抑制TGFβRI可促進心肌前驅細胞Survivin自泌與旁泌並抑制其Wnt訊息表現以達到心臟保護作用

Abstract

目前在全球由心肌梗塞所引起的心臟衰竭是主要致死及致病的因素。而在我們先前的研究中發現了在心肌梗塞受損後的小鼠中，施予A83-01後(一種乙型轉化生長因子第一型受體抑制劑)可有利於其心臟的修復，其中機制為增加Nkx2.5+心臟前驅細胞的數量。本研究的主旨是探討在受損的心臟中，A83-01如何透過調節自泌及旁泌因子的表現以達到心臟保護的作用。實驗利用Nkx2.5 enhancer-GFP基因轉殖報告鼠，初生小鼠藉由流式細胞分選儀辨識細胞自體螢光及表面抗體螢光分離三種心臟幹細胞包括Nkx2.5-GFP+ (Nkx2.5+)、 Sca1+ 及Nkx2.5+/Sca1+ 心臟前驅細胞進行培養。A83-01 主要可以透過MEK/ERK訊息傳遞路徑，活化Birc5 基因表現，促進三種心臟幹細胞的增殖。Survivin 為Birc5 基因轉錄轉譯後的小分子蛋白，當直接對培養的細胞施予Survivin時，可促進Nkx2.5+心臟前驅細胞的增殖，並增加體外培養之心肌細胞的存活率。同時，在體內實驗中也發現給與A83-01對心肌梗塞受損後的小鼠，可以維持並增加Birc5 基因的表現，以增加體內的心臟前驅細胞。另一方面，實驗中發現心臟在受損急性期後會持續增加Wnt3a的表現，Wnt3a抑制了心臟前驅細胞的生長，此作用可被A83-01所拮抗，藉由降低有關的Fzd6 及 WISP1 的表現，逆轉了Wnt3a活化之Wnt 訊息傳遞對心臟前驅細胞的作用。進一步在αMHC-cre/mTmG 小鼠，利用Tamoxifen 活化心肌細胞中α-肌球蛋白重鏈啟動子後的Cre 重組酶的表現，基因重組去除了紅色螢光蛋白(RFP)基因使心肌細胞表現綠色螢光蛋白(GFP)，未受重組之非心肌細胞則持續表現紅色螢光，在使用isoprenaline誘使受損後觀察，A83-01的心臟保護作用來自維持了較多的心肌細胞的存活(68.6±3.1% vs. 80.9±3.0%; P<0.05, n=6)，顯著減少纖維化的區域，少數的心肌細胞新生(0.026±0.005 vs. 0.062±0.008%; P<0.05, n=6)。總結而言，透過抑制乙型轉化生長因子第一型受體，會調節Survivin的自泌與旁泌，及抑制心臟前驅細胞中Wnt 訊息傳遞，以達到心臟保護及改善受損後心臟功能的作用。

Heart failure due to myocardial infarction (MI) is a major cause of morbidity and mortality in the world. We found previously that A83-01, a TGFβRI inhibitor, could facilitate cardiac repair in post-MI mice and induce the expansion of Nkx2.5+ cardiomyoblast population. The present study aimed to investigate the key autocrine/paracrine factors regulated by A83-01 in the injured heart and the mechanism of cardioprotection by this molecule. Using a previously described transgenic Nkx2.5 enhancer-GFP reporter mice, we isolated cardiac progenitor cells (CPC) including Nkx2.5-GFP+ (Nkx2.5+), Sca1+ and Nkx2.5+/Sca1+ cells. A83-01 was found to induce proliferation of these three subpopulations mainly through increasing Birc5 expression in the MEK/ERK-dependent pathway. Survivin, encoded by Birc5, could also directly proliferate Nkx2.5+ cells and enhance cultured cardiomyocytes viability. A83-01 could also reverse the down-regulation of Birc5 in post-injured mice hearts (n=6) to expand CPCs. Moreover, the increased Wnt3a in post-injured hearts could decrease CPCs, which could be reversed by A83-01 via inhibiting Fzd6 and WISP1 expressions in CPCs. Next, we used inducible αMHC-cre/mTmG mice to label cardiomyocytes with GFP and non-myocytes with RFP. We found A83-01 preserved more GFP+ myocytes (68.6±3.1% vs. 80.9±3.0%; P<0.05, n=6) and fewer renewed RFP+ myocytes (0.026±0.005 vs. 0.062±0.008%; P<0.05, n=6). It was in parallel with less cardiac fibrosis in isoprenaline-injected mice treated with A83-01. TGFβRI inhibition in an injured adult heart could both stimulate the autocrine/paracrine activity of survivin and inhibit Wnt in CPCs to mediate cardioprotection and improve cardiac function.