Circular RNA CHACR regulates pressure overload-induced cardiac hypertrophy and oxidative stress damage

doi:10.12307/2025.511

Abstract

Abstract: BACKGROUND: Pathological cardiac hypertrophy is a risk factor for various heart diseases, but its pathogenesis remains unclear. Circular RNAs are strongly associated with cardiac hypertrophy. However, the role of circular RNA CHACR in cardiac hypertrophy and its regulatory mechanisms have not been clarified.
OBJECTIVE: To investigate the role of circular RNA CHACR in pressure overload-induced cardiac hypertrophy and the underlying mechanisms.
METHODS: (1) Transverse aortic constriction was used to induce cardiac hypertrophy in vivo after in situ injection of cyclic RNA CHACR overexpressing lentivirus into the heart for 1 week. Heart mass/tibia length ratio and lung mass/tibia length ratio were calculated; cardiomyocyte surface area was measured; hypertrophic marker gene expression levels were detected; myocardial fibrosis degree was detected, and cardiac function was assessed. (2) H9c2 cardiomyocytes were treated with circular RNA CHACR overexpressing lentivirus for 72 hours, and then treated with 1 µmol/L angiotensin II for 24 hours to induce hypertrophy of cardiomyocytes. The hypertrophy was assessed by measuring the surface area of cardiomyocytes, the expression level of hypertrophic marker genes, and the protein /DNA ratio. Oxidative stress damage was assessed by detecting reactive oxygen species levels and mitochondrial membrane potential.
RESULTS AND CONCLUSION: (1) The expression level of circular RNA CHACR was significantly decreased in both in vivo and in vitro myocardial hypertrophy models (P < 0.01). (2) The overexpression of circular RNA CHACR significantly inhibited the cardiac hypertrophy induced by transverse aortic constriction, including reducing the enlarged heart volume, significantly decreasing the increased heart mass/tibia length ratio (P < 0.05), lung mass/tibia length ratio (P < 0.05), and cardiomyocyte surface area (P < 0.05), and decreasing the upregulated expression levels of hypertrophic markers atrial natriuretic peptide (P < 0.05) and brain natriuretic peptide (P < 0.05). (3) Cardiac fibrosis induced by transverse aortic constriction in mice was significantly inhibited by enforcing expression of circular RNA CHACR, as evidenced by reduced fibrotic area (P < 0.01) and decreased expression levels of the fibrosis marker gene Acta1 (P < 0.05). (4) Overexpression of circular RNA CHACR significantly improved cardiac function in mice, including significantly increased ejection fraction (P < 0.05) and fractional shortening (P < 0.01). (5) Enforced expression of circular RNA CHACR significantly inhibited angiotensin II-induced cardiomyocyte hypertrophy, including a significant reduction in cardiomyocyte surface area (P < 0.05), downregulation of atrial natriuretic peptide (P < 0.05), and brain natriuretic peptide (P < 0.05) expression levels, and a significant decrease in protein/DNA ratio (P < 0.05). (6) Overexpression of circular RNA CHACR significantly inhibited the elevation of reactive oxygen species levels (P < 0.001) and the decrease in mitochondrial membrane potential (P < 0.05) induced by angiotensin II. These results confirm that the expression level of circular RNA CHACR is significantly decreased in cardiac hypertrophy at both in vivo and in vitro myocardial hypertrophy models, and overexpression of circular RNA CHACR significantly inhibits cardiac hypertrophy, alleviates cardiac fibrosis, improves cardiac function, and significantly attenuates angiotensin II-induced oxidative stress damage.

Key words: cardicac, circular RNA, CHACR, angiotensin II, transverse aortic constriction, oxidative stress damage

CLC Number:

Wang Shuang, Han Yu, Yuan Min, Cao Jimin, Sun Teng. Circular RNA CHACR regulates pressure overload-induced cardiac hypertrophy and oxidative stress damage[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(25): 5362-5373.

