Sex differences in the effects of whole-life, low-dose cadmium exposure on postweaning high-fat diet-induced cardiac pathogeneses

Abstract

We previously showed the development of cardiac remodeling (hypertrophy or fibrosis) in mice with either post-weaning high-fat diet (HFD, 60% kcal fat) feeding or exposure to chronic low-dose cadmium. Here, we determined whether whole-life exposure to environmentally relevant, low-dose cadmium affects the susceptibility of offspring to post-weaning HFD-induced cardiac pathologies and function. Besides, we also determined whether these effects are sex-dependent. Male and female mice were exposed to cadmium-containing (0, 0.5, or 5 parts per million [ppm]) drinking water before breeding; the pregnant mice and dams with offspring continually drank the same cadmium-containing water. After weaning, the offspring were continued on the same regime as their parents and fed either a HFD or normal fat diet for 24 weeks. Cardiac function was examined with echocardiography. Cardiac tissues were used for the histopathological and biochemical (gene and protein expression by real-time PCR and Western blotting) assays. Results showed a dose-dependent cadmium accumulation in the hearts of male and female mice along with decreased cardiac zinc and copper levels only in female offspring. Exposure to 5 ppm, but not 0.5 ppm, cadmium significantly enhanced HFD cardiac effects only in female mice, shown by worsened cardiac systolic and diastolic dysfunction (ejection fraction, mitral E-to-annular e′ ratio), increased fibrosis (collagen, fibronectin, collagen1A1), hypertrophy (cardiomyocyte size, atrial natriuretic peptide, β-myosin heavy chain), and inflammation (intercellular adhesion molecule-1, tumor necrosis factor-α, plasminogen activator inhibitor type 1), compared to the HFD group. These synergistic effects were associated with activation of the p38 mitogen-activated protein kinases (MAPK) signaling pathway and increased oxidative stress, shown by 3-nitrotyrosine and malondialdehyde, along with decreased metallothionein expression. These results suggest that whole-life 5 ppm cadmium exposure significantly increases the susceptibility of female offspring to HFD-induced cardiac remodeling and dysfunction. The underlying mechanism and potential intervention will be further explored in the future.

Graphical Abstract

1. Introduction

Obesity has become a serious public health concern worldwide because it increases the risk of cardiovascular diseases (CVDs), including heart failure, coronary heart disease, arrhythmias, hypertension, and sudden cardiac death (Koliaki et al., 2019; Ortega et al., 2016). For instance, moderate-to-severe obesity lead to cardiac remodeling and heart failure with preserved ejection fraction (HFpEF) (Harada and Obokata, 2020; Koliaki et al., 2019; Packer, 2020; Tadic and Cuspidi, 2019). Consistent with human studies, in mouse models, high-fat diet (HFD)-induced obesity also induced cardiac hypertrophy and/or diastolic dysfunction (Sun et al., 2020; Wang et al., 2016). There are several possible mechanisms for obesity-induced cardiac remodeling and HFpEF, including enhanced cardiac output, increased inflammation and oxidative stress (OS), and increased collagen deposition and fibrosis (Harada and Obokata, 2020; Karason and Jamaly, 2020; Prenner and Mather, 2018; Tadic and Cuspidi, 2019).

With product innovative improvements and the human way of life and regular habitat changes, the world’s ecological contamination has turned into a danger to the general wellbeing in the previous years. Wastewater, air contamination, substantial metal presence, normal and manufactured natural contaminants have acquired worries (Sher et al., 2021; Rasheed et al., 2020b). Wastewater, including poisonous, mutagenic, and cancer-causing contaminations, can prompt numerous destructive issues, like organ failure, malignant growth, and other undesirable conditions (Rashid et al., 2021; Bagheri et al., 2021). With this idea, we have examined the wellbeing impact of heavy metal cadmium (Cd) (Turdi et al., 2013; Young and Cai, 2020) since it exists in water and air, both unavoidable to people.

Cd is a toxic heavy metal and is not an essential element for the human body; The World Health Organization (WHO) has classified Cd exposure as a significant environmental concern (W.H.O., 2019). Epidemiological studies have shown that chronic low-dose Cd exposure might have adverse effects on the cardiovascular system, including heart failure, cardiomyopathy, coronary heart disease, hypertension, and atherosclerosis (Lin et al., 2021; Tellez-Plaza et al., 2013; Tinkov et al., 2018; Vallée et al., 2020). In fact, due to its low antioxidant capacity, the heart is relatively sensitive to Cd exposure (Chen et al., 1994). The potential mechanisms of the effects of Cd exposure on the heart include excessive production of reactive oxygen and nitrogen species (ROS and RNS) and antioxidant defense alteration (Ferramola et al., 2012; Kukongviriyapan et al., 2016; Wang et al., 2004), increased inflammation, deoxyribonucleic acid (DNA) damage and repair abnormalities, and impaired cardiac energy homeostasis (Chen et al., 2015; Turdi et al., 2013; Yazıhan et al., 2011). In animal models, we have shown exposure to low-dose Cd (CdCl₂ 20 nmol/kg in saline, intraperitoneal injection, every other day for 4 weeks) induced cardiac contractile dysfunction in mice (Turdi et al., 2013). In addition to the direct impact of low-dose Cd exposure on the heart, there may also be a combined effect of low-dose Cd exposure and other pathogenic factors on the cardiovascular system. For instance, Cd exposure in drinking water (CdCl₂ 100 mg/l) for 12 weeks significantly enhanced the level of HFD-induced cardiac fibrosis and risk of heart failure in ApoE knock-out mice (Türkcan et al., 2015).

