Mitochondrial membrane potential instability on reperfusion after ischemia does not depend on mitochondrial Ca2+ uptake

Physiologic Ca2+ entry via the Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload but may also contribute to cell death during ischemia/reperfusion (I/R) injury. The MCU has been identified as the primary mode of Ca2+ import into mitochondria. Several groups have tested the hypothesis that Ca2+ import via MCU is detrimental during I/R injury using genetically-engineered mouse models, yet the results from these studies are inconclusive. Furthermore, mitochondria exhibit unstable or oscillatory membrane potentials (ΔΨm) when subjected to stress, such as during I/R, but it is unclear if the primary trigger is an excess influx of mitochondrial Ca2+ (mCa2+), reactive oxygen species (ROS) accumulation, or other factors. Here, we critically examine whether MCU-mediated mitochondrial Ca2+ uptake during I/R is involved in ΔΨm instability, or sustained mitochondrial depolarization, during reperfusion by acutely knocking out MCU in neonatal mouse ventricular myocyte (NMVM) monolayers subjected to simulated I/R. Unexpectedly, we find that MCU knockout does not significantly alter mCa2+ import during I/R, nor does it affect ΔΨm recovery during reperfusion. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCE) suppressed the mCa2+ increase during Ischemia but did not affect ΔΨm recovery or the frequency of ΔΨm oscillations during reperfusion, indicating that mitochondrial ΔΨm instability on reperfusion is not triggered by mCa2+. Interestingly, inhibition of mitochondrial electron transport or supplementation with antioxidants stabilized I/R-induced ΔΨm oscillations. The findings are consistent with mCa2+ overload being mediated by reverse-mode mNCE activity and supporting ROS-induced ROS release as the primary trigger of ΔΨm instability during reperfusion injury. Physiologic Ca2+ entry via the Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload but may also contribute to cell death during ischemia/reperfusion (I/R) injury. The MCU has been identified as the primary mode of Ca2+ import into mitochondria. Several groups have tested the hypothesis that Ca2+ import via MCU is detrimental during I/R injury using genetically-engineered mouse models, yet the results from these studies are inconclusive. Furthermore, mitochondria exhibit unstable or oscillatory membrane potentials (ΔΨm) when subjected to stress, such as during I/R, but it is unclear if the primary trigger is an excess influx of mitochondrial Ca2+ (mCa2+), reactive oxygen species (ROS) accumulation, or other factors. Here, we critically examine whether MCU-mediated mitochondrial Ca2+ uptake during I/R is involved in ΔΨm instability, or sustained mitochondrial depolarization, during reperfusion by acutely knocking out MCU in neonatal mouse ventricular myocyte (NMVM) monolayers subjected to simulated I/R. Unexpectedly, we find that MCU knockout does not significantly alter mCa2+ import during I/R, nor does it affect ΔΨm recovery during reperfusion. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCE) suppressed the mCa2+ increase during Ischemia but did not affect ΔΨm recovery or the frequency of ΔΨm oscillations during reperfusion, indicating that mitochondrial ΔΨm instability on reperfusion is not triggered by mCa2+. Interestingly, inhibition of mitochondrial electron transport or supplementation with antioxidants stabilized I/R-induced ΔΨm oscillations. The findings are consistent with mCa2+ overload being mediated by reverse-mode mNCE activity and supporting ROS-induced ROS release as the primary trigger of ΔΨm instability during reperfusion injury. Physiologic Ca2+ import into mitochondria is essential for matching energy supply with demand. Mitochondrial Ca2+ (mCa2+) activates three Ca2+-regulated dehydrogenases of the Krebs cycle (pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and isocitrate dehydrogenase) (1Denton R.M. Regulation of mitochondrial dehydrogenases by calcium ions.Biochim. Biophys. Acta. 