Adult Sprague Dawley male rats (275–325 g) were purchased from Taconic (Hillside, NJ, United States). All experiments were performed according to protocols approved by the UPR Medical Sciences Campus Institutional Animal Care and Use Committee and conformed to the National Research Council Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (2011, eighth edition).

H9c2 embryonic rat cardioblastic cells were cultured according to the manufacturer’s recommendations (American Type Culture Collection, Manassas, VA). Briefly, the cells were cultured in DMEM based modified media containing 4 mML-glutamine, 4.5 g/L glucose, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate supplemented with 10% fetal bovine serum and 1% antibiotic solution (HyClone) and maintained in 95% air and 5% CO2 at 37°C. Cells maintained within 80–90% confluence from passages 3–10 were used for experiments.

The isolation of mitochondria was adopted and modified from previous studies (Chapa-Dubocq et al., 2020). Briefly, heart ventricles were cut and homogenized using a Polytron homogenizer in ice-cold sucrose buffer containing (in mM): 300 sucrose, 20 Tris–HCl, and 2 EGTA, pH 7.2, and supplemented with 0.05% BSA. The heart homogenate was centrifuged at 2000 × g for 3 min to remove cell debris. The supernatant was centrifuged at 10,000 × g for 6 min to precipitate mitochondria and then washed again under the same conditions in sucrose buffer (BSA-free). The final pellet containing mitochondria was resuspended in 300 μl of sucrose buffer.

To isolate mitochondria from cultured cells, H9c2 cells were trypsinized and pelleted at 200 × g for 7 min (Jang and Javadov, 2018). Pellet was resuspended in ice-cold sucrose buffer containing (in mM): 300 sucrose, 10 Tris–HCl, and 2 EGTA; pH 7.4. Cells were centrifuged at 2,500 × g for 5 min at 4°C, the pellet was resuspended in the sucrose buffer. To disrupt the plasma membrane and expose mitochondria, cells were plunged using a 27G needle until all cells were successfully lysed. The cell lysate was then centrifuged at 400 × g for 5 min and the supernatant was collected. The mitochondria were concentrated by centrifugation at 10,000 × g for 5 min and finally dissolved with sucrose buffer.

The basic principles of the cell permeabilization technique for analysis of mitochondrial function in situ have been described in detail elsewhere (Kuznetsov et al., 2008; Dedkova and Blatter, 2012; Salabei et al., 2014). Cells were freshly harvested using trypsin–EDTA then permeabilized in sucrose buffer (300 mM sucrose, 10 mM Tris–HCl, 2 mM EGTA, pH 7.4) containing saponin or digitonin for 10 min on ice. After the permeabilization, cells were washed with equilibration buffer (100 mM sucrose, 10 mM Tris–HCl, 10 μM EGTA, pH 7.4), then resuspended in incubation buffer (200 mM sucrose, 10 mM Tris-MOPS, 5 mM α-ketoglutarate, 2 mM malate, 1 mM Pi, 10 μM EGTA-Tris, pH 7.4) containing 100 nM Calcium Green-5N.

To evaluate the effects of permeabilization on mitochondrial function, live H9c2 cells grown to 70–80% confluence were treated with saponin or digitonin for 10 min. Then, the cells were further incubated for 30 min with 300 nM DAPI for visualization of the nucleus and 30 nM Mitotracker Red, a membrane potential-dependent dye. As a positive control, 0.1% Triton X-100 was applied to induce irreversible permeabilization of the plasma membrane leading to structural collapse of the cells. Cell images were captured by an Olympus IX73 microscope with LUCPLFLN10X objective using Cellsense Dimension (Olympus) software.

The CRC was quantified by the Ca²⁺-sensitive fluorescence dyes Oregon Green 488 BAPTA-1, Calcium Green-5N, or Fluo-5N that measure extramitochondrial Ca²⁺ fluorescence in the assay buffer. Briefly, freshly isolated mitochondria (0.5 mg/ml) or permeabilized fresh cells were incubated at 37°C in 0.1 ml of incubation buffer (200 mM sucrose, 10 mM Tris-MOPS, 5 mM α-ketoglutarate, 2 mM malate, 1 mM Pi, 10 μM EGTA-Tris, pH 7.4) containing one of the fluorescence dyes. Calcium was added to increase matrix Ca²⁺ load and the fluorescence intensity was recorded by CLARIOStar microplate reader (BMG Labtech).

The swelling of mitochondria as an indicator of PTP opening in the presence or absence of Ca²⁺ was determined freshly isolated mitochondria (50 μg) by monitoring the decrease in light scattering at 525 nm as previously described, with minor modifications (Rodríguez-Graciani et al., 2020). The swelling buffer contained (in mM): 125 KCl, 20 MOPS, 10 Tris–HCl, 0.001 EGTA, and 2 KH₂PO₄, pH 7.1. The swelling curves were normalized to control and presented as an absorbance ratio.

Data values are presented as mean ± SE. Student’s t-test was used to compare differences between two groups. p < 0.05 was considered as statistically significant. The number of biological samples but not technical replicates were used as a sample size.

