The Mechanism Of CaMKⅡ Regulating Heart Failure And The Intervention Effect Of Qi-invigorating And Blood-activating Metho

BackgroundHeart failure（HF）refers to a group of complex clinical syndromes caused by abnormal changes in cardiac structure and/or function due to multiple factors,leading to ventricular systolic and/or diastolic dysfunction,mainly manifested as dyspnea,fatigue and fluid retention.Currently,there are about 23 million HF patients globally,of which HF with reduced ejection fraction（HFrEF）accounts for～50%.Although some progress has been made by Western medicine in the treatment of HFrEF,development to HFrEF still indicates high mortality and readmission rate.Therefore,prevention and treatment of HF,especially HFrEF is grim,which is currently still a hot and difficult issue.Traditional Chinese medicine（TCM）treatment has unique advantages in improving the quality of life and symptoms of HFrEF patients.The development of TCM lies in inheritance and innovation.The medication rules of distinguished doctors are the focus of TCM inheritance.Professor Qian Lin learnt from Professor Jiazhen Liao,an esteemed TCM master in Beijing.Prof.Lin is a famous TCM doctor in Beijing and "Qihuang scholar".She has been engaged in clinical practice,scientific research,and teaching of cardiovascular diseases treated by integration of traditional Chinese and Western medicine for more than 30 years,and achieved favorable effect on HFrEF using the methods of boosting qi and promoting blood circulation based on the theory of qi and blood in TCM.The analysis and summary of Professor Qian Lin’s medication rules in the treatment of HFrEF is particularly important in the inheritance of TCM treatment for HFrEF.Therefore,in this study,data mining was used to analyze and summarize her medication rules in the treatment of HFrEF,to provide a reference for the clinical application of TCM in the treatment of HFrEF,and to better inherit the essence of TCM.In clinical practice,Professor Lin has achieved satisfactory effect on HFrEF using boosting qi and promoting blood circulation.Previous studies by our team have shown that methods of boosting qi and promoting blood circulation can effectively improve the symptoms and cardiac function of patients with HFrEF,increase left ventricular（LV）ejection fraction（LVEF）,and ameliorate patients’ quality of life.Many fundamental studies have proved that Chinese herbal medicines（CHMs）and main components with the actions of boosting qi and promoting blood circulation may improve myocardial calcium homeostasis by regulating CaMKII,thereby alleviating HFrEF.Our team group also showed that Chinese herbal medicines with the actions of boosting qi and promoting blood circulation such as Dang Shen（Codonopsis Radix）,Huang Qi（Astragali Radix）,Dan Shen（Salviae Miltiorrhizae Radix Et Rhizoma）,and San Qi（Notoginseng Radix Et Rhizoma）can improve HFrEF by inhibiting the expression of CaMKII,but the underlying mechanism is still unclear.CaMKⅡδB and CaMKⅡδC are the main isoforms of CaMKⅡ in the heart,which play critical roles in HFrEF,and its overexpression can lead to HFrEF.Therefore,exploring the effect of boosting qi and promoting blood circulation on CaMKⅡδB and CaMKⅡδC is conducive to further explaining its potential mechanism for the treatment of HFrEF.In this study,the HFrEF model was established by constructing conditional overexpression of CaMKⅡδB and CaMKⅡδC transgenic mice,and the mechanism of CaMKII in HFrEF and the intervention effect of boosting qi and promoting blood circulation were thoroughly explored from the overall,tissue,cellular and molecular levels,to verify the hypothesis that qi boosting and blood circulation promoting can inhibit the overexpression of CaMKⅡδB and CaMKⅡδC and improve the expression of hypertrophic factors of cardiomyocytes and calcium pathway to prevent and treat HFrEF.PartⅠ Analysis of Professor Qian Lin’s medication rules and experience summary in the treatment of HF with reduced ejection fraction based on data mining technologyObjectiveTo discuss and summarize Professor Qian Lin’s medication rules in the treatment of HFrEF,to provide a reference for the clinical application of TCM in the treatment of HFrEF,and to better inherit the essence of TCM.MethodsThrough the database