Pyruvate dehydrogenase kinase 4 promotes ubiquitin–proteasome system‐dependent muscle atrophy

Abstract

Background Muscle atrophy, leading to muscular dysfunction and weakness, is an adverse outcome of sustained period of glucocorticoids usage. However, the molecular mechanism underlying this detrimental condition is currently unclear. Pyruvate dehydrogenase kinase 4 (PDK4), a central regulator of cellular energy metabolism, is highly expressed in skeletal muscle and has been implicated in the pathogenesis of several diseases. The current study was designed to investigated and delineate the role of PDK4 in the context of muscle atrophy, which could be identified as a potential therapeutic avenue to protect against dexamethasone-induced muscle wasting. Methods The dexamethasone-induced muscle atrophy in C2C12 myotubes was evaluated at the molecular level by expression of key genes and proteins involved in myogenesis, using immunoblotting and qPCR analyses. Muscle dysfunction was studied in vivo in wild-type and PDK4 knockout mice treated with dexamethasone (25 mg/kg body weight, i.p., 10 days). Body weight, grip strength, muscle weight and muscle histology were assessed. The expression of myogenesis markers were analysed using qPCR, immunoblotting and immunoprecipitation. The study was extended to in vitro human skeletal muscle atrophy analysis. Results Knockdown of PDK4 was found to prevent glucocorticoid-induced muscle atrophy and dysfunction in C2C12 myotubes, which was indicated by induction of myogenin (0.3271 ± 0.102 vs 2.163 ± 0.192, ****P < 0.0001) and myosin heavy chain (0.3901 ± 0.047 vs. 0.7222 ± 0.082, **P < 0.01) protein levels and reduction of muscle atrophy F-box (10.77 ± 2.674 vs. 1.518 ± 0.172, **P < 0.01) expression. In dexamethasone-induced muscle atrophy model, mice with genetic ablation of PDK4 revealed increased muscle strength (162.1 ± 22.75 vs. 200.1 ± 37.09 g, ***P < 0.001) and muscle fibres (54.20 ± 11.85% vs. 84.07 ± 28.41%, ****P < 0.0001). To explore the mechanism, we performed coimmunoprecipitation and liquid chromatography-mass spectrometry analysis and found that myogenin is novel substrate of PDK4. PDK4 phosphorylates myogenin at S43/T57 amino acid residues, which facilitates the recruitment of muscle atrophy F-box to myogenin and leads to its subsequent ubiquitination and degradation. Finally, overexpression of non-phosphorylatable myogenin mutant using intramuscular injection prevented dexamethasone-induced muscle atrophy and preserved muscle fibres. Conclusions We have demonstrated that PDK4 mediates dexamethasone-induced skeletal muscle atrophy. Mechanistically, PDK4 phosphorylates and degrades myogenin via recruitment of E3 ubiquitin ligase, muscle atrophy F-box. Rescue of muscle regeneration by genetic ablation of PDK4 or overexpression of non-phosphorylatable myogenin mutant indicates PDK4 as an amenable therapeutic target in muscle atrophy.

The Link between Mitochondrial Dysfunction and Sarcopenia: An Update Focusing on the Role of Pyruvate Dehydrogenase Kinase 4

“…During muscle atrophy, MuRF1 and MAFbx are overexpressed: inhibiting the function of MuRF1 and MAFbx suppresses muscle loss, attenuating muscle atrophy [ 60 , 61 ]. A recent study [ 62 ] indicates that overexpression or dexamethasone (Dex)-mediated induction of PDK4 enhances interaction between myogenin (MYOG) and MAFbx, but not between MYOG and MuRF1, which suggests that PDK4 promotes MAFbx recruitment to catalyze muscle atrophy-induced MYOG degradation ( Fig. 4 ); by contrast, Pdk4 −/− mice exert resistance against glucocorticoid-induced muscle atrophy.…”

“…4 ); by contrast, Pdk4 −/− mice exert resistance against glucocorticoid-induced muscle atrophy. In a Dex and cancer cachexia induced mouse model of atrophy, expression of PDK4 and MAFbx are upregulated, whereas knockdown of PDK4 reduces MAFbx expression [ 62 ]. Taken together, PDK4-dependent activation of the ubiquitin-proteasome pathway appears to underly the promotion of muscle atrophy by directly targeting the muscle-specific E3 ligase MAFbx.…”

“…4 ) [ 62 ]: PDK4 directly phosphorylates MYOG leading to MYOG ubiquitination and degradation. MYOG plays a crucial role in skeletal muscle differentiation and development by regulating expression of Mymk (myomaker, myoblast fusion factor), Mymx (myomixer, myoblast fusion factor), and myosin heavy chain, the key factors involved in the muscle differentiation and fusion process; as a result, suppressing PDK4 stabilizes MYOG and enhances muscle regeneration [ 62 ], while overexpression or induction of PDK4 by Dex decreases MYOG protein levels [ 62 ], suppressing myoblast differentiation and inhibiting myogenesis [ 5 ]. Thus, PDK4 has critical roles in regulating myogenic differentiation and muscle regeneration.…”

Single-Nuclei RNA-Sequencing of the Gastrocnemius Muscle in Peripheral Artery Disease

“…Consistent with these terms, both clusters displayed a significantly higher level of expression of PDK4 and TRIM63 in PAD muscles (Figure 2G), as well as FOXO1 (Figure S4C and S4D) genes all linked to muscle atrophy. 54,56 Cluster 4 from patients with PAD was also enriched for VEGFA (Figure S4E and S4F), a known regulator of angiogenesis. In addition to exploring PAD-specific subclusters, we also performed GSEA on type II myonuclei clusters that were observed in both non-PAD and PAD muscles (Figure S5).…”

Corrigendum to I.S. Sinam et al. J of Cachexia, Sarcopenia and Muscle, 13, 3122–3136. DOI: 10.1002/jcsm.13100

“…In the original full paper, 1 Figure 2D, we have inadvertently uploaded an incorrect figure. We came across this error during the process of data archiving.…”