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Published on: June 9, 2021
Mitochondria-associated apoptotic signalling in denervated rat skeletal muscle
1Laboratory of Muscle Biology an Sarcopenia, Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown WV 26506-9227, USA.
Abstract:
Apoptosis has been implicated in the regulation of denervation-induced muscle atrophy. However, the activation of apoptotic signal transduction during muscle denervation has not been fully elucidated. The present study examined the apoptotic responses to denervation in rat gastrocnemius muscle. Following 14 days of denervation, the extent of apoptotic DNA fragmentation as determined by a cytosolic nucleosome ELISA was increased by 100% in the gastrocnemius muscle. RT-PCR and immunoblot analyses indicated that Bax was dramatically upregulated while Bcl-2 was modestly increased; however, the Bax/Bcl-2 ratio was significantly increased in denervated muscles relative to control muscles. Analyses of ELISA and immunoblots from mitochondria-free cytosol extracts showed a significant increase in mitochondria-associated apoptotic factors, including cytochrome c, Smac/DIABLO and apoptosis-inducing factor (AIF). In addition to the upregulation of caspase-3 and -9 mRNA, pro-/cleaved caspase protein and proteolytic activity levels, the X-linked inhibitor of apoptosis (XIAP) protein level was downregulated. The cleaved product of poly(ADP-ribose) polymerase (PARP) was detected in muscle samples following denervation. Although we did not find a difference in the inhibitor of DNA binding/differentiation-2 (Id2) and c-Myc protein contents between the denervated and control muscles, the protein content of tumour suppressor p53 was significantly increased in both the nuclear and the cytosolic fractions with denervation. Moreover, denervation increased the protein content of HSP70, whereas the MnSOD (a mitochondrial isoform of superoxide dismutase) protein content was diminished, which indicated that denervation might have induced cellular and/or oxidative stress. Our data show that mitochondria-associated apoptotic signalling is upregulated during muscle denervation. We interpret these findings to indicate that apoptosis has a physiologically important role in regulating denervation-induced muscle atrophy.
Insights
Denervation significantly increases apoptosis in rat gastrocnemius muscle by upregulating pro-apoptotic factors and releasing mitochondrial apoptotic signaling molecules. This highlights apoptosis
Area of Science:
- Muscle physiology
- Cellular biology
- Molecular mechanisms of apoptosis
Background:
- Apoptosis plays a role in denervation-induced muscle atrophy.
- The precise apoptotic signaling pathways activated during muscle denervation remain unclear.
Purpose of the Study:
- To investigate the apoptotic responses to denervation in rat gastrocnemius muscle.
- To elucidate the molecular mechanisms of apoptosis activation during muscle denervation.
Main Methods:
- Rat gastrocnemius muscle denervation model.
- Cytosolic nucleosome ELISA for DNA fragmentation.
- RT-PCR and immunoblotting for gene and protein expression analysis.
- Mitochondrial fractionation and analysis of apoptotic factors.
Main Results:
- Denervation for 14 days increased DNA fragmentation by 100%.
- Bax was upregulated, Bcl-2 was modestly increased, leading to a higher Bax/Bcl-2 ratio.
- Mitochondria-associated apoptotic factors (cytochrome c, Smac/DIABLO, AIF) significantly increased.
- Caspase-3 and -9 were upregulated, XIAP downregulated, and cleaved PARP detected.
- Tumor suppressor p53 increased, while MnSOD decreased, suggesting oxidative stress.
Conclusions:
- Mitochondria-associated apoptotic signaling is upregulated during muscle denervation.
- Apoptosis plays a significant physiological role in regulating denervation-induced muscle atrophy.
