PHAF1/MYTHO is a novel autophagy regulator that controls muscle integrity

ABSTRACT

Skeletal muscles play key roles in movement, posture, thermogenesis, and whole-body metabolism. Autophagy plays essential roles in the regulation of muscle mass, function and integrity. However, the molecular machinery that regulates autophagy is still incompletely understood. In our recent study, we identified and characterized a novel Forkhead Box O (FoxO)-dependent gene, PHAF1/MYTHO (phagophore assembly factor 1/macro-autophagy and youth optimizer), as a novel autophagy regulator that controls muscle integrity. MYTHO/PHAF1 is upregulated in multiple conditions leading to muscle atrophy, and downregulation of its expression spares muscle atrophy triggered by fasting, denervation, cachexia and sepsis. Overexpression of PHAF1/MYTHO is sufficient to induce muscle atrophy. Prolonged downregulation of PHAF1/MYTHO causes a severe myopathic phenotype, which is characterized by impaired autophagy, muscle weakness, myofiber degeneration, mammalian target of rapamycin complex 1 (mTORC1) hyperactivation and extensive ultrastructural defects, such as accumulation of proteinaceous and membranous structures and tubular aggregates. This myopathic phenotype is attenuated upon administration of the mTORC1 inhibitor rapamycin. These findings position PHAF1/MYTHO as a novel regulator of skeletal muscle autophagy and tissue integrity.

Main

Autophagy function is crucial for the maintenance of cardiac and skeletal muscle mass and integrity. In the last ten years, researchers have shown that several muscle-associated diseases are induced by dysregulation of autophagy. An excessive autophagic flux, as emerge in many catabolic conditions such as cachexia, denervation, sepsis or fasting, triggers a decrease in the size of muscle cells, a process named atrophy, which is attributed to enhanced sarcomeric protein degradation and organelles removal. Conversely, a defective autophagic flux leads to an accumulation of membranous materials, protein aggregates, dysfunctional organelles such as mitochondria and enhanced oxidative stress, which altogether cause the dismantle of motor neuron synapses, muscle mass loss, drop in force and premature aging of the tissue.

The Forkhead Box-O (FoxO) proteins are master regulators of muscle wasting associated with catabolic conditions such as starvation, denervation, inactivity/disuse, cachexia, diabetes or sepsis, by regulating genes of autophagosome-lysosome and ubiquitin-proteome systems. However, our understanding of why catabolic conditions trigger muscle loss is incomplete since many players involved in muscle wasting remain largely uncharacterized. Moreover, a large bulk of the genes in the human genome is still poorly characterized. Therefore, we focused our attention on identifying new genes that may control autophagy. In this effort, we identified a novel FoxO-dependent gene, PHAF1/MYTHO (phagophore assembly factor 1/macro-autophagy and youth optimizer), that regulates autophagy and plays a major role in the maintenance of skeletal muscle integrity.[1] Subcellular localization studies showed that PHAF1/MYTHO is present on autophagosomes and autolysosomes, and that its transport to autolysosomes involves autophagosome fusion with lysosomes. The molecular details underlying the recruitment of PHAF1/MYTHO onto autophagosomes is still unclear, but bioinformatical analyses revealed several putative LC3 interacting regions (LIR) and its WD40 domains could explain such distribution. We next explored whether PHAF1/MYTHO may regulate autophagosome formation and the autophagy flux. By transfecting adult mouse muscles with shRNA targeting PHAF1/MYTHO, we found that downregulation of PHAF1/MYTHO decreased the autophagic flux. Importantly, and in accordance with the role played by autophagy activation during muscle loss, depletion of PHAF1/MYTHO was beneficial to spare muscle mass in different catabolic conditions such as denervation, fasting, cachexia and sepsis (Figure 1). However, when PHAF1/MYTHO depletion was prolonged for weeks, the chronic impairment of autophagy triggered an increase in muscle mass but with a concomitant important decline in muscle force. Morphological and ultrastructural studies showed accumulation of proteinaceous and membranous structures, tubular aggregates, abnormal mitochondrial morphology and dilated sarcoplasmic reticulum in myofibers. Moreover, muscles in which PHAF1/MYTHO was knocked down (PHAF1/MYTHO-KD) displayed severe myopathic features such as inflammation, fibers with central nuclei, necrotic fibers, heterogeneity of myofiber size with giant and smaller myofibers, and a fiber type switch toward glycolytic type IIb (Figure 1). When we compared these features with the structural and ultrastructural abnormalities of autophagy deficient mice lacking ATG7 (ATG7 KO mice), we observed some similarities but also several important differences. Indeed, the inhibition of PHAF1/MYTHO expression, which results in a reduction but not complete inhibition of the autophagy flux, resulted in a much more severe phenotype when compared with muscle-specific ATG7 KO mice. These observations suggest that PHAF1/MYTHO may play additional roles than just controlling autophagy. Because tubular aggregates were consistently observed in muscles with PHAF1/MYTHO-KD and because these structures are characteristic of some myopathies such as myotonic dystrophy type 1 (DM1) or myopathies with Ca²⁺ imbalance (e.g. mutations in the STIM1 (Stromal interaction molecule 1)/ORAI1 (ORAI Calcium Release-Activated Calcium Modulator 1) system), we tested whether Ca²⁺ homeostasis was perturbed. We found that PHAF1/MYTHO-KD muscles displayed aberrant STIM1 and SERCA (Sarco-Endoplasmic Reticulum Calcium ATPase) expression as well as mitochondrial Ca²⁺ retention dysregulation, confirming an abnormal Ca²⁺ homeostasis. Importantly, we also found that PHAF1/MYTHO is downregulated in muscle biopsies collected from DM1 patients.

