Effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat

Lyu Shangjun; Fan Ronghui; Wu Dan; Peng Xi

doi:10.3760/cma.j.cn501120-20210601-00208

Volume 37 Issue 12

Dec. 2021

Turn off MathJax

Article Contents

Abstract

References

Article Navigation > CHINESE JOURNAL OF BURNS AND WOUNDS > 2021 > 37(12): 1149-1157

Lyu SJ,Fan RH,Wu D,et al.Effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat[J].Chin J Burns,2021,37(12):1149-1157.DOI: 10.3760/cma.j.cn501120-20210601-00208.

Citation:

PDF( 4158 KB)

Effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat

doi: 10.3760/cma.j.cn501120-20210601-00208

1.
Department of Burns and Plastic Surgery, Shaanxi Armed Police Corps Hospital, Xi'an 710086, China
2.
Department of Burns and Plastic Surgery, Shaanxi Provincial People's Hospital, Xi'an 710068, China
3.
Clinical Medical Research Center, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing 400038, China

Funds:

General Program of National Natural Science Foundation of China 81971838

More Information

Corresponding author: Peng Xi, Email: pxlrmm@163.com
Received Date: 2021-06-01

Abstract

Abstract

Objective To investigate the effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat (hereinafter referred to as burn serum). Methods The experimental research method was applied. Ten gender equally distributed Wistar rats aged 7-8 months were taken to prepare normal rat serum (hereinafter referred to as normal serum), another twenty gender equally distributed Wistar rats aged 7-8 months were taken to prepare burn serum after full- thickness burn injury of 30% total body surface area, and primary cardiomyocytes were isolated and cultured from 180 Wistar rats aged 1-3 days by either gender and used in the following experiments. The cells were divided into normal serum group and burn serum group according to the random number table (the same grouping method below) and cultured with the corresponding serum. At post culture hour (PCH) 1, 3, 6, 9, and 12, trypanosoma blue test was used to detect the cell survival rate. The cells were divided into burn serum alone group, burn serum+4 mmol/L glutamine group, burn serum+8 mmol/L glutamine group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group, which were treated with burn serum alone or burn serum added with the corresponding final molarity of glutamine and cultured for the time screened in the experiment before, and then the cell survival rate was detected as before. The cells were divided into normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group and treated the same as before. After 30 min of culture, phosphorylation levels of mammalian target of rapamycin complex 1 (mTORC1), p70 ribosomal protein S6 kinase (p70 S6K), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) were detected by Western blotting. Cells were divided into normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group, and treated correspondingly. At PCH 1, 3, and 6, the expressions of heat shock protein 70 (HSP70) and metallothionein (MT), and the morphology of microtubule were determined with immunofluorescence method. The sample numbers in each index at each time point in each group were all 10. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference t test, least significant difference test, and Bonferroni correction. Results At PCH 1, 3, 6, 9, and 12, the cell survival rates in burn serum group were significantly lower than those in normal serum group (t=4.950, 16.752, 35.484, 34.428, 27.781, P<0.01). Compared within the group at PCH 1, the cell survival rate was significantly decreased in burn serum group at PCH 3, 6, 9, and 12 (P<0.05). Compared within the group at PCH 3, the cell survival rate was significantly decreased in burn serum group at PCH 6, 9, and 12 (P<0.05). Compared within the group at PCH 6 and 9, the cell survival rate was significantly decreased in burn serum group at PCH 12 (P<0.05). There were no statistically significant differences in the cell survival rates in burn serum group between PCH 6 and 9 (P>0.05). Thus PCH 6 was selected as the subsequent intervention time of burn serum. At PCH 6, compared with burn serum alone group, the cell survival rates in burn serum+4 mmol/L glutamine group, burn serum+8 mmol/L glutamine group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group were significantly increased (P<0.01). There were no statistically significant differences in cell survival rates between burn serum+12 mmol/L glutamine group and burn serum+16 mmol/L glutamine group (P>0.05). There were no statistically significant differences in cell survival rates in burn serum+16 mmol/L glutamine group and burn serum+20 mmol/L glutamine group (P>0.05). Thus 12, 16, and 20 mmol/L were selected as the subsequent intervention concentrations of glutamine. After 30 min of culture, the phosphorylation levels of mTORC1, p70 S6K, and 4E-BP1 of cells were respectively 1.001±0.042, 0.510±0.024, 0.876±0.022, 0.836±0.074, 0.856±0.041, 1.00±0.11, 0.38±0.09, 0.95±0.13, 0.96±0.13, 0.89±0.24, 1.00±0.07, 0.29±0.08, 0.87±0.27, 0.68±0.08, 0.60±0.21 in normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group. Compared with normal serum group, the phosphorylation levels of mTORC1, p70 S6K, and 4E-BP1 of cells were significantly decreased in the other 4 burn serum groups (P<0.01). Compared with those of burn serum alone group, the phosphorylation levels of mTORC1, p70 S6K, and 4E-BP1 of cells were significantly increased in the other 3 burn serum groups (P<0.01). The phosphorylation level of 4E-BP1 of cells in burn serum+12 mmol/L glutamine group was significantly higher than the levels in burn serum+16 mmol/L glutamine group and burn serum+20 mmol/L glutamine group (P<0.05). The expression of MT of cells in burn serum alone group was significantly lower than that in normal serum group at PCH 1 (P<0.05), while the expressions of MT of cells in burn serum alone group were significantly higher than those in normal serum group at the other time points (P<0.05). At PCH 1, 3, and 6, the expressions of HSP70 of cells in burn serum alone group were significantly higher than those in normal serum group (P<0.05), the expressions of HSP70 and MT of cells in burn serum+12 mmol/L glutamine group were significantly higher than those in burn serum alone group (P<0.05), and the expressions of HSP70 and MT of cells in burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group were significantly lower than those in burn serum+12 mmol/L glutamine group (P<0.01). The microtubular structures were intact, displaying grid alinement and uniform staining in cells of normal serum group at PCH 1, 3, and 6. In burn serum alone group, some microtubules showed fracture and irregular grid arrangement at PCH 1; the microtubular structures near the nucleus were clear, while the microtubules at the distal end of the nucleus were blurry at PCH 3; the microtubular structures were blurry at PCH 6. The microtubular damage of cells was alleviated in burn serum+12 mmol/L glutamine group as compared with that in burn serum alone group at each time point of culture. The morphology of microtubules of cells in burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group at each time point of culture was similar to that of burn serum alone group. Conclusions The burn serum can lead to damages to cardiomyocytes and significant decrease of cell survival rate in rats. Glutamine can exert cell protective function through the regulation of mTOR/p70 S6K/4E-BP1 signaling pathway, thus promoting the expressions of HSP70 and MT and stabilizing the microtubule structures.
- Burns,
- Glutamine,
- Myocytes, cardiac,
- Mammalian target of rapamycin signaling pathway

