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Carbonic Anhydrase III Attenuates Hypoxia-Induced Apoptosis and Activates PI3K/Akt/mTOR Pathway in H9c2 Cardiomyocyte Cell Line

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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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Data availability

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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Author notes

  1. Hua Li and Yibin Liu are co-first authors.

Authors and Affiliations

  1. Cardiac Ultrasonic Department, Traditional Chinese Medicine Hospital Affiliated to Xinjiang Medical University, No. 116 Huanghe Road, Shayibake District, Ürümqi, 830002, Xinjiang, China

    Hua Li, Sha Tang, Jie Hu, Qiuling Wu, Yang Wei & Ming Niu

  2. Ultrasonic Department, First Affiliated Hospital of Xinjiang Medical University, Ürümqi, 830011, Xinjiang, China

    Yibin Liu

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  1. Hua Li
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Contributions

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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Correspondence to Hua Li.

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The authors declare that they have no competing interests.

Ethical approval

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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Handling Editor: Y. James Kang.

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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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