The transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2) regulates the expression of genes involved in antioxidant defenses to modulate fundamental cellular processes such as mitochondrial function and GSH metabolism. Previous reports proposed that mitochondrial reactive oxygen species production and disruption of the GSH pool activate the Nrf2 pathway, suggesting that Nrf2 senses mitochondrial redox signals and/or oxidative damage and signals to the nucleus to respond appropriately. However, until now, it has not been possible to disentangle the overlapping effects of mitochondrial superoxide/hydrogen peroxide production as a redox signal from changes to mitochondrial thiol homeostasis on Nrf2. Recently, we developed mitochondria-targeted reagents that can independently induce mitochondrial superoxide and hydrogen peroxide production mitoParaquat (MitoPQ) or selectively disrupt mitochondrial thiol homeostasis MitoChlorodinitrobenzoic acid (MitoCDNB). Using these reagents, here we have determined how enhanced generation of mitochondrial superoxide and hydrogen peroxide or disruption of mitochondrial thiol homeostasis affects activation of the Nrf2 system in cells, which was assessed by the Nrf2 protein level, nuclear translocation, and expression of its target genes. We found that selective disruption of the mitochondrial GSH pool and inhibition of its thioredoxin system by MitoCDNB led to Nrf2 activation, whereas using MitoPQ to enhance the production of mitochondrial superoxide and hydrogen peroxide alone did not. We further showed that Nrf2 activation by MitoCDNB requires cysteine sensors of Kelch-like ECH-associated protein 1 (Keap1). These findings provide important information on how disruption to mitochondrial redox homeostasis is sensed in the cytoplasm and signaled to the nucleus. The transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2) regulates the expression of genes involved in antioxidant defenses to modulate fundamental cellular processes such as mitochondrial function and GSH metabolism. Previous reports proposed that mitochondrial reactive oxygen species production and disruption of the GSH pool activate the Nrf2 pathway, suggesting that Nrf2 senses mitochondrial redox signals and/or oxidative damage and signals to the nucleus to respond appropriately. However, until now, it has not been possible to disentangle the overlapping effects of mitochondrial superoxide/hydrogen peroxide production as a redox signal from changes to mitochondrial thiol homeostasis on Nrf2. Recently, we developed mitochondria-targeted reagents that can independently induce mitochondrial superoxide and hydrogen peroxide production mitoParaquat (MitoPQ) or selectively disrupt mitochondrial thiol homeostasis MitoChlorodinitrobenzoic acid (MitoCDNB). Using these reagents, here we have determined how enhanced generation of mitochondrial superoxide and hydrogen peroxide or disruption of mitochondrial thiol homeostasis affects activation of the Nrf2 system in cells, which was assessed by the Nrf2 protein level, nuclear translocation, and expression of its target genes. We found that selective disruption of the mitochondrial GSH pool and inhibition of its thioredoxin system by MitoCDNB led to Nrf2 activation, whereas using MitoPQ to enhance the production of mitochondrial superoxide and hydrogen peroxide alone did not. We further showed that Nrf2 activation by MitoCDNB requires cysteine sensors of Kelch-like ECH-associated protein 1 (Keap1). These findings provide important information on how disruption to mitochondrial redox homeostasis is sensed in the cytoplasm and signaled to the nucleus. Oxidative stress and damage are involved in the development and progression of many diseases (1Finkel T. Holbrook N.J. 