The wide and frequent depolarization events observed in the very active heart were evaluated and compared to another tissue, which under physiological conditions does not undergo such activity. Mitochondria were isolated from two different murine tissues namely, the liver or the heart from the same subject. In each sample, mitochondrial PT sensitivity and reversibility were evaluated measuring different activities.
Oxygen consumption
Different reports indicate that Ca2+ addition increases the rate of O2 consumption (OCR) in mitochondria and then when Ca2+ is withdrawn, the rate of O2 consumption returns to its basal level unless PTP remains open depleting ΔΨ(Batandier et al., 2004; Zhong et al., 2008) (Fig. 1). In LiverMit, 30 µM Ca2+ increased the rate of O2 uptake. Then EGTA addition reverted the increase in OCR at 30 sec but was ineffective at 60 sec or later (Fig. 1A; Fig. 2, latin letters). These results suggest that LiverMit PT became irreversible at some point after 30 sec. When the same experiment was conducted in HeartMit, 30 µM Ca2+ was also added and no effects on OCR were observed, EGTA had no further effects (Fig. 1B two upper traces). Raising added Ca2+ to 150 µM did increase OCR, although this increase reverted to below the basal rate in all cases with EGTA additions (Fig. 1B two lower traces; Fig. 2 greek letters). Thus, in LiverMit PT was triggered by 30 µM Ca2+ sometime between 30 and 60 sec, while in the heart acceleration of O2 consumption was observed only at 150 µM Ca2+ and even then, Ca2+ quenching returned the rate of O2 consumption to basal levels even after 180 sec suggesting that PT collapsed ΔΨ.
Statistics from triplicates of the above data (Fig. 1) were used to evaluate the significance of the results (Fig. 2). In the LiverMit (black columns) Ca2+ addition increased the rate of O2 consumption. EGTA addition after 30 sec reverted the increase, reaching basal levels. However, EGTA addition after 60 sec or later did not have any effects. In the HeartMit, the basal rate of O2 consumption was higher than in the LiverMit. Still, Ca2+ did increase the rate of O2 consumption and then the increase was always reverted by EGTA to levels below the basal rate. The results suggest that after 30 sec of Ca2+ exposure, PTP became open in the liver while it remained closed in the HeartMit (Figs. 1 & 2).
Ca 2+ driven mitochondrial swelling. Ca2+ is taken avidly by mitochondria (Carafoli; Halestrap & Davidson, 1990) and in the presence of high KCl PT evokes swelling, which can be observed spectrophotometrically (Jung et al., 1997). Thus, to evaluate PT, Ca2+ was added to mitochondria in the presence of 20 mM KCl. LiverMit did not swell spontaneously (Fig. 3A, trace a). However, sequential 5 µM Ca2+ additions led to rapid swelling beginning at the third addition (Fig. 3A, trace b). In the presence of Cyclosporine A (CsA) swelling was inhibited partially (Fig. 3A, trace c) suggesting that swelling was due to PT. In control HeartMit swelling was minimal (Fig. 3B, trace a). Under these conditions repeated addition of 10 µM Ca2+ induced mitochondrial swelling (Fig. 3B, trace b). Again, CsA inhibited HeartMit swelling (Fig. 3B, trace c). Thus, the results in Fig. 3 confirm that mitochondrial swelling was more evident and took place at lower Ca2+ concentrations in liver than in HeartMit; also, cardiac mitochondria did not undergo full swelling (for comparison, see Fig. 4B).
Reversibility of Ca 2+ -driven mitochondrial PTP opening. In the presence of KCl, mitochondria exhibit only a mild rate of swelling (Fig. 4, traces a); rapid mitochondrial swelling may be promoted by adding an adequate amount of Ca2+ (Fig. 4, traces b); CsA may prevent the Ca2+ effect, indicating that swelling is due to opening of PTP (Fig. 4, traces c). In all cases, Ca2+ quenching should stop swelling by closing PTP. This system was tested in LiverMit and HeartMit to explore PT reversibility (Fig. 4).
