Molecular Mechanisms Underlying The Pharmacological Effects Against Chronic Heart Failure Targeting At SUR2B/Kir6.1 Channel

Chronic heart failure(CHF)is a high fatal disease among cardiovascular diseases.Many incentive factors including pressure overload(PO),myocardial injury and weakened myocardial contractility can result from hypertension,myocardial ischemia(MI),arrhythmias,drugs(isoproterenol,ISO)and other common cardiovascular diseases.These causes lead to the imbalance of heart function regulation,which deteriorate cardiac function and eventually become chronic heart failure.At present,drugs for treating CHF have many side effects in the clinic,such as toxicity,narrow treatment window,increasing mortality for long-term treatment,and so on.Therefore,it is of great significance to seek new targeted,structure drugs and novel therapeutic approaches for the prevention and treatment of chronic heart failure.Endothelial cells are an important functional unit throughout the cardiovascular system.It is not only the first barrier of the blood vessel,but also can exchange material,release many biological active substances and regulate the tension of the blood vessel.Cardiovascular diseases are closely associated with the distributed integrity and normal function of endothelial cell on the vessel.Endothelial dysfunction can promote the occurrence and development of cardiovascular diseases.Similarly,cardiovascular diseases can lead to endothelial dysfunction and accelerate the development of diseases.Endothelial dysfunction is characterized by an imbalance between diastolic and contractile factors,such as NO and endothelin.It has been reported that endothelial dysfunction plays a vital role in PO-CHF,MI-CHF,ISO-CHF and vascular remodeling.NO is an important regulator of cardiac and vascular remodeling,and ET-1 is a promoting factor of myocardial hypertrophy and CHF,long-term imbalances between NO and ET system will promote cardiac remodeling,finally result in chronic heart failure.Therefore,it is a novel treatment approach for CHF to correct the imbalance of secretion substance from endothelial cells and to protect endothelial cell function.KATP channels are widely distributed in various tissues and organs,but there are obvious specificity from different subtype.SUR subunits are composed of SUR 1,SUR2 A and SUR2 B,and Kir subunits include Kir1.1,Kir6.1 and Kir6.2.The combination between SUR and Kir subunits has specific subtypes in the different tissues.The blood vessels were mainly located in SUR2B/Kir6.1 and SUR2B/Kir6.2 subtypes,among which SUR2B/Kir6.1 subtypes has more abundant expression in smooth muscle and endothelial cells.Our previous studies from research center had demonstrated that it was an important novel target for the treatment of cardiovascular diseases via activating the SUR2B/Kir6.1 channel in endothelial cells,correcting endothelial dysfunction and restoring endothelial function.Iptakalim(Ipt),a novel compound of fatty secondary amine with independent intellectual property rights,is ATP sensitive potassium channel opener(KCOs).At present,Ipt has entered the phage III clinical study on the treatment of hypertension.Natakalim(Nat),a derivative of Ipt,have the same parent nucleus and the different side chain structure.Our previous study has proved that Ipt and Nat can high selectively activate SUR2B/Kir6.1 channel by using patch clamp technique under normal physiological conditions.Moreover,Ipt and Nat have higher selectivity to SUR2B/Kir6.1 channel while energy substances were suppressed in the cell simultaneously.The studies provided a theoretical basis for SUR2B/Kir6.1 channel openers Nat and Ipt for treating CHF.According to the different factors resulting in CHF,we observed the pharmacological effect of Ipt and Nat in POCHF respectively,the effects of Nat were also observed in MI-CHF and ISO-CHF.The results showed that Ipt and Nat could restore endothelial function,protect the endothelial cells,and eventually improve the PO-CHF,MI-CHF and ISO-CHF.According to previous studies,we will provide the pharmacological evidence for treating CHF by targeting SUR2B/Kir6.1 channel in endothelial cells and explore the molecular mechanism of SUR2B/Kir6.1 channel openers.In the study,we further focus on the following questions:(1)Under the same experimental conditions,whether the highly selective SUR2B/Kir6.1 channel openers Nat or Ipt has equivalent effectiveness against CHF.(2)In vivo,whether the cardiovascular effects of Nat can be blocked by the highly selective KATP blocker(KCB)glibenclamide(Gli).(3)Whether the SUR2B/Kir6.1 channel openers or ACEI lisinopril(Lis)has the different mechanisms against ISO-CHF.