Abstract
Myotube apoptosis occurs normally during muscle development and aging but it can lead to destruction of skeletal muscle in neuromuscular diseases. Therefore, understanding how myotube apoptosis is regulated is important for developing novel strategies for treatment of muscle loss. We investigated the regulation of apoptosis in skeletal muscle and report a striking increase in resistance to apoptosis following differentiation. We find mitotic C2C12 cells (myoblast-like cells) are sensitive to cytosolic cytochrome c microinjection. However, differentiated C2C12 cells (myotube-like cells) and primary myotubes are markedly resistant. This resistance is due to endogenous X-linked inhibitor of apoptotic protein (XIAP). Importantly, the selective difference in the ability of XIAP to block myotube but not myoblast apoptosis is not due to a change in XIAP but rather a decrease in Apaf-1 expression. This decrease in Apaf-1 links XIAP to caspase activation and death. Our findings suggest that in order for myotubes to die, they may degrade XIAP, functionally inactivate XIAP or upregulate Apaf-1. Importantly, we identify a role for endogenous Smac in overcoming XIAP to allow myotube death. However, in postmitotic cardiomyocytes, where XIAP also restricts apoptosis, endogenous Smac was not capable of overcoming XIAP to cause death. These results show that as skeletal muscle differentiate, they become resistant to apoptosis because of the ability of XIAP to regulate caspase activation. The increased restriction of apoptosis in myotubes is presumably important to ensure the long term survival of these postmitotic cells as they play a vital role in the physiology of organisms.
Highlights
Skeletal muscle is a highly specialized tissue that is unique in its structure and development
To determine whether cytochrome c-mediated caspase activation becomes more restricted with skeletal muscle differentiation, we microinjected cytochrome c in C2C12 cells and myotubes
MC2C12 cells injected with yeast cytochrome c did not die, indicating that microinjection alone was not killing these cells (Fig. 1a, b)
Summary
Skeletal muscle is a highly specialized tissue that is unique in its structure and development. While the molecular details of this differentiation process are well understood, very little is known about whether fundamental biological processes such as apoptosis are altered during this process of differentiation Understanding this phenomenon is important because, following the developmental period, muscle loss can have deleterious effects. Apoptosis is a genetically regulated, evolutionarily conserved form of cell death It is characterized by the activation of caspase proteases that cleave numerous substrates within the cell to cause the demise of the cell [3]. Once free in the cytosol, cytochrome c binds to the adapter protein Apaf-1 This binding induces a conformational change in Apaf-1 in such a way that Apaf-1 oligomerizes as well as binds to procaspase 9 to form the apoptosome complex. Active caspase 3 is known as the executioner caspase because it cleaves various proteins leading to the death of the cell [4,5]
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