Cyclosporin A Increases Mitochondrial Buffering of Calcium: An Additional Mechanism in Delaying Mitochondrial Permeability Transition Pore Opening

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 (pH_m), and mitochondrial membrane potential (ΔΨ_m). Notably, we found that in PO₄^3- (P_i)-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 P_i just before pore opening in the P_i-depleted condition reinstated the Ca²⁺ buffering system and rescued mitochondria from mPTP opening. This study shows that CsA promotes P_i-dependent mitochondrial Ca²⁺ sequestration to delay mPTP opening and, concomitantly, maintains mitochondrial function.

Keywords: cyclosporin A; inorganic phosphate; mitochondria bioenergetics; mitochondria calcium buffering; mitochondria permeability transition pore.