of the Information Center of Dongzhimen Hospital of Beijing University of Chinese Medicine,outpatient prescriptions that met the criteria of inclusion were collected.After data cleaning and herbal name specification,the Ancient and Modern Medical Record Cloud Platform was used to calculate and analyze the numbers,frequency,four qi and five flavors of CHMs,and meridian entry.SPSS 20.0 software was used for cluster analysis using systematic clustering method;R Studio software was used for association analysis using Apriori algorithm;and finally,R Studio was used to visualize the results.Results1.Results of prescription inclusion:A total of 67 prescriptions from first-visit patients who met the criteria were included in this study,involving 80 herbs and a total of 1088 times.The top 10 most frequently used drugs were Huang Qi,Dan Shen,Dang Shen,Dang Gui（Angelicae Sinensis Radix）,Yuan Zhi（Polygalae Radix）,Hong Hua（Carthami Flos）,Ze Xie（Alismatis Rhizoma）,Zhu Ling（Polyporus）,Fu Ling（Poria）,and Tao Ren（Persicae Semen）.2.On the distribution of frequency of CHMs in the four qi,five flavors,and meridian entry,the top three four qi in CHMs were neutral（279）,warm（235）,and cold（182）.The top three five flavors in CHMs were sweet（608）,bitter（431）,and pungent（412）.The top three meridians entries in CHMs were Hear Meridian（437）,Lung Meridian（429）,and Spleen Meridian（422）.3.By association rules,top three pair herbs were Huang Qi-Dan Shen,Dan Shen-Dang Gui,and Dan Shen-Dang Shen.The top three tri-herbs were Dan Shen-Dang Gui-Huang Qi,Dan Shen-Dang Shen-Huang Qi,and Dan Shen-Huang Qi-Yuan Zhi.The top three four herbs in combination were Huang Qi-Hong Hua—Tao Ren-Ze Xie,Dan Shen-Dang Shen-Huang Qi-Yuan Zhi,and Dan Shen-Dang Gui-Dang Shen-Huang Qi.4.As for cluster analysis,five categories of herbs were obtained by using between-group connections in the cluster analysis.Category 1:Huang Qi,Yuan Zhi,Hong Hua,Ze Xie,Tao Ren,Zhu Ling,Ting Li Zi（Descurainiae Semen Lepidii Semen）,Sang Bai Pi（Mori Cortex）,Ze Lan（Lycopi Herba）,Fu Ling,Dan Shen,Dang Gui,Dang Shen,Bai Zhu（Atractylodis Macrocephalae Rhizoma）,and Yi Mu Cao（Leonuri Herba）;category 2:Huang Lian（Coptidis Rhizoma）,Rou Gui（Cinnamomi Cortex）,Suan Zao Ren（Ziziphi Spinosae Semen）,Shan Zhu Yu（Corni Fructus）,Zhi Mu（Anemarrhenae Rhizoma）;category 3:Zhi Gan Cao（Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle），Fu Zi（Aconiti Lateralis Radix Praeparata）;category 4:Chi Shao（Paeoniae Radix Rubra）,Chuan Xiong（Chuanxiong Rhizoma）,Bai Shao（Paeoniae Radix Alba）,Fa Ban Xia（Pinelliae Rhizoma Praeparatum）,Xuan Shen（Scrophulariae Radix）,Shan Zha（Crataegi Fructus）,Chai Hu（Bupleuri Radix）;category 5:Huo Ma Ren（Cannabis Fructus）.ConclusionBased on the theory of qi and blood,Professor Qian Lin believes that weakness of heart qi is the root,while blood stasis and internal retention of water rheum is the branch for HFrEF.The treatment strategy mainly focuses on boosting qi,promoting blood circulation and urination.Prescriptions with similar effects can be selected with the emphasis on strengthening the healthy qi and securing the root.On specific conditions,herbs with the action of tonifying qi can be used in a large dose.Following the principle of pattern identification and treatment,therapeutic methods of warming yang,soothing the liver,and clearing heat can be selected appropriately.Part Ⅱ Experimental partExperiment Ⅰ Construction of conditional overexpression of CaMKIIδB and CaMKⅡδc in transgenic mice and genotyping identificationExperiment Ⅱ Phenotype identification of conditional overexpression of CaMKⅡδB and CaMKⅡδSC in transgenic miceExperiment Ⅲ Intervention effect and mechanism of qi-boosting and blood circulation promoting herbs on HFrEF induced by conditional overexpression of CaMKⅡδB and CaMKⅡδCObjectiveTo explore the intervention effect and mechanism of CHMs including Dang Shen,Huang Qi,San Qi,and Dan Shen on HFrEF due to overexpression of CaMKⅡδB and CaMKⅡδC.MethodsExperiment Ⅰ1.The cDNA sequences of mouse CaMKⅡδB and CaMKⅡδC were obtained by searching the National Center for Biotechnology Information（NCBI）database and literature.By CRISPR/Cas9 technology and in vitro transcription,Cas9 mRNA and gRNA were obtained.The homologous recombination vector was constructed by inserting the CaMKⅡδB