FullText(HTML)

References(33)

References

[1]	彭曦. 烧伤高代谢机制的再认识及调控策略[J].中华烧伤杂志,2013,29(2):139-143. DOI: 10.3760/cma.j.issn.1009-2587.2013.02.012.
[2]	黄跃生. 自噬与严重烧伤后心肌缺血缺氧损害[J].中华烧伤杂志,2018,34(1):3-7. DOI: 10.3760/cma.j.issn.1009-2587.2018.01.002.
[3]	VermaA, SumiS, SeerviM. Heat shock proteins-driven stress granule dynamics: yet another avenue for cell survival[J]. Apoptosis, 2021,26(7/8):371-384. DOI: 10.1007/s10495-021-01678-w.
[4]	KurashovaNA, MadaevaIM, KolesnikovaLI. Expression of heat shock proteins HSP70 under oxidative stress[J]. Adv Gerontol, 2019,32(4):502-508.
[5]	吕尚军, 张勇, 孙勇, 等. 甘氨酰谷氨酰胺二肽对烧伤大鼠心功能的保护作用[J].中华烧伤杂志,2007,23(4):244-248. DOI: 10.3760/cma.j.issn.1009-2587.2007.04.002.
[6]	彭曦. 重视谷氨酰胺在烧伤临床的规范应用[J].肠外与肠内营养,2021,28(1):1-4. DOI: 10.16151/j.1007-810x.2021.01.001.
[7]	KimuraN, TokunagaC, DalalS, et al. A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway[J]. Genes Cells, 2003,8(1):65-79. DOI: 10.1046/j.1365-2443.2003.00615.x.
[8]	SaraviaJ, RaynorJL, ChapmanNM, et al. Signaling networks in immunometabolism[J]. Cell Res, 2020,30(4):328-342. DOI: 10.1038/s41422-020-0301-1.
[9]	RindomE, KristensenAM, OvergaardK, et al. Estimation of p70S6K Thr389 and 4E-BP1 Thr37/46 phosphorylation support dependency of tension per se in a dose-response relationship for downstream mTORC1 signalling[J]. Acta Physiol (Oxf), 2020,229(1):e13426. DOI: 10.1111/apha.13426.
[10]	HuangHL, LongLY, ZhouPP, et al. mTOR signaling at the crossroads of environmental signals and T-cell fate decisions[J]. Immunol Rev, 2020,295(1):15-38. DOI: 10.1111/imr.12845.
[11]	GonzálezA, HallMN, LinSC, et al. AMPK and TOR: the Yin and Yang of cellular nutrient sensing and growth control[J]. Cell Metab, 2020,31(3):472-492. DOI: 10.1016/j.cmet.2020.01.015.
[12]	MacFieJ, McNaughtC. Glutamine and gut barrier function[J]. Nutrition, 2002,18(5):433-434. DOI: 10.1016/s0899-9007(02)00766-9.
[13]	YangYJ, LiuMM, ZhangY, et al. Effectiveness and mechanism study of glutamine on alleviating hypermetabolism in burned rats[J]. Nutrition, 2020,79/80:110934. DOI: 10.1016/j.nut.2020.110934.
[14]	YuHD, ZhangYM, ZhangZY, et al. Towards identification of molecular mechanism in which the overexpression of wheat cytosolic and plastid glutamine synthetases in tobacco enhanced drought tolerance[J]. Plant Physiol Biochem, 2020,151:608-620. DOI: 10.1016/j.plaphy.2020.04.013.
[15]	XiaY, WenHY, YoungME, et al. Mammalian target of rapamycin and protein kinase A signaling mediate the cardiac transcriptional response to glutamine[J]. J Biol Chem, 2003,278(15):13143-13150. DOI: 10.1074/jbc.M208500200.
[16]	ShawRJ. mTOR signaling: RAG GTPases transmit the amino acid signal[J]. Trends Biochem Sci, 2008,33(12):565-568. DOI: 10.1016/j.tibs.2008.09.005.
[17]	KameiY, HatazawaY, UchitomiR, et al. Regulation of skeletal muscle function by amino acids[J]. Nutrients, 2020,12(1):261.DOI: 10.3390/nu12010261.
