Complementary RNA and protein profiling identifies iron as a key regulator of mitochondrial biogenesis

Abstract

Mitochondria are centers of metabolism and signaling whose content and function must adapt to changing cellular environments. The biological signals that initiate mitochondrial restructuring and the cellular processes that drive this adaptive response are largely obscure. To better define these systems, we performed matched quantitative genomic and proteomic analyses of mouse muscle cells as they performed mitochondrial biogenesis. We find that proteins involved in cellular iron homeostasis are highly coordinated with this process and that depletion of cellular iron results in a rapid, dose-dependent decrease of select mitochondrial protein levels and oxidative capacity. We further show that this process is universal across a broad range of cell types and fully reversed when iron is reintroduced. Collectively, our work reveals that cellular iron is a key regulator of mitochondrial biogenesis, and provides quantitative data sets that can be leveraged to explore posttranscriptional and posttranslational processes that are essential for mitochondrial adaptation.

Figure 1. Complementary RNA and Protein Profiling of PGC-1α-Induced Mitochondrial Biogenesis

(A) Experimental workflow for proteomic and microarray analysis of differentiated C2C12 mouse myotubes overexpressing PGC-1α and/or treated with 100 μM DFO. (B) Comparison of mRNA expression in GFP-treated cells and PGC-1α-treated cells (AU, arbitrary units). (C) Changes in protein expression during PGC-1α overexpression. Proteins are ordered from least to greatest fold change. (D) Comparison of mRNA expression in GFP-treated cells and DFO-treated cells. (E) Comparison of PGC-1α and DFO-induced changes in mRNA expression (59% of mitochondrial genes versus 18% of all genes are in the lower-right quadrant; p = 9.1 × 10⁻²²², χ² contingency test). (F) Comparison of OxPhos mRNA and protein expression during DFO treatment. (G) Mitochondrial mRNA and protein expression that show discordance during PGC-1α+DFO treatment. See also Figure S1 and Table S1.

Figure 2. Effect of Iron Deprivation on Nuclear and mtDNA-Encoded Gene Expression

(A) Level of the indicated proteins from samples used in the proteomic analyses as assessed by immunoblotting. (B) Abundance of the indicated transcripts from samples used in the microarray analyses as detected by real-time qPCR. Data are displayed as mean ± SD of triplicate measurements (*p < 0.05, ANOVA with Tukey's test). (C) Level of the indicated proteins in C2C12 myotubes after treatment with a range of DFO concentrations for 3 days as assessed by immunoblotting. (D) Level of the indicated proteins after DFO treatment for 24 hr in the indicated cell lines as assessed by immunoblotting. (E) Level of the indicated proteins after DFO treatment for 24 hr in C2C12 myoblasts as assessed by SDS-PAGE and immunoblotting. (F) Level of the indicated proteins after DFO treatment for 24 hr in myoblasts as assessed by BN-PAGE and immunoblotting (SC, respiratory supercomplex; NS, nonspecific band). (G) Mitochondrial mass in myoblasts after DFO treatment for 24hr as assessed by plate reader-based quantitation of MitoTracker Green FM or NAO fluorescence/cell number. Data are displayed as mean ± SD of triplicate measurements (NS signifies p ≥ 0.05, Student's t test). See also Figure S2.

Figure 3. The Mitochondrial Response to Iron Deprivation Is Independent of PGC-1α, PGC-1β, and HIF-1α

(A–I) Immunoblotting was used to assess protein abundance, and real-time qPCR was used to assess mRNA abundance. Data are displayed as mean ± SD of triplicate measurements (*p < 0.05, Student's t test). (A) Protein levels in HEK293 cells after 100 μM DFO treatment or ferroportin (Fpn-GFP) over-expression for 48 hr. Abundance of (B) Ndufs8, (C) Uqcrc1, and (D) Ppargc1a in C2C12 myoblasts following DFO treatment for the indicated times. (E) Level of the indicated proteins in wild-type or PGC-1α^−/− brown preadipocytes after 100 μM DFO treatment for 24 hr. (F) Abundance of Ppargc1b in myoblasts following DFO treatment for the indicated times. (G) Level of the indicated proteins in wild-type or PGC-1 b^f/f/MLC-Cre soleus muscle primary satellite cells after 100 μM DFO treatment for 24 hr. (H) Level of the indicated proteins after 100 μM DFO, 100 μM DP, or 500 μM DMOG treatment for 24 hr in myoblasts. (I) Level of the indicated proteins in wild-type or HIF-1α ^−/− MEFs after 100 μM DFO treatment for 24 hr. See also Figure S3.

Figure 4. Mitochondrial Gene Expression and Respiratory Function during Iron Deprivation and Recovery

(A) Experimental workflow for the analysis of mitochondrial gene expression and respiration during DFO response and recovery. (B) Level of the indicated proteins in C2C12 myoblasts treated with 100 μM DFO for 24 hr then passaged every 24 hr in DFO-free media as assessed by immunoblotting. (C) Mitochondrial respiratory profile of untreated myoblasts at day 1 of analysis. Data are displayed as mean ± SD of 14–16 replicates. (D–F) Basal OCR (pmol/min)/cell number (D), spare respiratory capacity (E), and coupling efficiency (F) of myoblasts treated with 100 μM DFO for 24 hr then passaged every 24 hr in DFO-free media. Data are displayed as mean±SD of 14–16 replicates (*p<0.05, Student's t test in D, ANOVA with Tukey's test in E and F). See also Figure S4.