expression cassette（CAG-LSLCaMKⅡδB-3xHA-IRES-EYFP）/CaMKⅡδC expression cassette（CAGLSL-CaMKⅡδC-3xFlag-IRES-tdTomato）into the Rosa26 gene site using In-Fusion cloning.2.Cas9 mRNA,gRNA,and homologous recombination vector were microinjected into fertilized eggs of C57BL/6J mice cultured in vitro.The fertilized eggs were transplanted into surrogate female C57BL/6J mice,and the mouse tail genotype was identified by polymerase chain reaction（PCR）to obtain F0 generation mice in positive expression.3.F1 generation mice were obtained by mating F0 generation mice with wild-type C57BL/6J mice.The F1 generation mice were mated with myocardial-specific Myh6-CreERT2 mice to obtain F2 generation mice,namely,myocardial-specific CaMKⅡδBoverexpression transgenic mice(Rosa26-LSL-CaMKⅡδB^+/-;Myh6-CreER⁺)and myocardialspecific CaMKⅡδC-overexpression transgenic mice(Rosa26-LSL-CaMKⅡδC^+/-;Myh6CreER⁺).Experiment Ⅱ1.Animal grouping:control group,CaMKⅡδB model group（B Model）,and CaMKⅡδC model group（C Model）.2.Phenotype induction:intraperitoneal injection of tamoxifen（final concentration 20 mg/mL）at the dose of 50 mg/kg was performed.Injection was performed every other day for five consecutive days.Phenotype test was conducted 7 days after the last injection.3.Detection method:Cardiac function was tested by echocardiography.Pathological changes were observed by Hematoxylin and eosin（HE）staining and Masson staining.Reverse transcription-quantitative PCR（RT-qPCR）,immunofluorescence and Western blot were used to detect the mRNA and protein expression of target genes（CaMKⅡδB and CaMKⅡδC）and their cellular localization.Experiment Ⅲ1.Modeling:intraperitoneal injection of tamoxifen（final concentration 20 mg/mL）at the dose of 50 mg/kg was performed.Injection was performed every other day for five consecutive days.Echocardiography was performed seven days after the last injection to identify the model.2.Grouping and administration:After successful modeling,mice that were survived were randomly assigned to each group subjected to four weeks of intervention.In control group,normal saline（NS）was used.In the B Model,NS was provided.In the CaMKⅡδB-treated CHMs group（B Treated）,prescriptions with the actions of boosting qi and promoting blood circulation such as Huang Qi,Dang Shen,Dan Shen,and San Qi were used.In the CaMKIIδB blocker group（B Inhibitor）,KN-93（10 μ mol/kg/d）was used.In the CaMKⅡδC model group（C Model）,NS was used.In the CaMK Ⅱδ C-treated CHMs group（C Treated）,prescriptions with the actions of boosting qi and promoting blood circulation such as Huang Qi,Dang Shen,Dan Shen,and San Qi were used.In the CaMK Ⅱδ C blocker group（C Inhibitor）,KN-93（10 μmol/kg/d）was offered.3.Detection methods:After 4 weeks of intervention,echocardiography was used to detect LV systolic function.HE staining and Masson staining were used to observe myocardial pathological changes.RT-qPCR was used to detect the mRNA expression of target genes and cardiac hypertrophy-related genes.Western blot was used to detect the expression of target proteins and calcium pathway-related proteins.Calcium transport was measured in acutely isolated cardiomyocytes.ResultsExperiment ⅠThe sequences of homologous recombinant plasmids,the genotypes of F0 generation mice,F1 generation mice,and F2 generation mice identified by PCR were consistent with prime sizes and sequences.Experiment ⅡCompared with the control group,the echocardiography of B Model showed that EF and fractional shortening（FS）were significantly decreased（P<0.05）,and LV end-diastolic diameter（LVEDd）was significantly increased（P<0.05）.HE staining showed that the ventricular cavity was evidently enlarged,and inflammatory cell infiltration and swelling were observed in myocardial cells.Masson staining showed slight fibrosis,and significant increase of cardiomyocyte cross-sectional area.The mRNA level of CaMKⅡδB was significantly increased by RT-qPCR（P<0.05）.The expression of CaMKⅡδB tag protein HA was increased,which was located in the nucleus by immunofluorescence.Echocardiography of C Model showed that EF and FS were significantly decreased（P<0.05）,and LVEDd was significantly increased（P<0.05）.HE staining showed that the ventricle was obviously enlarged,and disordered arrangement,inflammatory infiltration,and swelling were