COX7A2L genetic variants determine cardiorespiratory fitness in mice and human

Mitochondrial respiratory complexes form superassembled structures called supercomplexes. COX7A2L is a supercomplex-specific assembly factor in mammals, although its implication for supercomplex formation and cellular metabolism remains controversial. Here we identify a role for COX7A2L for mitochondrial supercomplex formation in humans. By using human cis-expression quantitative trait loci data, we highlight genetic variants in the COX7A2L gene that affect its skeletal muscle expression specifically. The most significant cis-expression quantitative trait locus is a 10-bp insertion in the COX7A2L 3′ untranslated region that increases messenger RNA stability and expression. Human myotubes harboring this insertion have more supercomplexes and increased respiration. Notably, increased COX7A2L expression in the muscle is associated with lower body fat and improved cardiorespiratory fitness in humans. Accordingly, specific reconstitution of Cox7a2l expression in C57BL/6J mice leads to higher maximal oxygen consumption, increased lean mass and increased energy expenditure. Furthermore, Cox7a2l expression in mice is induced specifically in the muscle upon exercise. These findings elucidate the genetic basis of mitochondrial supercomplex formation and function in humans and show that COX7A2L plays an important role in cardiorespiratory fitness, which could have broad therapeutic implications in reducing cardiovascular mortality.

Mitochondrial respiratory complexes form superassembled structures called supercomplexes. COX7A2L is a supercomplex-specific assembly factor in mammals, although its implication for supercomplex formation and cellular metabolism remains controversial. Here we identify a role for COX7A2L for mitochondrial supercomplex formation in humans. By using human cis-expression quantitative trait loci data, we highlight genetic variants in the COX7A2L gene that affect its skeletal muscle expression specifically. The most significant cis-expression quantitative trait locus is a 10-bp insertion in the COX7A2L 3′ untranslated region that increases messenger RNA stability and expression. Human myotubes harboring this insertion have more supercomplexes and increased respiration. Notably, increased COX7A2L expression in the muscle is associated with lower body fat and i mp ro ved c ar di or es pi ratory fitness in humans. Accordingly, specific reconstitution of Cox7a2l expression in C57BL/6J mice leads to higher maximal oxygen consumption, increased lean mass and increased energy expenditure. Furthermore, Cox7a2l expression in mice is induced specifically in the muscle upon exercise. These findings elucidate the genetic basis of mitochondrial supercomplex formation and function in humans and show that COX7A2L plays an important role in cardiorespiratory fitness, which could have broad therapeutic implications in reducing cardiovascular mortality.
Mitochondria are indispensable organelles as they are responsible for the production of the majority of ATP in the cell. Most cellular ATP is generated by oxidative phosphorylation (OxPhos), a process through which electrons are extracted from reducing equivalents and transferred through four different respiratory complexes (RCs) present in the mitochondria inner membrane (CI-CIV). Electron transfer is coupled with the generation of a proton gradient through the mitochondrial membrane that drives the phosphorylation of ADP to ATP by the ATP-synthase complex (also known as complex V).
Although mitochondrial RCs (MRCs) are often depicted as isolated entities, it is now widely accepted that isolated complexes coexist with superassembled structures called supercomplexes (SCs) [1][2][3] in the mitochondrial membrane. Within SCs, different MRCs physically interact with each other. There are different subtypes of SCs and their assembly may vary according to the organism, the metabolic state of the cell and the tissue type 4,5 ; however, the main SCs in mammalian mitochondria are composed of complex I, a complex III dimer and one or more copies of complex IV 2 , whereas complex II is mostly found isolated. These SCs are also called 'respirasomes' because they can 'respire' by themselves sequencing and RNA expression data of 54 tissues across approximately 1,000 individuals 34 (http://www.gtexportal.org/home/). We looked at expression quantitative trait loci (eQTLs) located in proximity (±1 Mb) of the COX7A2L transcription start site (TSS) (cis-eQTLs; Extended Data Fig. 1b). Considering an estimated average number of cis-eQTLs per gene of ~180 in GTEx 35 , we found a high number (1,427) of significant cis-eQTLs for COX7A2L (false discovery rate (FDR) < 0.05, P < 2.2 × 10 -4 ) in 32 different tissues (Fig. 1b and Supplementary Table 2; tissue full names and abbreviations used thereafter are described in Supplementary Table 3). We observed a large variation in the number of COX7A2L cis-eQTLs per tissue (range 1-1,100; Fig. 1b). As sample size significantly affects cis-eQTL mapping 35,36 , we tested whether the number of COX7A2L cis-eQTLs per tissue was dependent on the sample size. No significant correlation between the number of COX7A2L cis-eQTLs per tissue and the number of samples per tissue was observed (Extended Data Fig. 1c), suggesting tissue-specific regulation of COX7A2L expression. Notably, skeletal muscle harbors the highest number (1,100) and the most significant cis-eQTLs (Fig. 1b,c). Furthermore, muscle tissues (skeletal muscle and heart) are distinct from most other tissues in the effect of the cis-eQTLs alternative allele on COX7A2L expression; the majority of the cis-eQTLs downstream of the TSS are associated with opposite changes in COX7A2L expression in muscle versus most other tissues (Fig. 1c). Indeed, when looking at how the 20 tissues with the highest number of COX7A2L cis-eQTLs correlate on the basis of COX7A2L expression levels, the muscle correlates positively with the heart and negatively with most other tissues such as thyroid, testis, spleen, brain, skin, the intestinal tract (esophagus, ileum and colon) and liver (Fig. 1d,e). To assess tissue-specific regulation of COX7A2L expression, we looked at the proportion of cis-eQTLs that overlap in the top five tissues (muscle, thyroid, testis, esophagus mucosa and skin sun exposed). Skeletal muscle has double the amount of specific cis-eQTLs (18%) compared to the thyroid (9%) and testis (8%) and almost ten times more compared to the skin (2%) (Fig. 1f). A similarly higher proportion of tissue-specific cis-eQTLs was observed in the heart (Extended Data Fig. 1d), suggesting that regulation of COX7A2L expression is specific for muscle tissues. Altogether, our analysis of COX7A2L cis-eQTLs in the GTEx database revealed that several gene variants in human affect COX7A2L expression in different tissues; the skeletal muscle has a unique cis-eQTL profile and the regulation of COX7A2L expression is muscle tissue-specific.

