Effect Of Iptakalim On Pulmonary Arterial Hypertension And Cardiac Remodeling And Its Pharmacological Mechanisms

Objective: Pulmonary arterial hypertension (PAH) is a group of chronic and progressive diseasescharacterized by a sustained elevation of pulmonary vascular resistance leading to right ventricularfailure and premature death. The exact pathogenesis of PAH is not fully understood, but recentevidence demonstrates that elevated pulmonary artery pressure is the result of vasoconstrictionand structural remodeling of pulmonary vessels. There are multiple and complex mechanismsinvolving in the progression of PAH, including proliferation of pulmonary arterial smooth musclecells, endothelial dysfunction and inflammation.Iptakalim hydrochloride,2,3-dimethyl-N-(1-methylethyl)-2-butanamine hydrochloride, is anew structure type of adenosine triphosphate (ATP) sensitive potassium channel (KATP) opener(KCO) preferentially activating SUR2B/Kir6.1subtype without activation of SUR1/Kir6.2. Ourprevious studies showed that iptakalim inhibits the overexpression of monocyte chemoattractantprotein-1(MCP-1), Intercellular adhesive molecule-1(ICAM-1), and vascular cell adhesivemolecule-1(VCAM-1) mRNA induced by low-density lipoprotein, homocysteine, orhyperglycemia, prevents progression of cardiac hypertrophy to failure induced by a pressureoverload and ameliorate the remodeling of pulmonary artery in hypoxic pulmonary hypertensiverats,via augmenting the release of nitric oxide (NO) and inhibiting the endothelin-1(ET-1)system during endothelial dysfunction, thereby resulting in endothelial protection. Iptakalim alsoinhibited the ET-1-induced proliferation of cultured human pulmonary arterial smooth musclecells and rabbit pulmonary arterial smooth muscle cells, which suggested that iptakalim might be apromising candidate for the treatment of pulmonary arterial remodeling in pulmonary hypertension.Chronic hypoxia and injection of MCT can establish PH animal model, but the formationmechanism of two models is different, moreover, the performance of both models and the degreeof damage are also different. Hypoxic PH is usually only moderate and limited to medialhypertrophy with varying degrees of adventitial change, but may progress to extensive remodelingin some species. However, MCT induced PH is severe with endothelial degeneration orhyperplasia, hypertrophy of medial smooth muscle, and adventitial edema, which is used as astandard model for PAH.Thus, this study was designed to investigate the protective and therapeutic effect of Iptakalimhydrochloride on PAH induced by monocrotaline (MCT) in rats.Methods1Experimental protocol50adult male Wistar rats (180-220g) were randomly divided into five groups (n=10pergroup): model group, three iptakalim-treatment groups and control group. In the model group andthree iptakalim-treatment groups, rats were given a single intraperitoneal injection of MCT (50mg/kg), while the rats in the control group received an equal volume of physiological saline. Therats in the iptakalim-treatment and model groups were further assigned at random to receive eitheriptakalim (1,3, and9mg/kg/day, po, respectively) or an equal volume of physiological salinefrom the day of the monocrotaline injection up to the28th day.2Detection indexOn day28, all animals were weighed and anaesthetized with sodium pentobarbital. Afterstable anesthesia was obtained, two polyethylene catheters were inserted into the right ventriclethrough the right jugular vein and the left carotid artery respectively for hemodynamicmeasurements. Right ventricle systolic pressure, systemic blood pressure and heart rate weremeasured with a polygraph system. The blood samples were collected for investigating the changeof NO, ET-1and BNP in serum or plasma; the heart and lung were rapidly removed after animalswere killed, the left ventricles (including interventricular septum, LV+S) and right ventricular freewalls (RV) were collected separately and weighed. The right ventricular-weight index [RV/(LV+S)]was calculated. Right ventricle and left lung samples were fixed in10%buffered formalin andembedded in paraffin. The right lung was dissected into pieces, weighed and homogenized in0.8mL of physiological saline per200mg of tissue. The supernatant of the tissue sampleshomogenization was used for NO, ET-1and TNF-α measurement. Histopathological change Rightventricle and left lung samples were evaluated by hematoxylin and eosin staining. The medial thickness and external diameter were recorded and assessed microscopically for the degree ofarterial muscularization. The medial wall thickness was measured at two locations of each arteryand was expressed as the summation of the two points of (medial thickness/externaldiameter)×100%. In situ TUNEL was performed using assessment of the right ventricularmyocardial tissue cell apoptosis and the