Mitochondrial Ca2+ uptake and high-resolution respirometry

Abstract

Mitochondrial calcium homeostasis is involved in the regulation of calcium-dependent cellular processes. Mitochondria contribute to the buffering of cytosolic free calcium levels by uptake and release, or storage of osmotically inactive calcium phosphates in the mitochondrial matrix. Defective calcium homeostasis is a cause of bioenergetic stress, cell death, and diseases. Frequently, calcium retention capacity is measured as the amount of calcium titrated up to a threshold, when calcium uptake is reversed and permeability transition triggers the release of calcium, disruption of ion balance, dyscoupling, and inhibition of respiration. This is accompanied by leakage into the cytosolic space of signaling molecules such as cytochrome c. Since the interplay between mitochondrial Ca²⁺ uptake and oxidative phosphorylation is well recognized, an advanced integrated methodology to concurrently assess mitochondrial respiration and Ca²⁺ dynamics has the potential to enhance our understanding of their crosstalk under physiological and pathological conditions. Here, we present an optimized protocol for the simultaneous measurement of extramitochondrial Ca²⁺ using Calcium Green™-5N (CaG) in conjunction with high-resolution respirometry. We identified MiRCa medium containing CaG as suitable for concurrent assays without impairing respiration of isolated mouse liver mitochondria. A standardized Ca²⁺ calibration protocol was developed. This integrated assay enables real-time monitoring of mitochondrial Ca²⁺ handling and bioenergetic function, offering a valuable tool for exploring mitochondrial function in health and disease.

Cite:

Cecatto C, Cardoso LHD, Grings M, Gnaiger E (2025) Mitochondrial Ca²⁺ uptake and high-resolution respirometry. Bioenerg Commun 2025.7.  https://doi.org/10.26124/becprep.2025-0007