There are different sources of Cd exposure. Occupational exposure occurs as a result of smelting and refining, fossil fuel combustion, and recycling electronic waste (Fatima et al., 2019). In the general public, Cd exposure occurs through contaminated water and food, tobacco smoking, air contamination, and products such as jewelry, toys, and plastics. In the general, non-smoking population, exposure to environmental low-dose Cd is a major concern and needs to be given more attention. Cd has hydrochemical characteristics that result in its mobility in groundwater, rivers, and drinking water near mines and factories (Genchi et al., 2020). Cd-containing solders in water heaters, water coolers, taps, and fittings lead to increased Cd levels in drinking water, especially in regions that have soft water with a low pH that cause corrosion of the Cd-containing plumbing systems (W.H.O., 2019). Therefore, we focused on low-dose Cd exposure in drinking water in our study, which is similar to Cd exposure in real life. We have treated mice with Cd (5 parts per million [ppm]) in drinking water as a low-dose exposure, which caused Cd accumulation in the hearts of mice (Young et al., 2019; Liang et al., 2019). Therefore, we selected the 5 ppm exposure dose and a lower exposure dose of 0.5 ppm in the present study.

There are reports that Cd affects health more commonly in females than in males, which may be due to the higher body accumulation of Cd in females (Vahter et al., 2007; Järup and Akesson, 2009). However, the risk of CVD-related mortality is lower in females than in males in response to environmental Cd (Menke et al., 2009), which may be attributed to estrogen protection in females. Increasing evidence indicates that Cd has an estrogenic effect and can affect female offspring (Vahter et al., 2007). Therefore, addressing sex as a risk factor is an urgent requirement (Young and Cai, 2020).

Moreover, human and animal studies have shown maternal Cd exposure was associated with the increased risk of CVD in offspring (Jin et al., 2016; Young and Cai, 2020). Therefore, our aims were as follows. (1) We aimed to propose a mouse model of whole-life low-dose Cd exposure via breeding mice and offspring continuously exposed to Cd, combined with HFD feeding post-weaning for 24 weeks to detect the effect on the hearts. (2) Given the aforementioned sex differences among Cd exposure and CVD respectively, we also investigate whether there are different effects on the heart of female and male adult offspring when exposed to Cd combined with HFD-induced obesity. (3) Lastly, we also explored the possible mechanisms by which Cd and HFD impact heart health in offspring.

2. Material and methods

2.1. Animals and procedures

This study was performed in accordance with the Guideline of NIH on the Care and Use of Laboratory Animals, and regulations approved by the University of Louisville’s Institutional Animal Care and Use Committee. Parental mice (F0), 6 weeks old male and female C57BL/6J, were obtained from Jackson Laboratory (Bar Harbor, ME, USA), and were housed in a pathogen-free AAALAC-accredited laboratory condition and maintained on a 12 h light/dark cycle at a constant temperature (25 °C). To reduce metal contamination from tap water, these mice were given a standard laboratory diet ad libitum and provided with free access to deionized water for one week and then the standard chow was switched to an AIN-76A purified diet (Envigo TD 160377) to prevent diet contamination with other metals for 3 more weeks.

As shown in Fig. 1A, F0 mice began Cd exposure in the drinking water at 10 weeks old. Cd (CdCl₂, Alfa Aesar) stock solution was diluted in drinking water at a final concentration of 0, 0.5 ppm and 5 ppm: F0 male and females were divided into three groups: Control (Ctr), 0.5 ppm Cd (L-Cd), and 5 ppm Cd (H-Cd). At 12 weeks old, male and female F0 were mated (male/female ratio, 1:2) and continued to drink the same Cd-containing water. After weaning, the offspring continuously drank the deionized water containing different concentrations of Cd as their parents, and each of these groups of mice was fed either HFD (Research Diets D14020205– 60% fat) or a normal fat diet (ND, Research Diets D14020202– 10% fat) for 24 weeks post-weaning.

Fig. 1.

(A) Design of the study. Adult male and female C57BL/6 J mice (F0) were fed a low-fat diet and exposed to 0, 0.5, or 5 parts per million [ppm] cadmium in drinking water (red) for >2 weeks before mating. Female F0 mice were exposed to cadmium until offspring weaning. After weaning, offspring (F1) were continuously given the same cadmium exposure as F0. At weaning, female (yellow) and male (blue) offspring were given either a low- (green) or high-fat diet (orange) respectively for 24 weeks and then sacrificed. (B) Cardiac trace element dyshomeostasis. The upward arrow represented the level of metal concentration. The grey value shows the increased concentrations of cadmium, zinc, copper, and calcium in the hearts from light to dark grey as indicated by the grey arrow. Female offspring showed cardiac trace element dyshomeostasis when exposed to cadmium and high-fat diet feeding, which was not observed in male offspring.

2.2. Echocardiography

To assess the cardiac function of male and female offspring, transthoracic echocardiogram measurements were performed by ultrasonography using Vevo 2100 Imaging System (FUJIFILM Visual Sonics, Canada), as described elsewhere (Kang et al., 2020). The following parameters were assessed: interventricular septum, left ventricular (LV) internal dimension, LV posterior wall, LV mass, LV volume, fractional shortening, ejection fraction, E/A, and E/e′.

2.3. Metal analysis

Metal element quantification in the heart tissue was measured using the X Series II quadrupole inductively coupled plasma mass spectrometry (ICP-MS). 800 μl 70% nitric acid was added into heart tissue tubes, and incubated the samples at 85 °C shakers for 4 h. Then, the samples are allowed to cool down to room temperature, and centrifuged the tubes (3000 rpm, 1 min), and then diluted into 4% nitric acid with deionized water, vortexed, and further assayed.

2.4. Malondialdehyde levels analysis

Cardiac tissue malondialdehyde (MDA) content was measured using the thiobarbituric acid (TBA)-reactive substances method. Briefly, 25 μl sample was mixed with 8.1% sodium dodecyl sulfate (SDS, 10 μl), 20% acetic acid (75 μl), and 0.57% TBA (105 μl), and then heated for 70 min. After cooling on ice, the samples were diluted with ddH₂O, then removal of the precipitate was performed by centrifugation (4000g, 15 min). Absorbance was measured at 540 nm, and the MDA levels were calculated by measuring absorbance and concentration.

