Disturbed intracellular calcium homeostasis in neural tube defects in diabetic embryopathy

Abstract

Pregnancies complicated by preexisting maternal diabetes mellitus are associated with a higher risk of birth defects in infants, known as diabetic embryopathy. The common defects seen in the central nervous system result from failure of neural tube closure. The formation of neural tube defects (NTDs) is associated with excessive programmed cell death (apoptosis) in the neuroepithelium under hyperglycemia-induced intracellular stress conditions. The early cellular response to hyperglycemia remains to be identified. We hypothesize that hyperglycemia may disturb intracellular calcium (Ca²⁺) homeostasis, which perturbs organelle function and apoptotic regulation, resulting in increased apoptosis and embryonic NTDs. In an animal model of diabetic embryopathy, we performed Ca²⁺ imaging and observed significant increases in intracellular Ca²⁺ ([Ca²⁺]_i) in the embryonic neural epithelium. Blocking T-type Ca²⁺ channels with mibefradil, but not L-type with verapamil, significantly blunted the increases in [Ca²⁺]_i, implicating an involvement of channel type-dependent Ca²⁺ influx in hyperglycemia-perturbed Ca²⁺ homeostasis. Treatment of diabetic pregnant mice with mibefradil during neurulation significantly reduced NTD rates in the embryos. This effect was associated with decreases in apoptosis, alleviation of endoplasmic reticulum stress, and increases of anti-apoptotic factors. Taken together, our data suggest an important role of Ca²⁺ influx in hyperglycemia-induced NTDs and of T-type Ca²⁺ channels as a potential target to prevent birth defects in diabetic pregnancies.

1. Introduction

Diabetes mellitus in pregnancy has profound impact on the development of the embryo and fetus. One of the adverse outcomes is birth defects in newborn infants, a complication known as diabetic embryopathy [1,2]. Abnormalities in the central nervous system, including exencephaly and spina bifida, are commonly seen in affected infants. The anomalies are from the result of incomplete closure of the neural tube during early embryogenesis, and, thus, referred to as neural tube defects (NTDs) [3]. The failure of neural tube closure is intimately associated with excessive programmed cell death (apoptosis) in the neural epithelium [2]. Although the induction of apoptosis has not been completely understood, it has been observed that hyperglycemia causes profound changes in intracellular conditions in the embryo. Hyperglycemia perturbs the function of the endoplasmic reticulum (ER), resulting in abnormal protein folding and retention of misfolded proteins in the ER. The stress of the ER activates signaling cascades involving CHOP (C/EBP homologous protein), PERK (Protein kinase RNA-like endoplasmic reticulum kinase), and eIF2α (eukaryotic initiation factor 2α) in the so-called unfolded protein response to regulate protein quality control and cellular activities [4,5]. Hyperglycemia also affects mitochondrial apoptotic factors, including Caspase regulators and Bcl-2 family members, leading to increases in apoptosis [2,6]. However, the cell’s early response to hyperglycemia which triggers the profound intracellular events remains to be identified.

It has been shown that under maternal hyperglycemic conditions, glucose enters embryonic cells through glucose transporters [7]. Such activity may alter the properties of the cell membrane, leading to changes in ion transport and cellular ion homeostasis.

Calcium (Ca²⁺) is an important signaling regulator in cellular activities and embryogenesis [8,9]. Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) perturbs function of organelles and subsequently leads to cell apoptosis [10]. Free intracellular Ca²⁺ comes from two major sources: the extracellular space and intracellular stores [11]. Extracellular Ca²⁺ enters the cell through voltage-gated transmembrane channels, including low voltage-activated transient (T) and high voltage-activated long-lasting (L) types of channels [12,13]; intracellular Ca²⁺ is released from the ER and mitochondrion [14]. The aims of this study were to characterize changes in [Ca²⁺]_i in embryonic cells under high glucose conditions, and investigate a potential association between increased levels of [Ca²⁺]_i and diabetes-induced NTDs.

2. Materials and methods

2.1. Embryo culture and calcium imaging

This 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, and the protocol was approved by the Institutional Animal Care and Use Committee of University of Maryland, Baltimore.

