Rev-Erb co-regulates muscle regeneration via tethered interaction with the NF-Y cistrome

Abstract

ObjectiveThe loss of skeletal muscle mass and strength are a central feature of traumatic injury and degenerative myopathies. Unfortunately, pharmacological interventions typically fail to stem the long-term decline in quality of life. Reduced Rev-Erb-mediated gene suppression in cultured C2C12 myoblasts has been shown to stimulate myoblast differentiation. Yet the mechanisms that allow Rev-Erb to pleiotropically inhibit muscle differentiation are not well understood. In this study, we sought to elucidate the role of Rev-Erb in the regulation of muscle differentiation and regeneration in vivo.MethodsUsing Rev-Erbα/β shRNAs, pharmacological ligands, and Rev-Erbα null and heterozygous mice, we probed the mechanism of Rev-Erbα/β regulation of muscle differentiation and muscle regeneration.ResultsChIP seq analysis of Rev-Erb in differentiating myoblasts showed that Rev-Erbα did not transcriptionally regulate muscle differentiation through cognate Rev-Erb/ROR-response elements but through possible interaction with the cell fate regulator NF-Y at CCAAT-motifs. Muscle differentiation is stimulated by Rev-Erb release from CCAAT-motifs at promoter and enhancer elements of a number of myogenesis proteins. Partial loss of Rev-Erb expression in mice heterozygous for Rev-Erbα accelerated muscle repair in vivo whereas Rev-Erb knockout mice showed deficiencies in regenerative repair compared to wild type mice. These phenotypic differences between heterozygous and knockout mice were not apparently dependent on MRF induction in response to injury. Similarly, pharmacological disruption of Rev-Erb suppressive activity in injured muscle accelerated regenerative repair in response to acute injury.ConclusionsDisrupting Rev-Erb activity in injured muscle accelerates regenerative muscle repair/differentiation through transcriptional de-repression of myogenic programs. Rev-Erb, therefore, may be a potent therapeutic target for a myriad of muscular disorders.

Rev-erbα heterozygosity produces a dose-dependent phenotypic advantage in mice

“…In accord with previous studies, we observed that heterozygous REV-ERB mice in addition to possessing heightened regenerative capacity interestingly had a greater total average body mass than wildtype mice [16] (Fig 1A). Firstly, we quantified the expression of Rev-erbα in the SkM of Nr1d1 +/+ , Nr1d1 +/and Nr1d1 -/mice at CT8 and found that Rev-erbα gene expression was dose dependently linked to Nr1d1 genotype as partial loss of Rev-erbα resulted in a reduction in REV-ERBα expression ( Fig 1B).…”

“…The nuclear receptor REV-ERB is a key repressor of the molecular clock and modulates metabolic and developmental pathways. Our study wished to thoroughly understand how Rev-erb expression levels impacts metabolism and muscle morphology due to a previous observation that Nr1d1 heterozygous mice have superior muscle fiber size [16]. In this study we illustrate that Nr1d1-heterozygosity increased glucose clearance, fatty acid utilization, gluconeogenesis, and also boosted lean mass accumulation without generating higher adiposity in chow-fed mice.…”

“…As previously mentioned, compared to wildtype littermates null mice show a rapid decline in lean mass after reaching maturity [16]. We analyzed the CSA of soleus muscle from Nr1d1 +/and Nr1d1 -/mice and found that, surprisingly, both knockout and heterozygous mice displayed significantly larger myofibers than wildtype littermates at 6-weeks-of age (Fig 2A).…”

“…We have previously noted that Nr1d1 +/mice had larger myofiber diameters compared to wild-type and Nr1d1 -/mice, which implied that partial loss of REV-ERB uniquely stimulated muscle hypertrophy and or inhibited muscle degradation. Heterozygous mice also showed an enhanced regenerative response to injury compared to wildtype mice [16]. Rev-erb null mice are known to display elevated expression of mediators of muscle atrophy, impaired muscle function, and a progressive decline in myofiber size due to enhanced protein catabolism [19].…”

“…Previously, we have highlighted that REV-ERB directs myogenesis through tethered interaction with the transcription complex Nuclear Factor-Y [16]. Moreover, inhibiting REV-ERB with a synthetic antagonist enhanced muscle repair in an acute injury model and slowed disease progression in a model of Duchenne's muscular dystrophy [16,17]. Loss of Rev-Erb in contrast to Rev-Erb was also shown to stimulate skeletal muscle metabolism and fatty acid oxidation [18].…”