Figure 1.

PHAF1/MYTHO controls muscle integrity. Acute downregulation of PHAF1/MYTHO reduces the autophagic flux and protects from atrophy in conditions in which autophagy is induced, such as starvation, cachexia, denervation and sepsis. However, chronic downregulation of PHAF1/MYTHO increases mTORC1 activation, likely through increased growth signaling (increase in IGF2 expression and decrease in MSTN expression), which in turn triggers enhanced protein synthesis and further downregulation of the autophagic flux. These chronic changes lead to pathological muscle hypertrophy with severe myopathic features and impaired functionality. Created with BioRender.com..

Another interesting finding of our study is that chronic PHAF1/MYTHO-KD led to mammalian target of rapamycin complex 1(mTORC1) hyperactivation, with increased protein synthesis. mTORC1 hyperactivation probably contributes to the autophagy impairment and may have been caused by the upregulation of IGF2 (insulin-like growth factor 2) gene expression and the concomitant downregulation of MSTN (Myostatin) expression. Indeed, MSTN and IGF2 negatively and positively regulate the AKT (AKT serine-threonine kinase)/mTORC1 pathway, respectively. Importantly, rapamycin treatment ameliorated the myopathic features triggered by PHAF1/MYTHO-KD, supporting the notion of a role of mTORC1 hyperactivation in myofiber degeneration. These results are consistent with other studies showing a causal link between hyperactivation of mTORC1 and appearance of premature tissue aging and myopathic features. How PHAF1/MYTHO affects gene expression and impinges on mTORC1 complex and protein synthesis pathways, needs to be addressed in the future.

The generation of full body PHAF1/MYTHO knockout and conditional PHAF1/MYTHO knockout mouse lines would be useful not only for mechanistic studies, but also to identify tissues that require PHAF1/MYTHO function. Indeed, PHAF1/MYTHO is also relatively highly expressed in tissues such as liver, lungs, heart and brain. Our genetic approaches also did not address whether small amount of PHAF1/MYTHO protein may still preserve/maintain certain cellular functions/pathways.

Having found that PHAF1/MYTHO is a novel FoxO-dependent gene also opens new areas of investigation. FoxO is not only important in regulating genes involved in muscle mass loss, but it also modulates genes involved in metabolism, cell cycle and stress response, and is a master regulator of longevity. In C. elegans, Drosophila and mammals, FoxO transcription has been related to lifespan extension. Genetic variations within FoxO3 have been reported to be associated with human longevity. As alluded to in our study, we opted for the name MYTHO in part based on its ability to regulate longevity in C. elegans. However, the exact role that PHAF1/MYTHO plays in organismal aging and whether its impact on longevity is conserved and downstream of the insulin signaling system or of other longevity pathways, will require further studies.

Finally, the finding that PHAF1/MYTHO expression is downregulated in DM1 patients is associated with features also seen in mouse muscles with PHAF1/MYTHO-KD, such as the activation of mTORC1 signaling and impaired autophagy, suggests that low levels of PHAF1/MYTHO may contribute to the disease progression. Whether this is indeed the case and whether PHAF1/MYTHO also plays a role in other human diseases characterized by autophagy impairment, Ca²⁺ dysregulation and mTORC1 hyperactivation, will require further investigation.

Funding Statement

This work was supported by AFM-Telethon (22982), Foundation Leducq, AIRC (17388), AIRC (23257), H2020-MSCA-RISE-2014 project no 645648 ‘Muscle Stress Relief’, ASI (MARS-PRE), CARIPARO, Next Generation EU in the context of the National Recovery and Resilience Plan, Investment PE8 – Project Age-It: “Ageing Well in an Ageing Society”. This resource was co-financed by the Next Generation EU [DM 1557 11.10.2022] to MS. This work was also supported by the Natural Sciences and Engineering Council of Canada (NSERC, RGPIN-2021-03724) and Canadian Institutes of Health Research (CIHR) grants (GER-417022, MOV-399334 and MOV-438450). GG is supported by a Chercheur-boursier Junior 2 salary award from the Fonds de recherche du Québec en santé (FRQS-297877). JPLG was supported by a Postdoctoral Fellowship from the FRQS.

Disclosure statement

No potential conflict of interest was reported by the author(s).