*2.2. Animals*

Albino Hartley guinea pigs of both sexes weighing between 250 to 350 g were procured from Kuiper Rabbit Farm (Gary, IN, USA). All procedures were carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

## *2.3. Mitochondria Isolation*

Mitochondria were isolated from guinea pig hearts as described previously [29,35,36]. Briefly, the guinea pig was anesthetized with an intraperitoneal injection of 30 mg ketamine plus 700 units of heparin, for anticoagulation, and the heart was rapidly excised and minced in ice-cold isolation buffer containing 200 mM mannitol, 50 mM sucrose, 5 mM KH2PO4, 5 mM MOPS, 1 mM EGTA, and 0.1% bovine serum albumin (BSA) at pH 7.15 (adjusted with KOH). The suspension was homogenized at low speed for 20 s in ice-cold isolation buffer containing 5 U/mL protease (from *Bacillus licheniformis*) and the homogenate was centrifuged at 8000× *g* for 10 min. The supernatant was discarded, and the pellet was suspended in 25 mL isolation buffer, and centrifuged at 850× *g* for 10 min. The supernatant was centrifuged further at 8000× *g* to yield the final mitochondrial pellet, which was suspended in isolation buffer and kept on ice until experimentation. All isolation procedures were performed at 4 ◦C and all experiments were conducted at room temperature. Protein concentration was determined by the Bradford method and the final mitochondrial suspension was adjusted to 12.5 mg protein/mL with isolation buffer.

The functional integrity of mitochondria was determined by the respiratory control index (RCI) as described before [29,37]. Mitochondria were energized with pyruvic acid (PA, 0.5 mM; pH 7.15, adjusted with KOH) followed by ADP (250 μM) addition. RCI was defined as the ratio of state 3 (after added ADP) to state 4 respiration (after complete phosphorylation of the added ADP). Only mitochondrial preparations with RCIs ≥ 10 were used to conduct further experiments.

#### *2.4. Experimental Groups and Protocols*

Two protocols (Protocol A and Protocol B) were used to assess the effect of CsA and AdN on mitochondrial Ca2+ handling and bioenergetics in normal and Ca2+-overloaded mitochondria, as shown in Figure 1. Protocol A investigated the ability of CsA and AdN to modulate mitochondrial Ca2+ handling and delay mPTP opening. To further substantiate CsA-mediated buffering of matrix Ca2+, Protocol B was designed to test the effectiveness of CsA and AdN on rescuing a failing mitochondrial Ca2+ buffering system from imminent mPTP opening. There were five experimental groups: vehicle (DMSO), CsA, ADP, OMN, and OMN+ADP. Experiments were also conducted in the presence of deionized H2O as another vehicle (not shown). Each group was subjected to two different experimental protocols (Protocol A and Protocol B) that differed in the order of treatment and addition of CaCl2 boluses to the mitochondrial suspension in experimental buffer.

**Figure 1.** Schema of experimental timeline used to study the effect of Cyclosporin A (CsA) and adenine nucleotide (AdN) on mitochondrial Ca2+ handling and bioenergetics during repeated CaCl2 pulses. (**A**) In Protocol A, at t = 0 s, mitochondria (mito, 0.5 mg) were added to the Na<sup>+</sup>-free experimental buffer solution. The mitochondrial suspension was exposed to 0.5 μM CsA, 250 μM ADP, 10 μM oligomycin (OMN), or a combination of OMN+ADP at t = 30 s. Pyruvic acid (PA, 0.5 mM), was added at t = 60 s to energize mitochondria (state 2). At t = 180 s, 20 μM of CaCl2 was added, followed by sequential additions of 20 μM CaCl2 at every 300 s intervals until mPTP (mitochondrial permeability transition pore) opened or no further Ca2+ uptake was observed. (**B**) In Protocol B, the mitochondrial suspension was exposed to similar treatments as in Protocol A, but given after the last consecutive CaCl2 bolus preceding the imminent onset of mPTP opening.

Delayed opening of mPTP (Protocol A): At t = 0 s, the experiment was initiated by suspending 0.5 mg of isolated mitochondria into the experimental buffer containing 130 mM KCl, 5 mM K2HPO4, 20 mM MOPS, 1 mM EGTA, 0.1% BSA, and EGTA ~0.036–0.040 μM at pH 7.15 (adjusted with KOH). At t = 30 s, mitochondria were treated with DMSO (1 μM), ADP (250 μM), OMN (10 μM), OMN+ADP or CsA (0.5 μM); at t = 60 s, mitochondria were energized with PA (0.5 mM). At t = 180 s, CaCl2 bolus (20 μM final concentration) was added and subsequent CaCl2 boluses added at 5 min intervals until pore opening (Figure 1A). Note that all experiments were conducted under state 2 conditions, except in the ADP-and OMN+ADP-treated groups.

