Intervention Study Of Valsartan On The Expression Of Ca²⁺/Calmodulin-dependent Protein Kinaseii In Cardiac Myocytes Of The Juvenile Rat With Ventricular Dysfunction

Background: With the growing cardiovascular morbidity, thepathogenesis and treatment of cardiovascular disease has become aresearch focus of the medical profession at home and abroad. Maintaining acertain of blood rhodobryum，a term of heart failure that the contractionand （or） diastolic function of the heart decline resulting in apathophysiological process that can not absolutely or relatively meet thebody’s metabolic needs. As a pediatric emergency, heart failure is commonin children with congenital heart disease, myocarditis, cardiomyopathy orsevere respiratory illness.Various calcium homeostasis proteins presenting in the normalmyocardium have been widely regarded as playing a major role in theleading and promoting cardiac dysfunction, electrophysiological instabilityor arrhythmia events. Ca²⁺/calmodulin-dependent protein kinase Ⅱ（CaMKⅡ） is a serine-threonine protein kinase, as a regulatory protein inmyocardial calcium cycle with variety of biological functions. The richness of downstream target sites of CaMKⅡ plays an important supporting role incardiac excitation-contraction coupling （ECC） process. The activate ofCaMKⅡ in myocardial cells by increasing the concentration of intracellularcalcium. When cardiac overload, CaMKⅡ can excessive activate andpromote the development of heart failure and cardiac arrhythmias throughaffecting myocardial calcium cycle.In general, the phosphorylation of L-type calcium channel （LTCC） andryanodine receptor （RyR） mediated by CaMKⅡ regulate the normal processof ECC. In the initial stage of ECC, calcium flow through the active LTCCinto the myocardial cells, resulting in myocardial depolarization. The smallamount of calcium flow through LTCC into the myocardial cells furtheractivate the RyR receptors on cardiac sarcoplasmic reticulum （SR）releasing large amounts of calcium, which is the classic “calcium triggercalcium release”. A sufficient concentration of calcium ions released by SRin myocardial cells is the key to the myocardial contraction and otherimportant functions. At the beginning of diastole, the phospholamban （PLN）plays a negative regulatory role by disinhibition sarcoplasmic reticulumCa²⁺-ATPase （SERCA） on the myocardial SR and then SERCA retakecalcium ions in myocardial cells. In cardiovascular disease, theoverexpression of CaMKⅡ resulting hyperphosphorylation of LTCC, RyRand SERCA/PLN affects ECC process and cardiac function.Previous studies had found that the increased expression of CaMKⅡ in the adult animals with ventricular dysfunction, structural heart disease orcardiac electrophysiology disorders, but the research of CaMKⅡ in juvenileanimals with heart dysfunction is still blank. Recent studies have found thatreactive oxygen species （ROS）-mediated oxidation activate sites may existyet, as well as phosphorylation activate site of CaMKⅡ. Therefore, ourresearch group based on the model building of cardiac dysfunction inyoung rats, research the treatment of valsartan on the expression ofCaMKⅡ in juveniles with cardiac dysfunction, and attempt to furtherexplore the possible process of phosphorylation or oxidative activation ofCaMKⅡ. The study is divided into the following three parts:Part1The establishment of ventricular dysfunction model ofjuvenile ratsObjective: To construct ventricular dysfunction model of young rats.Method: Adopted abdominal aortic constriction （AAC） to build thecardiac dysfunction model in3-week-old male SD young rats （n=28）, andthe coarctation diameter was about0.06mm. At the same period the shamgroup of young rats were built with only the separation of the abdominalaorta （n=8）.4weeks after surgery, high-frequency echocardiography wasadopted to detect left ventricular-internal dimension systole （LVIDs）, leftventricular the-internal diastolic diameter （LVIDd）, left ventricularend-systolic volume （LVESV）, left ventricular end diastolicvolume（LVEDV）, left ventricular ejection fraction （LVEF）, left ventricular fractional shortening （LVFS） of the rats.Results: A total of28young rats with AAC,10of whom were deadduring the surgery or1week after operation. AAC surgery survival ratewas about73.68%. No mortality in sham group.4weeks after AAC, fromhigh-frequency ultrasound echocardiography, compared with normal youngrats, LVIDs、LVESV were increased （t=2.016, P=0.002; t=1.769, P=0.029）, LVEF、LVFS were lower （t=4.22, P <0.001; t=5.339, P <0.001）in young SD rats of AAC group, while no significant statistical differencewere found in LVIDd or LVEDV （P>0.05）.Conclusion: The successfully constructed cardiac dysfunction modelof young rats, with a certain