Briefly, female and male mice (C57BL/6J) were paired for mating in the afternoon. The presence of the vaginal plug the next morning was designated as embryonic (E) day 0.5. At E8.5, embryos were collected in cold minimal essential medium (MEM; Invitrogen). The embryos were cultured in MEM containing normal (150 mg/dl; NG) and high glucose (400 mg/dl; HG) at 37 °C for 4 h.

Ca²⁺ imaging was performed using fluorescent indicators, Fluo-4 AM or Indo-1 AM (Life Technologies, Carlsbad, CA). Fluo-4 can indicate both level and in situ localization of Ca²⁺ in the tissue. Indo-1 has two optimal emission wave lengths for free (485 nm) and Ca²⁺-bound (405 nm) forms. The ratio of fluorescent emission between 405 nm and 485 nm provides reliable quantification of intracellular Ca²⁺. Embryos were incubated with 1 µM Ca²⁺ indicator in the same media (NG or HG) for 30 min at room temperature. The embryos were then transferred to Ca²⁺ indicator-free media for imaging under an inverted microscope (Olympus IX-71) coupled to a Bio-Rad CellMap IC confocal imaging system and Ca²⁺ fluorometry apparatus. For Fluo-4 imaging, signals (excitation: 488 nm; emission: 505 nm) were captured using a × 20, 0.5 numerical aperture objective. For Indo-1 imaging, excitation light from a broadband UV source was filtered through a 380 ± 10 nm bandpass filter. Fluorescence emission was collected at 405 and 485 nm simultaneously, using two photomultipliers with a sampling rate of 2 kHz. The emission signals were digitized using a built-in AD/DA converter of an EPC10 amplifier and the acquisition software Patchmaster (HEKA Instruments Inc., Bellmore, NY). Five spots along the neuroepithelium per embryo were measured taken at different locations on the embryos.

The embryos were treated with the Ca²⁺ channel blockers, mibefradil (Mib; T-type; CAS 116644-53-2; Sigma-Aldrich) and verapamil (Ver; L-type; CAS 52-53-9; Sigma-Aldrich), dissolved in dimethyl sulfoxide (DMSO; vehicle = VEH) and added to media in a 1:1000 dilution for 4 h and then subjected to Ca²⁺ imaging.

2.2. Diabetic animal model and treatment

Female mice (C57BL/6J) were injected intravenously with streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO) in 0.1 M citrate buffer (pH 4.5; 65 mg/kg body weight) to eliminate insulin-producing β-cells in the pancreas. Diabetes mellitus (DM) was defined as blood glucose levels reaching ≥14 mM (250 mg/dl). Euglycemia (7–8 mM) was restored in the diabetic mice by subcutaneous implantation of insulin pellets (Linshin Canada, Scarborough, ON, Canada). At E5.5, insulin implants from the DM group were removed to make the female mice hyperglycemic again before the neurulation period (E6.5). The non-diabetic (ND) control group was STZ-injected mice whose insulin pellets remained implanted throughout the experiments.

Mib in DMSO (vehicle = VEH) was suspended in water in 1:1000 dilution and administered via gavage feeding to DM pregnant mice at 10 µg/kg body weight, once a day from E7.5 to E9.5. Maternal blood glucose levels were monitored every day from E6.5 to E10.5. At E10.5 (late neurulation), embryos from three groups, ND, DM + VEH, and DM + Mib, were collected for examination of NTDs.

2.3. TUNEL assay

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect apoptotic cells. Briefly, E10.5 embryos were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) at 4 °C for 24 h, and embedded in paraffin wax through dehydration with ethanol and xylene. Tissue sections (6-µm thick) were cut using a microtome. Tissue sections were dewaxed in xylene and rehydrated through a reverse ethanol concentration series to water.

Tissue sections were treated with proteinase K (20 µg/ml) for 15 min at room temperature, incubated with TUNEL reagents containing dUTP-fluorescein (Roche, Indianapolis, IN) for 2 h at 37 °C, and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The sections were observed under a fluorescent microscope (Zeiss, Thornwood, NY). Microscopic images were captured using a CCD camera connected to an image analysis station and arranged using the Adobe Photoshop software program. At least four embryos in each group were used for the assay.

