Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press REPORT Mitochondrial misreading in skeletal muscle accelerates metabolic aging and confers lipid accumulation and increased inflammation DIMITRI SHCHERBAKOV,1,4 STEFAN DUSCHA,1,4 REDA JUSKEVICIENE,1 LISA M. RESTELLI,2 STEPHAN FRANK,2 ENDRE LACZKO,3 and ERIK C. BÖTTGER1 1Institut für Medizinische Mikrobiologie, Universität Zürich, 8006 Zürich, Switzerland 2Division of Neuropathology, Institute of Medical Genetics and Pathology, Basel University Hospital, 4031 Basel, Switzerland 3Functional Genomics Center Zurich, ETH Zürich und Universität Zürich, 8057 Zürich, Switzerland ABSTRACT We have recently reported on an experimental model of mitochondrial mistranslation conferred by amino acid exchange V338Y in mitochondrial ribosomal protein MrpS5. Here we used a combination of RNA-seq and metabolic profiling of ho- mozygous transgenic Mrps5V338Y/V338Y mice to analyze the changes associated with the V338Y mutation in postmitotic skeletal muscle. Metabolome analysis demonstrated enhanced levels of age-associated metabolites in the mutant V338Y animals accompanied by increased glycolysis, lipid desaturation and eicosanoid biosynthesis, and alterations of the pentose phosphate pathway. In addition, transcriptome signatures of aged V338Y mutant muscle pointed to elevated inflammation, likely reflecting the increased levels of bioactive lipids. Our findings indicate that mistranslation-mediated impairment of mitochondrial function affects specific bioenergetic processes in muscle in an age-dependent manner. Keywords: mitochondria; misreading; skeletal muscle; aging; metabolome INTRODUCTION express a mtDNA mutator phenotype, with a threefold to fivefold increase in the levels of random point mutations A decline in mitochondrial function has been associated in mtDNA, display respiratory chain dysfunction and fea- with aging and complex age-related changes in metabo- tures of accelerated aging (Trifunovic et al. 2004; Kujoth lism (Sun et al. 2016; Kauppila et al. 2017). Alterations in et al. 2005). mitochondrial function including impaired oxidative phos- Increased error rates in mitochondrial protein synthesis phorylation, increased oxidative damage, reduced activity may have similar effects to that of heightened levels of sto- of metabolic enzymes, and changes in mitochondrial dy- chastic mtDNA mutations as both would increase the ran- namics and biogenesis have all been linked to various as- dom incorporation of missense mutations. Towards this pects of aging (Nunnari and Suomalainen 2012). It is now end, we recently demonstrated that the amino acid ex- widely accepted that mitochondria are not mere energy change V338Y in the mitochondrial ribosomal protein factories, but play central roles in metabolism and signal- MrpS5 confers mitoribosomal mistranslation (Akbergenov ing (Finkel 2015; Spinelli and Haigis 2018). et al. 2018). The identification of amino acid replacement The accumulation of somatic mutations in mitochondrial V338Y in mouse mitochondrial ribosomal protein MrpS5 DNA (mtDNA) in post-mitotic tissues is thought to be a key as ribosomal ambiguity mutation (ram) was based on the factor in the pathogenesis of age-related diseases (Wal- experimental demonstration that the human homolog lace 2005). mtDNA encodes for genes of oxidative phos- V336Y, while not affecting mitochondrial protein synthesis phorylation (OXPHOS) and corresponding mutations quantitatively, confers incorporation of near-cognate ami- result in a decline of OXPHOS activity, ultimately impairing no acids and stop-codon read-through in in-organello cellular bioenergetic capacity (Wallace 1999). Mice that translation assays using mitochondria from human HEK293 cells transfected with mutant V336Y Mrps5 4 These authors contributed equally to this work. Corresponding author: [email protected] Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna. © 2021 Shcherbakov et al. This article, published in RNA, is available 077347.120. Freely available online through the RNA Open Access under a Creative Commons License (Attribution 4.0 International), as option. described at http://creativecommons.org/licenses/by/4.0/. RNA (2021) 27:265–272; Published by Cold Spring Harbor Laboratory Press for the RNA Society 265 Downloaded from rnajournal.