**1. Introduction**

Regulation of intra-mitochondrial free calcium ([Ca<sup>2</sup>+]m) is critical in cardiac physiology and pathophysiology. Under physiological conditions, a moderate increase in [Ca<sup>2</sup>+]m is believed to stimulate key enzymes of the Krebs cycle and oxidative phosphorylation and to drive mitochondrial ATP production to match cellular energy demand [1,2]. In contrast, a pathological increase in [Ca<sup>2</sup>+]m causes opening of the mitochondrial permeability transition pore (mPTP), a key factor in initiation of cell death [3,4]. Pathophysiological dysregulation of [Ca<sup>2</sup>+]m is a primary mediator in cardiac ischemia and reperfusion (IR) injury, as Ca2+ overloading can lead to apoptosis [5–7].

[Ca<sup>2</sup>+]m is regulated by a dynamic balance between mitochondrial Ca2+ uptake, intra-mitochondrial Ca2+ bu ffering, and mitochondrial Ca2+ release. Mitochondrial Ca2+ uptake

is mediated primarily through the mitochondrial Ca2+ uniporter (MCU) [8–10], and is controlled by the large membrane potential (ΔΨm: −180 to −200 mV) across the inner mitochondrial membrane (IMM). The ΔΨm in turn is generated by the flow of electrons and proton pumping along the respiratory chain complexes [11]. When [Ca<sup>2</sup>+]m increases, this depolarizes ΔΨm, which is compensated by enhanced H<sup>+</sup> pumping/extrusion to alkalinize the matrix. Therefore, powerful, dynamic buffering of matrix pH (pHm) and Ca2+ are required to enable sufficient recovery of ΔΨm and to avoid overloading the matrix with a high [Ca<sup>2</sup>+]. Inorganic phosphate (Pi) has been recognized as a major player in maintaining the trans-matrix pH gradient when accompanied by the effective cotransport of H<sup>+</sup> [12] and buffering of matrix Ca2+ through the formation of amorphous calcium phosphate (Ca–Pi) granules [13–15]. The Ca–Pi buffer system sets the free Ca2+ at a steady-state level, enabling greater mitochondrial Ca2+ loading without impeding the Ca2+ uptake and affecting the efflux system [16–18]. The efflux systems that regulate [Ca<sup>2</sup>+]m are the Na+/Ca2<sup>+</sup>exchanger (NCLX) [17], and the putative Na<sup>+</sup>-independent Ca2+ exchanger/Ca2+-hydrogen exchanger (CHE) [19]. Any disruption in the uptake, and or impairment in the buffering or efflux of Ca2+ would disrupt the delicate balance of the [Ca<sup>2</sup>+]m and lead to impaired bioenergetics and to opening of the mPTP [3,4].

The opening of the high conductance mPTP channel is associated with a high degree of mitochondrial swelling, dissipation of ΔΨm, uncoupling of oxidative phosphorylation, membrane rupture and release of sequestered Ca2+, metabolites, and apoptotic signaling molecules [20–23]. Although the molecular components of the mPTP and its regulation remain largely unclear, cyclophilin D (Cyp D) is the only unambiguously recognized regulatory component of the mPTP. Cyp D is a mitochondrial matrix peptidyl-prolyl cis-trans isomerase (PPIase) that is translocated to the IMM during high matrix Ca2+ conditions; Cyp D is proposed to facilitate conformational changes in the putative mPTP core proteins thereby regulating pore opening [24–26].

Adenine nucleotides (AdN: ATP and ADP) have been implicated in the inhibition of Ca2+-dependent mPTP opening [27,28]. A previous study from our laboratory suggested that matrix AdN modulate [Ca<sup>2</sup>+]m, potentially by increased buffering of [Ca<sup>2</sup>+]m [29]. Oligomycin (OMN), an F0F1-ATP synthase inhibitor, influences the AdN (ATP/ADP) pool, and has been shown to modulate mPTP opening [30]. Cyclosporin A (CsA), a potent mPTP inhibitor is also believed to suppress pore opening by inhibiting matrix Cyp D, thereby preventing the Cyp D-induced conformational changes in mPTP core proteins [31,32]. CsA has long been known to desensitize mPTP from early opening during Ca2+ challenges by impeding Ca2+ interaction with Cyp D; however, the direct effects of CsA on the [Ca<sup>2</sup>+]m buffering system have not been investigated systematically. It is worth noting that in a previous study from Chalmers and Nicholls [14], it was proposed that CsA enhances the Ca2+ loading capacity of mitochondria without changing the relationship between free [Ca<sup>2</sup>+]m and total [Ca<sup>2</sup>+]m during continuous Ca2+ infusion in isolated rat liver and brain mitochondria. Altschuld et al. [33] proposed that CsA increases mitochondrial Ca2+ influx and reduces its efflux. Later, Wei et al. [34] demonstrated that although CsA had no effect on MCU activity, it inhibited NCLX activity at higher concentrations. Altogether, these findings raise important questions about how CsA delays Ca2+-induced mPTP opening while increasing net [Ca<sup>2</sup>+]m accumulation. Our study sought to answer these questions by (i) examining the effect of CsA during repeated CaCl2 challenges over an extended time-period on mitochondrial Ca2+ buffering, and (ii) by examining the underlying changes in bioenergetics during excessive Ca2+ overload.

To address our objective, we investigated systematically the effect of CsA on mitochondrial Ca2+ buffering and compared its effect with a known matrix buffering component, the AdN pool (OMN+ADP), by monitoring [Ca<sup>2</sup>+]e, [Ca<sup>2</sup>+]m, and key mitochondrial bioenergetics variables, ΔΨm, pHm, and NADH (redox state), under conditions of repeated Ca2+ loading. Furthermore, we determined the effect of CsA on the rescue of buffering capability and bioenergetics of failing mitochondria just before mPTP opening. We found that CsA enhanced the sequestration of mitochondrial Ca2+, maintained [Ca<sup>2</sup>+]m at a steady-state level, and markedly delayed mPTP opening. In addition, CsA preserved ΔΨm, NADH, and pHm during CaCl2 bolus challenges. However, in the absence of Pi, this

CsA-induced matrix Ca2+ sequestration was abrogated, and in turn led to the early mPTP opening. The results described herein reveal a novel way by which CsA modulates matrix Ca2+ sequestration to maintain [Ca<sup>2</sup>+]m, despite increased Ca2+ loading. CsA-mediated Ca2+ sequestration is likely achieved via a Pi-dependent [Ca<sup>2</sup>+]m buffering system that delays Ca2+-induced mPTP opening.

#### **2. Materials and Methods**
