Abstract
Regulation of mitochondrial free Ca2+ is critically important for cellular homeostasis. An increase in mitochondrial matrix free Ca2+ concentration ([Ca2+]m) predisposes mitochondria to opening of the permeability transition pore (mPTP). Opening of the pore can be delayed by cyclosporin A (CsA), possibly by inhibiting cyclophilin D (Cyp D), a key regulator of mPTP. Here, we report on a novel mechanism by which CsA delays mPTP opening by enhanced sequestration of matrix free Ca2+. Cardiac-isolated mitochondria were challenged with repetitive CaCl2 boluses under Na+-free buffer conditions with and without CsA. CsA significantly delayed mPTP opening primarily by promoting matrix Ca2+ sequestration, leading to sustained basal [Ca2+]m levels for an extended period. The preservation of basal [Ca2+]m during the CaCl2 pulse challenge was associated with normalized NADH, matrix pH (pHm), and mitochondrial membrane potential (ΔΨm). Notably, we found that in PO43− (Pi)-free buffer condition, the CsA-mediated buffering of [Ca2+]m was abrogated, and mitochondrial bioenergetics variables were concurrently compromised. In the presence of CsA, addition of Pi just before pore opening in the Pi-depleted condition reinstated the Ca2+ buffering system and rescued mitochondria from mPTP opening. This study shows that CsA promotes Pi-dependent mitochondrial Ca2+ sequestration to delay mPTP opening and, concomitantly, maintains mitochondrial function.
Highlights
Regulation of intra-mitochondrial free calcium ([Ca2+ ]m ) is critical in cardiac physiology and pathophysiology
To strengthen the thesis that cyclosporin A (CsA) increases the capacity of mitochondria to sequester Ca2+, we investigated the effect of CsA on mitochondrial bioenergetics. ∆Ψm, NADH, and pHm were assessed using the same protocols as described in Figure 1 for calcium retention capacity (CRC) to correlate changes in [Ca2+ ]m to changes in bioenergetics over time. mitochondrial permeability transition pore (mPTP)
Buffering capacity, which maintained ∆Ψm and the driving force for further Ca2+ uptake (Figure 6C). These results demonstrate that the magnitude of CsA-mediated increase in Ca2+ threshold for mPTP opening and maintenance of mitochondrial integrity is dependent on the [Ca2+ ]m level before
Summary
Regulation of intra-mitochondrial free calcium ([Ca2+ ]m ) is critical in cardiac physiology and pathophysiology. A moderate increase in [Ca2+ ]m is believed to stimulate key enzymes of the Krebs cycle and oxidative phosphorylation and to drive mitochondrial. A pathological increase in [Ca2+ ]m causes opening of the mitochondrial permeability transition pore (mPTP), a key factor in initiation of cell death [3,4]. [Ca2+ ]m is regulated by a dynamic balance between mitochondrial Ca2+ uptake, intra-mitochondrial Ca2+ buffering, and mitochondrial Ca2+ release. Cells 2019, 8, 1052 is mediated primarily through the mitochondrial Ca2+ uniporter (MCU) [8,9,10], and is controlled by the large membrane potential (∆Ψm : −180 to −200 mV) across the inner mitochondrial membrane (IMM). When [Ca2+ ]m increases, this depolarizes ∆Ψm , which is compensated by enhanced
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