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F U L L T I T L E : Nifedipine-induced AMPK activation alleviates senescence by increasing autophagy and suppressing of Ca2+ levels in vascular smooth muscle cells.
Highlights
• Calcium channel blocker, nifedipine suppresses senescence-related phenotypes.
• Oxidative stress-induced senescence was accompanied by Ca2+ elevation and impairment of autophagic flux.
• Nifedipine alleviates VSMC senescence by regulating Ca2+ influx and increasing autophagy.
• AMPK inhibition showed that the effect of nifedipine involved AMPK activation.
• Nifedipine-activated AMPK suppresses VSMC senescence by regulating Ca2+ levels and autophagic flux.
Abstract
Calcium (Ca2+) homeostasis is disrupted during aging in several cell types and this disruption leads to autophagy impairment. The mechanisms regarding Ca2+, senescence, and autophagy need to be elucidated. Therefore, we hypothesized that cellular senescence can be improved by regulating Ca2+ level and autophagy activity. We identified that hydrogen peroxide (H2O2)-induced senescence was accompanied by Ca2+ elevation, impairment of autophagic flux and increase of mammalian target of rapamycin (mTOR) phosphorylation in VSMCs. The treatment of nifedipine dose-dependently suppressed H2O2-induced senescence by reducing Ca2+ entry, autophagy impairment and mTOR signaling, and this suppression was found to be related to senescence-associated β-galactosidase (SA-β-gal) activity and the expressions of senescence marker protein 30 (SMP30), p53, and p21. Furthermore, H2O2-induced autophagy impairment also accelerated senescence and accumulations of ubiquitinated proteins. AMPK inhibition or transfection with AMPK siRNA showed that the anti-senescence effect of nifedipine involved AMPK activation. These results suggest nifedipine-inducted AMPK activation suppresses VSMC senescence by regulating autophagic flux and Ca2+ levels.
Abbreviations
AMPK: AMP-activated protein kinase
SMP30: senescence marker protein 30
3-MA: 3-methyladenine
Baf A1: bafilomycin A1
mTOR: mammalian target of rapamycin
C.C: compound C
SA-β-gal: senescence-associated β-galactosidase
Outline
1. Introduction
2. Materials and methods
2.1. Reagents and antibodies
2.2. Primary cell culture
2.3. Induction of senescence and drug treatment
2.4. Western blot analysis
2.5. Transfection of siRNA
2.6. Senescence-associated β-galactosidase (SA-β-gal) staining
2.7. Intracellular Ca2+ measurements
2.8. Measurement of intracellular reactive oxygen species (ROS)
2.9. Immunofluorescence analysis
2.10. Lysosomal staining assay
2.11. Statistical analysis
3. Results
3.1. Nifedipine inhibits oxidative stress-induced cellular senescence in VSMCs
3.2. Nifedipine attenuates [Ca2+]i levels and induces autophagy in senescent VSMCs
3.3. Inhibition of autophagy exacerbates VSMC senescence
3.4. Nifedipine regulates [Ca2+]i and autophagy activity through AMPK
3.5. Nifedipine alleviates VSMC senescence through AMPK-autophagy pathway
4. Discussion
Declaration of Competing Interest
Acknowledgments
References
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