Mitochondrial Permeability Transition Induces Atrophy in C2C12 Myotubes
Authors: Burke Sarah K. , Thome Trace T. , Ryan Terence E. , Hepple Russell T. 
Published: April 2020

INTRODUCTION

In advanced age (>75 yrs), muscle atrophy can lead to physical frailty, impaired mobility, and increased mortality. Impairment and functional decline of mitochondria within skeletal muscle are seen with aging, including a sensitization to mitochondrial permeability transition (MPT). MPT is an established pathological mechanism causing ischemic organ damage and has been observed in aged motoneurons and skeletal muscle. However, the role of MPT in age‐related muscle atrophy has not been determined. To better understand how MPT may induce skeletal muscle atrophy, we determined the impact of inducing MPT on myotube size in a cell culture model and the impact of modulating MPT on classical atrophy signaling transcripts. We hypothesized that induction of MPT in myotubes would cause atrophy, and MPT would modulate atrophy‐related signaling pathways.

METHODS

To determine if MPT can induce myotube atrophy, C2C12 myotubes were treated with doxorubicin (Dox; 1 μM), a known inducer of MPT, or DMSO (vehicle, 1 μM) on day 4 post‐differentiation for 12, 24, and 48 h. At each time point, cells were fixed/permeabilized in icecold methanol/acetone (1:1), blocked, and incubated with a primary antibody against sarcomeric myosin (MF‐20). Cells were incubated with a fluorescent secondary antibody and imaged at 20X on an inverted fluorescent microscope. Images (n=4 per well, 5 wells/treatment) were analyzed to quantify MF20+ area (myotube area) using a custom written program in CellProfiler.

To assess signaling mechanisms downstream of MPT, C2C12 myotubes were treated with Dox (1μM), Dox + an inhibitor of MPT (bongkrekic acid [BA]; DOX+BA, each 1 μM), or DMSO (vehicle;1 μM) for 12 h on day 4 post‐differentiation (n=3 biological replicates; n=4 technical replicates per treatment). At 12 h post‐treatment, cells were collected in TRIzol and their RNA isolated for RT‐PCR analysis of transcriptional regulators of muscle atrophy (FOXO1, NfKB1, Gadd45a, ATF4), an autophagy‐regulating gene (p62/SQSTM), and E3 ubiquitin ligases (Mafbx, MuRF). The comparative threshold cycle (CT) method was used to calculate fold changes in expression with 18s as an endogenous control. Relative fold changes in expression are presented as 2−ΔΔCT and normalized to DMSO‐treated samples.

Two‐tailed Student’s t‐tests were used to assess differences between DOX vs. DMSO, and DOX vs. DOX+BA, respectively. Significance was accepted at P<0.05.

RESULTS

DOX treatment significantly reduced myotube area by 24 h post‐treatment, and this persisted through 48 h (P<0.05). Inhibiting MPT with BA attenuated mRNA levels of transcription factors FOXO1 and ATF4, Gadd45a, the atrogene MuRF1, and the autophagy regulating gene p62 at 12 h post‐treatment (P<0.05 for all). There was no difference between DOX vs. DOX+BA for MAFbx (P=0.076) or NfKB1 (0.692) expression.

DISCUSSION

Consistent with our hypothesis, induction of MPT with DOX caused myotube atrophy. Additionally, inhibiting MPT in the presence of DOX attenuated mRNA levels of classical atrophy signaling pathway components, including FOXO1, ATF4, Gadd45α, MuRF1 and p62. Thus, our results suggest that MPT is a cause of atrophy in aging skeletal muscle.

Support or Funding Information

SB is supported by a T32 from the National Institute of Child Health and Human Development Grant T32HD043730.