出生後小鼠心臟Nkx2.5GFP+細胞分泌之外吐小體內微核糖核酸的角色與鑑定

Abstract

背景：全球心臟衰竭正在增加，不良的預後仍然是社會經濟負擔，因此促進心臟自我修復的新策略是很(令人感興趣的。使用攜帶Nkx2.5增強子和基本啟動子驅動GFP表達的轉基因轉殖小鼠（Nkx2.5GFP小鼠），我們可以鑑定辨識出在出生後小鼠中的Nkx2.5GFP +細胞群可以經由A83-01(一種乙型轉化生長因子第一型受體抑製劑)的作用在體外和體內進行擴增。我們之前的研究證明了出生後Nkx2.5GFP +細胞在受傷小鼠心臟中的旁分泌益處。然而，需要進一步研究以檢查旁泌介質並研究其潛在機制。 目的：本研究的目的是（1）檢測Nkx2.5GFP +細胞分泌的外吐小體（NkxExo）是否可以直接對成體心肌細胞發揮促進存活作用，（2）分析可能的心臟保護性外吐小體中的微核醣核酸，（3）評估外吐小體是否可以直接改善心臟損傷小鼠的心臟功能。 方法：從14天齡的Nkx2.5GFP小鼠中分離心臟Nkx2.5GFP +細胞，並經由細胞分選儀FACS Aria III分選出PI陰性/ sca1-APC陰性/ Nkx2.5GFP陽性細胞做進一步純化。將Nkx2.5GFP +細胞維持在含有A83-01並以IMDM為底的培養基中以增強細胞增殖。收取培養液透過ExoQuick-TC（SBI System Biosciences，MA，USA）純化外吐小體，透過Zymol RNA萃取試劑萃取外吐小體的RNA，並通過nanodrop 2000對總RNA進行定量。將含有1微克總RNA的外吐小體給予分離的成年小鼠心肌細胞。經由Hoechst 33342 + / SYTOX +染色死細胞來檢查外吐小體對心肌細胞存活率的影響。使用qPCR驗證心肌細胞中可能被外吐小體中微核醣核酸作用的目標基因的轉錄變化， 透過兩個心臟損傷動物模型（1）異丙腎上腺素（ISO）注射小鼠和（2）通過左前冠狀動脈結紮缺血（20分鐘）/再灌流（24小時）（I / R）損傷的小鼠測試外吐小體對損傷後心肌存活的直接影響。透過連 三次每日異丙腎上腺素劑量對小鼠進行心臟損傷後並在隔天給予外吐小體的一次性注射；在缺血/再灌流小鼠中會在灌流之前的缺血期期間，進行心肌內注射外吐小體到結紮區周圍的四個點。最後以心臟超音波評估異丙腎上腺素注射小鼠的心臟功能的變化，並且透過心電圖來測量有或沒有外吐小體處理的缺血/再灌流小鼠中ST波段升高的變化。 結果：在外吐小體處理組中，體外培養的成年小鼠心肌細胞的存活率有顯著增加，其從DMSO-或A83-處理的Nkx2.5GFP +細胞分泌的外吐小體的促進存活作用相似。通過微核醣核酸微陣列測定辨識外吐小體中的微核醣核酸。進一步分析在DMSO-或A83-處理的Nkx2.5GFP +細胞分泌的外來體中高度表達的微核醣核酸，從兩組的交集中獲得55個微核醣核酸。透過在線數據庫DIANA mirpath鑑定可能對心肌細胞存活有益的微核醣核酸，例如mmu-mir-466f，-466i-3p，-467f，-669c-3P和-669-3p。 Mmu-mir-467f，-669c-3P和-669-3p可能涉及調節HIPPO信號傳導途徑以增強YAP / TAZ媒介的促存活效應。在注射異丙腎上腺素的小鼠和I / R損傷的小鼠中分別評估外吐小體在受傷後心臟中的效益。在異丙腎上腺素注射的小鼠中，在最後一次異丙腎上腺素給藥後的第7天觀察到心衰竭。在施打外吐小體小鼠中可以觀察到動物存活率有顯著的改善，但左心室射血分數（LVEF）則沒有差異。在外吐小體處理的缺血/再灌流小鼠中ST段升高和QT區間延長的現象減弱。目前仍在進一步研究外吐小體如何改善心臟缺血/再灌流損傷的潛在機制。 結論：出生後心臟NkxGFP +細胞可以分泌外吐小體，調控受損後心臟的心臟保護作用。透過確定調控心臟保護效益的外吐小體微核醣核酸，就可以找到潛在的心衰竭標靶藥物。

