Tanshinone ⅡA Attenuated Hypoxic Pulmonary Hypertension Via Upgrading Kvchannels

Pulmonary hypertension （PH） is defined as a multifactorial, progressivedisease, and ultimately leading to severe pulmonary hypertension, rightventricular hypertrophy and right heart failure with a high morbidity andmortality. So far, there are still no effective treatments for PH. Currenttreatments focused on improving the pulmonary vascular dysfunction andvasoconstriction, which may slow the progression of PH but not halted it, andare still limited, expensive, often associated with significant sideeffects.Hypoxic pulmonary hypertension （HPH）, a common type of PH, oftenoccurs in plateau residents and respiratory diseases, such as chronic obstructivepulmonary disease （COPD）. The typical pathophysiological process of HPH ishypoxic pulmonary vasoconstriction （HPV） and gradual increased pulmonaryvascular structure remodeling （PVSR）. Generally, HPV is a uniquephysiological mechanism observed in pulmonary arteries. Acute hypoxia inducean active process of vasoconstriction to redistributes blood to optimallyventilated lung segments, which benefit gas exchange and maximizes oxygenation of venous blood in the pulmonary arterioles. However, sustainedHPV or chronic exposure to hypoxia can lead to elevation of pulmonary arteriespressure and PVSR.As it is well known, there are at least five functionally-distinguishablepotassium ion （K⁺） channels identified in pulmonary artery smooth muscle cells（PASMCs）. Among all K⁺channels, voltage-gated K⁺channels（KV） have beendemonstrated to contribute to regulating the resting membrane potential inPASMCs. Hypoxia can inhibit KVchannels, cause membrane depolarization,and then trigger PASMCs contraction. Studies also revealed that KVchannel inPASMCs play an important role in both the process of HPV and PVSR.Tanshinone IIA （TⅡA）, one of the major active component of Salviamiltiorrhiza Bunge （S. miltiorrhiza）, exhibits many vascular biological activitiesand has been commonly used in traditional oriental herbal medicine to treatcardiovascular diseases. Previous studies showed that T Ⅱ A attenuatedcardiomyocyte hypertrophy, induced coronary arteries vasorelaxation, activationvaries of K⁺channels. Our previous study showed that pharmacologicaltreatment with TⅡA played protective effects on HPH in vivo and stimulatedKV2.1expression in vitro. Therefore, in the current study, we investigatedwhether the protective effects of TⅡA against HPH were correlated withupgrading KVchannel under acute and chronic hypoxia exposure.MethodsAnimalAdult male Sprague-Dawley rats （200-250g weight） were obtained fromthe animal center （FourthMilitaryMedical University, Xi‘an, China）. Rats werekept in a temperature-controlled house with12-hour light-dark cycles.Chronic hypoxia experimentsModel and groupingRats were randomly divided into4groups（n=8）, i.e.,1) Control group and2) TⅡ A group: rat were housed continuously in room air and ambient barometric pressure（⁷18mmHg,21%oxygen） for4weeks with or without TⅡA （10mg/kg） via intraperitoneal injection;3) HPH group: rats were housedintermittently in a hypobaric hypoxia chamber depressurized to380mmHg andunder hypoxia exposure for8h per day continuing4weeks; and4) HPH/TⅡAgroup: rats also were housed intermittently in a hypobaric hypoxia chamberdepressurized to380mmHg and under hypoxia exposure for8h per daycontinuing4weeks, with or without TⅡA （10mg/kg） via intraperitonealinjection after the hypoxia exposure.Pulmonary hype rtension and PVSR assessmentAfter4weeks normoxia or hypoxia exposure with or without TⅡ Atreatment, rats were anesthetized with sodium pentobarbital （30mg/kg, i.p.）.Two heparin-filled blunt-ended polyethylene catheters connected to pressuretransducers were inserted into the right ventricle and the left carotid arteryrespectively, and then the right ventricular systolic pressure （RVSP） and themean carotid arterial pressure （mCAP） were recorded.Next, the hearts were dissected out, divided into right ventricle （RV）, leftventricle （LV） and septum （S）, blotted and weighed respectively to determinethe RV/（LV+S）%as an index of RV hypertrophy.At the end of experiments, the lungs at the lower lobe of the right lungwere fixed with10%formalin, and then were stained with hematoxylin-eosin.Microscopic evaluation was performed to characterize structure remodeling ofthe pulmonary arterioles. The pulmonary arterioles （external diameters of50-200μm） were chosen randomly