2.5. Pathological examination and immunohistochemistry

Cardiac tissues were placed into 10% formalin for 72 h. Then, we treated the samples with gradients of alcohol and xyleneand embedded the tissues in paraffin, from which 5-μm thick sections were obtained by microtome. To measure cardiac collagen accumulation and fibrosis, tissue sections were deparaffinized, rehydrated, and stained with picroSirius red. To measure the size of cardiomyocyte, sections were deparaffinized, digested with trypsin, blocked and stained with conjugated wheat germ agglutinin (WGA, Invitrogen, Waltham, MA, USA). Microscopic images were captured using an ECLIPSE E600 microscope (Nikon, Tokyo, Japan). Immunohistochemistry (IHC) staining was performed as described elsewhere (Jiang et al., 2013). The first IHC antibody in the present study was 3-nitrotyrosine (3-NT, 1:500; Millipore, Burlington, MA, USA). Quantitative results of staining were measured by Image J software.

2.6. Western blotting

Western blotting was performed according to the protocol previously described (Kang et al., 2020). Cardiac tissue was isolated from each heart and homogenized in a lysis buffer. Samples were centrifuged (12,000 rpm, 4 °C, 20 min) and then proteins were obtained. Electrophoresis was performed on 10% SDS-PAGE gels for 90 min and then proteins were transferred to PVDF membranes for 90 min at 4 °C. Blocking was performed with 5% nonfat milk for 1 h before incubation of the membranes with primary antibodies at 4 °C overnight. Then, the membranes were incubated with the secondary antibody (1:3000) for 1 h at room temperature. Metallothionein (MT) western blotting was performed as our previous protocol (Wang et al., 2006). Images were acquired using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). Band grayscale values were measured by the Image Lab software 6.0.1.

The primary antibodies were as follows: fibronectin (FN, 1:1000; Abcam, Cambridge, UK), collagen1A1 (COL1A1, 1:800; Santa Cruz Biotechnology, Dallas, TX, USA), intercellular adhesion molecule-1 (ICAM, 1:1000; Abcam), tumor necrosis factor-α (TNF-α, 1:2000; Santa Cruz Biotechnology), interleukin-1β (IL-1β, 1:800; Santa Cruz Biotechnology), plasminogen activator inhibitor type 1 (PAI-1, 1:2000; BD Biosciences, Franklin Lakes, NJ, USA), phosphorylation-P38/P38 (P-P38/P38, 1:1000; Cell Signaling Technology, Danvers, MA, USA), 3-nitrotyrosine (3-NT, 1:1000; Millipore), catalase (CAT, 1:2000; Santa Cruz Biotechnology), superoxide dismutase-2 (SOD2, 1:2000; Santa Cruz Biotechnology), Metallothionein (MT, 1:1000; Dako, Glostrup, Denmark), β-actin (1:4000; Santa Cruz Biotechnology), and GAPDH (1:3000; Abcam).

2.7. Real-time quantitative polymerase chain reaction

RNA was obtained from the heart tissue using Trizol reagent (Invitrogen). RNA concentration and quality were measured using a NanoDrop spectrophotometer. Complementary DNA (cDNA) was synthesized from RNA by reverse transcription (RT) reaction with the cDNA Synthesis Kit (Invitrogen). Quantitative polymerase chain reaction (PCR) was performed in a 10-μl reaction system, using the LightCycler 96 RT-PCR system (Roche Diagnostics, Indianapolis, IN, USA). Primers were acquired from Thermo Fisher (Grand Island, NY, USA) and were as follows: ANP (Mm01255748), β-MHC (Mm00600555), GATA4 (Mm00484689), MEF2c (Mm01340842), MT2 (Mm00809556), ZIP8 (Mm00470855), ZIP14 (Mm01317439), ZnT1 (Mm00437377), ZnT2 (Mm01185317), and DMT1 (Mm00558248).

2.8. Statistical analysis

Data are expressed as means ± standard deviation. Differences in variables between multiple groups were determined by one-way ANOVA and posthoc pairwise repetitive comparisons. P-value < 0.05 was assumed as statistically significant. Statistical analysis was performed with GraphPad Prism 8 Software (San Diego, CA, USA).

3. Results

3.1. Heart metal levels

ICP-MS metal analysis showed Cd accumulation in a dose-dependent manner in both female and male mice hearts (Supplemental Figs. 1 and 2). In female mice, the concentrations of zinc (Zn) and copper (Cu) were significantly decreased in L-Cd/HFD and H-Cd/HFD groups compared with the HFD group. The ratios of Zn/Cd and Cu/Cd were also decreased but the degree of decrease was greater in the H-Cd/HFD group than that in the L-Cd/HFD group. The concentration of calcium (Ca) was also lower and showed a trend decrease in female L-Cd/HFD and H-Cd/HFD groups compared with the HFD group. Other essential metal levels did not change significantly. These results suggested that HFD combined with Cd reduced the Zn, Cu, and Ca concentration in female hearts (Supplemental Fig. 1), but not in male hearts (Supplemental Fig. 2), which is summarized in Fig. 1B.

3.2. Cardiac function and structure

We first examined the effect of HFD on cardiac functional and structural changes. Echocardiographic analysis showed that, in female mice, HFD induced cardiac structural changes but not cardiac dysfunction. HFD/H-Cd mice, but not HFD/L-Cd, mice showed induced cardiac remodeling along with systolic and diastolic dysfunction (Table 1). These results suggested that there was a significant synergistic effect of Cd exposure and HFD on cardiac function and structure of female mice.

Table 1.

Echocardiographic analysis in female and male offspring.

