Takashi Urashima

Molecular and physiological characterization of RV remodeling in a murine model of pulmonary stenosis.

Right ventricular (RV) dysfunction is a common long-term complication in patients after the repair of congenital heart disease. Previous investigators have examined the cellular and molecular mechanisms of left ventricular (LV) remodeling, but little is known about the stressed RV. Our purpose was to provide a detailed physiological characterization of a model of RV hypertrophy and failure, including RV-LV interaction, and to compare gene alterations between afterloaded RV versus LV. Pulmonary artery constriction was performed in 86 mice. Mice with mild and moderate pulmonary stenosis (PS) developed stable hypertrophy without decompensation. Mice with severe PS developed edema, decreased RV function, and high mortality. Tissue Doppler imaging demonstrated septal dyssynchrony and deleterious RV-LV interaction in the severe PS group. Microarray analysis showed 196 genes with increased expression and 1,114 with decreased expression. Several transcripts were differentially increased in the afterloaded RV but not in the afterloaded LV, including clusterin, neuroblastoma suppression of tumorigenicity 1, Dkk3, Sfrp2, formin binding protein, annexin A7, and lysyl oxidase. We have characterized a murine model of RV hypertrophy and failure, providing a platform for studying the physiological and molecular events of RV remodeling. Although the molecular responses of the RV and LV to afterload stress are mostly concordant, there are several key differences, which may represent targets for RV failure-specific therapy.

Prognostic factors for the late onset Pompe disease with enzyme replacement therapy: From our experience of 4 cases including an autopsy case

We report 4 cases of late onset glycogen storage disease type II (GSD II) or Pompe disease (OMIM #232300), under enzyme replacement therapy (ERT) with recombinant human acid alpha glucosidase (rh-GAA, OMIM *606800). In these 4 cases, we focused on the case of a 28-years-old man, whose condition at the ERT starting was the worst and resulted in poor prognosis. The autopsy was done under his familys permission, and revealed severe accumulation of glycogen in his muscle, especially diaphragm or iliopsoas, and pulmonary veno-occlusive disease (PVOD) which resulted in severe pulmonary hypertension (PH). This is the first report of PVOD as the cause of PH in Pompe disease. We studied this case comparing to another 3 cases of late onset Pompe disease under the same course of ERT in our hospital, and the average data of the group of late onset Pompe disease with severe pulmonary insufficiency receiving ERT, supposed that low score of the body mass index (BMI) on the baseline, the presence of specific genotype (p.R600C), and signs of pulmonary dysfunction suggesting PH (tachypnea, ultrasound cardiography data) were factors that influenced the prognosis. For a better prognosis in the late onset Pompe disease, an early diagnosis for the early start of ERT before the onset of respiratory failure should be important, and the deliberate management and care should be needed even after the ERT start, especially for severe cases including pulmonary dysfunction.

Smooth Muscle Protein 22α–Mediated Patchy Deletion of Bmpr1a Impairs Cardiac Contractility but Protects Against Pulmonary Vascular Remodeling

Vascular expression of bone morphogenetic type IA receptor (Bmpr1a) is reduced in lungs of patients with pulmonary arterial hypertension, but the significance of this observation is poorly understood. To elucidate the role of Bmpr1a in the vascular pathology of pulmonary arterial hypertension and associated right ventricular (RV) dysfunction, we deleted Bmpr1a in vascular smooth muscle cells and in cardiac myocytes in mice using the SM22&agr;;TRE-Cre/LoxP;R26R system. The LacZ distribution reflected patchy deletion of Bmpr1a in the lung vessels, aorta, and heart of SM22&agr;;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutants. This reduction in BMPR-IA expression was confirmed by Western immunoblot and immunohistochemistry in the flox/flox group. This did not affect pulmonary vasoreactivity to acute hypoxia (10% O2) or the increase in RV systolic pressure and RV hypertrophy following 3 weeks in chronic hypoxia. However, both SM22&agr;;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox mutant mice had fewer muscularized distal pulmonary arteries and attenuated loss of peripheral pulmonary arteries compared with age-matched control littermates in hypoxia. When Bmpr1a expression was reduced by short interference RNA in cultured pulmonary arterial smooth muscle cells, serum-induced proliferation was attenuated explaining decreased hypoxia-mediated muscularization of distal vessels. When Bmpr1a was reduced in cultured microvascular pericytes by short interference RNA, resistance to apoptosis was observed and this could account for protection against hypoxia-mediated vessel loss. The similar elevation in RV systolic pressure and RV hypertrophy, despite the attenuated remodeling with chronic hypoxia in the flox/flox mutants versus controls, was not a function of elevated left ventricular end diastolic pressure but was associated with increased periadventitial deposition of elastin and collagen, potentially influencing vascular stiffness.

