Examining the Effect of Doxorubicin on Apoptotic Signaling in Skeletal Muscle of ARC KO Mice

Abstract

Apoptosis repressor with caspase recruitment domain (ARC) is an anti‐apoptotic protein that inhibits both the intrinsic and extrinsic apoptotic pathways. Along with its potent anti‐apoptotic actions, ARC has a unique tissue expression with high levels found in skeletal muscle, cardiac muscle, neurons, as well as some cancers. Within skeletal muscle, ARC content is fiber‐type specific with highest expression in slow‐twitch fibers. Despite its high expression, ARC's anti‐apoptotic role in skeletal muscle is not fully understood. To determine whether ARC contributes to apoptotic resistance, wild‐type (WT) and ARC knockout (KO) mice were given intraperitoneal injections of doxorubicin (DOX) (20 mg/kg) or saline. 24 hrs post injection, mice were sacrificed, the gastrocnemius was removed, and separated into red (RG) and white (WG) portions. DOX treated mice lost significantly more weight than saline controls (p<0.0001); however, whole gastrocnemius weights did not change. In RG, DOX increased ROS (p<0.01), with post hoc analysis revealing a significant increase between KO saline and KO DOX mice (p<0.05). DOX treatment significantly increased caspase‐9 (p<0.01) and calpain (p<0.05) activity; however, caspase‐8 and ‐3 activity remained unchanged. There was also a significant increase in calpain activity between KO saline and KO DOX treated mice (p<0.05). In WG, DOX had no influence on ROS levels. Interestingly, there was an increase in caspase‐3 activity in KO DOX and KO saline treated mice (p<0.05) despite no change in upstream initiator caspases. Taken together, 24 hr DOX treatment affected apoptotic signaling in RG to a greater extent than in WG. Furthermore, a lack of ARC led to higher ROS, as well as increased calpain activity in RG following DOX treatment.