Mitofusins Regulate Lipid Metabolism to Mediate the Development of Lung Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic lung disease, where lung function decline is gradual and the overall prognosis of patients with IPF is poor with a median survival after diagnosis of approximately 3.8 years. Growing evidence points to the fundamental role for the mitochondrion in alveolar type 2 (AEC2) cell dysfunction in the pathogenesis of IPF. Additionally, single-cell RNA sequencing (RNA-Seq) recently identified MFN2 mRNA as significantly upregulated in AEC2 cells from patients with IPF, suggesting a role for mitochondrial fusion in AEC2 cells and the development of IPF. To investigate the roles of mitofusin 1 (MFN1) and mitofusin 2 (MFN2) of AEC2 cells in the pathogenesis of pulmonary fibrosis, we utilized genetically modified mice harboring MFN1 and/or MFN2 flanked by two loxP sites, and crossed them with Sftpc-CreERT2 mice, in which the AEC2 cell specific Sftpc-promoter drives the expression of tamoxifen-responsive CreERT2. Using this approach, we were able to selectively delete MFN1, MFN2 and both MFN1 and MFN2 together in AEC2 cells of the lung. Profoundly, in the absence of both mitofusins in AEC2 cells, we observe significant morbidity and mortality associated with disordered mitochondrial dynamics, failure of surfactant lipid production and the development of spontaneous pulmonary fibrosis, reminiscent of usual interstitial pneumonitis observed in the lungs of patients with IPF. Furthermore, we showed that mice with MFN1 or MFN2 deletion in AEC2 cells would have deteriorated bleomycin-induced pulmonary fibrosis. The results together confirmed the critical roles of MFN1 and MFN2 in AEC2 cells in protecting the lung from fibrotic process. To explore the detailed transcriptomic responses, we isolated AEC2 cells from control mice (n=3) or mice with MFN1/2 loss (n=3) in AEC2 cells at 6 weeks after tamoxifen-induced target gene deletion. RNA of AEC2 cells was extracted using TRIzol reagent (Invitrogen), and was purified by the RNeasy Plus Mini Kit (Qiagen), together with DNA digestion with the RNA free DNase set (Qiagen). The RNA samples were then submitted to Genomic Resource Core Facility of Weill Cornell Medical College for next generation RNA sequencing. On the other hand, we isolated AEC2 cells from control mice, or mice with MFN1 or MFN2 loss in AEC2 cells, at 5 days after bleomycin or PBS intra-tracheal administration (n=4 per group). RNA extraction and the subsequent RNA sequencing were performed as described above.