| BackgroundMyocardial ischemia-reperfusion?IR?can increases cardiomyocyte apoptosis and jeopardise cardiac performance,which remains one of the most important factor affects the outcome of cardiovascular intervention.To reduce cardiomyocyte apoptosis and thus alleviate myocardial injury effectively has always been the hot spot for both clinicians and researchers.Recent studies have found myocardial IR can cause severe oxidative stress,resulting in a large number of reactive oxygen species?ROS?,triggering endoplasmic reticulum stress?ERS?,induced oxidized Ca2+/calmodulin-dependent protein kinase??oxCaMK??,leading to cardiomyocyte apoptosis.ox-CaMK? can induce intracellular Ca2+ overload and increase myocardial IR injury.Meanwhile,other scholars have found that a new gas molecules,hydrogen?Hydrogen,H2?,could act as a potential therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals.We hypothesized that H2 may exert protective effect on cardiomyocyte by alleviate cardiomyocyte apoptosis through inhibiting the production of ROS,mitigate ERS,decrease the activation of CaMK?,reduce [Ca2+]i,mitigate intracellular calcium overload.Objective1.To explore the protective effect of hydrogen in myocardial ischemia-reperfusion injury.2.To elucidate the molecular mechanism underlying the protective effect of hydrogen in myocardial ischemia-reperfusion injury.Materials and Methods1.Experimental Study on the Effect of H2 Regulating ERS on Cardiomyocytes ApoptosisThe neonatal rat cardiomyocytes were cultured for 30 min with hypoxia?1% oxygen + 5% carbon dioxide + 94% nitrogen?and then normal condition?5% carbon dioxide + 95% air?for 120 min to simulate myocardial hypoxia-reoxygenation?HR?model.The cells were randomly divided into 3 groups: Con group,HR group and H2 group?cultured with 0.6mM hydrogen-rich DMEM medium for 30 min firstly,then treated as same as the HR group?.The viability of cardiomyocytes was measured by CCK-8.The level of LDH in culture supernatant was measured by colorimetric assay,and the level of cTn I in culture supernatant was measured by ELISA.Flow cytometry was used to test the apoptotic rate of cardiomyocytes.Western blot was used to detect the relative expression level of CHOP?Bcl-2 and Bax.2.Study on Mechanism of H2 in Reducing Cardiomyocytes Apoptosis Mediated by ERS-CaMK?-Ca2+ Overload?1?Study on the effect of H2 on CaMK? oxidative activation mediated by CHOPRat CHOP overexpression lentiviral vector?Lv-CHOP?and CHOP gene short hairpin RNA?shRNA?lentiviral vector?Lv-shRNA?were constructed.The relative expression level of CHOP was up-regulated or down-regulated in isolated rat cardiomyocytes.The HR model of isolated cells was constructed in the same way as in Part 1.The cells were randomly divided into 7 groups: Con group,HR group,H2 group,HR+Lv-CHOP group?Lv-CHOP transfected cells firstly,then treated in the same way with HR group?,HR+Lv-CHOP+H2 group?Lv-CHOP transfected cells firstly,then treated in the same way with H2 group?and HR+Lv-shRNA group?Lv-shRNA transfected cells firstly,then treated in the same way with HR group?,HR+Lv-shRNA+H2 group?Lv-shRNA transfected cells firstly,then treated in the same way with H2 group?.The relative expression level of ox-CaMK? and CaMK?were detected by Western blot.?2?Study on the effect of H2 on intracellular Ca2+ overload mediated by CaMK?The HR model of isolated cardiomyocytes was established by the method of Part 1,and KN-93 was used to block CaMK? activation.The cells were randomly divided into 5 groups: Con group,HR group,H2 group,HR+KN-93 group?cells were cultured in DMEM medium containing 2?M KN-93 for 30 min firstly,then treated in the same way with HR group?and HR+H2+KN-93 group?cells were cultured in hydrogen-rich DMEM medium containing 2?M KN-93 for 30 min and then treated in the same way with HR group?.The intracellular Ca2+([Ca2+]i)and apoptosis rate of cardiomyocytes were detected by flow cytometry,and the relative expression level of Bcl-2 and Bax were detected by Western blot.3.Study on the Effect of H2 Regulating ERS on Cardiomyocytes Apoptosis in vivoThe myocardial IR model was established by ligating the left anterior descending coronary artery for 30 min and then reperfusion for 120 min.Thirty SD rats were randomly divided into three groups: Sham group?only thoracotomy?,IR group and H2 group?intraperitoneal injection of 10 m L/kg hydrogen-rich saline at 5min before reperfusion?.The left ventricular systolic pressure?LVSP?and maximum derivative of left ventricular pressure?