Selenium deficiency is a causative factor in heart failure and microRNAs (known as miRNAs or miRs) play an important role in numerous cardiovascular diseases. However, the changes of miRNA expression during selenium deficiency and whether selenium deficiency is involved in cardiac dysfunction remain unclear. In the present study, miRNA expression profiling was carried out in normal rats, selenium-deficient rats and selenium-supplemented rats by miRNA microarray. Cardiac function was evaluated by analyzing the plasma brain natriuretic peptide level, echocardiographic parameters and hemodynamic parameters. Cardiac glutathione peroxidase activity was assessed by spectrophotometry. The histological changes were examined by hematoxylin and eosin staining. Electrocardiograph was used to test the arrhythmia. The differentially expressed miRNAs were verified by reverse transcription-polymerase chain reaction. Additionally, the underlying mechanism associated with the Wnt/β-catenin signaling pathway was further explored. The cardiac dysfunction of the rat with selenium deficiency was mainly associated with five upregulated miRNAs, which were
Selenium deficiency is considered a causative factor in various types of heart failure, including Keshan disease (KD), which is prevalent in China and is also one of the most harmful endemic diseases (
microRNAs (miRNAs or miRs) are a class of endogenous non-coding small RNAs that are involved in the modulation of numerous biological processes by base-pairing, usually imperfectly, to the 3′ untranslated region of a target mRNA, leading to post-transcriptional inhibition and occasionally mRNA cleavage (
Wnt/β-catenin signaling is subjected to multiple levels of molecular control. The canonical Wnt/β-catenin signaling pathway is initiated when Wnt ligands bind to its receptor(s), Frizzled (
Given the potential role of miRNAs, their expression was profiled in selenium-deficient rats by microarray. The differentially expressed miRNAs were selected and validated. Due to the importance of Wnt/β-catenin signaling in cardiac function, it was further investigated in an attempt to provide a novel insight into the limited understanding of the biological process and mechanism of cardiac dysfunction in selenium-deficient rats.
A total of 60 male weaning Sprague-Dawley rats (3-weeks old; specific-pathogen-free class; body weight, 75±10 g) were raised under controlled-environmental conditions (12-h light-dark cycle; temperature, 25±1°C; humidity, 65±4%). The standard diet (containing 0.2 mg selenium/kg food) was produced by the Animal Experimental Center of Xi’an Jiaotong University (Xi’an, China) and the low-selenium diet (<0.02 mg selenium/kg food) was produced by Trophic Animal Feed High-tech Co. (Jiangsu, China) according to the AIN-93 M formula. The food was stored at 4°C and fresh tap water was allocated continuously. The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Xi’an Jiaotong University. All the surgeries were performed under chloral hydrate anesthesia, and all efforts were made to minimize suffering.
All the animals were randomly assigned into three groups: Control (n=20), low selenium (LS) (n=20) and selenium supplementation (SS) (n=20). In the control group, the animals were fed with the standard diet for 14 weeks and were treated by intraperitoneal injection of physiological saline every day for 21 days. In the LS group, the animals were fed with the low-selenium diet for 14 weeks and were treated by intraperitoneal injection of physiological saline every day for 21 days. In the SS group, the animals were fed with the low-selenium diet for 14 weeks and were treated by intraperitoneal injection of sodium selenite (0.05 mg/kg bodyweight; Sigma-Aldrich, St. Louis, MO, USA) as selenium supplementation every day for 21 days (
Samples of whole blood and hearts harvested from rats were wet-washed by mixed acids (nitric acid and perchloric acid) in borosilicate reaction tubes. Following removal of the excess acids, the samples were titrated to 10 ml by ultra-pure water (Aqulix 5 water purification system; Merck Millipore, Billerica, MA, USA). The selenium concentration was subsequently determined by the flameless atomic absorption spectrophotometry method using a Z-5000 spectrophotometer (Hitachi, Ltd., Tokyo, Japan) with a cathode lamp of Se (resonance line, 196.0 nm; Photron, Victoria, Australia). Standard selenium solutions were used to calibrate the results.
Cardiac glutathione peroxidase (GPx) activity in heart extract was assessed by a spectrophotometry method using the Glutathione Peroxidase Cellular Activity Assay kit (Sigma-Aldrich), following the manufacturer’s instructions. The assay is based on the reaction in which oxidation of glutathione (GSH) to oxidized glutathione (GSSG) is catalyzed by GPx. As NADPH is consumed when GSSG is recycled back to GSH, the decrease in NADPH absorbance at 340 nm (Tecan Sunrise Absorbance Reader, Tecan, Austria) can be utilized to calculate the activity of GPx indirectly.
