Key Laboratory of Remodeling-Related Cardiovascular Disease (Ministry of Education), Beijing Anzhen Hospital Affiliated to Captital Medical University, Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing 100029, China.
Chin Med J (Engl). 2018 Oct 5;131(19):2302-2309. doi: 10.4103/0366-6999.241794.
Myocardial ischemia injury is one of the leading causes of death and disability worldwide. Cardiac fibroblasts (CFs) have central roles in modulating cardiac function under pathophysiological conditions. Activating transcription factor 3 (ATF3) plays a self-protective role in counteracting CF dysfunction. However, the precise function of CF-specific ATF3 during myocardial infarction (MI) injury/repair remains incompletely understood. The aim of this study was to determine whether CF-specific ATF3 affected cardiac repair after MI.
Fifteen male C57BL/6 wild-type mice were performed with MI operation to observe the expression of ATF3 at 0, 0.5, 1.0, 3.0, and 7.0 days postoperation. Model for MI was constructed in ATF3TGfl/flCol1a2-Cre+ (CF-specific ATF3 overexpression group, n = 5) and ATF3TGfl/flCol1a2-Cre- male mice (without CF-specific ATF3 overexpression group, n = 5). In addition, five mice of ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- were subjected to sham MI operation. Heart function was detected by ultrasound and left ventricular remodeling was observed by Masson staining (myocardial fibrosis area was detected by blue collagen deposition area) at the 28 day after MI surgery in ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- mice received sham or MI operation. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect cell proliferation/cell cycle-related gene expression in cardiac tissue. BrdU staining was used to detect fibroblast proliferation.
After establishment of an MI model, we found that ATF3 proteins were increased in the heart of mice after MI surgery and dominantly expressed in CFs. Genetic overexpression of ATF3 in CFs (ATF3TGfl/flCol1a2-Cre+ group) resulted in an improvement in the heart function as indicated by increased cardiac ejection fraction (41.0% vs. 30.5%, t = 8.610, P = 0.001) and increased fractional shortening (26.8% vs. 18.1%, t = 7.173, P = 0.002), which was accompanied by a decrease in cardiac scar area (23.1% vs. 11.0%, t = 8.610, P = 0.001). qRT-PCR analysis of CFs isolated from ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- ischemic hearts revealed a distinct transcriptional profile in ATF3-overexpressing CFs, displaying pro-proliferation properties. BrdU-positive cells significantly increased in ATF3-overexpressing CFs than control CFs under angiotensin II stimuli (11.5% vs. 6.8%, t = 31.599, P = 0.001) or serum stimuli (31.6% vs. 20.1%, t = 31.599, P = 0.001). The 5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester assay showed that the cell numbers of the P2 and P3 generations were higher in the ATF3-overexpressing CFs at 24 h (P2: 91.6% vs. 71.8%, t = 8.465, P = 0.015) and 48 h (P3: 81.6% vs. 51.1%, t = 9.029, P = 0.012) after serum stimulation. Notably, ATF3 overexpression-induced CF proliferation was clearly increased in the heart after MI injury.
We identify that CF-specific ATF3 might contribute to be MI repair through upregulating the expression of cell cycle/proliferation-related genes and enhancing cell proliferation.
心肌缺血损伤是全球范围内导致死亡和残疾的主要原因之一。心脏成纤维细胞（CFs）在病理生理条件下调节心脏功能方面发挥着核心作用。激活转录因子 3（ATF3）在对抗 CF 功能障碍方面发挥着自我保护作用。然而，CF 特异性 ATF3 在心肌梗死（MI）损伤/修复过程中的精确功能仍不完全清楚。本研究旨在确定 CF 特异性 ATF3 是否影响 MI 后的心脏修复。
对 15 只 C57BL/6 野生型雄性小鼠进行 MI 手术，以观察术后 0、0.5、1.0、3.0 和 7.0 天时 ATF3 的表达情况。在 ATF3TGfl/flCol1a2-Cre+（CF 特异性 ATF3 过表达组，n=5）和 ATF3TGfl/flCol1a2-Cre-（无 CF 特异性 ATF3 过表达组，n=5）雄性小鼠中构建 MI 模型。此外，5 只 ATF3TGfl/flCol1a2-Cre+和 ATF3TGfl/flCol1a2-Cre-的小鼠进行假 MI 手术。在 MI 手术后 28 天，通过超声检测心功能，通过 Masson 染色观察左心室重构（通过蓝色胶原沉积面积检测心肌纤维化面积）。在接受假或 MI 手术的 ATF3TGfl/flCol1a2-Cre+和 ATF3TGfl/flCol1a2-Cre-小鼠中，使用定量实时聚合酶链反应（qRT-PCR）检测心脏组织中细胞增殖/细胞周期相关基因的表达。BrdU 染色用于检测成纤维细胞增殖。
在建立 MI 模型后，我们发现 MI 手术后小鼠心脏中的 ATF3 蛋白增加，主要在 CFs 中表达。CFs 中 ATF3 的遗传过表达（ATF3TGfl/flCol1a2-Cre+组）导致心脏功能改善，表现为射血分数增加（41.0% vs. 30.5%，t=8.610，P=0.001）和缩短分数增加（26.8% vs. 18.1%，t=7.173，P=0.002），同时心脏瘢痕面积减少（23.1% vs. 11.0%，t=8.610，P=0.001）。对 ATF3TGfl/flCol1a2-Cre+和 ATF3TGfl/flCol1a2-Cre-缺血心脏分离的 CFs 进行 qRT-PCR 分析显示，ATF3 过表达 CFs 具有明显的转录谱，表现出促增殖特性。在血管紧张素 II 刺激（11.5% vs. 6.8%，t=31.599，P=0.001）或血清刺激（31.6% vs. 20.1%，t=31.599，P=0.001）下，BrdU 阳性细胞在 ATF3 过表达 CFs 中明显增加。5(6)-羧基荧光素 N-羟基琥珀酰亚胺酯检测显示，在血清刺激 24 h（P2：91.6% vs. 71.8%，t=8.465，P=0.015）和 48 h（P3：81.6% vs. 51.1%，t=9.029，P=0.012）时，ATF3 过表达 CFs 的 P2 和 P3 代细胞数量更高。值得注意的是，MI 损伤后心脏中 ATF3 过表达诱导的 CF 增殖明显增加。
我们发现 CF 特异性 ATF3 通过上调细胞周期/增殖相关基因的表达和增强细胞增殖，可能有助于 MI 修复。