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Possibilities include that elevated mitochondrial superoxide production generates hydrogen peroxide that goes from the mitochondria to the cytosol to activate Nrf2 directly or indirectly. Alternatively, the redox changes within the mitochondria may lead to secondary signals to the cytosol that then activate Nrf2. Finally, there are also suggestions that mitochondrial dysfunction may activate Nrf2 through formation of a complex with Keap1 and the mitochondrial outer membrane serine/threonine protein phosphatase, phosphoglycerate mutase family member 5 (PGAM5) (28Kasai S. Shimizu S. Tatara Y. Mimura J. Itoh K. Regulation of Nrf2 by mitochondrial reactive oxygen species in physiology and pathology.Biomolecules. 2020; 10: 320Crossref Scopus (160) Google Scholar, 38O'Mealey G.B. Plafker K.S. Berry W.L. Janknecht R. Chan J.Y. Plafker S.M. A PGAM5–KEAP1–Nrf2 complex is required for stress-induced mitochondrial retrograde trafficking.J. 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Mimura J. Itoh K. Regulation of Nrf2 by mitochondrial reactive oxygen species in physiology and pathology.Biomolecules. 2020; 10: 320Crossref Scopus (160) Google Scholar), it has not been possible to distinguish between the effects of superoxide and hydrogen peroxide generation, and redox changes in mitochondria independently from those in the rest of the cell. Furthermore, many Nrf2 activators cause changes in both superoxide and hydrogen peroxide levels and in thiol homeostasis. However, these pathways interact closely and changes in thiol homeostasis can affect hydrogen peroxide levels, while conversely, increased levels of hydrogen peroxide can alter the thiol redox state via peroxidases (4Yamamoto M. Kensler T.W. Motohashi H. The KEAP1-NRF2 system: a thiol-based sensor-effector apparatus for maintaining redox homeostasis.Physiol. Rev. 2018; 98: 1169-1203Crossref PubMed Scopus (688) Google Scholar, 12Suzuki T. Motohashi H. Yamamoto M. 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Therefore, our challenge was to address the role in Nrf2 activation of mitochondrial redox changes independently of those from the cytosol while also distinguishing between the impact of mitochondrial superoxide and hydrogen peroxide production, and that of thiol redox changes. To do this, we used two mitochondria-targeted redox active agents. To investigate thiol redox state, we used a mitochondria-targeted disrupter of thiol redox homeostasis MitoChlorodinitrobenzoic acid (MitoCDNB), a 1-chloro-2,4-dinitrobenzene (CDNB) derivative that is selectively taken up by mitochondria within cells where it selectively depletes mitochondrial GSH largely, but not solely, by acting as a substrate for mitochondrial GSTs while also inhibiting the mitochondrial Trx system by inhibiting TRX reductases (Fig. 1A) (42Booty L.M. Gawel J.M. Cvetko F. Caldwell S.T. Hall A.R. Mulvey J.F. James A.M. Hinchy E.C. Prime T.A. Arndt S. Beninca C. Bright T.P. Clatworthy M.R. Ferdinand J.R. 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In addition, Nrf2 activation by MitoCDNB was greatly diminished by the thiol N-acetyl-L-cysteine (NAC) and in cells expressing mutant Keap1 that lacked particular sensor cysteine residues. These results indicate that elevated mitochondrial superoxide generation alone does not activate Nrf2 but provide confirmation of direct signaling to the cytosol as a stress response to disrupted mitochondrial thiol redox homeostasis. We first assessed if selective disruption of mitochondrial thiol redox homeostasis activated Nrf2. To do this, we used the mitochondria-targeted thiol reagent MitoCDNB, which we had previously shown selectively depletes GSH and inhibits TrxR2 within mitochondria, without directly affecting their cytosolic counterparts (42Booty L.M. Gawel J.M. Cvetko F. Caldwell S.T. Hall A.R. Mulvey J.F. James A.M. Hinchy E.C. Prime T.A. Arndt S. Beninca C. Bright T.P. Clatworthy M.R. Ferdinand J.R. Prag H.A. et al.Selective disruption of mitochondrial thiol redox state in cells and in vivo.Cell Chem. Biol. 2019; 26: 449-461Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Under control conditions, the Nrf2 protein is present at low levels and is only detectable in the cytosol of C2C12 mouse myoblasts (Fig. 2A). Treatment with H2O2 as a positive control (100 