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Isoflurane preconditioning confers cardioprotection by activation of ALDH2.

Lang XE, Wang X, Zhang KR, Lv JY, Jin JH, Li QS - PLoS ONE (2013)

Bottom Line: Pretreatment with isoflurane prior to ischemia reduced LDH and CK-MB levels and infarct size, while it increased phosphorylation of ALDH2, which could be blocked by the ALDH2 inhibitor, cyanamide.Hypoxia/reoxygenation (H/R) increased cardiomyocyte apoptosis and injury which were attenuated by isoflurane and forced the activation of ALDH2.In contrast, the effect of isoflurane-induced protection was almost abolished by knockdown of ALDH2.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, The First Clinical Medical College of Shanxi Medical University, Taiyuan, Shanxi, China.

ABSTRACT
The volatile anesthetic, isoflurane, protects the heart from ischemia/reperfusion (I/R) injury. Aldehyde dehydrogenase 2 (ALDH2) is thought to be an endogenous mechanism against ischemia-reperfusion injury possibly through detoxification of toxic aldehydes. We investigated whether cardioprotection by isoflurane depends on activation of ALDH2.Anesthetized rats underwent 40 min of coronary artery occlusion followed by 120 min of reperfusion and were randomly assigned to the following groups: untreated controls, isoflurane preconditioning with and without an ALDH2 inhibitor, the direct activator of ALDH2 or a protein kinase C (PKCε) inhibitor. Pretreatment with isoflurane prior to ischemia reduced LDH and CK-MB levels and infarct size, while it increased phosphorylation of ALDH2, which could be blocked by the ALDH2 inhibitor, cyanamide. Isolated neonatal cardiomyocytes were treated with hypoxia followed by reoxygenation. Hypoxia/reoxygenation (H/R) increased cardiomyocyte apoptosis and injury which were attenuated by isoflurane and forced the activation of ALDH2. In contrast, the effect of isoflurane-induced protection was almost abolished by knockdown of ALDH2. Activation of ALDH2 and cardioprotection by isoflurane were substantially blocked by the PKCε inhibitor. Activation of ALDH2 by mitochondrial PKCε plays an important role in the cardioprotection of isoflurane in myocardium I/R injury.

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ALDH2 knockdown blocks isoflurane-induced protection against hypoxia/reoxygenation.A. Schematic representation of adenoviruses encoding RFP (Ad-RFP) and ALDH2shRNA (Ad-Si-ALDH2-RFP, left panel); right panel, immunoblotting analysis of ALDH2 protein level. β-actin was used as a control. B. ALDH2 down-regulation inhibited attenuation of TUNEL positive staining level by isoflurane. (a) Apoptotic cells were examined using TUNEL assay for DNA fragmentation. Cardiomyoctes were photographed by fluorescence microscopy after 24 hours of hypoxia followed by 12 hours of reoxygenation. TUNEL-positive nuclei are shown in green and transfection efficiency of adenoviruses in red. (b) Quantitative analysis (percentage of apoptotic cells versus total) is shown in histogram. C. Representative Western blots of cleaved caspase-3 and full length caspase-3 (FL caspase-3) in (a), and 3 independent experiments were quantitated in (b). β-actin was used as a loading control. D. Cell lysates under each condition were quantitatively assayed for caspase-3 activity. E. LDH concentrations in cell culture media were analyzed. Values are means ± S.E.M., n = 5 in each group. *P<0.05, **P<0.01 vs. Control group, and #P<0.05, vs. the corresponding Ad-RFP group.
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pone-0052469-g003: ALDH2 knockdown blocks isoflurane-induced protection against hypoxia/reoxygenation.A. Schematic representation of adenoviruses encoding RFP (Ad-RFP) and ALDH2shRNA (Ad-Si-ALDH2-RFP, left panel); right panel, immunoblotting analysis of ALDH2 protein level. β-actin was used as a control. B. ALDH2 down-regulation inhibited attenuation of TUNEL positive staining level by isoflurane. (a) Apoptotic cells were examined using TUNEL assay for DNA fragmentation. Cardiomyoctes were photographed by fluorescence microscopy after 24 hours of hypoxia followed by 12 hours of reoxygenation. TUNEL-positive nuclei are shown in green and transfection efficiency of adenoviruses in red. (b) Quantitative analysis (percentage of apoptotic cells versus total) is shown in histogram. C. Representative Western blots of cleaved caspase-3 and full length caspase-3 (FL caspase-3) in (a), and 3 independent experiments were quantitated in (b). β-actin was used as a loading control. D. Cell lysates under each condition were quantitatively assayed for caspase-3 activity. E. LDH concentrations in cell culture media were analyzed. Values are means ± S.E.M., n = 5 in each group. *P<0.05, **P<0.01 vs. Control group, and #P<0.05, vs. the corresponding Ad-RFP group.

