【研究背景与目的】

病理性心肌肥厚是指在各种病理因素作用下心脏发生体积增大、质量增加、并伴有间质和血管周围纤维化和心功能下降的一种病理状态，是多种心血管疾病共同的病理过程。持续的病理性心肌肥厚最终可导致心力衰竭和猝死。高同型半胱氨酸血症是心血管疾病的独立危险因素，与心肌肥厚的发生相关，但目前对高同型半胱氨酸血症诱发心肌肥厚的分子机制的理解仍不充分，更缺乏对各机制之间相互关联的研究。既往研究分别报道了可溶性环氧化物水解酶（sEH）和经典瞬时受体电位3（TRPC3）通道在压力负荷性心肌肥厚中各自的作用，但sEH和TRPC3通道是否参与同型半胱氨酸诱导的心肌肥厚却不清楚。此外，sEH和TRPC3通道是否共同参与并关联介导心肌肥厚的发生目前尚未见研究报道。微小核糖核酸(microRNA，简称miRNA）作为真核生物体内一类重要的基因表达调控因子，在心肌肥厚的发展进程中起着正性或负性的调节作用。有关miRNA在同型半胱氨酸致心肌肥厚中的作用研究目前十分有限。因此，本研究旨在通过1）明确sEH和TRPC3通道在同型半胱氨酸诱导的心肌肥厚中的作用及两者间的信号传导关联机制，2）明确高同型半胱氨酸血症对心肌miRNA表达谱的影响并进一步探讨差异表达miRNA参与心肌肥厚的机制，推进对高同型半胱氨酸血症心肌肥厚发病机制的理解，为开发新的防治靶点提供实验研究依据。

【研究方法】

以2%高蛋氨酸饲料喂养大鼠诱导建立高同型半胱氨酸血症模型。以高浓度同型半胱氨酸（100 μmol/L）体外孵育H9c2细胞和原代乳鼠心肌细建立细胞模型。通过超声心动图、组织形态学和心肌肥大标志物表达检测综合评估动物心室肥厚情况，利用免疫荧光成像和肥大标志物评估细胞肥大情况。以全细胞膜片钳技术记录心肌细胞TRPC3通道电流。采用双荧光素酶报告基因检测实验检测基因启动子活性。采用DEseq2方法对miRNA高通量测序结果进行差异表达分析，并对差异表达miRNA进行qPCR实验验证。通过GO和KEGG富集分析，探索miRNA靶向基因的生物学过程、细胞组成成分、分子功能以及生物信号通路。利用miRNA模拟物和抑制剂，在细胞水平开展功能获得和功能丧失实验明确特定差异miRNA参与高同型半胱氨酸血症心肌肥厚发生的具体分子机制。

【研究结果】

高同型半胱氨酸血症大鼠及同型半胱氨酸孵育的心肌细胞出现心肌肥厚和细胞肥大的表型，伴有sEH和TRPC3通道表达和活性的增强。经sEH抑制剂TPPU治疗的高同型半胱氨酸血症大鼠其心脏体积缩小，心室重量减轻，心肌肥厚标志物的表达降低，间质胶原沉积减少，心功能改善，伴有心肌中EETs水平下降以及TRPC3通道mRNA和蛋白表达的明显下调。TPPU或敲低sEH抑制了心肌细胞中TRPC3的转录和翻译以及被同型半胱氨酸增强的TRPC3电流。TRPC3通道特异性抑制剂Pyr3可减弱同型半胱氨酸诱导的心肌细胞体积增大，降低肥大标志物的表达。外源性给予11,12-EET明显抑制同型半胱氨酸诱导的TRPC3表达和细胞肥大。此外，动物和细胞模型中均检测到C/EBPβ蛋白表达的增加。沉默C/EBPβ减弱了同型半胱氨酸诱导的心肌细胞肥大以及sEH和TRPC3的表达，而过表达C/EBPβ则促进了同型半胱氨酸诱导的肥大以及sEH和TRPC3的表达。双荧光素酶报告基因实验显示同型半胱氨酸诱导的sEH和TRPC3基因启动子激活分别被C/EBPβ敲低抑制，被C/EBPβ过表达增强。对高同型半胱氨酸血症心肌肥厚大鼠和对照组大鼠心肌的miRNA高通量测序共检出1287个miRNA分子，其中8个存在显著的表达差异，包括6个已知的miRNA和2个新型miRNA。在6个已知的miRNA分子中，rno-miR144-3p、rno-miR144-5p、rno-miR200a-3p、rno-miR3588和rno-miR378a-3p在高同型半胱氨酸血症心肌肥厚大鼠中表达下调，rno-miR125b-5p表达发生明显上调。qPCR验证实验进一步证实了这些miRNA的表达差异。生物信息学分析显示，这些差异miRNA的靶mRNA在多个KEGG通路中富集，包括卟啉与叶绿素代谢、抗坏血酸和醛糖二酸盐代谢、神经营养因子信号通路、MAPK信号通路和FOXO信号通路等。进一步针对miR-378a-3p的体外细胞实验结果显示，在同型半胱氨酸孵育的心肌细胞中miR-378a-3p表达水平同样降低，转染miR-378a-3p模拟物可抑制同型半胱氨酸诱导的心肌肥厚标志蛋白ANP和β-MHC的表达，同时降低心肌细胞中ERK1/2磷酸化水平和C/EBPβ蛋白含量，而转染miR-378a-3p抑制剂则可增强同型半胱氨酸促肥厚标志蛋白以及p-ERK1/2和C/EBPβ蛋白表达的作用。此外，抑制ERK1/2活性可消除miRNA-378a-3p抑制剂对同型半胱氨酸促C/EBPβ上调的增强作用。

