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  2. V-ATPase V0 subunit activation mediates maduramicin-induced methuosis through blocking endolysosomal trafficking in vitro and in vivo

V-ATPase V0 subunit activation mediates maduramicin-induced methuosis through blocking endolysosomal trafficking in vitro and in vivo

  • Food Chem Toxicol. 2023 Jun 30;178:113922. doi: 10.1016/j.fct.2023.113922.
Yuling Zheng 1 Jing Xiao 1 Junqi Wang 1 Bin Dong 1 Dawei Guo 1 Hui Ji 1 Haifeng Sun 2 Lin Peng 1 Shanxiang Jiang 1 Xiuge Gao 3
Affiliations

Affiliations

  • 1 Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, 1 Weigang, Nanjing, 210095, PR China; Center for Veterinary Drug Research and Evaluation, Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Nanjing Agricultural University, 1 Weigang, Nanjing, 210095, PR China.
  • 2 Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.
  • 3 Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, 1 Weigang, Nanjing, 210095, PR China; Center for Veterinary Drug Research and Evaluation, Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Nanjing Agricultural University, 1 Weigang, Nanjing, 210095, PR China. Electronic address: vetgao@njau.edu.cn.
Abstract

Methuosis, a novel cell death phenotype, is characterized by accumulation of cytoplasmic vacuolization upon external stimulus. Methuosis plays a critical role in maduramicin-induced cardiotoxicity despite the underlying mechanism is largely unknown. Herein, we aimed to investigate the origin and intracellular trafficking of cytoplasmic vacuoles, as well as the molecular mechanism of methuosis caused by maduramicin (1 μg/mL) in myocardial cells. H9c2 cells and broiler chicken were used and were exposed to maduramicin at doses of 1 μg/mL in vitro and 5 ppm-30 ppm in vivo. Morphological observation and dextran-Alexa Fluor 488 tracer experiment showed that endosomal compartments swelling and excessive macropinocytosis contributed to madurdamcin-induced methuosis. Cell counting kit-8 assay and morphology indicated pharmacological inhibition of macropinocytosis largely prevent H9c2 cells from maduramicin-triggered methuosis. In addition, late endosomal marker Rab7 and lysosomal associated membrane protein 1 (LAMP1) increased in a time-dependent manner after maduramicin treatment, and the recycling endosome marker Rab11 and ADP-ribosylation factor 6 (Arf6) were decreased by maduramicin. Vacuolar-H+-ATPase (V-ATPase) was activated by maduramicin, and pharmacological inhibition and genetic knockdown V0 subunit of V-ATPase restore endosomal-lysosomal trafficking and prevent H9c2 cells methuosis. Animal experiment showed that severe cardiac injury included the increase of creatine kinase (CK) and creatine kinase-MB (CK-MB), and vacuolar degeneration resembled methuosis in vivo after maduramicin treatment. Taken together, these findings demonstrate that targeting the inhibition of V-ATPase V0 subunit will prevent myocardial cells methuosis by restoring endosomal-lysosomal trafficking.

Keywords

Cardiotoxicity; Cytoplasmic vacuolization; Maduramicin; Methuosis; Nonapoptotic cell death; V-ATPase.

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