Tao Yu1, Mengli Wang1, Yadong Wen1, Yingya Cao1, Guanggui Shen1, Xiaogan Jiang1, Jingyi Wu1, Weihua Lu1, Xiaoju Jin2. 1. Department of Critical Care Medicine, Wannan Medical College First Affiliated Hospital, Yijishan Hospital, Wuhu, China. 2. Department of Critical Care Medicine, Wannan Medical College First Affiliated Hospital, Yijishan Hospital, Wuhu, China. Electronic address: jinxj2014@163.com.
Abstract
BACKGROUND: Our previous study demonstrated that ventilators increase diaphragmatic lipid accumulation in rabbits, but their cellular mechanism is poorly understood. Mammalian target of rapamycin (mTOR) plays an important role in atherosclerosis in rat vascular smooth muscle cells. The present study investigated the role of mTOR pathway activation in the diaphragmatic muscle of ventilated rats and hypoxia-induced C2C12 cells. MATERIALS AND METHODS: Male Sprague-Dawly rats were randomized into a control group (n = 8), controlled mechanical ventilation (CMV) group (n = 8), and CMV + Rapa group (n = 8). We evaluated the diaphragmatic contractility, lipid accumulation, and protein expression of the mTOR pathways. To explore the mechanism underlying ventilator-induced lipid accumulation, we observed protein expression of the mTOR and low-density lipoprotein receptor (LDLr) pathways in C2C12 cells under hypoxic and mTOR pathway inhibitor treatments. RESULTS: Compared with the control group, there was a significant decrease in the peak twitch and peak tetanic forces in the CMV group (384.24 ± 70.39 versus 496.33 ± 78.64 g/cm2, P < 0.05, and 869.24 ± 76.67 versus 1090.72 ± 118.91 g/cm2, P < 0.05, respectively). There was a significant increase in peak twitch and peak tetanic forces in the CMV + Rapa group compared with that in the CMV group (501.81 ± 23.15 versus 384.24 ± 70.39 g/cm2, P < 0.05, and 992.91 ± 88.99 versus 869.24 ± 76.67 g/cm2, P < 0.05, respectively). In the CMV group, there were significant increases in lipid accumulation (0.086 ± 0.009 versus 0.005 ± 0.002, P < 0.05) and expression of mTOR in diaphragmatic fibers compared with those in the control group (P < 0.05). Rapamycin prevented lipid accumulation in rats of the CMV + Rapa group compared with that in the CMV group rats (0.024 ± 0.004 versus 0.086 ± 0.009, P < 0.05). Compared with the CMV group, there was a significant decrease in the phosphorylated protein expression levels of mTOR in rats of the CMV + Rapa group (P < 0.05). Hypoxic conditions activated the mTOR and LDLr pathways in C2C12 cells, which were correlated with an increase in expression of the mTOR and LDLr pathways compared with the control group (P < 0.05). In C2C12 cells treated with hypoxia + rapamycin, activation of the mTOR and LDLr pathways was blocked compared with C2C12 cells treated with hypoxia (P < 0.05). CONCLUSIONS: These data suggest that CMV and hypoxia-induced activation of the mTOR pathway, resulting in lipid accumulation, and impaired the diaphragmatic contractile function. Therefore, pharmacologic agents that inhibit the mTOR pathway could potentially be useful for mitigating the diaphragmatic contractile dysfunction induced by mechanical ventilation.
BACKGROUND: Our previous study demonstrated that ventilators increase diaphragmatic lipid accumulation in rabbits, but their cellular mechanism is poorly understood. Mammalian target of rapamycin (mTOR) plays an important role in atherosclerosis in rat vascular smooth muscle cells. The present study investigated the role of mTOR pathway activation in the diaphragmatic muscle of ventilated rats and hypoxia-induced C2C12 cells. MATERIALS AND METHODS: Male Sprague-Dawly rats were randomized into a control group (n = 8), controlled mechanical ventilation (CMV) group (n = 8), and CMV + Rapa group (n = 8). We evaluated the diaphragmatic contractility, lipid accumulation, and protein expression of the mTOR pathways. To explore the mechanism underlying ventilator-induced lipid accumulation, we observed protein expression of the mTOR and low-density lipoprotein receptor (LDLr) pathways in C2C12 cells under hypoxic and mTOR pathway inhibitor treatments. RESULTS: Compared with the control group, there was a significant decrease in the peak twitch and peak tetanic forces in the CMV group (384.24 ± 70.39 versus 496.33 ± 78.64 g/cm2, P < 0.05, and 869.24 ± 76.67 versus 1090.72 ± 118.91 g/cm2, P < 0.05, respectively). There was a significant increase in peak twitch and peak tetanic forces in the CMV + Rapa group compared with that in the CMV group (501.81 ± 23.15 versus 384.24 ± 70.39 g/cm2, P < 0.05, and 992.91 ± 88.99 versus 869.24 ± 76.67 g/cm2, P < 0.05, respectively). In the CMV group, there were significant increases in lipid accumulation (0.086 ± 0.009 versus 0.005 ± 0.002, P < 0.05) and expression of mTOR in diaphragmatic fibers compared with those in the control group (P < 0.05). Rapamycin prevented lipid accumulation in rats of the CMV + Rapa group compared with that in the CMV group rats (0.024 ± 0.004 versus 0.086 ± 0.009, P < 0.05). Compared with the CMV group, there was a significant decrease in the phosphorylated protein expression levels of mTOR in rats of the CMV + Rapa group (P < 0.05). Hypoxic conditions activated the mTOR and LDLr pathways in C2C12 cells, which were correlated with an increase in expression of the mTOR and LDLr pathways compared with the control group (P < 0.05). In C2C12 cells treated with hypoxia + rapamycin, activation of the mTOR and LDLr pathways was blocked compared with C2C12 cells treated with hypoxia (P < 0.05). CONCLUSIONS: These data suggest that CMV and hypoxia-induced activation of the mTOR pathway, resulting in lipid accumulation, and impaired the diaphragmatic contractile function. Therefore, pharmacologic agents that inhibit the mTOR pathway could potentially be useful for mitigating the diaphragmatic contractile dysfunction induced by mechanical ventilation.