Effects And Mechanism Of Metallothionein On Protection From Obesity-induced Cardiac Damage In Mice

Background:Obesity is known to trigger metabolism disorders including glucose,lipid,blood pressure anomalies,which are the risk for cardiovascular diseases including hypertension,atherosclerosis,atrial fibrillation,cardiac hypertrophy,heart failure,etc.Obesity cardiomyopathy is defined as cardiac structural,functional,and metabolic abnormalities caused by obesity alone and is becoming an urgent public health problem that needs to be resolved worldwide.Up to now,the underlying mechanisms of obesity cardiomyopathy include oxidative stress,inflammation,metabolism disorders（insulin resistance and metabolism disturbances of glucose,lipid,and amino acid）,intracellular Ca²⁺dyshomeostasis,autophagy dysregulation,which can lead to cardiac fibrosis,electrical remodeling,microangiopathy,coronary artery endothelial dysfunction,impaired coronary flow reserve,and even heart failure.Notably,Obesity-related chronic inflammation plays a key role in the development of obesity cardiomyopathy.Based on the previous research of our group,high-fat diet（HFD）-induced obesity mice showed cardiac hypertrophy and impaired glucose tolerance,accompanied by increased cardiac inflammation（increased macrophages infiltration in the heart,increased expression of CARD9 and BCL10,increased activation of p38 MAPK and increased expression of cytokines TNF-αand IL-6）.Correspondingly,these adverse effects can be reversed by increased expression of MT induced by zinc supplementation.In in vitro mechanism study,we further found that the exacerbation of MT-I/II gene knockout on the palmitate-induced expression of BCL10 and activation of p38 MAPK,while zinc supplementation cannot block the palmitate-induced BCL10 expression and p38 MAPK activation as in wild type.Here,we further defined whether MT prevents obesity-induced cardiac remodeling via CARD9/BCL10/p38 MAPK pathway in vivo.In addition,compared with WT mice,CARD9-KO mice showed significant resistance to pressure overload-induced cardiac remodeling and dysfunction and exhibited NF-κB inactivation.So,we also investigate whether the activation of NF-κB（a classical downstream component of CARD9 in parallel with p38 MAPK）is involved in obesity cardiomyopathy.Aims:（1）To clarify whether MT-I/II gene knockout exacerbates obesity-induced cardiac damage in vivo.（2）To elucidate whether MT regulates obesity-induced cardiac damage via CARD/BCL10/MAPKs and/or CARD9/BCL10/NF-κB pathways-mediated inflammation.（3）To explore the potential trigger mechanism of the activation of CARD9 signaling influenced by MT.Methods:（1）MT-I/II knockout（MT-I/II KO）and 129S1 wild-type（WT）male mice were fed a normal diet（ND,10%kcal fat）or HFD（60%kcal fat）from 8 weeks old to 26 weeks old.Then we got four groups:1)ND/WT group:WT mice fed with ND;2)HFD/WT group:WT mice fed with HFD;3)ND/MT-I/II KO group:MT-I/II KO mice fed with ND;4)HFD/MT-I/II KO group:MT-I/II KO mice fed with HFD.（2）Body weight was measured every week during the dietary intervention.Glucose intolerance was measured by Intraperitoneal Glucose Tolerance Test at 18 weeks old and 26 weeks old.At the end of the 18-week HFD feeding,cardiac structure and function were evaluated using echocardiography.Then all mice were anesthetized and euthanized.And the blood and heart tissue were collected.Plasma triglyceride and cholesterol were determined using commercially available colorimetric kits.Cardiac hypertrophy and fibrosis of heart tissues were observed by WGA staining and Picro-Sirius Red staining.Inflammation in heart tissues was analyzed by anti-CD68 antibody staining of macrophage infiltration.Metallothionein（MT）expression was