(4)What protein molecules are involved in the molecular mechanisms underlying the SUR2B/Kir6.1 channel pathway actions against CHF.And(5)At present,miRNAs,a hot molecule in the cardiovascular system,is closely associated with CHF,and how miRNA molecules participate in the pharmacological effects of Nat through the regulatory networks of targeted gene.The aim of these studies is to provide experimental evidence for treating CHF through SUR2B/Kir6.1 channel in endothelial cells,and to provide an updated and in-depth understanding of the molecular mechanism of Nat against CHF.Part Ⅰ The effects for SUR2B/Kir6.1 channel openers against CHFBased on the previous CHF model,we established nine groups in the study: control,ISO model,Nat1,3,9 mg·kg-1·d-1 group,Ipt 3 mg·kg-1·d-1group,Lis 15 mg·kg-1·d-1 group,Gli 50 mg·kg-1·d-1 group,Gli combined with Nat treatment group.We compared the pharmacological effects of the same dose(3 mg·kg-1·d-1)of Nat and Ipt in the same model.We firstly observed the effect of KATP blocker Gli on Nat against CHF in vivo,and compared the mechanism of ACEI Lis and Nat for treating CHF.The results showed that,in terms of the hemodynamics,1,3,9 mg·kg-1·d-1 Nat could improve hemodynamic indexes including LVSP,+dp/dtmax,Vpm,Vmax and LVEDP,-dp/dtmax;the same dose(3 mg·kg-1·d-1)of Ipt and Nat had the same pharmacological effect;Gli could antagonize the effects of Nat.In respect of the cardiac structure remodeling,we showed that 1,3,9 mg·kg-1·d-1 Nat could reverse cardiac pathological changes based on the results of myocardial HE staining,TEM and immunohistochemistry.The same dose(3 mg·kg-1·d-1)of Ipt could also attenuate myocardial pathological changes.Gli had no effect on the established model,but it could antagonize the effect of Nat on reversing cardiac pathological changes;ACEI Lis could also improve pathological remodeling of myocardial structure.In CHF biomarkers,the same dose of Nat and Ipt could down-regulate the levels of CHF biomarkers NT-pro BNP level in the plasm and BNP expression in cardiac tissue,and Gli could block the effect of Nat;Lis could decrease their content in the plasma and cardiac tissue.On endothelial mechanism,compared with ISO group,Nat could increase e NOS m RNA expression in the myocardial tissue,while down-regulate the expression of i NOS and ET-1 m RNA.The same dose(3 mg·kg-1·d-1)of Ipt and Nat showed the equivalent pharmacological effect.Gli can antagonize the actions of Nat.Lis can decrease ET-1 level in the plasma and down-regulate ET-1 m RNA expression in myocardial tissue,but the effect of Lis on i NOS and e NOS m RNA expression was not statistically significant.These results suggested that Nat and Ipt could treat CHF by correcting the imbalance of endothelial function and protecting endothelial function through SUR2B/Kir6.1 channel.However,Lis could improve CHF not through correcting endothelial dysfunction.Therefore,the mechanism of Lis against CHF is completely different from that of Nat.Part Ⅱ The molecular mechanism of SUR2B/Kir6.1 channel opener Nat by i TRAQ technologyTo further explore Nat’s molecular mechanism,we selected the plasma as the study object,which was closely related to endothelial cells.Before and after Nat treatment,protein components in the plasma were detected by i TRAQ technology.We firstly found and screened out the key proteins closely associated with the effects of Nat against CHF.Seven hundred and twenty-four proteins were identified,among which 55 proteins were differences(P<0.05),compared to the ISO group,33 proteins were upregulated and 22 proteins were down-regulated.The results suggested that 55 proteins were closely related to action of Nat for treating CHF.We further analyzed 55 proteins by bioinformatics way including GO functional annotation,KEGG pathway enrichment analysis and protein-protein interaction analysis.GO enrichment analysis showed that the enriched proteins could be enriched into three categories: biological process,molecular function and cellular components.There are 42 proteins that are enriched and annotated,such as endothelial development(P=0.0230)and ATP decomposition(P=0.0024).GO analysis demonstrated that the molecular mechanism of Nat against CHF could be achieved by the above biological process or molecule function.KEGG pathway analysis indicated that the proteins could be enriched into 24 pathways including PRKAR2β,GAS6/e NOS/NO and NO/PKG/VASP pathway,which were closely associated with CHF.Protein-protein interaction networks were classified into four categories: the proteasome system,ATP function,energy metabolism,stress and c AMP dependent PKA signal pathway.Proteins and their combined clusters played a pivotal role in pathophysiology of CHF.We screened out ATP and five key proteins and validated them.The results by ELISA were consistent with the results of i TRAQ analysis.PRKAR2β,GAS6 and VASP proteins were involved in endothelial mechanism,and ATP,AGT and TIMP3 contributed to the cardiovascular effects of Nat.We further validated the relationship among Nat,NO and VASP by establishing HUVEC model,the results found that Nat could up-regulate VASP expression,NOS antagonist L-NAME could inhibit the effect of Nat on VASP expression,suggesting that Nat,a targeted to SUR2B/Kir6.1 channel opener,could treat CHF through e NOS/VASP pathway in endothelial cell.Part Ⅲ The effect of miR-1-3p involved in Nat against CHF and its molecular pathwaymicroRNA(miRNA or miR),a hot