μM, 30 min) increased the protein levels of Nrf2 within the cell ∼2-fold (Fig. 2A) and caused its redistribution to the nucleus, as assessed by immunocytochemistry (Fig. 2B), as did the positive control SFN (Fig. S1). Similarly, MitoCDNB (10 μM, 4 h) increased Nrf2 protein levels ∼2-fold (Fig. 2A) and led to its translocation to the nucleus, as assessed by immunocytochemistry (Fig. 2B) and by subcellular fractionation (Fig. 2C), with nearly 90% of cells having a clear nuclear distribution of Nrf2 upon MitoCDNB treatment (Fig. 2B). To determine whether this effect of MitoCDNB was due to its reaction with mitochondrial thiols, or by a nonspecific disruption of mitochondria by accumulation of the triphenylphosphonium-targeting group, we synthesized a MitoCDNB control compound (Fig. S2). The control compound is structurally very similar to MitoCDNB and is accumulated by mitochondria in response to the membrane potential, but lacks thiol reactivity (Fig. S2, A–C). This control compound had no effect on Nrf2 protein levels or nuclear translocation (Fig. 2, A–D). Thus, the activation of Nrf2 by MitoCDNB is dependent on its thiol reactivity and not a nonspecific interaction of the mitochondria-targeting moiety. To determine whether the nuclear accumulation of Nrf2 by MitoCDNB activates transcription of Nrf2-dependent genes, we assessed the levels of proteins known to be under Nrf2 control via the antioxidant response element. Immunoblotting showed that MitoCDNB, but not its corresponding control compound, led to a time-dependent increase in the levels of the Nrf2 downstream targets glutamate–cysteine ligase catalytic subunit (GCLC), GSH synthetase (GSS), and heme oxygenase-1 (HO-1). HO-1 was increased at 8 and 12 h after exposure, while GCLC and GSS levels increased later, at 12 h (Fig. 3, A–B). To further confirm Nrf2 activation, we used the quantitative NAD(P)H:quinone oxidoreductase-1 (NQO1) inducer assay (46Prochaska H.J. Santamaria A.B. Direct measurement of NAD(P)H:quinone reductase from cells cultured in microtiter wells: a screening assay for anticarcinogenic enzyme inducers.Anal. Biochem. 1988; 169: 328-336Crossref PubMed Scopus (458) Google Scholar, 47Fahey J.W. Dinkova-Kostova A.T. Stephenson K.K. Talalay P. The "Prochaska" microtiter plate bioassay for inducers of NQO1.Methods Enzymol. 2004; 382: 243-258Crossref PubMed Scopus (142) Google Scholar). The activity of this NAD(P)H:quinone oxidoreductase enzyme, which is involved in detoxification pathways, is a particularly sensitive indication of Nrf2 activation as transcription of its gene is primarily regulated by Nrf2, and thus, NQO1 is widely recognized as a classical Nrf2 target (48Dinkova-Kostova A.T. Talalay P. NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector.Arch. Biochem. Biophys. 2010; 501: 116-123Crossref PubMed Scopus (533) Google Scholar). The potency of MitoCDNB was defined as the concentration required to double (CD) the NQO1 enzyme-specific activity. For this Hepa1c1c7, cells were incubated with MitoCDNB, or its control, for 24 h, and NQO1 activity was assessed. MitoCDNB elicited a pronounced concentration-dependent NQO1 induction with a CD value of 12.5 μM that facilitates comparison of its potency with other inducers (Fig. 3C), whereas the control compound had no effect. We conclude that the selective disruption of mitochondrial thiol homeostasis by MitoCDNB activates Nrf2. It has been previously reported that increasing mitochondrial oxidative damage and/or stress activates the Nrf2 pathway (28Kasai S. Shimizu S. Tatara Y. Mimura J. Itoh K. Regulation of Nrf2 by mitochondrial reactive oxygen species in physiology and pathology.Biomolecules. 2020; 10: 320Crossref Scopus (160) Google Scholar). It was further suggested that mitochondrial oxidative damage and/or stress activated Nrf2 through kinase-dependent mechanisms such as the macrophage-stimulating 1 and macrophage-stimulating 2 systems (49Wang P. Geng J. Gao J. Zhao H. Li J. Shi Y. Yang B. Xiao C. Linghu Y. Sun X. Chen X. Hong L. Qin F. Li X. Yu J. et al.Macrophage achieves self-protection against oxidative stress-induced ageing through the Mst-Nrf2 axis.Nat. Commun. 