Selenium-Rich Diet Induces Myocardial Structural and Functional Abnormalities by Activating Caspase-9 and Caspase-3 in Gpx-1P198L-Overexpression Transgenic Mice.

BACKGROUND Selenium (Se) deficiency and supplementation result in multiple effects. GPx-1 (Pro198Leu) polymorphism is associated with Se deficiency. This study aimed to observe associations between Se-deficiency/supplement and GPx-1-198Leu overexpression in myocardial injuries. MATERIAL AND METHODS GPx-1P198L transgenic (Tg) mice and non-transgenic wild-type (WT) littermates were divided into Control (CON, 0.1-0.2 mg/kg), Se-deficiency (SD, <0.02 mg/kg), and Se-supplement (SS, 0.4 mg/kg) groups. Cardiac functions were observed with animal M-mode echocardiography. Se level was measured using 2,3-diamino Kenai fluorospectrophotometry. Total cardiac GPx activity was also measured. Myocardial histopathology was determined with HE and Masson's trichrome staining. Caspase-9 and caspase-3 were measured with Western blot analysis. RESULTS In WT Se-deficient mice, cardiac GPx activity was significantly decreased, and was not elevated by overexpression of GPx-1-198Leu gene. Increased GPx activity was observed in WT Se-supplemented mice and Tg Se-supplemented mice (much more). Se deficiency as well as supplementation resulted in cardiac systolic dysfunction, which was not affected by GPx-1-198Leu gene. Se deficiency led to myocardial fibrosis and pathological changes accompanied by increased activation of caspase-9 and caspase-3. Se supplementation significantly reduced pathological changes, as well as caspase-9 and caspase-3 levels in the presence of increased myocardial fibrosis. In Se-deficient mice, GPx-1-198Leu overexpression did not significantly decrease myocardial pathological injuries and fibrosis. In Se-supplemented Tg mice, myocardial fibrosis and caspase-9 level were increased, although pathological injuries and caspase-3 were similar to that in Se-supplemented WT mice. CONCLUSIONS Se deficiency as well as supplementation induced myocardial structural and functional abnormalities through activation of caspase-9 and caspase-3 in GPx-1P198L overexpression transgenic mice.

Background

Selenium (Se) is well established as an essential trace element that plays critical roles in chemo-protection against oxidative stress, but high concentrations of Se induce toxic and oxidative effects [1-3]. In fact, different levels of Se have different effects. It has been reported that Se at higher concentrations decreases cell proliferation and causes apoptosis in A20 and NB4 cells [4,5]. Dietary Se deficiency is considered to be a causative factor of the endemic cardiomyopathy prevalent in China, termed Keshan disease (KD) [6]. Lower blood Se levels are observed in patients with reduced left ventricular systolic heart failure [7,8]. Metes-Kosik et al. found that both Se deficiency and modest Se supplementation resulted in a similar phenotype of abnormal myocardial matrix remodeling and dysfunction in the normal heart [9]. Therefore, appropriate amounts of Se are required for optimal body health.

Mitochondria play an important role in apoptosis under various proapoptotic conditions. If mitochondria are exposed to proapoptotic signals, such as reactive oxygen species (ROS), mitochondrial cytochrome c is released into cytosol, binds to Apaf-1, and then participates in caspase-9 activation. The activated capase-9 consequently activates caspase-3, which in turn activates a caspase-activated DNase and leads to DNA degradation, a hallmark of apoptosis [10]. However, ROS production is part of the normal cellular metabolism and may function as signaling molecules necessary for cell growth and survival [11,12]. Accumulating evidence has indicated that ROS such as superoxide anion and hydrogen peroxide may be related to cell death and play an important role in a variety of oxidative stress-induced disorders [13-15]. GPx-1 an crucial antioxidant because it is located in both the cytosol and the mitochondrial matrix and it can reduce lipid peroxides and hydrogen peroxide into corresponding alcohols and water. As a Se-dependent enzyme, Gpx-1 activity decreases dramatically in Se deficiency and increases during Se supplementation. GPx-1 activity is also associated with GPx-1 (Pro198Leu) polymorphism and Se deficiency [16]. The Leu allele was found to be less responsive to increased Se levels than the Pro allele [17]. Moreover, a variety of evidence has indicated that the GPx-1 (Pro198Leu) polymorphism is associated with many oxidative stress-related diseases, such as endemic cardiomyopathy, cancers, and diabetic complications [16,18,19].

