Preventive Effect By Zinc Of High Fat Diet-induced Obesity Related Cardiomyopathy

Background:Obesity often leads to cardiovascular diseases,such as cardiomyopathy,in their adult age.Obesity-related cardiomyopathy(ORC),an early pathogenic change of the cardiomyopathy,is associated with cardiac oxidative stress and inflammation.Oxidative stress means either over-generation of reactive oxygen and nitrogen species(ROS/RNS)or down-regulation of antioxidant defense system in the cell or tissues.Zinc is structurally and functionally essential for many transcription factors and catalytic enzymes;Previously,the reaearchers have demonstrated that Zn protected against diabetes-induced damage in the heart,kidney,liver and testis,mainly through increasing insulin sensitivity and reducing oxidative stress and inflammation.However,its role in ORC and underlying mechanism(s)remain unclear and were explored here in mice with obesity induced with high-fat diet(HFD).Aims The present study aimed to investigate whether chronic treatment of obese mice with Zn can prevent the development and/or delay the progression of obesity-induced cardiovascular pathogenesis.Methods The first study: obesity was induced in 4-week old C57BL/6J mice fed with either high fat diet(HFD,60% kcal fat)or normal diet(ND,10% kcal fat)containing one of 3 different quantities: low zinc(ZD,10 mg zinc per 4057 kcal),normal zinc(ZN,30 mg zinc per 4057 kcal)or high zinc(ZS,90 mg zinc per 4057 kcal)for 3 or 6 months.Therefore,the first set of experiments contained six groups as follows: ND/ZN(n=10),ND/ZD(n=10),ND/ZS(n=12),HFD/ZN(n=14),HFD/ZD(n=16),and HFD/ZS(n=16).All the blood pressure and cardiac function were assessed at 3M and 6M.Cardiac inflammation(CD68,TNF-α,IL-6),fibrosis(CTGF,TGF-β),oxidative damage(3-NT,4-HNE),hypertrophy(ANP,BNP and β-MHC)by sirius red staining,western blot,wheat germ agglutinin(WGA)and real-time PCR method.Western blot was used to detect the expression of CARD9,BCL10,phospholated-p38MAPK,total-p38 MAPK,and MT.Co-immunoprecipitation was performed to detect the CARD9/BCL10 complex.The second study: A mechanistic study was performed on the mice in the following 5 groups: ND/ZN(n=6),HFD/ZN(n=6),HFD/ZN with the administration of the p38 MAPK inhibitor SB203580(1 mg/kg body weight,intraperitoneal injection,at the start of HFD treatment,HFD/ZN/SB,n=7),HFD/ZD(n=7)and HFD/ZD with SB203580 treatment(HFD/ZD/SB,n=8).These mice were anaesthetized as mentioned above and were sacrificed after ND or HFD feeding with and without SB for 3 months.Primary cardiomyocyte isolation,cell culture,palmitate and TPEN treatments and small interfering RNA transfection:C57BL/6J,MT-KO mice(the Jackson Laboratory,Bar Harbor,Maine),and 129S1 male mice aged 2-months were euthanized to isolate primary cardiomyocytes.Primary cultured cardiomyocytes were treated with palmitate at 100 μM to mimic HFD model in vivo and with TPEN,a Zn chelator,at 2 μM to mimic Zn deficiency model.Primary cultured cardiomyocytes were also pre-treated with specific small interfering RNA(si RNA)against p38MAPK(p38-si RNA)or BCL10(BCL10 si RNA).B6.BKS(D)-Leprdb/J(db/db)mice fed for six months on a normal diet containing three zinc levels(deficient,adequate,and supplemented),to explore the role of zinc in obesity related cardiomyopathy development and progression.The first 17 mice were fed a normal diet(10% calories from fat)with different amounts of Zn,including 10 mg(deficient,ZD;n = 7),30 mg(adequate,ZN;n = 6),and 90 mg(supplemented,ZS;n = 4)per 4057 Kcal,respectively,for six months.The fourth group of four db/db mice were fed ZS for the first three months and switched to ZN for the subsequent three months,the same as the db/db/ZS-N group.Meanwhile,seven C57BL/6J mice were fed a ZN diet for six months as a control group.After six months,all mice were sacrificed at 6M.Results HFD induced significant insulin resistance,hyperinsulinemia,and hypertriglyceridemia.Echocardiography demonstrated cardiac hypertrophy in obese mice,confirmed by increased heart weight,cardiomyocyte size,and hypertrophic molecular markers.HFD significantly increased cardiac inflammation,BCL10/CARD9 expression,and p38 MAPK and NF-κB activation.These obese effects were worsened in HFD/ZD mice and were attenuated in HFD/ZS group,respectively.Furthermore,Administration of p38 MAPK specific inhibitor in HFD mice or specific si RNA in palmitate-treated cardiomyocytes totally eliminated HFD-triggered,and zinc deficiency-further enhanced,activation of p38 MAPK,but did not significantly impact the expression of BCL10 and CARD9.In the cultured cardiomyocytes,inhibition of BCL10 expression by its si RNA prevented palmitate-increased p38 activation and ANP expression while inhibition of p38α MAPK only prevented ANP expression,but did not affect BCL10 expression.In addition,deletion of metallothionein completely abolished the protective effect of zinc on palmitate-induced up-regulation of BCL10 and phospho-p38.Zinc deficiency worsensed the cardiac dysfunction in the db/db mice.Conclusion These results suggest that HFD and zinc deficiency synergistically induce ORC via increasing oxidative stress-mediated activation of BCL10/CARD9/p38 MAPK signaling and zinc supplement ameliorates ORC through activation of metallothionein to repress oxidative stress-activated BCL10 expression and p38 MAPK activation.