DELE1 promotes translation-associated homeostasis, growth, and survival in mitochondrial myopathy

Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of Tfam, and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L). The preponderance of the response was directed at upregulating pathways for aminoacyl-tRNA biosynthesis, the intermediates for protein synthesis, and was similar in heart and skeletal muscle but more limited in brown adipose challenged with cold stress. Strikingly, models with early DELE1 mt-ISR activation failed to grow and survive to adulthood in the absence of Dele1, accounting for some but not all of OMA1’s protection. Notably, the DELE1 mt-ISR did not slow net protein synthesis in stressed striated muscle, but instead prevented loss of translation-associated proteostasis in muscle fibers. Together our findings identify that the DELE1 mt-ISR mediates a stereotyped response to diverse forms of mitochondrial stress and is particularly critical for maintaining growth and survival in early-onset mitochondrial myopathy.

The copyright holder for this preprint this version posted February 29, 2024.; https://doi.org/10.1101/2024.02.29.582673 doi: bioRxiv preprint DELE1 promotes growth and survival 55 (D) Heart to body weight ratio for C10 S59L mice; Dele1 KO mice and littermates at P140. (E) Heart to body weight ratio for Tfam mKO mice; Dele1 KO mice and littermates at P56. (F) Correlation between age at stress onset and DELE1 survival benefit among the models.* indicates genotypes for which some lifespan estimates were determined from prior studies (Nguyen et al, 2022;Shammas et al, 2022).
(G) Primary fibroblasts from Opa1 ∆s1/∆s1 mice treated with CCCP 20 M or vehicle only for 16 hrs.The c and e bands generated by OMA1 cleavage (from a and b, respectively) are reduced at baseline and following uncoupling with CCCP; the b band is also relatively retained with CCCP, together demonstrating that L-OPA1 ∆s1/∆s1 (a and b bands) is resistant to OMA1 cleavage.
(I) Immunoblot compares OPA1 cleavage by OMA1 and elevation of the mt-ISR marker protein MTHFD2 in C10 G58R animals with or without OMA1 or DELE1 that survived to 14 -16 weeks.Lysates from Oma1 KO animals are from samples that were previously generated and appeared in (Shammas et al, 2022).