Overexpression of GPx-1 can ameliorate myocardial remodeling and failure in diabetic mice, as well as in mice with myocardial infarction, via prevention of oxidative stress [20,21]. However, growing evidence has indicated that increased GPx-1 activity might be related to hyperglycemia, hyperinsulinemia, insulin resistance, and obesity observed in Gpx1-overexpressing mice [22,23]. Thus, the aim of the present study was to investigate the effects of different Se diets on cardiac structure and function, as well as activation of caspase-9 and -3, in mice overexpressing the human GPx-1-198leu gene.

Material and Methods

Transgenic mice

Our study was approved by the Institutional Animal Research Committee and conformed to the animal care guidelines of the Medical College of Xi’an Jiaotong University. Human mutated GPx-1-198Leu mRNA was recombined according to NCBI reference sequence NM_000581.2 and then inserted downstream of the human cytomegalovirus (CMV) immediate early promoter of plasmid pEGFP-N3. Part of GFP sequence was cut so that hGPx-1-198Leu could successfully and effectively be expressed. Identification of the transgenic vector was performed by PCR and sequencing. After linearization with AseI and AfIII, the transgenic piece including hGPx-1-198Leu, CMV promoter, and SV40 polyA was microinjected into fertilized eggs derived from C57BL/6J mice. The GPx-1-198Leu-overexpressing mice were designated as GPx-1P198L transgenic mice. The genotype of GPx-1P198L transgenic mice was detected by PCR, and hGPx-1-198Leu expression levels in the hearts of transgenic mice was determined by Western blot analysis. Four lines of transgenic mice carrying extra copies of the human GPx-1-198Leu gene were generated.

Diets and treatments

Twenty-seven 3-week old weanling transgenic mice were used and were randomly divided into 3 groups. Nine mice within each group (X 4, Y 5) were fed a Se-deficient diet (SD, <0.02 mg Se/kg) [24] with all the other nutrients at the standard level. The second group (X 4, Y 5) was fed a Se-supplemented diet (SS, the SD diet supplemented with sodium selenite 0.4 mg Se/kg) [24]. The last group (X 4, Y 5) was fed a standard diet as a control (CON, with a content of 0.1–0.2 mg Se/kg). Twenty-seven weaning non-transgenic wild-type C57BL/6J littermates (X 4, Y 5 for each group) were treated the same as transgenic mice. All animals were individually housed in an SPF animal room with a constant temperature (22°C) and a 12: 12 h light-to-dark cycle and were given free access to distilled water and chow for 12 weeks. The standard diet was supplied by the Animal Experimental Center of Xi’an Jiaotong University and the SD and SS diets were purchased from Trophic Animal Feed Hightech Co. (Jiangsu, China) according to the AIN-93M formula [25,26].

Echocardiographic measurements of cardiac function

For measurements of cardiac function, echocardiographic studies were performed in 6 anesthetized mice in each group with isoflurane 2–2.5 ml/min and spontaneous respiration. Parameters of interventricular septum wall thickness (IVSs, IVSd), left ventricular posterior wall thickness (LVPWs, LVPWd), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), ejection fraction (EF), and fractional shortening (FS) were evaluated using a M-mode echocardiography (Vevo2100, Canada).

Serum Se

After detection of cardiac function, whole blood from the 6 animals per group was collected for the determination of serum Se, which was analyzed by a flameless atomic absorption spectrophotometry method using a Z-5000 spectrophotometer (Hitachi, Japan) with a Se cathode lamp (resonance line, 196.0 nm, Photron, Australia).

Sample collection

After the above studies, the hearts were excised and the LV was immediately fixed in 10% formalin solution for 24 h, then embedded in paraffin. We cut 4-μm sections and stained them with hematoxylin and eosin for myocardial histopathology observation, as well as staining with Masson’s trichrome for collagen distribution determination. The remaining 3 mouse hearts were also excised, cut into 2 parts each, and stored at −80°C for protein extraction.

