以單細胞轉錄組分析與小鼠心臟再生功能關聯之巨噬細胞功能表型的轉換

Abstract

心衰竭所造成高死亡率的主要原因，是成人心臟在受損後無法進行心肌細胞的再生。近年來的研究顯示新生兒和初生小鼠心臟損傷後能夠進行再生，然而新生小鼠心臟的再生能力在出生後七天便急劇地下降。已知新生小鼠心臟組織常駐性巨噬細胞，在心肌受損後的修復過程具有必要的角色，並促使血管新生和心肌細胞增殖。然而新生小鼠心臟中，具有修復能力的巨噬細胞之族群生理特性及其調控機轉仍不清楚。本研究主要目的在於利用心肌梗塞小鼠模型，針對心臟巨噬細胞在不具有再生能力的成鼠和具有再生能力的新生小鼠中的異質性和功能進行比較。 首先我們在成鼠和新生小鼠誘導心肌梗塞後的第十天，從心臟組織裡分選出巨噬細胞。成鼠心臟裡主要的巨噬細胞是由單核球分化而來，而新生小鼠主要存在的是胚胎發育時期就已進駐心臟組織的巨噬細胞。在大量細胞定序分析 (Bulk sequencing) 中，成鼠和小鼠在心肌梗塞後的巨噬細胞分別有482 和 614個mRNA的表現量有顯著改變。分析其參與在訊息傳遞路徑中的功能，顯示在成鼠心肌梗塞後的巨噬細胞與Wnt傳遞路徑相關的基因功能調節異常; 另一方面，在新生小鼠心肌梗塞後的巨噬細胞則表現有關干擾素 γ的調控路徑。我們進一步利用做單細胞基因定序發現總共14群的巨噬細胞亞群，在心肌梗塞成鼠中的巨噬細胞亞群表現與發炎反應和細胞外基質的基因調控有關，顯示參與在成鼠心臟組織修復時的巨噬細胞偏向促進發炎反應和產生纖維化；相反地，新生小鼠在心肌梗塞後參與組織修復的巨噬細胞則表現出抑制與消除發炎反應、調節代謝功能並促進生物合成等反應發生。 本研究結果顯示心臟組織中的巨噬細胞亞群，對成鼠和新生小鼠心臟受損後的修復過程中產生不同的反應。大量細胞定序和單細胞基因體分析結果顯示心臟的巨噬細胞在心肌梗塞後的成鼠趨向表現發炎反應和纖維化; 而新生小鼠心臟則顯示具有強烈的氧化磷酸化反應和干擾素表現在受損的心肌組織。未來將藉此研究結果進一步探索，期能找到新型標的作為促進心臟再生與修復的治療策略。

Heart failure is an important cause of morbidity and mortality worldwide, in part owing to the inability of the human heart to replenish lost cardiomyocytes following cardiac injury. Recent studies showed that neonatal human and mouse can fully regenerate in response to injury. However, the regenerative capacity of cardiomyocytes in infant and neonataes is transient and age-dependent. The regenerative capacity of the neonatal mouse heart, for example, sharply declines by 7 days of age. It has been demonstrated that tissue-resident macrophages in neonatal hearts are required for cardiac repair and recovery from injuries by promoting angiogenesis and cardiomyocyte proliferation. The cellular and molecular determinants underlying the reparative/pro-regenerative properties of neonatal cardiac macrophages, however, remain elusive. This study aims to reciprocally compare macrophage heterogeneity and functionality that are critical for myocardial repair and regeneration following myocardial infarction (MI) injury in neonatal (regenerative) and adult (non-regenerative) mouse hearts. Cardiac macrophages (CD45+CD11b+CD64+) isolated from neonatal (P1-3) and adult (8 weeks) mouse heart at day 10 post-MI were subjected to bulk and single-cell RNA sequencing (scRNA-Seq) analyses. In response to MI injury, CCR2+ monocyte-derived macrophages predominate in adult, whereas CCR2- tissue-resident macrophages markedly expand in neonatal mouse hearts. Compared with the sham-operated controls, bulk RNA-seq revealed that the expression levels of a total of 482 and 614 mRNAs were significantly (absolute fold change > 2, P < 0.05) altered in adult and neonatal cardiac macrophages following MI, respectively. Pathway analyies showed that genes involved in the Wnt signaling were dysregulated in adults, whereas transcripts involved in oxidative phosphorylation and interferon-γ (IFN-γ) pathway were highly up-regulated in neonatal cardiac macrophages following MI. Finally, scRNA-Seq analysis identified 10 distinct cardiac macrophage clusters, 3 lymphocyte clusers and 1 stromal cell cluster in adult compared to those in neonatal mouse heart following MI. Adult-enriched macrophage clusters showed strong upregulation in inflammatory mediators (Saa3 and Ccl6) and extracellular matrix protein genes (Fn1 and Ecm), suggesting macrophage polarization towards a pro-inflammatory and fibrogenic phenotype in the post-injury adult heart. Neonate-enriched macrophage clusters, by contrast, showed altered cellular metabolism with enhanced oxidative phosphorylation (Atp5l, Cox6c, Ndufa1 and Ndufa3) and activated peptide biosynthesis, suggesting a metabolic reprogramming that favors tissue repair and regeneration in the neonatal heart following MI injury. The present study showed that distinct macrophage populations are present in neonatal and adult mouse hearts in response to MI injury. Bulk and scRNA-seq revealed divergent transcriptional signatures of cardiac macrophages in post-MI adult and neonatal mouse hearts. While Wnt signaling, inflammation and fibrogenic activities are increased in adult cardiac macrophages, oxidative phosphorylation, IFN-γ signaling and peptidyl biosynthesis are markedly activated in neonatal cardiac macrophages in response to injury. Phenotypic switching toward enhanced oxidative phosphorylation, IFN-γ signaling or translational activity in cardiac macrophages, therefore, could be the key to develop novel therapeutics to promote cardiac regeneration.