Background
Maternal type 1 and 2 diabetes mellitus are strongly associated with high rates of severe structural birth defects, including congenital heart defects. Studies in type 1 diabetic embryopathy animal models have demonstrated that cellular stress-induced apoptosis mediates the teratogenicity of maternal diabetes leading to congenital heart defect formation. However, the mechanisms underlying maternal type 2 diabetes mellitus–induced congenital heart defects remain largely unknown.
Objective
We aim to determine whether oxidative stress, endoplasmic reticulum stress, and excessive apoptosis are the intracellular molecular mechanisms underlying maternal type 2 diabetes mellitus–induced congenital heart defects.
Study Design
A mouse model of maternal type 2 diabetes mellitus was established by feeding female mice a high-fat diet (60% fat). After 15 weeks on the high-fat diet, the mice showed characteristics of maternal type 2 diabetes mellitus. Control dams were either fed a normal diet (10% fat) or the high-fat diet during pregnancy only. Female mice from the high-fat diet group and the 2 control groups were mated with male mice that were fed a normal diet. At E12.5, embryonic hearts were harvested to determine the levels of lipid peroxides and superoxide, endoplasmic reticulum stress markers, cleaved caspase 3 and 8, and apoptosis. E17.5 embryonic hearts were harvested for the detection of congenital heart defect formation using India ink vessel patterning and histological examination.
Results
Maternal type 2 diabetes mellitus significantly induced ventricular septal defects and persistent truncus arteriosus in the developing heart, along with increasing oxidative stress markers, including superoxide and lipid peroxidation; endoplasmic reticulum stress markers, including protein levels of phosphorylated-protein kinase RNA-like endoplasmic reticulum kinase, phosphorylated-IRE1α, phosphorylated-eIF2α, C/EBP homologous protein, and binding immunoglobulin protein; endoplasmic reticulum chaperone gene expression; and XBP1 messenger RNA splicing, as well as increased cleaved caspase 3 and 8 in embryonic hearts. Furthermore, maternal type 2 diabetes mellitus triggered excessive apoptosis in ventricular myocardium, endocardial cushion, and outflow tract of the embryonic heart.
Conclusion
Similar to those observations in type 1 diabetic embryopathy, maternal type 2 diabetes mellitus causes heart defects in the developing embryo manifested with oxidative stress, endoplasmic reticulum stress, and excessive apoptosis in heart cells.
Introduction
Both maternal type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) increase the risk that offspring will have cardiovascular malformations 5-fold compared with the general population, even with modern preconception care. The most common maternal diabetes-associated congenital heart defects (CHDs) include cardiac septation defects such as ventricular septal defects (VSDs) and conotruncal cardiac anomalies such as persistent truncus arteriosus (PTA). However, the mechanisms underlying these anomalies remain largely unknown. Because the number of women of reproductive age with T2DM is increasing rapidly, understanding the molecular pathways involved in maternal T2DM-induced CHDs is essential for developing new strategies to prevent these types of birth defects.
Oxidative stress is triggered by an imbalance in intracellular reduction-oxidation (redox) homeostasis, and is sustained by the generation of reactive oxygen species. Our previous studies have revealed that both T1DM and T2DM significantly induce oxidative stress by increasing the levels of superoxide and lipid peroxidation in the developing neuroepithelium (T1DM and T2DM) and the embryonic heart (T1DM). Overexpression of the antioxidant enzyme, superoxide dismutase 1 (SOD1), diminishes maternal T1DM-induced oxidative stress in developing neuroepithelium and the embryonic heart, and thus ameliorates neural tube defects (NTDs) and CHDs in offspring of diabetic dams.
Animal studies have shown that both maternal T1DM and T2DM trigger endoplasmic reticulum (ER) stress in neurulation-stage embryos (T1DM and T2DM) and in embryonic hearts (T1DM). ER stress occurs when misfolded proteins accumulate in the ER lumen and cause ER dysfunction. Both maternal T1DM and T2DM disrupt ER luminal homeostasis by enhancing transcription of the proapoptotic C/EBP homologous protein (CHOP), and increasing other ER stress markers, such as binding immunoglobulin protein (BiP) and calnexin, in the developing neuroepithelium. In addition, we have shown in vitro that the ER stress inhibitor, 4-phenylbutyric acid, inhibits NTD formation in cultured embryos exposed to high glucose.
