Intensive stretch-activated CRT-PMCA1 feedback loop promoted apoptosis of myoblasts through Ca²⁺ overloading

Mechanical stretch exerted pro-apoptotic effect on myoblasts, the mechanism of which is currently unknown. Intracellular Ca²⁺ accumulation has been implicated in stretch-induced apoptosis. calreticulin (CRT) and plasma membrane Ca²⁺ transporting ATPase 1 (PMCA1) are two critical components of Ca²⁺ signaling system participating in intracellular Ca²⁺ homeostasis. In this study, we explored the contribution of CRT and PMCA1 in mediating stretch-induced Ca²⁺ accumulation and apoptosis of myoblasts. Stretching stimuli elevated level of CRT while inhibited activity of PMCA1. Moreover, there were bidirectional regulations between CRT and PMCA1, which formed the positive feedback loop leading to continuous increment of CRT level and repression of PMCA1 activity, in stretched myoblasts. Specifically, increased CRT level inhibited PMCA1 activity via suppressing Calmodulin (CaM), while reduced PMCA1 activity promoted CRT expression through activating p38MAPK pathway. Thus, the CRT-CaM-PMCA1 and PMCA1-p38MAPK-CRT pathways constituted a close cycle comprising CRT, PMCA1, CaM and p38MAPK. Inhibition of both CaM and p38MAPK affected the other three factors in stretched myoblasts. Circulation of the vicious cycle resulted in escalated Ca²⁺ overloading in myoblasts under continuous stretching stimuli. CRT knock-down, PMCA1 overexpression, and p38MAPK inhibition all attenuated the raised intracellular Ca²⁺ level and ameliorated myoblast apoptosis in the stretching environment. Conversely, CRT overexpression, PMCA1 knock-down, and CaM inhibition all aggravated stretch-induced Ca²⁺ overloading and myoblast apoptosis. A positive feedback loop between CRT and PMCA1 was activated in stretched myoblasts, which contributed to intracellular Ca²⁺ accumulation and resultant myoblast apoptosis.