氧化三甲胺损害肥厚型心肌病小鼠心功能的机制研究

doi:10.3969/j.issn.1674-8115.2024.03.004

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (3): 325-333.doi: 10.3969/j.issn.1674-8115.2024.03.004

• 论著 · 基础研究 • 上一篇

氧化三甲胺损害肥厚型心肌病小鼠心功能的机制研究

靳步¹(), 陈汉章²(), 徐浒东¹, 陈婉玉¹, 袁颖¹, 赵婷婷¹, 黄晓蕾¹, 何佳璐¹, 于红¹^,³()

^1.上海交通大学医学院附属第一人民医院嘉定医院/上海市嘉定区江桥医院病理科，上海 201803
^2.上海市静安区闸北中心医院病理科，上海 200070
^3.上海交通大学医学院附属第一人民医院病理中心，上海 200080

收稿日期:2023-10-18 接受日期:2024-01-31 出版日期:2024-03-28 发布日期:2024-04-29
通讯作者: 于红 E-mail:progress1991@163.com;chz1970@163.com;1154879190@qq.com
作者简介:靳　步（1991—），女，病理学技术师，硕士；电子信箱：progress1991@163.com
陈汉章（1970—），男，主任医师，硕士；电子信箱：chz1970@163.com。第一联系人：（靳步、陈汉章列第一作者）
基金资助:
上海市卫生健康委员会卫生行业临床研究专项(20224Y0234);上海市嘉定区江桥医院科技创新项目(202228B)

Study on the mechanism of trimethylamine oxide damaging cardiac function in mice with hypertrophic cardiomyopathy

JIN Bu¹(), CHEN Hanzhang²(), XU Hudong¹, CHEN Wanyu¹, YUAN Ying¹, ZHAO Tingting¹, HUANG Xiaolei¹, HE Jialu¹, YU Hong¹^,³()

^1.Department of Pathology, Jiading Branch of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201803, China
^2.Department of Pathology, Zhabei Central Hospital of Shanghai Jingan District, Shanghai 200070, China
^3.Pathology Center, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China

Received:2023-10-18 Accepted:2024-01-31 Online:2024-03-28 Published:2024-04-29
Contact: YU Hong E-mail:progress1991@163.com;chz1970@163.com;1154879190@qq.com
Supported by:
Health Industry Clinical Research Project of Shanghai Municipal Health Commission(20224Y0234);Science and Technology Innovation Project of Shanghai Jiading District Jiangqiao Hospital(202228B)

摘要/Abstract

摘要：

目的·探讨氧化三甲胺（trimethylamine oxide，TMAO）对肥厚型心肌病（hypertrophic cardiomyopathy，HCM）小鼠心功能的影响和潜在分子机制。方法·以肌球蛋白重链6（myosin heavy chain 6，Myh6） c.1211G>A（p.R404Q^+/-）点突变的小鼠（HCM小鼠）为动物模型。根据分别向饲料中补充TMAO、TMAO抑制剂碘甲基胆碱（iodomethylcholine，IMC）进行喂养，将野生型（wild type，WT）小鼠、HCM小鼠分为WT组、HCM组（HCM-1组、HCM-2组）、WT+TMAO组、HCM+TMAO组及HCM+IMC组。采用超声心动图评估上述小鼠的左心室短轴缩短（left ventricular fraction shortening，FS）率和左心室后壁厚度（left ventricular posterior wall thickness，LVPW）。采用酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）检测HCM-1组和WT组小鼠血清TMAO浓度。采用苏木精-伊红染色（hematoxylin and eosin staining，HE staining，HE染色）评估小鼠心肌细胞排列规整度。采用马松（Masson）染色评估小鼠心肌纤维化比例。使用心肌蛋白激酶A（protein kinase A，PKA）试剂盒检测小鼠心肌组织PKA活性。采用蛋白质印迹法（Western blotting）检测小鼠心肌组织兰尼碱受体2（ryanodine receptor 2，RyR2）及p-RyR2（S2808）的表达。结果·超声心动图的结果显示，12月龄时WT+TMAO组、HCM+TMAO组小鼠的FS率分别低于对应的WT组、HCM-1组（均P<0.05）；HCM+TMAO组小鼠的LVPW高于HCM-1组，而HCM+IMC组小鼠的LVPW低于HCM-2组（均P<0.05）。ELISA结果显示，HCM-1组小鼠血清中TMAO浓度高于WT组（P<0.05）。HE染色和Masson染色的结果显示，HCM+TMAO组小鼠相较于HCM-1组的心肌细胞排列规整程度较低、纤维化比例较高，而HCM+IMC组小鼠相较于HCM-2组则相反（均P<0.05）。PKA的检测结果显示，经TMAO饲养后，WT+TMAO组、HCM+TMAO组小鼠的心肌组织PKA活性均有增加，而经IMC饲养的HCM+IMC组小鼠的心肌组织PKA活性则有下降（均P<0.05）。Western blotting的结果显示，经TMAO饲养后，WT+TMAO组、HCM+TMAO组小鼠的心肌组织中p-RyR2（S2808）表达升高，经IMC饲养的HCM+IMC组小鼠的p-RyR2（S2808）表达则降低（均P<0.05）；而RyR2的表达在各组间未见差异。结论·TMAO可增加心肌PKA活性，诱导RyR2色氨酸2808位点发生磷酸化，能够引起HCM小鼠心室重构并损害其心功能。

关键词: 肥厚型心肌病, 氧化三甲胺, 蛋白激酶A, 兰尼碱受体2

Abstract:

Objective ·To investigate the effects of trimethylamine oxide (TMAO) on cardiac function in mice with hypertrophic cardiomyopathy (HCM) and its potential molecular mechanism. Methods ·Mice with myosin heavy chain 6 (Myh6) c.1211G>A (p.R404Q^+/-) point mutation were used as the animal model. According to dietary supplementation of TMAO and TMAO inhibitor iodomethylcholine (IMC), the wild type (WT) mice and HCM mice were divided into WT group, HCM group (HCM-1 group, HCM-2 group), WT+TMAO group, HCM+TMAO group and HCM+IMC group, respectively. Left ventricular fraction shortening (FS) and left ventricular posterior wall thickness (LVPW) were assessed by echocardiography in all mice. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum TMAO concentration of mice in the HCM-1 group and WT group. The regularity of myocardial cell arrangement of mice was evaluated by hematoxylin and eosin staining (HE staining). The proportion of myocardial fibrosis was evaluated by Masson staining. The activity of protein kinase A (PKA) in mouse myocardial tissue was detected by PKA kit. The expression of ryanodine receptor 2 (RyR2) and p-RyR2(S2808) in mouse myocardial tissue was detected by Western blotting. Results ·The results of echocardiography showed that at 12 months of age, the FS of mice in the WT+TMAO group and HCM+TMAO group were lower than those in the corresponding WT group and HCM-1 group, respectively (P<0.05). The LVPW of mice in the HCM+TMAO group was higher than that in the HCM-1 group, while the LVPW of mice in the HCM+IMC group was lower than that in the HCM-2 group (P<0.05). ELISA results showed that the serum TMAO concentration of mice in the HCM-1 group was higher than that in the WT mice (P<0.05). The results of HE staining and Masson staining showed that the HCM+TMAO group had a lower degree of regular arrangement of cardiomyocytes and a higher proportion of fibrosis than the HCM-1 group, while the HCM+IMC group had a higher degree of regular arrangement of cardiomyocytes and a lower proportion of fibrosis than the HCM-2 group (P<0.05). The results of PKA assay showed that the PKA activity in the myocardial tissue of mice in the WT+TMAO group and HCM+TMAO group increased after TMAO treatment, while the PKA activity in the myocardial tissue of mice in the HCM+IMC group decreased (P<0.05). Western blotting results showed that the expression of p-RyR2(S2808) in the myocardial tissue of the WT+TMAO group and HCM+TMAO group mice increased, while it was decreased in the HCM+IMC group mice (P<0.05); however, there was no difference in RyR2 expression among the groups. Conclusion ·TMAO can increase the activity of PKA and induce the phosphorylation of RyR2 at S2808, which can cause ventricular remodeling and impair cardiac function in HCM mice.

Key words: hypertrophic cardiomyopathy (HCM), trimethylamine oxide (TMAO), protein kinase A (PKA), ryanodine receptor 2 (RyR2)

中图分类号:

R542.2

靳步, 陈汉章, 徐浒东, 陈婉玉, 袁颖, 赵婷婷, 黄晓蕾, 何佳璐, 于红. 氧化三甲胺损害肥厚型心肌病小鼠心功能的机制研究[J]. 上海交通大学学报（医学版）, 2024, 44(3): 325-333.

JIN Bu, CHEN Hanzhang, XU Hudong, CHEN Wanyu, YUAN Ying, ZHAO Tingting, HUANG Xiaolei, HE Jialu, YU Hong. Study on the mechanism of trimethylamine oxide damaging cardiac function in mice with hypertrophic cardiomyopathy[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(3): 325-333.

图/表 5

图1 TMAO和IMC对各组小鼠心功能和LPVW的影响Note：A. Representative echocardiographic pictures and statistical analysis charts of mice in the WT group, WT+TMAO group, HCM-1 group and HCM+TMAO group. B. Representative echocardiographic pictures and statistical analysis charts of mice in the HCM-2 group and HCM+IMC group.

Fig 1 Effects of TMAO and IMC on cardiac function and LPVW in each group of mice

图2 WT组和HCM-1组小鼠血清中TMAO浓度检测

Fig 2 Detection of serum TMAO concentration of mice in the WT group and HCM-1 group

图3 TMAO和IMC对各组小鼠心肌细胞排列规整度和心肌组织纤维化比例的影响Note： A. HE staining of mouse myocardium tissue in WT group, WT+TMAO group, HCM-1 group and HCM+TMAO group (×20). The blue arrows indicate disordered arrangement of cardiomyocytes. B. Masson staining of mouse myocardium tissue in WT group, WT+TMAO group, HCM-1 group and HCM+TMAO group (×20). Red represents the myocardium tissue and blue represents fibrosis of the myocardium tissue. C. HE staining and Masson staining of mouse myocardium tissue in HCM-2 group and HCM+IMC group (×20).

Fig 3 Effects of TMAO and IMC on the regularity of myocardial cell arrangement and the proportion of myocardial fibrosis in each group of mice

图4 各组小鼠心肌组织中PKA活性的差异Note： A. PKA activity in myocardium tissue of mice in WT group, WT+TMAO group, HCM-1 group and HCM+TMAO group. B. PKA activity in myocardium tissue of mice in HCM-2 group and HCM+IMC group.

Fig 4 Differences of PKA activity in myocardium tissue of mice in each group

图5 各组小鼠心肌组织中RyR2及p-RyR2(S2808)的表达情况检测Note： A. Detection of RyR2 and p-RyR2(S2808) expression in WT group, WT+TMAO group, HCM-1 group and HCM+TMAO group by Western blotting. B. Detection of RyR2 and p-RyR2(S2808) expression in HCM-2 group and HCM+IMC group by Western blotting.

Fig 5 Detection of RyR2 and p-RyR2(S2808) expression in myocardium tissue of mice in each group

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氧化三甲胺损害肥厚型心肌病小鼠心功能的机制研究

Study on the mechanism of trimethylamine oxide damaging cardiac function in mice with hypertrophic cardiomyopathy

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