Myocardial GPx activity

From the samples stored at −80°C, one part was homogenized in physiological saline on ice, centrifuged at 3000 rpm for 15 min, and supernatant was recollected. Myocardial GPx activity was spectrophotometrically measured using a cellular glutathione peroxidase activity assay kit (Nanjing Jiancheng, China), according to the manufacturer’s instructions.

Western blot analysis of caspase-9 and -3 levels

Western blot procedures followed previously described methods [27]. From the other part of the samples stored at −80°C, cytosol protein was also extracted using RIPA lysis (1% PMSF) on ice and centrifuged at 14 000 r/min for 20 min. After denaturation, the supernatant was stored at −20°C and set aside for analysis of apoptosis-related genes caspase-9 and -3. Rabbit anti-caspase-9 and rabbit anti-caspase-3 were diluted in PBST (1: 100 dilution, Wuhan Boster, China). GAPDH was detected as control (mouse anti-GAPDH, 1: 1000 dilution, Shanghai Abgent, China). Secondary antibodies used were HRP-conjugated goat anti-rabbit IgG (1: 5000 dilution, Xi’an Jingcai, China) and HRP-conjugated goat anti-mouse IgG (1: 4000 dilution, Xi’an Jingcai, China).

Statistical analysis

All of the data were analyzed using SPSS software 22.0 (SPSS Inc., Chicago, IL, USA). The quantitative data were recorded as mean ± standard deviation and analyzed using the t test for comparisons between 2 groups. Tukey’s post hoc test was used to validate ANOVA for comparing measurement data between groups. All the data were obtained from at least 6 independent tests or experiments. Statistical significance was defined as p<0.05.

Results

Serum Se concentrations and myocardial GPx activity

In the WT mice, serum Se level was significantly decreased in the SD group compared with the CON group (Figure 1, p<0.0001), and although the Se level in the SS group was higher, it was not significantly elevated compared with the CON group. Serum Se levels in the Tg groups were the same as in the WT groups. Cardiac GPx activity in the SD group was significantly lower compared with the CON group (Figure 2, p<0.05) not only in WT mice, but also in Tg mice. GPx activity in the SS group was significantly increased compared with the CON group (Figure 2, p<0.05) in WT mice as well as in Tg mice. GPx activity was not significantly different between WTCON and TgCON (Figure 2, p=0.206) or between WTSD and TgSD (Figure 2, p=0.625), but GPx activity in TgSS was significantly higher than that in the WTSS group (Figure 2, p<0.0001).

Figure 1

Effect of different diets on serum Se level in each group. (A) Effect on serum Se level. (B) Effect on cardiac GPx activity. WTCON – wild-type control mice, WTSD – wild-type Se-deficient mice, WTSS – wild-type Se-supplemented mice, TgCON – transgenic control mice, TgSD – transgenic Se-deficient mice, TgSS – transgenic Se-supplemented mice. * p<0.0001 vs. WTCON, WTSS, # p<0.0001 vs. TgCON, TgSS.

Figure 2

Effect of different diets on cardiac GPx activity in each group. WTCON – wild-type control mice, WTSD – wild-type Se-deficient mice, WTSS – wild-type Se-supplemented mice, TgCON – transgenic control mice, TgSD – transgenic Se-deficient mice, TgSS – transgenic Se-supplemented mice. * p<0.05 vs. WTCON, ** p<0.05 vs. WTCON, WTSD; & p<0.05 vs. TgCON, # p<0.05 pTgCON, TgSD, WTSS.

Echocardiographic measurements

The echocardiographic data of mice are shown in Table 1 and Figure 3. IVSs in WTSD was significantly decreased over that in WTCON (p=0.009). IVSs in TgSD and TgSS tended to decrease (p=0.049, 0.055). LVPWs and LVPWd in WTSD and TgSD were thinner but with no significant difference from those in the respective control group. LVESD in WTSD was significantly increased over that in WTCON (p<0.05). LVESD and LVEDD in TgSD was significantly increased over that in TgCON (p<0.05). LVEDV and LVESV in WTSD and TgSD were remarkably larger than those in the respective WTCON and TgCON (p<0.05). Moreover, LVESV in TgSS was also significantly increased over that in TgCON (p<0.05). EF and FS in SD and SS groups were significantly decreased over those in the CON group in WT mice as well as in Tg mice.

Table 1

Parameters of LV from mice in each group (n=6, mean ±SD).