COX7A2L lead cis-eQTL affects mRNA stability
To investigate the mechanism of genetic variation in COX7A2L expression in humans, we focused on the most significant cis-eQTL in the GTEx database (rs4181). Throughout this manuscript, we use the term 'lead' cis-eQTL to indicate the cis-eQTL that has the smallest association P value with COX7A2L expression or variants in linkage disequilibrium (LD) with the lead cis-eQTL. Notably, the lead COX7A2L cis-eQTL is located within the gene. Specifically, this variant corresponds to an insertion of 10 bp in the 3′ UTR of COX7A2L (Fig. 2a). The possible genotypes are −/− (reference allele in homozygosis; hereafter REF), −/AAATACACAC (heterozygous; hereafter HET) and AAATACACAC/ AAATACACAC (alternative allele in homozygosis; hereafter ALT) (Fig. 2a). This variant is very frequent in the population with a minor allele frequency (MAF) of 0.383 in the 1000 Genomes Project database 37 (Supplementary Table 4). Similar MAF is observed in the gnomAD database 38 (MAF = 0.393; Supplementary Table 4). The REF allele represents the minor allele in the general population with most individuals being either heterozygous or homozygous for the ALT allele (83%; Fig. 2b). To note, the frequency of this allele is not equal across populations: 80% of the individuals are homozygous for the ALT allele in the Mende population in Sierra Leone, whereas only 3% of the population has this genotype among the Peruvian in Lima 37 (Extended Data Fig. 2a and Supplementary Table 4), which suggests that one of the two alleles may be favored in certain environmental conditions. We found that (transfer electrons from NADH to molecular oxygen) 6 . In the current, most accepted model (the 'plasticity model') SCs assemble and disassemble dynamically to accommodate the cellular metabolic needs 4,5 ; however, how the assembly and disassembly of SCs are regulated is not yet fully understood and their functional relevance is still debated 7 . Evidence suggests that SCs are important for organizing flux of electrons and bolster substrate channeling [8][9][10] , to limit the leakage of electrons and reduce reactive oxygen species (ROS) production 4,11 and to facilitate the assembly and enhance the stability of the single complexes [12][13][14][15] . The assembly of SCs is compromised in various pathological conditions including cardiovascular diseases 16,17 , Barth syndrome 18,19 , neurological diseases 20,21 , diabetes 22 and aging 23 . We have previously shown that SCs are induced by exercise and involved in exercise adaptation in human 24 , suggesting a possible role in muscle physiology.
COX7A2L (also known as SCAF1) is the only known SC-specific assembly factor in mammals 10 . In particular COX7A2L is required for the stable interaction of complex III with complex IV 5 , thus it plays an important role in the stabilization of the respirasome in most tissues. Previous studies, including our own, have shown that Cox7a2l promotes SC formation as well as metabolic efficiency in zebrafish and mice 25,26 . COX7A2L expression is induced in certain stress conditions such as hypoxia, endoplasmic reticulum (ER) and nutrient stress and its induction promotes SC formation in mammalian cells [27][28][29] ; however, the function of COX7A2L in supercomplex formation and cellular metabolism is still controversial [30][31][32] .
In the present study we investigated COX7A2L function in human. We found that genetic variation significantly affects COX7A2L expression in several tissues with skeletal muscle showing the strongest signal. The most significant genetic variant, which is associated with increased COX7A2L expression specifically in muscle tissues, is a 3′ untranslated region (UTR) insertion that creates a short-repeated sequence and enhances COX7A2L expression by increasing messenger RNA (mRNA) stability. Human myotubes bearing this variant have increased amount of SCs and increased respiration. Notably, this highly frequent 3′ UTR variant is associated with lower body fat and improved cardiorespiratory fitness in humans. We validate these findings in C57BL/6J mice, which do not express Cox7a2l protein. Specific reconstitution of Cox7a2l expression in these mice leads to higher maximal oxygen consumption (VO 2 max), increased muscle mass and increased energy expenditure during the active phase. We further show that Cox7a2l and Cox7a2l-containing SCs are induced upon exercise specifically in the skeletal muscle. To summarize, we found that increased expression of the SC assembly factor, COX7A2L, enhances metabolic and cardiorespiratory fitness in mice and human.

Genetic variation affects COX7A2L expression in human muscle
To gain insights into COX7A2L function in humans we used the Gen-eBridge analysis platform (https://systems-genetics.org) 33 . GeneBridge integrates 1,337 datasets with over 265,000 human samples from different tissues and allows to interrogate a gene's function through its correlation or anticorrelation with other genes for which the Gene Ontology (GO) terms and biological pathway annotations are already known. We found that COX7A2L expression correlates positively with genes belonging to many mitochondria-related ontology terms and pathways (Fig. 1a), including several that were previously unknown, such as complex I biogenesis, cristae formation, mitochondrial translation and respiratory chain complex IV ( Fig. 1a and Supplementary Table 1). These associations were conserved across different human tissues (Extended Data Fig. 1a and Supplementary Table 1).
Given this strong association with mitochondrial function, we asked whether genetic variation affects COX7A2L expression and thus SC formation, in humans. We used the publicly available Genotype-Tissue Expression (GTEx) database v.8 release, which provides whole-genome      Table 3. c, COX7A2L cis-eQTLs by tissue plotted according to their distances from the COX7A2L TSS (x axis). Each dot represents one cis-eQTL and dot colors represent the effects of the alternative alleles on COX7A2L expression (NES, normalized effect size; blue, negative; red, positive). Only the 20 tissues with the highest number of COX7A2L cis-eQTLs are shown. d, Pearson pairwise correlations between tissues based on COX7A2L expression. Non-significant correlations (P > 0.05) are shown as blank squares. Only the 20 tissues with the highest number of COX7A2L cis-eQTLs are shown. e, Correlation of COX7A2L normalized expression in the muscle with expression in the liver (left) or heart (right), two-sided Pearson r and P value are shown above the graphs. f, COX7A2L cis-eQTLs overlap among the five tissues with the highest number of cis-eQTLs; the total numbers of cis-eQTLs per tissue are shown in parenthesis, percentages indicate the proportion of tissue-specific cis-eQTLs. Data source, GTEx database (https://www.gtexportal.org) v.8 release (b-f).
Article https://doi.org/10.1038/s42255-022-00655-0 rs4181 is a significant cis-eQTL in 19 different tissues in the GTEx database and has the most robust effect on expression in the muscle and thyroid (P = 1.1 × 10 −68 and 1.2 × 10 −50 , respectively) ( Fig. 2c,d). Consistent with our previous observation, the effect of this variant on COX7A2L expression is different according to the tissue: the association of the 10-bp insertion with COX7A2L expression is positive in the muscle, heart and cultured fibroblasts but negative in all other tissues (Fig.  2c,d). This finding again highlights the tissue-specific regulation of COX7A2L expression in humans. To test whether the rs4181 variant is sufficient to modulate expression, we cloned a portion of COX7A2L 3′ UTR with or without the 10-bp insertion in a luciferase reporter vector (Fig. 2e). An increase in luminescence was observed in HEK-293T cells transfected with the luciferase reporter harboring the 3′ UTR with the 10-bp insertion as in carriers of the ALT allele (Fig. 2f).
To dissect the molecular mechanism by which the 3′ UTR insertion increases expression in cultured cells, we first explored whether the 3′ UTR insertion is a transcriptional enhancer (whether this region increases expression by looping to the COX7A2L promoter). First, we genotyped HEK-293T cells and confirmed that this line is heterozygous for the rs4181 variant (has both REF and ALT alleles; Methods). We then took advantage of the CRISPR-Cas9 technology and co-transfected HEK-293T cells with a guide RNA targeting the COX7A2L promoter and a hemagglutinin (HA)-tagged dCas9 as described 39 (Extended Data Fig. 2b). We then performed chromatin immunoprecipitation using an HA-specific antibody and measured by qPCR the enrichment of COX7A2L promoter and the 3′ UTR region containing the rs4181 variant. We also tested another genomic region containing the cis-eQTL rs7572231 located ~20 kb upstream of rs4181. While we were able to immunoprecipitate and enrich for the COX7A2L promoter, no co-enrichment for the two alleles of rs4181 (REF and ALT) or rs7572231 genomic regions was observed (Extended Data Fig. 2c). We thus concluded that the 3′ UTR region of the COX7A2L gene does not loop to the promoter and may not act by increasing transcription itself.
The 3′ UTR regions harbor sequences that can be bound by RNA-binding proteins and modulate RNA levels by affecting transcript stability. To test this hypothesis, we obtained primary myoblasts from seven homozygous individuals (REF, n = 3; ALT, n = 4). These human myoblasts were cultured for 10 d in differentiation medium to induce myotubes differentiation, after which transcription was blocked with actinomycin D and RNA collected after 0 and 1 h. Myotubes homozygous for the REF allele had lower COX7A2L mRNA levels after actinomycin D treatment, suggesting increased mRNA degradation (Fig. 2g). To note, the 10-bp insertion creates a short-repeated sequence in the RNA 3′ UTR (Fig. 2h). Short-repeated sequences separated by 1-10 nucleotides are preferred by several RNA-binding proteins (RBPs) and might favor binding of RBPs harboring multiple RNA recognition motifs or cooperative binding of RBP dimers 40 .

