Acute molecular response of mouse hindlimb muscles to chronic stimulation

Abstract

Stimulation of the mouse hindlimb via the sciatic nerve was performed for a 4-h period to investigate acute muscle gene activation in a model of muscle phenotype conversion. Initial force production (1.6 +/- 0.1 g/g body wt) declined 45% within 10 min and was maintained for the remainder of the experiment. Force returned to initial levels upon study completion. An immediate-early growth response was present in the extensor digitorum longus (EDL) muscle (FOS, JUN, activating transcription factor 3, and musculoaponeurotic fibrosarcoma oncogene) with a similar but attenuated pattern in the soleus muscle. Transcript profiles showed decreased fast fiber-specific mRNA (myosin heavy chains 2A and 2B, fast troponins T(3) and I, alpha-tropomyosin, muscle creatine kinase, and parvalbumin) and increased slow transcripts (myosin heavy chain-1beta/slow, troponin C slow, and tropomyosin 3y) in the EDL versus soleus muscles. Histological analysis of the EDL revealed glycogen depletion without inflammatory cell infiltration in stimulated versus control muscles, whereas ultrastructural analysis showed no evidence of myofiber damage after stimulation. Multiple fiber type-specific transcription factors (tea domain family member 1, nuclear factor of activated T cells 1, peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -beta, circadian locomotor output cycles kaput, and hypoxia-inducible factor-1alpha) increased in the EDL along with transcription factors characteristic of embryogenesis (Kruppel-like factor 4; SRY box containing 17; transcription factor 15; PBX/knotted 1 homeobox 1; and embryonic lethal, abnormal vision). No established in vivo satellite cell markers or genes activated in our parallel experiments of satellite cell proliferation in vitro (cyclins A(2), B(2), C, and E(1) and MyoD) were differentially increased in the stimulated muscles. These results indicated that the molecular onset of fast to slow phenotype conversion occurred in the EDL within 4 h of stimulation without injury or satellite cell recruitment. This conversion was associated with the expression of phenotype-specific transcription factors from resident fiber myonuclei, including the activation of nascent developmental transcriptional programs.

Fig. 1.

Contractile characteristics for the stimulation time course. A: temporal changes in twitch force normalized to total body weight (in g/g body wt) and the associated changes in half-relaxation time (in ms) throughout a representative experiment reported in 10-min intervals. There was an initial decline in peak twitch force output and a concomitant prolongation of half-relaxation time after the onset of stimulation. Data are presented as means ± SE and were averaged for each 10-s period every 10 min over the time course. B and C: twitch at the start of the experiment (second twitch; B) and the next to last twitch at the end of the experiment (C) for the comparison of changes in the twitch profile throughout the duration of the experiment. Normalized force is presented on the ordinate, and time (in ms) is presented on the abscissa.

Fig. 2.

Histologic analysis of stimulated and resting extensor digitorum longus (EDL) muscles. Examples of serial cross sections from both stimulated and matching unstimulated contralateral EDL muscles are provided, showing the effects of the experimental protocol on glycogen content. These muscles exhibited a profound fast fiber type-specific response of glycogen depletion associated with chronic stimulation. A and C: representative sections stained with hematoxylin and eosin (H&E; A) and periodic acid Schiff (PAS; C) from a control EDL, respectively. B and D: results obtained from the matched contralateral EDL from the same animal subjected to stimulation (with H&E stain in B and PAS stain in D). The PAS (C and D) stain is specific to the presence of glycogen, which was diminished in D from the stimulated EDL muscle.

Fig. 3.

