Calcium Overload and Mitochondrial Metabolism

Abstract

Mitochondria calcium is a double-edged sword. While low levels of calcium are essential to maintain optimal rates of ATP production, extreme levels of calcium overcoming the mitochondrial calcium retention capacity leads to loss of mitochondrial function. In moderate amounts, however, ATP synthesis rates are inhibited in a calcium-titratable manner. While the consequences of extreme calcium overload are well-known, the effects on mitochondrial function in the moderately loaded range remain enigmatic. These observations are associated with changes in the mitochondria ultrastructure and cristae network. The present mini review/perspective follows up on previous studies using well-established cryo-electron microscopy and poses an explanation for the observable depressed ATP synthesis rates in mitochondria during calcium-overloaded states. The results presented herein suggest that the inhibition of oxidative phosphorylation is not caused by a direct decoupling of energy metabolism via the opening of a calcium-sensitive, proteinaceous pore but rather a separate but related calcium-dependent phenomenon. Such inhibition during calcium-overloaded states points towards mitochondrial ultrastructural modifications, enzyme activity changes, or an interplay between both events.

Keywords: bioenergetics; calcium overload; calcium phosphate; calcium precipitates; mitochondria; mitochondrial ATP production; mitochondrial function; mitochondrial ultrastructure; oxidative phosphorylation.

Figure 1

Calcium overload. In low amounts, calcium enhances mitochondrial function by activating several Ca²⁺–sensitive catabolic enzymes. In moderate amounts, depressed rates of oxidative phosphorylation become observable. In extreme amounts, mitochondria become structurally compromised and consume ATP in a futile attempt to restore homeostasis.

Figure 2

Mitochondrial ultrastructural changes associated with calcium overload. From left to right, IMOD [16] 3D mitochondrial reconstructions from cryo–electron tomography data where mitochondria were exposed to zero Ca²⁺, a bolus of 25 µM CaCl₂, a bolus of 50 µM CaCl₂ [2], and 1µM CsA. Calcium causes a decrease in cristae volume in a titratable manner. CsA leads to an expanded cristae volume and altered outer membrane morphology.

Figure 3

Method used to calculate the duration of oxidative phosphorylation. The transition is marked with an open circle and a shaded area for each condition that was used to estimate the point the majority of the O₂ flux switches from oxphos using the original ADP bolus to futile ATP cycling. This ATP cycling occurs when the rate of mitochondrial ATP efflux matches the ATP hydrolysis rate from extramitochondrial ATPase contaminants. All mitochondrial preparations when Mg²⁺ is present contain these contaminants. The arrows point to the start and end of oxphos for each condition.

Figure 4

Analysis of O₂ cost for each experimental condition. (A) Observed ADP consumed (ATP produced) per O₂ across a range of calcium loads with and without CsA. (B) Estimated ATP/O₂ ratios across a range of calcium loads with and without CsA. The theoretical PO₂ value for NADH linked substrates is approximately 5.46 assuming 8/3 + 1 H⁺ per ATP generated and exported and 20 H⁺ per O₂ consumed. (C) Calculated mean O₂ waste during the oxphos period used to correct apparent ADP per O₂ data shown in panel A to estimate the PO₂ values shown in panel B. (D) Calculated excess O₂ cost plotted against oxphos duration from panel C. (E) Oxphos duration estimated from data for each condition. Color key: blue, EGTA; orange, 40 nmol/mg, yellow, 250 nmol/mg, and 500 nmol/mg Ca²⁺ condition. Data are presented as the mean ± standard deviation for a sample size of n ≥ 8.