Mitochondrial superoxide–induced mitohormesis is mediated by citrate and cardioprotective

Figures

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  Fig. 1.. Transient increase in mitochondrial matrix superoxide during embryogenesis induces sustained mitohormetic adaptations in the adult heart. (A) Schematic depicting experimental design for inducible knockdown of superoxide dismutase 2 (Sod2) in iSOD2 mice during embryogenesis and postnatal adaptation. DOX is doxycycline. (B) Heatmap of significant differentially expressed genes between control and iSOD2 mice determined from RNA-seq of male and female 6-week-old heart tissue. Color depicts z-score. P adj. < 0.05 and log2FC > 0.25 (n = 5 mice per condition). (C) Top 25 enriched pathways in iSOD2 versus control mice determined with gene set enrichment analysis (GSEA) of RNA-seq dataset. FDR, false discovery rate, * depicts proposed mitohormetic pathways (n = 5 mice per condition). PPAR, peroxisome proliferator–activated receptor. (D) Western blots of heart lysates from 6-week-old control versus iSOD2 mice for transcriptional regulators of mitohormesis (n = 3 controls and n = 6 iSOD2 mice). (E and F) Western blot analysis confirming antioxidant and mitochondrial biogenesis transcriptional responses. (E) NRF2 and FOXO3 antioxidant targets and (F) OXPHOS proteins from heart lysates (n = 3…

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  Fig. 2.. Mitochondrial matrix superoxide–induced mitohormesis preserves mitochondrial content and limits oxidative injury during acute DIC. (A) Schematic depicting acute, high-dose DOXO injury regimen. (B) Heart weight normalized to tibia length in vehicle- or DOXO-treated control and iSOD2 female mice 48 hours postinjection (n = 5 to 6 mice per condition). (C) RT-qPCR analysis of cardiac injury marker Myh7 (normalized to 18S rRNA) in cardiac lysates from vehicle- or DOXO-treated control and iSOD2 mice (n = 5 to 6 mice per condition). (D) Principal components analysis (PCA) plot illustrating the variation in the transcriptome of hearts from vehicle- or DOXO-treated control or iSOD2 mice. (E) GSEA for significantly affected transcriptional processes different between control and iSOD2 mice treated with DOXO (n = 3 to 4 mice per condition). FDR, false discovery rate; NF-κB, nuclear factor κB. (F) Heatmap depicting expression of genes pertaining to mmu00190 OXPHOS pathway comparing control and iSOD2 mice treated with DOXO (n = 3 to 4 mice per condition). Color depicts z-score. (G) Western blot analysis for mitochondrial OXPHOS proteins normalized to total protein confirming the increase in…

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  Fig. 3.. Mitochondrial matrix superoxide–induced mitohormesis mitigates cardiac dysfunction and remodeling in chronic DIC. (A) Schematic illustrating dosing regimen and cardiac functional analysis timeline in a model of chronic DIC. (B) Ejection fraction and fractional shortening of control (n = 9) and iSOD2 (n = 8) female mice comparing baseline function to 6 weeks following first DOXO injection. (C) Representative left ventricular M-mode echocardiography images of control and iSOD2 mice at baseline and 6 weeks post-DOXO. (D) RT-qPCR analysis of heart failure markers Nppa, Nppb, and Myh7 (normalized to Hmbs) in untreated and DOXO-treated control and iSOD2 mice 10 weeks post–DOXO injection (n = 4 to 6 mice per condition). (E) Western blotting for apoptosis marker cleaved and total caspase-3 and quantification in untreated and DOXO-treated control and iSOD2 mice (n = 3 to 4 mice per condition). (F) Cardiac atrophy occurs in control mice treated with DOXO and not iSOD2 mice as depicted by heart weight normalized to tibia length (n = 5 to 6 mice per condition). (G to I) Representative images for (G) wheat germ agglutinin (WGA) staining illustrating cardiomyocyte shrinkage present in control…

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  Fig. 4.. Transient mitochondrial matrix superoxide accumulation in fibroblasts induces mitohormetic adaptations and confers resistance to DOXO-induced cell death. (A) Illustration of transient Sod2 loss, recovery, and stress challenge in mouse embryonic fibroblasts (MEFs). (B) Western blot analysis confirming SOD2 loss at 48 hours and induction of mitohormetic stress marker FOXO3a in siSod2 MEFs. KD is knockdown. (C) Live-cell microscopy measuring mitochondrial superoxide (MitoSOX) at 48 hours of Sod2 knockdown. (D) Gene expression analysis for mitochondrial biogenesis and mitochondrial genes 8 days following Sod2 loss (adapted) versus negative controls. (E) Western blotting confirming increased mitochondrial biogenesis and OXPHOS expression in adapted MEFs. (F) Relative mtDNA copy numbers in adapted MEFs normalized to nuclear B2M, presented as fold of negative control. (G) Mitochondrial oxygen consumption rates (OCRs) in adapted MEFs. Error bars represent SEM of six technical replicates. Graph is representative of three independent biological replicates. (H) RT-qPCR analysis of antioxidant enzyme expression in adapted MEFs. (I) Western blot analysis confirming increased antioxidant…

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  Fig. 5.. Mitochondrial citrate export mediates mitochondrial matrix superoxide–induced mitohormetic signaling. (A) Mitochondrial aconitase (ACO2) rate (nmoles per minute per microgram of mitochondria) in isolated mitochondrial from MEFs 48 hours post–Sod2 or Aco2 knockdown. KD is knockdown. Graph is technical replicates of one biological replicate (n = 2). (B) Total molar abundance of glucose-derived malate M+2 relative to citrate M+2 after 48 hours of Sod2 knockdown. (C) Representative live-cell microscopy and fluorescence quantification of a cytosolic targeted citrate biosensor, CitronCyto, 48 hours post–Sod2 knockdown (n = 91 control, n = 180 siSod2 cells, and n = 3 biological replicates). (D) Representative Western blots and quantification for citrate metabolic proteins in adapted MEFs, 8 days post–Sod2 knockdown. (E) Western blot analysis for citrate metabolic proteins in heart lysates of either untreated or 10-week post–chronic DOXO–treated control and iSOD2 mice (n = 3 to 4 mice per condition). (F) Quantification of increased CitronCyto fluorescence 48 hours post–Aco2 knockdown (n = 55 cells per condition and n = 3 biological replicates). (G) Western blot analysis of increased…

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  Fig. 6.. Mitochondrial matrix superoxide–induced mitohormesis increases histone acetylation via mitochondrial citrate export. (A) Representative Western blot illustrating increased lysine acetylation at proteins of 15 and 11 kDa in adapted MEFs 8 days posttransfection with Sod2 siRNA versus negative siRNA controls. (B and C) Representative Western blot for increased H3K27Ac normalized to total H3 in (B) adapted MEFs 8 days following transfection with Sod2 targeting siRNA and (C) adapted MEFs 2 days following Aco2 knockdown compared with negative controls. (D and E) Representative Western blots illustrating SLC25A1 dependence of both (D) Sod2 and (E) Aco2-mediated mitohormetic increase in histone acetylation. (F) Representative Western blot illustrating co-knockdown of ACO2 and ACLY compared with negative controls. (G) Representative Western blot depicting that the ACO2-mediated protection from DOXO (100 nM for 24 hours) is partially ACLY dependent. (H) Quantification of the number of total H3K27Ac peaks identified from CUT&RUN analysis of cells 48 hours following transfection with control or Aco2 targeting siRNA. (I) Quantification of the percent of genes in relevant mitohormetic gene…