Exogenous H2S regulates endoplasmic reticulum-mitochondria cross-talk to inhibit apoptotic pathways in STZ-induced type I diabetes
Abstract
The upregulation of reactive oxygen species (ROS) is a primary cause of cardiomyocyte apoptosis in diabetes cardiomyopathy (DCM). Mitofusin-2 (Mfn-2) is a key protein that bridges the mitochondria and endoplasmic reticulum (ER). Hydrogen sulfide (H2S)-mediated cardioprotection is related to antioxidant effects. The present study demonstrated that H2S inhibited the interaction between the ER and mitochondrial apoptotic pathway. This study investigated cardiac function, ultrastructural changes in the ER and mitochondria, apoptotic rate using TUNEL, and the expression of ER stress-associated proteins and mitochondrial apoptotic proteins in cardiac tissues in STZ-induced type I diabetic rats treated with or without NaHS (donor of H2S). Mitochondria of cardiac tissues were isolated, and MPTP opening and cytochrome c (cyt C) and Mfn-2 expression were also detected. Our data showed that hyperglycemia decreased the cardiac function by ultrasound cardiogram, and the administration of exogenous H2S ameliorated these changes. We demonstrated that the expression of ER stress sensors and apoptotic rates were elevated in cardiac tissue of DCM and cultured H9C2 cells, but the expression of these proteins was reduced following exogenous H2S treatment. The expression of mitochondrial apoptotic proteins, cyt C, and mPTP opening was decreased following treatment with exogenous H2S. In our experiment, the expression and immunofluorescence of Mfn-2 were both decreased after transfection with Mfn-2-siRNA. Hyperglycemia stimulated ER interactions and mitochondrial apoptotic pathways, which were inhibited by exogenous H2S treatment through the regulation of Mfn-2 expression.
diabetic cardiomyopathy (DCM) is one of the major cardiac complications that is independent of the coronary artery diseases and hypertension in diabetic patients (2, 3, 7, 21, 30, 37). Long-term hyperglycemia is a cellular insult that may induce the generation of excessive reactive oxygen species (ROS) in cardiomyocytes. Excessive ROS generation may be the pathological basis of diabetic cardiomyopathy (38). Mitochondria are likely the major source of ROS under hyperglycemic conditions. The metabolic abnormalities of diabetes cause mitochondrial ROS overproduction in the myocardium (16).
Previous results demonstrated the importance of mitochondrial ROS in the pathogenesis of diabetes mellitus and its complications through the modification of various cellular events in many important organelles (14).
The endoplasmic reticulum (ER) maintains calcium homeostasis and participates in protein folding and maturation (13). A variety of insults, such as oxidative stress, calcium overload, or unfolded proteins accumulation, lead to ER dysfunction and stress. Increased ROS generation often accompanies ER stress in DCM.
Recent studies suggest that ER stress is activated in diabetic hearts, and ER stress-mediated apoptosis is involved in the pathogenesis and development of DCM (5, 9, 10, 20, 39).
Increasing evidence, in the past few decades, demonstrates that the ER is not an isolated organelle, but it forms contact sites with many other cytoplasmic organelles, such as mitochondria, Golgi, and the plasma membrane (31). Some studies demonstrate that mitochondria and the ER are interconnected organelles that form an endomembrane network. The contact points where the ER communicates with mitochondria are referred to as mitochondria-associated membrane (MAM) (34). However, whether the ER and mitochondria act synergistically through a common domain in pathological conditions has not been determined.
Mitofusin-2 (Mfn-2) is a GTPase protein that is localized in the outer mitochondrial membrane and MAM (25). Mfn-2 participates in mitochondrial fusion and fission, but it also controls ER morphology and function (11). Increasing evidence shows that Mfn-2 is involved in mitochondrial function, but it also acts as a signaling molecule in the cardiovascular system. The upregulation of Mfn-2 induces cardiomyocyte apoptosis that is triggered by oxidative stress (32).
Hydrogen sulfide (H2S) has emerged as an important member of the family of gas transmitters, which also include nitric oxide and carbon monoxide. The physiological and pathophysiological roles of H2S in the regulation of cardiovascular function have received more attention (8, 22, 29, 40). Our previous studies demonstrated that H2S exerts its protective effects on myocardial injury through the amelioration of mitochondrial function (42). Therefore, the present study examined whether exogenous H2S inhibited apoptosis via regulation of E(S)R mitochondria-associated apoptotic pathways using a diabetic rat model in vivo and an H9C2 cell line in vitro. The possible mechanisms of these effects were also investigated.
MATERIAL AND METHODS
Reagents and Chemicals
Sodium hydrosulfide (NaHS) and streptozotocin were both purchased from Sigma Chemical (St. Louis, MO). Propidium iodide (PI), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), and Hoechst 33342 were obtained from Beyotime (Shanghai, China). N-acetylcysteine (NAC), Mitofusin-2, siRNA Mitofusin-2, and ER tracker Green were purchased from Cell Signaling Technology (Beverly, MA). Calcium green-5N and Lipofectamine 2000 were both obtained from Invitrogen (Carlsbad, CA). Caspase-12, cytochrome c, pro-caspase-3, cleavage-caspase 3, Bcl-2, Bax, ATF-6, GRP78, and CHOP antibodies were provided by Proteintech Group (Chicago, IL). DMEM-F12 medium and fetal bovine serum (FBS) were supplied by Hyclone (Logan, UT).
Diabetes Model and Treatment Protocols
Establishment of STZ-induced type I diabetes animal model.
All the experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the Chinese National Institutes of Health. The animal protocol was reviewed and adheres to the Declaration of Helsinki and International Ethical Guidelines for Biomedical Research. The study protocol was approved by the Animal Ethics Committee of Harbin Medical University.
Fifty male Wistar rats from the animal center of Harbin Medicine University (8 wk of age; mean body weight 240 ± 10 g) were used in this study. After 1 wk of acclimatization, streptozotocin (STZ; Sigma Chemical) dissolved in citrate buffer (pH 4.5) was administered intraperitoneally at a dose of 50 mg/kg body wt diluted in 0.3 ml of normal saline (0.9% NaCl injection USP, Baxter). To construct the Diabetic Cardiomyopathy Rat model, the 50 Wistar rats were sterilized by filtration. Hyperglycemia was confirmed by measuring the venous circulating plasma concentrations of glucose. Three days after STZ injection, blood samples were obtained from the rat tail vein after 12 h of fasting, and the glucose concentration was examined with an automatic analyzer (Roche Diagnostics, Indianapolis, IN). A blood glucose level of 16.7 mM was considered as a successful construction of the Type I diabetic rat model (6), whereas the value in the control group of rats injected with saline ranged from 5 to 10 mM. At 72 h following the STZ injection, blood samples were collected from the tail vein to measure blood glucose levels. Rats with fasting blood glucose levels > 16.7 mmol/l were considered as successful DM models and were used for further investigation (6). The normal rats that did not receive the treatment of STZ were randomly divided into the control group (treated with normal saline injection every day). By contrast, the rats that received the treatment of STZ were divided into two groups: the diabetes group, and the diabetes+ H2S (diabetic rats treated with concentration of NaHS; 100 μmol/kg). All rats were housed in individual cages with free access to normal rat diet and tap water. They were maintained under conditions of standard lighting (alternating 12:12-h light/dark cycle), temperature (20–22°C) and humidity (50–60%).
