Altered mRNA expression after long-term soleus electrical stimulation training in humans with paralysis

Abstract

In humans, spinal cord injury (SCI) induces deleterious changes in skeletal muscle that may be prevented or reversed by electrical stimulation muscle training. The molecular mechanisms underlying muscle stimulation training remain unknown. We studied two unique SCI subjects whose right soleus received >6 years of training (30 minutes/day, 5 days/week). Training preserved torque, fatigue index, contractile speed, and cross-sectional area in the trained leg, but not the untrained leg. Training decreased 10 mRNAs required for fast-twitch contractions and mRNA that encodes for myostatin, an autocrine/paracrine hormone that inhibits muscle growth. Conversely, training increased 69 mRNAs that mediate the slow-twitch, oxidative phenotype, including PGC-1α, a transcriptional coactivator that inhibits muscle atrophy. When we discontinued right soleus training, training-induced effects diminished slowly, with some persisting for >6 months. Training of paralyzed muscle induces localized and long-lasting changes in skeletal muscle mRNA expression that improve muscle mass and function.

Figure 1

Study design. Both subjects were paraplegic secondary to SCI and began training their right lower legs within six weeks of the acute injury. They continued to train the right lower legs for > 6 years prior to this study, which began at 0 months. At 0 and 6 months, we obtained bilateral soleus biopsies from each subject. We then discontinued right soleus training and obtained bilateral soleus biopsies two months and six months later.

Figure 2

Effect of right lower leg training on muscle physiology and cross-sectional area (CSA). (A-C) Soleus physiologic parameters for subject 1 (left column) and subject 2 (right column). (A) Representative examples of contractions 1 and 125 in trained and untrained conditions. (B) Soleus torque, with fatigue index (FI) values listed. (C) Normalized soleus torque rise time. (D) MRI images of trained and untrained lower legs from subject 2. The graph on the right quantifies plantar flexor CSA. Because of their single-joint attachment sites, the deep flexors also received the training stimulus.

Figure 3

Effects of long-term soleus training on soleus mRNA levels. (A-B) Global analysis of mRNA levels by exon expression arrays. Each right (trained) soleus mRNA level was normalized to the mRNA level in the paired left (untrained) soleus biopsy sample, which was arbitrarily set at 1. (A) The 10 most highly repressed mRNAs in the right soleus. (B) The 10 most highly induced mRNAs in the right soleus. (C-D) qPCR analysis of ACTN3, MSTN, MYH7 and MYL3 mRNA levels. Each right (trained) soleus mRNA level was normalized to the mRNA level in the paired left (untrained) soleus biopsy sample, which was arbitrarily set at 1 and indicated by the dashed lines. (A-D) Data are means ± SEM for both subjects from repeat baseline biopsy samples, and P ≤ 0.05 for all mRNAs shown.

Figure 4

Effects of soleus muscle training on levels of mRNAs that encode proteins involved in fast twitch muscle contraction (A), proteins involved in slow twitch muscle contraction (B), enzymes that promote glucose utilization (C), and subunits of the electron transport chain (D). Each right (trained) soleus mRNA level was determined using exon expression arrays, and normalized to the mRNA level in the paired left (untrained) soleus biopsy sample, which was set at 1. Data are means ± SEM for both subjects from repeat baseline biopsy samples. P ≤ 0.05 for all mRNAs shown.

Figure 5

Effect of two and six months detraining on right soleus mRNA levels for ACTN3 and MSTN (A) and for MYH7 and MYL3 (B). Each right (trained) soleus mRNA level was normalized to the mRNA level in the paired left (untrained) soleus biopsy sample, which was set at 1. Data are the mean for both subjects at each time point.

Figure 6

Effect of six months of right leg detraining on muscle physiology and cross-sectional area (CSA). (A-C) Soleus physiologic parameters for subject 1 (left column) and subject 2 (right column). (A) Representative examples of contractions 1 and 125 in trained and detrained conditions for the right limb. (B) Soleus torque, with fatigue index (FI) values listed. (C) Normalized soleus torque rise time. (D) MRI images of untrained (left) and detrained (right) lower legs from subject 2. The graph on the right quantifies plantar flexor CSA.