脂肪保护因子CTRP9调控自噬在阻塞性睡眠呼吸暂停致心肌损伤中的机制研究

Obstructive sleep apnea (OSA) induces myocardial injury, which is characterized by cardiomyocyte hypertrophy and apoptosis. Autophagy homeostasis imbalance leads to cardiomyocyte hypertrophy and apoptosis, which is involved in myocardial injury. However, the intrinsic protective mechanisms remain unclear. Several studies have shown that adipokines protect OSA-related myocardial injury. Our previous studies found that the serum levels of new C1q/tumor necrosis factor-related protein 9 (CTRP9) decreased in patients with OSA. OSA aggravated cardiomyocyte apoptosis in mice, and was accompanied by decreased levels of CTRP9 in plasma and myocardium. Recent studies showed that CTRP9 could modulate autophagy. Thus, we hypothesized that CTRP9 might modulate autophagy, inhibit cardiomyocyte apoptosis, and protect myocardial injury induced by OSA. In this program, we aimed to validate the correlation of OSA and its intervention with CTRP9 level in humans. Then, we established OSA model in wild type, CTRP9 knockout, and AdipoR1 knockout mice, in combination with CTRP9 over expression and autophagy inhibitor, to clarify the role of CTRP9 in modulating AdipoR1 and AMPK-dependent autophagy for protection of OSA-induced myocardial injury by echocardiography, pathological analysis, and tests of molecular biology. It is expected that this program may explore new therapeutic targets to clinically intervene OSA-induced myocardial injury.

阻塞性睡眠呼吸暂停（OSA）引起心肌损伤，表现为心肌细胞肥大、凋亡。自噬稳态失衡可致心肌细胞肥大及凋亡，参与心肌损伤，但内源性保护机制未明。研究显示，脂肪因子对OSA心肌损伤发挥保护作用。我们前期发现，OSA患者血清新型脂肪因子C1q肿瘤坏死因子相关蛋白9（CTRP9）水平明显下降；OSA小鼠有大量心肌细胞凋亡，伴随血浆和心肌CTRP9水平下降。新近研究提示CTRP9可调控自噬过程。据此我们假设：CTRP9可能通过调控自噬，抑制心肌细胞凋亡，保护OSA导致的心肌损伤。本项目拟在人群中验证OSA及其干预与CTRP9水平的相关性；同时在野生、CTRP9敲除和AdipoR1敲除小鼠中建立OSA模型，结合CTRP9过表达和自噬抑制剂等，通过超声、病理及分子生物学检测等，阐明OSA状态下，CTRP9通过AdipoR1调控AMPK依赖的自噬过程参与心肌损伤，以期为临床干预OSA引起的心肌损伤提供靶点。

1. 研究背景.阻塞性睡眠呼吸暂停（OSA）引起心肌损伤，表现为心肌细胞肥大、凋亡，但内源性保护机制未明。CTRP9可能通过调控自噬，抑制心肌纤维化，保护OSA导致的心肌损伤。.2. 研究内容.本研究入选北京安贞医院急诊危重症中心就诊的可疑OSA患者，行多导睡眠检测（PG）并留取患者静脉血标本。评价OSA对CTRP9表达水平的影响。.选择C57BL/6野生型（WT）小鼠，分别建立OSA（CIH）和常氧（normoxia，NOR）心肌梗死模型（暴露4周），两组分别于心梗后3、7、14、28日后采用小动物超声心动图评价心脏结构和功能及心脏采血。动物处死后，各组留取部分心肌标本，进行Masson染色、RT-PCR、ELISA、WesternBlot及免疫组化检测等。.选择C57BL/6 WT小鼠，尾静脉注射腺病毒表达载体CTRP9（Ad-CTRP9）以及Ad-β-gal，在CIH和NOR暴露4周后行冠状动脉左前降支结扎术建立小鼠心肌梗死模型，观察心脏结构、左室功能以及心肌纤维化等指标。.3. 研究结果.OSA患者MACCE发生率明显高于非OSA患者（Log-rank p=0.041）。在1年后的随访中，OSA是MACCE的独立预测因素（HR 1.59, 95% CI 1.01-2.50, p=0.043）。合并OSA的CAD患者血清CTRP-9水平与AHI（r=-0.238，P=0.003）和ODI（r=-0.234，P=0.004）呈负相关。.心肌梗死急性期CIH组小鼠心肌纤维化的面积比例明显小于NOR组（（0.79±0.0016）% vs（4.48±0.0167）%，P＜0.001）。但在心肌梗的后期，CIH可能通过上调miR-214-3p，抑制心脏CTRP9表达并加重心脏重塑。通过Western blot测定抑制miR-214-3p后的心脏CTRP9表达增加（P=3.69E-08）。重组CTRP9给药后可预防CIH诱发的心脏损伤，减少间质纤维化面积（P=0.0418，MI+CIH+对照vs. MI+CIH+gCTRP9）。.4. 研究结论.OSA可加重心肌梗死后心肌损伤，是远期不良预后的独立危险因素。本研究发现了MI+CIH可抑制心脏保护因子CTRP9，并且明确了导致其减少的分子机制。有望为早期阻断OSA与心肌损伤的分子关联探索新的治疗靶点。