Daily administration in the NaHS treatment groups.
In the NaHS-treatment group, the remaining 20 rats were given the intraperitoneal injection of NaHS (100 μM).
Age-matched diabetes rats and control rats were killed after 4 and 8 weeks of treatment. Under deep anesthesia with chloral hydrate (0.3 ml/100 g), animals were killed by decapitation. Hearts obtained from the left ventricular were isolated immediately and washed thoroughly with normal saline to flush the blood and then immediately processed for biochemical and morphological studies. Each heart was subjected to 3 tests: Western blotting for ER stress pathway-associated protein indexes, transmission electron microscope, and TUNEL staining.
Determination of Blood Glucose
Random, morning blood glucose nonfasting concentration obtained from tail veins was determined at 4- to 8-wk intervals in all groups by Roche AccuChek Inform (Roche Diagnostics, Indianapolis, IN).
Echocardiography Measurement
Cardiac functions were assessed using an echocardiography system (GEV1V1D7 10s). Echocardiograms were performed on self-breathing rats under anesthesia (intraperitoneal injection of 10% chloral hydrate at 0.3 ml/100 g body wt). Left ventricular (LV) systolic and diastolic function parameters measured included left ventricular end-systolic volume (LVESV), left ventricular end-diastolic (LVEDV), left ventricular posterior wall in diastole (LVPWD), and left ventricular posterior wall in systolic (LVPWS). All parameters represent the means of five consecutive cardiac cycles.
Electron Microscopy
The mixed cells and left ventricular myocardial tissues were collected and fixed in 2.5% glutaraldehyde under transmission electron microscopy. After dehydration and embedding, ultrathin sections were prepared with Reichert-Jung Ultracut Ultramicrotome (Leica, Vienna, Austria). Images were observed under an H7650 transmission electron microscope (Hitachi, Tokyo, Japan). All ultrastructural analyses were performed in a blinded and nonbiased manner from photomicrographs captured using the electron microscope. We analyzed the data using at least 3 biological replicates.
TUNEL Staining
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used to test the apoptosis of cardiomyocytes in rat hearts. The cardiac tissues (n = 3 for diabetes group and diabetes + NaHS group, respectively) were fixed in 4% formalin overnight, dehydrated, and then embedded in paraffin. The tissues were cut into 7-mm-thick slices. The percentage of apoptotic cells was calculated as the ratio of the TUNEL positive cells to the total cells. The number of TUNEL positive cells (apoptotic cells) was counted in five randomly fields selected by inverted Olympus IX70 microscope.
Western Blotting
Harvested organs were frozen in liquid nitrogen and stored at −80°C. Tissues were homogenized in RIPA buffer with protease and phosphate inhibitors after the indicated treatments, and H9C2 cells were harvested and rinsed with ice-cold lysis solution. After determination of concentration with BCA protein assay kit, proteins were separated on a gradient SDS-PAGE, transferred to a nitrocellulose membrane, and blocked with 5% milk buffer. After blocking for 2 h at room temperature the membrane was incubated with various primary antibodies at 4°C overnight. Following 3 washes with TBS-T, the membranes were incubated with 1:1,000 dilutions of anti-rabbit or anti-mouse IgG antibodies at room temperature for 1 h and washed three times in TBST, respectively. The volume of the protein bands was quantified by densitometry analysis of the scanned blots using Bio-Rad Quantity One software (Bio-Rad Laboratories, Hercules, CA).
Separation of Mitochondrial Protein from Cardiac Tissues
Isolation of mitochondrial protein from left ventricular tissue was performed according to the manufacture’s protocol (Beyotime, Nantong, China). The cardiac tissues (n = 4-5, per group) were washed twice with ice-cold PBS, resuspended in lysis buffer, and then homogenized by an homogenizer in ice/water. After removing the nuclei and cell debris by centrifugation at 1,000 g for 10 min at 4°C, the supernatants were further centrifuged at 10,000 g for 20 min at 4°C. The resulting mitochondrial pellets were resuspended in lysis buffer, which was stored at −80°C.
Calcium Retention Assay
Calcium retention capacities of rat mitochondria were measured in 2 ml incubation buffer without EGTA at 25°C using 2.5 mM malate and 5 mM glutamate for complex 1. Extramitochondrial calcium was detected with 0.5 μM calcium green−5N (Invitrogen, Carlsbad, CA) with a Cary Eclipse spectrophotometer at excitation wavelength of 500 nm and emission wavelength of 530 nm. Experiments were done in the presence and absence of 1.2 mM MgCl2 and 40 μM ADP as well as 1 mM cyclosporine A. Ten nanomoles CaCl2 was added once per minute until MPTP opening was indicated by an increase in calcium green fluorescence.
Cell Culture and Treatment
H9C2 cells (5 × 105/ml) were cultured in Hyclone medium supplemented with 10% FBS, and 1% penicillin-streptomycin solution in Dulbecco’s modified Eagle medium (DMEM) equilibrated with humidified air containing 5% CO2 at 37°C humidified atmosphere. Forty-eight hours after the cells were seeded, the cultured cardiomyocytes were randomly divided into the following four groups: control (low glucose, LG, 5.5 mM) group, high glucose (HG, 25.0 mM) group, HG + GYY4137 (100 μM) group, and HG + NAC (100 μM) group. Mannitol (25 mM) was used as the control to exclude the interference of the high-osmotic condition.
Evaluation of Intracellular ROS Formation
Cells were incubated with 10 μM probe dihydroethidium (DHE; Beyotime, Shanghai, China) in a 37°C humidified incubator for 30 min and washed twice with phosphate-buffered saline, pH 7.4. DHE expression level was detected every 24 h and 48 h after high glucose injury under fluorescence microscope (IX-71, Olympus, Tokyo, Japan) by using a 488-nm excitation beam.
Cell Apoptosis and Necrosis Examination
Cells were treated with different concentrations of glucose for indicated times, and washed with PBS solution twice. For Hoechst 33342/PI dual-staining, H9C2 cells were first stained with Hoechst 33342 (10 μM) for 30 min at 37°C and then stained with PI (10 μM) for 5 min. After being washed with PBS twice, the cell nucleus was observed under the fluorescence microscope.
Determination of MPTP Opening
MPTP opening in situ was determined as described by Di Lisa et al. (12). Calcein-AM is a nonfluorescent, cell-permeable, and hydrophilic compound that is widely used for detection of cell viability. In live cells, the hydrolysis of calcein-AM by intracellular esterase produces strongly green fluorescent calcein, a hydrophilic compound that is well retained in the cell cytoplasm. Shortly, cardiac cells were incubated for 20 min with the fluorescence dye, calcein-AM (1 μM), which accumulates in cytosolic compartments, including the mitochondria. Then the medium was changed to calcein-free medium containing 1 μM CoCl2 and incubated at 37 °C for 20 min in the dark. The fluorescence from cytosolic calcein was quenched by the addition of CoCl2 (1 mM) for 20 min, while that from the mitochondrial calcein was maintained. MPTP opening was induced by adding ionomycin (5 μM). Then the cells were washed with ice-cold PBS (pH 7.2) for four times and the fluorescence intensity of cells exposed to high glucose or untreated control cells was measured with excitation at 490 nm and emission at 520 nm. The percentage loss of calcein fluorescence was used to determine MPTP opening.
