Molecular Mechanisms Of Myocardial Fibrosis In Hypothyroidism Induced Heart Failure Rats

Hypothyroidism(HT) has to be considered a highly prevalent condition, mainly in females. The cardiovascular system is one of the most important targets of thyroid hormone. In recent years, hypothyroidism has been observed to be associated with heart failure(HF). Heart failure is a major public health problem, treatment of heart failure has been repeatedly observed. Precious studies showed that myocardial fibrosis is one of the pathological basis of heart failure. Fibrosis is an adaptive process in response to long-term cardiac overload. It is a common complication and pathological basis of a variety of cardiovascular diseases, and eventually lead to serious arrhythmias, heart failure. Therefore, prevention and reversal of this change has become an important goal of treatment. At home and abroad, an animal model of heart failure induced by hypothyroidism has not been established. The mechanism of heart failure is not clear. AMP-activated protein kinase(AMPK) is an protein kinases performancing regulation effect of energy metabolism in cell. Recent studies have found that AMPK gene mutation can lead to cardiac hypertrophy and arrhythmia, meanwhile activation of AMPK can negatively regulate mammalian target of rapamycin(m TOR)/p70S6 kinase(p70S6K) signaling pathway. Therefore, AMPK is expected to become a new therapeutic target to therapy myocardial fibrosis.This study intends to establish an animal model of heart failure induced by intraperitoneal injection of 131 I, research the targets and related molecular mechanisms leading to cardiac fibrosis in rats with hypothyroidism induced heart failure. The results of this study will help to clarify the mechanism of hypothyroidism induced heart failure, and provide new ideas and avenues of prevention and treatment of hypothyroidism induced heart failure. The experiments contain the following two parts:Part one Established an animal model of hypothyroidism induced heart failureObjective: To establish an animal model of hypothyroidism induced heart failure.Methods: Wistar male rats(200-220 g weight) were randomly assigned into seven groups: normal group, 100μCi group, 300μCi group, 500μCi group, 700μCi group, 900μCi group, 1100μCi group. The 131 I diluted with saline to obtain a mixture, each experimental group were intraperitoneally injected with 100μCi, 300μCi, 500μCi, 700μCi, 900μCi, 1100μCi equal volume 131 I mixture, the normal group received an equal volume of saline.After 4 weeks and 8 weeks feeding, cardiac ultrasound was performed to analysis the cardiac function, nuclide scan was used to evaluat thyroid polyiodides function, ELISA kits was performed to analysis serum TT3, TT4, thyroid stimulating hormone(TSH), B-type natriuretic peptide(BNP) levels.Results: 1 After 8 weeks feeding, compared with normal control group thyroid polyiodides function of rats was significantly decreased in the treatment group.2 Thyroid function of rats After 4 weeks and 8 weeks feeding, the level of TT3, TT4 were decreased in the treatment group(P<0.05). Compared with normal control group, the level of TSH was significantly increased in the treatment group(P<0.05). With the increase of dose, the difference was more significant, but compared with the 300μCi and 500μCi group 700μCi, 900μCi, 1100μCi group had no significant difference.3 Cardiac function of rats At 4th week, compared with normal control group interventricular septal(IVS) thickness was significantly increased in the treatment group(P<0.05). Compared with normal control group, 100μCi group, 300μCi group, 500μCi group, left ventricular end-diastolic dimension(LVEDD) of 700μCi group, 900μCi group, 1100μCi group showed significant difference(P<0.05). Compared with normal control group and 100μCi group, left ventriculus postterior wall dimension(LVPWD) and ejection fraction(EF) of 300μCi, 500μCi, 700μCi, 900μCi, 1100μCi group showed significant difference(P<0.05). Compared with normal control group, 100μCi and 300μCi group, left ventricular end-systolic dimension(LVESD) of 500μCi, 700μCi, 900μCi, 1100μCi group showed significant difference(P < 0.05). Fractional shortening(FS) were decreased in the treatment group(P<0.05).At 8th week, IVS, LVPWD of treatment group rats were increased(P<0.05). Compared with normal control group and 100μCi group, LVEDD, LVESD, EF, FS of 300μCi, 500μCi, 700μCi, 900μCi, 1100μCi group showed significant difference(P<0.05). With the increase of dose, the difference was more significant, but compared with the 300μCi and 500μCi group 700μCi, 900μCi, 1100μCi group had no significant difference.4 The level of BNP At 4th week, no significant difference of the level of BNP was observed in the treatment group and the normal group(P>0.05). At 8th week, compared with normal control group, the level of BNP of treatment group showed significant difference(P<0.05). With the increase of dose, the difference was more significant.Conclusions: Intraperitoneal injection of 131 I can cause hypothyroidism, at the same time the heart function of rats was reduced. 300μCi, 500μCi 131 I dose was the best model dose.Part two Molecular mechanisms of myocardial fibrosis in hypothyroidism induced heart failure ratsObjective: To assess morphological changes and myocardial fibrosis, observe AMPK and m TOR/p70S6 K pathway and explore the molecular mechanisms of myocardial fibrosis in hypothyroidism induced heart failure rats.Methods: 100μCi group, 300μCi group, 500μCi group and normal control group were selected from the first part, HE and sirius red staining were used to detect he myocyte area and collagen. RT-PCR were used to analysis the expression of transcripts of nuclear transcription factor kappa B(NF-κB), procollagen type I(CollagenⅠ), transforming growth factor β(TGF-β), tumor necrosis factor α(TNF-α), m TOR, EEF2, AMPK, p70S6 K. Western Blot was performed to evaluat the protein expression levels of cardiac AMPK, P-AMPK, EEF2, P-EEF2, p70S6 K, P-p70S6 K, m TOR, P-m TOR.Results: 1 Pathology results 1.1 HE staining resultsHE staining showed disorered arrangement and increased area of myocardial cell in the 100μCi group, 300μCi group, 500μCi group.1.2 Sirius red staining results Compared with normal control group, sirius red staining showed increased collagen content, disordered arrangement, increased collagen area and perivascular collagen hyperplasia.2 The expression of rats myocardial tissue gene after 8 weeks feeding: No significant changes of m TOR, EEF2, AMPKα2, p70S6 K gene expression were observed in the 100μCi group, 300μCi group, 500μCi group and normal control group(P>0.05). RT-PCR showed increased NFκB, TNF-α in the 100μCi group, 300μCi group, 500μCi group(P < 0.05), increased Collagen I, TGF-β in the 300μCi group, 500μCi group(P<0.05).3 The expression of rats myocardial tissue protein after 8 weeks feeding:Western blot showed no significant changes of m TOR, EEF2, AMPKα2, p70S6 K in the 100μCi group, 300μCi group, 500μCi group and normal control group(P>0.05), increasd P-m TOR, P-AMPKα2Ser491, P-p70S6KSer424 in the 100μCi group, 300μCi group, 500μCi group(P<0.05), decreased P-EEF2 in the 100μCi group, 300μCi group, 500μCi group(P<0.05).Conclusions: 1 Myocardial fibrosis was fromed in the animal model of hypothyroidism induced heart failure.2 AMPK and mTOR/p70S6 K signaling pathway was activated in the animal model of hypothyroidism induced heart failure.