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Shcherbakov et al. (Akbergenov et al. 2018). Accommodation of mismatched 1A). As evident from the heatmap (Fig. 1B), the expression aa-tRNAs by ram mutations increases the natural rate of of inflammation-related transcripts was also elevated in protein synthesis error frequency, resulting in random mis- aged wild-type animals, but more prominently in aged sense substitutions in primary amino acid sequences. Due V338Y mice. In addition to inflammation, the aged to the physical constraints of the mRNA–tRNA interaction, V338Y mutant mice showed enrichment for gene tran- accommodation of mismatched aa-tRNAs is limited to scripts associated with specific metabolic pathways, that near-cognate tRNAs, leading to missense amino acid sub- is, adipogenesis, fatty acid (FA) metabolism, eicosanoid stitutions that are conservative in nature because of the synthesis, pentose phosphate shunt, and glycolysis/gluco- way the genetic code is constructed (Woese 1965; Kurland neogenesis (Fig. 1A). et al. 1996; Zaher and Green 2009). Homozygous knock-in To further characterize the mutant MrpS5 V338Y-associ- C57/BL6 mice carrying the Mrps5 V338Y mutation showed ated changes in muscle tissue, we performed metabolic impaired oxidative phosphorylation and increased forma- profiling for 9 and 19-mo-old mice. For an initial assess- tion of ROS (Akbergenov et al. 2018). ment of differences, we performed a between group anal- To further dissect the organismal response to increased ysis (BGA). BGA simplifies a data set by reducing its mitochondrial misreading, we here combined unbiased dimensionality and defining axes that best discriminate RNA-seq and metabolome analysis to characterize the re- the groups. We assigned four groups (9 mo wild-type, 9 sponse to mitochondrial mistranslation in the skeletal mus- mo V338Y, 19 mo wild-type, 19 mo V338Y) and plotted cle of Mrps5V338Y/V338Y mutant mice. Our choice of tissue them into two-dimensional space using the first two was guided by muscle consisting of post-mitotic cells axes. BGA showed a similar metabolic pattern in wild- with high energy demands and vulnerability to defects in type and mutant V338Y mice at 9 mo of age (Fig. 2A). At mitochondrial function (Johnson et al. 2013; Boengler 19 mo, the metabolic profiles of both genotypes were sig- et al. 2017). We show that mitoribosomal misreading re- nificantly different from the 9 mo animals. In addition, the sults in pronounced age-related metabolite changes in 19 mo V338Y mice were also distinct from the age muscle, which are associated with alterations of the pen- matched wild-type controls (P < 0.0001). The observed tose phosphate pathway (PPP), increased glycolysis and metabolic profile indicates that both age and genotype enhanced lipid biosynthesis. Further, increased levels of cause the metabolic differences between these two bioactive lipids suggest a possible link to the transcrip- groups. Extraction of the most differentially up- and tome signature of enhanced inflammation observed in down-regulated metabolites associated with the horizon- aged Mrps5V338Y/V338Y mutant mice. tal axis (axis 1) included age-associated metabolites de- scribed previously (Supplemental Fig. S4A; Houtkooper et al. 2011), suggesting that axis 1 represents aging, as RESULTS this axis captures the largest fraction of age-related vari- Transcriptomes of skeletal muscle tissue samples from ance. Calculating the BGA on the basis of the published wild-type and Mrps5V338Y/V338Y mice were resolved using data set and projecting our animals on this model, corrob- RNA-seq. To study the impact of the V338Y mutation on orated our conclusion that the 19 mo Mrps5V338Y/V338Y an- age-related changes, mice were sampled at 3 and 19 mo imals showed enhanced age-associated metabolic of age. Considerable transcriptomic changes were detect- changes compared to age-matched wild-type controls ed by differential gene expression analysis of young (3 mo) (Supplemental Fig. S4B). and old (19 mo) muscle samples (Supplemental Fig. S1). The heatmap of the 40 metabolites with the highest cor- − With a threshold P-value <5 × 103 (FDR < 1 × 10 2) there relation to axis 1 (Fig. 2B) indicates reduced levels of late were 3343 and 4232 age-regulated transcripts in the glycolysis intermediates pyruvate, 3-phosphoglycerate wild-type and V338Y mutant data set, respectively. and phosphoenolpyruvate in the 19 mo Mrps5V338Y/V338Y Further analysis revealed the hallmarks of age-associated animals, whereas levels of early glycolysis intermediates transcriptomic alterations for both wild-type and mutant and lactate remain unchanged (not shown in the heatmap); animals—a decrease in mRNA transcripts related to gene the same pattern but to a lesser extent was observed in the annotations associated with mitochondrial function, pro- aged wild-type animals. Transcriptome profiles revealed teasome degradation, metabolism, and protein translation significant up-regulation (P < 0.05) of hexokinases Hk1 (Supplemental Fig. S2), for clustering of the experimental and Hk3 (Hk1/3), phosphofructokinase (Pfk), phosphoglyc- groups by BGA