Mentions: After 24 h of hypoxia followed by 12 h of reoxygenation we observed significant cardiomyocyte apoptosis demonstrated by increased DNA fragmentation using TUNEL staining, by laser scanning cytometry (LSC;) and caspase 3 activity in the vector control group. Pretreatment with isoflurane significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase 3 activity and leakage of LDH (Figure 3B–E). However, when ALDH2 was downregulated (Figure 3A) in cardiomyocytes by Ad-Si-ALDH2, increased TUNEL positive staining level (Figure 3B), more intense cleaved caspase-3 staining (Figure 3C), caspase 3 activity (Figure 3D) and LDH release (Figure 3E) were observed, which supports the hypothesis that phosphorylation of ALDH2 might play a critical role in isoflurane-induced cardioprotection. Immunoblotting analysis showed a substantial increase in ALDH2 level in Ad-CA-ALDH2-RFP-infected cells compared to Ad-RFP (Figure 4A). Constitutively active ALDH2 significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase 3 activity and leakage of LDH (Figure 4 B–E), which were not further increased by isoflurane treatment.


Isoflurane preconditioning confers cardioprotection by activation of ALDH2.

Lang XE, Wang X, Zhang KR, Lv JY, Jin JH, Li QS - PLoS ONE (2013)