2.4. Western blotting

Neural tissues in the brain region of E10.5 embryos were isolated and individually collected using fine scissors under a stereo-microscope in cold PBS (pH 7.4). The tissues were homogenized in a lysis buffer [25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)] containing protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL). The tissue homogenates were centrifuged at 14,000 rpm for 15 min at 4 °C to obtain supernatants.

Protein samples (20–40 µg) were resolved in 10% polyacrylamide gel using electrophoresis in the presence of SDS, and blotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking with 10% non-fat milk, the membranes were incubated with primary antibodies for 16 h at 4 °C. The primary antibodies against CHOP, phosphorylated (p) PERK, p-eIF2α, Survivin, and Bcl-xL were purchased from Cell Signaling Technology (Beverly, MA). This was followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. Signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Images were captured and density of the bands was measured using UVP Bioimage system (UVP, Upland, CA).

The same membranes were stripped using Restore Western Blot Stripping Buffer (Thermo Scientific) and probed again with an antibody against β-actin (Abcam, Cambridge, MA) to control for equal loading of protein samples. The values of β-actin band density were used to normalize those of the corresponding bands of interest.

2.5. Statistical analyses

NTD rate was calculated as a percentage of the total number of embryos. Log binomial models for clustered data were applied to compare the NTD rates between groups, with calculated confidence intervals. Normal distribution of Indo-1 fluorescence data was assessed using the Kolmogorov-Smirnov test. Ratios of fluorescence values at two different wavelengths and ratios of band density of interest to that of β-actin on Western blots, presented as mean ± standard deviation (SD), were analyzed using the Student t-test. A p-value of <0.05 was considered statistically significant.

3. Results

3.1. High glucose elevates intracellular Ca²⁺ in the neuroepithelium

To investigate whether hyperglycemia increases [Ca²⁺]_i, we examined the changes in fluorescence of Fluo-4 in E8.5 embryos treated with normal (150 mg/dl; NG) and high (400 mg/dl; HG) concentrations of glucose. Fluorescent intensities in the neuroepithelium were higher in the HG group than in the NG group (Fig. 1A and B). We further quantified the changes in [Ca²⁺]_i, using a ratio-metric Indo-1 indicator, which emits fluorescence at 405 nm (Ca²⁺-bound) and 485 nm (Ca²⁺-free) wavelengths (Fig. 1E). The ratios in fluorescent intensities between the two wavelengths indicated significant elevations of [Ca²⁺]_i in the embryos of the HG group, compared with those in the NG group (Fig. 1F).

Fig. 1.

Ca²⁺ imaging of mouse embryos. Intracellular Ca²⁺ was measured in the neuroepithelium of E8.5 embryos using Fluo-4 (A,B) and Indo-1 (E,F) indicators. (C,D) Transmitted light microscopic images of the embryos. (A,C) Normal glucose (NG; 150 mg/dl). (B,D) High glucose (HG; 400 mg/dl). (E) Real-time record of Indo-1 emissions at different wave lengths. (F) Quantification of intracellular Ca²⁺. Data are presented as mean ± SD. *p < 0.0001. n = 5 embryos (4 measures in each embryo).

3.2. Involvement of Ca²⁺ channels in the elevation of [Ca²⁺]_i in the neuroepithelium

Free intracellular Ca²⁺ can result from influxes of extracellular Ca²⁺ through transmembrane channels. To determine whether the elevated [Ca²⁺]_i we observed in HG embryos came from an extracellular source, we incubated NG and HG E8.5 embryos with Ca²⁺ channel blockers. The T-type channel blocker Mib, but not the L-type channel blocker Ver, significantly blunted the increase of intracellular Ca²⁺ in the embryos of HG group in a concentration-dependent manner (Fig. 2).

Fig. 2.

Effect of Ca²⁺ channel blockade on intracellular Ca²⁺ levels. E8.5 embryos cultured in high glucose were treated with verapamil (Ver; L-type) and mibefradil (Mib; T-type). Intracellular Ca²⁺ was measured using Indo-1 imaging. Data are presented as mean ± SD. *p < 0.0001, compared with vehicle (VEH) control. n = 5 embryos (4 measures in each embryo).