degree of postoperative survival, showed thatthe systolic function decline. Basic animal models were provided forfurther study about the therapeutic effect of valsartan in pups and theexpression of CaMKⅡ in heart failure.Part2The affect of valsartan on cardiac function and remodel inyoung rats with increased pressure overloadObjective: To study the therapeutic effect of valsartan on young ratswith heart function insufficiency.Methods:4weeks after operative, young rats with ventriculardysfunction were randomly divided into heart failure group （n=10） andtreatment group （n=8）.Young rats in treatment group were given valsartan30mg/（kg.d） for4weeks as treatment, at the same time heart failure group and sham-operated group given a placebo. At the end of the experiment, thehigh-frequency transthoracic echocardiography was employed to detect theLVIDs, LVIDd, LVESV, LVEDV,LVEF and LVFS among each groups. Leftventricular mass index （LVMI） and right ventricular mass index （RVMI）were calculated. The situation of myocardial fiber morphology andcollagen hyperplasia was observed by HE staining and Masson staining indifferent groups of young rats.Results:4weeks after treatment of valsartan, compared with thesham group （n=8）, LVIDs, LVESV were significantly increased （F=21.082, P <0.001; F=14.178,P=0.001）, LVEF, LVFS were significantlydecreased （F=103.766, P <0.001; F=62.953, P <0.001）, LVMI wasincreased significantly （F=13.272, P=0.01） in heart failure group （n=10）,though no significant statistical differences of LVIDd, LVEDV and RVMIwere found between2groups （p>0.05）. The difference of cardiac functionor ventricular mass index between sham group and treatment group was notstatistically significant （P>0.05）. Compared with the sham group ortreatment group，HE staining showed the more thickening of the cardiacmuscle fiber（sF=126.66，P <0.001；P <0.001）with disorder or even rupturein heart failure group. Compared with the sham group, Masson collagenstaining showed the collagen proliferate fibers in the heart failure group.Compared with heart failure group, easer myocardial thickening and lesscollagen fibers in the valsartan group. Compared with heart failure group， myocardial slimmer and less collagen fibers in the valsartan group.Conclusion: Compared with heart failure group, the heart dysfunctionhad been greatly improved after treatment with the decreased ventricularmass index and lighter myocardial remodeling in the treatment groupwhich indicated the therapeutic effectiveness in heart failure. This part ofthe research laid the foundation to further explore CaMKⅡ expression withventricular dysfunction and the possible mechanisms of valsartan treatmentfor heart failure.Part3The intervention study of valsartan on the expression andactivation of CaMKⅡ in cardiac dysfunction myocytesObjective: To study the expressions of CaMKⅡ and its subtypes incardiac myocytes after the treatment of valsartan for ventriculardysfunction,as well as the expressions of CaMKⅡ phosphorylation oroxidation activate sites. To exposure the possible mechanisms about thevalsartan treatment effect relating with local regulation of the CaMKⅡexpression in young rats with heart function insufficiency.Methods: Western blot was adopted to detect the expressions ofCaMKⅡ，phosphorylation sites （Thr286） and oxidation sites （Met281） incardiac myocytes.Results: Compared with the sham group，the expression of CaMKⅡwas increased1.95-fold （F=7.541, P=0.026）, CaMKⅡ subtypes （δB+δ9） expression was increased1.93-fold （F=7.042, P=0.029）,CaMKⅡδC expression was increased1.93-fold （F=6.714, P=0.045） in heart failuregroup. Very low CaMKⅡγ expression levels did not show significantdifferences between2groups （P>0.05）. Compared with the sham group,Thr286expression of CaMKⅡ was increased6.15-fold （F=16.388, P<0.001） in ventricular muscle cells of heart failure group. Met281expression slightly elevated but no significant difference was foundbetween2group （P>0.05）. After treatment with valsartan, no significantdifferences of the expressions of CaMKⅡ, CaMKⅡ subtypes and relatedactivation sites were showed between treatment group and the sham group（P>0.05）.Conclusion: When in heart failure, activated CaMKⅡ wasoverexpression. Valsartan improved pups heart function may be associatedwith lower CaMKⅡ overexpression. Excess expression of Subtypes （δB+δ9）, δCmaybe plays an important role in early heart dysfunction. LowCaMKⅡγ expression levels and found no significant difference between the3groups, so we require further experimental studies to explore whether thechange of CaMKⅡγ in the later development of heart failure. As CaMKⅡphosphorylation sites, activated Thr286is associated with the earlyactivation of CaMK Ⅱ instead of the oxidationsites Met281.