	Normal Diet (ND)			High Fat Diet (HFD)
	Ctrl	L-Cd	H-Cd	Ctrl	L-Cd	H-Cd
Females
IVS; d (mm)	0.70 ± 0.03	0.70 ± 0.02	0.72 ± 0.02	0.78 ± 0.04*	0.75 ± 0.03	0.73 ± 0.01&
LVID; d (mm)	3.60 ± 0.15	3.64 ± 0.10	3.65 ± 0.21	3.65 ± 0.22	3.50 ± 0.30	4.02 ± 0.08*
LVPW; d (mm)	0.69 ± 0.02	0.70 ± 0.02	0.73 ± 0.03	0.80 ± 0.02*	0.74 ± 0.03	0.73 ± 0.04&
IVS; s (mm)	0.97 ± 0.05	0.99 ± 0.05	0.94 ± 0.02	1.05 ± 0.02	0.99 ± 0.04	1.00 ± 0.06
LVID; s (mm)	2.27 ± 0.17	2.30 ± 0.09	2.35 ± 0.13	2.35 ± 0.29	2.31 ± 0.17	2.85 ± 0.17*,&
LVPW; s (mm)	0.96 ± 0.05	0.97 ± 0.06	0.96 ± 0.02	1.09 ± 0.07*	0.96 ± 0.03&	0.99 ± 0.04
LV Vol; d (ul)	54.75 ± 5.76	56.01 ± 3.78	53.36 ± 7.03	56.46 ± 7.90	51.63 ± 10.62	70.73 ± 3.45*
LV Vol; s (ul)	17.70 ± 3.43	18.13 ± 1.78	19.08 ± 2.43	19.70 ± 5.75	18.43 ± 3.28	31.00 ± 4.39*,&
%EF	67.58 ± 4.23	67.57 ± 3.09	63.99 ± 2.26	65.85 ± 5.80	63.65 ± 6.17	56.27 ± 5.24*
%FS	37.09 ± 3.44	36.87 ± 2.41	34.09 ± 1.71	35.70 ± 4.32	33.97 ± 4.51	29.12 ± 3.55*
LV mass (mg)	82.27 ± 7.29	84.39 ± 4.24	85.65 ± 8.47	99.51 ± 6.76*	86.17 ± 12.69	104.80 ± 4.83*
E/A	1.55 ± 0.21	1.40 ± 0.23	1.55 ± 0.16	1.31 ± 0.16	1.60 ± 0.19	1.73 ± 0.47
E/e′	20.43 ± 2.24	20.38 ± 2.26	21.92 ± 2.61	25.65 ± 2.60	23.93 ± 3.68	29.81 ± 4.83*
Males
IVS; d (mm)	0.71 ± 0.01	0.69 ± 0.03	0.69 ± 0.03	0.82 ± 0.02*	0.75 ± 0.04	0.84 ± 0.02*
LVID; d (mm)	3.88 ± 0.13	3.81 ± 0.12	3.84 ± 0.20	3.88 ± 0.10	3.82 ± 0.19	3.94 ± 0.12
LVPW; d (mm)	0.70 ± 0.02	0.72 ± 0.02	0.70 ± 0.01	0.82 ± 0.02*	0.77 ± 0.02&	0.86 ± 0.01*
IVS; s (mm)	0.96 ± 0.03	1.00 ± 0.08	0.95 ± 0.05	1.11 ± 0.07*	1.02 ± 0.02	1.12 ± 0.03*
LVID; s (mm)	2.42 ± 0.09	2.41 ± 0.11	2.52 ± 0.20	2.51 ± 0.11	2.44 ± 0.23	2.61 ± 0.16
LVPW; s (mm)	0.95 ± 0.03	0.95 ± 0.03	0.94 ± 0.07	1.11 ± 0.05*	1.01 ± 0.04	1.16 ± 0.05*
LV Vol; d (ul)	65.37 ± 5.07	62.33 ± 4.65	63.86 ± 7.75	65.33 ± 4.08	63.01 ± 7.17	67.61 ± 4.88
LV Vol; s (ul)	20.68 ± 1.87	20.41 ± 2.30	23.01 ± 4.40	22.68 ± 2.53	21.39 ± 4.73	25.01 ± 3.66
%EF	68.40 ± 0.74	67.23 ± 3.15	64.29 ± 2.87	65.37 ± 2.04	66.48 ± 4.08	63.21 ± 3.18
%FS	37.64 ± 0.55	36.74 ± 2.40	34.52 ± 2.02	35.33 ± 1.50	36.20 ± 3.00	33.81 ± 2.30
LV mass (mg)	93.02 ± 5.10	91.10 ± 3.93	91.29 ± 5.76	115.36 ± 3.44*	101.65 ± 9.21	124.51 ± 8.63*
E/A	1.59 ± 0.13	1.60 ± 0.09	1.60 ± 0.08	1.39 ± 0.02	1.46 ± 0.10	1.27 ± 0.03*
E/e′	19.10 ± 2.93	20.01 ± 1.18	19.65 ± 3.69	27.91 ± 1.88*	22.79 ± 2.39	33.24 ± 1.18*

IVS, d: end-diastolic interventricular septum; LVID, d: left ventricular (LV) end-diastolic diameter; LVPW, d: LV end-diastolic posterior wall; IVS, s: end-systolic inter-ventricular septum; LVID, s: LV end-systolic diameter; LVPW, s: LV end-systolic posterior wall; LV Vol, d: LV end-diastolic volume; LV Vol, s: LV end-systolic volume; EF: ejection fraction; FS: fractional shortening; LV mass: left ventricular mass; E/A: the ratio of early diastolic to late diastolic mitral inflow velocities; E/e′: mitral E-to-annular e′ ratio.

Data are presented as mean ± SD (n = 4 or 5).

^*p < 0.05 vs Ctr/ND group.

^&p < 0.05 vs Ctr/HFD group.

In male mice, the echocardiographic analysis showed no systolic dysfunction in any group. HFD not only induced cardiac hypertrophy but also induced mild diastolic dysfunction. L-Cd, H-Cd and L-Cd/HFD mice did not show any change in cardiac structure and function. In the H-Cd/HFD group, there was only a severity trend of diastolic dysfunction, shown by a further decreased E/A and increased E/e′. There was no LV diameter increase and EF% decrease in male mice, which was different from that in female mice (Table 1). Therefore, in the following sections, we will focus on the cardiac effects of whole-life Cd exposure at the 5 ppm level (H-Cd) with HFD feeding in offspring to explore the potential mechanisms in female mice and then compare the sex differences.