【研究结论】

本研究证明sEH和TRPC3通道表达和活性的增强共同参与了高同型半胱氨酸血症心肌肥厚的发生。sEH可通过11,12-EET调控TRPC3通道的表达和功能。C/EBPβ作为共同的转录因子介导了同型半胱氨酸对sEH和TRPC3基因表达的促进作用。首次揭示了sEH和TRPC3通道在心肌肥厚发生中的共同作用及两者间的信号传导关联机制。此外，高同型半胱氨酸血症对心肌miRNA表达的影响亦参与心肌肥厚的进程，其中miRNA-378a-3p下调可通过激活ERK增加心肌细胞C/EBPβ蛋白含量进而促发细胞肥大。这些研究结果丰富了对高同型半胱氨酸血症诱发心肌肥厚的分子机制的理解，为日后临床开发新的治疗干预靶点提供了科学依据。

Background and objectives:

Pathological cardiac hypertrophy occurs in response to various stimuli, which is manifested as an increase in heart size and mass, accompanied by interstitial and perivascular fibrosis and compromised cardiac function. It is a common pathological process of a variety of cardiovascular diseases. Long-term persistent pathological cardiac hypertrophy can lead to heart failure and sudden death. Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and is associated with the occurrence of cardiac hypertrophy. The molecular mechanisms underlying hyperhomocysteinemia-induced cardiac hypertrophy are not yet fully understood. Moreover, in mechanistic studies of cardiac hypertrophy, there is a lack of research addressing interrelations between different mechanisms.

Previous studies in pressure overload-induced cardiac hypertrophy models have suggested the individual role of soluble epoxide hydrolase (sEH) and canonical transient receptor potential channel 3 (TRPC3) . However, whether they are involved in homocysteine-induced cardiac hypertrophy remains unknown. Moreover, it remains unexplored whether sEH and TRPC3 channels may act jointly to mediate hypertrophic process.

As an important gene expression regulator in eukaryotic organism, microRNA (miRNA) can positively or negatively regulate initiation or progression of cardiac hypertrophy. So far, the role of miRNA in homocysteine-induced cardiac hypertrophy is barely studied. Therefore, this study aimed to 1) elucidate the role of, and interrelation between sEH and TRPC3 channels in homocysteine-induced cardiac hypertrophy, and 2) determine the changes in expression profile of miRNAs in the myocardium of hyperhomocysteinemic rats with further attempts to explore the mechanism of action of differentially expressed miRNAs in cardiac hypertrophy.