2009; 1787: 1309-1316Crossref PubMed Scopus (608) Google Scholar) and can regulate the activity of the ATP synthase (2Balaban R.S. The role of Ca(2+) signaling in the coordination of mitochondrial ATP production with cardiac work.Biochim. Biophys. Acta. 2009; 1787: 1334-1341Crossref PubMed Scopus (180) Google Scholar) and cytochrome oxidase (3Vygodina T. Kirichenko A. Konstantinov A.A. Direct regulation of cytochrome c oxidase by calcium ions.PLoS One. 2013; 8e74436Crossref PubMed Scopus (25) Google Scholar). Ca2+ overload is also a mainstay of current models of ischemia–reperfusion (I/R) injury. Excess mCa2+, facilitated by an increase in reactive oxygen species(ROS), damages mitochondria and triggers cell death, principally through the opening of the mitochondrial permeability transition pore (mPTP), with the irreversible collapse of the mitochondrial inner membrane potential (ΔΨm) (4Murphy E. Steenbergen C. Ion transport and energetics during cell death and protection.Physiology (Bethesda). 2008; 23: 115-123Crossref PubMed Scopus (99) Google Scholar). The mitochondrial calcium uniporter (MCU) has been identified as the primary mode of Ca2+ import into mitochondria (5Kirichok Y. Krapivinsky G. Clapham D.E. The mitochondrial calcium uniporter is a highly selective ion channel.Nature. 2004; 427: 360-364Crossref PubMed Scopus (1120) Google Scholar, 6Baughman J.M. 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Surprisingly, mice with germline knockout of MCU did not show any protection from I/R injury (10Pan X. Liu J. Nguyen T. Liu C. Sun J. Teng Y. et al.The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter.Nat. Cell Biol. 2013; 15: 1464-1472Crossref PubMed Scopus (511) Google Scholar). In addition, cardiac-specific overexpression of a dominant-negative MCU, which suppresses the activity of the endogenous channel, did not protect against I/R injury (11Rasmussen T.P. Wu Y. Joiner M.A. Koval O.M. Wilson N.R. Luczak E.D. et al.Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 9129-9134Crossref PubMed Scopus (124) Google Scholar). In contrast, mice with inducible cardiomyocyte-specific deficiency of MCU (12Luongo T.S. Lambert J.P. Yuan A. Zhang X. Gross P. Song J. et al.The mitochondrial calcium uniporter matches energetic supply with cardiac workload during stress and modulates permeability transition.Cell Rep. 2015; 12: 23-34Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 13Kwong J.Q. Lu X. Correll R.N. Schwanekamp J.A. Vagnozzi R.J. Sargent M.A. et al.The mitochondrial calcium uniporter selectively matches metabolic output to acute contractile stress in the heart.Cell Rep. 2015; 12: 15-22Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) or short-term inducible overexpression of MCUb (14Lambert J.P. Luongo T.S. Tomar D. Jadiya P. Gao E. Zhang X. et al.MCUB regulates the molecular composition of the mitochondrial calcium uniporter channel to limit mitochondrial calcium overload during stress.Circulation. 2019; 140: 1720-1733Crossref PubMed Scopus (72) Google Scholar) did protect against I/R injury. The discrepancies in outcomes have been attributed to possible adaptations of the organism to long-term inhibition of MCU that alter the mechanistic processes involved in myocyte damage (15Parks R.J. Menazza S. Holmström K.M. Amanakis G. Fergusson M. Ma H. et al.Cyclophilin D-mediated regulation of the permeability transition pore is altered in mice lacking the mitochondrial calcium uniporter.Cardiovasc. Res. 2019; 115: 385-394Crossref PubMed Scopus (58) Google Scholar). Alternative explanations for the failure, in some cases, of MCU knockout to protect against I/R injury could be that (i) MCU may not be the only mode of Ca2+ entry into mitochondria during ischemia (16Griffiths E.J. Ocampo C.J. Savage J.S. Rutter G.A. Hansford R.G. Stern M.D. et al.Mitochondrial calcium transporting pathways during hypoxia and reoxygenation in single rat cardiomyocytes.Cardiovasc. Res. 1998; 39: 423-433Crossref PubMed Scopus (98) Google Scholar), or (ii) mitochondrial damage on reperfusion