Study On Metallothionein Attenuation Of Cardiac Cell Death Via Suppression Of Endoplasmic Reticulum Stress In Diabetic Cardiomyopathy

DCM is a chronic and complex pathogenesis that is caused by abnormal cellular metabolism and defects in organelles such as myofibrils, mitochondria, and sarcolemma. Apoptotic cell death as an early cellular event in response to diabetes has been reported to play a critical role in DCM development. Although emerging evidence has indicated that ER stress-mediated apoptosis is involved in the pathogenesis of diabetic eye and kidney as well as non-diabetic heart failure, there was no direct evidence for the involvement of ER stress in diabetes-induced cardiac cell death and DCM. Metallothionein (MT) as a potent antioxidant was found to protect the heart from type 1 and type 2 diabetes. In terms of the cellular mechanisms involved in the prevention of MT against DCM, we found that MT protected the heart from diabetes mainly through inhibition of diabetic cardiac cell death. However, it is unclear how diabetes can induce cardiac cell death. The aim of this study was to investigate whether ER stress involves in the development of DCM with streptozotocin (STZ)-induced diabetic models using cardiac-specific MT-overexpression transgenic (MT-TG) mice and their wild-type (WT) mice.The present study shows an association of diabetes- and Ang II-induced cardiac cell death with cardiac ER stress, and also MT prevention of cardiac ER stress and associated cell death in diabetic, Ang II-treated, and chemical ER stressor-treated mouse models.Methods:1. Diabetic mouse model: STZ was dissolved in sodium citrate buffer (pH 4.5), and given intraperitoneally to both WT and MT-TG ten-week-old mice at 40 mg/kg body weight daily for 5 days, i.e.multiple low-dose STZ (MLD-STZ) model. Five days after last injection of STZ, whole blood glucose obtained from mouse tail-vein was detected using a SureStep complete blood glucose monitor. STZ-treated mice with whole blood glucose higher than 12 mmol/l were considered diabetic. Animals were sacrificed at 2 week (wk), and 2 and 5 months after the onset of diabetes.2. Ang II mouse model: Ang II was freshly prepared in a 154 mmol NaCl vehicle immediately prior to be used. MT-TG and WT mice received a single subcutaneous injection of Ang II at 1 mg/kg body weight. Control animals were given an equal volume of vehicle. Both control and Ang II-treated animals were sacrificed at 7 and 24 h later.3. Tunicamycin mouse model: Both MT-TG and WT mice were intraperitoneally given a single injection of chemical ER stress activator, tunicamycin at 1.5 mg/kg body weight. The tunicamycin was freshly prepared as a 0.05 mg/ml suspension in 150 mM dextrose. Twelve h after tunicamycin treatment, animals were sacrificed.4. Western blotting assay: Cardiac tissue homogenates were lysed and fractionated electrophoretically on sodium dodecyl (lauryl) sulfate polyacrylamide gel electrophoresis (10%-15% gradient gels), and proteins were transferred to a nitrocellulose membrane. The membrane was blocked with a 5% non-fat, dried milk for 1 h. The following antibodies were used: anti-ATF6 (1:2000,), anti-GRP78 (1:4000), anti-GRP94 (1:1000), anti-p-eIF2αand anti-CHOP (1:200 and 1:500, respectively), anti-cleaved caspase 3 (1:2000), and anti-cleaved caspase 12 (1:1000). Membranes were incubated with the primary antibodies overnight at 4°C. After the unbound antibodies were removed with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20, membranes were incubated with the secondary antibody for 1 h at room temperature. Antigen-antibody complexes were visualized with ECL system.5. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay: Heart tissue was fixed in 10% formalin, embedded in paraffin, and sectioned at 3-4μm. The slides were stained for TUNEL with the ApopTag Peroxidase In Situ Apoptosis Detection Kit. Each slide was deparaffinized and rehydrated, and treated with proteinase K (20 mg/L) for 15 min. The endogenous peroxidase was inhibited with 3% hydrogen peroxide for 5 min and then incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) and digoxigenin-11-dUTP for 1 h at 37°C. The TdT reaction was carried out in a humidified chamber at 37°C for 1 h, and then 3, 3-diaminobenzidine (DAB) chromogen was applied. Methyl green was used as counterstaining. Mouse testicular tissue was used as positive control. For negative control, TdT was omitted from the reaction mixture. The apoptotic cell death was quantitatively analyzed by counting the TUNEL-positive cells randomly selected from 5 fields from each of the 3 slides for each mouse.6. Cell culture: Embryonic rat heart derived cells (H9c2) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (50 U/ml penicillin and 50μg/ml streptomycin) at 37°C in an atmosphere of 95% air and 5% CO2. Both H9c2 and H9c2MT7 cells were treated with Ang II at 100 nM for 24 h. In some experiments, H9c2 cells were pre-treated with MnTMPyP, a cell-permeable superoxide dismutase mimic, at 50μM for 30 min or NAC, an antioxidant N-acetyl-L-cysteine at 100μM for 24 h and then exposed to Ang II at 100 nM with the presence of either MnTMPyP or NAC for 24 h.Results:1. Diabetes-induced cardiac ER stress and prevention by MTWestern blotting assay showed that both phosphorylated eIF2α(p-eIF2α) and GRP94 