第一部分  经桡动脉左心室心内膜心肌活检在心肌病患者中应用的有效性及安全性研究

研究背景

心内膜心肌活检（endomyocardial biopsy，EMB）是通过心肌活检钳经外周血管获取心内膜心肌组织的技术。EMB目前是非缺血性心肌病诊断的金标准，一些心脏疾病往往不能通过无创手段进行诊断，可以通过此项检查来确诊并优化治疗方案，如心肌炎、心脏移植术后排异反应、特殊类型心肌病和不明原因心力衰竭的病因诊断等。EMB迄今在临床上应用已超过50年，且相关技术不断更新。EMB可以分为左心室内膜心肌活检（left ventricular endomyocardial biopsy，LVEMB）和右心室内膜心肌活检（right ventricular endomyocardial biopsy，RVEMB），近年最新研究表明，LVEMB的有效性及安全性优于RVEMB。与传统的经股动脉途径LVEMB相比，经桡动脉途径LVEMB具备并发症发生率低、术后无需卧床等优点。以往报道的经桡动脉LVEMB的方法如Sheathless技术等，其操作过程都相对复杂，实际应用受限。

研究目的

评估一种全新的经桡动脉LVEMB方法的安全性及有效性，探索心肌病诊断的新技术，以及该技术在科研中应用的价值。

研究方法

评估一种经桡动脉LVEMB的方法，该方法应用桡动脉鞘管及指引导管。

（1）有效性指标：手术成功与否，心肌活检组织样本数量、质量（由相应病理科医生评估）以及病理诊断结果；心肌活检组织样本中DDX17（P72）的表达。

（2）安全性指标：手术时间，X射线暴露时间，X射线暴露剂量（包括剂量面积乘积及皮肤总量），造影剂用量，术后是否需卧床，手术结束至下床时间，手术并发症（包括桡动脉闭塞、动静脉瘘、假性动脉瘤、血栓栓塞、心脏穿孔、心包压塞、血红蛋白较术前下降20%或需治疗、死亡、恶性心律失常等）。

研究结果

（1）这项技术在53名患者中实施，手术成功率为100%（53/53），每位患者所取得活检组织样本数量中位数是5块，总共取得265块心肌组织标本，其中127块（48%）取自左室室间隔，138块（52%）取自左室游离壁，所有活检样本质量良好，均可为病理诊断提供线索。诊断扩张型心肌病32例（60%），肥厚型心肌病15例（28%），心肌炎3例（6%），心肌淀粉样变性2例（4%），线粒体心肌病1例（2%）；心肌组织中DDX17的RNA水平与左心室射血分数呈负相关。此外，所有患者经同一桡动脉鞘管行LV-EMB、左心室造影及冠状动脉造影。

（2）X射线暴露剂量：剂量面积乘积中位数为14535.00cGy•cm²，皮肤总量中位数为1382.00mGy）；X射线暴露时间：X线暴露时间中位数13.39min；平均手术时间63.5分钟（包含所有操作，如左心导管、冠脉造影等）；造影剂平均用量101.85ml；所有患者术后无需卧床；无严重并发症（心包填塞、危及生命的心律失常、脑血管意外或死亡），仅有1例患者发生轻度桡动脉痉挛，无严重的桡动脉痉挛。

研究结论

（1）应用桡动脉鞘管及指引导管行经桡动脉途径LVEMB的方法安全、有效。

（2）经桡动脉左心室心内膜心肌活检组织样本质量好，可以完成组织学、免疫组化、特殊染色、电镜等检查，有助于明确诊断。

（3）该技术操作简便，或优于目前现有方法，是一种心脏疾病的“一站式”介入诊断技术。

（4）随着心功能的减退，心力衰竭患者心肌细胞中线粒体排列紊乱、大小和形态异常以及线粒体嵴的破坏越明显。

（5）心力衰竭患者心肌组织中DDX17的RNA水平与左心室射血分数呈负相关。

第二部分 tRF-Glu-CTC-013与临床心肌肥厚相关性和作用机制研究

研究背景

tsRNAs是一类新的非编码RNA，在细胞RNA加工和蛋白质翻译调控中起重要作用，并且可能参与多种疾病的发生和发展。近期很多研究发现，tsRNA在多种心血管疾病中表达量发生改变，并且可能参与疾病的发生。心肌肥厚是慢性应激负荷引起左心室心肌一系列病理和结构改变的一种状态，与心肌缺血、梗死、心力衰竭等多种心脏疾病的发生密切相关。

