Effects Of Cardiac LPA/TRPV1 Signaling Mediating Ischemia-reperfusion Injury In Isolated Mouse Heart And Its Mechanisms

Background and objective Myocardial ischemia-reperfusion injury（IRI）commonly occurs in myocardial infarction,cardiopulmonary resuscitation,percutaneous coronary intervention（percutaneous coronary intervention,PCI）,major cardiovascular surgery under cardiopulmonary bypass and so on.The myocardium suffered from long term ischemia may be subject to further aggravation of myocardial injury while restoring blood perfusion.Although a large number of studies have focused on different aspects of the mechanism of IRI,in order to develop treatments for preventing post-ischemic myocardial injury,there is still a lack of effective myocardial protection strategies in clinical practice.Transient receptor potential vanilloid 1（TRPV1）is a kind of nociceptive molecule,which can be activated by a series of noxious stimuli.It plays an important role in atherosclerosis,hypertension,heart failure,vascular remodeling,and other cardiovascular diseases.TRPV1 receptor has become a vital target for the prevention and treatment of cardiovascular diseases.Lysophosphatidic acid（LPA）is an intermediate product of cell membrane phospholipid metabolism,which can be largely released during tissue injury or necrosis.LPA can directly bind to the Lysine（K）of 710 site（K711 in mouse）at the C-terminus of TRPV1,by which it induces acute pain.Currently,studies on the role and mechanism of LPA in ischemic heart diseases are mostly focused on the binding of LPA with its LPA receptors.There is still no report regarding the interaction of LPA with cardiac TRPV1 receptor in myocardial injury after ischemia.Therefore,this study is design to explore whether the interaction of LPA with cardiac TRPV1 receptor mediates myocardial ischemia-reperfusion injury,aiming to provide a theoretical basis and therapeutic target for the prevention and treatment of perioperative myocardial injury.Methods1.Effects of LPA on ischemia-reperfusion injury in isolated mouse heart.C57BL/6 mice were randomly divided into 4 groups with 9 mice in each group as following:sham operation group（Sham group）,ischemia-reperfusion group（IR group）,IR+LPA group and IR+HA130 group.The IRI model of isolated mouse heart was established by Langendorff device.The hearts in sham group were continuously perfused for 110 min.The hearts in IR group were stabilized for 20 min followed by no perfusion for 30 min and reperfusion for 60 min.Exogenous LPA was added in the K-H solution during IR in the IR+LPA group while HA130,an LPA synthesis inhibitor,was intraperitoneally injected before IR in the IR+HA130 group.The infarct volume was measured by TTC staining,the determination of LPA and LDH levels in coronary effluent and LPA concentration in serum was measured by ELISA method.Finally,western blot was used to determine the expression levels of p TRPV1/TRPV1 and Bcl-2/Bax in myocardial tissues.2.Effects of TRPV1 inhibitory peptide V1-cal on LPA-mediated myocardial ischemia-reperfusion injuryA peptide V1-Cal（YGRKKRRQRRRGSGRAITILDTEKS）and the control peptide TAT（YGRKKRRQRRRGSG）were synthetized to mimic the TRP domain sequence of TRPV1,and the TRP domain is the key structure that determines the opening of TRPV1channel.C57BL/6 mice were randomly divided into 3 groups with 6 mice in each group as following:IR+V1-cal group,IR+TAT group and IR+LPA+V1-cal group.The IRI model of isolated mouse heart was established by Langendorff device.The hearts in IR+V1-cal group and IR+TAT group were given exogenous V1-cal（1μmol/L）and TAT（1μmol/L）in addition to IR,and the hearts in IR+LPA+V1-cal group were given exogenous both V1-cal（1μmol/L）and LPA（10μmol/L）besides IR.The infarct volume was measured by TTC staining,and the LDH levels in coronary effluent was measured by ELISA method.3.Effects of TRPV1 K711N mutation on myocardial ischemia-reperfusion injuryTRPV1K711N point mutant mice were constructed via CRISPR/Cas9 technique,by which the lysine（K,Lys）at site 711 was mutated into asparagine（N,Asn）.Then,WT and TRPV1^K711N mice were divided into four groups with 6 mice in each group:WT+Sham group,WT+IR group,TRPV1^K711N+Sham group and TRPV1^K711N+IR group.The infarct volume was measured by TTC staining,and the LDH levels in coronary effluent was measured by ELISA method.The myocardial mitochondria of WT and TRPV1^K711Nmice were isolated and stimulated by LPA.The response of mitochondria to LPA was observed in WT and TRPV1^K711N mice.Results1.LPA aggravated ischemia-reperfusion injury in isolated mouse heart.When compared with the Sham group,IR caused evident myocardial infarction and increased the levels of LDH and LPA in coronary effluent.The increase of LPA was linearly correlated with myocardial infarction volume.In addition,the protein levels of p TRPV1 and TRPV1 in myocardium were increased,while the ratio of Bcl-2/Bax was decreased.The myocardial inury in IR+LPA group was aggravated as compared to those of IR group.LPA also up-regulated the protein levels of p TRPV1 and TRPV1 in myocardium.In contrast,myocardial IRI and the levels of p TRPV1 and TRPV1 were reversed in the IR+HA130 group.These results suggest that LPA mediates IRI in isolated mouse heart,which may be associated with the activation of TRPV1 signal.2.TRPV1 inhibitory peptide V1-cal blocks LPA-mediated myocardial ischemia-reperfusion injury.Administration of TRPV1 inhibitory peptide V1-cal before global ischemia and before reperfusion reduced myocardial infarction induced by IRI.However,when V1-cal peptide was perfused in company with LPA,the infarct volume and the level of LDH in coronary effluent were both reduced after reperfusion.This suggests that V1-cal may block the binding of LPA with TRPV1 receptor,thus attenuating LPA-mediated post-ischemic myocardial injury.3.TRPV1^K711N mutation attenuated myocardial ischemia-reperfusion injury and LPA-induced mitochondrial swelling.TRPV1^K711Npoint mutant mice were successfully constructed by CRISPR/Cas9technique and genotypes were confirmed by DNA sequencing.As compared with WT mice,the infarct volume and the level of LDH in coronary effluent caused by IRI were decreased in TRPV1^K711N mice.When the isolated myocardial mitochondria from WT and TRPV1^K711N mice were stimulated with LPA,the absorbance at 540 nm was markedly declined in WT mitochondria,indicating the swelling mitochondria,whereas the mitochondrial swelling in TRPV1^K711N mouse were alleviated.ConclusionIRI in isolated mouse heart increases the level of LPA,which can aggravate myocardial injury after myocardial ischemia by activating TRPV1 receptor.TRPV1inhibitory peptide V1-cal blocks the binding region of LPA with TRPV1 receptor and significantly reduces myocardial IRI induced by LPA.The K711N mutation of TRPV1receptor hinders the interaction of LPA with TRPV1,thus reducing myocardial IRI and LPA-mediated mitochondrial swelling.