2019; 10: 755Crossref PubMed Scopus (96) Google Scholar). However, it was unclear whether mitochondrial superoxide production alone could activate Nrf2 by generating hydrogen peroxide as a redox signal that was released from the organelle to the cytosol, or whether the putative redox signal was secondary to intramitochondrial alterations. Therefore, here we used the targeted redox cycler MitoPQ to generate superoxide selectively within mitochondria (40Wang W. Wang Y. Long J. Wang J. Haudek S.B. Overbeek P. Chang B.H.J. Schumacker P.T. Danesh F.R. Mitochondrial fission triggered by hyperglycemia is mediated by ROCK1 activation.Cell Metab. 2012; 15: 186-200Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), without directly affecting other mitochondrial processes, or the cytosolic redox environment of C2C12 cells (45Hinchy E.C. Gruszczyk A.V. Willows R. Navaratnam N. Hall A.R. Bates G. Bright T.P. Krieg T. Carling D. Murphy M.P. Mitochondria-derived ROS activate AMP-activated protein kinase (AMPK) indirectly.J. Biol. Chem. 2018; 293: 17208-17217Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The effects of MitoPQ were compared with its control compound, which is taken up by mitochondria within cells but does not generate superoxide (44Antonucci S. Mulvey J.F. Burger N. Di Sante M. Hall A.R. Hinchy E.C. Caldwell S.T. Gruszczyk A.V. Deshwal S. Hartley R.C. Kaludercic N. Murphy M.P. Di Lisa F. Krieg T. Selective mitochondrial superoxide generation in vivo is cardioprotective through hormesis.Free Radic. Biol. Med. 2019; 134: 678-687Crossref PubMed Scopus (31) Google Scholar). C2C12 cells were treated with 5-μM MitoPQ, a concentration that has been shown to robustly increase superoxide production within mitochondria, but not in the cytosol, in these cells (43Robb E.L. Gawel J.M. Aksentijević D. Cochemé H.M. Stewart T.S. Shchepinova M.M. Qiang H. Prime T.A. Bright T.P. James A.M. Shattock M.J. Senn H.M. Hartley R.C. Murphy M.P. Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat.Free Radic. Biol. Med. 2015; 89: 883-894Crossref PubMed Scopus (91) Google Scholar), as was confirmed here by showing that MitoPQ did not induce oxidative stress within the cytosol (Fig. S3A). MitoPQ did not cause an increase in Nrf2 protein levels (Fig. 4A) nor was there any localization of Nrf2 to the nucleus assessed by cell subfractionation followed by immunoblotting (Fig. 4B), in contrast to the positive control SFN (Fig. S1), or by immunofluorescence microscopy (Fig. 4, C–D), compared with the positive control H2O2. Under these conditions, MitoPQ did not elicit any changes in NQO1 activity (Fig. 4E) or in the expression of the Nrf2 targets, GCLC, GSS and HO-1 (Fig. 4F and Fig. S3B). Increasing MitoPQ concentrations 5- to 10-fold did not enhance cell levels of Nrf2 (Fig. S3C), indicating that the lack of effect on Nrf2 was not due to insufficient MitoPQ. We conclude that under these conditions, the selective production of superoxide and hydrogen peroxide within the mitochondrial matrix does not activate Nrf2. From the previous results, we observed that MitoCDNB, but not MitoPQ, led to Nrf2 stabilization, nuclear localization, and enhanced expression of its downstream targets. Previously (42Booty L.M. Gawel J.M. Cvetko F. Caldwell S.T. Hall A.R. Mulvey J.F. James A.M. Hinchy E.C. Prime T.A. Arndt S. Beninca C. Bright T.P. Clatworthy M.R. Ferdinand J.R. Prag H.A. et al.Selective disruption of mitochondrial thiol redox state in cells and in vivo.Cell Chem. Biol. 2019; 26: 449-461Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) it has been shown that MitoCDNB disrupts mitochondrial thiol antioxidant defenses and depletes mitochondrial GSH, which can disrupt mitochondrial thiol redox homeostasis, whereas the cytosolic GSH levels remained unchanged. In contrast, CDNB primarily disrupts cytosolic thiol antioxidant defenses (42Booty L.M. Gawel J.M. Cvetko F. Caldwell S.T. Hall A.R. Mulvey J.F. James A.M. Hinchy