Background: The global heart failure is growing and the ominous prognosis is still a socioeconomic burden. The novel strategy to facilitate cardiac self-repair is appealing. Using a transgenic mouse carrying the cassette of Nkx2.5 enhancer with basic promotor to drive GFP expression (Nkx2.5GFP mice), we can identify a population of Nkx2.5GFP+ cells in postnatal mice, which can be expanded by A83-01, a TGFβRI inhibitor, in vitro and in vivo. Our previous study demonstrated the paracrine benefit of postnatal Nkx2.5GFP+ cells in post-injured mice hearts. However, it needs further study to examine the paracrine mediators and to investigate the underlying mechanism. Aim: The objectives of the present study were (1) to examine whether the exosome secreted from Nkx2.5GFP+ cells (NkxExo) could directly exert pro-survival effect on adult cardiomyocytes, (2) to analyze the possible cardioprotective exosomal miRNAs, and (3) to assess whether NkxExo could directly improve cardiac function in heart-injured mice. Method: Cardiac Nkx2.5GFP+ cells were isolated from Nkx2.5GFP mice at the age of 14 days, and were further purified through sorting the PI negative/sca1-APC negative/Nkx2.5GFP positive cells by FACS Aria III. Nkx2.5GFP+ cells were maintained in IMDM-based medium in the presence of A83-01 to enhance cell proliferation. The culture medium was harvested to purify NkxExo by ExoQuick-TC (SBI System Biosciences, MA, USA). The exosomes containing 1 μg of total RNAs were administrated to the primary culture of the isolated adult mice cardiomyocytes to examine the effect of NkxExo on myocyte viability by staining the dead cell with Hoechst 33342+/SYTOX+. The transcriptional change of the possible exosomal miRNA-targeted gene in cardiomyocytes was validated by qPCR. Two cardiac injury animal models of (1) isoprenaline (ISO)-injected mice and (2) mice subjected to ischemia (20 min)/reperfusion (24h) injury (I/R) by left anterior coronary artery ligation were employed for testing the direct effect of NkxExo in myocardial survival after injury. One-shoot of NkxExo was administrated on the next day after the mice suffered from cardiac damage by consecutive three daily doses of ISO. Intramyocardial injection of NkxExo into four points around the ligation region was conducted in I/R mice during ischemia stage immediately before reperfusion. Echocardiography was performed to evaluate the change of cardiac function in ISO-injected mice and ECG was recorded to measure the change of ST elevation in I/R mice with or without NkxExo treatment. Results: The viability of the cultured adult mice cardiomyocyte in vitro was significantly increased in NkxExo-treated group. It was similar in the pro-survival effect of NkxExo secreted from DMSO- or A83- treated Nkx2.5GFP+ cells. Theexosomal miRNAs were identified by microRNA microarray assay. The miRNAs highly expressing in the exosomes secreted from DMSO- or A83- treated Nkx2.5GFP+ cells were further analyzed to get the 55 miRNAs in the intersection of both groups. The miRNAs such as mmu-mir-466f, -466i-3p, -467f, -669c-3P and -669-3p that may have beneficial to cardiomyocyte viability were identified by the online database DIANA mirpath. Mmu-mir-467f, -669c-3P and -669-3p may involve in regulating HIPPO signaling pathway to enhance YAP/TAZ-mediated pro-survival effect. In ISO-injected mice, heart failure was observed on day 7 after the last dose of ISO. The animal survival rate, but not left ventricular ejection fraction (LVEF), was significantly improved in one-shoot-NkxExo mice. In I/R mice, the phenomena of ST elevation and QT interval prolongation were attenuated in NkxExo-treated I/R mice. It remained further study to investigate the underlying mechanisms of how NkxExo could ameliorate cardiac I/R injury. Conclusion: Postnatal cardiac NkxGFP+ cells can secrete the exosomes to mediate cardioprotective benefit in post-injured hearts. Determining the exosomal miRNAs that mediate the pro-survival signaling pathway could identify the novel drugable targets/approaches for heart failure therapy.