+dp/dtmax?of rats were measured at 120 min after reperfusion.The area of myocardial infarction was measured by Evans-Blue/TCC staining.The positive expression index of 8-OHdG and 3-NT in myocardium was determined by immunohistochemistry.Serum LDH level was measured by dinitrophenylhydrazine spectrophotometry,and the level of cTn I in serum was measured by ELISA.The cardiomyocytes apoptosis index was determined by TUNEL.The relative expression of CHOP?ox-CaMK??CaMK??Bcl-2 and Bax were detected by Western blot.Results1.Experimental Study on the Effect of H2 Regulating ERS on Cardiomyocytes Apoptosis?1?Hydrogen can improve cardiomyocytes viabilityThe cardiomyocytes viability in different group were: Con 98.8±1.0%,HR 32.6±5.2% and H2 59.3±3.9%.The cardiomyocytes viability in H2 group was significantly higher than that in HR group?P <0.001?.Thus,hydrogen can improve cardiomyocytes viability.?2?Hydrogen can reduce cardiomyocytes injuryThe level of LDH in culture supernatant in different group were: Con 12.2±1.7U/L,HR 24.7±3.1U/L and H2 19.3±2.9U/L.The level of LDH in H2 group was significantly lower than that in HR group?P=0.047?.The level of cTn I in culture supernatant in different group were: Con 68.2±8.8ng/L,HR 127.8±16.6ng/L and H2 95.7±7.6ng/L.The level of cTnI in H2 group was significantly lower than that in HR group?P=0.015?.Thus,hydrogen can reduce cardiomyocytes HR injury.?3?Hydrogen can reduce cardiomyocytes apoptosisThe ratio of Bcl-2/Bax in HR group was significantly lower than that in Con group?P<0.001?,while that in H2 group was significantly higher than that of HR group?P<0.001?,suggesting that hydrogen can reduce cardiomyocytes apoptosis by increasing the Bcl-2/Bax ratio.The apoptotic rates of cardiomyocytes were: Con 7.7±1.6%,HR 87.7±9.0% and H2 68.9±9.6%.The apoptotic rates of cardiomyocytes in H2 group was significantly lower than that of HR group?P=0.024?.Thus,hydrogen can reduce cardiomyocytes apoptosis.?4?Hydrogen can regulate ERS and reduce CHOP expressionThe expression of CHOP in HR group was significantly higher than that in Con group?P<0.001?,while that in H2 group was significantly lower than that of HR group?P<0.001?.Thus,hydrogen can regulate ERS and reduce CHOP expression.2.Study on Mechanism of ERS-CaMK?-Ca2+ Overload Mediated H2 in Reducing Cardiomyocytes Apoptosis?1?Hydrogen can decrease oxidative activation of CaMK?mediated by CHOPThe ratio of ox-CaMK?/CaMK? in HR group was significantly higher than that in Con group?P<0.001?,while that in H2 group was significantly lower than that of HR group?P<0.001?,suggesting that hydrogen can decrease the level of CaMK? oxidation activation.The ratio of ox-CaMK?/CaMK? in HR+Lv-CHOP group was significantly increased than that in HR group?P=0.002?,and that in HR+Lv-CHOP+H2 group was significantly decreased than that in HR+Lv-CHOP group?P<0.001?,while there was no significant difference between HR+Lv-CHOP+H2 group and HR group?P=0.163?.The ratio of oxCaMK?/CaMK? in HR+Lv-shRNA group was significantly decreased than that in HR group?P<0.001?,and that in HR+Lv-shRNA+H2 group was significantly decreased than that in HR+Lv-shRNA group?P<0.001?,and there was no significant difference between HR+Lv-shRNA+H2 group and H2 group?P=0.940?.Thus,hydrogen can decrease oxidative activation of CaMK?mediated by CHOP.?2?Hydrogen can decrease [Ca2+]i mediated by CaMK?The [Ca2+]i in HR group was significantly higher than that in Con group?P<0.001?,while that in H2 group was significantly lower than that of HR group?P<0.001?,suggesting that hydrogen can decrease [Ca2+]i.The [Ca2+]i in HR+KN-93 group was significantly decreased than that in HR group?P<0.001?,and that in HR+KN-93+H2 group was significantly decreased than that in HR+KN-93 group?P=0.001?,and there was no significant difference between HR+KN-93+H2 group and H2 group?P=0.143?.Thus,hydrogen can decrease [Ca2+]i mediated by CaMK?.?3?Hydrogen can reduce cardiomyocytes apoptosis mediated by Ca MK?The Bcl-2/Bax ratio in HR+KN-93 