[18]	LaplanteM, SabatiniDM. mTOR signaling in growth control and disease[J]. Cell, 2012,149(2):274-293. DOI: 10.1016/j.cell.2012.03.017.
[19]	TafurL, KefauverJ, LoewithR. Structural insights into TOR signaling[J]. Genes (Basel), 2020,11(8):885.DOI: 10.3390/genes11080885.
[20]	SzwedA, KimE, JacintoE. Regulation and metabolic functions of mTORC1 and mTORC2[J]. Physiol Rev, 2021,101(3):1371- 1426. DOI: 10.1152/physrev.00026.2020.
[21]	SaxtonRA, SabatiniDM. mTOR signaling in growth, metabolism, and disease[J]. Cell, 2017,168(6):960-976. DOI: 10.1016/j.cell.2017.02.004.
[22]	ParkJH, LeeG, BlenisJ. Structural insights into the activation of mTORC1 on the lysosomal surface[J]. Trends Biochem Sci, 2020,45(5):367-369. DOI: 10.1016/j.tibs.2020.02.004.
[23]	Francois-VaughanH, AdebayoAO, BrilliantKE, et al. Persistent effect of mTOR inhibition on preneoplastic foci progression and gene expression in a rat model of hepatocellular carcinoma[J]. Carcinogenesis, 2016,37(4):408-419. DOI: 10.1093/carcin/bgw016.
[24]	YangHJ, RudgeDG, KoosJD, et al. mTOR kinase structure, mechanism and regulation[J]. Nature, 2013,497(7448):217-223. DOI: 10.1038/nature12122.
[25]	ZhuJ, WangYF, ChaiXM, et al. Exogenous NADPH ameliorates myocardial ischemia-reperfusion injury in rats through activating AMPK/mTOR pathway[J]. Acta Pharmacol Sin, 2020,41(4):535-545. DOI: 10.1038/s41401-019-0301-1.
[26]	ItoN, RueggUT, TakedaS. ATP-induced increase in intracellular calcium levels and subsequent activation of mTOR as regulators of skeletal muscle hypertrophy[J]. Int J Mol Sci, 2018,19(9):2804.DOI: 10.3390/ijms19092804.
[27]	ParrottaL, CrestiM, CaiG. Heat-shock protein 70 binds microtubules and interacts with kinesin in tobacco pollen tubes[J]. Cytoskeleton (Hoboken), 2013,70(9):522-537. DOI: 10.1002/cm.21134.
[28]	YuanAT, KorkolaNC, WongDL, et al. Metallothionein Cd4S11 cluster formation dominates in the protection of carbonic anhydrase[J]. Metallomics, 2020,12(5):767-783. DOI: 10.1039/d0mt00023j.
[29]	YangLF, MaJP, TanY, et al. Cardiac-specific overexpression of metallothionein attenuates L-NAME-induced myocardial contractile anomalies and apoptosis[J]. J Cell Mol Med, 2019,23(7):4640-4652. DOI: 10.1111/jcmm.14375.
[30]	党永明, 房亚东, 胡炯宇, 等.成体大鼠心肌细胞微管解聚对线粒体分布及能量代谢的影响[J] . 中华烧伤杂志,2010,26(1): 18-22. DOI: 10.3760/cma.j.issn.1009-2587.2010.01.006.
[31]	XiangF, MaSY, LvYL, et al. Tumor necrosis factor receptor- associated protein 1 regulates hypoxia-induced apoptosis through a mitochondria-dependent pathway mediated by cytochrome c oxidase subunit Ⅱ[J/OL]. Burns Trauma, 2019,7:16[2021-06-01]. https://pubmed.ncbi.nlm.nih.gov/31143823/.DOI: 10.1186/s41038-019- 0154-3.
[32]	滕苗, 黄跃生, 郑霁, 等. 微管干预剂对大鼠缺氧心肌细胞能量生成的影响[J].中华烧伤杂志,2007,23(3):164-167. DOI: 10.3760/cma.j.issn.1009-2587.2007.03.002.
[33]	BuruteM, KapiteinLC. Cellular logistics: unraveling the interplay between microtubule organization and intracellular transport[J]. Annu Rev Cell Dev Biol, 2019,35:29-54. DOI: 10.1146/annurev-cellbio-100818-125149.