Respiration Buffer Research Articles

Mitochondrial transplantation following cardiopulmonary resuscitation improves neurological function in rats by inducing M2-type MG/MΦ polarization

AimExplore the effects of mitochondrial transplantation (MT) after cardiopulmonary resuscitation (CPR) on the polarization of microglia/macrophages (MG/MΦ) and neurological function.MethodsSeventy-five Sprague-Dawley rats were randomly divided into five groups: sham, normal saline (NS), vehicle, mitochondria (Mito), and non-functional mitochondria (N-Mito) group. Rats in sham group underwent surgical procedures without cardiac arrest, while the other four groups underwent cardiac arrest and CPR, and then received NS, respiration buffer, mitochondrial suspension or non-functional mitochondria, immediately after the restoration of spontaneous circulation (ROSC). The number of mitochondria in the hippocampus, the morphology and structure of mitochondria in MG/MΦ, the phenotype of MG/MΦ, and hippocampal tissue injury, neuroinflammation, and neuronal apoptosis were detected on days 1 and 3 after ROSC. Neurodeficit score (NDS) was performed on days 1, 3, 7, 15 and 30 after ROSC.ResultsCompared with other groups, the number of mitochondria in the hippocampus was increased, and the morphology and structure of mitochondria in MG/MΦ were significantly improved in the Mito group. Our results show higher expression of M2-type markers in MG/MΦ and decreased hippocampal tissue damage in the Mito group. Levels of NSE and S100β in serum, and TNF-α, IL-6 in the hippocampus were decreased, while the levels of TGF-β and IL-10 were increased in the Mito group. Apoptosis rate of neurons in the Mito group was decreased and the NDS of the Mito group was higher than the other groups.ConclusionsExogenous MT can improve neurological function after CPR by promoting the polarization of MG/MΦ to M2-type cells, and this could be a potential method for brain protection after CPR.

Mitochondrial transplantation combined with coenzyme Q10 induces cardioprotection and mitochondrial improvement in aged male rats with reperfusion injury.

Ischaemic heart diseases (IHD) are among the major causes of mortality in the elderly population. Although timely reperfusion is a common treatment for IHD, it causes additional damage to the ischaemic myocardium known as ischaemia-reperfusion (IR) injury. Considering the importance of preventing reperfusion injuries, we aimed to examine the combination effect of mitochondrial transplantation (MT) and coenzyme Q10 (CoQ10 ) in myocardial IR injury of aged male rats. Seventy-two aged male Wistar rats were randomly divided into six groups: Sham, IR, CoQ10 , MT, combination therapy (MT+CoQ10 ) and vehicle. Myocardial IR injury was established by occlusion of the left anterior descending coronary artery followed by reopening. Young male Wistar rats were used as mitochondria donors. Isolated mitochondria were injected intraventricularly (500µL of a respiration buffer containing 6×106 ± 5×105 mitochondria/mL) in MT-receiving groups at the onset of reperfusion. CoQ10 (10mg/kg/day) was injected intraperitoneally for 2weeks before IR induction. Twenty-four hours after reperfusion, haemodynamic parameters, myocardial infarct size (IS), lactate dehydrogenase (LDH) release and cardiac mitochondrial function (mitochondrial reactive oxygen species (ROS) generation and membrane potential) were measured. The combination of MT and CoQ10 improved haemodynamic index changes and reduced IS and LDH release (P<0.05). It also decreased mitochondrial ROS generation and increased membrane potential (P<0.05). CoQ10 also showed a significant cardioprotective effect. Combination therapy displayed greater cardioprotective effects than single treatments. This study revealed that MT and CoQ10 combination treatment can be considered as a promising cardioprotective strategy to reduce myocardial IR injury in ageing, in part by restoring mitochondrial function.

Abstract 165: Immediate Internalization of Transplanted Mitochondria in Circulatory Immune Cells After Cardiac Arrest Resuscitation in Rats