Following femoral artery puncture, whole blood samples were collected using EDTA-Na2 vacuum blood collection tubes. The supernatant was collected after centrifugation at 377 × g for 20 min. The samples were processed by the Triage BNP assay (Biosite, San Diego, CA, USA) within 1 h after collection at room temperature, according to the manufacturer’s instructions.
ECG in lead II was recorded (PowerLab 4/25; ADInstrument, New South Wales, Australia) during the experiment time. Prior to electrocardiography, all the rats were anesthetized by intraperitoneal injection of chloral hydrate (10%, 0.03 ml/kg bodyweight). The left upper limb, right upper limb and right lower limb electrodes were placed for leads I. Subsequently, the ECG was analyzed for changes in the ST segment, T wave, AV block and arrhythmia (premature ventricular contraction, ventricular tachycardia and fibrillation and atrial fibrillation). Cardiac conduction of rats was evaluated by the number of ventricular arrhythmic events (VAEs) within 15 min.
Echocardiographic tests were performed according to the instructions described in previous studies (
Hemodynamic determination was conducted according to methods described in a previous study (
The animals were anesthetized with chloral hydrate (10%, 0.03 ml/kg bodyweight) intraperitoneally. Subsequently, they were sacrificed by removing the hearts following complete anesthesia. The harvested hearts were rinsed in phosphate-buffered saline, fixed in 4% paraformaldehyde for 24 h, embedded in paraffin and cross-sectioned into 10-μm slices. The sections were stained with hematoxylin and eosin (HE) for cells alignment according to the general procedure. The morphological structures of the heart were observed by light microscopy.
Total RNA was isolated using TRIzol (Invitrogen) and the miRNeasy mini kit (Qiagen) according to the manufacturer’s instructions, which efficiently recovered all RNA species, including miRNAs. RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies, Wilmington, DE, USA) and RNA integrity was determined by gel electrophoresis. Following RNA isolation from the samples, the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer’s guideline for miRNA labeling. Each sample (1 μg) was 3′-end-labeled with the Hy3TM fluorescent label, using T4 RNA ligase as follows: RNA in 2.0 μl water was combined with 1.0 μl calf intestinal alkaline phosphatase (CIP) buffer and CIP (Exiqon). The mixture was incubated for 30 min at 37°C, and was terminated by incubation for 5 min at 95°C. Subsequently, 3.0 μl labeling buffer, 1.5 μl fluorescent label (Hy3TM), 2.0 μl dimethyl sulfoxide and 2.0 μl labeling enzyme were added into the mixture. The labeling reaction was incubated for 1 h at 16°C, and terminated by incubation for 15 min at 65°C. Subsequent to stopping the labeling procedure, the Hy3TM-labeled samples were hybridized on the miRCURYTM LNA Array (v.16.0) (Exiqon) according to the array instructions. The total mixture (25 μl) from Hy3TM-labeled samples with 25 μl hybridization buffer were first denatured for 2 min at 95°C, incubated on ice for 2 min and hybridized to the microarray for 16–20 h at 56°C in a 12-Bay Hybridization System (Hybridization System-Nimblegen Systems, Inc., Madison, WI, USA), which provides an active mixing action and constant incubation temperature to improve hybridization uniformity and to enhance the signal. Following hybridization, the slides were obtained, washed several times using Wash buffer kit (Exiqon), and dried by centrifugation for 5 min at 100 × g. Subsequently, the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA). The scanned images were imported into the GenePix Pro 6.0 software (Axon Instruments) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs with intensities >50 in all the samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization (
GO analysis was applied in order to organize genes into hierarchical categories and uncover the miR-gene regulatory network on the basis of biological process and molecular function. In detail, two-side Fisher’s exact test was used to classify the GO category, and the false discovery rate (FDR) was calculated to correct the P-value. Only GOs that had a P-value of <0.001 and an FDR of <0.05 were chosen. Within the significant category, the enrichment rare earth (Re) was: Re=(
Differentially expressed miRNAs of
Frozen cardiac tissue was homogenized in radioimmunoprecipitation assay lysis buffer system (Santa Cruz Biotechnology, Inc., Dallax, TX, USA) with phenylmethylsulfonyl fluoride (Santa Cruz Biotechnology, Inc.). All the procedures followed manufacturer’s instructions. The sample protein concentration was detected by using bicinchoninic acid protein assay kit (Santa Cruz Biotechnology, Inc.). The sample protein was boiled in 1x SDS-PAGE loading buffer, separated by electrophoresis in 10% SDS-polyacrylamide gel and subsequently transferred to a polyvinylidene fluoride membrane. Antibodies against Wnt (ab15251) and β-catenin (ab6302) (Abcam, Cambridge, MA, USA) were applied to incubate the bolts at 4°C overnight. Tris-buffered saline (containing 0.02% Tween 20) was used to wash the membranes, which were subsequently incubated with goat polyclonal secondary antibody to rabbit immunoglobulin G conjugated to horseradish peroxidase (Abcam). The membranes were developed using Super Signal West Pico chemiluminescence reagent (Thermo Scientific, Waltham, MA, USA) and were visualized on X-ray films.