proteins were significantly increased in the heart of WT diabetic mice at 2 wk, but not 2 and 5 months, after diabetes onset . Cleaved ATF6 protein was significantly increased both at 2 wk and 2 months, but not at 5 month. In contrast to the above changes, GRP78 protein level was increased in the heart of diabetic mice at all three time-points with the highest expression at 2 wk after diabetes . More importantly, none of these increased ER chaperones in the hearts of WT diabetic mice were observed in the heart of MT-TG diabetic mice.2. Diabetes-induced cardiac MT up-regulationWestern blotting assay revealed that diabetes induced a significant increase in MT contents of heart from 2 wk to 5 months with a rapid increase at 2 wk and then gradually decrease until 5 months after diabetes, but it remains significantly higher than control levels. Interestingly, diabetes-induced cardiac MT synthesis in the heart of WT mice was not observed in the heart of MT-TG diabetic mice.3. Diabetes-induced cardiac cell death and prevention by MTwe examine the cardiac cell death from 2 wk to 2 and 5 months after diabetes onset with TUNEL assay for apoptotic cells and Western blotting assay for CHOP, the activated form of caspase-3 and caspase-12. Both assays delineate that diabetes mainly induced cardiac cell death at the early stage of diabetes (2 wk), but not in the late stages (2 and 5 months). However, there was no apoptotic cell death in the hearts of MT-TG diabetic mice.4. Ang II-induced cardiac ER stress and prevention by MTTreatment of mice with Ang II significantly increased the expression of p-eIF2α, cleaved ATF6 and GRP78 except for GRP94. The up-regulated ER chaperone proteins by Ang II were not observed in the heart of MT-TG mice.5. Ang II-induced cardiac MT up-regulationAng II significantly induced cardiac MT synthesis only in the WT mice, as observed in the hearts of diabetic mice.6. Ang II -induced cardiac cell death and prevention by MTAng II significantly induced cardiac cell death only in the WT mice, as observed in the hearts of diabetic mice. The association of Ang II-induced cardiac cell death with ER stress was also favored by significant increases in CHOP protein expression and caspase-12 activation.7. Tunicamycin-induced cardiac ER stress and prevention by MTWestern blotting assay showed that proteins of p-eIF2α, cleaved ATF6 and GRP78 all were significantly up-regulated, except for GRP94 protein that was not changed. Similar to the above experiments in diabetic and Ang II-infusion models, MT significantly prevented tunicamycin-up-regulated ER chaperone proteins. 8. Tunicamycin-induced cardiac MT up-regulationIt is noted that tunicamycin also significantly induced cardiac MT synthesis, as shown in the hearts of other ER-stress models.9. Tunicamycin-induced cardiac cell death and prevention by MTCardiac cell death was significantly increased in the heart of Tu-treated WT mice, but not Tu-treated MT-TG mice, examined by Western blotting for caspase-3 activation and TUNEL assay, and confirmed by the increased CHOP protein expression and caspase-12 cleavage.10. Ang II-induced ER stress was attenuated by MT in cultured cardiac cellsTreatment with Ang II at 100 nM for 24 h significantly induced ER stress, shown by increased expressions of cleaved ATF6, p-eIF2α, GRP78 and GRP94. All these ER-stress effects were not observed in H9c2MT7 cells, suggesting the protection of cardiac cells by MT from Ang II-induced ER stress.11. Ang II-induced cell death was attenuated by MT in cultured cardiac cellsTreatment with Ang II at 100 nM for 24 h also significantly induced apoptotic cell death in H9c2 cells, measured by CHOP protein expression and caspase-12 activation, but not in H9c2MT7 cells. This in vitro finding is consistent with those of in vivo study.12. antioxidants-induced ER stress was attenuated by MT in cultured cardiac cellsPre-exposure of H9c2 cells to MnTMPyP at 50μM for 30 min or NAC at 50μM for 24 h significantly prevented Ang II-induced ER stress, while same pretreatments did not have any effect on normal H9c2 cells and H9c2MT7 cells.13. antioxidants-induced cell death was attenuated by MT in cultured cardiac cellsPre-exposure of H9c2 cells to MnTMPyP at 50μM for 30 min or NAC at 50μM for 24 h significantly prevented Ang II-induced apoptosis, while same pretreatments did not have any effect on normal H9c2 cells and H9c2MT7 cells.Conclusion:1. We successfully set up streptozotocin (STZ)-induced diabetic, Ang II and tunicamycin- induced mice models, using cardiac-specific MT-overexpression transgenic (MT-TG) mice and their wild-type (WT) mice.2. Cardiac cell death mediated by ER stress is one of the nosogenesis of DCM;3. MT can attenuate diabetes-induced cardiac cell death via suppression of cardiac ER stress;4. Oxidative stress plays a pivotal role in Ang II-induced both ER stress and associated apoptotic effects;5. Prevention of Ang II-induced ER stress and associated apoptotic effects by MT is most likely mediated by its antioxidant action.Innovative findings:1. Diabetes induces cardiac ER stress along with induction of cardiac cell death;2. MT can attenuate diabetes-, Ang II- and chemical ER stressor-induced cardiac cell death via suppression of cardiac ER stress;3. MT can protect DCM by suppression of endoplasmic reticulum stress.