	WT			Tg
	CON	SD	SS	CON	SD	SS
IVSd (mm)	0.98±0.09	0.96±0.1	0.95±0.25	1±0.08	1.06±0.15	0.95±0.15
IVSs (MM)	1.5±0.15	1.13±0.18	1.49±0.18	1.45±0.12	1.38±0.16	1.24±0.08
LVPWd (mm)	1.19±0.2	0.87±0.44	1±0.52	1±0.52	0.85±0.5	1.9±0.59
LVPWs (mm)	1.76±0.16	1.18±0.06	1.49±0.15	1.36±0.43	1.13±0.37	2.23±0.67
LVEDD (mm)	4.21±0.3	4.79±0.08	4.46±0.69	3.85±0.56	5.33±0.71	4.62±0.9
LVESD (MM)	3.03±0.28	4.34±0.01	3.61±0.53	2.9±0.55	4.8±0.75	3.9±0.89
LVEDV (μl)	59.4±11.22	127.13±13.96	92.57±33.61	61.94±22.74	139.44±42.16	102.12±43.02
LVESV (μl)	27.48±15.84	84.77±2.58	55.98±19.32	26.9±9.8	110.05±38.8	69.18±33.75
EF (%)	53.91±1.56	20.71±3	39.36±3.42	56.59±1.65	21.94±4.31	34.04±6.71
FS (%)	28.13±1.59	9.43±1.46	19±1.93	24.99±3.98	10.12±2	16.17±3.32

LV – left ventricle; IVSd – interventricular septum diastolic thickness. IVSs – interventricular septum systolic thickness; PWd – posterior diastolic wall thickness; PWs – posterior systolic wall thickness; ESD – end-systolic diameter; EDD – end-diastolic diameter; ESV – end-systolic volume; EDV – end-diastolic volume; EF – ejection fraction; FS – fractional shortening; WTCON – wild-type control mice; WTSD – wild-type Se-deficient mice; WTSS – wild-type Se-supplemented mice; TgCON – transgenic control mice; TgSD – transgenic Se-deficient mice; TgSS – transgenic Se-supplemented mice.

p<0.05 vs. WTCON, WTSS;

p<0.05 vs. WTSS, TgCON, TgSD;

p<0.05 vs. WTCON;

p<0.05 vs. TgCON.

Figure 3

M-mode echocardiography of mice in each group. (A) Wild-type control mice. (B) Wild-type Se-deficient mice. (C) Wild-type Se-supplemented mice. (D) Transgenic control mice. (E) Transgenic Se-deficient mice. (F) Transgenic Se-supplemented mice.

Myocardial histopathology

In the WT mice in the SD group, myocytes were small and arranged in a wave-like pattern, more inflammatory cells were found, and gaps between myocytes were widened compared with the CON group. In the SS group, myocytes were larger and arranged in a wave-like pattern, more inflammatory cells were observed, and gaps between myocytes were narrowed compared with the CON group. In the Tg mice, the pathological changes in SD and SS groups were similar to the respective WTSD and WTSS groups (Figure 4).

Figure 4

Myocardial HE staining of mice in each group. (A) Wild-type control mice. (B) Wild-type Se-deficient mice. (C) Wild-type Se-supplemented mice. (D) Transgenic control mice. (E) Transgenic Se-deficient mice. (F) Transgenic Se-supplemented mice. Scar bar=100 μm.

Myocardial fibrosis

Masson’s trichrome staining showed that myocardial fibrosis in WTSD and WTSS groups were significantly increased compared with the WTCON group (Figure 5, p<0.05), and myocardial fibrosis was not significantly different between WTSD and WTSS. In the Tg mice, myocardial fibrosis in SD and SS groups were also significantly increased compared with the CON group (Figure 5, p<0.05). Myocardial fibrosis was higher in TgSD but not significantly different from that in TgSS, while myocardial fibrosis in TgSS was significantly increased compared with that in the WTSS group (Figure 5, p<0.05).