might occur through Ca2+-independent mechanisms, such as through ROS-induced ROS release (RIRR) (17Zorov D.B. Juhaszova M. Sollott S.J. Mitochondrial ROS-induced ROS release: an update and review.Biochim. Biophys. Acta. 2006; 1757: 509-517Crossref PubMed Scopus (878) Google Scholar). With regard to the first point, a previous report suggested that the mCa2+ increase in cardiomyocytes during hypoxia is mediated by the reversal of the mitochondrial Na+/Ca2+ exchanger (mNCE), and not by the MCU, based on its sensitivity to clonazepam but not ruthenium red (16Griffiths E.J. Ocampo C.J. Savage J.S. Rutter G.A. Hansford R.G. Stern M.D. et al.Mitochondrial calcium transporting pathways during hypoxia and reoxygenation in single rat cardiomyocytes.Cardiovasc. Res. 1998; 39: 423-433Crossref PubMed Scopus (98) Google Scholar). On the latter point, there is ample evidence that reversible ΔΨm loss or oscillation can occur through ROS-dependent and Ca2+-independent mechanisms during metabolic or oxidative stress. For example, our group has shown that oscillations of ΔΨm and redox potential can be triggered in local clusters of mitochondria, or synchronize across the entire mitochondrial network of a cardiomyocyte (18Aon M.A. Cortassa S. Marbán E. O'Rourke B. Synchronized whole cell oscillations in mitochondrial metabolism triggered by a local release of reactive oxygen species in cardiac myocytes.J. Biol. Chem. 2003; 278: 44735-44744Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 19Romashko D.N. Marban E. O'Rourke B. Subcellular metabolic transients and mitochondrial redox waves in heart cells.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1618-1623Crossref PubMed Scopus (184) Google Scholar), in response to various methods to induce oxidative stress (20Aon M.A. Cortassa S. Maack C. O'Rourke B. Sequential opening of mitochondrial ion channels as a function of glutathione redox thiol status.J. Biol. Chem. 2007; 282: 21889-21900Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), even when cellular Ca2+ is depleted. Similarly, single mitochondrion superoxide "mitoflashes" (21Gong G. Liu X. Zhang H. Sheu S.-S. Wang W. Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart.Am. J. Physiol. Heart Circ. Physiol. 2015; 309: H1166-H1177Crossref PubMed Scopus (33) Google Scholar), redox/pH transients (22Breckwoldt M.O. Armoundas A.A. Aon M.A. Bendszus M. O'Rourke B. Schwarzländer M. et al.Mitochondrial redox and pH signaling occurs in axonal and synaptic organelle clusters.Sci. Rep. 2016; 623251Crossref PubMed Scopus (21) Google Scholar), or local RIRR events induced by a laser (17Zorov D.B. Juhaszova M. Sollott S.J. Mitochondrial ROS-induced ROS release: an update and review.Biochim. Biophys. Acta. 2006; 1757: 509-517Crossref PubMed Scopus (878) Google Scholar), may be modulated by, but are not obligatorily dependent on, Ca2+. This leaves open the possibility that mitochondrial ΔΨm instability and cellular damage could be primarily driven by ROS rather than mCa2+, at least during early reperfusion. The post-ischemic heart is also prone to ventricular arrhythmias that have been linked to RIRR-triggered ΔΨm instability but were not sensitive to the mPTP inhibitor cyclosporine A (23Akar F.G. Aon M.A. Tomaselli G.F. O'Rourke B. The mitochondrial origin of postischemic arrhythmias.J. Clin. Invest. 2005; 115: 3527-3535Crossref PubMed Scopus (273) Google Scholar, 24Brown D. O'Rourke B. Cardiac mitochondria and arrhythmias.Cardiovasc. Res. 2010; 88: 241-249Crossref PubMed Scopus (175) Google Scholar). Treatment with ligands of the mitochondrial benzodiazepine receptor prevented ventricular arrhythmias on reperfusion (23Akar F.G. Aon M.A. Tomaselli G.F. O'Rourke B. The mitochondrial origin of postischemic arrhythmias.J. Clin. Invest. 2005; 115: 3527-3535Crossref PubMed Scopus (273) Google Scholar) and stabilized ΔΨm oscillations (25Solhjoo S. O'Rourke B. Mitochondrial instability during regional ischemia–reperfusion underlies arrhythmias in monolayers of cardiomyocytes.J. Mol. Cell Cardiol. 