3.3. Pathophysiological changes induced by Cd and HFD: Synergistic effect in female mice

3.3.1. Cd/HFD synergistically induced cardiac fibrosis

Sirius red staining revealed a higher degree of myocardial interstitial collagen accumulation in the H-Cd/HFD group compared with the Ctr/ND and Ctr/HFD groups (Fig. 2A). Western blotting analysis revealed higher protein expression of the pro-fibrotic markers fibronectin (FN, Fig. 2B) and collagen1A1 (COL1A1, Fig. 2C) in H-Cd/HFD female mice compared with Ctr or HFD female mice. These fibrotic responses were associated with H-Cd/HFD-induced cardiac dysfunction (Table 1).

Fig. 2.

Sirius red staining and protein expression of pro-fibrosis markers in female offspring hearts when exposed to 5 ppm cadmium and high-fat diet (HFD) feeding. (A) Sirius red staining results in female mice hearts. (B) Fibronectin (FN) protein expression in female mice hearts. (C) Collagen 1A1 (COL1A1) protein expression in female mice hearts (yellow, female control [Ctr] group; yellow with grid, female HFD group; orange, female 5 ppm cadmium group; orange with grid, female 5 ppm cadmium/HFD group; n = 5; *p < 0.05 versus [vs.] Ctr/normal fat diet [ND] group; ^&p < 0.05 vs. Ctr/HFD group). H-Cd, 5 ppm Cd.

3.3.2. H-Cd exposure exacerbated HFD-induced cardiac hypertrophy

The ratio of heart weight to tibial length was increased in Ctr/HFD and H-Cd/HFD female mice (Fig. 3A). This was consistent with the echocardiographic finding that HFD increased the wall thickness, suggesting HFD-induced cardiac hypertrophy. H-Cd/HFD mice exhibited a higher degree of morphological hypertrophy than Ctr/HFD mice, as determined by analysis of the cardiomyocyte size via WGA staining (Fig. 3B). Consistent with pathological remodeling changes, the mRNA expression of GATA4 and MEF2c, two transcriptional factors of cardiac hypertrophy mediators, were increased in HFD mice and further increased in H-Cd/HFD mice (Fig. 3C, D), along with the mRNA expression of hypertrophic molecules ANP and β-MHC in similar patterns (Fig. 3E, F).

Fig. 3.

The ratio of heart weight to tibial length, cardiomyocyte size, and mRNA expression of hypertrophic markers in female offspring hearts when exposed to 5 ppm cadmium and high-fat diet (HFD) feeding. (A) The ratio results of heart weight to tibial length. (B) Cardiomyocyte size showed by wheat germ agglutinin (WGA) staining in female mice and statistical results of cardiomyocyte size in female mice. (C) mRNA expression of GATA4. (D) mRNA expression of MEF2c. (E) mRNA expression of ANP. (F) mRNA expression of β-MHC. (yellow, female control [Ctr] group; yellow with grid, female HFD group; orange, female 5 ppm cadmium group; orange with grid, female 5 ppm cadmium/HFD group; n = 5; *p < 0.05 versus [vs.] Ctr/normal fat diet [ND] group; ^&p < 0.05 vs. Ctr/HFD group). H-Cd, 5 ppm Cd.

3.3.3. H-Cd exposure exacerbated HFD-induced cardiac inflammation and P38 MAPK activation

P38 MAPK is an important cardiac inflammatory and remodeling signaling pathway. Western blotting showed increased phosphorylation of P38 in the HFD group, and this further increased in H-Cd/HFD mice (Fig. 4A, B). Protein expressions of cardiac pro-inflammatory makers ICAM (Fig. 4A, C), TNF-α (Fig. 4A, D), PAI-1 (Fig. 4A, E), and IL-1β (Fig. 4A, F) were increased in HFD mice and further increased in the H-Cd/HFD group.

Fig. 4.

Phosphorylation of P38/P38 and inflammatory protein expression levels in female mice hearts when exposed to 5 ppm cadmium and high-fat diet (HFD) feeding. (A) Western blotting (WB) for phosphorylation-P38/P38 and ICAM, TNF-α, PAI-1, and IL-1β. (B) WB statistical analysis of the ratio of phosphorylation-P38 to P38. (C) WB statistical analysis of ICAM. (D) WB statistical analysis of TNF-α. (E) WB statistical analysis of PAI-1. (F) WB statistical analysis of IL-1β. (yellow, female control [Ctr] group; yellow with grid, female HFD group; orange, female 5 ppm cadmium group; orange with grid, female 5 ppm cadmium/HFD group; n = 5; *p < 0.05 versus [vs.] Ctr/normal fat diet [ND] group; ^&p < 0.05 vs. Ctr/HFD group). H-Cd, 5 ppm Cd.

3.3.4. H-Cd exposure exacerbated HFD-induced cardiac oxidative stress and alleviated the antioxidant defense

HFD-fed female mice displayed an increased MDA level (Fig. 5A) and 3-NT (Fig. 5B, C) protein expression in the hearts, suggesting that HFD induced cardiac oxidative damage, were further exacerbated in H-Cd/HFD mice. CAT protein expression increased in the Ctr/HFD and H-Cd/HFD groups, but there was no synergistic effect of H-Cd and HFD (Fig. 5D). Additionally, there was no change of SOD2 protein expression in any of the groups (Fig. 5D). MT is an important free radical scavenger and its expression at the protein and mRNA level was markedly decreased in H-Cd/HFD mice compared with HFD mice (Fig. 5E, F). These results suggest that there is an imbalance between oxidative stress and anti-oxidative capacity in the hearts of H-Cd/HFD mice.

Fig. 5.

Oxidative stress and antioxidant levels in female offspring hearts when exposed to 5 ppm cadmium and high-fat diet (HFD) feeding. (A) Cardiac tissue malondialdehyde (MDA) level in female mice. (B) Immunohistochemistry staining of 3-nitrotyrosine (3-NT) results in female mice hearts. (C) 3-NT protein expression. (D) Protein expression of the antioxidants catalase (CAT) and superoxide dismutase-2 (SOD2). (E) Cardiac MT2 mRNA expression level. (F) Cardiac metallothionein (MT) protein expression. (yellow, female control [Crt] group; yellow with grid, female high-fat diet [HFD] group; orange, female 5 ppm cadmium group; orange with grid, female 5 ppm cadmium/HFD group; n = 5; *p < 0.05 versus [vs.] Ctr/normal fat diet [ND] group; ^&p < 0.05 vs. Ctr/HFD group). H-Cd, 5 ppm cadmium.