group was significantly higher than that in HR group?P=0.004?,and that in HR+KN-93+H2 group was significantly increased than that in HR+KN-93 group?P=0.030?,and there was no significant difference between HR+KN-93+H2 group and H2 group?P=0.372?.Thus,hydrogen can regulate Bcl-2/Bax ratio mediated by CaMK?.The apoptotic rate of cardiomyocytes in HR+KN-93 group was significantly lower than that in HR group?P<0.001?,and that in HR+KN-93+H2 group was significantly lower than that in HR+KN-93 group?P<0.001?,and there was no significant difference between HR+KN-93+H2 group and H2 group?P=0.272?.Thus,hydrogen can reduce cardiomyocytes apoptosis mediated by CaMK?.3.Study on the Effect of H2 Regulating ERS on Cardiomyocytes Apoptosis in vivo?1?Hydrogen can reduce the level of hydroxyl radical?·OH?and peroxynitrite anion?ONOO-?in vivoThe positive expression indices of 8-hydroxy deoxyguanine?8-OHdG?reflecting the level of ·OH were:Sham 4.3±0.8%,IR 89.1±0.8% and H2 54.9±5.0%,and the positive expression indices of 8-OHdG in H2 group was significantly lower than that in IR group?P<0.001?.The positive expression indices of 3-nitrotyrosine?3-NT?reflecting the level of ONOO-were: Sham 9.7±0.8%,IR 86.7±5.2%,H2 63.4±7.4%.Similarly,the positive expression indices of 3-NT in H2 group was significantly lower than that in IR group?P<0.001?.Thus,hydrogen can reduce the level of ·OH and ONOO-in vivo.?2?Hydrogen can regulate ERS and decrease CHOP expression in vivoThe expression level of CHOP in IR group was significantly higher than that in Sham group?P<0.001?,while that of H2 group was significantly lower than that in IR group?P=0.001?.Thus,hydrogen can regulate ERS and decrease the expression of CHOP in vivo.?3?Hydrogen can decrease oxidative activation of CaMK? in vivoThe ratio of ox-CaMK?/CaMK? in IR group was significantly higher than that in Sham group?P<0.001?,while that of H2 group was significantly lower than that in IR group?P<0.001?.Thus,hydrogen can decrease oxidative activation of CaMK? in vivo.?4?Hydrogen can reduce cardiomyocytes apoptosis in vivoThe ratio of Bcl-2/Bax in IR group was significantly lower than that in Sham group?P<0.001?,while that of H2 group was significantly higher than that in IR group?P=0.003?.Thus,hydrogen can reduce cardiomyocytes apoptosis by regulating Bcl-2/Bax in vivo.The apoptosis index?AI?were: Sham 4.6±1.1%,IR 42.7±5.7% and H2 28.1±6.9%.The AI in H2 group was significantly lower than that of HR group?P=0.001?.Thus,hydrogen can reduce cardiomyocytes apoptosis in vivo.?5?Hydrogen can reduce myocardial injuryThe level of LDH in serum in different group were: Sham 2195.4±167.6U/L,IR 4159.7±243.1U/L and H2 3699.4±182.7U/L.The level of LDH in H2 group was significantly lower than that in HR group?P=0.003?.The level of cTnI in serum in different group were: Sham 35.4±3.5ng/L,IR 150.5±18.4ng/L and H2 121.6±13.8ng/L.The level of cTnI in H2 group was significantly lower than that in HR group?P=0.005?.Thus,hydrogen can reduce myocardial IR injury.?6?Hydrogen can reduce myocardial infarct areaThe ratio of INF/AAR in H2 group was significantly lower than that of IR group?22.4±2.9 vs 48.4±3.5 %,P<0.001?.Thus,hydrogen can reduce myocardial infarct area in myocardial IR injury.?7?Hydrogen can improve cardiac performance in myocardial IR injuryThe LVSP and the +dp/dtmax were significantly lower in IR group than those in Sham group?92.4±6.3 vs 131.8±8.1mmHg,P<0.001;3601.2±339.6 vs 8563.4±514.6mmHg/s,P<0.001?,while the LVSP and the +dp/dtmax in H2 group were significantly higher than those in IR group?120.0±9.7 vs 92.4±6.3mm Hg,P<0.001;5801.0±816.8 vs 3601.2±339.6mmHg/s,P<0.001?.Thus,hydrogen can improve left ventricular systolic capacity and improve cardiac performance in myocardial IR injury.Conclusions1.Myocardial IR can activate ERS and increase cardiomyocytes apoptosis.2.Hydrogen can down-regulate the CHOP protein expression by extenuating ERS in myocardial IR,which can ease the oxidative activation of CaMK ? and alleviate cardiomyocyte apoptosis.3.The mechanism of hydrogen on reducing the cardiomyocyte apoptosis in myocardial IR involves the rebalance of intracellular calcium hemostasis by easing the oxidative activation of CaMK?. |
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