and were analyzed using animage-processing program. The outside diameter and inside diameter ofpulmonary arterioles were measured, and the medial wall thickness, the crosssectional area of medial wall, and the total cross sectional vessel area wereobtained, following which the two indexes were calculated: the ratio of medialwall thickness （MT%）=100×（medial wall thickness）/（vessel semidiameter）;the ratio of medial wall area （MA%）=100×（cross-sectional medial wall area）/ （total cross-sectional vessel area）. All the morphological analysis was conductedin a double-blind method.RT-PCR analysis and western blottingThe lungs from rats exposed to normoxia or hypoxia for4weeks with orwithout TⅡA treatment were dissected and immediately placed on ice. Thepulmonary arteries （1th-3th divisions） were carefully isolated, and theadventitial tissues were removed. Total RNA was isolated by using Trizolreagent. The primer pairs were designed by primer premier. The primer pairs forKv2.1, Kv1.5, and β-actin as following:（1） Kv2.1[Genebank numberNM013186] forward:5‘-AGG CCG AAC TGT GTC TAC TC-3‘; reverse:5‘-GTC CTC TGC ACC CTC CTA AC-3‘,557bp.（2） Kv1.5[Genebanknumber M27158] forward:5‘-ATG CAG GGT CAC TCC ATC–; reverse:5‘-GGC TTC TCC TCT TCC TTG-3‘,340bp.（3） β-actin [Genebank numberNM031144] forward:5‘-TAA AGA CCT CTA TGC CAA CAC AGT-3‘;reverse:5‘-CAC GAT GGA GGG CCG GAC TCA TC-3‘;240bp.（4） β-actin[Genebank number NM031144] forward:5‘-CAT CTC TTG CTC GAA GTCCA-3‘; reverse:5‘-ATC ATG TTT GAG ACC TTC AAC A-3‘, size ofamplifiedproduct:318bp. PCR products were analyzed by agarose gel electrophoresis.An invariant mRNA quantity of β-actin was used as an internal control toquantify PCR products.Total proteins were extracted. Protein concentrations were determined bycoomassie brilliant blue assay. Western blot analysis was performed by usingprimary antibodies for Kv1.5（1:200）, Kv2.1（1:200）, and β-actin （1:5000）.Immunoreactivity was visualized with corresponding peroxidase-conjugatedsecondary antibodies and the relative content of target proteins was detected bychemiluminescence.PASMCs isolation and electrophysiology recordPASMCs were dispersed from normal rats‘IPA branches （2th-3th division）as previously described. Isolated PASMCs were placed in recording chamber for whole-cell patch-clamping study. After a brief period to allow partial adherenceto the bottom of the recording chamber, cells were continuously perfused （1ml/min） with a solution composed of （in mM）: NaCl135, KCl4.7, MgCl₂1,EGTA1, HEPES10, glucose10, tetrodotoxin0.001and TEA5, heated to34°C（pH7.35-7.45）. The IKVcurrent of PASMC was determined under normoxic orhypoxic conditions in the absence or presence of TⅡA （25ug/ml）. Whole-cellrecordings were made with borosilicate glass pipettes of3-6MΩ resistancecontaining （in mM）: KCl125, MgCl₂4, Na2ATP2, EGTA10and HEPES10（pH7.3; osmolarity285-290）. Cells were voltage-clamped at-70mV and currentswere evoked in20mV steps from-70mV to+70mV using400ms pulses.Whole-cell currents were normalized by cell capacitance and expressed ascurrent density.Acute hypoxia experimentsIntrapulmonary arteriole rings isolation and organ bath expe rimentsNormal rats were anesthetized with sodium pentobarbital （30mg/kg, i.p.）.The lungs were rapidly removed and placed in ice-cold Krebs-Henseleit solution,which contained （M）: NaCl118.4, KCl4.7, CaCl₂2.5, MgCl₂1.2, KH₂PO₄1.2,NaHCO₃25.0, and dextrose11.1. The endothelium-denuded intrapulmonaryarterioles （IPA,200-500μm in intraluminal diameter,3th division） rings wereprepared. Ring segments （3mm） were cut and suspended vertically betweenhooks in organ baths （6ml） containing modified Krebs-Henseleit solution,which was maintained at pH of7.4, heated to37°C, and bubbled with a mixturegas of95%O₂and5%CO₂. Tension was recorded with a force-displacementtransducer and amplifier, and then was analyzed by MacLab/400and Chartsoftware. The IPA rings were stretched under a predetermined optimal restingtension of750mg. The rings were treated with10-6M PE again. When theplateau attained, the rings were exposed to acute hypoxia. Tension changes weremeasured in the absence or presence of TⅡA (10^-5M) or4-AP (10^-6M)pre-treatment. PASMCs isolation and electrophysiology recordThe IKVcurrent of PASMC was determined under normoxic or hypoxicconditions in the absence or presence of T Ⅱ A （25ug/ml）. Whole-cellpatch-clamping were used to analyse IKVcurrents as previously described.Statistical analysisData are