Real-Time Fluorescence Measurements of ROS and [Ca²⁺] in Ischemic / Reperfused Rat Hearts:Detectable Increases Occur only after Mitochondrial Pore Opening and Are Attenuated by Ischemic Preconditioning

Mitochondrial permeability transition pore (mPTP) opening is critical for ischemia / reperfusion (I/R) injury and is associated with increased [Ca2+] and reactive oxygen species (ROS). Here we employ surface fluorescence to establish the temporal sequence of these events in beating perfused hearts subject to global I/R. A bespoke fluorimeter was used to synchronously monitor surface fluorescence and reflectance of Langendorff-perfused rat hearts at multiple wavelengths, with simultaneous measurements of hemodynamic function. Potential interference by motion artefacts and internal filtering was assessed and minimised. Re-oxidation of NAD(P)H and flavoproteins on reperfusion (detected using autofluorescence) was rapid (t0.5 < 15 s) and significantly slower following ischemic preconditioning (IP). This argues against superoxide production from reduced Complex 1 being a critical mediator of initial mPTP opening during early reperfusion. Furthermore, MitoPY1 (a mitochondria-targeted H2O2-sensitive fluorescent probe) and aconitase activity measurements failed to detect matrix ROS increases during early reperfusion. However, two different fluorescent cytosolic ROS probes did detect ROS increases after 2-3 min of reperfusion, which was shown to be after initiation of mPTP opening. Cyclosporin A (CsA) and IP attenuated these responses and reduced infarct size. [Ca2+]i (monitored with Indo-1) increased progressively during ischemia, but dropped rapidly within 90 s of reperfusion when total mitochondrial [Ca2+] was shown to be increased. These early changes in [Ca2+] were not attenuated by IP, but substantial [Ca2+] increases were observed after 2-3 min reperfusion and these were prevented by both IP and CsA. Our data suggest that the major increases in ROS and [Ca2+] detected later in reperfusion are secondary to mPTP opening. If earlier IP-sensitive changes occur that might trigger initial mPTP opening they are below our limit of detection. Rather, we suggest that IP may inhibit initial mPTP opening by alternative mechanisms such as prevention of hexokinase 2 dissociation from mitochondria during ischemia

The role of oxidized cytochrome c in regulating mitochondrial reactive oxygen species production and its perturbation in ischaemia

Oxidized cytochrome c is a powerful superoxide scavenger within the mitochondrial IMS (intermembrane space), but the importance of this role in situ has not been well explored. In the present study, we investigated this with particular emphasis on whether loss of cytochrome c from mitochondria during heart ischaemia may mediate the increased production of ROS (reactive oxygen species) during subsequent reperfusion that induces mPTP (mitochondrial permeability transition pore) opening. Mitochondrial cytochrome c depletion was induced in vitro with digitonin or by 30 min ischaemia of the perfused rat heart. Control and cytochrome c-deficient mitochondria were incubated with mixed respiratory substrates and an ADP-regenerating system (State 3.5) to mimic physiological conditions. This contrasts with most published studies performed with a single substrate and without significant ATP turnover. Cytochrome c-deficient mitochondria produced more H2O2 than control mitochondria, and exogenous cytochrome c addition reversed this increase. In the presence of increasing [KCN] rates of H2O2 production by both pre-ischaemic and end-ischaemic mitochondria correlated with the oxidized cytochrome c content, but not with rates of respiration or NAD(P)H autofluorescence. Cytochrome c loss during ischaemia was not mediated by mPTP opening (cyclosporine-A insensitive), neither was it associated with changes in mitochondrial Bax, Bad, Bak or Bid. However, bound HK2 (hexokinase 2) and Bcl-xL were decreased in end-ischaemic mitochondria. We conclude that cytochrome c loss during ischaemia, caused by outer membrane permeabilization, is a major determinant of H2O2 production by mitochondria under pathophysiological conditions. We further suggest that in hypoxia, production of H2O2 to activate signalling pathways may be also mediated by decreased oxidized cytochrome c and less superoxide scavenging