Mfn-2 Short Interfering RNA Transfection
Mitofusin-2 siRNA (a pool of 3 target-specific, 19–25 nucleotide siRNAs designed to knockdown the gene expression of mitofusin-2 in rat) and scrambled control were purchased from Cell Signaling Technology (Beverly, MA). Transient transfection was initially standardized to improve knockdown efficiency. Subsequently, all the transient transfections were performed with 150 nM mitofusin-2 siRNAs using Lipofectamine 2000 (Invitrogen) as per manufacturer's protocol, and after 6 h, transfection mixtures were replaced with regular medium. Forty eight hours after transfection, cells were incubated with medium containing 5 or 25 mM glucose for 24 h. Cells were the processed for assessment of mitochondrial network and protein expression.
Immunofluorescence
H9C2 cells were cultured on chamber slides (Thermo Fisher Scientific) until 70% confluent (2–3 days). Cells were stained using antibodies specific for Mitofusin 2 (Cell Signaling Technology, Beverly, MA). Alexa Fluor 488 nm goat anti-rabbit (Invitrogen) secondary antibodies were used and nuclei were stained using DAPI. Staining was visualized by immunofluorescent microscopy, and representative images from a minimum of 10 high powered fields were captured to characterize phenotypic properties of H9C2 cell cultures.
Staining Procedures and Microscopic Experiments
For all microscopic experiments, 0.5 µl of ER tracker green DMSO stock solution (1 mM) was added into 2 ml of prewarmed culture medium and then incubated at 37°C, 5% CO2 for 30 min. After incubation, washing with fresh prewarmed PBS solution was carried out once. For costaining experiments, the final concentration of ER tracker Green and DAPI were 500 nM and 1 µM, respectively.
Statistical Analysis
All values are given as means ± SE for at least three independent experiment replicates. Statistical analysis was analyzed with Newman t-test or ANOVA as appropriate using Graph Pad Prism 5 for Windows platform (Graph Pad Software, San Diego, CA). P < 0.05 was considered statistically significant.
RESULTS
Establishment of the STZ-Induced Type I Diabetic Cardiomyopathy Model
A well-established type 1 diabetes mellitus model of STZ injection was used in this study. Random blood glucose levels were measured for 8 wk to determine the successful construction of the type I diabetic cardiomyopathy rat model (Fig. 1A). Blood glucose levels in the 4-wk and 8-wk diabetic groups were significantly higher compared with that in the control group (*P < 0.05). Blood glucose levels in the 4-wk and 8-wk diabetes rats were higher compared with the 4-wk and 8-wk diabetes + NaHS groups (P < 0.05), respectively.
Fig. 1.A: blood glucose levels (mg/dl) obtained via tail-vein puncture during the study period at 4- to 8-wk intervals. Diabetic and Diabetic + NaHS groups were compared with the corresponding values of the control group. Experimental groups were compared with their corresponding values after 4 and 8 wk. Values are given as means ± SE; n = 8. *P < 0.05 vs. control group; #P < 0.05 vs. 4-wk Diabetes group;▽P < 0.05 vs. 8-wk Diabetes group. Echocardiographic assessment of NaHS on cardiac left ventricle function: left ventricular end-diastolic volume (LVEDV; B); left ventricular end-systolic volume (LVESV; C); LV posterior wall in systole (LVEDs; D); and LV posterior wall in diastole (LVPWd; E). *P < 0.05 vs. control group; #P < 0.05 vs. Diabetes group, respectively (n = 3).
The LV posterior wall in diastole (LVPWd), LV posterior wall in systole (LVPWs), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) were examined using echocardiography. LVEDV decreased significantly in the 4-wk diabetes group compared with the control group. LVEDV and LVESV (Fig. 1, B and C) increased significantly in the 8-wk diabetes group compared with the control group. LVESV decreased in the 8-wk diabetes + NaHS group compared with the 8-wk diabetes group. No significant differences in LVESV were identified among the 4-wk diabetes and the diabetes + NaHS groups. LVPWd and LVPWs both decreased in diabetic groups compared with control, and these two indexes both increased in the diabetes + NaHS group compared with diabetic groups.
Exogenous H2S Supplementation Ameliorated Myocardial Tissue Injury in Diabetic Rats
Ultrastructural examination of the myocardium was observed using electron microscopy to demonstrate ultrastructural injury of cardiac tissues induced by type I diabetes. The mitochondrion (MIT) and sarcoplasmic reticulum (SR) are shown in Fig. 2 as white bold font. A linear arrangement of myocardiofibers and well-organized myocardial mitochondria and sarcoplasmic reticulum (SR) (Fig. 2A1) was observed in the control group. The myocardial SR was evidently elongated in the 4- and 8-wk diabetic groups, and the mitochondria were swollen and deformed. Mitochondrial cristae were also fractured in these groups (Fig. 2, A2 and A3). The dilation of myocardial SR was barely discernible in the diabetes + NaHS group, and mitochondrial structure was relatively normal (Fig. 2, A4 and A5).
Fig. 2.A: the ultrastructural changes in myocardium. TEM revealed that in rats in control group (A1), the structure of myocardial mitochondria (MIT) and sarcoplasmic reticulum (SR) was clearly observed; in the 4- and 8-wk Diabetes group (A2, A3), myocardial SR was evidently dilated, and mitochondria were swollen; whereas in the Diabetes + NaHS group (A4, A5), parts of the mitochondria were mildly swollen, and the SR showed moderate expansion. The outlined box represents the area that is magnified beside. White arrows represent mitochondrion or endoplasmic reticulum. B: the statistically analyzed data are shown together with images. *P < 0.05. C: representative illustration of TUNEL staining in the different groups of cardiomyocytes extracted from rat hearts. Normal heart as the control group is shown on the top of the picture (C1). Nuclei with brown staining indicate TUNEL-positive cells. Diabetic rats at 4 and 8 wk (C2, C3); Diabetes + NaHS at 4 and 8 wk (C4, C5) (magnification, ×400). Bar graph, quantitative results of TUNEL staining of rat hearts after different treatments. Values are means ± SE (n = 3). *P < 0.05 vs. control group; #P < 0.05 vs. 4-wk Diabetes group;▽P < 0.05 vs. 8 -wk Diabetes group.
Apoptotic cells were identified using the TUNEL assay. Brown-stained nuclei indicated apoptotic cells, and blue-green or tan shades signified nonapoptotic cells in diabetic rat hearts. Few apoptotic cells were observed in the control group, and cardiomyocytes were the major apoptotic cells in STZ-induced rats. The number of apoptotic cells (TUNEL-positive nuclei) increased significantly in 4- and 8-wk STZ-induced rats (Fig. 2, C2 and C3) compared with that of control group (Fig. 2, C1). Exogenous H2S administration in 4- and 8-wk (Fig. 2, C4 and C5) reduced apoptosis (P < 0.05) (Fig. 2D).
Exogenous H2S Supplementation Downregulated Myocardial ER-Related Proteins Expression in Diabetic Rats
The expression of ER stress-associated proteins was analyzed by Western blotting to elucidate whether the cytoprotective effect of H2S reduced ER stress against hyperglycemia-induced injury. The expression of GRP78 and p60 ATF-6 in cardiac tissues increased in 4- and 8-wk diabetes groups compared with control (P < 0.05). The expression of myocardial GRP78 and p60 ATF-6 decreased significantly in the Diabetes + NaHS groups compared with the diabetes groups (P < 0.05) after 4 and 8 wk of exogenous H2S treatment (Fig. 3A). The expression of CHOP and cleaved caspase-12 increased significantly in diabetic rats compared with control rats (P < 0.05), and exogenous H2S treatment decreased their expressions (P < 0.05) (Fig. 3B).
Fig. 3.Cardiac tissues were obtained from normal, Diabetes, and Diabetes+NaHS groups after 4 or 8 wk. Western blots of ER chaperone GRP78, p60 ATF6 (A), and the ER-associated apoptosis protein CHOP, cleaved caspase-12 (B) were examined. The top trace of each group shows representative blots of the respective protein, and the bottom panels show the bar graphs summarizing the immunoblot data. Densitometric results are expressed as a fold increase (*P < 0.05 vs. control group; #P < 0.05 vs. diabetes group). C: calcium retention capacity in control, Diabetes, and Diabetes + NaHS groups after 4 and 8 wk. At least 3 rats were included in each study group. The results are expressed as means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. Diabetes group.
Hyperglycemia-Induced Apoptosis is Associated with MPTP Opening, Which is Inhibited by NaHS Treatment
We investigated MPTP opening in isolated myocardial mitochondria from diabetic and NaHS-treated diabetic rats (Fig. 3C). MPTP opening was measured by a calcium retention assay using calcium green. The calcium concentration that was needed to induce MPTP opening was significantly lower in diabetic mitochondrial fractions than the control and NaHS treatment groups (P < 0.05).
H2S Supplementation Ameliorated High Glucose-Induced Injury in H9C2 Cells
The ultrastructure of cardiomyocytes was examined to further elucidate the effect of H2S treatment on hyperglycemia-induced cardiomyocyte injury. H9C2 cells in the control group were cultured in 5.5 mM DMEM, which was the low glucose culture group. Nuclear contour was an ellipse, and the nuclear envelope was both intact and relatively smooth. The nuclei-cytoplasmic ratio was uniform, and chromatin was distributed uniformly in the control group. Mitochondrion was well defined, and the ER appeared as flat vesicles that were lined up in a regular manner (Fig. 4A). Cells in the high glucose group were treated with 25 mM DMEM for 48 h. The nuclear contour was deformed, and the nuclear envelope was fractured and rugged. Mitochondria were swollen, and mitochondrial cristae were fractured and dissolved. The ER was significantly dilated and showed round vesicles. Cells in the HG + GYY4137 (100 μM) group were pretreated with the H2S donor GYY4137 (100 μM) for 30 min followed by coincubation with 25 mM DMEM for 48 h. The nuclear envelopes in these cells were relatively intact, and no discernible aggregation of chromatin was observed. Mitochondria were slightly swollen, and the ER was mildly dilated (Fig. 4C).
Fig. 4.The ultrastructural changes in H9C2 cells cultured after 48 h. TEM revealed that cells in 5.5 mM glucose (LG) exhibited clearly observed structures of H9C2 cells (A); in 25 mM glucose group (HG), the nuclear contour was deformed, and mitochondria were swollen (B), whereas in the HG + NaHS group, mitochondria were slightly swollen, and the ER was mild dilated (C). N represents cellular nuclear, n represents nucleolus, M represents mitochondria, and ER represents endoplasmic reticulum. D: effects of NaHS and NAC on hyperglycemia-induced overproduction of reactive oxygen species (ROS) in H9C2 cells. Random micrographs of DHE-derived fluorescence in H9C2 cells. LG, 5.5 mM glucose; HG, 25 mM glucose; HG + GYY4137, H9C2 cells were pretreated with 100 µM GYY4137 for 30 min; HG + NAC, cells were treated with 100 µM NAC. All groups were treated for 24 or 48 h, and quantitative analysis of the mean fluorescence intensity was obtained in the indicated groups. Data are means ± SE (n = 3). *P < 0.05 compared with LG, #P < 0.05 compared with HG. E: cell apoptosis was assessed using Hoechst/PI staining for mannitol group; LG, 5.5 mM glucose; HG, 25 mM glucose; HG + GYY4137, H9C2 cells were pretreated with 100 µM GYY4137 for 30 min; HG + NAC, cells were treated with 100 µM NAC. All groups were treated for 24 h or 48 h. Summary data showing Hoechst-positive cells (%total counted cells) and PI-positive cells from 10 visual fields of 5 different areas. Values are means ± SE. *P < 0.05 vs. the LG group. #P < 0.05 vs. the HG group (n = 3). Original magnification, ×200.
Exogenous H2S and NAC Attenuated Hyperglycemia-Induced Apoptosis Mediated by ROS in H9C2 Cells
The quantity of ROS in H9C2 cells cultured with high glucose for 48 h was determined by DHE (Fig. 4D). As expected, exogenous H2S treatment significantly reduced high glucose-induced intracellular ROS levels (P < 0.05). H9C2 cells cultured in high glucose were also treated with the ROS scavenger NAC (100 μM). Intracellular ROS levels increased significantly 48 h after exposure to 25 mM DMEM. The NAC treatment group also reduced hyperglycemia-induced ROS accumulation (*P < 0.05 vs. HG group).
The effects of H2S against hyperglycemia-induced apoptosis were further examined using the Hoechst 33342/PI staining assay. Representative photomicrographs of nuclei morphology of H9C2 cells are shown in Fig. 4E. High glucose culture induced condensed and fragmented nuclei, which are characteristic of apoptosis. Treatment with GYY4137 and NAC at 100 μM significantly attenuated the increased cell apoptosis and necrosis compared with the high glucose cultured group (Fig. 4D) after 24 and 48 h in high glucose culture. These results confirmed the protective effects of H2S against hyperglycemia-induced apoptosis in H9C2 cells via suppression of ROS generation.
Exogenous H2S Modulated the Hyperglycemia-Induced Stimulatory Effects of ER Stress-Related Protein Expression in H9C2 Cells
Oxidative stress is the primary cause of high glucose-induced cardiomyocyte insults (13, 15). The effects of H2S and NAC on the expression of GRP78, ATF-6, and PERK in H9C2 cells were detected to assess whether the cytoprotective effect of H2S against hyperglycemia-induced cell apoptosis was associated with the inhibition of ER stress. H9C2 cells treated with GYY4137 and NAC for 48 h exhibited significantly attenuated expression of hyperglycemia-induced GRP78, PERK, and p60 ATF6 (Fig. 5A) (P < 0.05). These data suggest that hyperglycemia induced oxidative stress that upregulated the expression of ER stress-related proteins in H9C2 cells.
Fig. 5.Western blots of indicated proteins in H9C2 cells. LG, low glucose; HG, high glucose; HG + GYY4137, H9C2 cells were pretreated with 100 µM GYY4137 for 30 min; HG + NAC, cells were treated with 100 µM NAC (A and B). The top trace of each group shows representative blots of the respective proteins, and the bottom panels show the bar graphs summarizing the immunoblot data. Densitometric results are expressed as a fold increase. (*P < 0.05 vs. LG group; #P < 0.05 vs. HG group). C: high glucose induces apoptosis via caspase-3 activation and cleavage with increases in cytochrome c (cyt C) release to cytosol. H9C2 cells were treated with 5.5 mM glucose (lane 1), 25 mM glucose (lane 2), 100 μM GYY4137 (lane 3), and 100 µM NAC (lane 4) for 48 h. β-Actin served as the loading control. A representative Western blot using a cyt C antibody to assay cyt C content in the cytosol from H9C2 treated with GYY4137 (100 μM) or NAC (100 μM) cultured in high glucose. Data represent the best of 3 separate experiments. D: imaging of mitochondrial permeability transition pore (mPTP) opening with calcein. H9C2 cells were treated with LG, 5.5 mM glucose; HG, 25 mM glucose; HG + GYY4137(100 µM); HG + NAC(100 µM); and then further incubated for 48 h. The MPTP opening was measured by staining with calcein-AM and CoCl2 (n = 5 independent preparations).
Our purpose was to investigate whether intracellular ROS was associated with hyperglycemia-induced apoptosis in cardiomyocytes. Nakagawa et al. (24) demonstrated that caspase-12 is confined to the cytoplasmic side of the ER, and it plays an important role in ER stress-mediated cell death. GYY4137 (100 μM) treatment dramatically inhibited the increased expression of caspase-12. NAC treatment also decreased CHOP and caspase-12 expression (Fig. 5B).
The protein expression of mitochondrial cytochrome c indicated that the NaHS-treated group experienced a low amount of leakage of cytochrome c from the mitochondria to the cytoplasm compared with the HG group (Fig. 5C). We examined caspase-3 to determine whether increased apoptotic signaling was associated with hyperglycemia insult. Pro-caspase-3 is cleaved by active caspase-9, and caspase-3 plays a crucial role in apoptosis (23). Figure 5C also shows that high glucose significantly induced pro-caspase-3 cleavage to its 17-kDa subunits in H9C2 cells. Our data confirmed that high glucose activated caspase-3 in H9C2 cells, and H2S suppressed this activation. These results suggest that the inhibition of ER stress may be involved in the cytoprotective effects of H2S in hyperglycemia-induced cell apoptosis in H9C2 cells.
High Glucose-Induced Apoptosis Is Related to MPTP Opening in H9C2 Cells
Since MPTP has been recognized as one of the major target of Ca2+, and opening of MPTP marks the irreversible point of the cell death process (4), to examine whether calcium-dependent apoptosis induced by ER stress is mediated through MPTP, we stained H9C2 cells for calcein/Co2+ to test the opening of MPTP. We found the mitochondrial calcein in the cytoplasm decreased gradually after high glucose treatment. Treatment with GYY4137 significantly inhibited MPTP opening at 48 h after high glucose whereas treatment with NAC did not (Fig. 5D).
H2S Reversed Hyperglycemia-Induced Changes in Specific Mitochondrial Protein Content in H9C2 Cells
We examined the protein content of Bax and Bcl-2 in H9C2 cells. Bcl-2 is an antiapoptotic protein that prevents cyt C release from mitochondria. Bax is a proapoptotic protein that promotes cyt C release. The translocation of Bax to the outer mitochondrial membrane during apoptosis induces mitochondrial membrane permeabilization. Bcl-2 protein blocks this process (1). Therefore, the effects of H2S on Bcl-2 and Bax protein levels were also examined. Western blotting indicated that high glucose significantly decreased basal levels of Bcl-2 but increased Bax protein expression. The ratio of Bcl-2 over Bax decreased in high glucose cultured cells (Fig. 6). GYY4137 (100 μM) markedly attenuated this effect (P < 0.05 vs. high glucose).
Fig. 6.Apoptosis markers were evaluated in H9C2 cells. Western blotting analysis of the ratio of Bcl-2 over Bax of H9C2 cells in different condition. Data are means ± SE (n = 3). *P < 0.05 compared with low glucose; #P < 0.05 compared with high glucose.
H2S inhibits ROS-induced apoptosis via regulatlion of BCL-2 family proteins.
Mfn-2 Expression Increased In High Glucose-Induced H9C2 Cell Injury
Previous results showed that human Mfn-2 and its homologs localized to the mitochondrial outer membrane and played a crucial role in mitochondrial fusion 25. We found that Mfn-2 expression was increased in extracted cardiac mitochondria after 4 and 8 wk in diabetic rats (Fig. 7). H9C2 cells were treated with high glucose and GYY4137 for 48 h to induce ER stress to demonstrate a link between ER stress and the expression of proteins involved in mitochondrial dynamics. Mfn-2 expression levels were higher in the high glucose-cultured group than those in the GYY4137 treatment group, as determined using quantitative Western blotting (Fig. 8A). We also used immunofluoresence to verify that Mfn-2 was increased in the high glucose-cultured group. We use immunofluorescence to examine the expression of Mfn-2 in H9C2 cells (Fig. 8C). Figure 8D shows that mean fluorescence intensity of Mfn-2 was increased in H9C2 cells in the high glucose-cultured group, and Mfn-2 was decreased after 48 h of GYY4137 administration.
Fig. 7.Mfn-2 expression in extracted cardiac mitochondria in 4- and 8-wk diabetic groups. Western blots of Mfn-2 protein were examined. The top trace of each group shows a representative blot of Mfn-2 protein, and the bottom panel shows the bar graph summarizing the immunoblot data. Densitometric results are expressed as a fold increase. Data are means ± SE (n = 3). *P < 0.05 vs. control group; #P < 0.05 vs. Diabetes group.
Fig. 8.A: Mfn-2 protein levels in LG, HG, HG + GYY4137 (100 μM), or NAC (100 μM). B: H9C2 cells were transfected with Mfn-2 siRNA and cultured under high glucose. H9C2 cells were treated with 5.5 mM glucose (lane 1), 25 mM glucose (lane 2), HG + control-siRNA (lane 3), and HG + 150 nM Mfn-2siRNA (lane 4) for 48 h. β-Actin served as the loading control. The top trace of each group shows representative blots of the respective proteins, and the bottom panels show the bar graphs summarizing the immunoblot data. Densitometric results are expressed as a fold increase. *P < 0.05 vs. LG group; #P < 0.05 vs. HG group. C: images of intracellular localization using immunofluoresence in low glucose, high glucose, and high glucose with GYY4137. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) and antibodies for Mfn-2 (green). D: analysis of MFI in H9C2 cells.*P < 0.05 vs. control group. #P < 0.05 vs. high glucose group. E: cell apoptosis was assessed using Hoechst/PI staining for mannitol group; LG, 5.5 mM glucose; HG, 25 mM glucose; HG + control siRNA; HG+Mfn-2 siRNA, cells were treated with 150 nM mitofusin-2 siRNA. All groups were treated for 48 h. Summary data showing Hoechst-positive cells (% total counted cells) and PI-positive cells from 10 visual fields of 5 different areas. Values are means ± SE. *P < 0.05 vs. the LG group. #P < 0.05 vs. the HG group (n = 3). Original magnification, ×200.
Mfn-2 Silencing Blocked Hyperglycemia-Induced Apoptosis in H9C2 Cells
We determined whether Mfn-2 was necessary for increased ROS-induced cell apoptosis in the presence of high glucose using siRNA-mediated silencing of Mfn-2 in H9C2 cells. Figure 8B shows that the expression of Mfn-2 was reduced 48 h after transfection with siRNA targeting Mfn-2. To examine the apoptosis/necrosis data after transfection, we divided H9C2 cells into 4 groups: low glucose, high glucose, high glucose + control siRNA, and high glucose + Mfn-2 siRNA. Our results showed that the ratio of apoptosis and necrosis in H9C2 cells was reduced 48 h after transfection with siRNA targeting Mfn-2 compared with both the HG group and the HG + control siRNA group (Fig. 8E).
Notably, exogenous H2S after transfection with Mfn-2 siRNA fully protected H9C2 cells against hyperglycemia-induced endoplasmic reticulum stress compared with the high glucose-cultured group, as measured by ER-tracker (Fig. 9). Mfn-2 triggers cardiomyocyte apoptosis via the primary mitochondrial apoptotic pathway (32). We found that cytosolic cyt C and Bax were markedly elevated in cells treated with high glucose (25 mM for 48 h), but treatment with GYY4137 and Mfn-2 siRNA reduced the expression of Bax and cyt C. Notably, GYY4137 and Mfn-2 siRNA fully protected H9C2 cells against high glucose-induced apoptosis (Fig. 10). Taken together, these results suggest that exogenous H2S downregulates Mfn-2 expression to reduce H9C2 cell apoptosis.
Fig. 9.H9C2 cells were cultured in different conditions and treatments. A: LG, low glucose (5.5 mM). B: HG, high glucose (25 mM). C: HG + GYY4137 (100 μM). D: transfected with Mitofusin-2 siRNA in high glucose culture. H9C2 cells were stained with ER-tracker (green), and the nucleus was stained with blue-fluorescent DAPI. Fluorescence was observed under a nonconfocal fluorescence microscope. Original magnification, ×200. E: The analysis of MFI in H9C2 cells. Data are means ± SE. *P < 0.05 vs. the LG group. #P < 0.05 vs. the HG group (n = 3).
Fig. 10.The mitochondrial apoptotic pathway is essentially involved in Mfn-2-mediated H9C2 cell apoptosis. H9C2 cells were treated with 5.5 mM glucose (lane 1), 25 mM glucose (lane 2), 100 μM GYY4137 (lane 3), and 100 µM NAC (lane 4) for 48 h. β-Actin served as the loading control. A representative Western blot using cyt C and Bax antibody to assay protein content in H9C2 cells treated with GYY4137 and Mfn-2 siRNA cultured in high glucose. Data represent the best of 3 separate experiments. *P < 0.05 vs. the LG group. #P < 0.05 vs. the HG group (n = 4).
DISCUSSION
Diabetic cardiomyopathy is characterized by a decrease in cardiac systole (30) and an increase in cardiomyocyte apoptosis. Hyperglycemia is the key initiating factor in the development of DCM, and it generates a cascade of events in the heart that eventually result in chronic heart failure (30). Oxidative stress plays a major role in DCM. This study addressed the potential of exogenous H2S to decrease ROS-mediated cardiomyocyte apoptosis through the SR and mitochondria subjected to hyperglycemia and high glucose. The following results are the main findings of this study: 1) exogenous H2S inhibited hyperglycemia and high glucose-mediated cardiomyocyte apoptosis via regulation of the ER and mitochondrial apoptotic pathway; 2) Mfn-2 mediated oxidative stress-induced cardiomyocyte apoptosis via the ER and mitochondrial apoptotic pathway; and 3) H2S protected against diabetic cardiomyopathy via suppression of Mfn-2 expression to decrease cardiomyocyte apoptosis.
The overproduction of ROS in DCM is a direct consequence of hyperglycemia or high glucose, which is the unifying upstream mechanism of diabetic complications. Increasing evidence indicates that ER stress-mediated apoptosis is involved in the progression of DCM, but there is no direct evidence for the involvement of ER stress in diabetes-induced cardiomyocyte apoptosis and DCM. Several lines of evidence demonstrated that increased extracellular glucose rapidly stimulated the generation of intracellular ROS. Recent studies confirmed that ROS are crucial regulators of ER function and UPR (unfolded protein response) activation. ER stress and increased ROS production occur concurrently (41). Verfaillie and colleagues (35) demonstrated that ROS production in the ER, as detected using a photosensitizer, was also associated with the induction of UPR genes, which suggests the occurrence of ER stress. Our ultrastructural examination showed that myocardial SR was evidently enlarged in the diabetic group, the mitochondria were swollen and deformed, and mitochondria cristae fractured and dissolved (Fig. 2A). Grp78 is an ER-chaperon protein that exhibits increased expression during ER stress, and this protein is likely a biomarker of ER stress. The unfolded protein loads in the endoplasmic reticulum exceed the processing capacity of the endoplasmic reticulum, and cellular apoptosis may be initiated and accompanied by the increased expression of CHOP and caspase-12 (18). Our results showed that hyperglycemia and high glucose upregulated caspase-12 and CHOP, which are markers of the ER stress-mediated apoptotic pathway in STZ-induced diabetic cardiac tissues (Fig. 3B) and H9C2 cells (Fig. 5B), and this upregulation was concomitant with increased apoptosis.
The ER and mitochondria are spatially close organelles that exhibit a network structure, which facilitates the formation of interorganellar communications. Mitochondria-ER associated membranes (MAMs) are specific domains where the ER and mitochondria join together as contact sites. MAMs provide a platform to coordinate calcium transfer, inflammasome formation, and the provision of membranes for autophagy (17). However, some studies revealed that ROS modulate ER-mitochondrial cross-talk during ER stress-evoked apoptosis (33). Bcl-2 is an antiapoptotic protein that is vital for the integrity of the mitochondrial membrane because it prevents the release of proapoptotic proteins, such as cytochrome c. Our results demonstrated that H2S treatment increased Bcl-2 protein expression after high glucose injury and inhibited apoptosis, as revealed by decreasing levels of cleaved caspase-3 in H2S-treated H9C2 cells (Figs. 5C and 6). H2S was previously shown to protect the diabetic cardiomyopathy by preserving mitochondrial function (42). Therefore, it is possible that H2S mediates the preservation of mitochondrial function after high glucose injury via the upregulation of Bcl-2 expression. H2S is an endogenous gas molecule that exerts different effects in various tissues. Recent studies demonstrated that H2S served as an antioxidant gas in the cardiovascular system (22). The physiological concentration of H2S is involved in the protective effects on mitochondrial functions and the promotion of the survival of myocardial cells.
