Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by increased activation of fibroblasts/myofibroblasts. Previous reports have shown that IPF fibroblasts are resistant to apoptosis, but the mechanisms remain unclear. Since inhibition of the mitochondrial permeability transition pore (mPTP) has been implicated in the resistance to apoptosis, in this study, we analyzed the role of mitochondrial function and the mPTP on the apoptosis resistance of IPF fibroblasts under basal conditions and after mitomycin C-induced apoptosis. We measured the release of cytochrome c, mPTP opening, mitochondrial calcium release, oxygen consumption, mitochondrial membrane potential, ADP/ATP ratio, ATP concentration, and mitochondrial morphology. We found that IPF fibroblasts were resistant to mitomycin C-induced apoptosis and that calcium, a well-established activator of mPTP, is decreased as well as the release of pro-apoptotic proteins such as cytochrome c. Likewise, IPF fibroblasts showed decreased mitochondrial function, while mPTP was less sensitive to ionomycin-induced opening. Although IPF fibroblasts did not present changes in the mitochondrial membrane potential, we found a fragmented mitochondrial network with scarce, thinned, and disordered mitochondria with reduced ATP levels. Our findings demonstrate that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and that altered mPTP opening contributes to this resistance. In addition, IPF fibroblasts show mitochondrial dysfunction evidenced by a decrease in respiratory parameters.

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Mitochondrialbc1complex from yeast has ten subunits, but only Cytochromeb(Cytb) subunit is encoded in the mitochondrial genome. Cytbhas eight transmembrane helices containing two hemesbfor electron transfer. Cbp3 and Cbp6 assist Cytbsynthesis, and together with Cbp4 induce Cytbhemylation. Subunits Qcr7/Qcr8 participate in the first steps of assembly, and lack of Qcr7 reduces Cytbsynthesis through an assembly-feedback mechanism involving Cbp3/Cbp6. Since Qcr7 resides near the Cytbcarboxyl-region, we wondered whether this region is important for Cytbsynthesis/assembly. Although deletion of the CytbC-region did not abrogate Cytbsynthesis, the assembly-feedback regulation was lost, so Cytbsynthesis was normal even if Qcr7 was missing. Mutants lacking the CytbC-terminus were non-respiratory due to absence of fully assembledbc1complex. By performing complexome profiling, we showed the existence of aberrant early-stage subassemblies in the mutant. In this work we demonstrate that the C-terminal region of Cytbis critical for regulation of Cytbsynthesis andbc1complex assembly.