observed in myocardial cells.Masson staining showed that slight fibrosis,and significant increase of cardiomyocyte cross-sectional area.The mRNA level of CaMKⅡδC detected by RT-qPCR was significantly increased（P<0.05）,and the expression of the tag protein Flag of CaMKⅡδC was increased,which was located in the cytoplasm by immunofluorescence.Experiment Ⅲ1.Effects of qi-boosting and blood circulation-promoting CHMs on the cardiac function and structure of mice in each group:Compared with the control group,the EF and FS of B Model and C Model groups were significantly decreased（P<0.05）,and the LVEDd was significantly increased（P<0.05）.There was no difference in LV anterior wall thickness at diastole（LVAWd）and LV posterior wall thickness at diastole（LVPWd）.Compared with B Model group,B Treated group could significantly increase EF and FS（P<0.05）.There was no difference in LVEDd（P<0.05）,but a downward trend could be observed.There was no difference in EF,FS,or LVEDd of the B Inhibitor group（P>0.05）.Compared with C Model group,C Treated group could significantly increase EF and FS（P<0.05）,and reduce LVEDd（P<0.05）,while no difference in EF,FS,LVEDd was observed in the C Inhibitor group（P>0.05）.2.Effects of qi-boosting and blood circulation-promoting CHMs on the heart tissue of mice in each group:Compared with the control group,HE staining showed disordered myocardial arrangement,extensively swollen myocardial cells,vacuoles,and severe infiltration of large amounts of inflammatory cells in the B Model group.Masson staining displayed mild myocardial fibrosis in the B Model group with collagen volume fraction（CVF）and myocardial cell cross-sectional area increasing significantly（P<0.05）.In the B Treated group,pathological changes were improved and myocardial cell cross-sectional area was reduced（P<0.05）,but failed to reduce CVF.In the B Inhibitor group,the pathological conditions were improved,but failed to reduce the cross-sectional area of cardiomyocytes.In the C Model group,HE staining showed disorder myocardial arrangement,less clear myocardial cell space,extensive cellular swollen,and inflammatory cell infiltration and necrotic cells.Masson staining showed mild myocardial fibrosis in the C Model group,and significant increase of CVF and myocardial cell cross-sectional area（P<0.05）.In the C Treated group,the above-mentioned pathological conditions were improved to certain degree.The cross-sectional area of cardiomyocytes and CVF were significantly reduced（P<0.05）.In the C Inhibitor group,the above conditions were improved,and CVF was significantly reduced（P<0.05）,but failed to reduce the crosssectional area of cardiomyocytes.3.Effects of qi-boosting and blood circulation-promoting CHMs on the expression of overexpressed genes,cardiac hypertrophy-related genes mRNA,proteins and calcium pathwayrelated proteins in mice in each group.Overexpressed genes:After 4 weeks of intervention,compared with the control group,the mRNA and HA protein expressions of CaMKⅡδB in the B Model group were significantly increased（P<0.05）,and the mRNA and Flag protein expressions of CaMKⅡδC in the C Model group were significantly increased（P<0.05）.Compared with the B Model group,the expressions of CaMKⅡδB mRNA and HA protein in the B Treated group were significantly decreased,but no difference in the B Inhibitor group（P>0.05）.Compared with the C Model group,the expressions of CaMKIISC mRNA and Flag protein were significantly decreased in the C Treated group,but no difference in the C Inhibitor group（P>0.05）.Hypertrophy-related genes:After 4 weeks of intervention,compared with the control group,the mRNA levels of B-type natriuretic peptide（BNP）,myocytes enhancer factor 2（MEF2）,and atrial natriuretic factor（ANF）in the B Model group were significantly increased（P<0.05）,and the mRNA levels of CaMKⅡδC and MEF2 in the C Model group were significantly increased（P<0.05）.The mRNA level of BNP showed an increase trend,but there was no statistical significance（P>0.05）.Compared with the B Model group,the mRNA levels of BNP,MEF2 and ANF in the B Treated group were significantly reduced（P<0.05）.In the B Inhibitor group,a decreased tendency to reduce the mRNA levels of BNP、MEF2 and ANF could be observed,but the difference was not statistically significant（P>0.05）.Compared