Oxidative stress– and ER stress-induced cell apoptosis are causative events in maternal T1DM- and T2DM-induced NTDs. Apoptosis is a precisely controlled cellular event that is essential to many biological processes. However, our previous studies have demonstrated that maternal T1DM and T2DM induces excessive apoptosis, leading to defective neurulation and failed neural tube closure. Furthermore, we have found that deletion of the gene for apoptosis signal-regulating kinase 1 significantly ameliorates diabetes-induced NTDs. Therefore, we hypothesize that oxidative stress, ER stress, and subsequent excessive apoptosis are contributing factors for maternal T2DM-induced CHDs.
Here, we use a high-fat diet (HFD)-induced T2DM mouse model to explore whether oxidative stress and ER stress-induced excessive apoptosis are present in T2DM-induced CHDs. A previous study demonstrated that this murine T2DM model exhibits modest hyperglycemia, glucose intolerance, insulin resistance, and hyperinsulinemia, all of which are characteristics of human T2DM. We showed that the levels of oxidative stress, ER stress, and apoptosis markers were increased in hearts of embryos from T2DM dams. We also found that maternal T2DM specifically induced VSDs and PTA. By investigating the role of abnormal cellular processes in embryonic heart development, we elucidated possible mechanisms in T2DM-induced CHDs.
Materials and Methods
Animal model of embryopathy
The procedures for animal use were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee. The mouse model of T2DM was established as previously described. Four-week-old female C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were maintained in a temperature-controlled room on a 12-hour light-dark cycle. After arrival, mice were divided into 2 groups and fed either a HFD (Research Diets Inc, New Brunswick, NJ) or a normal diet (Harlan Laboratories, Indianapolis, IN) for 15 weeks. The HFD contained 20% protein, 20% carbohydrate, and 60% fat. The normal diet contained 20% protein, 70% carbohydrate, and 10% fat. HFD mice and mice fed the normal diet were mated with lean male mice. During pregnancy, mice in the normal diet group were either maintained on the normal diet (control group 1) or subsequently fed HFD to serve as the high circulating free fatty acid control group (control group 2). Male and female mice were paired at 3:00 pm , and day 0.5 (E0.5) of pregnancy was established at noon of the day when a vaginal plug was present. Only CHDs were examined in these animals. NTDs were not the subject of the present study.
India ink injection and hematoxylin-eosin staining
After euthanizing the pregnant dams at E17.5, fetuses were excised from uteri then the rib cage was removed and the hearts were exposed, morphologically examined, and imaged under a dissecting microscope (Leica, Wetzlar, Germany) while remained attaching to the spine. India ink was injected into the left ventricle of the heart using the μTIP (TIP10TW1-L, World Precision Instrument Inc, Sarasota, FL) tip, and images were taken for the examination of outflow tract defects. Subsequently, embryonic hearts were harvested and fixed in methacarn (methanol, 60%; chloroform, 30%; glacial acetic acid, 10%), embedded in paraffin, and cut into 8-μm sections. After deparaffinization and rehydration, all specimens then underwent hematoxylin-eosin staining in a standard procedure. All heart sections were photographed and examined for heart defects.
Dihydroethidium staining
Dihydroethidium (DHE) staining (Thermo Scientific, Rockford, IL) was used to detect the level of superoxide. DHE reacts with superoxide that is bound to cellular components including protein and DNA, and exhibits bright red fluorescence. E12.5 embryonic hearts were fixed in 4% paraformaldehyde for 30 minutes, washed 3 times with phosphate-buffered saline (PBS) (5 min/wash), and then embedded in optimum cutting temperature compound (Tissue-Tek; Sakura Finetek USA Inc, Torrance, CA). We incubated 10-μm frozen embryonic sections with 1.5 μmol/L DHE for 5 minutes at room temperature, then washed them 3 times with PBS for 5 min/wash. Sections were counterstained with 4′,6-diamidino-2-phenylindole (Sigma, St Louis, MO) and mounted with aqueous mounting medium (Sigma).