2015; 78: 90-99Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), potentially implicating Inner Membrane Anion Channels (26Ponnalagu D. Singh H. Anion channels of mitochondria.Handb Exp. Pharmacol. 2017; 240: 71-101Crossref PubMed Scopus (61) Google Scholar) or the outer membrane Translocator Protein (27Šileikytė J. Blachly-Dyson E. Sewell R. Carpi A. Menabò R. Di Lisa F. et al.Regulation of the mitochondrial permeability transition pore by the outer membrane does not involve the peripheral benzodiazepine receptor (Translocator Protein of 18 kDa (TSPO)).J. Biol. Chem. 2014; 289: 13769-13781Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) in this process (28Seidlmayer L.K. Hanson B.J. Thai P.N. Schaefer S. Bers D.M. Dedkova E.N. PK11195 protects from cell death only when applied during reperfusion: succinate-mediated mechanism of action.Front. Physiol. 2021; 12628508Crossref PubMed Scopus (1) Google Scholar). These data support the possibility that alternative, mPTP-independent, mitochondrial targets may contribute to ΔΨm instability and electrophysiological abnormalities during reperfusion. Here, we critically examine whether MCU-mediated mitochondrial Ca2+ uptake during I/R is involved in triggering ΔΨm oscillations or sustained mitochondrial depolarization during reperfusion. Employing acute knockout of MCU in a cellular model of I/R in neonatal mouse ventricular myocytes, we show that MCU deficiency has no effect on ΔΨm recovery or oscillation on reperfusion. Moreover, MCU knockout did not affect mCa2+ import during I/R, while inhibition of the mitochondrial Na+/Ca2+ exchanger (mNCE) did, challenging the paradigm that MCU is the primary mode of mCa2+ import during ischemia. Ca2+ uptake into the mitochondria is driven by the electrochemical Ca2+ gradient and the negative membrane potential inside mitochondria (29Gunter T.E. Pfeiffer D.R. Mechanisms by which mitochondria transport calcium.Am. J. Physiol. Cell Physiol. 1990; 258: C755-C786Crossref PubMed Google Scholar, 30Fieni F. Bae Lee S. Jan Y.N. Kirichok Y. Activity of the mitochondrial calcium uniporter varies greatly between tissues.Nat. Commun. 2012; 3: 1-12Crossref Scopus (175) Google Scholar). MCU is the primary mode of Ca2+ entry into the mitochondria and is responsible for the rapid uptake of mCa2+ (5Kirichok Y. Krapivinsky G. Clapham D.E. The mitochondrial calcium uniporter is a highly selective ion channel.Nature. 2004; 427: 360-364Crossref PubMed Scopus (1120) Google Scholar, 8Chaudhuri D. Sancak Y. Mootha V.K. Clapham D.E. MCU encodes the pore conducting mitochondrial calcium currents.eLife. 2013; 2e00704Crossref PubMed Scopus (144) Google Scholar, 30Fieni F. Bae Lee S. Jan Y.N. Kirichok Y. Activity of the mitochondrial calcium uniporter varies greatly between tissues.Nat. Commun. 2012; 3: 1-12Crossref Scopus (175) Google Scholar). Because we were interested in the functional effects of acute knockout of the MCU gene, while minimizing potential systemic adaptative responses, we utilized a viral gene transfer method to express Cre recombinase to achieve MCU knockout (∼80% decrease) within 5 days in culture (Fig. 1, C and D). We first measured mCa2+ levels in neonatal mouse ventricular myocytes (NMVMs) using the genetically encoded MitoCam (4mtd3cpv) FRET probe (Fig. 2A). We recorded baseline mCa2+ levels in unstimulated cells for 10 min, acquiring an image every 15 s. We found no difference between matrix resting Ca2+ levels in MCU-WT and KO cells, similar to observations in several other MCU knockout studies (12Luongo T.S. Lambert J.P. Yuan A. Zhang X. Gross P. Song J. et al.The mitochondrial calcium uniporter matches energetic supply with cardiac workload during stress and modulates permeability transition.Cell Rep. 2015; 12: 23-34Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 13Kwong J.Q. Lu X. Correll R.N. Schwanekamp J.A. Vagnozzi R.J. Sargent M.A. et al.The mitochondrial calcium uniporter selectively matches metabolic output to acute contractile stress in the heart.Cell Rep. 2015; 12: 15-22Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 31Kwong J.Q. Huo J. Bround M.J. Boyer J.G. Schwanekamp J.A. Ghazal N. et al.The mitochondrial calcium uniporter underlies metabolic fuel preference in skeletal muscle.JCI Insight. 