Article
https://doi.org/10.1038/s42255-022-00655-0 Taken together, these results demonstrated that COX7A2L lead cis-eQTL is a 3′ UTR insertion that is sufficient to increase gene expression in vitro by increasing mRNA stability.

COX7A2L lead cis-eQTL affects cellular respiration
Next, we assessed the metabolic phenotype of human myoblasts harboring the REF and ALT allele in homozygosis. We first confirmed that differentiated myotubes homozygous for the ALT allele have higher expression of COX7A2L (Fig. 3a). To note, the difference in COX7A2L expression between genotypes becomes only apparent in myotubes, suggesting that it is specific for differentiated muscle cells (Fig. 3a). We then asked whether differences in COX7A2L expression in human myotubes translate in differences in SC formation. To address this, we extracted mitochondria from differentiated human myotubes cultured in glucose-free medium (10 mM galactose) for 48 h and performed blue native (BN)-PAGE. As expected, human myotubes homozygous for the ALT allele had increased SCs (Fig. 3b). We then tested whether increased COX7A2L expression and increased supercomplex formation affects cellular respiration. For this, we measured respiration by Seahorse flux analyzer (Agilent) in differentiated myotubes cultured in glucose-free medium for 48 h. Myotubes homozygous for the ALT allele had higher basal, ATP-linked and maximal respiration (Fig. 3c,d). No significant difference in cellular respiration was observed when cells were cultured in high-glucose medium (25 mM glucose) (Extended Data Fig. 3a,b). Consistent with these results, myotubes homozygous for the ALT allele had higher COX7A2L protein expression, compared to myotubes homozygous for the REF allele, when cultured in glucose-free medium ( Fig. 3f), whereas no differences were observed when cells were cultured in high glucose (Extended Data Fig. 3c). Cells in glucose-free medium are forced to rely on mitochondrial respiration to produce ATP and this has been linked to increased COX7A2L expression and increased MRC rearrangement into SCs 27 . To rule out increased mitochondrial biogenesis driving the observed differences in the ALT allele myotubes, we measured other MRC subunits as well as mitochondrial biogenesis markers both at the protein and mRNA levels in the two genotypes (Fig. 3e,f and Extended Data Fig. 3d) and found no significant differences. Additionally, we measured mitochondrial DNA copy number as a proxy of mitochondrial content in the REF and ALT allele myotubes and found no significant differences (Extended Data Fig. 3e).
These data show that differentiated myotubes with higher COX7A2L expression have higher SC formation and are able to adapt better to nutrient deprivation by increasing mitochondrial respiration.

COX7A2L lead cis-eQTL affects cardiorespiratory fitness
We next tested whether COX7A2L 3′ UTR insertion is associated with metabolic and cardiorespiratory fitness-related phenotypes in human cohorts. We first looked into the IEU GWAS database (https://gwas. mrcieu.ac.uk/datasets/) 41 , which contains summary statistics from, Article https://doi.org/10.1038/s42255-022-00655-0 among others, the UK Biobank (UKBB), which is a biobank that contains genotype data, as well as a wide variety of phenotypic data for about 500,000 individuals 42 . We performed two-sample Mendelian randomization, which is a method that allows testing for causal effects of gene expression on phenotypes through a genetic variant 43,44 . The lead cis-eQTL for COX7A2L in GTEx, rs4181 (previously annotated as rs56873751), is a small insertion that is not present in most GWAS studies. For this reason, we investigated the second-most significant cis-eQTL, rs10183278, a single-nucleotide polymorphism (SNP) that is in high LD with rs4181 (r 2 = 0.952; Fig. 4a). rs10183278 was significantly associated with several metabolic phenotypes (FDR < 0.05, P < 0.02; Supplementary Table 5 and Fig. 4b).
Notably, an increase in COX7A2L expression was associated with reduced body weight, reduced body fat and body mass index (BMI) and increased usual walking pace (Fig. 4b). The UKBB has only a limited number of phenotypes that allow assessing cardiorespiratory fitness and muscle function. Examples include walking pace 45 , an indicator of overall physical fitness and grip strength, which is more specific for upper body strength. To validate the role of COX7A2L in overall cardiorespiratory fitness, we hence analyzed participants from the Trøndelag Health (HUNT) study. The HUNT study is a large population-based cohort study that includes questionnaire data, clinical measurements and biological samples from more than 120,000 individuals 46 . In a sub-study of HUNT (HUNT3 fitness study 47,48 ), maximal oxygen uptake (VO 2 max) was measured in 4,631 individuals. VO 2 max measurement is the gold standard for assessing cardiorespiratory fitness 49 . Again, as the most significant skeletal muscle cis-eQTL for COX7A2L in GTEx, rs4181, is not present in the HUNT cohort, we investigated the second-most significant cis-eQTL, rs10183278 and showed it to be significantly associated with VO 2 max (Fig. 4c). To further validate these findings, we also analyzed participants from an additional independent cohort, the Helsinki Birth Cohort Study (HBCS). In this cohort several tests were performed to assess physical fitness in 1,078 individuals aged 67-77 years 50 , of which 606 took part in the Urho Kaleva Kekkonen (UKK) 2-km walk test. This test evaluates cardiorespiratory fitness without the requirements for maximal physical efforts 51,52 and is a valid substitute for maximal physical efforts tests (VO 2 max) in elderly individuals and patients with obesity 53,54 . Mendelian randomization analysis of rs10183278 indicated that increased COX7A2L expression in the muscle was associated with several HBCS phenotypes (FDR < 0.2; Supplementary  Fig. 4e). To summarize, using three independent human cohorts, we showed that COX7A2L lead cis-eQTL is associated with metabolic and cardiorespiratory fitness-related phenotypes.

