (100 mg/kg) had noeffect on these parameters after MCT or saline injection. Bosentanraised plasma ET-1 levels but had no effect on lung ET-1 levels.Bosentan (200 mg/kg) also had no effect on wet-to-dry lung weightratios 6 days after MCT injection. When given during the last 10 days,but not the first 11 days of a 3-wk period after MCT injection,bosentan reduced RV/(LV+S) compared with MCT-injected controls. Weconclude that ET-1 contributes to the pathogenesis of MCT-inducedpulmonary hypertension and acts mainly during the later inflammatoryrather than the acute injury phase after injection.

     

Fasudil dichloroacetate (FDCA), an orally available agent with potent therapeutic efficiency on monocrotaline-induced pulmonary arterial hypertension rats

Given the therapeutic efficacy of fasudil hydrochloride (F) and dichloroacetate (DCA) on pulmonary arterial hypertension (PAH), a new salt fasudil dichloroacetate (FDCA) was designed, synthesized and biologically evaluated. FDCA exhibited comparable ROCK II inhibitory activity relative to fasudil hydrochloride, and suppressed the expression of TNF-α and IL-6 in both PDGF-BB and hypoxia-treated pulmonary arterial smooth muscle cells (PASMCs) and endothelial cells (PAECs). Significantly, FDCA lowered mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP), and decreased right ventricular hypertrophy (RVH) in monocrotaline (MCT)-induced PAH rats. Meanwhile, FDCA remarkably decreased pulmonary artery medial thickness (PAMT) and hyperplasia, restoring the elasticity of elastic fiber, reduced cardiac hypertrophy, and attenuated fibrosis of heart and lung. Collectively, FDCA exhibited triple activities of pulmonary vasodilation, vascular remodeling inhibition and RVH inhibition, suggesting that it may be a promising agent for PAH intervention.     

PDE5A inhibition attenuates bleomycin-induced pulmonary fibrosis and pulmonary hypertension through inhibition of ROS generation and RhoA/Rho kinase activation

Pulmonary hypertension frequently complicates interstitial lung disease, where it is associated with a high mortality. Patients with this dual diagnosis often fare worse than those with pulmonary arterial hypertension (PAH) alone and respond poorly to standard PAH therapy, often dying of right ventricular (RV) failure. We hypothesize that nitric oxide synthase (NOS) uncoupling is important in the pathogenesis of interstitial lung disease-associated pulmonary hypertension, and this process can be abrogated by phosphodiesterase type 5 (PDE5) inhibition to improve pulmonary vascular remodeling and right ventricular function. Intratracheal bleomycin (4 U/kg) or saline control was administered to C57/BL6 mice after anesthesia. After recovery, animals were fed a diet of sildenafil (100 mg.kg(-1).day(-1)) or vehicle for 2 wk when they underwent hemodynamic measurements, and tissues were harvested. Survival was reduced in animals treated with bleomycin compared with controls and was improved with sildenafil (100.0 vs. 73.7 vs. 84.2%, P < 0.05). RV/LV+S ratio was higher in bleomycin-alone mice with improvement in ratio when sildenafil was administered (33.00 +/- 0.01% vs. 20.98 +/- 0.01% P < 0.05). Histology showed less pulmonary vascular and RV fibrosis in the group cotreated with sildenafil. Bleomycin was associated with a marked increase in superoxide generation by DHE histological staining and luminol activity in both heart and lung. Treatment with sildenafil resulted in a concomitant reduction in superoxide levels in both heart and lung. These data demonstrate that PDE5 inhibition ameliorates RV hypertrophy and pulmonary fibrosis associated with intratracheal bleomycin in a manner that is associated with improved NOS coupling and a reduction in reactive oxygen species signaling.     

Rho-kinase inhibition alleviates pulmonary hypertension in transgenic mice expressing a dominant-negative type II bone morphogenetic protein receptor gene

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by a sustained elevation in the pulmonary artery pressure and subsequent right heart failure. The activation of Rho/Rho-kinase activity and the beneficial effect of Rho-kinase inhibition have been demonstrated in several experimental models of pulmonary hypertension. However, it remains unclear whether Rho-kinase inhibitors can also be used against pulmonary hypertension associated with mutations in the type II bone morphogenetic protein receptor (BMPRII) gene. Transgenic mice expressing a dominant-negative BMPRII gene (with an arginine to termination mutation at amino acid 899) in smooth muscle by a tetracycline-gene switch system (SM22-tet-BMPR2(R899X) mice) were examined. They developed an elevated right ventricular systolic pressure (RVSP), right ventricular (RV) hypertrophy, muscularization of small pulmonary arteries, and an associated disturbed blood flow in their lungs. The Rho/Rho-kinase activity and Smad activity were determined by a Western blot analysis by detecting GTP-RhoA and the phosphorylation of myosin phosphatase target subunit 1, Smad1, and Smad2. In the lungs of SM22-tet-BMPR2(R899X) mice, the Rho/Rho-kinase activity was elevated significantly, whereas the Smad activity was almost unchanged. Fasudil, a Rho-kinase inhibitor, significantly decreased RVSP, alleviated RV hypertrophy and muscularization of small pulmonary arteries, and improved blood flow in SM22-tet-BMPR2(R899X) mice, although it did not alter Smad signaling. Our study demonstrates that Rho/Rho-kinase signaling is activated via a Smad-independent pathway in an animal model of pulmonary hypertension with a BMPRII mutation in the cytoplasmic tail domain. Rho-kinase inhibition is therefore a possible therapeutic approach for the treatment of PAH associated with genetic mutation.