3.3.5. Zn transporter mRNA expression changes

As shown in Fig. 6A, MT also plays a key role in storing intracellular Zn by binding and releasing Zn under reductive and oxidative states (or stress), respectively (Kassim et al., 2013; Krężel and Maret, 2021). The intracellular Zn level is regulated by two kinds of Zn transporters that are either able to import or export Zn in/out of the cell. However, Cd can compete with Zn for transporters (Pabis et al., 2018). Therefore, we examined whether what we observed — the Zn level was significantly decreased and Cd level was markedly increased in H-Cd/HFD mice compared with Ctr/HFD mice (Fig. 1B, Supplemental Fig. 1) — associated with these transporters. mRNA expression levels of the Zn transporters in the heart of female mice examined by qRT-PCR measurement. The results displayed that mRNA expression of ZIP8 and ZIP14, which are involved in both Zn and Cd uptake, tended to increase or increase in the H-Cd group (Fig. 6B, C), and markedly increased in H-Cd/HFD mice compared with the Ctr/HFD group (Fig. 6B, C). Regarding other Zn transporters, the mRNA expression level of ZnT1 was also increased in H-Cd/ND and further increased in the H-Cd/HFD group (Fig. 6D). ZnT2 mRNA expression did not significantly change between the treated groups (Fig. 6E) whereas the mRNA expression of DMT1, which mainly transports toxic divalent metal ions, was increased only in the hearts of H-Cd/HFD female mice (Fig. 6F). Giving the fact that both ZIP8 and ZIP14 significantly and even DMT1 trendily increased, all of which are transporters to potentially increase Cd intracellular uptake, while there is only ZnT1 as one of transporters that potentially export Cd outside of cells, these changes may explain the reason why cardiac accumulation of Cd was significantly increased in the groups of mice with both Cd/HFD.

Fig. 6.

Zn transporter mRNA expression in female mice hearts when exposed to 5 ppm cadmium and high-fat diet (HFD) feeding. (A) Brief sketch (Zn transporters that also possibly transport Cd). (B) Statistic analysis of ZIP8 mRNA expression. (C) Statistic analysis of ZIP14 mRNA expression. (D) Statistic analysis of ZnT1 mRNA expression. (E) Statistic analysis of ZnT2 mRNA expression. (F) Statistic analysis of DMT1 mRNA expression. (yellow, female control [Ctr] group; yellow with grid, female HFD group; orange, female 5 ppm Cd group; orange with grid, female 5 ppm Cd/HFD group; n = 5 in ZIP8, ZIP14, ZnT1, and DMT1 analyses. For ZnT2 analysis, 5 samples were not detected in mRNA expression. N = 3 in Ctr group, n = 3 in HFD group, n = 4 in H-Cd group, n = 5 in H-Cd/HFD group. *p < 0.05 versus [vs.] Ctr/normal fat diet [ND] group; ^&p < 0.05 vs. Ctr/HFD group). H-Cd, 5 ppm cadmium.

3.4. Pathophysiological changes in male hearts

In male mice, the ratio of heart weight to tibial length was increased in the HFD and H-Cd/HFD groups (Fig. 7A). Sirius red staining revealed that HFD induced more myocardial interstitial collagen accumulation in male mice (Fig. 7B), along with a higher degree of pro-fibrosis proteins (FN, COL1A1, Fig. 7C), compared with Ctr/ND group, which was different from that in the HFD-fed female mice (Fig. 2). However, no statistically significant differences were observed in the protein expressions of collagen or FN between the H-Cd/HFD and Ctr/HFD groups (Fig. 7C). Cardiomyocyte size measured by WGA staining (Fig. 7D) and mRNA expression of the hypertrophic gene β-MHC were also increased in HFD and H-Cd/HFD male mice (Fig. 7F). ANP mRNA expression showed an increasing trend in the HFD group and significantly increased in the H-Cd/HFD group (Fig. 7E). However, no difference was observed in cardiomyocyte size and β-MHC mRNA expression between the H-Cd/HFD and Ctr/HFD groups (Fig. 7D, F). Overall, in male mice, the synergistic detrimental effects of Cd and HFD were not obvious, as seen in female mice.

Fig. 7.

Sirius red staining, pro-fibrosis markers, and cardiac hypertrophic markers in male offspring hearts when exposed to 5 ppm cadmium and high-fat diet (HFD). (A) The ratio results of heart weight to tibial length in male mice. (B) Sirius red staining analysis in male mice hearts. (C) Pro-fibrosis markers (fibronectin and collagen1A1) protein expression in male mice hearts. (D) Cardiomyocyte size showed by wheat germ agglutinin (WGA) staining. (E) mRNA expression of ANP. (F) mRNA expression of β-MHC. (blue, male control [Ctr] group; blue with grid, male HFD group; pink, male 5 ppm cadmium group; pink with grid, male 5 ppm cadmium/HFD group; n = 4; *p < 0.05 versus Ctr/normal fat diet [ND] group).

4. Discussion

In the current study, we used a model of HFD feeding combined with whole-life low-dose Cd exposure to investigate the effects on the hearts of adult male and female mice. We showed that there was a dose-dependent Cd accumulation in the hearts of both male and female mice. Exposure to 5 ppm Cd with HFD induced cardiac trace element dyshomeostasis only in female mice. Furthermore, there was an obvious synergistic effect of Cd exposure and HFD feeding on female hearts, as shown by cardiac dysfunction and structural remodeling along with increased inflammatory response and oxidative stress decreased MT protein expression level and activated P38 MAPK function.