Functional and cardioprotective effects of simultaneous and individual activation of protein kinase A and Epac

BACKGROUND AND PURPOSE: Myocardial cAMP elevation confers cardioprotection against ischaemia/reperfusion (I/R) injury. cAMP activates two independent signalling pathways, PKA and Epac. This study investigated the cardiac effects of activating PKA and/or Epac and their involvement in cardioprotection against I/R. EXPERIMENTAL APPROACH: Hearts from male rats were used either for determination of PKA and PKC activation or perfused in the Langendorff mode for either cardiomyocyte isolation or used to monitor functional activity at basal levels and after 30 min global ischaemia and 2 h reperfusion. Functional recovery and myocardial injury during reperfusion (LDH release and infarct size) were evaluated. Activation of PKA and/or Epac in perfused hearts was induced using cell permeable cAMP analogues in the presence or absence of inhibitors of PKA, Epac and PKC. H9C2 cells and cardiomyocytes were used to assess activation of Epac and effect on Ca(2+) transients. KEY RESULTS: Selective activation of either PKA or Epac was found to trigger a positive inotropic effect, which was considerably enhanced when both pathways were simultaneously activated. Only combined activation of PKA and Epac induced marked cardioprotection against I/R injury. This was accompanied by PKCε activation and repressed by inhibitors of PKA, Epac or PKC. CONCLUSION AND IMPLICATIONS: Simultaneous activation of both PKA and Epac induces an additive inotropic effect and confers optimal and marked cardioprotection against I/R injury. The latter effect is mediated by PKCε activation. This work has introduced a new therapeutic approach and targets to protect the heart against cardiac insults

A Unifying Mechanism for Mitochondrial Superoxide Production during Ischemia-Reperfusion Injury.

Ischemia-reperfusion (IR) injury occurs when blood supply to an organ is disrupted--ischemia--and then restored--reperfusion--leading to a burst of reactive oxygen species (ROS) from mitochondria. It has been tacitly assumed that ROS production during IR is a non-specific consequence of oxygen interacting with dysfunctional mitochondria upon reperfusion. Recently, this view has changed, suggesting that ROS production during IR occurs by a defined mechanism. Here we survey the metabolic factors underlying IR injury and propose a unifying mechanism for its causes that makes sense of the huge amount of disparate data in this area and provides testable hypotheses and new directions for therapies.Work in our laboratories is supported by the Medical Research Council (UK) and the British Heart Foundation. E.T.C. is supported by a Human Frontiers Science Program fellowship.This is the author accepted manuscript. The final version is available from Cell Press via http://dx.doi.org/10.1016/j.cmet.2015.12.00

Hearts from Mice Fed a Non-Obesogenic High-Fat Diet Exhibit Changes in Their Oxidative State, Calcium and Mitochondria in Parallel with Increased Susceptibility to Reperfusion Injury

High-fat diet with obesity-associated co-morbidities triggers cardiac remodeling and renders the heart more vulnerable to ischemia/reperfusion injury. However, the effect of high-fat diet without obesity and associated co-morbidities is presently unknown.To characterize a non-obese mouse model of high-fat diet, assess the vulnerability of hearts to reperfusion injury and to investigate cardiac cellular remodeling in relation to the mechanism(s) underlying reperfusion injury.Feeding C57BL/6J male mice high-fat diet for 20 weeks did not induce obesity, diabetes, cardiac hypertrophy, cardiac dysfunction, atherosclerosis or cardiac apoptosis. However, isolated perfused hearts from mice fed high-fat diet were more vulnerable to reperfusion injury than those from mice fed normal diet. In isolated cardiomyocytes, high-fat diet was associated with higher diastolic intracellular Ca2+ concentration and greater damage to isolated cardiomyocytes following simulated ischemia/reperfusion. High-fat diet was also associated with changes in mitochondrial morphology and expression of some related proteins but not mitochondrial respiration or reactive oxygen species turnover rates. Proteomics, western blot and high-performance liquid chromatography techniques revealed that high-fat diet led to less cardiac oxidative stress, higher catalase expression and significant changes in expression of putative components of the mitochondrial permeability transition pore (mPTP). Inhibition of the mPTP conferred relatively more cardio-protection in the high-fat fed mice compared to normal diet.This study shows for the first time that high-fat diet, independent of obesity-induced co-morbidities, triggers changes in cardiac oxidative state, calcium handling and mitochondria which are likely to be responsible for increased vulnerability to cardiac insults

