The Protective Role And Mechanism Of CaMKⅡ Inhibition In Hyperthyroid Heart Disease

Part Ⅰ Cardiac CaMKⅡ inhibition ameliorates the progression of hyperthyroidism heart disease(HHD)Objective:To explore the role of cardiac CaMKⅡ inhibition on the development and progression of hyperthyroidism heart disease(HHD).Methods:1.AC3-Ⅰ mice and WT mice were intraperitoneally administrated with L-thyroxine(T4)for 7 days or 30 days to develop experimental hyperthyroidism.Next,we determined whether there were any differences in heart changes between WT and AC3-Ⅰ mice.2.The serum T4 levels of the two groups were detected by ELISA for hyperthyroidism evaluation.3.The parameters of heart weight(HW),body weight(BW),tibia length(TL),and ratios of HW/BW and HW/TL were detected to determine the HHD progression and heart enlargement.Results:1.The serum T4 levels of AC3-Ⅰ and WT mice were similar and had increased synchronically during T4 treatment.The T4 levels reached three and ten times on days 7 and 30 compared to baseline,respectively2.The heart weights(HW)of AC3-Ⅰ and WT mice had increased gradually during T4 treatment,but the HW of AC3-Ⅰ mice was significantly higher than that of WT mice on day 30.3.The body weights(BW)of AC3-Ⅰ and WT mice were similar and had increased slightly on day 7 and decreased slightly on day 30 without significant difference between the two groups at three time points.4.The tibial lengths(TL)of AC3-Ⅰ and WT mice had no significant change during T4 treatment and were not significantly different between the two groups.Conclusions:T4 administration intraperitoneally can lead to hyperthyroidism and HHD progression in mice after 30 days of treatment,which is characterized by an increase in heart weight(HW).In contrast,cardiac CaMKⅡ inhibition significantly ameliorates HHD progression in mice with relieved cardiac enlargement.Part Ⅱ Cardiac CaMKⅡ inhibition protects against arrhythmias and adverse myocardial remodeling in hyperthyroidism heart disease(HHD)Objective:To explore the role of cardiac CaMKⅡ inhibition in preventing arrhythmias and adverse myocardial remodeling in hyperthyroidism heart disease(HHD).Methods:1.The surface and telemetry electrocardiogram(ECG)of AC3-Ⅰ and WT mice were recorded to detect the ECG parameters and arrhythmias susceptibility in HHD.2.Isoproterenol(ISO)was administrated intraperitoneally to AC3-Ⅰ and WT mice to induce potential arrhythmias in HHD.3.Echocardiography was performed to detect the changes in heart structure and function in AC3-Ⅰ and WT mice during HHD.4.The adverse myocardial remodeling of AC3-Ⅰ and WT mice,including atrial fibrosis,ventricular hypertrophy,and perivascular fibrosis,was determined by tissue analysis.Results:1.The heart rate(HR)of WT mice increased depending on T4 treatment time and manifested as sinus tachycardia,and was significantly higher than that of AC3-Ⅰ mice on day 30.The PR intervals of AC3-Ⅰ and WT mice shortened during T4 treatment,but the PR interval of AC3-Ⅰ mice was always longer than that of WT mice regardless of T4 treatment time.The QRS intervals of the two groups were similar before T4 treatment and became shorter during T4 administration,but the QRS interval of WT mice was significantly shorter than that of AC3-Ⅰ mice after T4 treatment.The QT90 intervals of AC3-Ⅰ and WT mice shortened during T4 treatment,but the QT90 interval of AC3-Ⅰ mice was always shorter than that of WT mice during T4 treatment.2.Atrial and ventricular arrhythmias were not found in hyperthyroidism mice by telemetry ECG at rest conditions.However,the tachycardia arrhythmias had been induced by ISO only in WT mice,including atrial fibrillation(AF),premature ventricular contraction(PVC),ventricular premature bigeminy(VPB),and polymorphic ventricular tachycardia(PVT).In contrast,AC3-Ⅰ mice had ISO-induced type-Ⅱ atrial-ventricular block(ⅡAVB),defined by progressively prolongated PR interval.3.The WT mice had developed heart dysfunction characterized by high cardiac output during T4 treatment,while the heart function of compared AC3-Ⅰ mice remained stable.The EF,FS,SV,and CO of WT mice had increased rapidly and significantly higher than those of compared AC3-Ⅰ mice on day 7 after T4 treatment.However,the SV and CO of WT mice had decreased to similar levels compared with AC3-Ⅰ mice on day 30 post T4 treatment.Meanwhile,the EF and FS of WT mice were still significantly higher than those of AC3-Ⅰ mice.In addition,WT mice had more severe ventricular hypertrophy than AC3-Ⅰ mice in HHD after T4 treatment.4.The cardiac structure was similar between the WT and AC3-Ⅰ mice before T4 treatment.The relative cardiomyocyte diameter and the ratio of PVCA/VA of WT mice increased depending on treatment time and were significantly larger than those of compared AC3-Ⅰ mice during T4 treatment.The atrial fibrotic area of WT mice was significantly higher than that of AC3-Ⅰ mice in HHD on day 30 post T4 treatment.Conclusions:The HHD mice suffer from sinus tachycardia and are susceptible to atrial and ventricular tachycardia arrhythmias induced by ISO.The HHD mice also have heart dysfunction with high cardiac output,concomitant with maladaptive myocardial remodeling,including atrial fibrosis and ventricular hypertrophy.However,cardiac CaMKⅡ inhibition can ameliorate HHD and preserve heart function by fighting against arrhythmias and myocardial remodeling.Part Ⅲ Cardiac CaMKⅡ inhibition ameliorates myocardial remodeling by blunting the HDAC4/MEF2a pathway in hyperthyroidism heart disease(HHD)Objective:To explore the mechanism of cardiac CaMKⅡ inhibition in protecting against adverse myocardial remodeling in hyperthyroidism heart disease(HHD).Methods:1.The ventricular tissue of mice during T4 treatment was extracted for western blot and detecting protein expressions of CaMKⅡ,p-CaMKⅡ,HDAC4,p-HDAC4,MEF2a,and ANP.2.Cardiac H9C2 cells were cultured and prepared for T4 stimulation.The protein expressions were detected by western blot,including CaMKⅡ,p-CaMKⅡ,HDAC4,p-HDAC4,MEF2a,and ANP.Then KN93,the inhibitors of CaMKⅡ,and LMK235,the inhibitor of HDAC4,were added to T4 treated H9C2 cells for further validation of HDAC4/MEF2a signaling activation.Results:1.The expression of CaMKⅡ and p-CaMKⅡ of WT heart increased during T4 treatment,thus activating HDAC4,and derepressing MEF2a and its downstream hypertrophic ANP expression,contributing to adverse myocardial remodeling.2.The expression of CaMKⅡ and p-CaMKⅡ in H9C2 cells increased after T4 treatment,and the expression of p-HDAC4,MEF2a,and ANP also increased.After being treated with CaMKⅡ inhibitor KN93 and HDAC4 inhibitor LMK235,respectively,cardiac H9C2 cells showed downregulated expression of p-HDAC4,MEF2a,and ANP.Conclusions:The increased CaMKⅡ expression in HHD leads to HDAC4/MEF2a pathway activation for downstream hypertrophic genes transcription and contributes to adverse myocardial remodeling.Cardiac CaMKⅡ inhibition would blunt the HDAC4/MEF2a pathway to ameliorate maladaptive myocardial remodeling in HHD.