Methods:

Rats were fed methionine-enriched diet to induce hyperhomocysteinemia. H9c2 cells and neonatal rat cardiomyocytes (NRCMs) were incubated with homocysteine (100 μmol/L). Cardiac hypertrophy was evaluated by echocardiography, histological examination, immunofluorescence imaging, and expressions of hypertrophic markers. Epoxyeicosatrienoic acids (EETs) were determined by ELISA. TRPC3 channel current was recorded by patch-clamp in whole-cell mode. Gene promotor activity was measured using dual-luciferase reporter assay. The expression profile of miRNAs in the myocardium was revealed by small RNA sequencing. The differential expression was determined by DEseq2 method and validated by qPCR. Through GO and KEGG enrichment analysis, the biological process, cellular component, molecular function, and biological signaling pathways of miRNA-targeted genes were explored. Further attempts were made to understand the action of a certain differentially expressed miRNA in the development of cardiac hypertrophy induced by homocysteine.

Results:

The heart of hyperhomocysteinemic rats and the cardiomyocytes exposed to homocysteine both exhibited hypertrophy phenotype, accompanied by an increased expression and enhanced activity of sEH and TRPC3 channels. Inhibition of sEH by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) reduced ventricular mass, lowered the expression of hypertrophic markers, decreased interstitial collagen deposition, and improved cardiac function in hyperhomocysteinemic rats, associated with restoration of EETs levels and TRPC3 mRNA and protein expression levels in myocardium. TPPU or knockdown of sEH suppressed TRPC3 transcription and translation as well as TRPC3 current that were enhanced by homocysteine. Pyr3, the specific inhibitor of TRPC3 channel, attenuated homocysteine-induced cardiomyocyte enlargement and decreased the expression of hypertrophy markers. Exogenous 11,12-EET inhibited homocysteine-induced TRPC3 expression and cellular hypertrophy. In addition, C/EBPβ protein expression was increased in both animal and cell models. Silencing C/EBPβ attenuated, while overexpressing C/EBPβ promoted homocysteine-induced hypertrophy and expressions of sEH and TRPC3, resulting respectively from inhibition or activation of sEH and TRPC3 gene promoters. Small RNA sequencing revealed that out of 1287 miRNAs identified 8 miRNAs are differentially expressed in the myocardium between hyperhomocysteinemic rats and control rats, including 6 known miRNAs and 2 novel miRNAs. Among the six known miRNAs, rno-miR144-3p, rno-miR144-5p, rno-miR200a-3p, rno-miR3588 and rno-miR378a-3p were down-regulated in hyperhomocysteinemic rats and the expression of rno-miR125b-5p was significantly up-regulated. The expression differences of these miRNAs were further confirmed by qPCR. Bioinformatics analysis showed that the target mRNAs of these differentially expressed miRNAs are enriched in multiple KEGG pathways, including porphyrin and chlorophyll metabolism, ascorbate and aldarate metabolism, neurotrophin signaling pathway, MAPK signaling pathway, and FOXO signaling pathway. Further in vitro study on miR-378a-3p showed that the expression of miR-378a-3p is also decreased in homocysteine-incubated NRCMs. miR-378a-3p mimics suppressed homocysteine-induced expression of the hypertrophy marker ANP and β-MHC, inhibited the phosphorylation of ERK1/2, and lowered the protein level of C/EBPβ, while miR-378a-3p inhibitor exhibited the opposite effect. Furthermore, inhibition of ERK1/2 activity abolished the enhancement of C/EBPβ up-regulation caused by miRNA-378a-3p inhibitor in homocysteine-exposed NRCMs.  

Conclusions：

We for the first time demonstrated that sEH and TRPC3 channels jointly contribute to homocysteine-induced cardiac hypertrophy through interrelated mechanisms. sEH activation leads to an upregulation of TRPC3 channels via a 11,12-EET-dependent manner. Homocysteine transcriptionally activates sEH and TRPC3 genes through a common regulatory element C/EBPβ. In addition, this study identified changes in miRNA expression profile in myocardium and indicated possible involvement of the differentially expressed miRNAs in hyperhomocystenemia-induced cardiac hypertrophy. Decrease of miRNA-378a-3p activates ERK, leading to an increase of C/EBPβ in cardiomyocytes, which serves as a key mediator in the development of cardiac hypertrophy. Results derived from this study enriched our understanding of the molecular mechanism underlying homocysteine-induced cardiac hypertrophy and provided a scientific basis for the development of new therapeutic strategies .