Ultrastructural analysis of stimulated and control EDL muscles. A–D: scanning electron micrographs obtained from thin sections of unstimulated EDL muscles; E–H: comparable regions from contralateral stimulated EDL muscles. All images were obtained at the same original direct magnification (×2,650). Scale bars = 2 μm, which applies to each image. A and E: membrane and myofibrillar architecture from both EDL muscles. There were unusual accumulations of mitochondria (Mito) near the end of EDL muscle fibers in some but not all animals, as demonstrated in B and F. C and G: satellite cells commonly identified in both muscles [satellite cell nucleus (SatN) and satellite cell membrane (SCM)]. The resident myofiber nuclei (MyoN) populating these muscle fibers were similar in appearance and located in juxtaposition to the sarcolemma, consistent with the morphology of normal skeletal muscle fibers (D and H).

Fig. 4.

Myofiber phenotype-specific transcripts altered by stimulation. Fast and slow muscle contractile gene transcripts obtained after 4 h of stimulation are shown and were associated with the process of myofiber phenotype transformation. Data are presented as fold changes (means ± SE) relative to the contralateral muscle (resting control) for each animal in paired comparison. Decreased values are depicted as −1/%change to compare fold changes in equivalent scalar magnitude regardless of directional change. *Statistically significant fold changes as detected by ANOVA followed by a post hoc paired t-test except for myosin heavy chain (MyHC)-2B, where all subjects demonstrated a decrease (P = 0.057), and MyHC-1β/slow, where all subjects demonstrated increased transcript levels (P = 0.103). MyHC-1β/slow was detected at background levels in the control muscle, and the stimulated values reflected “turn on” of this transcript. TROPN, troponin; TROPOMY 3G, tropomyosin 3γ; MYLC, myosin light chain; mCK, muscle creatine kinase; SOL, soleus muscle.

Fig. 5.

Immediate-early gene/stress response transcripts. Each of the bars indicates an increase of ≥1.5-fold change in the EDL muscle for the mean value ± SE of transcripts associated with an immediate-early gene response, including FOS, JUN-B, and early growth response 4 (EGR4). B cell translocation gene 2 (BTG2), musculoaponeurotic fibrosarcoma oncogene (MAFK), Dme1/homer1 (HOMER1), immediate-early response 5 (IER5), ephrin A₁ (EFNA1), JUN-D₁ (JUND), and heterogenous ribonucleoprotein D (HNRPD) were transcripts associated with a stress-activated response to extracellular stimulation. *Statistically significant change in either the EDL or soleus muscles.

Fig. 6.

Transcription factors associated with myofiber transformation. All displayed values (means ± SE) were statistically significant (P < 0.05) in the EDL muscle, including a cutoff of >1.5-fold change. The value for eyes absent 1 homolog (EYA1) represents a graphical maximum where fold change values were >20-fold based on expression levels in the unstimulated EDL muscle at or below threshold levels. PPARG, peroxisome proliferative activated receptor-γ; PPARGC1a, PPAR-γ coactivator 1α; PPARGC1b, PPAR-γ coactivator 1β; CLOCK, circadian locomotor output cycles kaput; BHLHB2, basic helix-loop-helix domain containing B2; HIF1a and HIF3a, hypoxia-inducible factor 1α and 3α; NFATc1 and NFATc4, nuclear factor of activated T cells 1 and 4; TEAD1, tea domain family member 1. *Statistically significant changes for transcripts in the soleus muscle.

Fig. 7.

Transcription factors associated with organogenesis/histogenesis induced by sciatic nerve stimulation. The classification of cellular development was significantly overrepresented among all transcription factors. These genes are characteristically involved in functions associated with tissue and organismal development, cell proliferation, and embryo formation. The transcripts presented were significantly elevated in the stimulated EDL muscle by >1.5-fold change but not in the soleus muslce. Kruppel-like factor 4 (KLF4), SRY box-containing gene 17 (SOX 17), inhibitor of DNA binding (ID), transcription factor 15 (TCF15), embryonic lethal, abnormal vision (ELAV), PBX/knotted 1 homeobox (PKNOX1), and tuberous sclerosis factor 1 (TSC1) were significantly elevated in stimulated EDL muscles. CRIPTO and TWISTED are murine homologs of Drosophila genes critically associated with mesoderm pattern formation, and transcripts for these molecules were significantly increased by stimulation.