E.C. Prime T.A. Arndt S. Beninca C. Bright T.P. Clatworthy M.R. Ferdinand J.R. Prag H.A. et al.Selective disruption of mitochondrial thiol redox state in cells and in vivo.Cell Chem. Biol. 2019; 26: 449-461Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). CDNB itself (5 μM) does increase Nrf2 protein levels (Fig. 5A) and its translocation to the nucleus (Fig. 5B) in C2C12 cells. Hepa1c1c7 cells exposed to CDNB and MitoCDNB both increased NQO1 activity (Fig. 5C). As MitoCDNB acts in part by depleting mitochondrial GSH levels, we next assessed the effect of NAC, a GSH precursor that also increases cell thiol levels and can thereby directly ameliorate cellular oxidative stress that impacts thiols (50Mukherjee T.K. Mishra A.K. Mukhopadhyay S. Hoidal J.R. High concentration of antioxidants N-acetylcysteine and mitoquinone-Q induces intercellular adhesion molecule 1 and oxidative stress by increasing intracellular glutathione.J. Immunol. 2007; 178: 1835-1844Crossref PubMed Scopus (50) Google Scholar), on Nrf2 activation by MitoCDNB. NAC (1 mM) added 1 h before, or simultaneously with, MitoCDNB (10 μM, 4 h) prevented the induction by MitoCDNB of Nrf2 nuclear localization (Fig. 5D). As expected, NAC did not have an influence on SFN-induced Nrf2 nuclear localization (Fig. S4A). The MitoCDNB-mediated induction of NQO1 was also diminished when cells were either pretreated (24 h) or cotreated with NAC (Fig. 5E). Treatment with NAC at concentrations up to 10 mM had no effect on the activity of NQO1 (Fig. S4B). As NAC and MitoCDNB do not interact directly (Fig. S5A), this suggests that NAC prevents the MitoCDNB-mediated Nrf2 activation by boosting thiol defenses within the cell. This was confirmed by showing that NAC did prevent depletion of whole-cell GSH by CDNB and MitoCDNB (Fig. S5B) and increased mitochondrial GSH even in the presence of MitoCDNB (Fig. S5C). To investigate further how MitoCDNB affected the cytosol, we used CellROX, which is a fluorescent probe that responds to a wide range of oxidative processes, enabling us to measure changes in whole-cell oxidative stress, but did not observe an increase with MitoCDNB (Fig. 5F). The above analysis indicates that MitoCDNB, but not MitoPQ, generates signals that activate Nrf2. Under homeostatic conditions, Nrf2 is bound to Keap1 and targeted for ubiquitination and proteolysis by the proteasome. The canonical pathway for the activation of Nrf2 by oxidants or electrophiles is via the reaction of the activators with thiols on Keap1, with specific thiols likely having particular reactivity with different species. These reactions disrupt the substrate adaptor function of Keap1 and enable Nrf2 to escape ubiquitination, migrate to the nucleus, and activate gene expression. To interrogate the involvement of Keap1 in the mechanism of Nrf2 activation by MitoCDNB, we used mouse embryonic fibroblast (MEF) cells expressing two different Keap1 cysteine mutants or their WT counterpart (41Suzuki T. Muramatsu A. Saito R. Iso T. Adachi S. Kawaguchi S.-I. Iwawaki T. Suda H. Morita M. Baird L. Yamamoto M. Molecular mechanism of cellular oxidative stress sensing by Keap1.Cell Rep. 2019; 28: 746-758Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Specifically, these mutants were Keap1C151S and Keap1C226S/C613S (Fig. 6A). Cys151 on Keap1 is the main sensor for SFN, 4-hydroxynonenal, and nitric oxide, whereas Cys226 and Cys613 respond to H2O2 (Fig. 6A) (51Dayalan Naidu S. Dinkova-Kostova A.T. KEAP1, a cysteine-based sensor and a drug target for the prevention and treatment of chronic disease.Open Biol. 2020; 10: 200105Crossref PubMed Scopus (40) Google Scholar). The MEF cells were incubated with MitoCDNB, and NQO1 activity was measured 24 h later (Fig. 6B). In WT cells, MitoCDNB induced NQO1 in a concentration-dependent manner, but this induction was diminished in MEF cells expressing either of the Keap1 mutants (Fig. 6B). The NQO1 inducer potency of the classical Nrf2 activator SFN was also greatly reduced in the Keap1C151S mutant cells in comparison with their WT or Keap1C226S/C613S counterparts, in agreement with previous reports that Cys151 is the main sensor for SFN (7Saito R. Suzuki T. Hiramoto K. Asami S. Naganuma E. Suda H. Iso T. Yamamoto H. Morita M. Furusawa Y. Negishi T. Ichinose M. Yamamoto M. Characterizations of three major cysteine sensors of Keap1 in stress response.Mol. Cell. Biol. 2015; 36: 271-284Crossref PubMed Scopus (169) Google Scholar,52Zhang D.D. Hannink M. Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress.Mol. Cell Biol. 2003; 23: 8137-8151Crossref PubMed Scopus (1106) Google Scholar) (Fig. 6C). The use of Keap1 mutants confirms that MitoCDNB activates Nrf2 through multiple cysteine sensors in Keap1 and further suggests the involvement of both electrophiles and oxidants as potential mediators. Nrf2 plays a central role in the cytoprotective response to oxidative stress and is critical for the maintenance of mitochondrial redox homeostasis (13Dinkova-Kostova A.T. Abramov A.Y. The emerging role of Nrf2 in mitochondrial function.Free Radic. Biol. Med. 2015; 88: 179-188Crossref PubMed Scopus (549) Google Scholar, 28Kasai S. Shimizu S. Tatara Y. Mimura J. Itoh K. Regulation of Nrf2 by mitochondrial reactive oxygen species in physiology and pathology.Biomolecules. 2020; 10: 320Crossref Scopus (160) Google Scholar). However, how mitochondrial oxidative stress and damage generate the signals that lead to Nrf2 activation in the cytosol is not understood. To address this, we used selective chemical biology approaches that enabled us to interrogate separately the effects of mitochondrial superoxide and hydrogen peroxide production and disruption of thiol redox homeostasis. We demonstrate that disrupting mitochondrial thiol redox homeostasis leads to Nrf2 activation, whereas enhanced mitochondrial superoxide production alone does not.
Abstract The naked mole-rat Heterocephalus glaber is a eusocial mammal exhibiting extreme longevity (37-year lifespan), extraordinary resistance to hypoxia and absence of cardiovascular disease. To identify the mechanisms behind these exceptional traits, metabolomics and RNAseq of cardiac tissue from naked mole-rats was compared to other African mole-rat genera (Cape, Cape dune, Common, Natal, Mahali, Highveld and Damaraland mole-rats) and evolutionarily divergent mammals (Hottentot golden mole and C57/BL6 mouse). We identify metabolic and genetic adaptations unique to naked mole-rats including elevated glycogen, thus enabling glycolytic ATP generation during cardiac ischemia. Elevated normoxic expression of HIF-1α is observed while downstream hypoxia responsive-genes are down-regulated, suggesting adaptation to low oxygen environments. Naked mole-rat hearts show reduced succinate levels during ischemia compared to C57/BL6 mouse and negligible tissue damage following ischemia-reperfusion injury. These evolutionary traits reflect adaptation to a unique hypoxic and eusocial lifestyle that collectively may contribute to their longevity and health span.
Cell models of cardiac ischemia-reperfusion (IR) injury are essential to facilitate understanding, but current monolayer cell models poorly replicate the in vivo IR injury that occurs within a three-dimensional tissue. Here we show that this is for two reasons: the residual oxygen present in many cellular hypoxia models sustains mitochondrial oxidative phosphorylation; and the loss of lactate from cells into the incubation medium during ischemia enables cells to sustain glycolysis. To overcome these limitations, we incubated isolated adult mouse cardiomyocytes anoxically while inhibiting lactate efflux. These interventions recapitulated key markers of in vivo ischemia, notably the accumulation of succinate and the loss of adenine nucleotides. Upon reoxygenation after anoxia the succinate that had accumulated during anoxia was rapidly oxidized in association with extensive mitochondrial superoxide/hydrogen peroxide production and cell injury, mimicking reperfusion injury. This cell model will enable key aspects of cardiac IR injury to be assessed in vitro.
