A minimal dose of electrically induced muscle activity regulates distinct gene signaling pathways in humans with spinal cord injury

Abstract

Paralysis after a spinal cord injury (SCI) induces physiological adaptations that compromise the musculoskeletal and metabolic systems. Unlike non-SCI individuals, people with spinal cord injury experience minimal muscle activity which compromises optimal glucose utilization and metabolic control. Acute or chronic muscle activity, induced through electrical stimulation, may regulate key genes that enhance oxidative metabolism in paralyzed muscle. We investigated the short and long term effects of electrically induced exercise on mRNA expression of human paralyzed muscle. We developed an exercise dose that activated the muscle for only 0.6% of the day. The short term effects were assessed 3 hours after a single dose of exercise, while the long term effects were assessed after training 5 days per week for at least one year (adherence 81%). We found a single dose of exercise regulated 117 biological pathways as compared to 35 pathways after one year of training. A single dose of electrical stimulation increased the mRNA expression of transcriptional, translational, and enzyme regulators of metabolism important to shift muscle toward an oxidative phenotype (PGC-1α, NR4A3, IFRD1, ABRA, PDK4). However, chronic training increased the mRNA expression of specific metabolic pathway genes (BRP44, BRP44L, SDHB, ACADVL), mitochondrial fission and fusion genes (MFF, MFN1, MFN2), and slow muscle fiber genes (MYH6, MYH7, MYL3, MYL2). These findings support that a dose of electrical stimulation (∼10 minutes/day) regulates metabolic gene signaling pathways in human paralyzed muscle. Regulating these pathways early after SCI may contribute to reducing diabetes in people with longstanding paralysis from SCI.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. A representative example of the phenotype for a trained and untrained human paralyzed muscle.

(A) A representative example of the torque produced during the stimulation of a chronically paralyzed human soleus muscle, contractions 1, 15, 60, and 120 during the first bout of electrical stimulation are illustrated. (B) The ratio of muscle to adipose tissue from several MR images slices of the proximal shank and distal thigh after >7 years of unilateral soleus electrical stimulation training in subject 1. A representative MR Image slice of the trained and untrained lower leg before (C) and after (D) implementing the muscle and fat tissue segmentation algorithm. Immunofluorescence stain for collagen IV (green) in a chronically trained (E) and untrained (F) paralyzed muscle. Note the loss of collagen IV (green) in the chronically trained muscle. Immunofluorescence stain (green) for mitochondrial distribution in a trained (G) and untrained (H) paralyzed muscle.

Figure 2. Gene Ontology(GO) Biological Process pathways upregulated by gene set enrichment analysis(GSEA) compared to non-stimulated limb.

117 pathways, primarily consisting of intra- and inter- cellular signaling pathways were upregulated 3-hours after a single session of exercise using electrical muscle stimulation (Acute Soleus Stimulation). 35 pathways, primarily consisting of metabolic pathways, were upregulated in subjects that trained >7 years using electrical muscle stimulation (Chronic Soleus Stimulation).

Figure 3. Expression of transcription factor, fast-twitch fiber, and slow-twitch fiber genes following acute or chronic stimulation.

PGC-1α was increased 3 hours after a dose of muscle stimulation (5.46±0.64, p1 year of muscle training (1.73±0.09, p1 year of muscle training (0.79±0.06, p = 0.046) (B). ABRA was increased after a single dose of muscle stimulation (5.98±0.40, p1 year of soleus training (0.66±0.18, p1 year of muscle training (0.33±0.03, p1 year of muscle training (E, F, and G). There was no difference detected 3 hours after a dose of muscle stimulation for MYL5 (1.09±0.083, p = 0.45). MYL6 (0.95±0.048, p = 0.32), and ACTN3 (0.99,0.095, p = 0.72) (E, F, and G). PVALB was increased after a single dose of muscle stimulation (1.47±0.22, p = 0.074), but was decreased after >1 year of muscle training (0.26±0.19, p = 0.047) (H). MYH6 (6.76±2.50, p = 0.030), MYH7 (11.69±4.93, p = 0.025), MYL2 (2.78±0.80, p = 0.063), and MYL3 (9.07±3.75, p = 0.046) were increased after >1 year of muscle training, while they were decreased 3 hours after single session of muscle stimulation (0.81±0.04, p = 0.0073, 0.77±0.073, p = 0.030, 0.92±0.036, p = 0.066, 0.76±0.078, p = 0.037; respectively) (I, J, K, and L). † indicates a p-value