Figures/Tables 7

2.6 过表达circ-CHACR抑制横向主动脉缩窄手术诱导的心脏纤维化为了检测circ-CHACR在横向主动脉缩窄手术诱导的小鼠心脏纤维化中的作用，通过天狼猩红染色检测心脏组织的纤维化面积。结果显示，与假手术组相比，横向主动脉缩窄手术组的纤维化程度加重，纤维化面积增多(P < 0.05)和纤维化相关基因Acta1的表达水平升高(P < 0.000 1)。而当心脏原位注射circ-CHACR过表达慢病毒后，与阴性对照组相比，circ-CHACR过表达后显著降低了心肌纤维化面积(P < 0.01)和Acta1的表达水平(P < 0.05)，见图6A-C。以上结果表明，过表达circ-CHACR显著抑制了横向主动脉缩窄手术诱导的小鼠心脏纤维化。 2.7 过表达circ-CHACR改善横向主动脉缩窄手术诱导的心功能障碍为了检测circ-CHACR对横向主动脉缩窄手术诱导的小鼠心功能的影响，通过超声心动图检测小鼠心脏功能。结果显示，与假手术组相比，横向主动脉缩窄手术组小鼠的射血分数和短轴缩短率显著降低(P < 0.000 1)。而当心脏原位注射circ-CHACR过表达慢病毒后，与阴性对照组相比，circ-CHACR过表达后显著抑制了射血分数 (P < 0.05)和短轴缩短率(P < 0.01)的降低，见图7A，B。以上结果表明，过表达circ-CHACR明显改善了横向主动脉缩窄手术诱导的心功能障碍。 2.8 过表达circ-CHACR抑制血管紧张素Ⅱ诱导的心肌细胞肥大为了检测circ-CHACR在血管紧张素Ⅱ诱导的心肌细胞肥大模型中的作用，构建了circ-CHACR过表达的慢病毒。分别用circ-CHACR过表达慢病毒和其阴性对照慢病毒处理H9c2细胞72 h，通过实时荧光定量PCR实验检测circ-CHACR的表达水平。结果显示，与circ-CHACR阴性对照组相比，circ-CHACR的表达水平明显升高(P < 0.01)，表明circ-CHACR在体外被成功过表达，可用于后续实验研究，见图8。"

为了进一步检测circ-CHACR是否参与了血管紧张素Ⅱ诱导的心肌细胞肥大机制调控，继续展开研究。结果显示，与对照组相比，血管紧张素Ⅱ处理后，心肌细胞的表面积明显增大(P < 0.01)，见图9A，B，心房利钠肽(P < 0.05)和脑钠肽(P < 0.05)的mRNA表达水平显著升高，见图9C，D，蛋白质/DNA比值增大(P < 0.05)，见图9E。而当加入circ-CHACR过表达慢病毒后，与阴性对照组相比，血管紧张素Ⅱ诱导的心肌细胞肥大效应被明显减轻，具体表现在：F-actin染色后细胞表面积减小(P < 0.05)，心房利钠肽(P < 0.05)和脑钠肽(P < 0.05)等肥大标志基因的表达水平降低，蛋白质/DNA比值减小(P < 0.05)，见图9A-E。以上结果表明，过表达circ-CHACR显著抑制了血管紧张素Ⅱ诱导的心肌细胞肥大。 2.9 过表达circ-CHACR减轻血管紧张素Ⅱ诱导的氧化应激为了检测circ-CHACR在血管紧张素Ⅱ诱导的心肌细胞氧化应激中的作用，通过DCFH-DA荧光探针检测活性氧水平。结果显示，与对照组相比，血管紧张素Ⅱ处理后，活性氧水平显著升高(P < 0.05)，而当加入circ-CHACR过表达慢病毒后，与阴性对照组相比，circ-CHACR过表达明显抑制了血管紧张素Ⅱ诱导的活性氧水平升高(P < 0.001)，见图10A，B。由于氧化应激会引起线粒体功能障碍，线粒体膜发生去极化，膜电位降低，这是线粒体功能丧失的标志，所以通过JC-1荧光探针检测线粒体膜电位水平。JC-1是一种广泛用于检测线粒体膜电位的荧光探针，在健康细胞中，JC-1可以选择性地以JC-1聚集体的形式进入线粒体，并发出红色荧光。当线粒体膜电位降低时，JC-1弥散到细胞质中，形成JC-1单体，发出绿色荧光。因此，JC-1的红/绿荧光比表示线粒体膜电位的变化[32-33]。结果显示，与对照组相比，血管紧张素Ⅱ处理后增加了绿色荧光强度，但降低了红色荧光强度，表明细胞的线粒体膜电位显著降低(P < 0.01)，而当加入circ-CHACR过表达慢病毒后，与阴性对照组相比，circ-CHACR过表达明显抑制了线粒体膜电位的降低(P < 0.05)，见图10C，D。以上结果表明，过表达circ-CHACR显著减轻了血管紧张素Ⅱ诱导的氧化应激。"

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