All the results were expressed as mean ± standard deviation. Statistical analysis was performed with one-way analysis of variance for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.
The Se concentration in blood is shown in
As shown in
The Triage BNP assay showed that the plasma BNP level increased significantly in the LS and SS groups compared to the control group (P<0.05). A clearer increase of plasma BNP was found in the LS group. Following Se supplementation by intra-peritoneal injections, a reduction in the plasma BNP level in the SS group was identified compared to the LS group (P<0.05) (
ECG of rats in the control group was normal (
The echocardiographic parameters, including LVEDD, LVESD, LVEF and LVFS, were detected and calculated in the present study as shown in
The hemodynamic parameters, including LVEDP, LVSP, LVdp/dtmax and LVdp/dtmin, were detected and calculated in the study as shown in
HE staining showed that the structure of myocardial tissue in the control group was clear, including well-connected intercalated discs and normally arranged filaments (
Using the miRCURY LNA Array platform, the miRNAs expression profiles were assessed in Se-deficient rats. The expression profiles of 116 miRNAs were regulated between the control, LS and SS groups, and the samples were separated into biologically interpretable groups. Among these, 5 miRNAs were identified to be upregulated >5-fold in the LS group compared to the SS group, whereas 3 miRNAs were less than the threshold level (3-fold) set during the progression of Se deficiency, but following selenium supplementation these miRNAs were >1.5-fold compared to Se deficiency. These 8 miRNAs were validated to be significantly different (P<0.05). As shown in
According to the threshold of GOs that were significantly regulated by miRNAs, the P-value and FDR was <0.001 and <0.05, respectively. The high-enrichment GOs targeted by upregulated miRNAs included signal transduction, transport, cell differentiation and response to stress. By contrast, the significant GOs corresponding to downregulated miRNAs appeared to include signal transduction, cell differentiation, transport and cell proliferation. Among these, the maximum-enriched-GO associated with signal transduction, together with the numerous miRNAs that interacted with signal transduction-related genes, suggested them to have an important role in the activation of selenium deficiency (
Western blotting was employed to detect the changes of Wnt and β-catenin expression in the present study. As shown in
In China, KD is the most detrimental and widely distributed endemic cardiomyopathy. In an observational epidemiological study and from population-based intervention trial results, it was concluded that Se deficiency is the critical etiological factor for KD (
Increasing evidence has confirmed miRNA as one of the most significant and critical factors controlling gene expression. Thus far, miRNAs have been noted to play an extremely important role in cardiac development, homeostasis and pathobiology (
Molecular mechanisms underlying cardiac dysfunction caused by Se deficiency should be intensively studied. Wnt signaling is required for various features of cardiac and vascular development, such as myocardial specification, cardiac morphogenesis and cardiac valve formation, as well as endothelial and vascular smooth muscle cell proliferation (
In conclusion, Se deficiency is associated with eight miRNAs:
The present study was supported by the Natural Science Foundation of China (NSFC) (grant no. 30972557). The company Genminix provided technical assistance.
Se concentrations for each group. Se concentrations in the blood of rats were analyzed by spectrophotometry. *P<0.05 vs. control; #P<0.05 vs. LS. LS, low selenium; SS, selenium supplementation.
Effects of Se deficiency/supplementation on GPx activity. A spectrophotometry method was utilized to examine the activity of GPx. Values indicated by columns in the graph are presented as mean ± standard deviation. *P<0.05 vs. control; #P<0.05 vs. LS. LS, low selenium; SS, selenium supplementation; GPx, glutathione peroxidase.
Effects of Se deficiency/supplementation on plasma BNP level. Plasma BNP concentration (pg/ml) was examined by the Triage BNP Assay. The values indicated by the columns in the graph are presented as mean ± standard deviation. *P<0.05 vs. control; #P<0.05 vs. LS. LS, low selenium; SS, selenium supplementation; BNP, brain natriuretic peptide.
ECG features. (A) Normal ECG in the control group, (B) premature ventricular contraction in the LS group, (C) paroxysmal supraventricular tachycardia in the LS group and (D) premature atrial contraction in the SS group. (E) Analysis of VAEs (a,#P<0.001 vs. control; b,#P<0.001 vs. LS). ECG, electrocardiography; LS, low selenium; SS, selenium supplementation; VAE, ventricular arrhythmic event.
