Short‐ and Long‐Term Hindlimb Immobilization and Reloading: Profile of Epigenetic Events in Gastrocnemius

Abstract

Skeletal muscle dysfunction and atrophy are characteristic features accompanying chronic conditions. Epigenetic events regulate muscle mass and function maintenance. We hypothesized that the pattern of epigenetic events (muscle-enriched microRNAs and histone acetylation) and acetylation of transcription factors known to signal muscle wasting may differ between early- and late-time points in skeletal muscles of mice exposed to hindlimb immobilization (I) and recovery following I. Body and muscle weights, grip strength, muscle-enriched microRNAs, histone deacetylases (HDACs), acetylation of proteins, histones, and transcription factors (TF), myogenic TF factors, and muscle phenotype were assessed in gastrocnemius of mice exposed to periods (1, 2, 3, 7, 15, and 30 days, I groups) of hindlimb immobilization, and in those exposed to reloading for different periods of time (1, 3, 7, 15, and 30 days, R groups) following 7-day immobilization. Compared to non-immobilized controls, muscle weight, limb strength, microRNAs, especially miR-486, SIRT1 levels, and slow- and fast-twitch cross-sectional areas were decreased in mice of I groups, whereas Pax7 and acetylated FoxO1 and FoxO3 levels were increased. Muscle reloading following splint removal improved muscle mass loss, strength, and fiber atrophy, by increasing microRNAs, particularly miR-486, and SIRT1 content, while decreasing acetylated FoxO1 and FoxO3 levels. In this mouse model of disuse muscle atrophy, muscle-enriched microRNAs, especially miR-486, through Pax7 regulation delayed muscle cell differentiation following unloading of gastrocnemius muscle. Acetylation of FoxO1 and 3 seemed to drive muscle mass loss and atrophy, while deacetylation of these factors through SIRT1 would enable the muscle fibers to regenerate. J. Cell. Physiol. 232: 1415-1427, 2017. © 2016 Wiley Periodicals, Inc.

Muscle regeneration potential and satellite cell activation profile during recovery following hindlimb immobilization in mice

“…The rationale to use such methodologies was to identify a pure population of muscle progenitors as demonstrated in previous studies (Yin, Rice, & Rudnicki, ). In fact, one of the main goals in the study was to characterize the gene expression profile of the isolated progenitor cells during recovery following a period of hindlimb muscle disuse for 7 days, which was defined as a key time‐point to analyze early and late events as previously shown in this experimental model (Chacon‐Cabrera et al, ; Chacon‐Cabrera, Gea, et al, ). Furthermore, the use of methodologies such as histological procedures enabled us to define the localization and numbers of active and proliferating satellite cells in the study muscles.…”

“…Mice were exposed to unilateral hindlimb immobilization as previously described to reproduce a model of disuse muscle atrophy (Chacon‐Cabrera et al, ; Chacon‐Cabrera, Gea, et al, ). Briefly, the left hindlimb was shaved with clippers and was enveloped using surgical tape.…”

“…In all the study animals, food intake was measured daily, and food and water were supplied ad libitum for the entire duration of the immobilization or recovery periods. In all mice, body weight and limb strength were determined on day 0, and right at the end of the immobilization or recovery time‐points (day 7, as described above) using a scale and a grip strength meter (Bioseb, Vitrolles, France), respectively, as previously reported (Barreiro & Gea, ; Chacon‐Cabrera et al, , , ; Chacon‐Cabrera, Gea, et al, ; Chacon‐Cabrera, Mateu‐Jimenez, et al, ). In the two groups of animals, total body weight and limb strength gain variables were calculated as the percentage of the measurements performed at the end of the study period (7‐day I or 7‐day R) with respect to the same measurements obtained at baseline (day 0 or on day 7 following the immobilization protocol) in the 7‐day I and 7‐day R study groups, respectively.…”

“…Recently, we have demonstrated that a specific program of molecular and cellular events characterized by increased muscle proteolysis and injury was observed in the gastrocnemius of mice following hindlimb unloading for several time‐points, while reloading of the muscle improved all those alterations (Chacon‐Cabrera et al, ). Additionally, in the same mouse model, muscle‐enriched microRNAs, especially miR‐486, through paired box (pax) seven regulation was shown to delay muscle cell differentiation, probably via acetylation of fork‐head box (FoxO) 1 and 3, in the unloaded gastrocnemius (Chacon‐Cabrera, Gea, et al, ). Interestingly, reloading of the muscle after splint removal induced a rise of the histone deacetylase sirtuin‐1 along with a decrease in acetylation levels of the transcription factors FoxO 1 and 3, thus enabling the muscle fibers to regenerate in the mice (Chacon‐Cabrera, Gea, et al, ).…”

“…Additionally, in the same mouse model, muscle‐enriched microRNAs, especially miR‐486, through paired box (pax) seven regulation was shown to delay muscle cell differentiation, probably via acetylation of fork‐head box (FoxO) 1 and 3, in the unloaded gastrocnemius (Chacon‐Cabrera, Gea, et al, ). Interestingly, reloading of the muscle after splint removal induced a rise of the histone deacetylase sirtuin‐1 along with a decrease in acetylation levels of the transcription factors FoxO 1 and 3, thus enabling the muscle fibers to regenerate in the mice (Chacon‐Cabrera, Gea, et al, ). We concluded that the kinetics of the biological events leading to skeletal muscle mass loss and recovery were tightly regulated in the gastrocnemius of mice exposed to different time‐points of muscle unloading and reloading (Chacon‐Cabrera et al, ; Chacon‐Cabrera, Gea, et al, ).…”