molecular in the cardiovascular system,has the most extensive regulation function and is closely related to cardiovascular diseases.miRNA-mediated gene expression silencing had previously been reported to be important in many physiological and pathological processes of cardiovascular diseases.Many miRNAs express specifically in blood vessels and heart and play a key role in regulating the physiological function.Based on the online website database(NCBI and GO)and previous reports in the literature,we selected 13 miRNAs and detected the expression level of these miRNAs by q RT-PCR in myocardial tissue.The results showed that the expression of miR-1-3p,miR-133a-3p and miR-29a-3p in myocardial tissue significantly increased after Nat treatment,and the expression of miR-423-5p,miR-125a-5p,miR-125b-5p and miR-199a-3p decreased.Among them,the difference fold of miR-1-3p was more significant.Hence,miR-1-3p was further regarded as the object of study in the experiment.We predicted the target genes of miR-1-3p in miRDB and Target Scan databases and then took their intersection,183 target genes were predicted in two databases simultaneously.GO and KEGG enrichment analysis on targeted genes showed that many target genes of miR-1-3p could be enriched to biological processes or pathways associated with cardiovascular disease.Therefore,it is reasonable to speculate that miR-1-3p is involved in the regulation effect of Nat on the cardiovascular system.The prediction of binding site revealed that there was a complemental sequence of miR-1-3p located at ET-1 m RNA 3’ UTR 158-165 sequence.To verify the binding site,we successfully constructed a double luciferase vector of ET-1 m RNA 3’ UTR wild type and mutant type.The estabolished vectors and miR-1-3p mimic were co-transfected in HEK 293 cells,fluorescence method proved the existance of binding between miR-1-3p and ET-1 mRNA 3’ UTR.To furthere xplain the relationship among Nat,miR-1-3p and ET,we successfully constructed the lentivirus vector of miR-1-3p overexpression(PDS087-pri-miRNA-1-3p)and miR-1-3p inhibition expression(p L6.3-GFP-Tu D2-rno-miR-1-3),respectively, and then successfully completed the lentiviral packaging in the HEK 293 T cells.In rat primary cardiac microvascular endothelial cells(RPCMEC)model,we observed the effect of Nat on the expression of miR-1-3p.The results showed that Nat could increase the expression of miR-1-3p,which was consistent with the results of animal models.RPCMEC were infected by the lentivirus containing overexpression or inhibition expression vector of miR-1-3p.The results demonstrated that there was a reverse relation between ET-1 and miR-1-3p expression.Therefore,it is reasonable to suggest that Nat can regulate ET-1 expression and correct the endothelial function dysfunction via miR-1-3p in endothelial cells,and then achieve therapeutic effects for CHF.In summary,the study can draw the following conclusions:(1)In the study,under the condition of the same model and the same dose,SUR2B/Kir6.1 channel openers Nat and Ipt with the same parent nucleus and different chain structures have the equivalent pharmacological effects for CHF treatment.Their molecular mechanisms are closely related to correcting the disorder of endothelial function and protecting endothelial cells in the blood vessel.The action mechanisms of ACEI Lis against CHF is completely different from that of Nat.Gli can antagonize the action of Nat against CHF in vivo,suggesting that Nat can highly select SUR2B/Kir6.1 channel and activate the channel open for treating CHF.The results provide systematic pharmacological evidence for a novel approach for CHF treatment through SUR2B/Kir6.1 channel.(2)In this study,ATP and five key proteins were screened out by proteomics method and then validated them.Bioinformatics analysis showed that VASP protein had strong relationship with NO system after Nat treatment.The results in vivo and in vitro demonstrated that Nat could improve CHF through the e NOS/VASP pathway in endothelial cells,which provided new insights into the pharmacological mechanism of Nat against CHF.(3)In the study,we found that miR-1-3p expression was closely associated with the action of Nat in vivo and in vitro.The prediction and validation of target gene proved the existence of the combination between miR-1-3p and ET-1 m RNA 3 ’UTR.We successfully constructed the lentivirus with miR-1-3p overexpression and inhibition expression vector,and then RPCMEC were infected.The results demonstrated that Nat could regulate endothelial function and have a therapeutic effect on CHF through the miR-1-3p/ET-1 pathway,which provided a new direction for the molecular mechanism of Nat against CHF.In conclusion,this paper provides a comprehensive and systematic pharmacological evidence for the novel approach to treat CHF through SUR2B/Kir6.1 channel,which provides guiding significance for the development of new drugs targeted SUR2B/Kir6.1 channel against CHF.Moreover,the study provides a novel insight and new directions into the molecular mechanism of Nat against CHF.