Histological changes of the rat hearts. (A) Control, (B–E) LS and (F) SS groups. (A) Normal structure of myocardial tissue (magnification: ×400). (B) Swelling and disorganized muscle fibers (magnification, ×400). (C) Myocardial necrosis and nuclear condensation (magnification, ×1000). (D) Intermittent muscle fibers (magnification, ×400). (E) Small myocardial necrosis stove accompanied by inflammatory infiltration (magnification, ×200). (F) Mild swelling and orderly arrangement myocardial fibers (magnification, ×400). LS, low selenium; SS, selenium supplementation.
miRNA profiles differentiate rats with normal diet from rats with Se deficiency or Se supplementation. Five upregulated (
GO of significant microRNAs. The gray column shows the GOs targeted by upregulated miRNAs and the black column shows the GOs targeted by downregulated miRNAs. All these GOs show increased enrichment. The vertical axis is the GO category and the horizontal axis is the enrichment of GO. GO, gene ontology.
Effects of Se deficiency and Se supplementation on protein expression of Wnt and β-catenin in cardiac tissue. Sample protein was extracted, separated and detected by specific antibodies against Wnt and β-catenin. The expression of Wnt and β-catenin were normalized by β-actin in the same sample. The darkness of the immunoblots represents the expression level of the protein. (A) Western blotting of Wnt and β-catenin. (B) Analysis of the relative expression of Wnt and (C) β-catenin normalized by β-actin. *Values are significantly different from LS (P<0.05). LS, low selenium; SS, selenium supplementation.
Primers used in TaqMan RT-qPCR.
Gene primer | Product size, bp | Number gene primer (5′→3′) |
---|---|---|
62 | F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′ | |
68 | F: 5′-CTCGGATGGATATAATACA-3′ | |
62 | F: 5′-GGGGTAGCAGCACGTAAATA-3′ | |
94 | F: 5′-CTTCTGGAGATCCTGCTC-3′ | |
78 | F: 5′-AACTCTCCTGGCTCTAGC-3′ | |
86 | F: 5′-GGGCAGTCTTTGCTACTG-3′ | |
96 | F: 5′-GGACATTACCTACCCAA-3′ | |
70 | F: 5′-GGACTGGTGCGGAAAGG-3′ | |
70 | F: 5′-AGAGATGCGGAGCTGTT-3′ |
RT-qPCR, reverse transcription-quantitative polymerase chain reaction; bp, base pair; F, forward primer; R, reverse primer.
Echocardiographic parameters of the rats in the different groups.
Group | No. | LVEDD, mm | LVESD, mm | LVEF, % | LVFS, % |
---|---|---|---|---|---|
Control | 10 | 4.14±0.38 | 1.13±0.77 | 85.60±11.02 | 75.64±8.30 |
LS | 10 | 5.29±0.26a* | 2.77±0.35a,c | 68.93±10.92a,c | 50.44±5.73a,d |
SS | 10 | 4.92±0.79b* | 2.05±0.23b,c | 79.20±9.41b,c | 97.73±6.28b,d |
Cardiac functions (left ventricular functions) were assessed by echocardiographic detection. All the results are presented as mean ± standard deviation. aValues are significantly different from the control (P<0.05) (cP<0.05; dP<0.01). bValues are significantly different from the LS group (P<0.05) (cP<0.05; dP<0.01). LVEDD, left ventricular end-diastole diameter; LVESD, left ventricular end-systole diameter; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening.
Hemodynamic parameters of the rats in the different groups.
Group | No. | LVEDP, mmHg | LVSP, mmHg | LVdp/dtmax, mmHg/sec | LVdp/dtmin, mmHg/sec |
---|---|---|---|---|---|
Control | 7 | 47.62±6.41 | 185.32±13.28 | 3794.55±127.47 | 2887.65±154.13 |
LS | 8 | 67.81±5.50a* | 157.69±14.24a,c | 2640.31±144.20a,c | 2216.28±138.69a,c |
SS | 8 | 53.29±5.13b** | 162.30±14.68b,c | 2948.19±152.35b,c | 2677.00±142.98b,c |
Cardiac functions (left ventricular functions) were assessed by hemodynamic tests using a cardiac intubation method. All the results are presented as mean ± standard deviation. aValues are significantly different from the control (cP<0.05). bValues are significantly different from the LS group (cP<0.05). LS, low selenium; SS, selenium supplementation; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastole pressure; LVdp/dtmax, maximum rising rate of left ventricular pressure; LVdp/dtmin, maximum dropping rate of left ventricular pressure.