A Cox7a2l mutation affects cardiorespiratory fitness in mice
Human genetic analyses have the limitation that several confounding factors, including environmental factors or other genetic variants co-inherited with the variant of interest, may affect the observed association of an SNP with an outcome. Environmental factors are difficult to control and correct for in human but can be tightly controlled in mice. To determine whether specific changes in Cox7a2l expression are associated with metabolic and cardiorespiratory fitness-related phenotypes, we took advantage of the presence of a known Cox7a2l genetic polymorphism in common mouse inbred strains. C57BL/6J mice have a six-nucleotide deletion in the Cox7a2l gene that truncates the protein by two amino acids and renders it unstable 10 Table 5). c, Association between the second-most significant COX7A2L cis-eQTL (rs10183278) with VO 2 max measured in the HUNT3 study. d, Mendelian randomization analysis between the second-most significant COX7A2L cis-eQTL (rs10183278) and the 2-km walk test (UKK) in the HBCS (Supplementary Table 5). e, Fitness index of the HBCS participants. Individuals are grouped by genotype. The number of participants per genotype is indicated. Data are represented as mean ± s.e. (b-d) and violin plots and Tukey's box and whiskers plots (superimposed) (e). Box plots center represents the median, lower and upper hinges represent the first and third quartile (25th and 75th percentiles). The whiskers extend to the largest and smallest value up to 1.5 × IQR from the hinge. Data points beyond the whiskers are plotted individually. Statistical analysis used to determine the P value is described in Methods (b-d).
Article https://doi.org/10.1038/s42255-022-00655-0 derived from crosses between C57BL/6J and DBA/2J mice 26,55-57 (www. genenetwork.org). This population segregates for the Cox7a2l polymorphism. Specifically, the BXD strains bearing the C57BL/6J allele for Cox7a2l (BB allele) have higher basal levels of Cox7a2l mRNA, compared to the strains bearing the DBA/2J allele (DD allele), likely due to a compensatory mechanism (Extended Data Fig. 4b, left), but as expected have lower levels of Cox7a2l protein 5 (Extended Data Fig. 4b, middle). In agreement with the original observation in C57BL/6J mice 10 , we previously showed that BXD strains carrying the Cox7a2l BB allele lack some of the SCs and that this trait maps to a QTL containing the Cox7a2l gene 26 . Here we observed that, on a high-fat diet, strains with the DD allele (long Cox7a2l form) have higher VO 2 max after training (Extended Data Fig. 4b, right). No difference in VO 2 max between mice carrying the BB and DD allele was observed in the BXD population at baseline or on chow diet.
C57BL/6J mice are also homozygous for a loss-of-function mutation in the nicotinamide nucleotide transhydrogenase (Nnt) gene 58 that renders them more susceptible to metabolic dysfunctions 59 . To rule out an effect of the Nnt mutation on the observed differences in VO 2 max between the BXD strains with the long and short Cox7a2l allele, we checked how the Nnt allele distributes between the two groups. We found an about equal distribution of DD (wt) and BB (null) Nnt allele among the two Cox7a2l genotypes (Extended Data Fig. 4c). Specifically, among the strains with the Cox7a2l DD allele, 45% have the Nnt BB allele and 50% have the Nnt DD allele. While among the strains with the Cox7a2l BB allele, 36% have the Nnt BB allele and 57% have the Nnt DD allele. Thus, the observed difference in VO 2 max is not due to differences in the Nnt gene.
To address the specific contribution of Cox7a2l to phenotypic differences, we used congenic C57BL/6J mice expressing the long (functional) form of Cox7a2l (thereafter referred to as C57BL/6J Cox7a2l ) 5 (Fig. 5a). Consistent with our previous findings in the BXD strains 26 (Extended Data Fig. 4b), as well as with the phenotypic associations found with COX7A2L cis-eQTLs in the human genetic analysis of the UKBB, HBCS and HUNT datasets (Fig. 4b-e), C57BL/6J Cox7a2l mice have higher VO 2 max (with a tendency for an increased run distance), lower body weight, more muscle mass and less fat deposits compared to C57BL/6J mice (Fig. 5b-e). To address Cox7a2l-driven metabolic differences, we housed C57BL/6J and C57BL/6J Cox7a2l mice in Promethion metabolic cages with or without access to a running wheel (Methods). We did not observe changes in total activity and wheel activity between the two genotypes (Extended Data Fig. 5a,b). Notably, C57BL/6J Cox7a2l mice had increased energy expenditure specifically during the active dark phase and this difference was more pronounced when mice had access to a running wheel ( Fig. 5f and Extended Data Fig. 5c,d). While the body temperature of C57BL/6J Cox7a2l was lower than C57BL/6J at rest, after exercise C57BL/6J Cox7a2l had a higher body temperature increase (+2.6 °C versus +1 °C, respectively; Fig. 5g). This temperature shift was significantly higher also when expressing it as percentage of the initial body temperature (+7.1% versus +2.7%; Fig. 5h). These results suggest that mice with the long Cox7a2l allele can increase energy expenditure more when energy demands rise, compared to mice with the short Cox7a2l allele. Consistent with the observed increase in energy expenditure during the active phase, C57BL/6J Cox7a2l mice had higher total and relative food intake (Fig. 5i), despite having lower body weight (Fig. 5c). Therefore, all together, these results confirm a specific role of COX7A2L/Cox7a2l in cardiorespiratory fitness in both human and mice.

Cox7a2l-containing SCs are induced upon exercise
Given the specific role of COX7A2L in cardiorespiratory fitness and energy expenditure during physical activity and as SCs are induced by exercise in humans 24 , we addressed the role of Cox7a2l in exercise. C57BL/6J and C57BL/6J Cox7a2l mice were given free access to a running wheel for 5 weeks and then killed. Both Cox7a2l total protein and Cox7a2l-containing SCs were increased in the skeletal muscle of C57BL/6J Cox7a2l mice after 5 weeks of training, but were absent in C57BL/6J mice (Fig. 6a-f). Notably, only C57BL/6J Cox7a2l mice showed an induction of the higher respirasome bands and III 2 + IV SC upon exercise (Fig. 6c-e). This increase was likely muscle tissue-specific as no changes were observed in Cox7a2l protein expression and the levels Article https://doi.org/10.1038/s42255-022-00655-0 of Cox7a2l-containing SCs upon exercise in the liver (Extended Data Fig. 6a,b). The increase in Cox7a2l expression upon exercise was not dependent on the genetic background, as it was also observed in DBA/2J mice and was proportional to the duration of the training (Extended Data Fig. 6c). Given the observed association of Cox7a2l with other mitochondrial ontologies (Fig. 1a) we assessed possible differences in mitochondrial gene expression and content in the two genotypes. We measured levels of individual mitochondrial complexes by western blot and measured PGC1a and TFAM as markers of mitochondrial biogenesis both at mRNA and protein levels (Fig. 6a,b and Extended Data Fig. 6e).
We found an overall increase in the amount of mitochondrial complexes as well as PGC1a and TFAM protein levels upon endurance exercise, this was expected as endurance exercise is known to increase mitochondrial biogenesis (Fig. 6a,b). The increase in the mitochondrial complexes, PGC1a and TFAM was equal in both genotypes. Thus, upon exercise, the observed increase in the Cox7a2l-containing respirasome bands in the mice expressing the long isoform of Cox7a2l is not simply due to an overall increase in the single complexes but depends on Cox7a2l. Furthermore, C57BL/6J Cox7a2l mice, but not C57BL/6J mice, had decreased mitochondrial DNA (mtDNA)/nuclear DNA (nDNA) ratio in the muscle (but not the liver) after exercise ( Fig. 6g and Extended Data Fig. 6d). This phenomenon was also observed in humans 60,61 and might suggest increased mitochondrial turnover and remodeling with long-term training (Discussion). Altogether, these data suggest a Cox7a2l-dependent increase in respirasome formation upon exercise.