Over-expression of mitochondrial creatine kinase in the murine heart improves functional recovery and protects against injury following ischaemia–reperfusion

Aims Mitochondrial creatine kinase (MtCK) couples ATP production via oxidative phosphorylation to phosphocreatine in the cytosol, which acts as a mobile energy store available for regeneration of ATP at times of high demand. We hypothesized that elevating MtCK would be beneficial in ischaemia–reperfusion (I/R) injury. Methods and results Mice were created over-expressing the sarcomeric MtCK gene with αMHC promoter at the Rosa26 locus (MtCK-OE) and compared with wild-type (WT) littermates. MtCK activity was 27% higher than WT, with no change in other CK isoenzymes or creatine levels. Electron microscopy confirmed normal mitochondrial cell density and mitochondrial localization of transgenic protein. Respiration in isolated mitochondria was unaltered and metabolomic analysis by 1 H-NMR suggests that cellular metabolism was not grossly affected by transgene expression. There were no significant differences in cardiac structure or function under baseline conditions by cine-MRI or LV haemodynamics. In Langendorff-perfused hearts subjected to 20 min ischaemia and 30 min reperfusion, MtCK-OE exhibited less ischaemic contracture, and improved functional recovery (Rate pressure product 58% above WT; P < 0.001). These hearts had reduced myocardial infarct size, which was confirmed in vivo: 55 ± 4% in WT vs. 29 ± 4% in MtCK-OE; P < 0.0001). Isolated cardiomyocytes from MtCK-OE hearts exhibited delayed opening of the mitochondrial permeability transition pore (mPTP) compared to WT, which was confirmed by reduced mitochondrial swelling in response to calcium. There was no detectable change in the structural integrity of the mitochondrial membrane. Conclusions Modest elevation of MtCK activity in the heart does not adversely affect cellular metabolism, mitochondrial or in vivo cardiac function, but modifies mPTP opening to protect against I/R injury and improve functional recovery. Our findings support MtCK as a prime therapeutic target in myocardial ischaemia

The role of the mitochondrial permeability transition pore in heart disease

AbstractLike Dr. Jeckyll and Mr. Hyde, mitochondria possess two distinct persona. Under normal physiological conditions they synthesise ATP to meet the energy needs of the beating heart. Here calcium acts as a signal to balance the rate of ATP production with ATP demand. However, when the heart is overloaded with calcium, especially when this is accompanied by oxidative stress, mitochondria embrace their darker side, and induce necrotic cell death of the myocytes. This happens acutely in reperfusion injury and chronically in congestive heart failure. Here calcium overload, adenine nucleotide depletion and oxidative stress combine forces to induce the opening of a non-specific pore in the mitochondrial membrane, known as the mitochondrial permeability transition pore (mPTP). The molecular nature of the mPTP remains controversial but current evidence implicates a matrix protein, cyclophilin-D (CyP-D) and two inner membrane proteins, the adenine nucleotide translocase (ANT) and the phosphate carrier (PiC). Inhibition of mPTP opening can be achieved with inhibitors of each component, but targeting CyP-D with cyclosporin A (CsA) and its non-immunosuppressive analogues is the best described. In animal models, inhibition of mPTP opening by either CsA or genetic ablation of CyP-D provides strong protection from both reperfusion injury and congestive heart failure. This confirms the mPTP as a promising drug target in human cardiovascular disease. Indeed, the first clinical trials have shown CsA treatment improves recovery after treatment of a coronary thrombosis with angioplasty

