Calcium phosphate precipitation inhibits mitochondrial energy metabolism

Early studies have shown that moderate levels of calcium overload can cause lower oxidative phosphorylation rates. However, the mechanistic interpretations of these findings were inadequate. And while the effect of excessive calcium overload on mitochondrial function is well appreciated, there has been little to no reports on the consequences of low to moderate calcium overload. To resolve this inadequacy, mitochondrial function from guinea pig hearts was quantified using several well-established methods including high-resolution respirometry and spectrofluorimetry and analyzed using mathematical modeling. We measured key mitochondrial variables such as respiration, mitochondrial membrane potential, buffer calcium, and substrate effects for a range of mitochondrial calcium loads from near zero to levels approaching mitochondrial permeability transition. In addition, we developed a computer model closely mimicking the experimental conditions and used this model to design experiments capable of eliminating many hypotheses generated from the data analysis. We subsequently performed those experiments and determined why mitochondrial ADP-stimulated respiration is significantly lowered during calcium overload. We found that when calcium phosphate levels, not matrix free calcium, reached sufficient levels, complex I activity is inhibited, and the rate of ATP synthesis is reduced. Our findings suggest that calcium phosphate granules form physical barriers that isolate complex I from NADH, disrupt complex I activity, or destabilize cristae and inhibit NADH-dependent respiration.早期研究表明，适度钙超载（calcium overload）可降低氧化磷酸化（oxidative phosphorylation）速率。然而，此前针对这些发现的机制阐释仍不够充分。尽管学界已充分明确重度钙超载对线粒体功能（mitochondrial function）的影响，但针对轻度至中度钙超载所引发后果的相关报道却极为匮乏，几乎无相关研究。为弥补这一研究空白，本研究采用高分辨率呼吸测定法（high-resolution respirometry）、荧光分光光度法（spectrofluorimetry）等多种成熟方法对豚鼠心脏的线粒体功能进行定量检测，并结合数学建模（mathematical modeling）开展分析。我们针对从接近零到接近线粒体通透性转换（mitochondrial permeability transition）水平的一系列线粒体钙负荷，测定了呼吸活性、线粒体膜电位（mitochondrial membrane potential）、缓冲液钙浓度以及底物效应等关键线粒体变量。此外，我们开发了一个可紧密模拟实验条件的计算机模型，并利用该模型设计实验，以排除数据分析中产生的诸多假说。随后我们完成了上述实验，并阐明了钙超载过程中线粒体ADP刺激呼吸显著降低的具体机制。我们发现，当磷酸钙水平而非基质游离钙达到足够阈值时，复合物I（complex I）的活性会受到抑制，ATP合成速率也会随之降低。本研究结果表明，磷酸钙颗粒会形成物理屏障，将复合物I与NADH隔离开来，破坏复合物I活性，或使线粒体嵴（cristae）不稳定，进而抑制NADH依赖的呼吸活性。