2018; 3e121689Crossref PubMed Scopus (52) Google Scholar). Blocking the mitochondrial Na+/Ca2+ exchanger with the mNCE inhibitor CGP-37157 (CGP;10 μM) also did not alter baseline mCa2+ levels (Fig. 2A). However, when we measured beat-to-beat mCa2+ transient amplitudes in MCU-WT and MCU-KO myocytes (Example traces in Fig. S1), we observed a 55% decrease in MCU-KO myocytes compared to WT (Fig. 2B). To determine if there was any difference in mCa2+ uptake in response to a large rise in cytosolic Ca2+, we initiated caffeine-induced SR-Ca2+ release (32Hobai I.A. O'Rourke B. Enhanced Ca 2+ -activated Na + -Ca 2+ exchange activity in canine pacing-induced heart failure.Circ. Res. 2000; 87: 690-698Crossref PubMed Scopus (147) Google Scholar). Cells were superfused with Na+- and Ca2+ -free buffer to prevent Ca2+ extrusion via the sarcolemmal Na+/Ca2+ exchanger (NCX). Then 20 mM Caffeine was then added to release the SR-Ca2+ stores. Under these conditions, Ca2+ accumulation in the mitochondria was measured. We found that mCa2+ uptake into the mitochondria was significantly reduced by ∼80% in MCU-KO cells (Fig. 2C). Calibration of the MitoCam probe in both WT and KO cells to obtain the minimum (Rmin) and maximum (Rmax) YFP/CFP FRET ratios showed no differences in Rmin and Rmax between MCU-WT and KO cells (Fig. 2, D and E). Examples of the calibration traces are shown in Fig. S2. Further, since the mitochondria contribute to beat-to-beat buffering of systolic Ca2+ transients (33Drago I. De Stefani D. Rizzuto R. Pozzan T. Mitochondrial Ca2+ uptake contributes to buffering cytoplasmic Ca2+ peaks in cardiomyocytes.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 12986-12991Crossref PubMed Scopus (172) Google Scholar) via MCU, we also measured cytosolic Ca2+ transients using Fura-2. MCU-KO monolayers displayed a ∼37% increase in cytosolic Ca2+ transient amplitude compared to WT. Adding 10 μM CGP to WT cells increased cytosolic transient amplitude by ∼24% compared to controls (Fig. 2F). While it is presently unclear why we observe this increase, this demonstrates that CGP does not inhibit cytosolic Ca2+ cycling (important for the subsequent interpretation of its effect on I/R Ca2+). These results show that, although there is no difference in matrix Ca2+ levels at baseline between MCU-WT and KO, fast mCa2+ uptake is significantly reduced in MCU-KO cells.Figure 2MCU is required for rapid Ca2+ uptake into mitochondria. A, mCa2+ levels at baseline in Neonatal Mouse Ventricular Myocytes using the MitoCam probe. mCa2+ levels are represented as a ratio of the FRET signal (YFP) to CFP. Baseline mCa2+ for MCU-WT, MCU-KO, as well as MCU-WT or MCU-KO in the presence of CGP-37157 (CGP; 10 μM). B, mCa2+ transient amplitude in unstimulated cells in MCU-WT and KO. C, mCa2+ uptake measured when SR-Ca2+ is released by caffeine in the presence of 0 mM Na+ (Welch's t test, WT= 7, KO= 5 cells). D and E, Rmin and Rmax levels for MitoCam signal in both groups. F, Cytosolic Ca2+ transients measured using Fura-2, with and without CGP. N > 18 cells (Kruskal Wallis non-parametric test, with Dunn's Multiple comparison). Experiments (and calibrations) were repeated at least 3 times. Mean ± SEM is shown. KO, knockout; MCU, mitochondrial calcium uniporter; WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Excessive Ca2+ influx into mitochondria during metabolic stress can trigger cell death pathways via mPTP (34Halestrap A.P. Clarke S.J. Javadov S.A. Mitochondrial permeability transition pore opening during myocardial reperfusion—a target for cardioprotection.Cardiovasc. Res. 2004; 61: 372-385Crossref PubMed Scopus (1016) Google Scholar), leading to the hypothesis that preventing or reducing Ca2+ influx into mitochondria during ischemia could be beneficial. Nevertheless, there have been conflicting reports of the role of MCU on in vivo I/R injury (10Pan X. Liu J. Nguyen T. Liu C. Sun J. Teng Y. et al.The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter.Nat. Cell Biol. 2013; 15: 1464-1472Crossref PubMed Scopus (511) Google Scholar, 11Rasmussen T.P. Wu Y. Joiner M.A. Koval O.M. Wilson N.R. Luczak E.D. et al.Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 9129-9134Crossref PubMed Scopus (124) Google Scholar, 12Luongo T.S. Lambert J.P. Yuan A. Zhang X. Gross P. Song J. et al.The mitochondrial calcium uniporter matches energetic supply with cardiac workload during stress and modulates permeability transition.Cell Rep. 2015; 12: 23-34Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 13Kwong J.Q. Lu X. Correll R.N. Schwanekamp J.A. Vagnozzi R.J. Sargent M.A. et al.The mitochondrial calcium uniporter selectively matches metabolic output to acute contractile stress in the heart.Cell Rep. 2015; 12: 15-22Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), so we next assessed the impact of genetic knockout of MCU on mitochondrial Ca2+ uptake during I/R injury. Particularly, we wanted to understand the mechanisms of Ca2+ import into mitochondria during I/R while simultaneously monitoring ΔΨm. To monitor mitochondrial Ca2+, an adenovirus expressing MitoCam was transduced into these cells at least 48 h prior to imaging. mCa2+ and ΔΨm were monitored during 1 h of Ischemia (induced by placing a coverslip) followed by 1 h of reperfusion (removing the coverslip). We analyzed mCa2+ levels during I/R by determining the YFP (FRET acceptor emission)/CFP (donor emission) ratio for each cell in the microscopic field (∼100) and repeating the experiment on a minimum of 5 different preparations. We observed that there was no correlation between MitoCam expression and the characteristics of the mCa2+ response, ruling out possible differences due to direct buffering by MitoCam. Example traces of mCa2+ during I/R (included in Fig. S3) show the consistent behavior of all cardiomyocytes in the NMVM monolayer. We observed a rise in Ca2+ levels during early ischemia up to ∼25 min, after which mCa2+ levels declined in both MCU-WT and MCU-KO cells (Fig. 3, A and C), in parallel with loss of ΔΨm (Fig. 3E). At the end of ischemia, mCa2+ levels were lower than at baseline. No significant differences were observed in mCa2+ levels between MCU-WT and MCU-KO during the early (2–3 min), maximum, and end (last minute) of the ischemic phase (Fig. 3C). Immediately upon reperfusion, mCa2+ influx was observed in both MCU-WT and MCU-KO cardiomyocytes. No significant differences were found in mCa2+ levels between MCU-WT and MCU-KO cells at early (first minute), mid (29–31 min), or end (last minute) of the reperfusion phase (Fig. 3D). We then tested whether inhibition of mNCE had effects on mCa2+ and ΔΨm during I/R. We found that CGP significantly abolished the rise of mCa2+ in early ischemia and greatly suppressed mCa2+ influx during reperfusion (although this suppression was not significantly different between CGP-treated and untreated cells) (Fig. 3, B and C). Although the ischemia-induced early rise in mCa2+ levels was suppressed, mCa2+ at the end of Ischemia was not significantly different in CGP-treated versus untreated cells. We also measured cytosolic Ca2+ with a genetically encoded cytoplasmic Ca2+ probe (d3cpv), with or without CGP, and confirmed that the CGP effect was not attributable to inhibition of cytoplasmic Ca2+ levels during I/R (Fig. S4). To determine if CGP was indirectly suppressing mCa2+ uptake by disrupting ΔΨm, we also monitored ΔΨm changes simultaneously with mCa2+ during the I/R period. The observed changes in mCa2+ uptake in CGP-treated cells were not due to altered ΔΨm responses. These results indicate that the mNCE mediates mitochondrial Ca2+ uptake during Ischemia. ΔΨm changes were assessed by plotting the TMRM dispersion for each individual cell during 1 h of ischemia and 1 h of reperfusion. This measure minimizes potential artifacts due to dye loading variability and fluorescence decay over the experimental