Discussion
The assembly of MRCs in SCs is widely accepted; however, their functional importance has so far remained elusive. While several studies have found that SC assembly may be beneficial to enhance electron transport efficiency, channel substrates, reduce ROS production and stabilize single MRCs 4,8,9,[11][12][13]15,62 , other studies have challenged these observations 63,64 . COX7A2L is required for the interaction of complex III with complex IV and is important for the formation of the respirasome 5,26 ; however, these findings have also been contested 31,32,65-68 . Most structural models of the mammalian respirasome come from mitochondria isolated from heart at rest; however, it is now clear that SC assembly and the role of assembly factors may be dependent on the tissue type and metabolic status of the cell. Although only a few studies have investigated the physiological function of COX7A2L, a recent study suggested that it may promote metabolic efficiency in zebrafish and mice 25 . Nonetheless, the possible function COX7A2L in cellular bioenergetics is still controversial 30 and its role in human is unknown.
To shed light on this conundrum, we sought to determine the function of COX7A2L in human. We found that genetic variation strongly affects COX7A2L expression. We identified the highest number and most significant cis-eQTLs in the skeletal muscle. Although several other tissues also had a high number of significant cis-eQTLs with high overlap between tissues, the effect of the alternative allele was opposite in muscle tissues compared to most other tissues. This suggests a muscle-specific mechanism mediating the effect of the genetic variant on gene expression. COX7A2L lead cis-eQTL is a 3′ UTR insertion that is sufficient by itself to increase expression in cultured cells. We identify a possible mechanism by which this 3′ UTR variant affects expression. The insertion creates a short-repeated sequence that increases mRNA stability, possibly through increased binding of an mRNA stabilizing factor or through disruption of a microRNA binding site. Further investigations are needed to identify the detailed molecular mechanism and to determine why this variant might have a negative effect on gene expression in most other tissues such as the liver and thyroid. In our analysis we found other independent variants that affect COX7A2L expression, thus we cannot exclude that other independent causal variants exist and act through different mechanisms.
Increased COX7A2L expression in human myoblasts leads to increased amounts of SCs and increased cellular respiration when cells are cultured in galactose medium; this is in line with previous reports showing that SCs may play a role in certain metabolic stress conditions and that COX7A2L may be important when cells are forced to use oxidative phosphorylation to produce ATP 27 . Physical exercise causes metabolic stress in muscle tissues. The increased energy demand from muscle contraction leads to a surge in cellular AMP levels and to a decline in oxygen levels that triggers mitochondrial adaptations to decreased energy levels and hypoxia. We show that Cox7a2l is induced in skeletal muscle upon exercise in mice and that humans with higher expression of COX7A2L have improved metabolic and cardiorespiratory phenotypes. While some studies have demonstrated a rearrangement of mitochondrial RCs into supercomplexes upon exercise training 24,69 , one recent study reported that increased mitochondrial biogenesis, rather than rearrangement of mitochondrial complexes into supercomplexes is responsible for improved muscle bioenergetics upon exercise 70 . Here, while we observe an increase in mitochondrial biogenesis and in the amount of OxPhos complexes upon exercise in mice, we demonstrate that the absence of Cox7a2l protein and the lack of respirasome formation affect exercise performance and energy expenditure. Of note, mice expressing Cox7a2l have a decreased mtDNA/nDNA ratio after exercise. mtDNA synthesis is an energy-consuming process that may be less favored in situations of reduced cellular ATP, such as during or after physical effort. Furthermore, exercise leads to increased ROS production and oxidative stress in the muscle that may damage mtDNA 71,72 . Thus, it is plausible that improved mitochondrial efficiency, associated with mitochondrial turnover and remodeling, rather than overall increase in mtDNA synthesis, may be favored in certain conditions of metabolic stress. Such a decrease in mtDNA/nDNA ratio in skeletal muscle upon exercise was also observed in humans 60,61 , suggesting a conserved adaptive response. Our findings suggest that individuals having genetic variants associated with increased COX7A2L expression in the muscle may be able to better adapt to situations of increased metabolic stress and physical effort demand, such as during physical activity. Notably, we found that the frequency of COX7A2L lead eQTL is remarkably different in certain populations (Extended Data Fig. 2a), suggesting a possible adaptive role of SCs in different environmental conditions; however, further research is needed to support this hypothesis. Previous studies have identified several human genetic polymorphisms in nuclear and mtDNA affecting mitochondrial gene expression 73 , metabolism 74 and exercise performance 75,76 as well as exercise-induced changes in mitochondrial gene expression 77 . Co-inheritance of these genetic polymorphisms with the COX7A2L variants may be used in the future for metabolic risk prediction.
The findings in our study not only shed light on the genetic underpinnings of how variants in COX7A2L affect mitochondrial supercomplex formation in human muscle, but also demonstrate the importance of COX7A2L and SCs for cardiometabolic fitness and human health. Cardiorespiratory fitness is an important predictor of cardiovascular and metabolic health 78,79 . Improving cardiorespiratory fitness and exercise capacity is a highly promising approach to reduce all-cause and cardiovascular mortality 79 .

Helsinki Birth Cohort
The HBCS includes 13,345 individuals born in Helsinki between 1934 and 1944. The clinical study includes 2003 participants and the protocol was approved by the Ethics Committee of Epidemiology and Public Health of the Hospital District of Helsinki and Uusimaa. Written informed consent was obtained from each participant before any study procedure was initiated. The UKK 2-km walk test has been validated against maximal effort tests by treadmill or bicycle ergometry in multiple populations, including individuals with obesity 53 and elderly individuals 54 . During the test, participants are instructed to walk a 2-km course on flat ground as fast as possible. The test results are Article https://doi.org/10.1038/s42255-022-00655-0 expressed as a fitness index, which takes into account the individual's age, sex, BMI, time spent in walking and heart rate. The senior fitness test, which describes the physical fitness of the study participants was performed in 695 individuals and was described previously 80 . Isometric grip strength of the dominating hand was tested by a Newtest Grip Force dynamometer (Newtest Oy). The maximum value of three squeezes was used in analyses of linear regression. Weight and height were measured at the time of the grip strength measurements and BMI was calculated as weight (kg) divided by height (m) squared.
DNA was isolated from blood and genotyping was carried out with the modified Illumina 610k chip by the Wellcome Trust Sanger Institute. Genotype imputation was performed using the 1000 Genomes Project Phase I integrated variant set (v.3/April 2012; NCBI build 37/hg19) and IMPUTE2 software. Before genotype imputation some quality control filters were applied: SNP clustering probability for each genotype >95%; call rate >95% individuals and markers (99% for markers with MAF < 5%); MAF > 1%; and Hardy-Weinberg equilibrium P > 1 × 10 −6 . Moreover, heterozygosity, sex checks and relatedness checks were performed and any discrepancies were removed. Phenotype association analyses in HBCS were performed in individuals who had available genotype and senior fitness test data, approximately 400 individuals for the analysis. The scores of the fitness tests were classified based on fifth percentile range, with a score of 1 being the worst performance (score below fifth percentile); 2 the score from the fifth to ninth percentile; and 20 the best performance (in or above the 95th percentile) as described 50 . We performed linear regressions with SNPtest 81 assuming an additive genetic model. We adjusted all models for age, sex, highest education achieved (basic or less/upper secondary/lower tertiary/upper tertiary) and smoking (yes/no).