Background: Mitochondrial transplantation (MTx) is an innovative technology that has the potential effect for attenuating ischemia reperfusion injury. We previously demonstrated that MTx significantly improves survival and neurological outcomes after cardiac arrest (CA) and resuscitation in rats. However, the underlying mechanism by which MTx enhances CA outcomes remains unclear. Aim: To investigate the hypothesis that injected donor mitochondria are rapidly internalized by circulatory immune cells, particularly monocytes/macrophages, which play crucial roles in post-CA inflammatory signaling. Methods and Results: Adult male Sprague-Dawley rats underwent sham operation or 10 minutes of asphyxial CA followed by resuscitation with CPR. Either a respiration buffer as a vehicle or donor mitochondria labeled with MitoTracker Deep Red (MTDR) was intravenously injected to animals of both groups. In the CA groups, the injection was performed upon achieving spontaneous circulation. Serial blood samples were collected at 30, 75, and 150 seconds after injection. Flow cytometry analysis revealed that a marked increase of MTDR-positive ratios, peaking at 30 seconds, in CD45 + leukocytes, CD3 + T lymphocytes, and CD11b/c + monocytes/macrophages in the MTx-treated CA group compared to the other groups (Figure A). Notably, in sham-operated rats with donor mitochondrial injection, only minimal donor mitochondria were found in the circulating immune cells at those time points. Brain and spleen 1 h post-CA with MTx showed enhanced signals of MTDR measured using an in vivo imaging system, indicating successful transfer of injected mitochondria to organs (Figure B). Conclusion: Our findings demonstrate rapid internalization of injected mitochondria by circulatory immune cells after MTx in post-CA rats, suggesting a pivotal role for circulating immune cells in the mechanism underlying the beneficial effects of MTx in CA resuscitation.

Abstract 108: Mitochondria-Targeted S-Nitrosothiol MitoSNO Improves Neurological Recovery and Survival After Asphyxial Cardiac Arrest in Rats

Introduction: Mitochondria-targeted S-nitrosothiol MitoSNO offers protection against ischemia-reperfusion injury by attenuating the reverse electron transport of mitochondrial complex I during reperfusion. This study aimed to assess whether administration of MitoSNO after ROSC improves neurological and survival outcomes following cardiac arrest (CA). Methods: Adult male rats were subjected to 10 minute of asphyxial CA followed by mechanical ventilation and manual CPR. After successful CPR, animals were assigned to one of two experimental groups: receiving respiration buffer as a vehicle or MitoSNO (100 ng per dose). Based on our preliminary study, two doses of each drug were intravenously injected via femoral vein: immediately after ROSC and at 60 minutes post-ROSC. The survival time and neurological function score (NFS) were recorded for up to 72 h. Surgery, CPR, drug injections, echocardiography, and immunohistochemistry were performed in a blind manner. Results: MitoSNO significantly improved NFS and survival rate ( Fig A ). Echocardiography revealed that MitoSNO improved left ventricular ejection fraction 2 h post-ROSC compared to the vehicle. In another set of experiments, relative cerebral blood flow (rCBF) compared to baseline was invasively monitored at 2 h post-CA using a laser speckle imaging. The imaging showed CA caused a reduction in rCBF 2 h post-CA in the vehicle group, which was markedly improved by MitoSNO ( Fig B ). Western blotting assay showed, in brain 2 h post-CA, MitoSNO significantly mitigated protein levels of 3-NT, a marker for nitrogen free radical-modified proteins, and cleaved Caspase-3, an indicator of programmed cell death. Immunohistochemistry revealed that MitoSNO suppressed neuronal degeneration in vulnerable brain regions of surviving animals. Conclusions: Our study demonstrated that MitoSNO inhibited the production of oxidative stress in the brain and improved neurological and survival outcomes following CA in rats.

Mitochondrial transplantation protects against sepsis-induced myocardial dysfunction by modulating mitochondrial biogenesis and fission/fusion and inflammatory response.

Sepsis-induced myocardial dysfunction is associated with worse clinical outcomes and high mortality, but no effective therapeutic intervention has been explored, reinforcing the urgent need to develop innovative strategies. Mitochondrial dysfunction underlies the pathogenesis of sepsis-induced myocardial dysfunction. Herein, we assessed the effect of mitochondrial transplantation on sepsis-induced myocardial dysfunction in a rat model of cecal ligation and puncture (CLP)-induced sepsis. Male Wistar rats (n = 80, 12 weeks old, 250-300g) were divided into groups with/without CLP-induced sepsis receiving mitochondrial transplantation in single or two repetitive injections (1h or 1 and 7h post-CLP, respectively). Mitochondria were isolated from donor rats and injected intravenously (400µl of mitochondrial suspension containing 7.5 × 106 mitochondria/ml of respiration buffer) in recipient groups. Twenty-four hours post-operation, LDH and cTn-I levels, mitochondrial functional endpoints, expression of mitochondrial biogenesis (SIRT-1 and PGC-1α) and fission/fusion (Drp1/Mfn1 and Mfn2) genes, and inflammatory cytokines (TNF-α, IL-1β, and IL-6) levels were evaluated. Survival was tested over 72h post-operation. Mitotherapy significantly improved 72-hours survival (P < .05) and decreased LDH and cTn-I levels (P < .01). It also restored mitochondrial function and expression of mitochondrial biogenesis and fusion genes, and decreased the expression of mitochondrial fission gene and the levels of inflammatory cytokines (P < .05 to P < .01). Mitotherapy with repetitive injections at 1 and 7h post-CLP provided noticeable mitoprotection in comparison with the group receiving mitotherapy at single injection. Mitotherapy improved mitochondrial function, biogenesis, and dynamic associated with SIRT-1/PGC-1α network and suppressed inflammatory response in CLP-induced sepsis model, therefore, offers a promising strategy to overcome life-threatening sepsis challenge.