Lipid hydroperoxide quantification
As previously described, the degree of lipid peroxidation in E12.5 hearts was quantitatively assessed using the Calbiochem lipid hydroperoxide assay kit (Millipore, Bedford, MA) by following the manufacturer’s instructions. Lipid hydroperoxides of embryonic hearts were extracted by deoxygenated chloroform, and then measured by the absorbance of 500 nm after reaction with chromogen. The results were expressed as μmol/L lipid hydroperoxides/μg protein. Protein concentrations were determined by the BioRad detergent compatible protein assay kit (Hercules, CA).
Immunoblotting
Immunoblotting was performed as described by Yang et al. To extract protein, the lysis buffer (Cell Signaling Technology, Danvers, MA) containing a protease inhibitor cocktail (Sigma) was used. Equal amounts of protein and the Precision Plus Protein Standards (BioRad) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto Immunobilon-P membranes (Millipore). Membranes were incubated in 5% nonfat milk for 45 minutes, and then were incubated for 18 hours at 4°C with the following primary antibodies at dilutions of 1:1000 in 5% nonfat milk: phosphorylated protein kinase RNA-like endoplasmic reticulum kinase; protein kinase RNA-like endoplasmic reticulum kinase; p-eIF2α; eIF2α; CHOP; BiP; IRE1α (cell signaling); p-IRE1α (Abcam, Cambridge, United Kingdom); caspase 8 (mouse specific) (Enzo Life Sciences, Farmingdale, NY); and caspase 3 (Millipore). Membranes were exposed to horseradish peroxidase–conjugated goat antirabbit, goat antimouse, or goat antirat (Millipore) secondary antibodies. Signals were detected using SuperSignal West Femto maximum sensitivity substrate kit (Thermo Scientific), and chemiluminescence emitted from the bands was directly captured using a Bioimage EC3 system (UVP Inc, Upland, CA). Densitometric analysis of chemiluminescence signals was performed using software (VisionWorks LS; UVP Inc). To ensure that equivalent amounts of protein were loaded among samples, membranes were stripped and incubated with a β-actin antibody (Abcam). All experiments were repeated in triplicate with the use of independently prepared tissue lysates.
Detection of XBP1 messenger RNA splicing
Messenger RNA (mRNA) was extracted from E12.5 embryonic hearts and reverse-transcribed using the QuantiTect reverse transcription kit (Qiagen, Venlo, The Netherlands). The polymerase chain reaction (PCR) primers for X-box binding protein 1 were as follows: forward, 5’-GAACCAGGAGTTAAGAACACG-3’ and reverse, 5’-AGGCAACAGTGTCAGAGTCC-3’. If no XBP1 mRNA splicing occurred, a 205-base pair (bp) band was produced. When XBP1 splicing occurred, a 205-bp band and a 179-bp main band were produced.
Real-time quantitative PCR
Using the Rneasy mini kit (Qiagen), mRNA was isolated from E12.5 embryonic hearts, and then reverse-transcribed using the high-capacity complimentary DNA archive kit (Applied Biosystems, Grand Island, NY). Real-time PCR for BiP, CHOP, calnexin, IRE1α, protein disulphide isomerase A, 94 kDa glucose-regulated protein, and β-actin were performed using the Maxima SYBR green/ROX quantitative PCR Master Mix assay (Thermo Scientific) in the StepOnePlus system (Applied Biosystems). Primer sequences for real-time PCR are listed in Table 1 .
| Primers name | Primer sequences |
|---|---|
| BiP | Forward primer 5’-ACTTGGGGACCACCTATTCCT-3’ |
| Reverse primer 5’-ATCGCCAATCAGACGCTCC-3’ | |
| CHOP | Forward primer 5’-CGGAACCTGAGGAGAGAGTG-3’ |
| Reverse primer 5’-CTGTCAGCCAAGCTAGGGAC-3’ | |
| Calnexin | Forward primer 5’-ATGGAAGGGAAGTGGTTACTGT-3’ |
| Reverse primer 5’-GCTTTGTAGGTGACCTTTGGAG-3’ | |
| IRE1α | Forward primer 5’-ACACCGACCACCGTATCTCA-3’ |
| Reverse primer 5’-CTCAGGATAATGGTAGCCATGTC-3’ | |
| PDIA | Forward primer 5’-CGCCTCCGATGTGTTGGA-3’ |
| Reverse primer 5’-CAGTGCAATCCACCTTTGCTAA-3’ | |
| GRP94 | Forward primer 5’-TCGTCAGAGCTGATGATGAAGT-3’ |
| Reverse primer 5’-GCGTTTAACCCATCCAACTGAAT-3’ | |
| β-Actin | Forward primer 5’-GAACCAGGAGTTAAGAACACG-3’ |
| Reverse primer 5’-AGGCAACAGTGTCAGAGTCC-3’ |
TUNEL assay
The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was performed using the ApopTag fluorescein in situ apoptosis detection kit (Millipore) as previously described. Briefly, 10-μm frozen heart sections were fixed with 4% paraformaldehyde in PBS and incubated with TUNEL reaction agents. Three embryonic hearts from 3 different dams (n = 3) per group were used, and 2 sections per heart were examined.