The Trøndelag Health study
The HUNT study and HUNT3 sub-study (HUNT3 fitness study 47,48 ) were described previously 48 . The HUNT3 fitness study was approved by the Regional Committee for Medical Research Ethics (2012/1672/REK nord), the Norwegian Data Inspectorate and the National Directorate of Health and is in compliance with the Helsinki Declaration. Written informed consent was obtained from all participants. The 4,631 healthy adult participants performed a measure of VO 2 max. Exclusion criteria for the HUNT3 fitness study were described previously 48 . VO 2 max measurement for this study was described previously 48 . The 4,463 participants who reached VO 2 max were included in the analyses.
DNA was extracted from blood samples, as described previously 82 . Genotyping of the HUNT study was described previously 83 . In brief, genotyping was performed with one of three different Illumina Human-CoreExome arrays (HumanCoreExome12 v.1.0, HumanCoreExome12 v.1.1 and UM HUNT Biobank v.1.0) according to standard protocols. The genotypes from different arrays had quality control performed separately and were reduced to a common set of variants across all arrays. Samples that failed to reach a 99% call rate, had contamination >2.5% as estimated by BAF Regress, large chromosomal copy number variants, lower call rate of a technical duplicate pair and twins, gonosomal constellations other than XX and XY or whose inferred sex contradicted the reported sex were excluded. The genomic position, strand orientation and reference allele of genotyped variants were established by aligning the probe sequences against the human genome using the BLAT tool 84 . With PLINK v.1.90 (ref. 85 ), variants were excluded if probe sequences could not be perfectly mapped and cluster separation was <0.3, Gentrain score <0.15, showed divergence from Hardy-Weinberg equilibrium in unrelated samples of European ancestry (P < 0.0001), had call rates <99% or another array with higher call rate genotyped the same variant. Ancestry of samples was derived from projecting genotyped samples into the principal components of the Human Genome Diversity Project reference panel. Different arrays were matched by narrowing to a set of overlapping variants and eliminating variants with frequency differences >15% between datasets, or that were monomorphic in one and had MAF > 1% in another dataset. These genotype data were phased using Eagle2 (v.2.3.8) 86 . Imputation was performed on samples of recent European ancestry using Minimac3 (v.2.0.1; http://genome.sph.umich. edu/wiki/Minimac3) 87 and a merged reference panel built on the Haplotype Reference Consortium panel 88 (release v.1.1) together with a local reference panel, which included 2,202 whole-genome sequenced HUNT study participants. Relatively unrelated individuals (kinship coefficient <0.0884) were chosen using KING 89 and FastIndep 90 tools. BOLT-LMM algorithm 91 was employed to execute statistical analyses while variants with MAF < 0.0011 92 were excluded from the analysis. This algorithm computes statistics for testing association between phenotype and genotypes using a linear mixed model. The analysis was adjusted for sex and age. Genetic principal components were included as covariates in the analysis to control for residual population structure. The study was in conformity with Norwegian laws and the Helsinki Declaration and signed informed consent was obtained from all participants.

Mouse lines
C57BL/6J mice expressing the long functional Cox7a2l allele were generated as described 5 . The C57BL/6J OlaHsd used to generate the C57BL/6J Cox7a2l mice carried the wild-type allele of the Nnt gene. For genotyping, DNA was extracted from tails and PCR amplified using the KAPA2G mix (KK5103, Kapa Biosystems) following manufacturer's instructions (Supplementary Table 6 lists the primer sequences). PCR products were run on a QIAxcel instrument to identify different molecular weight bands indicating presence or absence of the six-nucleotide deletion (50 or 56 bp). For all experiments, littermates of the same sex were randomly assigned to experimental groups. All experiments were performed on male mice at 10-14 weeks of age. All mice were fed a chow diet (SAFE 150) and were kept in standard housing conditions (12-h light-12-h dark cycle and temperature of 22 °C). All animals had ad libitum access to food and water. All animal experiments were performed according to Swiss ethical guidelines and were approved by the Service de la Consommation et des Affaires Vétérinaires of the Canton de Vaud (license VD3419)

DNA extraction and genotyping of cell lines
DNA extraction was performed using NucleoSpin Tissue mini kit (Macherey-Nagel, 740952). For genotyping, primers flanking the rs4181 10-bp insertion site were used for PCR amplification (primer sequences are listed in Supplementary Table 6). PCR was performed using the KAPA2G mix (KK5103, Kapa Biosystems) following manufacturer's instructions. PCR products were run on a QIAxcel instrument to identify different molecular weight bands indicating presence or absence of the insertion (425 or 435 bp). PCR products were further purified using the PCR clean-up gel extraction kit (Macherey-Nagel, 740609) and sequenced by Sanger sequencing (Microsynth) to confirm electrophoresis results. Only homozygous myoblast lines were used for experiments. Article https://doi.org/10.1038/s42255-022-00655-0

GeneBridge analysis
Ontology terms and pathways correlated to COX7A2L in humans were identified using the publicly available GeneBridge tool (https:// systems-genetics.org). First, an analysis was performed with all available tissues in the database. GO terms and pathways with GMAS ± 0.268 were considered significantly associated with COX7A2L; the threshold of significance was determined as previously described 33 (Fig. 1a). Then, 13 main tissues were selected and the same analysis was repeated for each tissue separately. GO terms and pathways with score ± 0.268 in at least one of the 13 tissues are represented in Extended Data Fig. 1a.

Genotype-Tissue Expression analyses
For the cis-eQTL analysis we used publicly available data from the GTEx Consortium 34 (https://www.gtexportal.org) v.8 release. Only cis-eQTLs (eQTLs within 1 Mb from the COX7A2L gene) are included in the analysis. For the correlation analyses in GTEx, we obtained the publicly available gene transcripts per million and the covariates used in the cis-eQTL analysis (dbGAP study accession no. phs000424.v8). To avoid correlations due to known or unknown factors and batch effects, we used the removeBatch-Effect effect function from the limma R package v.3.38.3 (ref. 93 ) along with covariates as calculated and released by the GTEx consortium.