In the first set of experiments, we assessed the sensitivity of three different fluorescent dyes to Ca²⁺ in isolated heart mitochondria. The CRC was measured in 50 μg of mitochondria by using Oregon Green 488 BAPTA-1, Calcium Green-5N or Fluo-5N that possess a dissociation constant (K_d for Ca²⁺) of 0.17 μM, 15 μM, or 90 μM, respectively (Figure 1). Results exhibited a different sensitivity of the dyes to Ca²⁺. The Oregon Green 488 BAPTA-1 dye was the most sensitive and CRC reached the maximum at ~60 μM Ca²⁺ (Figure 1A) whereas Calcium Green-5N demonstrated the maximum CRC at 200 μM. (Figure 1B). The CRC of mitochondria was less sensitive to Fluo-5N and could not reach the maximum at 280 μM (Figure 1A). Moreover, inhibition of the mitochondrial swelling by sanglifehrin A, a PTP blocker that inhibits cyclophilin D activity, affected differently the fluorescence intensity of the dyes; inhibition of the CRC was more obvious in the presence of Calcium Green-5N or Fluo-5N (Figures 1B,C) suggesting that the inhibitory effect of sanglifehrin A on mitochondrial swelling depends on the sensitivity of the dyes to Ca²⁺ as well as on the concentration of Ca²⁺. It should be noted that decreased CRC was associated with increased PTP opening in the isolated cardiac mitochondria (Figures 1D–F). Ca²⁺ induces inner membrane expansion and outer membrane rupture (Strubbe-Rivera et al., 2021), leading to the swelling of mitochondria. The increase in matrix volume is accompanied by a decrease in the intensity of light scattered (Tedeschi and Harris, 1955). Thus, these data demonstrate that due to differences in K_d for Ca²⁺, the different Ca²⁺-sensitive fluorescent dyes can be applied for measurement of the CRC at low (Oregon Green 488 BAPTA-1: K_d = 170 nM), medium (Calcium Green-5N: K_d = 14 μM), or high (Fluo-5N: K_d = 90 μM) concentrations of Ca²⁺. Moreover, we experimentally demonstrated the relationship between mitochondrial swelling and Ca²⁺ efflux in response to Ca²⁺-induced PTP opening.

Based on the sensitivity to Ca²⁺ (Figures 1A–C), we used Calcium Green-5N to assess the permeabilization capabilities, potential effects of digitonin and saponin, and the CRC in H9c2 cardioblasts. DAPI, a nuclear staining dye that does not enter non-permeabilized cells, was used to estimate the effectiveness of the detergents to permeabilize the cells. Analysis of permeabilization capabilities demonstrated that both biological detergents at high concentrations have toxic effects on mitochondria, inducing loss of membrane potential (Figures 2A,C,D). The optimal concentrations of the detergents to induce permeabilization of the cells with no effects on mitochondrial functional activity were different for saponin and digitonin. Quantification of DAPI fluorescence to total cells found that saponin levels of 25 μg/ml or higher and all digitonin levels used were capable of permeabilizing the cellular membrane (Figures 2A–C). Analysis of Mitotracker Red displayed mitochondria viability ranging from 5 to 100 μg/ml of saponin as well as digitonin levels between 5 and 10 μg/ml. Saponin effectively permeabilized the cells at concentrations 25, 50, and 100 μg/ml (Figure 2A), whereas optimal permeabilization of the cells by digitonin was observed at 5 and 10 μg/ml (Figure 2C).

To determine whether the detergents have interference with Calcium Green-5N (Figure 3; Supplementary Figures S2A,B), the fluorescence intensity of the dye was measured in the assay buffer (no cells) by adding digitonin or saponin at concentrations of 50, 100, and 200 μg/ml. Saponin at 200 μg/ml demonstrated interference with Calcium Green-5N as evidenced by saturated fluorescence signal in the absence of Ca²⁺ (Figure 3A; Supplementary Figure S2A) whereas digitonin had no interference with the dye at all concentrations (Figure 3B; Supplementary Figure S2B).

Next, we analyzed the CRC in permeabilized cells in situ and in isolated mitochondria in vitro to choose the technique which can be used for accurate quantification of mitochondrial CRC in cultured cells. First, we measured the CRC in H9c2 cardiomyocytes permeabilized by saponin or digitonin (Figure 4A; Supplementary Figure S2C). The cells permeabilized by digitonin at all concentrations (10, 50, and 100 μg/ml) had lower CRC than saponin-permeabilized cells. The cells permeabilized with 50 and 100 μg/ml saponin required approximately 9 and 10 nmol Ca²⁺, respectively, to induce a massive release of Ca²⁺ whereas nearly 6, 7, and 8 nmol Ca²⁺ were required to open the PTP/Ca²⁺ release from mitochondria of cells permeabilized by 10, 50, and 100 μg/ml of digitonin, respectively.

Considering the negative effects of digitonin on the CRC of mitochondria (Figure 4A; Supplementary Figure S2C), in the next set of experiments, we used saponin-permeabilized cells for comparison of mitochondrial CRC in the permeabilized cells versus isolated mitochondria. An equal number of H9c2 cells (0.6 × 10⁶) were used for permeabilization of cells and isolation of mitochondria. Notably, mitochondrial mass was not decreased significantly (<7%, p < 0.974) in isolated mitochondria in comparison with the permeabilized cells as evidenced by citrate synthase activities in both samples. Results demonstrated that the CRC in permeabilized cells was higher compared to the isolated mitochondria (Figure 4B; Supplementary Figure S2D); 10 nmol Ca²⁺ was required to trigger PTP opening/massive Ca²⁺ release in the cells permeabilized with 50 μg/ml saponin whereas isolated mitochondria started swelling at 6 nmol Ca²⁺ indicating at their lower CRC. Thapsigargin, a non-competitive inhibitor of the SERCA, was used to estimate the contribution of sarcoplasmic reticulum to the CRC of mitochondria. The mitochondrial CRC of saponin-permeabilized cells was slightly reduced in the presence of 1 μM thapsigargin. As expected, it had no effect on the CRC of the isolated mitochondria (Figure 4B; Supplementary Figure S2D).