Downs R M, Hughes M A, Kinsey S T, Johnson M C, Baumgarner B L
Division of Natural Sciences and Engineering, University of South Carolina Upstate, Spartanburg, SC, USA.
Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, NC, USA.
Biochem Biophys Res Commun. 2016 Nov 4;480(1):61-68. doi: 10.1016/j.bbrc.2016.10.008. Epub 2016 Oct 4.
Caffeine is a widely consumed stimulant that has previously been shown to promote cytotoxic stress and even cell death in numerous mammalian cell lines. Thus far there is little information available regarding the toxicity of caffeine in skeletal muscle cells. Our preliminary data revealed that treating C2C12 myotubes with 5 mM caffeine for 6 h increased nuclear fragmentation and reduced basal and maximal oxygen consumption rate (OCR) in skeletal myotubes. The purpose of this study was to further elucidate the pathways by which caffeine increased cell death and reduced mitochondrial respiration. We specifically examined the role of c-Jun N-terminal kinase (JNK), which has previously been shown to simultaneously increase caspase-dependent cell death and reduce mitochondrial respiration in other mammalian cell lines. We found that caffeine promoted a dose-dependent increase in cell death in multinucleated myotubes but did not in mononucleated myoblasts. The addition of 10 μM Z-DEVD-FMK, a specific inhibitor of executioner caspases, completely inhibited caffeine-dependent cell death. Further, the addition of 400 μM dantrolene, a specific ryanodine receptor (RYR) inhibitor, prevented the caffeine-dependent increase in cell death and the reduction in basal and maximal OCR. We also discovered that caffeine treatment significantly increased the phosphorylation of JNK and that the addition of 30 μM SP600125 (JNKi), a specific JNK inhibitor, partially attenuated caffeine-induced cell death without preventing the caffeine-dependent reduction in basal and maximal OCR. Our results suggest that JNK partially mediates the increase in caspase-dependent cell death but does not contribute to reduced mitochondrial respiration in caffeine-treated skeletal muscle cells. We conclude that caffeine increased cell death and reduced mitochondrial respiration in a calcium-dependent manner by activating the RYR and promoting reticular calcium release.
咖啡因是一种被广泛摄入的兴奋剂，此前已证明它能在多种哺乳动物细胞系中引发细胞毒性应激甚至细胞死亡。到目前为止，关于咖啡因对骨骼肌细胞毒性的信息很少。我们的初步数据显示，用5 mM咖啡因处理C2C12肌管6小时会增加细胞核碎片化，并降低骨骼肌肌管的基础和最大氧消耗率（OCR）。本研究的目的是进一步阐明咖啡因增加细胞死亡和降低线粒体呼吸的途径。我们特别研究了c-Jun氨基末端激酶（JNK）的作用，此前已证明它在其他哺乳动物细胞系中能同时增加半胱天冬酶依赖性细胞死亡并降低线粒体呼吸。我们发现咖啡因能促进多核肌管中细胞死亡的剂量依赖性增加，但对单核成肌细胞则无此作用。添加10 μM Z-DEVD-FMK（一种执行半胱天冬酶的特异性抑制剂）可完全抑制咖啡因依赖性细胞死亡。此外，添加400 μM丹曲林（一种特异性兰尼碱受体（RYR）抑制剂）可防止咖啡因依赖性细胞死亡增加以及基础和最大OCR的降低。我们还发现咖啡因处理显著增加了JNK的磷酸化，添加30 μM SP600125（JNKi，一种特异性JNK抑制剂）可部分减轻咖啡因诱导的细胞死亡，但不能阻止咖啡因依赖性基础和最大OCR的降低。我们的结果表明，JNK部分介导了半胱天冬酶依赖性细胞死亡的增加，但对咖啡因处理的骨骼肌细胞中线粒体呼吸的降低没有作用。我们得出结论，咖啡因通过激活RYR并促进内质网钙释放，以钙依赖性方式增加细胞死亡并降低线粒体呼吸。
