Gender Differences In The Effects Of Whole-life,Low-dose Cadmium Exposure On Postweaning High-fat Diet-induced Cardiac Pathogeneses And Underlying Mechanisms

Backgroud:Obesit is a very important public health problem in both developing and development countries.Obesity can cause a varity of complications related to cardiovascular diseases,and is an important risk factor for cardiovascular diseases(mainly including myocardial remodeling and heart failure).Epidemiological data and experimental studies suggest a direct association between obesity and myocardial remodeling and heart failure.With the development of social economy and increasing of human activities,Cadmium(Cd)contamination is also an environmental health concern.Epidemiological studies have shown the association between cadmium exposure and cardiovascular disease.Studies have suggested that cadmium can accumulate in the heart and has various effects on the heart.However,most of the studies showed that the exposure dose of cadmium was higher than the environmentally relevant dose.In addition,there are few studies to observe the effects of environmental dose cadmium exposure combined with high-fat diet on the heart,and the gender difference is not clear.Therefore,it is necessary and meaningful to investigate the effects of whole-life,environmental dose cadmium exposure on high-fat diet induced cardiac pathogeneses and to define gender differences.Objective:We want to establish a whole-life,low-dose cadmium exposure model through multiple-generation cadmium exposure,and the offspring were fed with high-fat diet after weaning,to observe the effects of whole-life,low-dose cadmium exposure on postweaning high-fat diet-induced cardiac pathogeneses,as well as gender differences and related mechanisms.Methods:Female and male C57BL/6J mice aged 6 weeks were selected as parental mice.VI The parental mice were given a standard laboratory diet and deionized water for one week,and then fed with an AIN-76 A purified diet for three weeks.Parental mice began Cd exposure in drinking water at 10 weeks old.Male and femal parental mice were divided into 3 groups respectively: Control group(Ctr),0.5 parts per million(0.5ppm,L-Cd)group and 5 ppm(H-Cd)group.At 12 weeks old,male and female parent mice were mated and then continued to drink water with different concentrations of cadmium.The offsprings were given breastfeed for three weeks,after weaning,divided into male and female groups.The offspring continued to drink deionized water containing different concentrations of cadmium as their parents,and each group were fed a high-fat diet(HFD,60% fat)or a normal diet(ND,10% fat)for 24 weeks after weaning.Therefore,the offspring mice were divided into three cadmium exposure doses groups,two diets and two gender groups,totally twelve groups(Female offspring group,n=5;Male offspring group,n=4).To evaluate cardiac structure and function of female and male offspring,echocardiographic measurements were performed.Then we collected the data of heart weight and tibial length,and measure the metal content in the heart.We prepared cardiac tissue pathological slices for sirius red staining to evaluate the degree of myocardial fibrosis,and wheat germ agglutinin staining to observe the myocardial cell size.The protein expression of pro-fibrotic markers and m RNA expression of hypertrophic markers were observed.The above results were used to evaluate the sex differences of offspring.Then,the mechanism of synergistic effect of Cd and HFD on the female hearts was further defined.Western blotting was used to detect myocardial pro-inflammatory markers(TNF-α,IL-1β,PAI-1,ICAM)and the protein expression level of phosphorylated P38.Malondialdehyde(MDA)content was detected in heart tissue to measure lipid peroxidation level.Oxidative stress and antioxidant stress markers(3-NT,CAT,SOD2,MT)were detected by Western blotting.m RNA expression of MT2,ZIP8,ZIP14,Zn T1,Zn T2 and DMT1 were further evaluated by q RT-PC.Results:1.Gender differences in metal content,echocardiography analysis,and cardiac fibrosis and hypertrophy(1)Determination of metal content: Cd accumulated in a does-dependent manner both in female and male offspring hearts.In female mice hearts,the concentrations of zinc,copper and calcium in H-Cd/HFD group were significantly decreased or trendly decreased compared with HFD group.(2)Echocardiography analysis:Echocardiogram results showed that in female offspring,L-Cd exposure did not induce cardiac structural and functional abnormalities.H-Cd only induced cardiac ejection trended decrease.HFD induced cardiac hypertrophy,but there was no cardiac dysfunction.There was no cardiac structural and functional change between ND and HFD groups when exposed to L-Cd.H-Cd and HFD synergically induced cardiac remodeling,systolic and diastolic dysfunction,including left ventricular end-diastolic and systolic diameter increased(3.60±0.15 vs.4.02±0.0,P<0.05;2.27±0.17 vs.2.85±0.17,P<0.050),left ventricular ejection fraction decreased(67.58±4.23 vs.56.27±5.24,P<0.05),and mitral E-to-annular e’ ratio increased(20.43±2.24 vs.29.81±4.83,P<0.05).In male offspring,Cd exposure also did not induce cardiac structural and functional abnormalities.HFD induced not only hypertrophy but also mild diastolic dysfunction.In the H-Cd/HFD group,there was only a severity trend of diastolic dysfunction.The synergistic effect was not so obvious as in female offspring.(3)Cardiac fibrosis and hypertrophyIn female offspring,H-Cd and HFD synergically induced myocardial fibrosis.Sirus red staining suggested increased myocardial interstitial collagen accumulation in the H-Cd/HFD group.Western blotting showed that higher protein expression of fibronectin and collagen1A1 in H-Cd/HFD group compared with Ctr or HFD groups.H-Cd exposure aggravated HFD-induce cardiac hypertrophy in female offspring.HFD induced cardiac hypertrophy along with morphological hypertrophy and hypertropic markers m RNA expression increased.Compared with HFD groups,myocardial cell size further increased(wheat germ agglutinin staining).m RNA levels of upstream transcription factors(GATA4,MEF2C)and cardiomyocyte hypertrophy factors(β-MHC)further increased in H-Cd/HFD group.In male offspring,HFD induced hypertrophy(myocardial cell size,hypertrophy factors expression)and fibrosis(collagen accumulation,protein expression of fibronectin and collagen1A1).However,there was no further aggravation of cardiac fibrosis and hypertrophy in H-Cd/HFD group compared with the HFD group.2.The underlying mechanism of synergistic effect of 5 ppm Cd exposure combined with HFD in female offspring hearts(1)H-Cd exposure further activated HFD-induced activation of P38 MAPK pathway and aggravated HFD-induced cardiac inflammation(ICAM,TNF-α,PAI-1protein expression further increased).(2)H-Cd exposure exacerbated HFD-induced oxidative stress damage(malondialdehyde level,3-nitrotyrosine expression further increased).The antioxidant capacity(expression of metallothionein protein and m RNA decreased)was decreased in H-Cd/HFD group.(3)The mRNA expressions of ZIP8,ZIP14,and Zn T1 were increased or tended increased in H-Cd group compared with Ctr group,and significantly increased in H-Cd /HFD group.DMT1 m RNA expression was also increased in H-Cd/HFD group.Conclusion:(1)There was cardiac trace element dyshomeostasis in female offspring hearts when exposed to Cd combined with HFD.(2)H-Cd and HFD synergistically induced systolic and diastolic dysfunction,and myocardial fibrosis in female offspring.H-Cd exposure exacerbated HFD-induced cardiac hypertrophy,inflammation,and oxidative stress.(3)Whole-life 5 ppm cadmium exposure significantly increased the susceptibility of female offspring to HFD-induced cardiac remodeling and dysfunction.