Figure 5

Masson’s trichrome staining and statistical analysis of mice in each group. (A) Wild-type control mice. (B) Wild-type Se-deficient mice. (C) Wild-type Se-supplemented mice. (D) Transgenic control mice. (E) Transgenic Se-deficient mice. (F) Transgenic Se-supplemented mice. (G) Comparison of interstitial fibrosis of mice in each group. WTCON – wild-type control mice, WTSD – wild-type Se-deficient mice, WTSS – wild-type Se-supplemented mice, TgCON – transgenic control mice, TgSD – transgenic Se-deficient mice, TgSS – transgenic Se-supplemented mice. * P<0.05 vs. WTCON, ** P<0.05 vs. WTCON, TgSS, # P<0.05 vs. TgCON. Scar bar=100 μm.

Caspase-9 and caspase-3 expression levels

Caspase-9 in SD and SS groups was significantly higher than that in CON groups in WT mice and in Tg mice (Figure 6, p<0.05). Caspase-9 in WTSD was significantly increased compared with the WTSS group (Figure 6, p<0.05), while caspase-9 in TgSD was significantly lowered than that in WTSD group (Figure 6, p<0.05). Caspase-9 in TgSS was highly elevated over that in WTSS and TgSD groups (Figure 6, p<0.05).

Figure 6

Expression level of caspase-9 in each group. (A) Image results of caspase-9. (B) Relative expression level of caspase-9. W1 – wild-type control mice, W2 – wild-type Se-deficient mice, W3 – wild-type Se-supplemented mice, T1 – transgenic control mice, T2 – transgenic Se-deficient mice, T3 – transgenic Se-supplemented mice. * P<0.05 vs. W1; ** P<0.05 vs. W1, W2, & P<0.05 vs. W2, T1; # P<0.05 vs. W3, T1, T2.

Caspase-3 in the SD group was significantly increased over that in the CON group in WT mice and in Tg mice (Figure 7, p<0.05), and caspase-3 in SS groups was higher but was not significantly different from than that in the CON groups. Caspase-3 in TgSD and TgSS groups was slightly lowered compared with the respective WTSD and WTSS groups (Figure 7).

Figure 7

Expression level of caspase-3 in each group. (A) Image results of caspase-9. (B) Relative expression level of caspase-3. W1 – wild-type control mice, W2 – wild-type Se-deficient mice, W3 – wild-type Se-supplemented mice, T1 – transgenic control mice, T2 – transgenic Se-deficient mice, T3 – transgenic Se-supplemented mice. * P<0.05 vs. W1, W3, # P<0.05 vs. T1, T3.

Discussion

It is well known that Se deficiency is associated with reversible heart failure that can be improved by Se supplementation [28-30]. Lei first found that the single-nucleotide polymorphism (Pro198Leu) in GPx-1 gene of KD patients is associated with Se deficiency as well as decreased GPx-1 activity. To further elucidate the hypothesis that both Se and the mutated GPx-1 gene might increase the risk of KD, the GPx-1P198L transgenic mice were generated and fed diets with different concentrations of Se. In the present study, Serum Se levels and cardiac GPx activity were remarkably decreased by Se deficiency in both WT and Tg mice. Serum Se levels in Se-supplemented groups were elevated but the difference was not significant, which was consistent with a previous study [9]. However, in WT mice, Se supplementation significantly increased GPx activity, which was even higher in Tg mice.

Our study provides evidence that overexpression of human GPx-1-198Leu does not exert protection against Se-deficiency-reduced cardiac remodeling and dysfunction. Cardiac systolic dysfunction was observed in Se-deficient mice and in Se-supplemented mice. The genotype did not work on improvement of cardiac function. As shown in Table 1 and Figure 3, the interventricular septum wall thickness in systole was significantly decreased in Se-deficient wild-type mice. Strangely, the ventricular posterior wall thickness was significantly increased in Se-supplemented transgenic mice. The left ventricle was heavily expanded, as evidenced by increased LVEDD and LVESD resulting from Se-deficient and Se-supplemented wild-type and transgenic mice, which indicates that the walls move poorly and the left ventricle diameter varies little from diastole to systole. Thus, cardiac systolic function was impaired by Se deficiency and Se supplement, not only in WT but also in Tg mice, which was also manifested as significantly lower EF and FS. Cardiac systolic dysfunction due to Se deficiency was not improved by overexpression of GPx-1-198Leu, which might be secondary to Ca2+ transport abnormalities of sarcoplasmic reticulum resulting from Se deficiency [31]. Unexpectedly, Se supplementation also led to systolic dysfunction. Metes-Kosik found that Se supplementation resulted in diastolic dysfunction [9]. Thus, we hypothesized that systolic dysfunction in Se-supplemented mice was secondary to chronic diastolic dysfunction. The elevated LVEDD and LVESD detected in Se-supplemented mice indicates that too much blood returned to the heart and the heart could not pump the increased blood volume into the systemic circulation; consequently, cardiac systolic dysfunction occurred.

