3.1 Daily drinking water and Cd intake
The daily drinking water ingestion rates of four groups of mice are shown in Fig. 1A. As shown in Fig. 1A, the water ingestion rates for blank control and Cd control mice (normal mice) looked stable during 25 weeks with a mean of 0.115 mL/g body weight/day, whose water ingestion rates showed a slight decrease in first 12 weeks and kept stable in following time. However, the water ingestion rates for diabetic control and experimental mice (diabetic mice) were not stable. For diabetic control mice, the water ingestion rate was slightly decreased in first four weeks (from 0.1 mL/g body weight/day to 0.077 mL/g body weight/day), and increased from 0.077 mL/g body weight/day to 0.32 mL/g body weight/day during 4-12nd weeks, and kept stable in the last 12 weeks. For experimental mice, the regulation is same to diabetic control mice in the early exposure time, whose water ingestion rate also showed a slight decrease in the first 4 weeks, and had a great increase from 4nd to 12nd weeks. However, unlike diabetic control mice, water ingestion rate of experimental mice showed an obvious decrease in the last time (12–24 weeks), indicating Cd exposure decreased the drinking water ingestion of diabetic mice. However, this phenomenon not occurred in normal mice, meaning diabetic mice might be more sensitive than normal mice during Cd exposure. In addition, the average water ingestion rate of diabetic mice was higher than normal mice. There are several reasons for above phenomenon. The Cd exposure level is calculated by daily water consumption and body weight. Thus, first reason is diabetic mice tend to drink more water than normal mice, and second is that diabetic mice have lower body weight. At the beginning of the study, the body weight had no significant difference between Cd control and experimental group mice. After injection of STZ, the weight of diabetic mice gradually decreased. Finally, for the reason of decreased cadmium exposure in the last time is still unclear, previous study found the similar phenomenon that the average intake of the cadmium in the early phase of treatment was higher than that in the subsequent[30].
For daily Cd exposure, blank and diabetic control mice were negligible due to without Cd in drinking water. For Cd control and experimental mice, the regulation of daily Cd exposure level is same to drinking water ingestion rate. The daily Cd exposure levels of Cd control and experimental group were 1.74–2.45 and 1.37–3.58 mg/kg body weight/day respectively, the level has been translated from mice to human being.
3.2 Cd aggravated the diabetes-induced injury of cardiac function
Defective cardiac contractility is one of the characteristic abnormalities of diabetic cardiomyopathy. Echocardiographic imaging is used to assess the cardiac physiology and architecture. It could effectively calculate the EF% and then detected cardiac contractility, which is closely related to cardiac function [31, 32]. Hence, we used echocardiographic imaging to examine the impact of chronic Cd exposure on heart function. As shown in Fig. 2 and Table 2, the EF% and FS% of mice in experimental, diabetic control and Cd control group were significantly lower than blank control (P < 0.05), and the lowest value occurred in experimental group. Due to diabetic cardiomyopathy is consist of two major components: (1) physiological adaptation to metabolic alterations shortly; (2) the capacity of myocardium for repair is limited and lead to degenerative changes [33, 34]. In early stage of DCM, overt functional abnormalities were not happened, and ejection fraction was normal in myocytes. With the development of disease, myocyte apoptosis and necrosis were increased, resulting in myocyte injury and myocardial fibrosis, which caused a slight decrease of EF%, while the obvious changes in cardiac structure and function will gradually show after that[33].