Early stroke detection and treatment are critical for improving patient outcomes. Optical brain pulse monitoring (OBPM) uses red and infrared light to capture brain pulse waveforms reflecting arteriole-to-venous pressure levels driving microvascular blood flow. This study assessed OBPMs potential to detect middle cerebral artery occlusion (MCAo) and reperfusion in a clinically relevant sheep model. Stroke was induced in 11 Merino wethers via 4-hour occlusion of the right MCA, followed by 6 hours of reperfusion. OBPM recordings were taken at baseline, MCAo, early and late reperfusion. The OBPM brain pulse waveform classes were classified based on the presence of arterial or central venous circulation wave features. Magnetic resonance imaging assessed infarct volume at 2 hours post-reperfusion. Invasive brain tissue oxygen and intracranial pressures were also monitored. The OBPM brain pulse waveform classes changed during MCAo and reperfusion (p <0.0001). MCAo was associated brain pulses with venous circulation features (p = 0.0007). Reperfusion was associated with the return of arterial circulation features (p = 0.001). Early reperfusion was also associated with an increase in the brain pulse amplitude (p < 0.05) and the respiratory wave amplitude (p < 0.05). OBPM may aid in early stroke detection and reperfusion assessment following intervention.
There is considerable interest in developing drugs and probes targeted to mitochondria in order to understand and treat the many pathologies associated with mitochondrial dysfunction. The large membrane potential, negative inside, across the mitochondrial inner membrane enables delivery of molecules conjugated to lipophilic phosphonium cations to the organelle. Due to their combination of charge and hydrophobicity, quaternary triarylphosphonium cations rapidly cross biological membranes without the requirement for a carrier. Their extent of uptake is determined by the magnitude of the mitochondrial membrane potential, as described by the Nernst equation. To further enhance this uptake here we explored whether incorporation of a carboxylic acid into a quaternary triarylphosphonium cation would enhance its mitochondrial uptake in response to both the membrane potential and the mitochondrial pH gradient (alkaline inside). Accumulation of aryl propionic acid derivatives depended on both the membrane potential and the pH gradient. However, acetic or benzoic derivatives did not accumulate, due to their lowered pKa. Surprisingly, despite not being taken up by mitochondria, the phenylacetic or phenylbenzoic derivatives were not retained within mitochondria when generated within the mitochondrial matrix by hydrolysis of their cognate esters. Computational studies, supported by crystallography, showed that these molecules passed through the hydrophobic core of mitochondrial inner membrane as a neutral dimer. This finding extends our understanding of the mechanisms of membrane permeation of lipophilic cations and suggests future strategies to enhance drug and probe delivery to mitochondria.
Abstract Reducing infarct size during a cardiac ischaemic‐reperfusion episode is still of paramount importance, because the extension of myocardial necrosis is an important risk factor for developing heart failure. Cardiac ischaemia‐reperfusion injury (IRI) is in principle a metabolic pathology as it is caused by abruptly halted metabolism during the ischaemic episode and exacerbated by sudden restart of specific metabolic pathways at reperfusion. It should therefore not come as a surprise that therapy directed at metabolic pathways can modulate IRI. Here, we summarize the current knowledge of important metabolic pathways as therapeutic targets to combat cardiac IRI. Activating metabolic pathways such as glycolysis (eg AMPK activators), glucose oxidation (activating pyruvate dehydrogenase complex), ketone oxidation (increasing ketone plasma levels), hexosamine biosynthesis pathway (O‐GlcNAcylation; administration of glucosamine/glutamine) and deacetylation (activating sirtuins 1 or 3; administration of NAD + ‐boosting compounds) all seem to hold promise to reduce acute IRI. In contrast, some metabolic pathways may offer protection through diminished activity. These pathways comprise the malate‐aspartate shuttle (in need of novel specific reversible inhibitors), mitochondrial oxygen consumption, fatty acid oxidation (CD36 inhibitors, malonyl‐CoA decarboxylase inhibitors) and mitochondrial succinate metabolism (malonate). Additionally, protecting the cristae structure of the mitochondria during IR, by maintaining the association of hexokinase II or creatine kinase with mitochondria, or inhibiting destabilization of F O F 1 ‐ATPase dimers, prevents mitochondrial damage and thereby reduces cardiac IRI. Currently, the most promising and druggable metabolic therapy against cardiac IRI seems to be the singular or combined targeting of glycolysis, O‐GlcNAcylation and metabolism of ketones, fatty acids and succinate.
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