Sample Preparation as a Critical Aspect of Blood Platelet Mitochondrial Respiration Measurements-The Impact of Platelet Activation on Mitochondrial Respiration.

Blood platelets are considered as promising candidates as easily-accessible biomarkers of mitochondrial functioning. However, their high sensitivity to various stimulus types may potentially affect mitochondrial respiration and lead to artefactual outcomes. Therefore, it is crucial to identify the factors associated with platelet preparation that may lead to changes in mitochondrial respiration. A combination of flow cytometry and advanced respirometry was used to examine the effect of blood anticoagulants, the media used to suspend isolated platelets, respiration buffers, storage time and ADP stimulation on platelet activation and platelet mitochondria respiration. Our results clearly show that all the mentioned factors can affect platelet mitochondrial respiration. Briefly, (i) the use of EDTA as anticoagulant led to a significant increase in the dissipative component of respiration (LEAK), (ii) the use of plasma for the suspension of isolated platelets with MiR05 as a respiration buffer allows high electron transfer capacity and low platelet activation, and (iii) ADP stimulation increases physiological coupling respiration (ROUTINE). Significant associations were observed between platelet activation markers and mitochondrial respiration at different preparation steps; however, the fact that these relationships were not always apparent suggests that the method of platelet preparation may have a greater impact on mitochondrial respiration than the platelet activation itself.

Measurement of Mitochondrial Respiration ‐ Comparison of Two Respirometers: MitoCellMt200A versus O2k

Background Mitochondrial function is a critical determinant of cardiomyocyte survival after ischemia/reperfusion (I/R) and thus an important target of cardioprotection. A reliable measurement of mitochondrial respiration is therefore essential for cardioprotection research. Methods Mitochondria were isolated by differential centrifugation from naïve rat hearts (n=8-14) or isolated buffer-perfused rat hearts subjected to 30 min global zero-flow ischemia and 10 min reperfusion (n=8-14). Mitochondrial respiration was measured in 100 µg/ml mitochondria at 37°C during magnetic stirring using two different Clark-type electrode respirometers, the MitoCellMt200A from Strathkelvin (Glasgow, UK) and the O2k from Oroboros (Innsbruck, AT). Two different respiration buffers (incubation buffer and MiR05 buffer) were used as recommended: in mmol/L; incubation buffer: 125.0 KCl, 10.0 3-(N-morpholino) propane sulfonoic acid, 2.0 MgCl2, 5.0 KH2PO4, and 0.2 ethylene glycol tetra-acetic acid; MiR05 buffer: 0.5 ethylene glycol tetra-acetic acid, 3.0 MgCl2, 60.0 lactobionic acid, 20.0 taurine, 10.0 KH2PO4, 20.0 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, and 110.0 D-sucrose. Glutamate and malate (5.0 mmol/L, each) were added as complex I substrates. Complex I respiration was measured after adding adenosine diphosphate (ADP, 1.0 mmol/L). Complex IV respiration was stimulated by adding N,N,N′,N′-tetra-methyl-p-phenylene diamine (TMPD, 300 µmol/L) and ascorbate (3 mmol/L). Mitochondrial respiration was normalized to mitochondrial protein content. Results Baseline, ADP-stimulated complex I and complex IV respiration were higher in mitochondria isolated from naïve rat hearts than in those isolated from rat hearts, which had undergone global zero-flow ischemia and reperfusion - independently of the used respirometers and the used respiration buffers (see Table 1). Baseline respiration was l higher in the MitoCellMt200A respirometer - independently of the respiration buffer used (see Table 1). ADP-stimulated complex I respiration was comparable between the respirometers, whereas the MiR05 buffer resulted in higher respiration than the incubation buffer (see Table 1). Complex IV respiration, as an indicator for equal loading of the chambers with viable mitochondria, was comparable between the respirometers and the buffers but, as expected, reduced after ischemia/reperfusion (see Table 1). Conclusion Independently of the Clark-type electrode respirometers or respiration buffers used, we identify a reduction of mitochondrial respiration after myocardial I/R. However, the respiration buffer composition determines the magnitude of mitochondrial ADP-stimulated complex I respiration. A lower sensitivity of the MitoCellMt200A respirometer in comparison to the O2k high-resolution respirometer at low oxygen consumption may affect the accuracy of baseline respiration measurements.