Table 2
Echocardiographic assessment of left ventricle structural and functional data in mice
|
Blank Control
|
Cd Control
|
DM Control
|
Experiment
|
EF(%)
FS(%)
LVID;d(mm)
LVID;s(mm)
LVAW;d(mm)
LVAW;s(mm)
LVPW;d(mm)
LVPW;s(mm)
LV Vol;d(µL)
LV Vol;s(µL)
LV mass(mg)
LVM/BW
|
57.39 ± 3.76
29.59 ± 2.46
3.70 ± 0.27
2.61 ± 0.22
0.74 ± 0.09
0.99 ± 0.07
0.74 ± 0.08
0.98 ± 0.14
58.48 ± 10.00
25.00 ± 5.47
75.02 ± 14.08
0.00248
|
46.84 ± 5.35 **
23.13 ± 3.13 **
3.96 ± 0.39
3.04 ± 0.36 *
0.76 ± 0.15
0.95 ± 0.15
0.74 ± 0.09
0.96 ± 0.11
69.07 ± 15.37
36.97 ± 10.02 *
85.16 ± 21.30
0.00270
|
50.17 ± 6.17 **
25.19 ± 3.78 **
3.94 ± 0.22
2.95 ± 0.24*
0.68 ± 0.10
0.90 ± 0.11
0.76 ± 0.05
1.02 ± 0.06
67.70 ± 9.38
33.80 ± 6.68*
80.26 ± 9.94
0.00311
|
44.46 ± 6.27 ***
21.65 ± 3.63 ***
3.73 ± 0.34
2.93 ± 0.32*
0.66 ± 0.09
0.82 ± 0.09 *&
0.61 ± 0.06 ***&&&$$$
0.84 ± 0.05* &$$$
59.97 ± 13.02
33.48 ± 9.08*
62.05 ± 8.32 &&$
0.00276
|
Data are presented as the mean ± SD. EF: ejection fraction; FS: fractional shortening; LV: left ventricular; LVIDd: LV internal diastolic diameter; LVIDs: LV internal systolic diameter; LVAW;d: End-diastole LV anterior wall thickness; LVAWs: End-systolic LV anterior wall thickness; LVPWd: End-diastole LV posterior wall thickness; LVPWs: End-systolic LV posterior wall thickness; LV Vold: LV end-diastolic volume; LV Vols: LV end-systolic volume; LVM: LV mass; BW: body weight. “*, ** and ***” meaning the P value lower than 0.05, 0.01 and 0.001 respectively (compared with blank control). &P < 0.05, &&P < 0.01, &&&P < 0.001 (compared with Cd control); $P < 0.05, $$P < 0.01, $$$P < 0.001 (compared with group diabetic mice (DM) control). |
In addition, echocardiographic data showed that the left ventricular wall thickness had no significant differences among four groups. However, Cd and diabetes increased LV mass (LVM)/body weight (BW) ratio of mice. The lower ejection fraction and higher LVM/BW in Cd control and diabetic control group indicated that the heart was overburdened and the myocardium compensates were enlarged and hypertrophied to overcome the increased resistance, which ensure the ejection volume and maintain the normal cardiac output for a long time[35]. Meanwhile, compared with diabetic control group, the LVM/BW ratio and EF% of mice was decreased in experimental group (p < 0.05) which means the metabolic alteration is overburdened and Cd aggravates cardiac dysfunction and exceeds stage of DCM in diabetic mice.
3.3 Cd increased the level of fibrosis and cardiac injury in diabetic mice
To explore the mechanism of Cd-induced cardiac dysfunction, HE staining, the cTnT, BNP, ANP and β-MHC of mice were measured. In general, for normal mice, Cd increased the mRNA levels of ANP and BNP and the serum concentrations of cTnT (Fig. 3). As shown in Fig. 3, the cTnT content of experimental group mice was two times higher than diabetic control group mice, but the mRNA expression level of BNP and β-MHC were decreased (Fig. 3). cTnT is a specific marker for cardiac injury and it is regarded as more sensitive and specific than other cardiac biomarkers[36]. After myocardial injury, cTnT is released from death cell within 2–4 h and remained in the blood stream probably more than 10 days[37]. In this study, we found that Cd increased the cTnT concentration in diabetic and normal mice, which suggested that Cd could damage the myocardium and aggravated the cardiac injury in diabetic mice. HE staining showed the degree of myocardial tissue impairment in mice models (Fig. 4A), and the myocardial cells were disordered and destroyed in Cd control and DM control group. For experimental group, the morphology of cardiomyocytes was further damaged and there was obvious rupture.