Statistical analyses
Data were presented as means ± SE. Statistical differences were determined by 1-way analysis of variance using software (SigmaStat 3.5, Systat Software Inc, San Jose, CA). In 1-way analysis of variance analysis, Tukey test was used to estimate the significance of the results ( P < .05). The χ 2 test was used to estimate the significance of difference in VSDs and PTA rates among experimental and control groups.
Materials and Methods
Animal model of embryopathy
The procedures for animal use were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee. The mouse model of T2DM was established as previously described. Four-week-old female C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were maintained in a temperature-controlled room on a 12-hour light-dark cycle. After arrival, mice were divided into 2 groups and fed either a HFD (Research Diets Inc, New Brunswick, NJ) or a normal diet (Harlan Laboratories, Indianapolis, IN) for 15 weeks. The HFD contained 20% protein, 20% carbohydrate, and 60% fat. The normal diet contained 20% protein, 70% carbohydrate, and 10% fat. HFD mice and mice fed the normal diet were mated with lean male mice. During pregnancy, mice in the normal diet group were either maintained on the normal diet (control group 1) or subsequently fed HFD to serve as the high circulating free fatty acid control group (control group 2). Male and female mice were paired at 3:00 pm , and day 0.5 (E0.5) of pregnancy was established at noon of the day when a vaginal plug was present. Only CHDs were examined in these animals. NTDs were not the subject of the present study.
India ink injection and hematoxylin-eosin staining
After euthanizing the pregnant dams at E17.5, fetuses were excised from uteri then the rib cage was removed and the hearts were exposed, morphologically examined, and imaged under a dissecting microscope (Leica, Wetzlar, Germany) while remained attaching to the spine. India ink was injected into the left ventricle of the heart using the μTIP (TIP10TW1-L, World Precision Instrument Inc, Sarasota, FL) tip, and images were taken for the examination of outflow tract defects. Subsequently, embryonic hearts were harvested and fixed in methacarn (methanol, 60%; chloroform, 30%; glacial acetic acid, 10%), embedded in paraffin, and cut into 8-μm sections. After deparaffinization and rehydration, all specimens then underwent hematoxylin-eosin staining in a standard procedure. All heart sections were photographed and examined for heart defects.
Dihydroethidium staining
Dihydroethidium (DHE) staining (Thermo Scientific, Rockford, IL) was used to detect the level of superoxide. DHE reacts with superoxide that is bound to cellular components including protein and DNA, and exhibits bright red fluorescence. E12.5 embryonic hearts were fixed in 4% paraformaldehyde for 30 minutes, washed 3 times with phosphate-buffered saline (PBS) (5 min/wash), and then embedded in optimum cutting temperature compound (Tissue-Tek; Sakura Finetek USA Inc, Torrance, CA). We incubated 10-μm frozen embryonic sections with 1.5 μmol/L DHE for 5 minutes at room temperature, then washed them 3 times with PBS for 5 min/wash. Sections were counterstained with 4′,6-diamidino-2-phenylindole (Sigma, St Louis, MO) and mounted with aqueous mounting medium (Sigma).
Lipid hydroperoxide quantification
As previously described, the degree of lipid peroxidation in E12.5 hearts was quantitatively assessed using the Calbiochem lipid hydroperoxide assay kit (Millipore, Bedford, MA) by following the manufacturer’s instructions. Lipid hydroperoxides of embryonic hearts were extracted by deoxygenated chloroform, and then measured by the absorbance of 500 nm after reaction with chromogen. The results were expressed as μmol/L lipid hydroperoxides/μg protein. Protein concentrations were determined by the BioRad detergent compatible protein assay kit (Hercules, CA).