Supplemental Figure 3. Comparison of OXPHOS subunit expression in heart mitochondria from diverse models of mitochondrial myopathy/cardiomyopathy.
(A) Scatterplots depicting relative abundance of OXPHOS complexes I -V subunits, mito-ribosome, and Coenzyme Q from the indicated genotypes.Data are from the same datasets represented in (Fig. 3F -H), replotted to compare disease models.For statistics, a one-way ANOVA was performed followed by post-hoc testing, corrected for the multiple comparisons depicted within the graph with Dunnett's test.
All values are relative to littermate controls except for C2/C10 DKO which are matched to unrelated agematched controls.Data from these proteomics datasets also appear in Fig. 5G -I.
Enrichment analysis mitochondrial proteins that significantly changed in Tfam mKO vs. control mitochondria isolated from hearts, using Tier 3 MitoPaths from MitoCarta 3.0.SupplementalFigure 4. TEM of myocardium and ultrastructural features of mitochondria in C10 G58R on Dele1+/-and Dele1 KO backgrounds.(A) Kernel density plots showing distribution of mitochondrial areas for indicated genotypes, measured from TEM images of heart mitochondria.Median values and, in parentheses, interquartile ranges are reported adjacent to curves.N = 2 animals per genotype except for Tfam mKO; Dele1 KO, where only 1 animal was available.> 600 mitochondria were measured per animal.(B) Bar graph comparing the areas of segmented and non-segmented types of electrolucent mitochondria that were obtained from analysis of C10 G58R animals and littermates in (A).Statistics were performed using Mann-Whitney test.(C -E) Representative TEM images acquired at 2000x direct magnification show areas of myocardium of indicated genotype used for analysis of mitochondria.Scale bar = 5 µm.(F) Image of the subarea boxed yellow in D, acquired at 5000x direct magnification and representative of the images used to quantify ultrastructural features of mitochondria detailed in Figure 4. Scale bar = 2.5 µm.(G) Examples of inclusions observed in C10 G58R mutant mitochondria (black arrows).(H) Examples of two types of electrolucent mitochondria characterized by an enlarged matrix area absent of electron-dense substance and fewer cristae.(Top) A uniformly electrolucent mitochondrion.(Bottom) a segmented mitochondrion that has an electrolucent part (white arrow) separated from a 105 and is also made available for use under a CC0 license.(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted February 29, 2024.; https://doi.org/10.1101/2024.02.29.582673 doi: bioRxiv preprint DELE1 promotes growth and survival 57 portion of normal-looking matrix and cristae by a cut-through cristae.Open black arrowhead indicates the junction between electrolucent and normal portions of the segmented mitochondria.(I) Mitochondria that are fully wrapped by electron-dense phagosome membranes (black arrows).(J) Mitochondria with ruptured OMMs.Open white arrowheads indicate sites where the intact IMM is visible, but OMM is absent.Scale bar in J = 500 nm and applies to G-J.Supplemental Figure 5. TEM of myocardium and ultrastructural features of mitochondria in C2/C10 DKO.(A-B) Representative TEM images acquired at 2000x direct magnification show areas of myocardium of indicated genotype used for analysis of mitochondria.Scale bar = 5 µm.(C) Image of the subarea boxed yellow in B, acquired at 5000x direct magnification and representative of the images used to quantify ultrastructural features of mitochondria detailed in Figure 4. Scale bar = 2.5 µm.(D) Examples of inclusions observed in C2/C10 DKO mitochondria (black arrows).Scale bar = 500 nm.(E) Examples of electrolucent mitochondria characterized by an enlarged matrix area absent of electrondense substance and fewer cristae.Scale bar = 500 nm.Supplemental Figure 6.TEM of myocardium and ultrastructural features of mitochondria in C10 S59L on Dele1+/-and Dele1 KO backgrounds.(A-C) Representative TEM images acquired at 2000x direct magnification show areas of myocardium of indicated genotype used for analysis of mitochondria.Scale bar = 5 µm.
105 and is also made available for use under a CC0 license.(whichwas not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USCThe copyright holder for this preprint this version posted February 29, 2024.; https://doi.org/10.1101/2024.02.29Image of the subarea boxed yellow in B, acquired at 5000x direct magnification and representative of images used to quantify ultrastructural features of mitochondria detailed in Figure 4. Scale bar = 2.5 µm.(E) Examples of mitochondria that are partially or fully enclosed by electron-dense phagosome membranes (black arrows).Open black arrowhead indicates a portion of the mitochondria that is not enclosed.Partially enclosed mitochondria with ruptured OMMs were also observed.Open white arrowheads indicate sites where the intact IMM is visible, but the OMM is absent.(F) Examples of mitochondria with ruptured OMMs.Open white arrowheads indicate sites where the intact IMM is visible, but an OMM is absent.Scale bar in F = 500 nm and applies to E. (G) Serial sections through a 250 nm diameter mitochondrion show that it is a spherical nanomitochondrion spanning fewer than five 60-nm sections (< 300 nm in Z).Top row shows the five serial sections without colorization, bottom row shows the same serial sections with the nano-mitochondrion shaded yellow.Yellow dotted lines indicate absence of the mitochondrion in neighboring serial sections.Scale bar = 200 nm.(H) Five serial sections of 60-nm thickness show a 100 nm-wide tubule-shaped mitochondrion.Top row shows five serial sections through the tubular nano-mitochondrion, bottom row shows the same serial sections with the tubular nano-mitochondrion shaded yellow.The yellow dotted lines indicate absence of the mitochondrion in the neighboring section.Scale bar = 200 nm.Supplemental Figure 7. TEM of myocardium and ultrastructural features of mitochondria in Tfam mKO on Dele1+/-and Dele1 KO backgrounds.105 and is also made available for use under a CC0 license.(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted February 29, 2024.; https://doi.org/10.1101/2024.02.29C)Representative TEM images acquired at 2000x direct magnification show areas of myocardium of indicated genotype used for analysis of mitochondria.Yellow star in B indicates a myocyte with milder structural phenotype compared to neighboring myocytes, illustrating the observed mosaicism of the phenotype.Scale bar = 5 µm.(D) Image of the subarea boxed yellow in B, acquired at 5000x direct magnification and representative of images used to quantify ultrastructural features of mitochondria detailed in Figure 4. Scale bar = 2.5 µm.(E) Tfam mKO mitochondria displayed populations of closely aligned "stacked" cristae (black arrows) and sparse areas filled with a granular matrix material and few cristae (open white arrowheads).Scale bar = 500 nm.(F) Examples of crumpled cristae (white arrows) that occurred in Tfam mKO mitochondria.Scale bar = 500 nm.(G) Stacked cristae boxed in E and crumpled cristae boxed in F are shown enlarged in G. Scale bar = 250 nm.Supplemental Figure 8. DELE1 mt-ISR transcriptional response in heart is similar in response to diverse mitochondrial stressors.(A) Heat map of Log 2 FC for DELE1-dependent DEGs detected in hearts of two out of three myopathy/cardiomyopathy models.(B) Venn diagram showing intersection of stress-induced DEGs in heart among the 3 models of myopathy/cardiomyopathy.
105 and is also made available for use under a CC0 license.(whichwas not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.(whichwas not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted February 29, 2024.; https://doi.org/10.1101/2024.02.29.582673 doi: bioRxiv preprint