Supplementation of H2S in the present study reduced total cytoplasmic ROS levels and alleviated myocardial injury. Ultrastructural examination demonstrated that the structure of the myocardial SR was restored to its normal state, and mitochondria recovered to their original shape (Fig. 2A). Further research demonstrated that H2S supplementation decreased the expression of GRP78, caspase-12, and CHOP in the myocardium of diabetic rats. The reduced expression of GRP78 and caspase-12 by H2S is consistent with the effects of H2S on stress-related hyperhomocysteinemia (36). H2S may regulate the expression of ER stress-associated markers in diabetic rats to promote myocardial cell survival.
We also performed in vitro studies to further elucidate the direct effects of H2S on high glucose-induced ER stress. The H9C2 cell line is derived from rat embryonic heart, and it is used to study the exclusive molecular mechanisms of myocardial cells (19). Ultrastructural examination revealed that 25 mM high glucose damaged H9C2 cells. The ER was significantly dilated, and it was seen as round vesicles. Mitochondria were swollen or compact (Fig. 4, A, B, and C). These changes were consistent with our in vivo study. Cells treated with 25 mM high glucose showed an increased expression of CHOP in the cytoplasm. Caspase-12 is a specific proapoptotic ER stress marker. Our data demonstrated that 25 mM high glucose increased caspase-12 expression, which further confirmed that ER stress was an important result of high glucose (28).
Mitofusin-2 (Mfn-2; also called hyperglycemia suppressor gene) is a member of the mitofusin family, and it is a large GTPase that is essential for mitochondrial fusion during embryonic development and neuronal differentiation (26). Mfn-2 is predominantly expressed in the heart, and Mfn-2 is associated with the fragmentation of the mitochondrial network that promotes early apoptotic events (28).
Increasing evidence indicates that Mfn-2 is a major determinant of oxidative stress-mediated cardiomyocyte apoptosis. Xiao and coworkers revealed that H2O2-induced oxidative stress increased Mfn-2 expression and apoptosis in cultured neonatal rat cardiomyocytes (28, 32). However, mounting evidence indicates that Mfn-2 resides on ER membranes, and this dual localization may facilitate the transfer of Ca2+ from the ER into the adjacent mitochondria. Walsh et al. (27) demonstrated that Mfn-2 knock down in cardiomyocytes protected these cells from the local generation of ROS.
Our studies revealed an important functional role of Mfn-2 in the mediation of oxidative stress-induced H9C2 cell apoptosis via the Bcl-2/Bax cell signaling mechanism. First, high glucose promoted apoptosis of H9C2 cells via the elevation of Mfn-2 expression (Fig. 8A), and siRNA-mediated Mfn-2 silencing protected cells against oxidative stress-induced apoptosis (Fig. 8, B and E). Second, the mitochondrial apoptotic pathway is essentially involved in Mfn-2-mediated H9C2 cell apoptosis (Fig. 10). Our results indicated that myocardial oxidative stress induced Mfn-2 expression, which plays an essential role in oxidative stress-mediated apoptosis of H9C2 cells.
GRANTS
This study was supported by grants from the National Natural Science Foundation of China (81370421, 81370330,81670344) and the Graduate Innovation Foundation of Harbin Medical University (YJSCX2014-05HYD).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
F.Y., X.Y., T.L., Jianjun Wu, J.L., A.S., X.Z., F.L., and W.Z. performed experiments; F.Y., X.Y., T.L., Jianjun Wu, and J.L. analyzed data; F.Y., X.Y., and Jichao Wu interpreted results of experiments; F.Y., Jichao Wu, and X.Z. prepared figures; F.Y., S.D., F.L., and W.Z. drafted manuscript; F.Y., Y.Z., and W.Z. edited and revised manuscript; Y.Z., X.Z., C.X., and W.Z. approved final version of manuscript; W.Z. conceived and designed research.
REFERENCES
- 1. . The Bcl-2 protein family: arbiters of cell survival. Science 281: 1322–1326, 1998. doi:10.1126/science.281.5381.1322.
Crossref | PubMed | Web of Science | Google Scholar - 2. . Role of changes in cardiac metabolism in development of diabetic cardiomyopathy. Am J Physiol Heart Circ Physiol 291: H1489–H1506, 2006. doi:10.1152/ajpheart.00278.2006.
Link | Web of Science | Google Scholar - 3. . The pathogenesis of myocardial fibrosis in the setting of diabetic cardiomyopathy. J Am Coll Cardiol 47: 693–700, 2006. doi:10.1016/j.jacc.2005.09.050.
Crossref | PubMed | Web of Science | Google Scholar - 4. . Calcium elevation in mitochondria is the main Ca2+ requirement for mitochondrial permeability transition pore (mPTP) opening. J Biol Chem 284: 20796–20803, 2009. doi:10.1074/jbc.M109.025353.
Crossref | PubMed | Web of Science | Google Scholar - 5. . Myocardial ultrastructural changes in alloxan-induced diabetes in rabbits. Acta Anat (Basel) 125: 195–200, 1986. doi:10.1159/000146161.
Crossref | PubMed | Google Scholar - 6. . Ameliorative effect of quercetin on memory dysfunction in streptozotocin-induced diabetic rats. Neurobiol Learn Mem 94: 293–302, 2010. doi:10.1016/j.nlm.2010.06.008.
Crossref | PubMed | Web of Science | Google Scholar - 7. . Diabetic cardiomyopathy, causes and effects. Rev Endocr Metab Disord 11: 31–39, 2010. doi:10.1007/s11154-010-9131-7.
Crossref | PubMed | Web of Science | Google Scholar - 8. . The novel proangiogenic effect of hydrogen sulfide is dependent on Akt phosphorylation. Cardiovasc Res 76: 29–40, 2007. doi:10.1016/j.cardiores.2007.05.026.
Crossref | PubMed | Web of Science | Google Scholar - 9. . Role of endoplasmic reticulum stress in apoptosis of differentiated mouse podocytes induced by high glucose. Int J Mol Med 33: 809–816, 2014. doi:10.3892/ijmm.2014.1642.
Crossref | PubMed | Web of Science | Google Scholar - 10. . Beta-blocker timolol alleviates hyperglycemia-induced cardiac damage via inhibition of endoplasmic reticulum stress. J Bioenerg Biomembr 46: 377–387, 2014. doi:10.1007/s10863-014-9568-6.
Crossref | PubMed | Web of Science | Google Scholar - 11. . Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456: 605–610, 2008. doi:10.1038/nature07534.
Crossref | PubMed | Web of Science | Google Scholar - 12. . Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart. J Biol Chem 276: 2571–2575, 2001. doi:10.1074/jbc.M006825200.
