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.Author summary: Mitochondrial calcium handling has been studied for nearly half a century. As we understand it today, low concentrations (1–10 nmol/mg mitochondria) of calcium exert beneficial effects on energy transduction. And high concentrations (>500 nmol/mg mitochondria) lead to respiratory uncoupling and membrane damage. But relatively little is known about the effect of moderate calcium concentrations (10–500 nmol/mg mitochondria) on mitochondrial function. At these concentrations, mitochondrial membrane integrity remains intact and energized, while ATP synthesis becomes significantly impaired. Prior studies have postulated several possible mechanisms, but the precise consequence of calcium overload on mitochondrial ATP production remained obscure. In this study, we combine experimental and computational approaches to show that calcium phosphate precipitation, as opposed to matrix free calcium, inhibits respiratory function at complex I just enough to limit proton pumping during oxidative phosphorylation and decrease ATP synthesis rates.