Pathological changes in mice in different groups were detected to offer direct histomorphometry evidence for the above myocardial abnormalities. HE staining results showed that myocytes in Se-deficient mice became small with wave-like arrangement, gaps between cells were widened, and more inflammatory cells were observed. In Se-supplemented mice, myocytes were large with wave-like arrangement and crowded, and more inflammatory cells were also detected. Interstitial fibrosis was significantly increased due to Se deficiency and supplementation in WT and Tg mice. Fortunately, this provided us evidence that cardiac dysfunction was probably attributable to cardiac fibrosis and remodeling.

Lower Se level is associated with increased reactive oxygen species (ROS) production, a condition termed oxidative stress. Mitochondria are an important source of ROS within most mammalian cells [32-35]. Accumulated ROS in mitochondria can lead to oxidative damage to mitochondrial proteins, membranes, and DNA, impairing the metabolic functions of mitochondria and contributing to a variety of pathologies [36-39]. In addition, mitochondrial ROS also plays an important role in redox signaling from the organelle to the rest of the cell [40-42]. Mitochondrial oxidative damage can also increase the release of cytochrome c to the cytosol and thereby activate the cell apoptotic pathway. Se functions as an important antioxidant to detoxify ROS via selenoproteins, one of which is GPx-1, the major layer reducing lipid peroxides and hydrogen peroxide. Both Se deficiency and excess result in abnormal cellular defence mechanisms against oxidative stress [43,44]. Thus, in the present study, activation of caspase-9 and -3 were detected in Se-deficient WT mice. With no significant decrease in caspase-3, overexpression of GPx-1-198Leu gene in Se-deficient mice significantly attenuated the activation of caspase-9, which was still significantly higher than in control mice. Consistent with previous reports [44], Se supplementation in WT mice also led to increased caspase-9, which was much more highly elevated in Se-supplemented Tg mice. Caspase-3 in Se-supplemented mice was higher than in control animals but the difference was not significant, indicating that Se deficiency and supplementation can cause myocardial abnormalities via the mitochondria apoptotic pathway. Overexpression of GPx-1-198Leu did increase cardiac GPx activity and had some effects of inhibition to Se deficiency. However, the protection was not strong enough to exert significant improvement on cardiac structural and functional abnormalities. This provides evidence that the decreased activity of GPx-1-198Leu gene as well as Se deficiency might play a synergistic role in the pathogenesis of KD. High concentrations of Se may lead to assembly of the apoptotic molecules and ROS, which induce mitochondrial membrane permeabilization and caspase activation [5]. It has been reported that lipid peroxidation (LPO) was significantly increased in the liver of mice fed a 0.4-Se/kg diet compared to the animals fed a 0.1 Se/kg diet [45]. Since GPx-1 is considered to be the body storage form of Se and to serve a homeostatic function in Se metabolism [46], more Se might have been deposited in the Tg mice. In the presence of GSH, high concentrations of Se can react to become selenide anion (RSe−), hydrogen selenide (H2Se) [47], and GSSe- [1], which react with O2 or GSH and then more super-oxidative anion are produced, and finally the mitochondria apoptotic pathway is activated [1,44,47]. Decreases in activity of superoxide dismutase (SOD) have been reported both in Se-deficient and Se-excess diet-fed mice [48]. Thus, it is understandable that accumulated superoxide was not successfully detoxified in Se-deficient or in supplemented animals, although GPx activity in Se-supplemented animals was elevated.

Conclusions

We have demonstrated that both Se deficiency and supplementation induced myocardial structural and functional abnormalities via activation of caspase-9 and -3, but overexpression of human GPx-1-198Leu could not attenuate cardiac injuries caused by Se deficiency and Se supplementation. Blocking the mitochondrial apoptotic pathway might impede the occurrence and development of Se-related cardiomyopathy.