Our previous studies have shown that feeding HFD post-weaning feeding (60% kcal fat) for 3 and 6 months induced cardiac hypertrophy in mice (Wang et al., 2017; Wang et al., 2016). In the present study, we showed that HFD feeding for 24 weeks induced hypertrophy in the hearts of both female and male mice, but only induced diastolic dysfunction in male mice. Here, we detected cardiac diastolic function that was different from our previous study, which only detected cardiac systolic function. This was the first sex difference in our study; HFD feeding induced cardiac diastolic dysfunction only in male mice. This obesity-induced sex difference result was consistent with that of previous studies (Böhm et al., 2013; Louwe et al., 2012). A possible explanation for this sex difference in HFD-obesity-induced cardiac dysfunction is the difference in the susceptibility to obesity. In our previous study, low-dose Cd exposure for 4 weeks induced cardiac remodeling in mice (Turdi et al., 2013). However, in the present study, we did not observe cardiac remodeling in response to Cd exposure in either male or female mice. This could be because we used different doses and exposure routes: we provided Cd exposure with drinking water in the present study, and not by intraperitoneal injection as before (Turdi et al., 2013). Our previous research reported that there was a sex difference in the synergistic cardiac remodeling effect when exposed to 5 ppm Cd combined with HFD feeding (42% kcal fat) for 10 weeks (Liang et al., 2019). Our data indicate that in female mice hearts, exposure to 5 ppm Cd combined with HFD for 24 weeks, has a stark synergistic effect, along with cardiac systolic and diastolic dysfunction and remodeling. However, the synergistic effect was not so obvious in male mice which is consistent with our previous finding (Liang et al., 2019). Therefore, we further investigated and discussed the mechanisms of HFD feeding and 5 ppm Cd exposure in damaging the hearts of female mice.

Zn is an essential nutrient for living organisms because of its key role in the proper function and structure of more than 2700 enzymes (Choi et al., 2018). Zn deficiency is associated with cardiac hypertrophy and heart failure probably due to the increased inflammatory response and oxidative stress (Cao et al., 2020; Malekahmadi et al., 2020; Rosenblum et al., 2020a, 2020b; Yu et al., 2018). Similarly, Cu deficiency is also associated with these cardiac pathologies (Malekahmadi et al., 2020; Mandour et al., 2021; Medeiros, 2017; Olivares et al., 2019; Thatipaka et al., 2020). Herein, we found that the Zn and Cu concentrations decreased and the Ca concentration showed a trend decrease in H-Cd/HFD female mice hearts when compared with HFD-fed female mice hearts. Furthermore, H-Cd/HFD induced cardiac dysfunction (decreased shortening fraction and ejection fraction) and remodeling (including increased chamber dilation, and increased FN and COL1A1). These changes might be attributed to the synergistic effect of H-Cd and HFD, and the induced effects of Zn and Cu deficiencies on female hearts, which were different from those in male mice. Moreover, it is well established that contraction and diastole of the heart are controlled by changes in the cytoplasmic Ca concentration (Eisner, 2018). Therefore, the decreased Ca concentration in heart tissue might also be associated with cardiac dysfunction in female mice hearts.

To date, no specific Cd transporters have been confirmed; Cd has similar chemical properties to Zn and can compete with Zn for transporters to enter cells (Limaye and Shaikh, 1999; Pabis et al., 2018; C. Yu et al., 2021; H.T. Yu et al., 2021). Two carrier families, 30 (SLC30; ZnT) and 39 (SLC39; ZIP) transport proteins are involved in maintaining intracellular Zn homeostasis. ZIP transporters import extracellular Zn into the cytoplasm or release vesicular Zn into cytosol while ZnT transporters export Zn from cells or promote Zn uptake into vesicles (Schweigel-Röntgen, 2014). Both ZIP8 and ZIP14 transporters can transport Zn and Cd into cells (C. Yu et al., 2021; H.T. Yu et al., 2021), and their mRNA expression levels were significantly increased in female H-Cd/HFD mice. In addition, the ZnT1 transporter was reported to take part in Zn and Cd transport (Ohana et al., 2006), and in our study, ZnT1 mRNA expression was also induced by 5 ppm Cd exposure in the hearts of female mice when combined with HFD feeding. Moreover, DMT1 mediates the transport of several metal ions, including iron, Cd, Zn, and manganese (Illing et al., 2012). We detected the mRNA expression of DMT1, which was also induced in H-Cd/HFD female mice hearts. As the consequence, the heart metal concentration results indicated that the Zn/Cd and Cu/Cd ratios decreased in the H-Cd/HFD group compared with the HFD group. Therefore, Cd exposure with HFD resulting in these metal transporter alterations may attribute to the essential metal dyshomeostasis, leading to chronic inflammation and oxidative stress in the hearts of H-Cd/HFD mice, as illustrated in Fig. 8.

Fig. 8.

Schematic representation of 5 ppm cadmium (H-Cd) exposure, high-fat diet (HFD), and H-Cd/HFD effects on the female mice hearts. (left) Whole-life H-Cd exposure caused mRNA expression of ZIP14 and ZnT1 to increase and ZIP8 tend to increase, which might attribute to essential metal dyshomeostasis, including concentrations of zinc (Zn), copper (Cu), calcium (Ca) trended decrease, and ratios of Zn/Cd and Cu/Cd decreased. There was no significant cardiac remodeling but trended slight decrease in cardiac ejection, which might be associated with decreased Ca concentration in the female mice hearts. (right) Postweaning HFD induced oxidative stress and P38 MAPK activation, leading to increased cardiac inflammation as well as activation of p38 MAPK-dependent hypertrophic signaling. Both chronic inflammation and p38 MAPK-dependent hypertrophic signaling contribute to cardiac hypertrophic and/or fibrotic responses with mild (not significant yet) cardiac dysfunction. (middle) H-Cd exposure combined with HFD have significant synergistic effects on the heart, by increasing oxidative stress, P38 MAPK activation, and cardiac inflammation, leading to significantly cardiac remodeling (hypertrophic and/or fibrotic response), and combined the essential metal dyshomeostasis-mediated decrease in cardiac contractibility, resulting in the significant cardiac dysfunction in female mice. ZIP8/14, ZnT1 and DMT1 expression increase might also be associated with cardiac dysfunction.