time course. ΔΨm decreased biphasically during ischemia, consistent with visual observations of TMRM redistribution within the cells. Upon reperfusion, ΔΨm repolarized within the first minute of coverslip removal, followed by heterogeneous ΔΨm oscillation across the microscopic field (Video S1). When we compared the ΔΨm response in MCU-WT to MCU-KO NMVMs, or to CGP-treated monolayers, we observed no significant difference in the pattern of behavior between the different groups (Fig. 3, E and F). Although MCU-KO myocytes showed somewhat higher average dispersion values on reperfusion, closer examination of the images indicated that this was due to an increase in the spatial heterogeneity of the mitochondrial network within cells, rather than an actual increase in ΔΨm of polarized mitochondria in the MCU-KO group, that is, there was a slight increase in variation in signal across the cell as a consequence of abundant mitochondrial ΔΨm oscillations (Video S2). It is important to note that the dispersion measurement gives us a broad representation of ΔΨm across the coverslip but does not facilitate the identification of the transition states of ΔΨm polarization and depolarization in individual cells. Hence, we tracked each cell's TMRM fluorescence and assessed ΔΨm changes at the single-cell level during ischemia and reperfusion, applying MitoWave, a wavelet transform signal processing method we recently developed (35Ashok D. O'Rourke B. MitoWave: spatiotemporal analysis of mitochondrial membrane potential fluctuations during I/R.Biophys. J. 2021; 120: 3261-3271Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar), to automatically detect transition points during ischemia and frequency components of ΔΨm instability on reperfusion. Interestingly, MCU knockout was found to accelerate the time to ΔΨm loss during ischemia indicating greater susceptibility, while CGP delayed the time to Ischemic ΔΨm loss in MCU-KO cells. This ischemic prolongation effect by CGP was not observed in MCU-WT cells (Fig. 3G). This increased susceptibility to CGP in MCU-KO monolayers could suggest an increase in the expression of NCLX, the protein underlying mNCE; however, NCLX expression levels did not differ between MCU-KO and WT cells (Fig. S10). Next, we determined if modulating mCa2+ influx affects ΔΨm instability during reperfusion. ΔΨm instability during reperfusion is a hallmark of mitochondrial damage that could translate to higher organ-level arrhythmias (23Akar F.G. Aon M.A. Tomaselli G.F. O'Rourke B. The mitochondrial origin of postischemic arrhythmias.J. Clin. Invest. 2005; 115: 3527-3535Crossref PubMed Scopus (273) Google Scholar). Since ΔΨm oscillations during reperfusion are non-stationary, we also used the MitoWave method (35Ashok D. O'Rourke B. MitoWave: spatiotemporal analysis of mitochondrial membrane potential fluctuations during I/R.Biophys. J. 2021; 120: 3261-3271Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar) for an unbiased approach to quantitatively analyze and categorize ΔΨm oscillatory behavior during reperfusion. Continuous wavelet transform enabled the determination of the dominant frequencies displayed by individual mitochondrial clusters over the duration of reperfusion (Fig. 4). We further classified ΔΨm oscillatory behaviors by separating them into frequency bands. High-frequency oscillators fell into the top two bands of 45 to 8.6 and 8.6 to 4.3 mHz (oscillations periods of 22–116 s and 116–230 s, respectively), moderately fast frequencies ranging from 4.3 to 2.2 mHz (230–450 s), and low-frequency oscillators of less than 2.2 mHz (>450 s), largely representing stable polarized mitochondria. The time at which a mitochondrion underwent irreversible ΔΨm collapse during reperfusion was also included in this analysis. Figure 4, A–D shows scalograms of representative individual mitochondria from WT, KO, WT+CGP, or KO+CGP NMVMs during reperfusion. High coefficient peaks are present in the low scale range of 1 to 10 with all the interventions. We next plotted the frequency components