Cardiac pressure overload hypertrophy is differentially regulated by β-adrenergic receptor subtypes

In isolated myocytes, hypertrophy induced by norepinephrine is mediated via α(1)-adrenergic receptors (ARs) and not β-ARs. However, mice with deletions of both major cardiac α(1)-ARs still develop hypertrophy in response to pressure overload. Our purpose was to better define the role of β-AR subtypes in regulating cardiac hypertrophy in vivo, important given the widespread clinical use of β-AR antagonists and the likelihood that patients treated with these agents could develop conditions of further afterload stress. Mice with deletions of β(1), β(2), or both β(1)- and β(2)-ARs were subjected to transverse aortic constriction (TAC). After 3 wk, β(1)(-/-) showed a 21% increase in heart to body weight vs. sham controls, similar to wild type, whereas β(2)(-/-) developed exaggerated (49% increase) hypertrophy. Only when both β-ARs were ablated (β(1)β(2)(-/-)) was hypertrophy totally abolished. Cardiac function was preserved in all genotypes. Several known inhibitors of cardiac hypertrophy (FK506 binding protein 5, thioredoxin interacting protein, and S100A9) were upregulated in β(1)β(2)(-/-) compared with the other genotypes, whereas transforming growth factor-β(2), a positive mediator of hypertrophy was upregulated in all genotypes except the β(1)β(2)(-/-). In contrast to recent reports suggesting that angiogenesis plays a critical role in regulating cardiac hypertrophy-induced heart failure, we found no evidence that angiogenesis or its regulators (VEGF, Hif1α, and p53) play a role in compensated cardiac hypertrophy. Pressure overload hypertrophy in vivo is dependent on a coordination of signaling through both β(1)- and β(2)-ARs, mediated through several key cardiac remodeling pathways. Angiogenesis is not a prerequisite for compensated cardiac hypertrophy.

Novel nonsense mutation of the BMPR-II gene in a Japanese patient with familial primary pulmonary hypertension

Familial PPH locus ( PPH1 ) was mapped on chromosome 2q33. 2,3 It has been assumed that familial PPH is transmitted as an autosomal dominant trait. Several mutations were found for both familial PPH 4–6 and sporadic PPH 7 in one of the components of the transforming growth factor beta (TGFβ ) family, namely the bone morphogenetic protein receptor type II (BMPR-II). Bone morphogenetic proteins (BMP) signal through BMPR-II. Certain BMP have been shown to inhibit vascular smooth-muscle cell proliferation and to induce apoptosis in some cell types in culture. 8

Pulmonary hypertension due to left heart disease causes intrapulmonary venous arterialization in rats

Objective: A rat model of left atrial stenosis–associated pulmonary hypertension due to left heart diseases was prepared to elucidate its mechanism. Methods: Five‐week‐old Sprague–Dawley rats were randomly divided into 2 groups: left atrial stenosis and sham‐operated control. Echocardiography was performed 2, 4, 6, and 10 weeks after surgery, and cardiac catheterization and organ excision were subsequently performed at 10 weeks after surgery. Results: Left ventricular inflow velocity, measured by echocardiography, significantly increased in the left atrial stenosis group compared with that in the sham‐operated control group (2.2 m/s, interquartile range [IQR], 1.9‐2.2 and 1.1 m/s, IQR, 1.1‐1.2, P < .01), and the right ventricular pressure‐to‐left ventricular systolic pressure ratio significantly increased in the left atrial stenosis group compared with the sham‐operated control group (0.52, IQR, 0.54‐0.60 and 0.22, IQR, 0.15‐0.27, P < .01). The right ventricular weight divided by body weight was significantly greater in the left atrial stenosis group than in the sham‐operated control group (0.54 mg/g, IQR, 0.50‐0.59 and 0.39 mg/g, IQR, 0.38‐0.43, P < .01). Histologic examination revealed medial hypertrophy of the pulmonary vein was thickened by 1.6 times in the left atrial stenosis group compared with the sham‐operated control group. DNA microarray analysis and real‐time polymerase chain reaction revealed that transforming growth factor‐&bgr; mRNA was significantly elevated in the left atrial stenosis group. The protein levels of transforming growth factor‐&bgr; and endothelin‐1 were increased in the lung of the left atrial stenosis group by Western blot analyses. Conclusions: We successfully established a novel, feasible rat model of pulmonary hypertension due to left heart diseases by generating left atrial stenosis. Although pulmonary hypertension was moderate, the pulmonary hypertension due to left heart diseases model rats demonstrated characteristic intrapulmonary venous arterialization and should be used to further investigate the mechanism of pulmonary hypertension due to left heart diseases.