研究目的与方法

本研究采用血管紧张素II (Ang II)诱导的小鼠心肌肥厚模型。通过tsRNA转录组测序筛选出差异表达的tsRNA，并对其生物学功能进行预测。并在心肌肥厚患者的心肌活检组织中进行临床验证。

研究结果

通过对乳鼠心肌细胞的验证，我们发现5个可能参与心脏肥厚发展的上调tsRNAs，其中tRF-Glu-CTC-013的表达显著上调，且差异表达最为显著。进一步的功能分析发现tRF-Glu-CTC-013过表达可以抑制AngII刺激造成心肌细胞面积增加，并且显著抑制了炎症、纤维化和心肌肥厚标志物的增加。荧光素酶报告基因实验显示，tRF-Glu-CTC-013直接靶向TAS1R3的3'UTR，导致心肌细胞中自噬水平升高，抑制心肌肥厚的发生和发展。进一步的临床研究发现，心肌肥厚患者左心室心肌组织活检样本的tRF-Glu-CTC-013表达水平与患者血清BNP的表达水平呈显著正相关。这些结果表明，tsRNA可以参与心脏肥厚的病理生理过程，其中tRF-Glu-CTC-013作为一种保护性tsRNA，具有抑制心肌肥厚和心脏保护作用。

研究结论

tRF-Glu-CTC-013在心肌肥厚发生时表达明显增高，可能作为临床心肌肥大的生物学标志物。此外，tRF-Glu-CTC-013还可以靶向抑制TAS1R3，在病理状况下促进心肌自噬，减轻心肌肥厚的发生。本研究对心脏肥厚的发病机制和新的治疗靶点的筛选提供了理论依据。

Part I. Efficacy and safety of transradial left ventricular endomyocardial biopsy in patients with cardiomyopathy

Background

Endomyocardial biopsy (EMB) is a technique for obtaining endomyocardial tissue through peripheral blood vessels through myocardial biopsy forceps. EMB is currently the gold standard for the diagnosis of non-ischemic cardiomyopathy. Some heart diseases can not be diagnosed by noninvasive means. This examination can be used to diagnose and optimize the treatment plan, such as myocarditis, heart transplantation rejection, special types of cardiomyopathy and etiological diagnosis of unknown heart failure. EMB has been used in clinical practice for more than 50 years, and the related technology is constantly updated. EMB can be divided into left ventricular endocardial biopsy (LVEMB) and right ventricular endocardial biopsy (RVEMB). Recent studies have shown that LVEMB is more effective and safe than RVEMB. Compared with the traditional transfemoral approach, transradial approach has the advantages of low incidence of complications and no need to stay in bed after operation. The previously reported transradial LVEMB methods, such as Sheathless technology, are relatively complex in operation process and limited in practical application.

Purpose

Our aim is to evaluate the safety and effectiveness of a new transradial LVEMB method, explore the new technology of cardiomyopathy diagnosis, and the value of this technology in scientific research.

Methods

To evaluate a method of transradial LVEMB using radial artery sheath and guide catheter.

(1) Effectiveness indicators: whether the operation was successful or not, the number and quality of myocardial biopsy tissue samples (evaluated by the corresponding pathologist) and pathological diagnosis results; Expression of DDX17 (P72) in myocardial biopsy tissue samples.

(2) Safety indicators: operation time, X-ray exposure time, X-ray exposure dose (including dose area product and total skin), dosage of contrast agent, whether to stay in bed after operation, time from the end of operation to getting out of bed, surgical complications (including radial artery occlusion, arteriovenous fistula, false aneurysm, thromboembolism, heart perforation, pericardial tamponade, hemoglobin decreased by 20% or needed treatment, death, malignant arrhythmia, etc.).

