**Cyclosporin A Increases Mitochondrial Bu** ff**ering of Calcium: An Additional Mechanism in Delaying Mitochondrial Permeability Transition Pore Opening**

**Jyotsna Mishra 1, Ariea J. Davani 1, Gayathri K. Natarajan 1, Wai-Meng Kwok 1,2,3,4, David F. Stowe 1,2,5,6 and Amadou K.S. Camara 1,2,4,6,\***


Received: 6 July 2019; Accepted: 3 September 2019; Published: 7 September 2019

**Abstract:** Regulation of mitochondrial free Ca2+ is critically important for cellular homeostasis. An increase in mitochondrial matrix free Ca2+ concentration ([Ca<sup>2</sup>+]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<sup>+</sup>-free bu ffer conditions with and without CsA. CsA significantly delayed mPTP opening primarily by promoting matrix Ca2+ sequestration, leading to sustained basal [Ca<sup>2</sup>+]m levels for an extended period. The preservation of basal [Ca<sup>2</sup>+]m during the CaCl2 pulse challenge was associated with normalized NADH, matrix pH (pH m), and mitochondrial membrane potential (ΔΨ m). Notably, we found that in PO4 3− (Pi)-free bu ffer condition, the CsA-mediated bu ffering of [Ca<sup>2</sup>+]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+ bu ffering 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.

**Keywords:** cyclosporin A; mitochondria calcium bu ffering; mitochondria bioenergetics; mitochondria permeability transition pore; inorganic phosphate
