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

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Abstract

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.

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Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

CRT:

Calreticulin

PMCA1:

Plasma membrane Ca2+ transporting ATPase 1

ER:

Endoplasmic reticulum

TUNEL:

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling

DAPI:

4′,6-Diamidino-2-phenylindole

FACS:

Fluorescence activated cell sorting

FITC:

Fluorescein isothiocyanate

CaM:

Calmodulin

OE:

Overexpressing:

KD:

Knock-down

EGTA:

Ethylene glycol tetra-acetic acid

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Acknowledgements

The authors thank the laboratory members of Central Laboratory of Affiliated Hospital of Qingdao University for their help and instructions.

Funding

This conduct of this work was financially supported by the National Natural Science Foundation of China (No. 31870929).

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Authors and Affiliations

  1. Department of Stomatology Medical Center, Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China

    Dapeng Ren, Ran liu, Xiao Yan, Qiang Zhang, Xuemin Zeng & Xiao Yuan

  2. Central Laboratory of Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China

    Dapeng Ren, Ran liu, Xiao Yan, Qiang Zhang, Xuemin Zeng & Xiao Yuan

  3. Department of Orthodontics, School of Stomatology, Qingdao University, Qingdao, China

    Dapeng Ren, Xiao Yan, Qiang Zhang, Xuemin Zeng & Xiao Yuan

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  1. Dapeng Ren
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  2. Ran liu
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  4. Qiang Zhang
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  5. Xuemin Zeng
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Contributions

Conceptualization: YX, RD; Experiment performance: LR, YX and ZQ; Material preparation, data collection and analysis: ZX. The first draft of the manuscript was written by RD and all authors commented on previous versions of the manuscript.

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Correspondence to Xiao Yuan.

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Ren, D., liu, R., Yan, X. et al. Intensive stretch-activated CRT-PMCA1 feedback loop promoted apoptosis of myoblasts through Ca2+ overloading. Apoptosis 27, 929–945 (2022). https://doi.org/10.1007/s10495-022-01759-4

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