Study On The Mechanism Of Calcium Regulatory Protein Involving In NLRP3 Inflammasome Activation In Heart Failure With Preserved Ejection Fraction Induced By Hypertension

Background:Hypertension is a common cause of heart failure with preserved ejection fraction（HFpEF）,and activation of the NLRP3 inflammasome plays an important role in the development of Hfp EF.When intracellular Ca²⁺overload leads to mitochondrial dysfunction and structural changes,reactive oxygen species（ROS）production increases,and these stimuli lead to NLRP3 inflammasome assembly,which in turn triggers the activation and maturation of downstream inflammatory factors IL-1and IL-18,amplifying the body’s inflammatory response leads to heart damage.Current studies have shown that calmodulin plays an important role in the activation of NLRP3inflammasome,but the mechanism of calmodulin involved in the activation of NLRP3inflammasome in hypertension-induced HFpEF is still unclear.Therefore,this study hypothesized that calmodulin promotes the occurrence and development of HFpEF by participating in the activation of the NLRP3 inflammasome.Objective:In order to clarify that the activation of NLRP3 inflammasome may promote the occurrence and development of HFpEF,the effect of calmodulin on NLRP3 inflammasome and HFpEF and its possible mechanism were explored.Methods:In this study,12-week-old female spontaneously hypertensive rats （SHR）（n=60）and the same week-old female Wistar Kyoto rat（WKY）（n=15）were used as normal control groups,and they were divided into five groups.They were:WKY group;SHR+Diltiazem（SHR+Dil）group;SHR+NLRP3 inflammasome inhibitor（SHR+16673）group;SHR+mito-Tempo（SHR+Tempo）group and SHR group.The following assays were performed after 8 weeks of drug intervention:（1）Measure the blood pressure of rat tail in each group,including systolic blood pressure（SBP）,diastolic blood pressure（DBP）and mean blood pressure（MBP）.（2）The BNP levels in the plasma of the rats in each group were determined by ELISE method.（3）The following values were determined by transthoracic echocardiography:diastolic ventricular septal thickness（IVS d）、left ventricular posterior wall thickness（LVPW）、left ventricular end-systolic diameter（LVESD）、left ventricular end-diastolic diameter（LVEDD）、Blood fraction（EF）、short-axis shortening rate（FS）、early diastolic mitral valve blood flow velocity（E）、late diastolic mitral valve blood flow velocity（A）、early diastolic mitral valve annular tissue velocity（E’）and late diastolic mitral annular tissue velocity（A’）.（4）Determination of oxidative stress-related indicators include:the level of IL-1βin plasma,the content of malondialdehyde（MDA）in myocardial tissue,and the level of nicotinamide adenine dinucleotide phosphate oxidase 4（NOX4）in myocardial tissue.（5）Western-blot technique was used to detect the levels of NLRP3inflammasome-related proteins NLRP3 and ASC in rat myocardial tissue.（6）Western-blot technique was used to detect the levels of calcium regulatory proteins in rat myocardial tissue,including calcium ion/calmodulin-dependent protein kinase IIδ（CaMKIIδ）and Ca²⁺-ATPase 2a（SERCA2a）.（7）HE staining was used to observe the morphological changes of rat cardiomyocytes in each group.Results:（1）Changes in blood pressure of rats in each group:Before intervention,compared with WKY rats,SBP,DBP and MBP in SHR group were significantly increased（P<0.05）.After 8 weeks of intervention,compared with the SHR group,the SBP,DBP and MBP of the SHR+Dil and SHR+16673 groups were decreased,and the results were statistically significant（P<0.05）,while the blood pressure of the SHR+Tempo group did not decrease significantly,and the difference was not statistically significant academic significance（P>0.05）.（2）Compared with the WKY rats,the BNP level in the plasma of the SHR group was significantly increased（P<0.05）.Compared with the SHR group,the BNP level of the SHR+Dil,SHR+16673and SHR+Tempo groups decreased,and the difference was statistically significant.Significance（P<0.05）.（3）Among the structural indicators of the heart,compared with WKY rats,the IVS d and LVPW of the SHR group were increased,and the results were statistically significant（P<0.05）.Compared with the SHR group,SHR+Dil,SHR+16673 and SHR+Tempo group both decreased LVPW,and the results were statistically significant（P<0.05）,but the IVS d did not change significantly,and the difference was not statistically significant（P>0.05）.Among cardiac function indexes,compared with WKY rats,the E/A ratio and E/E’ratio of SHR group were increased（P<0.05）.Compared with SHR group,SHR+Dil,SHR+16673 and the E/A ratio and E/E’ratio of the SHR+Tempo group were both decreased,and the results were statistically significant（P<0.05）.There was no difference in EF,FS,LVESD and LVEDD among the groups（P>0.05）.（4）The detection results of oxidative stress indicators showed that compared with WKY rats,the content of MDA in myocardial tissue,the level of NOX4 protein and the level of IL-1βin plasma increased in the SHR group,and the results were statistically significant（P<0.05）,compared with the SHR group,the SHR+Dil,SHR+16673 and SHR+Tempo groups had lower levels of MDA,NOX4 protein and plasma IL-1βin myocardial tissue（P<0.05）.（5）Compared with WKY rats,the levels of NLRP3 and ASC in the myocardial tissue of the SHR group were significantly increased（P<0.05）.Compared with the SHR group,the levels of NLRP3 and ASC in the SHR+Dil,SHR+16673 and SHR+Tempo groups decreased,the result was statistically significant（P<0.05）.（6）Compared with WKY rats,the level of CaMKIIδin myocardial tissue of SHR group was significantly increased（P<0.05）.The difference was statistically significant（P<0.05）.Compared with WKY rats,the level of SERCA2a in the myocardial tissue of the SHR group was decreased,with statistical significance（P<0.05）.Statistical significance（P<0.05）,but there was no significant difference between the SHR+Tempo group and the SHR group（P>0.05）.（7）HE staining results showed that compared with WKY rats,cardiomyocytes in SHR group were hypertrophic and disordered.Compared with SHR group,cardiomyocytes in SHR+Dil,SHR+16673 and SHR+Tempo groups were slightly less hypertrophic.Conclusion:Calmodulins CaMKIIδand SERCA2a are involved in the activation of NLRP3 inflammasome,and the activated NLRP3 inflammasome promotes the occurrence and development of HFpEF.