長鏈非編碼核醣核酸lnc-Synpo透過表觀遺傳抑制SERCA2a損害心肌收縮功能與鈣離子恆定

Abstract

根據WHO的資料顯示，心臟疾病至今仍是造成全球死亡的最主要原因之一，儘管現代醫學取得了許多進展，但與心臟衰竭相關的患病率和死亡率仍高居不下，為全球社會帶來沉重負擔。RNA定序(RNA-seq)發現心衰竭病人左心室調控鈣離子恆定的基因表現量顯著降低，其中心肌肌漿網鈣泵(SERCA2)的主要亞型SERCA2a表現量的下降，會導致肌膜鈣離子恆定異常及心肌細胞收縮力受損。因此，於心衰竭期間維持SERCA2a的表現量和功能，被認為是治療心臟衰竭的潛在策略。我們利用RNA定序取得人類心臟內所有可檢測到的mRNA / lncRNA建立加權基因共表達網絡分析（WGCNA），並發現了坐落於蛋白編碼基因SYNPO2的3’端非轉錄區的長鏈非編碼RNA ”lnc-Synpo”是SERCA2a的潛在調控者。 為了確定lnc-Synpo對人類心臟鈣離子調控和心肌收縮的作用，以了解lnc-Synpo調控SERCA2a表達的分子機制。我們在人類誘導多能幹細胞(hiPSC)分化出來的心肌細胞中，發現敲低lnc-Synpo能顯著增加SERCA2a的mRNA和蛋白的表達量，而過量表達lnc-Synpo則會降低SERCA2a的表達。此外，lnc-Synpo的過量表達會損害心肌細胞的收縮能力、鈣離子瞬時峰值和肌漿網中鈣離子的回收速率，而降低lnc-Synpo的表達則會逆轉這種現象。此外，我們發現敲低lnc-Synpo能促使SERCA2a啟動子的表觀遺傳標記H3K4me3增加和H3K27me3減少。而透過ChIRP的實驗也證實，lnc-Synpo能直接與此段啟動子DNA結合。另外，RNA免疫沉澱(RIP)以及RNA pull down皆顯示lnc-Synpo能與多梳抑制複合體2(PRC2)中的關鍵蛋白EZH2結合。綜上所述，我們的數據證明lnc-Synpo能夠引導與之結合的EZH2和PRC2到SERCA2a的啟動子來改變其表觀遺傳修飾進而導致SERCA2a轉錄活性降低。因此，靶向lnc-Synpo可能是改善衰竭心臟中鈣離子恆定和心臟收縮功能的新型治療方法。

Despite the advances in modern medicine, the prevalence, morbidity, and mortality associated with heart failure (HF) remain high, which imposes a considerable burden on the global society. There is an urgent need to identify novel mediators/pathways of HF to develop new therapeutics for HF patients. The abnormal sarcolemmal Ca2+ handling and impaired systolic contractility owing to decreased SERCA2a, a major isoform of SERCA, are pathognomonic hallmarks of HF. Finding novel ways to increase SERCA pump expression and activity during HF has been considered a potentially useful therapeutic strategy for HF. Exploiting Weighted Gene Co-expression Network Analysis (WGCNA) of all detectable human cardiac mRNA/lncRNA from an RNASeq dataset, lnc-Synpo, a fragment of 3’ untranslated region of protein-coding gene SYNPO2, has been identified as a potential regulator of SERCA2a expression. To determine the role of lnc-Synpo in cardiac Ca2+ homeostasis and contractile function, knockdown and overexpression experiments were conducted to reveal the molecular mechanisms through which lnc-Synpo modulates SERCA2a gene expression. In vitro, knocking down lnc-Synpo with antisense oligonucleotide in hiPSC-CM significantly increased the mRNA and protein level of SERCA2a, whereas overexpression lnc-Synpo decreases SERCA2a expression. Meanwhile, forced expression of lnc-Synpo impaired myocyte contraction ability, Ca2+ transient peak height, and sarcoplasmic reticulum Ca2+ uptake, while depletion of lnc-Synpo reverse such phenomenon. Mechanistically, knockdown of lnc-Synpo led to increased transcriptional activity of SERCA2a, as evidenced by increased H3K4me3 and reduced H3K27me3 epigenetic marks revealed by ChIP-qPCR. Chromatin isolation by RNA purification (ChIRP) showed that lnc-Synpo binds directly to SERCA2a promoter DNA. In addition, RNA immunoprecipitation showed that lnc-Synpo binds to EZH2, a critical component of the polycomb repressor complex 2 (PRC2). Taken together, our data suggest that lnc-Synpo represses SERCA2a transcript expression by recruiting EZH2 and PRC2 complex to the promoter of SERCA2a, leading to epigenetic silencing of SERCA2a transcriptional activity. Targeting lnc-Synpo, therefore, could be a novel therapeutic approach to improve Ca2+ homeostasis and contractile function in failing human heart.