Pharmacological inhibition of the mitochondrial Ca2+ uniporter: Relevance for pathophysiology and human therapy
Graphical abstract
Section snippets
Why is it important to target mtCU?
In unstimulated cells, the mitochondrial matrix [Ca2+] ([Ca2+]m) is similar to the cytoplasmic [Ca2+] ([Ca2+]c), and upon exposure of mitochondria to an uncoupler that releases their entire Ca2+ content, little or no increase in [Ca2+]c is detected [1]. These results suggest that mitochondria do not serve as intracellular Ca2+ stores. However, during the response to physiological Ca2+-mobilizing agonists, [Ca2+]m shows transient increases, leading to the stimulation of energy metabolism through
Molecular composition of the mtCU
Cytoplasmic Ca2+ has to cross both the outer and inner mitochondrial membranes (OMM, IMM) to enter the matrix (Fig. 1). The predominant pathways for Ca2+ diffusion across the OMM are the Voltage Dependent Anion-selective Channels (VDACs) [33,34]. Because of their high density and high conductance for cations in their “closed” state, VDACs allow rapid Ca2+ diffusion. The primary driving force for Ca2+ entry into the matrix is the inside-negative membrane potential (ΔΨm) across the IMM maintained
Genetic targeting of the mtCU
The first MCU-deficient mouse was created on a mixed genetic background. The heterozygous breeding of these mice yielded KO/KO mice below the Mendelian ratio, but the few KO/KO offspring showed normal development and had only minor functional problems like the adaptation of striated muscle to work. Mitochondria harvested from the tissues failed to take up added Ca2+ [59]. By contrast, homozygous MCU-deficient mice created on the C57BL/6 background failed to give any viable pups [60,61].
Pharmacological targeting
Many pharmacological inhibitors of the mtCU are divalent/trivalent cations or polycations. Divalents/trivalents can either traverse mtCU and act as competitive inhibitors of Ca2+ like Sr2+ or bind to the channel without permeation like Mg2+ or La3+ [35]. These inhibitors have been useful to explore the biophysical properties of the mtCU but offer little help in intact cell or tissue paradigms. The most frequently used is Ruthenium Red (RuRed), an oxo-bridged triruthenium polycation stain that
Lessons from MCU-i11
MCU-i11 and MCU-i4 are two small-molecule compounds, identified as mtCU inhibitors effective in intact HeLa cells, mouse embryonic fibroblasts and skeletal muscle fibers in a MICU1-dependent manner [91] (Fig. 1). MCU-i4 causes mitochondrial depolarization, making the interpretation of the phenotypes in terms of mtCU-dependence difficult so our further analysis here is focused on MCU-i11 (Fig. 3, Fig. 4). The experiments described below also provide some guidance for testing future candidates
Future directions
The demand has been growing for means to target the mtCU for research and, potentially, for clinical applications (see Fig. 1). Genetic targeting has succeeded for each component of the mtCU in cells and mice. The phenotype seems to be more severe for deletion of the main Ca2+ sensing regulator, MICU1, than for the pore-forming unit, MCU, though outcomes depend on the genetic background. The loss of gatekeeping sensitizes cells to mitochondrial Ca2+ overload and cell death/tissue injury.
Declaration of ethical approval
Some experiments involve the use of animal subjects, here we state that all procedures were performed in compliance with the relevant laws and institutional guidelines - Institutional Animal Care & Use Committee (IACUC) – and that the appropriate institutional committee(s) have approved them (Protocol No. 01338).
Declaration of Competing Interest
The authors declare no competing interest.
Acknowledgements
The authors thank Justin Wilson and Joshua Woods for providing Ru265, Suresh K Joseph for providing MCUKO HeLa cells and comments on the manuscript, the entire Foundation Leducq “Mitocardia” team (Tish Murphy, Paolo Bernardi, Fabio di Lisa, Jeff Molkentin, Michel Cohen, Michael Ovize, and Mike Forte) and Diego De Stefani and Cristina Mammucari for the stimulating discussions on the subject of this review and David Weaver, György Csordás and Erin L. Seifert for comments on the manuscript. The
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Equal contribution.