Cyclosporine‐A Enhances Mitochondrial Calcium Buffering to Delay mPTP Opening

Abstract

Regulation of intra‐mitochondrial (matrix) free calcium ([Ca2+]m) is critical for cellular Ca2+ homeostasis. While mitochondria have the capacity to store a large amount of Ca2+, the availability of [Ca2+]m is maintained within a sub‐micro molar range at steady‐state by the dynamic balance between Ca2+ uptake, buffering, and efflux. Excessive matrix Ca2+ with oxidative stress induces permanent opening of a nonselective inner membrane pore, the mitochondrial permeability transition pore (mPTP), which leads to cell death. Cyclosporine A (CsA), a well‐known inhibitor of the mPTP, is thought to suppress pore opening, in part, by binding to cyclophilin D (CypD) and inhibiting its peptidyl‐prolyl cis‐trans isomerase (PPIase) activity in the presence of high [Ca2+]m. It is not known if the CsA mediated delay in mPTP opening involves regulation of [Ca2+]m as a potential mechanism for mPTP inhibition. In the present study, we tested a novel hypothesis that CsA enhances matrix Ca2+ buffering and consequently modulates the [Ca2+]m threshold for mPTP opening. To test this hypothesis, we investigated the effects of CsA on mitochondrial Ca2+ handling and bioenergetics in K+ pyruvate energized mitochondria isolated from the guinea pig hearts. We quantitatively determined mitochondrial Ca2+ uptake by monitoring both the disappearance of the extra‐matrix ([Ca2+]e) and the increase in [Ca2+]m, using the fluorescent dyes Fura‐4 penta‐K+ salt and Fura‐4 AM, respectively. Mitochondria suspended in Na+ free buffer, to obviate mitochondrial Na+/Ca2+ exchanger, containing 40 μM EGTA, were challenged with repeated boluses of CaCl2 (20 μM) every 5 minutes until mitochondria could not take up or retain the additional Ca2+. Key mitochondrial bioenergetics variables, membrane potential (ΔΨm), matrix pH and NADH (redox state), were also monitored using specific fluorescent dyes. We found that treatment of mitochondria with CsA, before adding boluses of CaCl2, significantly increased the amount of Ca2+ uptake and sequestration, maintained [Ca2+]m at a steady‐state level, and markedly delayed mPTP opening. The extent of matrix Ca2+ buffering, as determined by the bound/free Ca2+ ratio, was strikingly higher for CsA treated mitochondria vs. control. In addition, by enhancing matrix Ca2+ buffering, CsA preserved ΔΨm, matrix pH, and redox state. Interestingly, when CsA was added to mitochondrial suspension just before the onset of mPTP opening, it inhibited pore opening, in part, by promoting the buffering of [Ca2+]m, which reduced the [Ca2+]m and rescued bioenergetics variables to normal values. Since inorganic phosphate (Pi) is vital for Ca2+ sequestration in the matrix, we conducted experiments in Pi‐depleted mitochondria and in Pi free buffer condition. We found that in the absence of Pi, the CsA mediated buffering of [Ca2+]m was abrogated, and mitochondrial bioenergetics were compromised. These results suggest that CsA delays mPTP opening by a novel mechanism that involves enhanced matrix Ca2+ buffering primarily by induction of a PO43− ‐dependent matrix Ca2+ sequestration system.Support or Funding InformationNational Institutes of Health (R01 HL131673‐01A1) and the Veterans Administration (BX‐002539‐01).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.