Results

(1) This technique was implemented in 53 patients with a success rate of 100% (53/53). The median number of biopsy tissue samples obtained for each patient was 5. A total of 265 myocardial tissue samples were obtained, of which 127 (48%) were from the left ventricular septum and 138 (52%) were from the left ventricular free wall. All biopsy samples were of good quality, which could provide clues for pathological diagnosis. There were 32 cases of dilated cardiomyopathy (60%), 15 cases of hypertrophic cardiomyopathy (28%), 3 cases of myocarditis (6%), 2 cases of myocardial amyloidosis (4%), and 1 case of mitochondrial cardiomyopathy (2%); The RNA level of DDX17 in myocardial tissue was negatively correlated with left ventricular ejection fraction. In addition, all patients underwent LVEMB, left ventriculography and coronary angiography through the same radial artery sheath.

(2) X-ray exposure dose: the median of dose area product was 14535.00cgy • cm2, and the median of total skin volume was 1382.00mgy; X-ray exposure time: the median X-ray exposure time was 13.39min; The average operation time was 63.5 minutes (including all operations, such as left cardiac catheterization, coronary angiography, etc.); The average dosage of contrast agent was 101.85ml; all patients did not need to stay in bed after operation; There were no serious complications (pericardial tamponade, life-threatening arrhythmia, cerebrovascular accident or death), only one patient had mild radial artery spasm, and no severe radial artery spasm.

Conclusions

(1) The method of transradial LVEMB using radial artery sheath and guiding catheter is safe and effective.

(2) Myocardial tissue from transradial LVEMB can be completed with histological, immunohistochemical, special staining, electron microscopy and other examinations, which is helpful for the diagnosis.

(3) This technique is simple to operate, and may be superior to the existing methods, and is a "one-stop" interventional diagnosis technology for heart disease.

(4) With the decline of cardiac function, the disorder of mitochondrial arrangement, abnormal size and morphology, and the destruction of mitochondrial cristae are more obvious in patients with heart failure.

(5) The RNA level of DDX17 in myocardial tissue of patients with heart failure was negatively correlated with left ventricular ejection fraction.

Part II. Study of the correlation and regulatory mechanism of tRF-Glu-CTC-013 with clinical cardiac hypertrophy

Abstract

tsRNAs are a new class of non-coding RNAs that play important roles in regulating RNA processing and protein translation, and may be involved in the onset and progression of many diseases. Recent studies have found that the expression of tsRNAs is altered in various cardiovascular diseases and may contribute to their development. Myocardial hypertrophy is a state involving a series of pathological and structural changes in the left ventricular myocardium, induced by chronic stress loads. It is closely related to various cardiac diseases, such as myocardial ischaemia, infarction, and heart failure. A mouse model of Angiotensin II (Ang II) -induced cardiac hypertrophy was used in this study. Differentially expressed tsRNAs were selected by tsRNA transcriptome sequencing and their biological functions were predicted. By validating neonatal mouse ventricular cardiomyocytes, we identified five tsRNAs that were upregulated and that may be involved in the development of cardiac hypertrophy. Among these, the expression of tRF-Glu-CTC-013 was found to be significantly increased, with the greatest differential expression. Further functional analysis revealed that tRF-Glu-CTC-013 reduced cardiomyocyte cell area and significantly suppressed the increase in inflammation, fibrosis, and cardiac hypertrophy markers. Luciferase reporter gene assays revealed that tRF-Glu-CTC-013 directly targets the 3'UTR of TAS1R3, thereby increasing autophagy levels in neonatal mouse ventricular cardiomyocytes. Further clinical correlation studies revealed that the expression level of tRF-Glu-CTC-013 in left ventricular myocardial tissue biopsy samples from patients with cardiac hypertrophy was significantly and positively correlated with patients' serum BNP expression levels. These results suggest that tsRNAs are involved in regulating cardiac hypertrophic responses, with tRF-Glu-CTC-013 acting as a protective tsRNA with anti-inflammatory and anti-fibrotic cardioprotective effects. In summary, the expression of tRF-Glu-CTC-013 increases significantly at the onset of cardiac hypertrophy, suggesting that it could be used as a biological marker for the condition. Furthermore, tRF-Glu-CTC-013 can target and inhibit TAS1R3, thereby promoting myocardial autophagy and reducing the progression of cardiac hypertrophy in pathological states. This study lays the groundwork for a better understanding of the pathogenesis of cardiac hypertrophy and the discovery of new therapeutic targets.