Crossref | PubMed | Web of Science | Google Scholar - 13. . Oxidative stress in diabetes - circulating advanced glycation end products, lipid oxidation and vascular disease. Ann Clin Biochem 51: 125–127, 2014. doi:10.1177/0004563213508747.
Crossref | PubMed | Web of Science | Google Scholar - 14. . Mitochondrial reactive oxygen species in cell death signaling. Biochimie 84: 131–141, 2002. doi:10.1016/S0300-9084(02)01369-X.
Crossref | PubMed | Web of Science | Google Scholar - 15. . Diabetic cardiomyopathy [ix.]. Endocrinol Metab Clin North Am 35: 575–599, 2006. doi:10.1016/j.ecl.2006.05.003.
Crossref | PubMed | Web of Science | Google Scholar - 16. . Oxidative stress and diabetic complications. Circ Res 107: 1058–1070, 2010. doi:10.1161/CIRCRESAHA.110.223545.
Crossref | PubMed | Web of Science | Google Scholar - 17. . Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell 141: 656–667, 2010. doi:10.1016/j.cell.2010.04.009.
Crossref | PubMed | Web of Science | Google Scholar - 18. . Endoplasmic reticulum stress and the development of diabetes: a review. Diabetes 51, Suppl 3: S455–S461, 2002. doi:10.2337/diabetes.51.2007.S455.
Crossref | PubMed | Web of Science | Google Scholar - 19. . Stable high level expression of a transfected human HSP70 gene protects a heart-derived muscle cell line against thermal stress. J Mol Cell Cardiol 26: 695–699, 1994. doi:10.1006/jmcc.1994.1084.
Crossref | PubMed | Web of Science | Google Scholar - 20. . A functional and ultrastructural analysis of experimental diabetic rat myocardium. Manifestation of a cardiomyopathy. Diabetes 34: 876–883, 1985. doi:10.2337/diab.34.9.876.
Crossref | PubMed | Web of Science | Google Scholar - 21. . Conundrum of pathogenesis of diabetic cardiomyopathy: role of vascular endothelial dysfunction, reactive oxygen species, and mitochondria. Mol Cell Biochem 386: 233–249, 2014. doi:10.1007/s11010-013-1861-x.
Crossref | PubMed | Web of Science | Google Scholar - 22. . Hydrogen sulfide (H2S) - the third gas of interest for pharmacologists. Pharmacol Rep 59: 4–24, 2007.
PubMed | Web of Science | Google Scholar - 23. . An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. J Biol Chem 277: 34287–34294, 2002. doi:10.1074/jbc.M204973200.
Crossref | PubMed | Web of Science | Google Scholar - 24. . Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403: 98–103, 2000. doi:10.1038/47513.
Crossref | PubMed | Web of Science | Google Scholar - 25. . Mitofusin-2 maintains mitochondrial structure and contributes to stress-induced permeability transition in cardiac myocytes. Mol Cell Biol 31: 1309–1328, 2011. doi:10.1128/MCB.00911-10.
Crossref | PubMed | Web of Science | Google Scholar - 26. . Mitofusin-2 maintains mitochondrial structure and contributes to stress-induced permeability transition in cardiac myocytes. Mol Cell Biol 31: 1309–1328, 2011. doi:10.1128/MCB.00911-10.
Crossref | PubMed | Web of Science | Google Scholar - 27. . Mitofusins and the mitochondrial permeability transition: the potential downside of mitochondrial fusion. Am J Physiol Heart Circ Physiol 303: H243–H255, 2012. doi:10.1152/ajpheart.00185.2012.
Link | Web of Science | Google Scholar - 28. . Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis. Cardiovasc Res 77: 387–397, 2008. doi:10.1093/cvr/cvm029.
Crossref | PubMed | Web of Science | Google Scholar - 29. . Endogenous hydrogen sulfide and the cardiovascular system-what’s the smell all about? Clin Invest Med 29: 146–150, 2006.
PubMed | Web of Science | Google Scholar - 30. . Diabetic cardiomyopathy: the search for a unifying hypothesis. Circ Res 98: 596–605, 2006. doi:10.1161/01.RES.0000207406.94146.c2.
Crossref | PubMed | Web of Science | Google Scholar - 31. . Microdomains of intracellular Ca2+: molecular determinants and functional consequences. Physiol Rev 86: 369–408, 2006. doi:10.1152/physrev.00004.2005.
Link | Web of Science | Google Scholar - 32. . Mitofusin-2 is a major determinant of oxidative stress-mediated heart muscle cell apoptosis. J Biol Chem 282: 23354–23361, 2007. doi:10.1074/jbc.M702657200.
Crossref | PubMed | Web of Science | Google Scholar - 33. . Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep 7: 880–885, 2006. doi:10.1038/sj.embor.7400779.
Crossref | PubMed | Web of Science | Google Scholar - 34. . Phospholipid synthesis in a membrane fraction associated with mitochondria. J Biol Chem 265: 7248–7256, 1990.
Crossref | PubMed | Web of Science | Google Scholar - 35. . PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress. Cell Death Differ 19: 1880–1891, 2012. doi:10.1038/cdd.2012.74.
Crossref | PubMed | Web of Science | Google Scholar - 36. . Hydrogen sulfide attenuates hyperhomocysteinemia-induced cardiomyocytic endoplasmic reticulum stress in rats. Antioxid Redox Signal 12: 1079–1091, 2010. doi:10.1089/ars.2009.2898.
Crossref | PubMed | Web of Science | Google Scholar - 37. . Contributions of inflammation and cardiac matrix metalloproteinase activity to cardiac failure in diabetic cardiomyopathy: the role of angiotensin type 1 receptor antagonism. Diabetes 56: 641–646, 2007. doi:10.2337/db06-1163.
Crossref | PubMed | Web of Science | Google Scholar - 38. . Glucose autoxidation and protein modification. The potential role of ‘autoxidative glycosylation’ in diabetes. Biochem J 245: 243–250, 1987. doi:10.1042/bj2450243.
Crossref | PubMed | Web of Science | Google Scholar - 39. . Diabetes- and angiotensin II-induced cardiac endoplasmic reticulum stress and cell death: metallothionein protection. J Cell Mol Med 13: 1499–1512, 2009. doi:10.1111/j.1582-4934.2009.00833.x.
Crossref | PubMed | Web of Science | Google Scholar - 40. . The possible role of hydrogen sulfide on the pathogenesis of spontaneous hypertension in rats. Biochem Biophys Res Commun 313: 22–27, 2004. doi:10.1016/j.bbrc.2003.11.081.
Crossref | PubMed | Web of Science | Google Scholar - 41. . Endoplasmic reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response. Cell 124: 587–599, 2006. doi:10.1016/j.cell.2005.11.040.
Crossref | PubMed | Web of Science | Google Scholar - 42. . Exogenous hydrogen sulfide attenuates diabetic myocardial injury through cardiac mitochondrial protection. Mol Cell Biochem 371: 187–198, 2012. doi:10.1007/s11010-012-1435-3.
Crossref | PubMed | Web of Science | Google Scholar