expressed as mean±S.D., and statistical analysis was performedwith analysis of variance （ANOVA）, followed by a LSD-t test for multiplecomparisons. A statistical difference was accepted as significant if P <0.05.ResultsTⅡA ameliorated pulmonary hypertension and PVSR in chronic HPHratsThere were no difference in mCAP in all groups, and TⅡA treatment hadno effect on mCAP. There were no significant changes in RVSP and RVHI in TⅡA group and control group. In contrast, rats subjected to hypoxia for4weekshad pulmonary hypertension, evidenced by a significant increase in RVSP andRVHI. However, this elevated RVSP and RVHI were strikingly decreased in theHPH/TⅡA group （P <0.05）.We also observed the pulmonary arterioles histological changes bymicroscope after4weeks intermittent hypoxia exposure. There were normalpulmonary arterioles structure and clear pulmonary alveoli in the Control groupand T Ⅱ A group. Chronic hypoxia exposure caused a marked PVSR,characterized by the small pulmonary arteriole vessel wall thickened, the vessellumina deflated, congestion, edema, and the sequestration of inflammatory cells.However, with TⅡA treatment these changes were less pronounced comparedwith the HPH group. Furthermore, the medial wall thickness （MT）%and themedial wall area （MA）%of the arterioles were dramatically elevated afterchronic hypoxia exposure versus the control group （P <0.01, respectively）. Incontrast, TⅡA treatment significantly diminished these changes （P <0.05, respectively）. Nevertheless, both （MT）%and （MA）%were still no sgnificantdifference between control group and TⅡA group. Above results demonstratedthat TⅡ A normalizes the pulmonary hypertension and PVSR in chronichypoxia-induced HPH and had no effects on systemic hemodynamics andnormal pulmonary arterioles structure.TⅡA stabilized chronic hypoxia-induced the expression of KV1.5andKV2.1Then, we measured the mRNA and protein expressions of KV1.5and KV2.1in the pulmonary arteries from rats exposed to normoxia or chronic hypoxiafor4weeks with or without TⅡA treatment. Results showed that both KV1.5and KV2.1mRNA and protein levels were observably decreased after c hronichypoxia exposure （P <0.01, respectively）. TⅡA treatment partially recoveredthe down-regulation of KV1.5and KV2.1（P <0.05, respectively）, and TⅡAdidn‘t affect the levels in the control group and TⅡA group.TⅡA attenuated acute hypoxia-induced IPA rings vasoconstrictionThere were biphasic vasocontractions: an initial transient contraction, thena transient vasorelaxation （maximum vasodilation）, and finally a delayedsustained contraction （phase II vasoconstriction） when stimulated by acutehypoxia. Pre-incubation with T Ⅱ A (10^-5M) eliminated the acutehypoxia-induced initial transient contraction, potentiated followed maximumvasorelaxation （P <0.05）, and attenuated the phase II vasoconstriction （P <0.01）.Moreover, acute hypoxia-induced phase II vasoconstriction reversed to sustainedrelaxation （phase II vasodilation） soon after. Pre-incubation with TⅡA (10^-5M)and4-AP (10^-6M) also removed the acute hypoxia-induced initial transientcontraction. But the maximum vasorelaxation was suppressed （P <0.05） and thephase II vasoconstriction was significantly increased （P <0.01） compared withTⅡ A pre-treatment. These results showed that TⅡ A treatment improvedrelaxation and concentration dysfunction in chronic hypoxia-induced pulmonary arterioles.T Ⅱ A reversed hypoxia-induced IKVcurrent down-regulation inPASMCsIn chronic hypoxia experiments, TⅡA treatment did not affect the meanIKVcurrents in PASMCs from the Control group and TⅡA group rats. Chronichypoxia leaded to a dramatically decrease in the IKVcurrents compared with theControl group （P <0.01）. TⅡA treatment protocols also reversed the IKVcurrents （P <0.05）. In acute hypoxia experiments, TⅡA did not affect the IKVcurrents under normoxia exposure. However, acute hypoxia markedlysuppressed the IKVcurrents, which was partially reversed in the presence of TⅡA. Above results indicated that TⅡA markedly improved acute and chronichypoxia-induced IKVcurrents down-regulation.ConclusionOur current study investigated that TⅡA significantly attenuated HPV,inhibited PVSR, improved the relaxation and contraction function abnormalityof small pulmonary arterioles, and reduced RVSP increasing and RVhypertrophy, which were probably through upgrading the down-regulation of KVchannels. We demonstrated that TⅡA should be a very attractive potentialtherapy for HPH.