Prevention of mitochondrial pH gradient dissipation: a novel role for cyclosporin A on inhibiting calcium‐hydrogen exchange activity in cardiac isolated mitochondria
Authors: Stowe David F , Heisner James S , Camara Amadou KS , Lambert David S , Glait Lyall , Bevers Kyle , Mishra Jyotsna , Kwok Wai‐Meng 
Published: June 2021

Calcium‐hydrogen exchange (CHE) can be induced in cardiac isolated mitochondria (m) either indirectly, by dinitrophenol (DNP)‐induced proton leakage into the mitochondrial matrix when proton pumping by complex V ATPase is inhibited by oligomycin, or directly by acidifying the extra‐mitochondrial buffer after adding excess CaCl2. Because the matrix efflux of Ca2+ by mCHE is partially matched by its reuptake via the mitochondrial Ca2+ uniporter (MCU), adding the MCU blocker ruthenium red after adding CaCl2 exposes the total release of Ca2+ via the mCHE. In addition to mCHE two other Ca2+ efflux pathways are the well‐known, the sodium‐hydrogen exchanger (mNCE) and the purported transient, low conductance, mitochondrial permeability transition pore (mPTP), through which ions and small molecules might exit the mitochondria in a “flickering” manner. We questioned if the effects of cyclosporine A (CsA) to block Ca2+ efflux resulted from transient mPTP opening rather than Ca2+ efflux via mCHE. We isolated Guinea pig heart mitochondria and tested if 500 nM CsA, a known inhibitor of mPTP opening, inhibits mCa2+ efflux induced after adding CaCl2 to an acidic (pH 6.9) or alkaline (pH 7.6) medium. We used a Na+ free mitochondrial buffer that contained ≈40 μM EGTA, carried over from the isolation buffer, to block mNCE. A single bolus of 40 μM CaCl2 was added to the buffer. Ca2+ uptake by mitochondria was assessed by the disappearance of Ca2+ from the buffer using the fluorescent dye Fura‐4 penta‐K+ salt. We also examined for buffer pH‐induced differences in Ca2+ retention capacity (CRC), identified by adding consecutive boluses of 10 μM CaCl2 every 90 s until external Ca2+ was no longer taken up by mitochondria. We found that CsA, in all pH conditions, delayed mPTP opening as shown by the greater CRCs. However, compared to without CsA, external (buffer) [Ca2+]e did not rise, and matrix pHm and membrane potential (ΔΨm) did not fall over time in acidic buffer when CsA was added before adding CaCl2. In addition, with or without CsA, CRC was greater at external pHe 7.6, and least at pHe 6.9. Although we found that CsA blocked mCa2+ efflux, we hypothesize that this was not mediated via mPTP flickering. CsA did not directly prevent the ΔΨm depolarization that occurs during matrix acidification or after addition of 30 μM DNP alone. In the absence of CsA, the mCHE‐mediated changes in pHm, external [Ca2+]e, and ΔΨm, induced by adding CaCl2 at extra‐matrix pH 6.9, occurred very slowly over 25‐30 min; this time course is indicative of slow cation exchange activity. Moreover, full ΔΨm depolarization was incomplete. Our results suggest a novel role of CsA in which, directly or indirectly, it prevents dissipation of the mitochondrial pH gradient induced when the extra‐matrix pH is low. Potentially, this may be due to inhibition of mCHE activity via mitochondrial Ca2+ buffering.

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VA Merit 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.