Impairment of Excitation-Contraction Coupling in Right Ventricular Hypertrophied Muscle with Fibrosis Induced by Pulmonary Artery Banding.

Interstitial myocardial fibrosis is one of the factors responsible for dysfunction of the heart. However, how interstitial fibrosis affects cardiac function and excitation-contraction coupling (E-C coupling) has not yet been clarified. We developed an animal model of right ventricular (RV) hypertrophy with fibrosis by pulmonary artery (PA) banding in rats. Two, four, and six weeks after the PA-banding operation, the tension and intracellular Ca2+ concentration of RV papillary muscles were simultaneously measured (n = 33). The PA-banding rats were clearly divided into two groups by the presence or absence of apparent interstitial fibrosis in the papillary muscles: F+ or F- group, respectively. The papillary muscle diameter and size of myocytes were almost identical between F+ and F-, although the RV free wall weight was heavier in F+ than in F-. F+ papillary muscles exhibited higher stiffness, lower active tension, and lower Ca2+ responsiveness compared with Sham and F- papillary muscles. In addition, we found that the time to peak Ca2+ had the highest correlation coefficient to percent of fibrosis among other parameters, such as RV weight and active tension of papillary muscles. The phosphorylation level of troponin I in F+ was significantly higher than that in Sham and F-, which supports the idea of lower Ca2+ responsiveness in F+. We also found that connexin 43 in F+ was sparse and disorganized in the intercalated disk area where interstitial fibrosis strongly developed. In the present study, the RV papillary muscles obtained from the PA-banding rats enabled us to directly investigate the relationship between fibrosis and cardiac dysfunction, the impairment of E-C coupling in particular. Our results suggest that interstitial fibrosis worsens cardiac function due to 1) the decrease in Ca2+ responsiveness and 2) the asynchronous activation of each cardiac myocyte in the fibrotic preparation due to sparse cell-to-cell communication.

Low cardiac output leads hepatic fibrosis in right heart failure model rats

Background Hepatic fibrosis progresses with right heart failure, and becomes cardiac cirrhosis in a severe case. Although its causal factor still remains unclear. Here we evaluated the progression of hepatic fibrosis using a pulmonary artery banding (PAB)-induced right heart failure model and investigated whether cardiac output (CO) is responsible for the progression of hepatic fibrosis. Methods and Results Five-week-old Sprague-Dawley rats divided into the PAB and sham-operated control groups. After 4 weeks from operation, we measured CO by echocardiography, and hepatic fibrosis ratio by pathological examination using a color analyzer. In the PAB group, CO was significantly lower by 48% than that in the control group (78.2±27.6 and 150.1±31.2 ml/min, P<0.01). Hepatic fibrosis ratio and serum hyaluronic acid, an index of hepatic fibrosis, were significantly increased in the PAB group than those in the control group (7.8±1.7 and 1.0±0.2%, P<0.01, 76.2±27.5 and 32.7±7.5 ng/ml, P<0.01). Notably, the degree of hepatic fibrosis significantly correlated a decrease in CO. Immunohistological analysis revealed that hepatic stellate cells were markedly activated in hypoxic areas, and HIF-1α positive hepatic cells were increased in the PAB group. Furthermore, by real-time PCR analyses, transcripts of profibrotic and fibrotic factors (TGF-β1, CTGF, procollargen I, procollargen III, MMP 2, MMP 9, TIMP 1, TIMP 2) were significantly increased in the PAB group. In addition, western blot analyses revealed that the protein level of HIF-1α was significantly increased in the PAB group than that in the control group (2.31±0.84 and 1.0±0.18 arbitrary units, P<0.05). Conclusions Our study demonstrated that low CO and tissue hypoxia were responsible for hepatic fibrosis in right failure heart model rats.