Cardiac Remodeling, Intracellular Calcium Handling, And Underlying Molecular Mechanisms In Rabbits With Experimental Hyperthyroid Cardiomyopathy

Objective:To study the molecular mechanisms of myocardial remodeling and the alteration of intracellular calcium handling in hyperthyroid cardiomyopathy induced by Levothyroxine(L-Thy). Methods:A rabbit model of hyperthyroidism was established by daily intraperitoneal injections of L-Thy (45μg/kg per day, 28 days). Forty New Zealand rabbits were randomly divided into fours groups: control group (n=10), L-Thy group(L-Thy only,n=10),Imidapril group ( L-Thy+ imidapril, n=10), and Valsartan group(L-Thy+valsartan,n=10). All the rabbits were followed for 4 weeks. Every 7 days blood samples were obtained. Serum TT3 and TT4 were measured with Radioimmunoassays (RIAs). Electrocardiogram(ECG) was performed to determine heart rate(HR). At the end of the follow-up period all the rabbits underwent echocardiography and IVS, LV,LVPW thickness and LVEF were measured. Ventricular tissues were collected at 4 weeks. Cardic hypertrophy index, cardiomyocyte diameter, structural and ultrastructural changes were detected. Plasm and Cardiac angiotensin II concentration were measured with RIAs. Cardiac fibrosis was displayed by Masson's stain and collegen volume fraction(CVF)was measured using pathological image analytic system. Ventricular myocytes were acutely isolated by enzymatic digestion and intracellular Ca2+ concentration was detected with the fluoresent Ca2+ indicator Fluo3/AM and laser scanning confocal microscopy. Activity of Sarco/Endoplasmic Ca2+ ATPase(SERCA)was evaluated with p-NPP method. mRNA expression of ACE, AT1R, AT2R, L-type Ca2+ channel (LCC), ryanodine receptor (RyR),and SERCA was semi-quantified with RT-PCR. Expression of ACE, Erk, and p-Erk protein was evaluated with Western blot analysis. Protein of IP3R were localized by immunostaining and semi- quantified with pathological image analytic system. Results: Compared with control group, rabbits merely treated with L-Thy displayed remarkable myocardial hypertrophy, extracellular matrix fibrosis, and morphological changes in both structure and ultrastructure. Increased plasm/tissue AngII, increased intracellular Ca2+ concentration, and decreased SERCA activity were detected in L-Thy group. RT-PCR, Western blot analysis and/or immunohistochemistry revealed enhanced mRNA expression of ACE, AT1R,AT2R, LCC , RyR,SERCA and increased protein expression of ACE,Erk, p-Erk, IP3, RyR. Erk/p-Erk protein expression correlated positively well with both cardiomyocyte diameter and CVF. Our study demonstrated that both imidapril and Valsartan alleviated cardiomyocyte hypertrophy, extracellular matrix fibrosis, and structural damage induced by L-Thy. Compared with L-Thygroup, decreased Erk/p-Erk expression, decreased intracellular Ca2+ concentration, and elevated SERCA activity were found in both imidapril and Valsartan groups. Compared with L-Thy and Valsartan group, Imidapril group showed significantly lower plasm /tissue AngII concentration and more effective inhibition of extracellular matrix fibrosis. Expression of AT1R, AT2R, ACE, LCC, IP3R, RyR, and SERCA were unchanged. Plasm concentration of AngII was markedly higher in valsartan group compared with L-Thy group, whereas tissue AngII concentration was not significantly different between the two groups. AT1R and AT2R mRNA expressions are significantly upregulated, whereas Valsartan didn't alter expression of ACE, LCC, IP3R, RyR, and SERCA. Conclusion: Renin-angiotension system(RAS),MAPK/Erk signaling pathway, and intracellular Ca2+overload may play important roles in hyperthyroid cardiomyopathy. Activated RAS is possibly responsible for activation of MAPK/Erk signaling pathway and decreased SERCA activity may contribute to intracellular Ca2+overload induced by L-Thy. Imidapril and Valsartan may exert benefical effects on hyperthyroid cardiomyopathy via retarding myocardial remodeling and restoring SERCA activity.