Rescue of mitochondria from mPTP opening (Protocol B): The mitochondrial suspension was exposed to repetitive boluses of CaCl2 (20 μM) as described in Protocol A; rescue of mitochondria from mPTP opening with the different treatments was carried out at 1 min of the last CaCl2 bolus in which mitochondria Ca2+ uptake was observed before pore opening (Figure 1B). The onset of mPTP opening was predicted based on calcium retention capacity (CRC) of the DMSO (control)-treated group for each day's experiment. The pulse preceding mPTP opening observed in the control was the pulse chosen for targeted intervention in all subsequent experiments. In all experiments, extrusion of Ca2+ via the Na+/Ca2<sup>+</sup> exchanger (NCLX) was prevented by conducting all the experiments in Na<sup>+</sup>-free conditions. That is, the respiration buffer, mitochondrial substrates, and all reagents/drugs were Na<sup>+</sup>-free to prevent activation of the NCLX. Some experiments were conducted in the presence of 10 μM CGP 37157 (Tocris Bioscience), an NCLX inhibitor, which ascertained there was no potential Na<sup>+</sup> contamination in the respiration buffer from other sources [35,38,39].

#### *2.5. Mitochondrial Function Measurements*

Fluorescence spectrophotometry (Qm-8, Photon Technology International, Horiba, Birmingham, NJ, USA) was used to measure mitochondrial function, including mitochondria extra- and intra-matrix free [Ca<sup>2</sup>+] ([Ca<sup>2</sup>+]e and [Ca<sup>2</sup>+]m, respectively), ΔΨm, redox state (NADH), and pHm. Fura-4F penta-potassium salt (1 μM, Invitrogen™, Eugene, OR) was used to measure [Ca<sup>2</sup>+]e. For [Ca<sup>2</sup>+]m measurements, mitochondria were incubated with Fura-4F AM (5 μM, Invitrogen™, Eugene, OR) for 30 min at room temperature (25 ◦C) followed by a final spin and resuspension to remove any residual dye. ΔΨm was assessed using the cationic lipophilic dye TMRM (1 μM, Invitrogen™, Eugene, OR, USA) in a ratiometric excitation approach [40]. NADH was measured by tissue autofluorescence, and matrix pH (pHm) was assessed by incubating mitochondria in 5 μM BCECFAM (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature (25 ◦C) followed by a final spin and resuspension [29,35,38,39].

#### *2.6. Measurements of Free Ca2*+

Quantification of [Ca<sup>2</sup>+]e and [Ca<sup>2</sup>+]m were made using the fluorescent Ca2+ indicator probe Fura-4F with dual-excitation wavelengths (λex) at 340/380 nm and a single emission wavelength (λem) at 510 nm. Ca2+ fluorescent intensities with Fura-4F are not influenced by background noise (e.g., NADH autofluorescence), so a background subtraction was unnecessary [38]. Fura-4F fluorescence ratios (F340/F380) were used to calculate [Ca<sup>2</sup>+] using the equation described by Grynkiewicz: [41].

$$\left[\text{Ca}^{2+}\right] = \text{K}\_{\text{d}} \frac{\text{S}\_{\text{f2}}}{\text{S}\_{\text{b2}}} \frac{\left(\text{R} - \text{R}\_{\text{min}}\right)}{\left(\text{R}\_{\text{max}} - \text{R}\right)}.\tag{1}$$

The Kd value for Fura-4F binding to Ca2+ is 890 nM, which was described by us previously [38]. R is the ratio of the fluorescence intensities at λex 340 and 380 nm, Sf2/Sb2 is the ratio of fluorescence intensities measured at λex 380 nm in Ca2+-free (f)/Ca2+-saturated (Ca<sup>2</sup>+-bound, b) conditions. Rmin (Ca<sup>2</sup>+-free) and Rmax (Ca<sup>2</sup>+-saturated) are R values for Fura 4F, carried out after mPTP opening, adding 1 mM CaCl2, followed by 10 mM EGTA, pH 7.1. The free [Ca<sup>2</sup>+] in the buffer was calculated using an online version of MaxChelator program (http://www.stanford.edu/~{}cpatton/maxc.html) and accordingly, a standard curve was generated for the Fura-4F signal to the free [Ca<sup>2</sup>+] in the experimental solution by fitting to the Grynkiewicz equation, as described above in Equation 1 [41].