Our previous study confirmed that Zn deficiency exacerbated obesity-related cardiac hypertrophy by increasing P38 MAPK pathway activation (Wang et al., 2016). The P38 MAPK pathway as one of the MAPKs is responsive to various stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock. Persistent activation of the p38 MAPK pathway turned on cardiac inflammation and pathological remodeling signaling pathways (Romero-Becerra et al., 2020; Zhang and Cai, 2020). For these effects, the P38 MAPK pathway can activate its downstream transcription factors such as NF-κB, which can bind to the promoter regions of genes encoding inflammatory cytokines, including mRNA or/and protein expression of TNFα, IL-1β, and IL-6 and regulated IL-8 expression, and also MEF2 and GATA4, resulting in up-regulation of cardiac hypertrophy, cardiomyopathy, and remodeling (Nakamura and Sadoshima, 2018; Turner and Blythe, 2019; Romero-Becerra et al., 2020; Zhang and Cai, 2020). In the current study, we observed that HFD-fed alone could activate the P38 MAPK pathway, but 5 ppm Cd exposure alone did not; however, exposure to 5 ppm Cd significantly increased HFD-induced activation of the P38 MAPK pathway and the associated cardiac inflammation and hypertrophy (increased cardiomyocyte sizes, expression of hypertrophic transcription factors GATA4 and MEF2 as well as their downstream hypertrophic genes ANP and β-MHC) in H-Cd/HFD female mice compared with HFD female mice alone. These synergistic effects may be attributed to Zn deficiency (Fig. 8), as it was previously reported that Zn deficiency exacerbated HFD cardiac effects (Wang et al., 2016).

Oxidative stress plays an important role in cardiac pathogenesis induced by obesity or HFD (Jeong et al., 2016; Muthulakshmi and Saravanan, 2013). Oxidative stress and damage occur because cells either generate excess ROS and RNS or lose their anti-oxidative capacity. Reportedly oxidative stress can occur at the condition of either Cd exposure or Zn deficiency (Cao et al., 2020; Ferramola et al., 2012; C. Yu et al., 2021; H.T. Yu et al., 2021), both are seen in the present model. MT is a family of cysteine-rich proteins with about 1/3 amino acid as cysteine and localized to the membrane of the Golgi apparatus (Cai, 2006), To date, MT remains undiscovered by any enzyme function and direct involvement of any signaling pathway; however MT’s high content of cysteine makes it able (1) to bind both physiological (such as zinc, copper, selenium) and xenobiotic (such as cadmium, mercury, silver, arsenic) heavy metals through the thiol group of its cysteine residues, and (2) to scavenge various free radicals, including superoxide, hydroxyl radicals, hydrogen peroxide, and peroxynitrite (Thornalley and Vasák, 1985; Quesada et al., 1996; Cai et al., 2000; Cai, 2006). Previous studies from the authors and others reported that cardiac-specific transgenic overexpression of MT of mice displayed a protective role in HFD-induced dysfunction and Zn deficiency exacerbated HFD (6 months)-induced hypertrophy by decreasing MT and increased oxidative stress (Dong et al., 2007; Wang et al., 2017; Wang et al., 2016). Consistent with these results, the present study showed that MT protein and mRNA expressions were markedly decreased in the HFD/H-Cd group compared to the control or HFD groups, suggesting that its antioxidant capacity was reduced. Although CAT was increased in both the HFD and H-Cd/HFD groups, CAT can only catalyze the decomposition of hydrogen peroxide to water, while MT can scavenge various ROS and RNS (Cai, 2006). Therefore, decreased MT expression in the heart of H-Cd/HFD female mice may explain the induction of oxidative stress and damage.

Furthermore, environmental pollution has been paid more and more attention now. Fossil fuel combustion induced air pollution including Cd released, therefore waste to energy (WtE) has been viewed as an economical and sustainable way for energy recovery with reduced air pollution (Rasheed et al., 2021a). In addition, carbon nanotubes and magnetic solid-phase extraction may be promising absorption materials to remove various environmental pollutants (Rasheed et al., 2020a; Ali et al., 2021). The main sources of toxic and dangerous pollutants are heavy metals and drug residues, and industrial effluents sources include pharmaceutical effluents, fertilizers, foods and dairy processing wastes and others (Ali et al., 2021; Rashid et al., 2021). Regarding pharmaceutical pollutants properties, nanomaterials or other materials are also considered for removing the contaminants (Rasheed et al., 2021a, 2021b; Teixeira et al., 2021).

5. Conclusions

In brief, this study is the first to use a whole-life low-dose Cd exposure and post-weaning HFD (60% kcal fat) induced-obesity mouse model to investigate the sex-different effects on the heart of the offspring at 27 weeks old. We found a dose-dependent cadmium accumulation in the hearts of male and female mice along with decreased cardiac zinc and copper levels only in female offspring. Exposure to 5 ppm cadmium significantly enhanced HFD cardiac effects in females, but not in males, shown by worsened cardiac systolic and diastolic dysfunction, increased cardiac fibrosis, hypertrophy, and inflammation, compared to the HFD group alone. Activation of the P38 MAPK signaling pathway and down-regulation of the antioxidant MT appears to be the underlying mechanism for the observed outcomes in the hearts of female mice. These discoveries may provide new therapeutic perspectives for Cd exposure combined with obesity-induced cardiomyopathy. Although environmental dose Cd exposure did not induce significant cardiac dysfunction both in male and female mice but when combined with HFD induced cardiac dysfunction. For environmental dose Cd exposure, either from wastewater or air pollution, newly-developed methods to reduced associated environmental problems will make meaningful and beneficial improvements for human health globally.

HIGHLIGHTS.

• Cardiac effects of whole-life exposure to low-dose cadmium in male and female mice

• Dose-dependent outcomes of whole-life cadmium exposure for the heart of offspring

• Cadmium exposure affects postweaning high-fat diet (HFD) induced cardiac dysfunction

• Cadmium exposure with HFD induced cardiac trace metal dyshomeostasis in female mice

• Synergistic effects on P38 MAPK pathway in the mice with both high cadmium and HFD