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Myofibril breakdown during atrophy is a delayed response requiring the transcription factor PAX4 and desmin depolymerization.

ABSTRACT: A hallmark of muscle atrophy is the excessive degradation of myofibrillar proteins primarily by the ubiquitin proteasome system. In mice, during the rapid muscle atrophy induced by fasting, the desmin cytoskeleton and the attached Z-band-bound thin filaments are degraded after ubiquitination by the ubiquitin ligase tripartite motif-containing protein 32 (Trim32). To study the order of events leading to myofibril destruction, we investigated the slower atrophy induced by denervation (disuse). We show that myofibril breakdown is a two-phase process involving the initial disassembly of desmin filaments by Trim32, which leads to the later myofibril breakdown by enzymes, whose expression is increased by the paired box 4 (PAX4) transcription factor. After denervation of mouse tibialis anterior muscles, phosphorylation and Trim32-dependent ubiquitination of desmin filaments increased rapidly and stimulated their gradual depolymerization (unlike their rapid degradation during fasting). Trim32 down-regulation attenuated the loss of desmin and myofibrillar proteins and reduced atrophy. Although myofibrils and desmin filaments were intact at 7 d after denervation, inducing the dissociation of desmin filaments caused an accumulation of ubiquitinated proteins and rapid destruction of myofibrils. The myofibril breakdown normally observed at 14 d after denervation required not only dissociation of desmin filaments, but also gene induction by PAX4. Down-regulation of PAX4 or its target gene encoding the p97/VCP ATPase reduced myofibril disassembly and degradation on denervation or fasting. Thus, during atrophy, the initial loss of desmin is critical for the subsequent myofibril destruction, and over time, myofibrillar proteins become more susceptible to PAX4-induced enzymes that promote proteolysis.

SUBMITTER: Volodin A

PROVIDER: S-EPMC5338431 | biostudies-other | 2017 Feb

REPOSITORIES: biostudies-other

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Project description:Background Mutations in ?B-crystallin result in proteotoxic cardiomyopathy with desmin mislocalization to protein aggregates. Intermittent fasting ( IF ) is a novel approach to activate transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in the myocardium. We tested whether TFEB activation can be harnessed to treat advanced proteotoxic cardiomyopathy. Methods and Results Mice overexpressing the R120G mutant of ?B-crystallin in cardiomyocytes ( Myh6-Cry ABR 120G) were subjected to IF or ad-lib feeding, or transduced with adeno-associated virus- TFEB or adeno-associated virus-green fluorescent protein after development of advanced proteotoxic cardiomyopathy. Adeno-associated virus-short hairpin RNA-mediated knockdown of TFEB and HSPB 8 was performed simultaneously with IF . Myh6-Cry ABR 120G mice demonstrated impaired autophagic flux, reduced lysosome abundance, and mammalian target of rapamycin activation in the myocardium. IF resulted in mammalian target of rapamycin inhibition and nuclear translocation of TFEB with restored lysosome abundance and autophagic flux; and reduced aggregates with normalized desmin localization. IF also attenuated left ventricular dilation and myocardial hypertrophy, increased percentage fractional shortening, and increased survival. Adeno-associated virus- TFEB transduction was sufficient to rescue cardiomyopathic manifestations, and resulted in reduced aggregates and normalized desmin localization in Myh6-Cry ABR 120G mice. Cry ABR 120G-expressing hearts demonstrated increased interaction of desmin with ?B-crystallin and reduced interaction with chaperone protein, HSPB 8, compared with wild type, which was reversed by both IF and TFEB transduction. TFEB stimulated autophagic flux to remove protein aggregates and transcriptionally upregulated HSPB 8, to restore normal desmin localization in Cry ABR 120G-expressing cardiomyocytes. Short hairpin RNA-mediated knockdown of TFEB and HSPB 8 abrogated IF effects, in vivo. Conclusions IF and TFEB activation are clinically relevant therapeutic strategies to rescue advanced R120G ?B-crystallin mutant-induced cardiomyopathy by normalizing desmin localization via autophagy-dependent and autophagy-independent mechanisms.

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Myofibril breakdown during atrophy is a delayed response requiring the transcription factor PAX4 and desmin depolymerization.

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