#### *2.7. Calculation of Mitochondrial Ca2*+ *Bu*ff*ering Capacity*

The ability of mitochondria to sequester Ca2+ is an index of its Ca2+ loading capacity, without altering mitochondrial function. Here we calculated mitochondrial Ca2+ buffering capacity (mβCa) using the model described by Bazil et al. [42]. Briefly, experimental data for extra-and intra-matrix Ca2+ were fit with smooth trend curves satisfying the equation:

$$\mathbf{y}(\mathbf{t}) = \mathbf{p}\_1 + \mathbf{p}\_2 \mathbf{e}^{\frac{(\mathbf{t} - \mathbf{p}\_3)}{\mathbb{P}4}} + \mathbf{p}\_5 \mathbf{t},\tag{2}$$

where y(t) was either [Ca<sup>2</sup>+]e or [Ca<sup>2</sup>+]m at any given time, t. Global trend-fits were performed in MATLAB (Mathworks, Inc., MA) and parameters p1 (offset value), p2 (pre = exponential constant), p3 (time lag), p4 (decay time constant), and p5 (steady-state slope) were estimated and optimized using the lsqnonlin and fmincon functions.

Mitochondrial Ca2+ buffering capacity for the second Ca2+ pulse (a cumulative of 40 μM added Ca2+) was then calculated [42] as:

$$\mathbf{m}\beta\_{\rm Ca} = -\beta\_{\rm Ca,e}\mathbf{V}\_{\rm r}\frac{\mathbf{d}[\rm Ca^{2+}]\_{e}}{\rm dt} / \frac{\mathbf{d}[\rm Ca^{2+}]\_{m}}{\rm dt},\tag{3}$$

where, <sup>m</sup>βCa, is the intra-mitochondrial Ca2+ buffering power, βCa,e is the extra-mitochondrial Ca2+ buffering power determined by:

$$\beta\_{\rm Ca,c} = 1 + \frac{\partial [\rm CaEGTA]\_{\rm e}}{\partial [\rm Ca^{2+}]\_{\rm e}}.\tag{4}$$

Vr is the volume ratio of the extra-mitochondrial space and matrix space (~2000), d[Ca<sup>2</sup>+]e/dt and d[Ca<sup>2</sup>+]m/dt are the rates of change of extra-and intra-mitochondrial free [Ca<sup>2</sup>+], respectively. d[Ca<sup>2</sup>+]e/dt and d[Ca<sup>2</sup>+]m/dt were estimated by evaluating the analytical derivative of Equation (2) using parameter estimates obtained from the trend fits [42].

Trend fits for data in Figure S4 were performed in Origin 2017 (OriginLab Corporation, Northampton, MA, USA).

#### *2.8. Measurement of* ΔΨ*m, Redox State (NADH) and Matrix pH*

Membrane potential was assessed by the dual-excitation ratiometric approach using the fluorescent dye, TMRM, as described by Scaduto and Grotyohann [40] and in our published work [35,38,39]. Fluorescence changes were determined by two excitations, λex 546 and 573 nm, and a single emission λem 590 nm. The calculated ratio of λex 573/546 is proportional to ΔΨ m and has the advantage of a broader dynamic range when compared to a single wavelength technique. Changes in mitochondrial redox state (NADH) were determined by autofluorescence (i.e., by exciting the energized mitochondria at λex 350 nm and collecting data at λem 456 nm). An increase in the signal reflects an increase in the redox ratio of NADH to NAD+ (i.e., a shift to a more reduced state). Matrix pH was assessed using BCECFAM (5 μM) at λex 504 nm and λem 530 nm. This fluorescent probe emits less fluorescence in an acidic environment, thus a decrease in signal indicates matrix acidification and an increase in signal indicates matrix alkalization [29].

#### *2.9. Depletion of Endogenous Mitochondrial Phosphate*

Given the important role of Pi in the mitochondrial Ca2+ buffering system [14,29], we tested the effect of Pi in CsA-induced mitochondrial Ca2+ buffering. Isolated cardiac mitochondria were depleted of endogenous Pi by pre-incubating mitochondria for 10 min at room temperature with 0.75 units/mL hexokinase, 1 mM glucose, 0.5 mM ADP, 1 mM MgCl2, and 5 mM PA, as previously described [14,43,44].