The role of Hexokinase in cardioprotection - mechanism and potential for translation

Mitochondrial permeability transition pore (mPTP) opening plays a critical role in cardiac reperfusion injury and its prevention is cardioprotective. Tumour cell mitochondria usually have high levels of hexokinase isoform 2 (HK2) bound to their outer mitochondrial membranes (OMM) and HK2 binding to heart mitochondria has also been implicated in resistance to reperfusion injury. HK2 dissociates from heart mitochondria during ischaemia, and the extent of this correlates with the infarct size on reperfusion. Here we review the mechanisms and regulations of HK2 binding to mitochondria and how this inhibits mPTP opening and consequent reperfusion injury. Major determinants of HK2 dissociation are the elevated glucose‐6‐phosphate concentrations and decreased pH in ischaemia. These are modulated by the myriad of signalling pathways implicated in preconditioning protocols as a result of a decrease in pre‐ischaemic glycogen content. Loss of mitochondrial HK2 during ischaemia is associated with permeabilization of the OMM to cytochrome c, which leads to greater reactive oxygen species production and mPTP opening during reperfusion. Potential interactions between HK2 and OMM proteins associated with mitochondrial fission (e.g. Drp1) and apoptosis (B‐cell lymphoma 2 family members) in these processes are examined. Also considered is the role of HK2 binding in stabilizing contact sites between the OMM and the inner membrane. Breakage of these during ischaemia is proposed to facilitate cytochrome c loss during ischaemia while increasing mPTP opening and compromising cellular bioenergetics during reperfusion. We end by highlighting the many unanswered questions and discussing the potential of modulating mitochondrial HK2 binding as a pharmacological target. LINKED ARTICLES: This article is part of a themed section on Conditioning the Heart – Pathways to Translation. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue‐

Extent of Mitochondrial Hexokinase II Dissociation During Ischemia Correlates With Mitochondrial Cytochrome c Release, Reactive Oxygen Species Production, and Infarct Size on Reperfusion

BACKGROUND: The mechanisms by which ischemic preconditioning (IP) inhibits mitochondrial permeability transition pore opening and, hence, ischemia–reperfusion injury remain unclear. Here we investigate whether and how mitochondria‐bound hexokinase 2 (mtHK2) may exert part of the cardioprotective effects of IP. METHODS AND RESULTS: Control and IP Langendorff‐perfused rat hearts were subject to ischemia and reperfusion with measurement of hemodynamic function and infarct size. Outer mitochondrial membrane (OMM) permeabilization after ischemia was determined by measuring rates of respiration and H(2)O(2) production in the presence and absence of added cytochrome c in isolated mitochondria and permeabilized fibers. IP prevented OMM permeabilization during ischemia and reduced the loss of mtHK2, but not Bcl‐x(L), observed in control ischemic hearts. By contrast, treatment of permeabilized fibers with glucose‐6‐phosphate at pH 6.3 induced mtHK2 loss without OMM permeabilization. However, metabolic pretreatments of the perfused heart chosen to modulate glucose‐6‐phosphate and intracellular pH(i) revealed a strong inverse correlation between end‐ischemic mtHK2 content and infarct size after reperfusion. Loss of mtHK2 was also associated with reduced rates of creatine phosphate generation during the early phase of reperfusion. This could be mimicked in permeabilized fibers after mtHK2 dissociation. CONCLUSIONS: We propose that loss of mtHK2 during ischemia destabilizes mitochondrial contact sites, which, when accompanied by degradation of Bcl‐x(L), induces OMM permeabilization and cytochrome c loss. This stimulates reactive oxygen species production and mitochondrial permeability transition pore opening on reperfusion, leading to infarction. Consequently, inhibition of mtHK2 loss during ischemia could be an important mechanism responsible for the cardioprotection mediated by IP and other pretreatments