Elevated calcium and reactive oxygen species (ROS) are responsible for the bulk of cell death occurring in a variety of clinical settings that include acute coronary events, cerebrovascular accidents, and acute kidney injury. It is commonly believed that calcium and ROS participate in a viscous cycle during these events. However, the precise feedback mechanisms responsible are unknown. In this study, we quantitatively demonstrate that, on the contrary, calcium does not stimulate free radical production but suppresses it. Isolated mitochondria from guinea pig hearts were energized with a variety of substrates and exposed to calcium concentrations designed to induce moderate calcium overload conditions associated with ischemia/reperfusion injury but do not elicit the well-known mitochondrial permeability transition phenomenon. Metabolic function and free radical emission were simultaneously quantified using high-resolution respirometry and fluorimetry. In parallel, membrane potential, high amplitude swelling, and calcium dynamics were also quantified. Our results reveal that calcium overload does not lead to excessive ROS emission but does decrease ADP stimulated respiration rates for NADH-dependent pathways. Moreover, we developed an empirical model of mitochondrial free radical homeostasis to identify the processes that are different for each substrate and calcium condition. In summary, we show that in healthy guinea pig mitochondria, calcium uptake and free radical generation do not contribute to a viscous cycle and that the relationship between net free radical production and oxygen concentration is hyperbolic. Altogether, these results lay out an important foundation necessary to quantitatively determine the role of calcium in IR injury and ROS production.

中文翻译：

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钙过载减少心脏线粒体中的净自由基排放

升高的钙和活性氧 (ROS) 是导致各种临床环境中发生的大部分细胞死亡的原因，包括急性冠状动脉事件、脑血管意外和急性肾损伤。人们普遍认为，在这些事件中，钙和 ROS 参与了一个粘性循环。然而，负责的精确反馈机制尚不清楚。在这项研究中，我们定量地证明，相反，钙不会刺激自由基的产生，而是抑制它。从豚鼠心脏中分离出的线粒体被各种底物激发并暴露于钙浓度，该浓度旨在诱导与缺血/再灌注损伤相关的中度钙过载条件，但不会引起众所周知的线粒体通透性转变现象。代谢功能和自由基排放同时使用高分辨率呼​​吸测定法和荧光测定法进行量化。同时，膜电位、高振幅肿胀和钙动力学也被量化。我们的结果表明，钙超载不会导致过量的 ROS 排放，但会降低 ADP 刺激的 NADH 依赖性途径的呼吸速率。此外，我们开发了线粒体自由基稳态的经验模型，以确定每种底物和钙条件不同的过程。总之，我们表明，在健康的豚鼠线粒体中，钙吸收和自由基的产生对粘性循环没有贡献，净自由基产生与氧浓度之间的关系是双曲线的。共，