Mechanistic Study Of Hypothyroidism-related Heart And Liver Damag

Hypothyroidism induced cardiac dysfunction and its underlying mechanismsBackgroundThyroid function is closely related to the cardiovascular system.Several clinical studies have confirmed that hypothyroidism is an independent risk factor for heart failure,myocardial infarction,and dilated cardiomyopathy.The low T3 level and the elevated TSH are clearly related to the poor prognosis of cardiovascular disease.The heart is one of the organs with the most vigorous energy metabolism in the human body,and lipid metabolism plays a major role in the energy metabolism of the heart.Lipid metabolism disorder is an important pathological mechanism in cardiovascular diseases,especially in heart failure.Hypothyroidism is also clearly related to dyslipidemia.However,there is no clear conclusion on the lipid metabolism status of the myocardium in hypothyroidism.It is still unknown whether lipid metabolism disorder plays an important pathological role in hypothyroid heart failure.SIRT6 is an NAD+dependent deacetylase belonging to the sirtuin protein family and plays an important role in glucose and lipid metabolism,aging,and DNA repair.The relationship of sirtuin protein family and thyroid hormone has been reported.Several researches have shown that SIRT6 plays an important role in cardiovascular diseases.However,the role of SIRT6 in hypothyroid heart failure remains unknown.Methods1.The 8-week-old female mice were orally given propylthiouracil drinking water for 3 months to construct hypothyroidism.The cardiac function was assessed by echocardiography and the thyroid function was detected by serum radioimmunoassay.2.The myocardial lipid deposition was observed by oil red O staining,electron microscopy,and BODIPY staining.The myocardial lipotoxicity damage was evaluated by MitoSOX and 4-HNE staining.3.The myocardial sirtuins,key enzymes and transcription factors of lipid metabolism were detected by Western Blot.The combination of SIRT6 and PPARa was confirmed by co-IP.The downstream β-oxidation enzyme CPT1b was also evaluated.4.In the cell experiments,the neonatal mice ventricular myocytes(NMVMs)were treated with TSH to imitate hypothyroidism.The lipid metabolism of cardiomyocytes was observed.The Ad-SIRT6 virus and si-SIRT6 were used to up-regulate or down-regulate the expression of SIRT6,respectively.the metabolic changes of mitochondria oxidative phosphorylation were observed by Seahorse.5.Based on the modeling of hypothyroidism,myocardial specific SIRT6 knockout mice(SIRT6cKO mice)and the AAV9-cTnT-SIRT6 cardiac injection were used in vivo to observe their effects on lipid metabolism and cardiac function in hypothyroidism.Results1.The hypothyroidism mice exhibited significant myocardium lipid toxicity damage.Compared with the euthyroidism group,the systolic function was significantly reduced,and there was significant lipid deposition and lipid toxicity damage in the hypothyroid myocardium.2.The myocardial SIRT6 level was decreased and the fatty acids β-oxidation was inhibited in hypothyroidism group.Compared with the euthyroidism group,the myocardial SIRT6 levels in the hypothyroid group were significantly reduced.The combination of SIRT6 and PPARa was also decreased.The expression of CPT1b was also decreased significantly(all P<0.05).3.The TSH treatment induced significant lipid toxicity damage in cardiomyocytes.The NRVMs exhibited lipid metabolism disorder and lipid deposition after TSH treatment.The SIRT6 and CPT1b levels were significantly decreased(all P<0.05).4.The SIRT6 enhanced mitochondrial oxidative respiration and reduced lipotoxicity damage in TSH treatment.Overexpression of SIRT6 by Ad-SIRT6 enhanced myocardial mitochondrial oxidative respiration and partially restored lipid toxicity damage by TSH treatment.Downregulation of SIRT6 inhibited myocardial mitochondrial oxidative respiration and exacerbated lipid toxicity damage(all P<0.05).5.The myocardial specific SIRT6 knockout leads to further exacerbation of cardiac lipotoxicity damage and deterioration of hypothyroid heart failure.In hypothyroidism,SIRT6cKO mice exhibited lower levels of CPT1b compared to the SIRT6fl/fl group.The cardiac function was further decreased,and the lipid deposition was aggravated in hypothyroidism(all P<0.05).6.The myocardial overexpression of SIRT6 improved myocardial lipotoxicity damage and hypothyroid heart failure.The myocardial AAV9-cTnT-SIRT6 injection was used to enhance the expression of myocardial SIRT6.The results showed that the binding relationship of SIRT6 with PPARa was strengthened and the level of CPT1b was significantly increased.The myocardial lipid deposition,myocardial and lipid toxicity damage were reduced,and the cardiac function were significant improvement(all P<0.05).ConclusionIn the hypothyroidism heart failure,the SIRT6 level was significantly reduced,and its downstream PPARα-CPT1b signal was suppressed,leading to myocardial β-oxidation inhibition,decreased mitochondrial oxidative respiration,lipid deposition and lipid toxicity damage in the myocardium.Targeting SIRT6 overexpression could promote PPARα-CPT1b signaling,which improved mitochondrial oxidative respiration,inhibit lipid deposition and lipid toxicity damage,and finally improved cardiac function in