Abstract
Myocardial ischemia can cause insufficient oxygen and functional damage to myocardial cells. Carbonic anhydrase III (CAIII) has been found to be closely related to the abnormality of cardiomyocytes. To investigate the role of CAIII in the apoptosis of myocytes under hypoxic conditions and facilitate the strategy for treating hypoxia-induced damage, in vitro experiments in H9c2 were employed. The protein expression of CAIII in H9c2 cells after hypoxia or normoxia treatment was determined by western blotting and immunohistochemistry. MTT assay was employed for cells viability measurement and LDH release was monitored. The apoptotic cells were observed using immunofluorescence assay, flow cytometric analysis, and TUNEL assay. CAIII-overexpression or -knockdown cells were constructed to determine the role of CAIII in regulating apoptosis-related proteins caspase-3, Bax, Bcl-2, and anti-apoptosis pathway PI3K/Akt/mTOR. The mRNA levels of CAIII and genes related to CAIII synthesis including REN, IGHM, APOBEC 3F, and SKOR2 were significantly upregulated in hypoxia fetal sheep. The expression of CAIII protein and content of apoptotic H9c2 cells were increased at 1, 3, 6, and 12 h after hypoxia treatment. Overexpression of CAIII significantly upregulated Bcl2 level and downregulated Bax and caspase-3 cleavage levels, while its knockdown led to the contrary results. Overexpressed CAIII promoted the HIF-1α level and activated the PI3K/Akt/mTOR pathway, thereby exerting an inhibitory effect on hypoxia-induced apoptosis. In conclusion, our findings revealed that CAIII could protect cell from hypoxia-apoptosis of H9c2 cells, in which, activated PI3K/Akt/mTOR signaling pathway may be involved.
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All data generated or analyzed during this study are included in this manuscript.
Abbreviations
- CAIII:
-
Carbonic anhydrase III
- AMI:
-
Acute myocardial infarction
- MI:
-
Myocardial ischemia
- CA:
-
Carbonic anhydrase
- qRT-PCR:
-
Real-time reverse transcription quantitative PCR
- FBS:
-
Fetal bovine serum
- H/N:
-
Hypoxia/Normoxia
- DAPI:
-
4',6-Diamidino-2-phenylindole
- AV/PI:
-
Annexin V-FITC/propidium iodide
- DMSO:
-
Dimethyl sulfoxide
- LDH:
-
Lactate dehydrogenase
- SD:
-
Standard deviation
- ANOVA:
-
Analysis of variance
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Funding
This research was supported by “The effects of hypoxia during pregnancy on fetal cardiovascular development and its related mechanisms” (Grant No.: 81560291).
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HL conceived and designed the entire study; HL and YBL collected and analyzed the data, and they were responsible for data interpretation; they were co-first authors; ST, JH, QLW, YW, and MN performed statistical analysis, literature research, and data visualization; HL and YBL wrote the manuscript. HL revised it critically for important intellectual content. All authors have read and approved the final manuscript.
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This study was approved by the Ethics Committee of General Hospital of Xinjiang Military Region of the People's Liberation Army (No. 2017.301).
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Li, H., Liu, Y., Tang, S. et al. Carbonic Anhydrase III Attenuates Hypoxia-Induced Apoptosis and Activates PI3K/Akt/mTOR Pathway in H9c2 Cardiomyocyte Cell Line. Cardiovasc Toxicol 21, 914–926 (2021). https://doi.org/10.1007/s12012-021-09683-w
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DOI: https://doi.org/10.1007/s12012-021-09683-w