detected by the anti-MT antibody of Western blotting.Western blotting and RT-q PCR were performed to detect the cardiac pro-fibrotic factors（Fn,col1a1,col3a1,TGF-β）,macrophage markers（CD68）,chemokines（CCL2）,adhesion molecules（VCAM-1,ICAM-1）,inflammation cytokines（TNF-α,IL-6,IL-1β）,inflammatory pathway molecules（CARD9,BCL10,phospho-NF-κB p65/p-p65,p38 MAPK,p-p38 MAPK,JNK,p-JNK,Erk1/2,p-Erk1/2）,oxidative stress damage markers（3-NT,4-HNE）and antioxidant enzymes（SOD2,CAT,GPX4）.ICP-MS was used to measure essential trace elements concentrations（zinc,copper,and iron）.Results:（1）MT-I/II knockout exacerbated HFD-induced obesity and systemic lipid metabolic disorders.1)Compared to the ND/WT group,the body weight and perineal white adipose weight were increased in the HFD/WT group,but lower than the HFD/MT-I/II KO group.2)HFD led to glucose intolerance both in WT mice and MT-I/II KO mice;MT-I/II gene knockout aggravated obesity-induced increased plasma cholesterol.（2）Knockout of MT-I/II gene combined with obesity led to cardiac remodeling but did not influence cardiac systolic function.1)MT-I/II gene knockout led to left ventricular dilation（as shown by increased LVID,d,and LV Vol,d）in both ND and HFD intervention mice;MT-I/II gene knockout did not influence left ventricular systolic function（EF%and FS%）in both ND and HFD intervention mice.2)Compared to the HFD/WT group and ND/MT-I/II KO group,the IVS,LVPW,HW/Tibia ratio,and myocyte area were increased in the HFD/MT-I/II KO group.3)Compared to the HFD/WT group and ND/MT-I/II KO group,the collagen content,m RNA expression of Fn,col1a1,col3a1,and TGF-β,and protein expression of COL1A1 and TGF-β1 were increased in the HFD/MT-I/II KO group.（3）Knockout of MT-I/II gene enhanced obesity-induced macrophage infiltration and inflammation in mice heart.1)Compared to the ND/WT group,m RNA and protein expression of CD68 and quantitative analysis of CD68 IHC staining was increased or showed an increased trend in the HFD/WT group and ND/MT-I/II KO group,but all lower than HFD/MT-I/II KO group.2)Compared to the ND/WT group,m RNA expression of CCL2,protein expression of ICAM-1,VCAM-1,TNF-α,IL-6,and IL-1βwere increased or showed an increased trend in the HFD/WT group and ND/MT-I/II KO group,but all significantly lower than HFD/MT-I/II KO group.3)Compared to the ND/WT group,protein expression of CARD9,BCL10,p-p65 were increased in the HFD/WT group and ND/MT-I/II KO group,but all significantly lower than HFD/MT-I/II KO group.（4）Knockout of MT-I/II gene exacerbated obesity-induced cardiac oxidative stress.1)Compared to the ND/WT group,protein expression of 3-NT,4-HNE were increased in the HFD/WT group and ND/MT-I/II KO group,but all significantly lower than the HFD/MT-I/II KO group.2)Compared to the ND/WT group,protein expression of MT was decreased in the HFD/WT group and no expression in those two MT-I/II KO groups;Compared to the ND/WT group,protein expression of CAT and GPX4 were increased in the HFD/WT group and ND/MT-I/II KO group,but all significantly lower than the HFD/MT-I/II KO group.3)Compared to the ND/WT group,the ratio of Cu/Zn and Cu/Fe were increased in the HFD/WT group and slightly increased in the ND/MT-I/II KO group,while the significantly higher Cu/Fe ratio or a higher trend of Cu/Zn ratio was found in HFD/MT-I/II KO group.Conclusions:（1）MT prevents the heart against obesity-induced cardiac damage to cardiac remodeling.（2）MT protects the heart against obesity-induced oxidative stress and inflammation and consequent development of cardiac hypertrophy and fibrosis.（3）MT prevents obesity-induced cardiac remodeling,possibly via inhibiting cardiac macrophage infiltration,expression of CARD9 and BCL10,and activation of NF-κB,but not MAPKs.