ALDH2 knockdown blocks isoflurane-induced protection against hypoxia/reoxygenation.A. Schematic representation of adenoviruses encoding RFP (Ad-RFP) and ALDH2shRNA (Ad-Si-ALDH2-RFP, left panel); right panel, immunoblotting analysis of ALDH2 protein level. β-actin was used as a control. B. ALDH2 down-regulation inhibited attenuation of TUNEL positive staining level by isoflurane. (a) Apoptotic cells were examined using TUNEL assay for DNA fragmentation. Cardiomyoctes were photographed by fluorescence microscopy after 24 hours of hypoxia followed by 12 hours of reoxygenation. TUNEL-positive nuclei are shown in green and transfection efficiency of adenoviruses in red. (b) Quantitative analysis (percentage of apoptotic cells versus total) is shown in histogram. C. Representative Western blots of cleaved caspase-3 and full length caspase-3 (FL caspase-3) in (a), and 3 independent experiments were quantitated in (b). β-actin was used as a loading control. D. Cell lysates under each condition were quantitatively assayed for caspase-3 activity. E. LDH concentrations in cell culture media were analyzed. Values are means ± S.E.M., n = 5 in each group. *P<0.05, **P<0.01 vs. Control group, and #P<0.05, vs. the corresponding Ad-RFP group.
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pone-0052469-g003: ALDH2 knockdown blocks isoflurane-induced protection against hypoxia/reoxygenation.A. Schematic representation of adenoviruses encoding RFP (Ad-RFP) and ALDH2shRNA (Ad-Si-ALDH2-RFP, left panel); right panel, immunoblotting analysis of ALDH2 protein level. β-actin was used as a control. B. ALDH2 down-regulation inhibited attenuation of TUNEL positive staining level by isoflurane. (a) Apoptotic cells were examined using TUNEL assay for DNA fragmentation. Cardiomyoctes were photographed by fluorescence microscopy after 24 hours of hypoxia followed by 12 hours of reoxygenation. TUNEL-positive nuclei are shown in green and transfection efficiency of adenoviruses in red. (b) Quantitative analysis (percentage of apoptotic cells versus total) is shown in histogram. C. Representative Western blots of cleaved caspase-3 and full length caspase-3 (FL caspase-3) in (a), and 3 independent experiments were quantitated in (b). β-actin was used as a loading control. D. Cell lysates under each condition were quantitatively assayed for caspase-3 activity. E. LDH concentrations in cell culture media were analyzed. Values are means ± S.E.M., n = 5 in each group. *P<0.05, **P<0.01 vs. Control group, and #P<0.05, vs. the corresponding Ad-RFP group.
Mentions: After 24 h of hypoxia followed by 12 h of reoxygenation we observed significant cardiomyocyte apoptosis demonstrated by increased DNA fragmentation using TUNEL staining, by laser scanning cytometry (LSC;) and caspase 3 activity in the vector control group. Pretreatment with isoflurane significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase 3 activity and leakage of LDH (Figure 3B–E). However, when ALDH2 was downregulated (Figure 3A) in cardiomyocytes by Ad-Si-ALDH2, increased TUNEL positive staining level (Figure 3B), more intense cleaved caspase-3 staining (Figure 3C), caspase 3 activity (Figure 3D) and LDH release (Figure 3E) were observed, which supports the hypothesis that phosphorylation of ALDH2 might play a critical role in isoflurane-induced cardioprotection. Immunoblotting analysis showed a substantial increase in ALDH2 level in Ad-CA-ALDH2-RFP-infected cells compared to Ad-RFP (Figure 4A). Constitutively active ALDH2 significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase 3 activity and leakage of LDH (Figure 4 B–E), which were not further increased by isoflurane treatment.

Bottom Line: Pretreatment with isoflurane prior to ischemia reduced LDH and CK-MB levels and infarct size, while it increased phosphorylation of ALDH2, which could be blocked by the ALDH2 inhibitor, cyanamide.Hypoxia/reoxygenation (H/R) increased cardiomyocyte apoptosis and injury which were attenuated by isoflurane and forced the activation of ALDH2.In contrast, the effect of isoflurane-induced protection was almost abolished by knockdown of ALDH2.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, The First Clinical Medical College of Shanxi Medical University, Taiyuan, Shanxi, China.

ABSTRACT
The volatile anesthetic, isoflurane, protects the heart from ischemia/reperfusion (I/R) injury. Aldehyde dehydrogenase 2 (ALDH2) is thought to be an endogenous mechanism against ischemia-reperfusion injury possibly through detoxification of toxic aldehydes. We investigated whether cardioprotection by isoflurane depends on activation of ALDH2.Anesthetized rats underwent 40 min of coronary artery occlusion followed by 120 min of reperfusion and were randomly assigned to the following groups: untreated controls, isoflurane preconditioning with and without an ALDH2 inhibitor, the direct activator of ALDH2 or a protein kinase C (PKCε) inhibitor. Pretreatment with isoflurane prior to ischemia reduced LDH and CK-MB levels and infarct size, while it increased phosphorylation of ALDH2, which could be blocked by the ALDH2 inhibitor, cyanamide. Isolated neonatal cardiomyocytes were treated with hypoxia followed by reoxygenation. Hypoxia/reoxygenation (H/R) increased cardiomyocyte apoptosis and injury which were attenuated by isoflurane and forced the activation of ALDH2. In contrast, the effect of isoflurane-induced protection was almost abolished by knockdown of ALDH2. Activation of ALDH2 and cardioprotection by isoflurane were substantially blocked by the PKCε inhibitor. Activation of ALDH2 by mitochondrial PKCε plays an important role in the cardioprotection of isoflurane in myocardium I/R injury.

Show MeSH
Related in: MedlinePlus