Figure 4. Expression of glycolysis and fatty acid oxidation genes following acute or chronic stimulation.

PDK4 was increased 3 hours after a single session of muscle stimulation (3.37±0.83, p = 0.008), but was unchanged after >1 year of soleus training (1.55±0.35, p = 0.21) (A). PDHA1 (1.60±0.057, p1 year of muscle training, but were unchanged 3 hours after a single session of muscle stimulation (1.05±0.05, p = 0.46, 1.11±0.09, p = 0.35, 1.09±0.13, p = 0.59; respectively) (B, C, and D). ACADVL (0.93±0.03, p = 0.064) was decreased 3 hours after a single session of muscle stimulation, but was increased after >1 year of muscle training (1.63±0.049, p = 0.049) (E). ACADL (0.94±0.031, p = 0.098) was decreased 3 hours after a single session of muscle stimulation and after >1 year of muscle training (0.80 and ACADL (0.94±0.031, p = 0.098) were decreased 3 hours after a single session of muscle stimulation 0.044, p = 0.025) (F). ACAD8 (1.33±0.089, p = 0.023) and ACAD9 (1.16±0.023, p = 0.006) were increased after >1 year of muscle training, but were unchanged 3 hours after a single dose of muscle stimulation (0.96±0.042, p = 0.33, 0.96±0.06, p = 0.39; respectively) (G and H). † indicates a p-value

Figure 5. Expression of tricarboxylic acid cycle, oxidative phosphorylation, and mitochondrial fission/fusion genes following acute or chronic stimulation.

BRP44 (1.55±0.17, p = 0.034), BRP44L (1.55±0.19, p = 0.036), OGDH (1.50±0.092, p = 0.007), and SDHB (1.54±0.081, p = 0.004) were increased after >1 year of muscle training, but were unchanged 3 hours after a single dose of muscle stimulation (1.14±0.099, p = 0.25, 1.02±0.093, p = 0.97, 0.97±0.067, p = 0.58, 1.10±0.16, p = 0.74; respectively) (A, B, C, and D). NDUFB1 (1.22±0.088, p = 0.067), NDUFA2 (1.40±0.11, p = 0.022), and CYC1 (1.34±0.13, p = 0.066) were increased after >1 year of muscle training, but were unchanged 3 hours after a single dose of muscle stimulation (0.98±0.02, p = 0.28, 1.03±0.05, p = 0.72, 0.95±0.03, p = 0.10; respectively) (E, F, and G). COQ10A was increased after >1 year of muscle training (1.49±0.14, p = 0.024), but was decreased 3 hours after a single dose of muscle stimulation (0.79±0.021, p1 year of muscle training, but were unchanged 3 hours after a single dose of muscle stimulation (0.95±0.31, p = 0.31, 1.00±0.78, p = 0.77, 0.97±0.54, p = 0.54); respectively) (I, J, and L). MFN1 was unchanged after >1 year of muscle training (1.36±0.25, p = 0.22) and 3 hours after a single dose of muscle stimulation (1.18±0.16, p = 0.42) (K). † indicates a p-value

Figure 6. Confirmatory qPCR of a subset of genes important for metabolic and hypertrophy pathways in muscle.

qPCR analysis of mean ABRA (A), EGR1(B), NR4A3(C), MYH7(D), and MSTN(E) levels for acutely stimulated muscles (black) and chronically trained muscles (white). (A–E) Data represented as mean ± standard error.