with the C Model group,the mRNA level of MEF2 in the C Treated group was reduced,but the difference was not statistically significant（P>0.05）.In the C Inhibitor group,decreased mRNA level of MEF2 could be observed,but the difference was not statistically significant（P>0.05）.Calcium pathway-related proteins:After 4 weeks of intervention,compared with the control group,the SERCA2a protein expression of the B Model and C Model groups was significantly reduced（P<0.05）,and the PLB protein expression of the B Model and C Model groups showed a decrease trend,but the difference was not statistically significant（P>0.05）.Compared with the control group,no difference in PLB-T17 protein expression in the B Model group was observed（P>0.05）,while increased significantly in the C Model group（P<0.05）.Compared with the B Model group,SERCA2a protein expression increased significantly in the B Treated group（P<0.05）,and there was an increasing trend of PLB protein expression,but did not differ statistically（P>0.05）.In the B Inhibitor group,increasing trend of SERCA2a level could be observed,but the difference was not statistically significant（P>0.05）.Compared with the C Model group,the level of SERCA2a in the C Treated group was significantly increased（P<0.05）,and an increasing trend of SERCA2a expression in the C Inhibitor group was also observed,but without significant difference（P>0.05）.4.Cardiomyocyte calcium transport:After 4 weeks of intervention,acutely isolated mouse cardiomyocytes were tested for calcium transport.Compared with the control group,the systolic calcium transient amplitude,calcium storage capacity of the Model B and Model C groups were significantly decreased（P<0.05）and calcium leakage level of the Model B and Model C groups were significantly increased（P<0.05）.Compared with the B Model group,the systolic calcium transient amplitude and calcium storage capacity were significantly increased in the B Treated group（P<0.05）,and a decrease trend of the level of calcium leakage was observed,but the difference was not statistically significant（P>0.05）.In the B Inhibitor group,a trend of increasing the systolic calcium transient amplitude and calcium storage capacity,and reducing the calcium leakage level was observed,but the difference was not statistically significant（P>0.05）.Compared with the C Model group,systolic calcium transient amplitude and calcium storage capacity were significantly increased in the C Treated group（P<0.05）,and calcium leakage was significantly reduced（P<0.05）.In the C Inhibitor group,calcium leakage was significantly reduced（P<0.05）,and an increase trend of systolic calcium transient amplitude and calcium storage capacity could be observed,but the difference was not statistically significant（P>0.05）.ConclusionExperiment ⅠRosa26-LSL-CaMKⅡδB^+/-,Myh6-CreER⁺,Rosa26-LSL-CaMKⅡδC^+/-,and Myh6CreER⁺mice models were successfully constructed.Experiment ⅡRosa26-LSL-CaMKⅡδB^+/-,Myh6-CreER⁺,Rosa26-LSL-CaMKⅡδC^+/-,Myh6-CreER⁺mice have the phenotype of HFrEF.Mouse phenotype expression can be successfully induced by tamoxifen（50 mg/kg,injected every other day,for 5 consecutive days）,which is safe and can be validated by the mRNA and protein expressions and the location of protein expression location.Experiment Ⅲ1.The conditional overexpression of CaMKⅡδB may be mainly through the activation of cardiac hypertrophy-related factors,which may cause HFrEF.It is also related to the increase of calcium leakage and decreased expression of SERCA2a,which lead to abnormal calcium reuptake.The mechanism of qi-boosting and blood circulation promoting herbs to improve HFrEF may be related to inhibiting the overexpression of CaMKⅡδB,reducing the expression of hypertrophic factors,increasing the expression of SERCA2a and the calcium reserves.2.The conditional overexpression of CaMKⅡδC may cause HFrEF mainly due to increased calcium leakage and decreased expression of SERCA2a resulting in abnormal calcium reuptake.It is also related to activation of cardiac hypertrophy-related factors.The mechanism of qi-boosting and blood circulation promoting herbs to improve HFrEF may relate to inhibition of CaMKⅡδC overexpression,reduction of calcium leakage,and increase of SERCA2a expression,which can increase calcium reserve.