Expression profiles of protein markers regulating the mitochondrial lifecycle in skeletal muscle of acute spinal cord transected rats
Authors: Hyatt Jon‐Philippe K. , Pullman Beau R. 
Published: May 2022

Spinal cord transection (ST) inactivates the neuromuscular complex and triggers progressive muscular atrophy of the affected skeletal muscles. Disruption in neural input also leads to a reduction in mitochondrial volume. The mitochondria lifecycle can be generally divided into processes of biogenesis, expression of OXPHOS proteins, fusion, fission, and mito/autophagy; specifically, the balance between biogenesis vs. mitophagy dictates the rate of formation and destruction, respectively. Here, we examined the expression profiles of the markers associated with the mitochondrial lifecycle up to one month following ST in phenotypically slow (soleus; SOL) and mixed (plantaris; PLT) skeletal muscle. We hypothesized that ST would induce a reduction in mitochondrial DNA (mtDNA) copy number, decrease expression in markers associated with biogenesis, and increased proteins that regulate mito/autophagy. Adult female Sprague Dawley rats were randomly divided into control (CON; n=6), 1 day (1dST; n=5), 8 day (8dST; n=8), and 28 day post‐ST (28dST; n=8). Compared to CON relative SOL and PLT muscle masses (absolute mass / body mass) were 98, 65, and 62% and 85, 60, and 73% at 1, 8 and 28d post‐ST, respectively (p<0.05). Compared to CON, no significant changes were observed in mtDNA copy number in ST muscles following amplification using standard end‐point PCR protocols for mtDNA (ND1, COX1, or ATP6) and nuclear (β2M) genes (p>0.05). Total protein was isolated from SOL and PLT, separated using standard SDS‐PAGE protocol, and probed for markers associated with various stages of the mitochondrial lifecycle using standard western blot protocols. Upstream markers known to influence mitochondrial lifecycle signaling (Rev‐Erbα, AMPK) showed a varied temporal and muscle‐specific response: in 1dST SOL muscles, Rev‐Erbα increased 74% from CON then decreased 43% at 28dST, whereas 8dST PLT muscle exhibited a 44% increase (p<0.05). In 8dST SOL and PLT muscles, the pAMPK:AMPK ratio was elevated 366 and 85% from CON, respectively (p<0.05). Markers for mitochondrial biogenesis (PGC‐1α, NRF1, NRF2) in SOL and PLT muscles were largely unchanged, although a ~50% decrease in Tfam protein expression was observed at each ST time point compared to CON (p<0.05). Mitofusion2, a marker for mitochondrial fusion, was unchanged (p>0.05). However, for markers detecting mitochondrial fission (MFF, Drp1, Fis1), we observed a ~60% increase in MFF expression in 1dST SOL and PLT muscles (p<0.05). As expected markers for mitophagy (Parkin, PINK, p62, LC3 I/II) were significantly elevated: Parkin increased ~70% at 28dST (p<0.05), whereas LC3 I/II increased 65% and 112% at 1dST in in SOL and PLT muscles, respectively (p<0.05), and remained significantly elevated in PLT muscles at 8dST (27%) and 28d (37%) (p<0.05). Taken together, these data suggest that mitochondrial volume and expression of markers regulating mitochondrial biogenesis and fusion are not influenced by acute ST despite a decrease in muscle mass. However, selective markers for fission and mitophagy are significantly elevated in both SOL and PLT muscles, suggesting that mitochondrial destruction mechanism are activated acutely following ST injury. Lastly, selective proteins of the OXPHOS protein complex significantly increased in 1dST SOL and PLT muscles suggesting possible activity‐dependent regulation of mitochondrial gene expression.