Immunoblotting
Immunoblotting was performed as described by Yang et al. To extract protein, the lysis buffer (Cell Signaling Technology, Danvers, MA) containing a protease inhibitor cocktail (Sigma) was used. Equal amounts of protein and the Precision Plus Protein Standards (BioRad) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto Immunobilon-P membranes (Millipore). Membranes were incubated in 5% nonfat milk for 45 minutes, and then were incubated for 18 hours at 4°C with the following primary antibodies at dilutions of 1:1000 in 5% nonfat milk: phosphorylated protein kinase RNA-like endoplasmic reticulum kinase; protein kinase RNA-like endoplasmic reticulum kinase; p-eIF2α; eIF2α; CHOP; BiP; IRE1α (cell signaling); p-IRE1α (Abcam, Cambridge, United Kingdom); caspase 8 (mouse specific) (Enzo Life Sciences, Farmingdale, NY); and caspase 3 (Millipore). Membranes were exposed to horseradish peroxidase–conjugated goat antirabbit, goat antimouse, or goat antirat (Millipore) secondary antibodies. Signals were detected using SuperSignal West Femto maximum sensitivity substrate kit (Thermo Scientific), and chemiluminescence emitted from the bands was directly captured using a Bioimage EC3 system (UVP Inc, Upland, CA). Densitometric analysis of chemiluminescence signals was performed using software (VisionWorks LS; UVP Inc). To ensure that equivalent amounts of protein were loaded among samples, membranes were stripped and incubated with a β-actin antibody (Abcam). All experiments were repeated in triplicate with the use of independently prepared tissue lysates.
Detection of XBP1 messenger RNA splicing
Messenger RNA (mRNA) was extracted from E12.5 embryonic hearts and reverse-transcribed using the QuantiTect reverse transcription kit (Qiagen, Venlo, The Netherlands). The polymerase chain reaction (PCR) primers for X-box binding protein 1 were as follows: forward, 5’-GAACCAGGAGTTAAGAACACG-3’ and reverse, 5’-AGGCAACAGTGTCAGAGTCC-3’. If no XBP1 mRNA splicing occurred, a 205-base pair (bp) band was produced. When XBP1 splicing occurred, a 205-bp band and a 179-bp main band were produced.
Real-time quantitative PCR
Using the Rneasy mini kit (Qiagen), mRNA was isolated from E12.5 embryonic hearts, and then reverse-transcribed using the high-capacity complimentary DNA archive kit (Applied Biosystems, Grand Island, NY). Real-time PCR for BiP, CHOP, calnexin, IRE1α, protein disulphide isomerase A, 94 kDa glucose-regulated protein, and β-actin were performed using the Maxima SYBR green/ROX quantitative PCR Master Mix assay (Thermo Scientific) in the StepOnePlus system (Applied Biosystems). Primer sequences for real-time PCR are listed in Table 1 .
| Primers name | Primer sequences |
|---|---|
| BiP | Forward primer 5’-ACTTGGGGACCACCTATTCCT-3’ |
| Reverse primer 5’-ATCGCCAATCAGACGCTCC-3’ | |
| CHOP | Forward primer 5’-CGGAACCTGAGGAGAGAGTG-3’ |
| Reverse primer 5’-CTGTCAGCCAAGCTAGGGAC-3’ | |
| Calnexin | Forward primer 5’-ATGGAAGGGAAGTGGTTACTGT-3’ |
| Reverse primer 5’-GCTTTGTAGGTGACCTTTGGAG-3’ | |
| IRE1α | Forward primer 5’-ACACCGACCACCGTATCTCA-3’ |
| Reverse primer 5’-CTCAGGATAATGGTAGCCATGTC-3’ | |
| PDIA | Forward primer 5’-CGCCTCCGATGTGTTGGA-3’ |
| Reverse primer 5’-CAGTGCAATCCACCTTTGCTAA-3’ | |
| GRP94 | Forward primer 5’-TCGTCAGAGCTGATGATGAAGT-3’ |
| Reverse primer 5’-GCGTTTAACCCATCCAACTGAAT-3’ | |
| β-Actin | Forward primer 5’-GAACCAGGAGTTAAGAACACG-3’ |
| Reverse primer 5’-AGGCAACAGTGTCAGAGTCC-3’ |
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