Mendelian randomization
For the Mendelian randomization analysis, we made use of the cis-eQTL data from GTEx 34 , which are freely available from https://storage.googleapis.com/gtex_analysis_v8/single_tissue_qtl_data/GTEx_Analysis_v8_ eQTL.tar. rs4181 was not genotyped in the UKBB and the HBCS, therefore, we selected the second-most-significant muscle cis-eQTL, rs10183278, as the instrumental variable for our analysis. This cis-eQTL is in high LD with rs4181 (r 2 = 0.952). As with rs4181, rs10183278 is also significantly linked with EML4 expression, a gene whose 3′ terminus lies only 997 bp upstream of COX7A2L, but this association is much weaker; the effect size on COX7A2L expression in muscle is 0.44 (P = 3.7 × 10 −68 ) versus an effect size of −0.12 for EML4 (P = 2.6 × 10 −5 ). We also verified rs10183278 in PhenoScanner v.2 (http://www.phenoscanner.medschl.cam.ac.uk/) 94 and found no obvious confounders. The results of this analysis are in Supplementary Table 7. Muscle gene expression of COX7A2L was used as the exposure variable. We used the available_outcomes function from the TwoSampleMR v.0.5.3 R package to obtain 188 body weight-and health-related outcome summary statistics from the IEU GWAS database (https://gwas.mrcieu.ac.uk/datasets/) 41 (Supplementary Table 5). For the HBCS, we used the summary statistics of the additive genetic model (see section on Helsinki Birth Cohort). Statistical inference was performed through Wald ratio tests as implemented in the harmonise_data function of the TwoSampleMR package and P values were converted to q values with the Benjamini-Hochberg FDR procedure.

Body composition
Whole-body composition was measured by NMR using a Minispec instrument (Bruker).

Metabolic cages
Energy expenditure, VO 2 , VCO 2 , food intake, cage and wheel activity were measured using the Promethion system (Sable Systems International). Mice were housed in metabolic cages for 4 d and had free access to a running wheel. To measure energy expenditure and cage activity in standard housing conditions, the wheel was kept blocked for the first 2 d of recording.

VO 2 max test in mice
VO 2 max in mice was measured with a calorimetric treadmill (Columbus Instruments) with an incremental speed protocol. To avoid anxiety created by finding themselves in a new environment, mice were habituated to the device for 10 min before starting the test. The run distance, VO 2 and VCO 2 were measured. The experiment was stopped when mice were exhausted or VO 2 max was reached (when VO 2 levels failed to increase despite increasing running velocity or when respiratory exchange ratio was =1.0).

Long-term training
For long-term training experiments mice were housed singularly and had access to a running wheel for 5 weeks. After a first estimation of the average distance run per day by the first mouse cohort, the maximum allowed distance was set on this average (1,800 m per day) after which the wheel was blocked for all mice to avoid large variability between mice and between experiments. The meters run on the wheel was monitored for each mouse and for all the duration of the experiment. There were no significant differences in the total distance run between the two genotypes. Mice in control groups were also housed singularly and had access to a wheel; however, the wheel was blocked for the entire duration of the experiment.

Treadmill exercise and body temperature measurement
Body temperature was measured using a rectal probe before and after 30 min of treadmill run at incremental speed (10 min at 20 cm s −1 , 10 min at 23 cm s −1 and 10 min at 26 cm s −1 ). The mice were acclimatized to the treadmill for 3 d before the experiment. All animals satisfactorily reached the end of the test.

RNA extraction and qRT-PCR
RNA was extracted using TriPure Isolation Reagent (Roche, 11667165001). For the RNA extraction from myoblasts and differentiated myotubes, two wells of a six-well plate were pooled for each sample and 1 μl of glycogen (R0551, Thermo Fisher) was added during the extraction for maximum recovery of the RNA and visualization of the pellet. For reverse transcription, PrimeScript RT Reagent kit with gDNA Eraser (Takara, RR047B) was used following manufacturer's instructions. For qPCR TB Green Premix Ex Taq (Takara, RR420W) was used and plates were run on a LightCycler 480 instrument (Roche). Primer sequences are listed in Supplementary Table 6. All qRT-PCR fold changes were calculated using Gapdh/GAPDH as housekeeping gene. The average of three technical replicates was used for each data point.

MtDNA/nucDNA ratio
MtDNA abundance was measured with the ΔΔCt method as previously described 97 . Briefly, ~15 mg of frozen liver or muscle tissues were used to Article https://doi.org/10.1038/s42255-022-00655-0 extract total cellular DNA using NucleoSpin Tissue kit (Macherey-Nagel, 740952). For qPCR, 20 ng of DNA was used. qPCR was carried out as described above. For mtDNA amplification, primers against the 16S rRNA were used (Supplementary Table 6). For nuclear DNA amplification primers against the β2M gene were used (Supplementary Table 6). For mtDNA copy number estimation in human myoblasts the Human Mitochondrial DNA Monitoring Primer Set (Takara, 7246) was used.

Luciferase reporter assay
A 100-bp fragment of the human COX7A2L 3′ UTR surrounding the rs4181 insertion variant with (3′ UTR-ALT) or without (3′ UTR-REF) the 10-bp insertion was cloned in a pUC57 vector by Genscript (for the insert sequence see Supplementary Table 6) flanked by XbaI and FseI restriction sites at the 5′ and 3′ respectively. The 100-bp fragment was excised and cloned in the pGL3-promoter vector (Promega) downstream of the luciferase gene using XbaI and FseI restriction enzymes (NEB). The obtained plasmids were sequenced to check the correct directionality of the inserted sequence (Microsynth). For the luciferase assay, HEK-293T cells were transfected in 96-well plates with pGL3-promoter-3′ UTR-REF, pGL3-promoter-3′ UTR-ALT or pGL3-promoter-empty vectors (190 ng per well) together with a pRL-CMV vector (10 ng per well) to control for transfection efficiency. Transfection was performed using JetPEI transfection reagent (Polyplus) with the reverse transfection method following manufacturer's instructions. Briefly, cells were trypsinized, the JetPEI-DNA mix was added to the cell suspension and ~30,000 cells were then plated per well. A JetPEI-DNA ratio of 2:1 was used. At 24 h after transfection both luciferase and Renilla luminescence was measured using the Dual-Glo Luciferase Assay System (Promega, E2920). A minimum of eight wells per conditions were measured.

COX7A2L promoter tagging with dCAS9-HA
COX7A2L promoter tagging was performed as described earlier 98 with some modifications. Guide RNAs for COX7A2L promoter were designed using the online GPP web portal tool (https://portals.broadinstitute. org/gpp/public/analysis-tools/sgrna-design-crisprai?mechanism= CRISPRa) using Streptococcus pyogenes PAM sequence (NGG). Two gRNAs having the best predicted on-and off-target scores were selected (gRNA1and gRNA2; see also Supplementary Table 6). Both gRNAs were cloned into phU6 plasmids using BbsI restriction enzyme (Genewiz). Insertion of the gRNA was verified by sequencing (Microsynth). HEK-293T cells cultured in 15-cm dishes were co-transfected with 75 μg of dCas9-HA (Addgene, 61355) and 27 μg of the empty phU6 plasmid (EV) or its subclone expressing the gRNA sequence. Transfection was performed using Lipofectamine 3000 (Invitrogen, L3000001). At 24 h after transfection, Opti-MEM medium (Gibco, 31985070) was replaced with normal culture medium and 24 h later cells were collected for chromatin immunoprecipitation. gRNA1 gave the highest enrichment for COX7A2L promoter and was therefore selected for the experiment in Extended Data Fig. 2c.

