运动神经元通过启动肌肉中的动作电位来控制肌肉收缩。运动神经元的肌肉去神经导致肌肉萎缩,这与线粒体功能障碍有关。众所周知,去神经化会促进肌肉中线粒体活性氧(ROS)的产生,而神经支配肌肉中线粒体ROS产生的最初原因仍然难以捉摸。由于去神经将肌肉与运动神经元隔离开来,并剥夺了它的任何电刺激,没有动作电位启动,因此,无神经支配的肌纤维内不产生生理Ca2+瞬变。我们测试了生理Ca2瞬变的损失是否是导致失神经支配的骨骼肌线粒体功能障碍的初始原因。
表达线粒体靶向生物传感器(mt-cpYFP)的转基因小鼠模型允许在去神经后实时测量与ROS相关的线粒体代谢功能,被称为“mitoflash”。\"使用活细胞成像,电生理学,药理学,和生化研究,我们研究了去神经支配后启动ROS相关线粒体功能障碍的潜在分子机制.
我们发现,去神经支配后24小时,肌纤维的线粒体活性增加了四倍。去神经支配诱导的线粒体闪光活性可能与线粒体通透性转换孔(mPTP)的活性增加有关,因为应用环孢素A使mitoflasm活性减弱。电刺激可迅速降低假肌纤维和去神经肌纤维的mitoflasm活性。我们进一步证明,线粒体内的Ca2水平遵循胞质Ca2瞬时的时间过程,并且Ru360对线粒体Ca2摄取的抑制作用会阻止电刺激对线粒体闪光活性的影响。
由于去神经支配而导致的胞质Ca2+瞬变的损失导致线粒体Ca2+摄取的下游缺失。我们的研究表明,这可能是骨骼肌中mPTP相关线粒体ROS生成增强的初始触发因素。
Motor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological Ca2+ transients are generated inside denervated muscle fibers. We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle.
A transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed \"mitoflash.\" Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation.
We found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity.
The loss of cytosolic Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle.