Muscle-type specific stress responses and conflicting fuel choices underlie mitochondrial external ophthalmoplegia

Mitochondrial dysfunctions elicit progressive tissue-specific stress responses that can be protective or deleterious. Here, we show that even different muscles of an individual react differently to mitochondrial disease. In mitochondrial myopathy (MM), the extraocular muscles (EOMs) are affected first, followed by exercise intolerance in the large lower limb muscles. Both muscle types show clear signs of respiratory chain deficiency. However, the limbs upregulate the mitochondrial integrated stress response (ISRmt) that drives glucose carbons to anabolic repair pathways, while EOMs present no signs of ISRmt. In contrast, in EOMs, Pdk4 activation inhibits pyruvate metabolism, while beta-oxidation of fatty acids is induced - despite the reliance of beta-oxidation on a functional respiratory chain for ATP production. The data suggest that the inability to upregulate ISRmt and consequent deleterious fuel choices sensitize EOMs to early weakness and atrophy, explaining ophthalmoplegia, the most common MM sign. The distinct responses to disease even in muscles of a single individual predict different responses to treatment, which is essential knowledge when designing interventions. Introduction Mitochondrial muscle disease (MM) is the most common form of adult-onset mitochondrial diseases. A clinical hallmark of the disorder is chronic progressive external ophthalmoplegia (CPEO) and ptosis, which are consequences of early manifestation of the extra-ocular muscles (EOMs). The EOMs show progressive weakness and atrophy that leads to generalized restriction of eye movements (ophthalmoplegia) and drooping of eyelids (ptosis), which disturb vision, closure of eyes during sleep and social life. The disease progresses to large skeletal muscles and manifests as exercise intolerance and weakness of limb muscles (Goldstein & Falk, 2019). MM is typically caused by disorders of mitochondrial DNA (mtDNA) expression: mutations in mitochondrial tRNAs and sporadic large-scale single or multiple mtDNA deletions. The latter are secondary consequences of nuclear gene mutations in mtDNA maintenance genes, such as the replicative helicase or polymerase of mtDNA (Twinkle, TWNK; DNA polymerase gamma, POLG) or different enzymes regulating mitochondrial nucleotide pools (Spelbrink et al, 2001; Suomalainen & Isohanni, 2010; Van Goethem et al, 2001). All of these dysfunctions lead to respiratory chain (RC) deficiency (Gorman et al, 2016). However, mutations affecting directly RC enzyme subunits typically cause a brain disease, not CPEO (Gorman et al, 2016) indicating that the metabolic responses to MM are not directly caused by RC deficiency. Indeed, data from humans and mouse models indicate that large limb muscles affected by MM upregulate a stage-wise mitochondrial integrated stress response, ISRmt (Nikkanen et al, 2016; Bao et al, 2016; Forsström et al, 2019; Khan et al, 2017; Kühl et al, 2017; Tyynismaa et al, 2010) that remodels metabolism in the whole cells. The early activation of ISRmt is marked by robust transcriptional upregulation of ATF-transcription factor target genes encoding mitochondrial folate cycle, metabokines FGF21 and GDF15. The second stage is dependent on prior induction of FGF21 and induces synthesis of serine (PHGDH, PSAT1), nucleotides, and the transsulfuration pathway (CTH, CBS), and modifies one-carbon metabolism remarkably. mTORC1 (mechanistic target of rapamycin complex1) is active in the most affected fibers that also show stalled mitochondrial recycling (Khan et al, 2017; Mito et al, 2022). While molecular responses of large muscles to MM are already quite well understood, little is known why EOMs are especially sensitive to mtDNA expression defects. Human studies on EOMs in MM patients have been dependent on autopsy samples, where muscle atrophy is already close to total. Therefore, good animal models are needed. The “Deletor” mouse model, which expresses constitutively a dominant Twinkle patient mutation (Tyynismaa et al, 2005) replicates well the morphological and molecular characteristics of adult-onset MM. Indeed, these mice were the basis of the original characterization of ISRmt, later replicated to be fully conserved in patients with the same disease. Here we used the Deletor mice to elucidate EOM responses compared to the large limbs. We report that stress responses of the small oxidative EOMs differ from large thigh muscles, with opposite fuel choices (glucose vs lipids) promoted upon mitochondrial disease. The data indicate high tissue-specificity of mitochondrial stress responses even between different muscles.