Chromatin immunoprecipitation followed by qPCR
Chromatin immunoprecipitation was performed as described 39   Beads were then washed three times with 150 mM wash buffer (1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, pH 8.0 and 20 mM Tris-HCl, pH 8.0) and twice with 500 mM wash buffer (1% Triton X-100, 0.1% SDS, 500 mM NaCl, 2 mM EDTA, pH 8.0 and 20 mM Tris-HCl, pH 8.0). After the washes, the beads were resuspended in 120 μl of elution buffer (1% SDS and 0.1 M NaHCO 3 ) and incubated for 1 h at 65 °C with shaking. The supernatant was collected in new tubes and both supernatant and input were incubated overnight at 65 °C to reverse crosslinking. Immunoprecipitated DNA was purified using MinElute PCR Purification kit (QIAGEN, 28004) and assayed by qPCR. For qPCR, LightCycler 480 SYBR Green I Master (Roche, 04887352001) and primers were added to 3 μl of the purified DNA. Primer sequences are listed in Supplementary Table 6. Fold enrichment over IgG was calculated based on the percent input recovered in anti-hemagglutinin and IgG samples.

RNA stability assay
The RNA stability assay in differentiated myotubes was performed as described previously 99 . Briefly, myoblasts were grown in differentiation medium for 10 d, at day 10 of differentiation, actinomycin D (Sigma, A1410) was added to the medium at a concentration of 10 μg ml −1 and cells were collected in TriPure Isolation Reagent (Roche, 11667165001) after 0 and 1 h of treatment. RNA extraction and qRT-PCR were performed as described above. RNA abundance at each time point was calculated relatively to time 0.

Quantification and statistical analysis
No statistical methods were used to predetermine sample size. The exact value of n, the statistical methods used to determine significance and error bars are described in the figure legends. All replicates represent biological replicates. A two-tailed Student's t-test was used to calculate statistical differences between the means of two groups. One-way or two-way ANOVA tests were used to determine statistical differences between multiple groups. Statistical tests were performed using GraphPad Prism 7 or R. In the Promethion experiment, mice that did not spontaneously run on the running wheel were excluded from the analysis (n = 1-2 per group).

Materials availability
Plasmids generated in this study will be made available upon request to the corresponding author.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
The data generated in this study are available with the published manuscript and/or were deposited at Mendeley Data (https://doi. org/10.17632/8fcntj63x6.1) and will be publicly available upon publication. The GTEx gene expression and eQTL data are publicly available at http://www.gtexportal.org/home/. GO terms and pathways correlated to COX7A2L can be found through the publicly available GeneBridge tool (https://systems-genetics.org). Genotype frequencies data from the 1000 Genomes Project Phase 3 and gnomAD are publicly available at https://www.ensembl.org/. The IEU GWAS database is publicly available at https://gwas.mrcieu.ac.uk/datasets/. Source data are provided with this paper. , VO 2 (c) and VCO 2 (d) measured using the Promethion metabolic cages in standard housing condition or with free access to a running wheel (yellow bar). a-d. Left panels: hour averages, right panels: day and night averages (C57BL/6 J n = 15, C57BL/6J Cox7a2l n = 15). In a-d (left panels) data are represented as mean ± SEM. In a-d (right panels) data are represented as boxes and whiskers. Box plots center represents the median, lower and upper hinges represent the first and third quartile (25 th and 75 th percentiles). The whiskers extend to the largest and smallest value. Statistical analysis for a-d: three-way ANOVA and Sidak's multiple comparisons test.

Nature Metabolism
Article https://doi.org/10.1038/s42255-022-00655-0 Extended Data Fig. 6 | Cox7a2l is induced upon exercise specifically in the skeletal muscle. a. Western blot analysis of Cox7a2l protein expression at rest or after 5 weeks of wheel running exercise in liver. b. BN-PAGE immunoblot with total OxPhos immunostaining (left) and Cox7a2l immunostaining (right) in isolated mitochondria from liver at rest or after 5 weeks of wheel running exercise. c. Cox7a2l relative mRNA expression in the quadriceps of DBA/2 J mice in basal conditions (sedentary) or after 2 and 4 weeks of exercise training (n = 5). d. Mitochondrial to nuclear DNA ratio measured in liver at rest or after 5 weeks of wheel running exercise. e. mRNA expression of the indicated genes measured by qRT-PCR in gastrocnemius muscle at rest or after 5 weeks of wheel running exercise. In c and e data are represented as mean ± SEM. In d data are represented as box and whiskers. Box plots center represents the median, lower and upper hinges represent the first and third quartile (25 th and 75 th percentiles). The whiskers extend to the largest and smallest value. Statistical analysis for c: one-way ANOVA and Dunnett's multiple comparisons test, statistical analysis for d and e: two-way ANOVA and Sidak's multiple comparison test.

March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. The data generated in this study are available with the published manuscript and/or were deposited at Mendeley Data (doi: 10.17632/8fcntj63x6.1) and will be publicly available upon publication. The GTEx gene expression and eQTL data are publicly available at http://www.gtexportal.org/home/. Ontology terms and pathways correlated to COX7A2L can be found through the publicly available GeneBridge tool (https://systems-genetics.org). Genotype frequencies data from the 1000 genomes project phase 3 and gnomAD are publicly available at https://www.ensembl.org/. The ieu gwas database is publicly available at https:// gwas.mrcieu.ac.uk/datasets/.
No statistical method was used to determine sample size. Sample sizes were chosen based on studies with similar experimental design and on the known variability of the assay. For example, VO2max and energy expenditure measurements in the mice have high variability, therefore groups of 15-19 mice were analysed. The number of myoblast lines was chosen based on the available homozygous lines out of a library of 15 different lines.
Mice that showed signs of disease, predefined by the animal authorizations, were euthanized and removed from the study. In the Promethion experiment mice that did not spontaneously run on the running wheel were excluded from the analysis The experiments were repeated at least twice, and replication attempts were successful. All the replicates represent biological replicates. The mice used for experiments were from multiple different litters.
For all the in vivo and in vitro experiments, groups were randomly assigned. The human genetic association analysis were adjusted for sex and age.
Scientists performing the mouse phenotyping experiments were blinded to the experimental groups. The in vitro experiments were performed by a single investigator and blinding was not possible The HUNT3 fitness study was approved by the Regional committee for medical research ethics (2012/1672/REK nord), the Norwegian Data Inspectorate and the National Directorate of Health, and is in compliance with the Helsinki declaration. Written informed consent was obtained from all participants.
The clinical Helsinki Birth Cohort study protocol was approved by the Ethics Committee of Epidemiology and Public Health of the Hospital District of Helsinki and Uusimaa. Written informed consent was obtained from each participant before any study