In LiverMit full swelling was observed at 30 µM Ca2+ while HeartMit needed 150 µM Ca2+. In LiverMit Cyclosporine-sensitive Ca2+-mediated mitochondrial swelling was triggered with 30 µM Ca2+ (Fig. 4A, trace b). Then, EGTA was added at different times, and it was observed that swelling stopped at 30 sec (Fig. 4A, trace d). When EGTA was added at 60 sec (Fig. 4A, trace e) swelling continued for 5 sec and then stopped. Lastly, at 120 sec (Fig. 4A, trace f) swelling was not inhibited. When the same experiment was conducted in HeartMit, 150 µM Ca2+ was needed to trigger similar swelling (Fig. 4B, trace b). In addition, at all times evaluated, 30, 60 or 120 sec EGTA stopped swelling immediately (Fig. 4B, traces d, e and f). In addition, while hepatic mitochondrial PTP opening lost reversibility after 30 sec, cardiac mitochondrial PTP remained reversible after addition at all three times tested.
Ca 2+ -retention assays using Arsenazo III. Many reports indicate that sequential Ca2+ additions trigger mitochondrial PT (Scarpa et al., 1978; Uribe & Devlin, 1994). As expected from Figs. 3 & 4, in LiverMit the fourth to fifth 5 µM Ca2+ addition triggered PT and the release for Ca2+ (Fig. 5A, trace a), while CsA delayed PT to about twice as many additions (Fig. 5A, trace b). In contrast, in HeartMit, repeatedly adding 5 µM Ca2+ with or without CsA did not trigger PT (Fig. 5B). Only when adding aliquots of 20 µM and 30 µM Ca2+ did PTP open at 100 µM Ca2+ (Fig. 5C, trace a). In addition, CsA delayed PT, even when 300 µM Ca2+ was added (Fig. 5C, trace b). CsA effects on the ability to buffer Ca2+ were observed which suggest that CsA interacts with mitochondria at sites other than PT (Schote et al., 2002). Again, our results indicate that PTP in HeartMit withstands much higher Ca2+ loading than LiverMit and it remains reversible for much longer.
Ca 2+ -retention assays using Ca 2+ -Green. In order to confirm our data with Arsenazo III, Ca2+ flows were measured with the fluorescent dye Ca2+ Green-5N. In addition, a different reaction mixture with high KCl, and without previous chelex-100 was used (Correa et al., 2017) (Fig. 1S). Sequential additions of 20 µM Ca2+ were tested: LiverMit presented PT at the fifth addition (Fig. 1SA, trace a) while HeartMit resisted almost three times as many Ca2+ additions before undergoing PT (Fig. 1SA, trace b). When a higher Ca2+ (50 µM additions) was tested (Fig. 1SB), PT was exhibited at the second addition by LiverMit (Fig. 1SB, trace a) while HeartMit underwent PT only the sixth to seventh addition (Fig. 1SB, trace b). Thus, the results were similar to those performed with Arsenazo III (Fig. 5). Additionally, it was also confirmed that CsA inhibited PT in both cases (Fig. 1SC); except that in Fig. 1SC, an upward shift of Ca2+ was observed in HeartMit (Fig. 1SC, trace b) which was not present in the Arsenazo III experiment (Fig. 5B). This was probably due to the much higher Ca2+ used in the Ca2+ Green experiment and an active Ca2+ antiport in the heart. RuR inhibited Ca2+ uptake (Fig. 1SD) by both LiverMit (Fig. 1SD, trace a) and HeartMit (Fig. 1SD, trace b).
In addition to releasing ions from the matrix, PTP has been proposed to work as a physiological uncoupling mechanism that prevents mitochondrial ROS over-production (Morales-García et al., 2021). Here, in LiverMit PTP opening did not affect ROS concentration, but instead, after 2 to 4 min ROS increased slightly as compared to the control (Fig. 5A). In contrast, in HeartMit PTP opening led a small decrease in ROS, which was not statistically significant (Fig. 5B). Further experiments are needed to determine whether in the heart PTP opening does decrease ROS production.