3.3. Reduction of NTD rate and apoptosis by T-type Ca²⁺ channel blockade

Based on our results suggesting that T-type Ca²⁺ channel is involved in hyperglycemia-induced Ca²⁺ influx to disturb intracellular Ca²⁺ homeostasis, we further explored whether blocking T-type Ca²⁺ channels could prevent embryonic malformations under maternal diabetic conditions. When diabetic pregnant mice were treated with Mib during early neurulation (from E6.5 to E9.5), NTD rates were significantly decreased in their embryos, compared with embryos from dams treated with VEH (Fig. 3A–C; Table 1). In association with the decreases in NTD rate, Mib treatment also reduced apoptosis in the neuroepithelium of the embryos in diabetic litters (Fig. 3D–L).

Fig. 3.

Effects of T-type Ca²⁺ channel blockade on NTD formation and apoptosis. (A–C) NTDs in E10.5 embryos. (A) non-diabetic (ND), (B) diabetic (DM)+vehicle (VEH), and (C) DM+Mib. Arrow indicates open neural tube in the brain. Scale bar = 200 µm, applied to A–C. (D–L) Apoptosis in the neuroepithelium of E9.5 embryos. (D–F) ND. (G–I) DM + VEH. (J–L) DM+Mib. (D,G,J) TUNEL assay (signals in green), (E,H,K) DAPI, (F,I,L) Merged. Scale bar = 100 µm, applied to D–L.

Table 1.

Effect of calcium channel blockade on NTD rate in diabetic embryopathy.

Groups	Total number of Embryos (litters)	Embryos with NTDs	NTD rate (%)	Blood glucose (mg/dl; mean ± SD)
DM+VEH	78 (10)	15	19.23	297.6 ± 8.21
DM+Mib (10 µg/kg)	61 (8)	2	3.28*	305.1 ± 12.92

DM, diabetes mellitus; Mib, mibefradil; VEH, vehicle.

^*p < 0.05.

3.4. Effects of Ca²⁺ channel blockade intracellular stress and apoptotic regulation

Elevation of [Ca²⁺]_i may perturb calcium homeostasis in the ER and the function of the organelle, leading to activation of apoptotic process. Examination of ER stress factors, Chop, p-Perk, p-eIF2α, in embryos showed significant increases in the DM+VEH group, compared with those in the ND group (Fig. 4A–C). Treatment with Mib significantly reduced the levels of these factors in the embryos of the DM+Mib group, compared with those in the DM+VEH group (Fig. 4A–C).

Fig. 4.

Effects of T-type Ca²⁺ channel blockade on ER stress and anti-apoptotic factors in the neuroepithelium. (Upper images) Western blots. β-actin used as loading control. (Lower bar graphs) Quantification of the bands on the Western blots. Data are presented as mean ± SD. *p < 0.05, compared with ND or DM+Mib. n = 4 embryos. (A) Chop, (B) p-Perk, (C) p-eIF2α, (D) Survivin, (E) Bcl-xL.

The expression of anti-apoptotic regulators, Survivin (Caspase inhibitor) and Bcl-xL, in the embryos was significantly decreased in DM+VEH group, compared with that in the ND group (Fig. 4D and E). Treatment with Mib significantly upregulated the expression of Survivin and Bcl-xL in the DM+Mib group, compared with that in the DM+VEH group (Fig. 4D and E).

4. Discussion

In diabetic embryopathy, maternal hyperglycemia causes dramatic elevation of [Ca²⁺]_i in the neural epithelium of the embryo. The source of Ca²⁺ elevation is largely from the extracellular space via influx through T-type Ca²⁺ channels. The increases in intracellular Ca²⁺ levels are associated with dysfunctions in the ER and mitochondria, manifested as ER stress and altered apoptotic regulation. Importantly, blockade of T-type Ca²⁺ channels alleviated intracellular stress conditions, suppressed apoptosis, and significantly reduced NTD rate in the embryos of diabetic mice.