β-MHC and BNP are two significant hypertrophy markers. Once cardiomyocytes become stretched in response to mechanical strain, BNP would be largely synthesized and secreted by ventricular myocytes, which associated with overloaded pressure and ventricular volume expansion [38]. And in current guidelines, the increase of plasma BNP level is in proportion to disease severity in patients with heart failure and cardiac dysfunction which is different with the results in present study[39, 40]. For this, we supposed two possible reasons. At first, fibrosis could deposit extra cellular matrix (ECM) and leads to stiffness of ventricular wall, which blocked the stretching of cardiomyocytes [41]. In this study, the myocardial sections were dyed with Masson staining and the myocardial collagen fibers were stained blue, and the myocardial fibers were stained red. As shown in Fig. 4B, Cd and DM deposited amounts of collagen when compared with blank group. For diabetic mice, cadmium disordered the arrangement of myocardial fibers and accelerated the fibrosis on myocardial fibers. Furthermore, Cd and diabetes increased the level of TGF-β1, while mRNA level of FN almost unchanged. And the mRNA expressions of TGF-β and FN in experimental mice were approximately two times higher than that in diabetic control mice. This phenomenon might be due to that with the development of cardiopathy, accumulated fibrosis inhibited cardiomyocytes stretching, and then blocked the secretion of BNP. Besides this conjecture, previous study suggested that the mRNA expression levels of ANP, BNP and β-MHC were different in atrium and ventricle[42]. This is a limitation of this study, we did not distinguish the cardiac structural clearly based on the measured mRNA expression of ANP, BNP and β-MHC. Further investigation is required for elucidating the significant of ANP, BNP and β-MHC mRNA levels in cardiomyopathy.
3.4 Cd increased the level of inflammatory cytokine in diabetic mice.
With the development of cardiac disease, the inflammatory cytokine response would be activated and result in continuous deleterious effects on the heart and vasculature, finally lead to the progression of cardiac dysfunction and heart failure[43]. Thus, the mRNA levels of TNF-α (tumor necrosis factor-alpha), IL-1 (Interleukin-1) and MCP-1 (monocyte chemotactic protein 1) were detected to investigate the effect of inflammatory cytokine on cardiac injury. MCP-1 plays a critical role in heart disease, it can recruit peripheral leucocytes to tissues and result in the development of chronic inflammation[43]. TNF-α could be produced by cardiac cells while they are in the situation of pressure and volume overload, which contributes to the progressive LV wall thinning and adverse cardiac remodeling[44]. In addition, TNF-α can increase the production of IL-1 and they exacerbates cardiac myocyte contractile dysfunction[44, 45]. As showed in Fig. 5, for normal mice, the expressions of TNF-α and IL-1 were slightly increased, and the expression of MCP-1 was increased after Cd exposure. For diabetic mice, the mRNA expressions of TNF-α, IL-1 and MCP-1 in experimental group were two-three times higher than the diabetic control mice. Inflammation is one of the earliest events in cardiac stress situations and involved in myocardial remodeling[46]. Ventricular remodeling contributes to ventricular dilation and dysfunction, which includes myocyte hypertrophy and extracellular matrix remodeling[47]. The constant remodeling of extracellular matrix is regulated by matrix metalloproteinases, which controlled by cytokines including TNF-α and IL-1β[48]. As Fig. 5 showed, the diabetic mice were more sensitive than normal mice when exposed to Cd and showed severer inflammation and extracellular matrix remodeling (Fig. 4B). Additional, MCP-1 plays a causative role in experimental diabetic cardiopathy, and the heart failure was attenuated in MCP-1 deficient animal models [49–51]. These results suggested that Cd could promote myocardial inflammatory processes in diabetic mice, finally contributes to the adverse ventricular remodeling.