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Elevated expression of the molecular clock suppressors Period 1 and Period 2 promote myotube atrophy in vitro with implications to the loss of limb muscle mass following androgen deprivation

ABSTRACT: Low testosterone levels in men (hypogonadism) are associated with loss of limb muscle mass. We previously showed molecular clock function is disrupted in limb muscle by androgen deprivation. That clock disruption coincided with higher protein levels of the molecular clock suppressor, Period 2 (Per2). The primary purpose of this study was to assess whether Per2 regulates muscle size. Transducing fully differentiated C2C12 myotubes with adenovirus overexpressing Per2 was sufficient to lower myotube diameter. Moreover, deletion of Per2 in male mice partially maintained mass of the tibialis anterior (TA) muscle following castration-induced androgen deprivation. The partial maintenance of mass was specific to the TA even though molecular clock disruption is evident in other limb muscles. Thus, the secondary purpose of this study was to assess whether Per1 also regulates muscle size. Overexpression of the other primary molecular clock suppressor, Per1, in C2C12 myotubes was also sufficient to decrease myotube diameter. The lower myotube diameter in response to Per1 or Per2 overexpression was due in part to an autocrine-mediated inflammatory signals as conditioned media from Per1 or Per2 overexpressing myotubes contained elevated cytokines/chemokines, and the Per conditioned media was sufficient to lower C2C12 myotube diameter. The mRNA contents of inflammatory genes altered following Per1 or Per2 overexpression in vitro were altered in limb muscle in vivo following castration. These data define a novel muscle mass/size regulatory function of the primary molecular clock suppressors with direct implications for their role in regulating limb muscle mass loss in response to low androgen levels

ORGANISM(S): Mus musculus

PROVIDER: GSE299632 | GEO | 2025/08/06

REPOSITORIES: GEO

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Project description:Muscle spindles are skeletal muscle stretch receptors that mediate axial and limb position sensation (proprioception) to the central nervous system. Defective proprioception, often characterized by gait ataxia and poor coordination, is present in a variety of human sensory and motor neuropathies having diverse etiologies. Spindles contain specialized intrafusal muscle fibers that are induced during development by sensory innervation. The molecular events mediating the morphogenetic induction process are poorly characterized but depend upon neuregulins and their cognate ErbB receptor signaling. Recently, we demonstrated that the transcription factor Egr3 is induced and regulated by intrafusal muscle fiber sensory innervation during spindle induction. Moreover, Egr3-deficient mice have impaired spindle morphogenesis and profound gait ataxia. Thus, Egr3 mediated transcription is critical for muscle stretch receptor development and potentially for myotube fate specification.,Egr3 may mediate spindle morphogenesis induced by Neuregulin/ErbB signaling after myotubes are contacted by sensory axons during development. As a starting point for understanding the specific role for Egr3 in this process, we will use a genome wide expression analysis to identify genes regulated (either directly or indirectly) by Egr3 in primary myotubes. It is not practical to examine Egr3 mediated gene expression directly within spindles since they comprise less than 0.1% of the muscle mass. Therefore, gene expression analysis will be examined in primary myotubes grown in vitro. Primary myoblasts isolated from mice and differentiated in vitro are biochemically most similar to extrafusal muscle fibers which do not express Egr3. To identify genes regulated by Egr3 in myotubes, gene expression in myotubes enforced to express full length Egr3 will be compared to gene expression in myotubes enforced to express truncated (transcriptionally inactive) Egr3.,Egr3-deficient mice lack muscle spindles and have profound proprioceptive deficits. Egr3 is specifically expressed in a subset of developing primary myotubes after innervation by sensory axons. Spindle induction is dependent upon sensory/myotube contact which involves sensory axon produced neuregulin and ErbB receptor signaling in the myotube. Shortly after or during induction, Egr3 expression is upregulated and maintained in the nascent myotube/spindle as one of the earliest molecular events know to occur during spindle morphogenesis. We hypothesize that Egr3 mediated transcription is critical for engaging gene programs specific to and essential for, muscle spindle formation. Moreover, Egr3 may be involved in fate specification of myotubes that develop into biochemically and physiologically distinct intrafusal muscle fibers within spindles. To date, the target genes regulated by Egr3 during spindle morphogenesis have not been characterized.,Myoblasts from wild type newborn mice will be isolated and purified. Myoblasts will be plated on collagen coated plates and differentiated in vitro for 7 days. During in vitro differentiation, myoblasts fuse to form multinucleated myotubes that exhibit contractile properties. Egr3 is not expressed in myotubes in vitro, which is consistent with their similarity to extrafusal muscle fibers that also do not express Egr3 in vivo. Only intrafusal fibers of muscle spindles express high levels of Egr3 in this muscle fiber context. Adenoviruses that express full length Egr3 (Egr3wt; transcriptionally active) and truncated Egr3 (Egr3tr; transcriptionally inactive) have been generated and characterized in our laboratory. After 7 days of in vitro differentiation, primary myotubes will be infected with either Egr3wt or Egr3tr adenoviruses. 100% infection is routinely obtained which is confirmed by coexpression of enhanced green fluorescent protein (EGFP). Care will be taken to minimize viral induced toxicity of the myotubes after infection. Myotubes are maintained after infection for 24 hours, after which total RNA is extracted from the cell lysates. The two RNA samples will be used for microarray analysis to identify genes that are upregulated and downregulated by Egr3 in the myotube cellular context. We will repeat the infection, RNA isolation and microarray analysis three times to minimize identifying false positive differentially expressed genes. Differentially expressed genes will be confirmed using real-time PCR. Interesting differentially expressed genes will be examined by in situ hybridization to determine if they are specifically expressed in wild type spindles. Since the identified differentially expressed genes will represent both indirect and direct target genes, we will perform Chromatin Immunoprecipitation (ChIP) from Egr3wt infected myotubes and screen the ChIP DNA library by PCR to determine which gene promoters are directly bound by Egr3.

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Elevated expression of the molecular clock suppressors Period 1 and Period 2 promote myotube atrophy in vitro with implications to the loss of limb muscle mass following androgen deprivation

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