hypothyroidism.Hypothyroidism induced hepatic steatosis and its underlying mechanismsBackgroundThe liver is the main metabolic organ in the human body,and thyroid function is closely related to liver metabolism.The thyroid hormone(T3,T4)and thyroid hormone(TSH)regulate liver lipid metabolism and cholesterol metabolism by regulating the expression of various metabolic enzymes.Multiple cohorts’ studies have confirmed a clear correlation between hypothyroidism and non-alcoholic fatty liver disease(NAFLD),and hypothyroidism is an independent risk factor for NAFLD.As is well known,NAFLD is closely related to cardiovascular disease and is an independent risk factor for coronary heart disease.The study of hepatic steatosis induced by hypothyroidism is of great significance for in-depth study of hypothyroidism induced cardiovascular metabolic changes.The NAFLD is mainly manifested as liver steatosis with lipid droplet deposition,while the main causes of steatosis were increased fatty acid and triglyceride uptake,synthesis or reduced lipolysis and oxidation degradation.Lipid droplet autophagy(lipophagy)is the crucial step in liver lipolysis.Lipid droplet encapsulation proteins perilipins(Plins)are key molecules that regulate lipid storage,lipid homeostasis,and lipid droplet autophagy.In lipophagy,there are two mechanisms:macroautophagy and chaperone mediated autophagy y(CMA).The macroautophagy way directly engulfs lipid droplets and transport lipid droplet through autophagy lysosomes,while the CMA mediated way is labeled with heat shock protein HSC70,which bind to LAMP2A and transferring lipid droplets directly to lysosomes.Plins is the most important recognition protein in lipophagy.The Plins family including Plin1-5.Among which Plin2,Plin3,and Plin5 play predominant roles in the liver.The pathological role of Plins mediated lipophagy in hypothyroid hepatic steatosis has not been reported yet.Methods1.The 8-week-old female mice were orally given propylthiouracil drinking water for 3 months to construct hypothyroidism.The pathological morphology of liver tissue was assessed by HE staining.The hepatic lipid deposition and lipophagy were evaluated by Oil red O staining,BODIPY staining,and electron microscopy.and the thyroid function was detected by serum radioimmunoassay.2.The hepatic expression of Plin2,Plin3,Plin5 were assessed by western blot.The expression of HSC70,LAMP2A,LAMP1,LC3,and p62 were also detected,as well as the phosphorylation level of upstream AMPK and ULK1.3.At the cellular level,C3A liver cells were treated with TSH to simulate hypothyroidism.AICAR were used to stimulate AMPK.The expression of Plin3 and AMPK-ULK1 mediated lipophagy signal were observed.The hepatocyte lipophagy was also observed by LC3 and BODIPY immunofluorescence staining.4.AICAR intraperitoneal administration was used to stimulate AMPK and lipophagy in vivo.The downstream ULK1 phosphorylation,LAMP1,LC3,p62,and Plin3 expression were observed.The hepatic lipid deposition and lipophagy were evaluated by Oil red O staining,BODIPY staining,and electron microscopy.Results1.The hepatic lipophagy was decreased in hypothyroidism,and the hypothyroid exhibited lipid deposition and hepatic steatosis.Compared with the euthyroidism group,the hypothyroidism group exhibited a significant decrease in liver lipophagy,with lipid droplet deposition,The serum ALT,AST,TC and TG levels were elevated.The mice exhibited abnormal glucose tolerance(all P<0.05).2.The AMPK mediated lipophagy was inhibited in hypothyroidism group,and the Plin3 level was increased.Compared with the euthyroidism group,the hepatic Plin3 level in the hypothyroid group was increased,and there was no significant difference in Plin2 and Plin5 expression levels.There was also no significant difference in HSC70 and LAMP2A levels.The phosphorylation of AMPK and downstream ULK1 were decreased,while the expression levels of LAMP1 LC3 and p62 were decreased(all P<0.05).3.TSH treatment decreased the lipophagy levels and increased cell lipid deposition.The phosphorylation of AMPK and ULK1 were inhibited,and the expression levels of LAMP1,LC3 and p62 were significantly reduced,which leading to the inhibition of Plin3 degradation after TSH treatment.The lipid content in hepatocytes were significantly increased(all P<0.05).4.AMPK activation by AICAR up-regulated lipophagy and improve hepatic steatosis in hypothyroidism.The AMPK specific activator AICAR was used to stimulated AMPK in vivo.The results showed an increase in AMPK and ULK1 phosphorylation levels,as well as the LAMP 1,LC3 and p62 expression.The Plin3 level was significant decreased,which indicated an enhanced lipophagy.Histological experiments also showed that AMPK activation significantly increased lipophagy and improved liver lipid deposition.The ALT,AST,TC,TG were reduced compared to the euthyroidism group,and abnormal glucose tolerance was improved(all P<0.05).ConclusionHypothyroidism could induce hepatic steatosis directly,and the decreased lipophagy may be one of the main causes.The underlying mechanism may be related to the decrease of AMPK-ULK1 mediated lipophagy signaling.The degradation of lipophagy key protein Plin3 was disrupted,leading to abnormal liver lipid deposition.AMPK activation by AICAR could stimulate lipophagy and significantly improve liver steatosis.