**5. Conclusions**

The salient observation of this study is that CsA mitigated mPTP opening by promoting the maintenance of a low [Ca<sup>2</sup>+]m, by stimulating and/or potentiating MCBS. Specifically, we showed that the presence of CsA, (i) significantly delayed the mPTP opening when compared to ADP or OMN+ADP (Protocol A); (ii) overturned the high amplitude increase in [Ca<sup>2</sup>+]m (Protocol B); (iii) maintained pH m, redox state (NADH) and basal ΔΨ m, which maintains the driving force for more Ca2+ uptake and sequestration; and (iv) activates Pi-dependent mitochondrial Ca2+ sequestration to delay mPTP opening.

Our study provides a novel insight into how CsA-mediates a delay in mPTP opening by activating the MCBS, which lowers ss[Ca<sup>2</sup>+]m below the threshold for mPTP activation. This concept is shown in the scheme presented in Figure 9. Our finding supports the notion that CsA facilitates Pi-dependent matrix Ca2+ bu ffering, which maintains matrix free Ca2+ and enables massive Ca2+ loading capacity, without diminishing the driving force for Ca2+ influx by maintaining ΔΨ m. CsA may delay mPTP opening by enhancing Pi-dependent matrix Ca2+ bu ffering and by inhibiting Cyp D [31,32]. The culmination of these two mechanisms, and possibly others not ye<sup>t</sup> identified, might be responsible for CsA protection against mitochondrial Ca2+ overload. Together, these findings add to our understanding of the mechanism of CsA-mediated modulation of mPTP. Importantly, we believe that therapeutic approaches targeted at regulating [Ca<sup>2</sup>+]m homeostasis represent a promising strategy to reduce cardiac injury due to Ca2+ overload by delaying mPTP opening and preventing induction of apoptosis.

**Figure 9.** Schema of the potential mechanism by which CsA mediates delay in Ca2+-induced mPTP opening. Pathological conditions, like cardiac ischemia-reperfusion injury, leads to an increase in cytosolic Ca2+ ([Ca<sup>2</sup>+]c). This in turn increases [Ca<sup>2</sup>+]m and generation of reactive oxygen species (ROS), impairs respiration and substrate utilization, and leads to uncoupling of oxidative phosphorylation. Lower ΔΨm, oxidized redox state, and dissipation of the pH m gradient, together induces mPTP opening which triggers apoptosis. These detrimental consequences that underlie IR injury could be mollified by CsA, which allows the mitochondria to maintain their basal [Ca<sup>2</sup>+]m via enhanced Pi-dependent matrix Ca2+ bu ffering, in addition to, or through, Cyp D inhibition. Sustained low [Ca<sup>2</sup>+]m maintains mitochondrial integrity and function and delays mPTP opening.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/8/9/1052/s1. Figure S1: Quantification of calcium retention capacity (CRC) for each treatment, DMSO (control), ADP, oligomycin (OMN), OMN+ADP, and CsA during Protocol A (A) and Protocol B (B). Figure S2: Average Ca2+ added before mPTP opening, ΔΨm collapse, NADH oxidation, and matrix acidification. Figure S3: Trend-fits for calculation of buffering rate. Figure S4: Fura-4F ratio representing the change in [Ca<sup>2</sup>+]e before and after adding a 20 μM CaCl2 bolus in the absence or presence of 250 μM ADP in the mitochondria-free experimental buffer. Figure S5: Representative raw traces of extra-matrix Ca2+ fluorescence (Fura-4F ratio) and Ca2+ uptake in mitochondria pretreated with 0.5% DMSO (control), OMN+ADP, and CATR (carboxyatractyloside)+OMN+ADP prior to mPTP opening.

**Author Contributions:** J.M. and A.K.S.C., conceptualized and designed the experiments; J.M. and A.J.D., performed experiments; J.M., A.J.D. and G.K.N., analyzed data; J.M., W.-M.K., D.F.S. and A.K.S.C., interpreted results; J.M. and A.K.S.C., drafted the manuscript and figures; J.M., G.K.N., W.-M.K., D.F.S. and A.K.S.C., critically read/edited the manuscript. All authors have read and approved the manuscript.

**Funding:** This project was supported by the Veterans Administration (Merit Review BX-002539-01).

**Acknowledgments:** The authors are thankful to James S. Heisner for technical assistance.

**Conflicts of Interest:** The authors declare no conflicts of interest.