Increased apoptosis in the neuroepithelium has been shown to be associated with the formation of NTDs in the embryos of diabetic animals [2]. Signaling systems that likely regulate apoptosis have been previously characterized [15]. However, the early responses of embryonic cells to hyperglycemia, which potentially trigger apoptotic execution, remain to be identified. This report shows that a change in Ca²⁺ homeostasis may be one of the early responses to maternal hyperglycemia.

Ca²⁺ is an important ion that regulates a large number of intracellular signaling systems and cellular activities, including mitosis and apoptosis [10]. Changes in [Ca²⁺]_i are a sensitive response to intracellular and extracellular stimulations [16]. The increases in [Ca²⁺]_i in the cells of embryos from diabetic pregnant dams indicate the involvement of Ca²⁺-regulated signaling in diabetic embryopathy.

Elevation of [Ca²⁺]_i disturbs intracellular Ca²⁺ homeostasis. The consequences include perturbation of organelles that are highly sensitive to Ca²⁺ changes, such as the ER and mitochondria [17]. Dysfunction of the ER results in defects in protein folding and subsequent retention misfolded proteins in the ER [18]. Under stress conditions, the ER activates signaling systems, known collectively as the unfolded protein response (UPR), to regulate cell mitosis and apoptosis [19,20]. The involvement of ER stress in diabetic embryopathy is indicated by previous observations that PERK and eIF2a are upregulated under high glucose conditions [4,5]. The results in this report show that inhibition of the hyperglycemia-induced elevation of [Ca²⁺]_i leads to alleviation of ER stress. The direct action of high Ca²⁺ on ER stress remains to be further demonstrated.

Intracellular Ca²⁺ homeostasis is maintained by regulation of Ca²⁺ entry through Ca²⁺ channels and Ca²⁺ release from intracellular stores in the ER and mitochondria [11]. Our experiments identified a route of intracellular Ca²⁺ elevation, which is transmembrane Ca²⁺ channels. However, Ca²⁺ release from the intracellular stores is not ruled out and requires investigations.

It has been well characterized that glucose entry opens transmembrane Ca²⁺ channels in various types of cell [21,22]. However, the question of how hyperglycemia alters Ca²⁺ channel activity in diabetic embryopathy remains to be answered. One possible mechanism is that hyperglycemia disturbs intracellular conditions, leading to alteration of Ca²⁺ channel activity [21]. It is also possible that glucose entry through glucose transporters may alter membrane properties and, thus, influence the activities of transmembrane Ca²⁺ channels [7].

Our experiments also demonstrate that hyperglycemia stimulates Ca²⁺ entry into embryonic cells via a Ca²⁺ channel typespecific manner. T-type, but not L-type, Ca²⁺ channels play an important role in the disturbance of intracellular Ca²⁺ homeostasis in embryos. T-type Ca²⁺ channels are more sensitive to stimulation than other types of Ca²⁺ channels and require less dramatic changes in membrane potential to open [23,24]. In human diabetic embryopathy, slight and brief increases in glucose level could have dramatic impact on embryogenesis [25]. This raises a question of whether this is associated with involvement sensitive Ca²⁺ channels in disturbance of intracellular Ca²⁺ homeostasis.

T-type Ca²⁺ channels are expressed in the neuroepithelium and play a role in Ca²⁺regulation during early embryogenesis [26–28]. In diabetic embryopathy, blocking T-type Ca²⁺ channels during neurulation significantly decreased the NTD rate. These data suggest that increases in [Ca²⁺]_i in embryonic cells are sufficient to disrupt normal development of the neural epithelium and cause NTDs. These results also imply that selective targeting of certain types of Ca²⁺ channels can be a potential strategy to effectively protect the embryos from hyperglycemic insult.

Ca²⁺ channel blockers are commonly used in pregnancy to ameliorate maternal hypertension and inhibit preterm birth [29–32]. Treatments with Ca²⁺ channel blockers during early pregnancy have not been found to increase teratogenic risk [33,34]. Because embryonic malformations occur during early organogenesis, attenuation of T-type Ca²⁺ channel activity during the short vulnerable period of embryogenesis can be a practical approach for preventing fetal abnormalities in diabetic pregnancies.