number of TUNEL-positive cardiac myocyte nuclei wasexpressed as percentage of the total number of cardiac myocyte nuclei and called the apoptosisindex. The streptavidin-peroxidase conjugate (SP) method was performed in sections stained witha primary polyclonal antibody (ab19645, abcam) to BNP protein expression in the right ventricularmyocardial tissue and the degree of BNPexpression was assessed by the mean optical density.3Statistical analysisData are expressed as mean±SD. Differences between groups were assessed by ANOVA andStudent Newman–Keuls post hoc test for multiple comparisons. Statistical significance wasaccepted at P<0.05.Results:1Hemodynamic effects of iptakalimon day28after MCT administration, RVSP in monocrotaline-induced model group waselevated markedly compared with that in control group. However, HR and MAP inmonocrotaline-induced model group were decreased significantly compared with control group.Iptakalim at the daily oral doses of1,3, and9mg/kg for28days ameliorated the increase in RVSPand reduction in HR and MAP.2Iptakalim prevents the right ventricular hypertrophy induced by monocrotalineon day28after MCT administration, ratio of RV weight to LV plus septum weight (RV/LV+S)in monocrotaline-induced model group were elevated markedly compared with control groupvalues, however, body weight (BW) in monocrotaline-induced model group were decreasedsignificantly compared with control group. Iptakalim at the daily oral doses of1,3, and9mg/kgfor28days ameliorated the increase in RV/LV+S. In addition, the body weight of theMCT-injected group did not differ from control with iptakalim treatment at the daily oral doses of1,3, and9mg/kg for28days.3Iptakalim downregulates BNPprotein expression in the right ventricular myocardial cellsBecause it was well recognized that BNP is a molecular marker of heart failure,wedetermined the effects of iptakalim on the expression of BNP in myocardial cells. In the modelgroup, the mean optical density of right ventricular myocardial cell BNP protein expressiondramatically increased when compared to control rats. These changes could be prevented completely by treatment with iptakalim at the dose of9mg/kg/d for4weeks.4Iptakalim inhibits pathological changes in lung tissues and right ventricular tissuesOn day28after MCT administration, alveolar mononuclear cell infiltration was observed inlung tissue consolidation fibroblasts region. The wall of pulmonary artery was thickened, andintimal smooth muscle cell disorder, hyperplasia, stenosis, and occlusion were also observed. Thecapillaries in lung tissues were expanded slightly and congested. The secretions of bronchia wereincreased, and necrotic epithelial cells, red blood cells and edema fluid were found in bronchialcavity. Histological examinations demonstrated that fibrosis of the heart occurred in the rightventricle myocardium. The numbers of monocytes and lymphocytes increased in myocardialinterstitial tissues. Scattered, small sheet of multiple foci of myocardial necrosis were alsoobserved in the right ventricle myocardium derived from model group. All above mentionedpathological changes in lung tissues and right ventricular tissues could be prevented by treatmentwith iptakalim at the dose of9mg/kg/day for4weeks.MCT-treated rats had significantly increased medial wall thickness of muscular pulmonaryarteries corresponding to terminal bronchioles compared with that of control group. Iptakalim atthe dose of9mg/kg/day treatment could reduce the MCT-induced increase in medial wallthickness significantly.5Iptakalim attenuated the right ventricular myocardial tissue cell apoptosis induced byMCTOn day28after MCT administration, in the model group, the rate of the right ventricularmyocardial tissue cell apoptosis dramatically increased compared with the control rats. The cellapoptosis induced by MCT could be attenuated by treatment with iptakalim at the dose of9mg/kg/day for4weeks.6Iptakalim down-regulated serum NO production, plasma BNPand ET-1levelsThe concentrations of serum NO, plasma BNPand ET-1were increased at the end of4weeksafter MCT administration. Iptakalim treatment could significantly prevent these effects.7Iptakalim suppressed TNF-α, NO and ET-1Levels in lung tissuesOn day28after MCT administration, the lung TNF-α, NO, ET-1levels of the model groupwere increased significantly compared with those of control group, respectively. These changescould be prevented by treatment with iptakalim at the dose of1,3,9mg/kg/day for4weeks.Conclusions: Iptakalim, a new KCO, reduced right ventricle remodeling and RVSP, increased theMAP and HR, inhibited pathological NO, ET-1, BNP and TNF-α release,improved thepathological changes of the right ventricle and pulmonary tissue in MCT animals. Results of this study suggest that iptakalim might be a promising candidate for the treatment of PAH and rightheart failure.