Mitochondrial Transplantation for Myocardial Protection in Ex-Situ Perfused Hearts Donated after Cardio-Circulatory Death

Donation after circulatory death (DCD) offers an additional source of cardiac allografts, potentially allowing expansion of the donor pool. In this study, we investigated the efficacy of mitochondrial transplantation to improve myocardial function in DCD hearts. Cardiocirculatory death was induced in Yorkshire pigs (40-50 kg, n=22) by cessation of mechanical ventilation. After 20 minutes of warm ischemia, cardioplegia was administered. The hearts were then reperfused on an ex-situ blood perfusion system for 240 minutes. Following 15 minutes of ex-situ perfusion, mitochondria (5 × 109 in respiration buffer, Mitochondria, n=8) or respiration buffer alone (Vehicle, n=8) were delivered as a single bolus to the coronary arteries. A control group (Sham, n=6) was not subjected to warm ischemia. Hearts were perfused in a resting mode for 120 minutes before converting the system to a continuous isovolumic loading mode. Left ventricular (LV) global function, myocardial oxygen consumption, and arterial lactates were compared by two-way repeated-measures analysis of variance (ANOVA) with Bonferroni adjustment. At the end of 240 minutes of reperfusion, LV peak developed pressure (LVdevP) and dP/dt max were significantly higher in the Sham group than in Vehicle (p<0.001 both) and Mitochondria (p=0.002 and p=0.022, respectively). Hearts treated with mitochondria showed a significantly greater LVdevP (p=0.002), dP/dt max (p<0.001) and myocardial oxygen consumption (p=0.004) compared to Vehicle (Figure A-C). There was no significant difference in myocardial oxygen consumption between Sham and Mitochondria (p>0.05), which was significantly lower in Vehicle (p=0.004). No differences were found in arterial lactate levels (Figure D) (p> 0.05). Mitochondrial transplantation significantly preserves myocardial function and oxygen consumption in DCD hearts, thus providing an additional possible benefit for expanding heart donor pool.

Mitochondrial transplantation prolongs cold ischemia time in murine heart transplantation.

Cold ischemia time (CIT) causes ischemia‒reperfusion injury to the mitochondria and detrimentally effects myocardial function and tissue viability. Mitochondrial transplantation replaces damaged mitochondria and enhances myocardial function and tissue viability. Herein we investigated the efficacy of mitochondrial transplantation in enhancing graft function and viability after prolonged CIT. Heterotopic heart transplantation was performed in C57BL/6J mice. Upon heart harvesting from C57BL/6J donors, 0.5 ml of either mitochondria (1 × 108 in respiration buffer; mitochondria group) or respiration buffer (vehicle group) was delivered antegrade to the coronary arteries via injection to the coronary ostium. The hearts were excised and preserved for 29 ± 0.3 hours in cold saline (4°C). The hearts were then heterotopically transplanted. A second injection of either mitochondria (1 × 108) or respiration buffer (vehicle) was delivered antegrade to the coronary arteries 5 minutes after transplantation. Grafts were analyzed for 24 hours. Beating score, graft function, and tissue injury were measured. Beating score, calculated ejection fraction, and shortening fraction were significantly enhanced (p < 0.05), whereas necrosis and neutrophil infiltration were significantly decreased (p < 0.05) in the mitochondria group as compared with the vehicle group at 24 hours of reperfusion. Transmission electron microscopy showed the presence of contraction bands in vehicle but not in mitochondria grafts. Mitochondrial transplantation prolongs CIT to 29 hours in the murine heart transplantation model, significantly enhances graft function, and decreases graft tissue injury. Mitochondrial transplantation may provide a means to reduce graft failure and improve transplantation outcomes after prolonged CIT.

A multi-sensor system for measuring bovine embryo metabolism

This paper presents the development of a multi-sensor platform capable of simultaneous measurement of dissolved oxygen (DO) concentration, glucose and lactate concentrations in a micro-chamber for real-time evaluation of metabolic flux in bovine embryos. A micro-chamber containing all three sensors (DO, glucose, and lactate) was made to evaluate metabolic flux of single oocytes or embryos at different stages of development in ≤ 120 µL of respiration buffer. The ability of the sensor to detect a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis was demonstrated in embryos by an ablation of oxygen consumption and an increase in lactate production following addition of oligomycin, an inhibitor of mitochondrial adenosine triphosphate (ATP) synthesis. An increased reliance upon glycolysis relative to OXPHOS was demonstrated in embryos as they developed from morula to hatched blastocysts by a progressive increase in the lactate/oxygen flux ratio, consistent with isolated metabolic assessments reported previously. These studies highlight the utility of a metabolic multi-sensor for integrative real-time monitoring of aerobic and anaerobic energy metabolism in bovine embryos, with potential applications in the study of metabolic processes in oocyte and early embryonic development.

Monitoring oocyte/embryo respiration using electrochemical-based oxygen sensors

This paper presents a disposable three-electrode, Clark-type biosensor suitable for mitochondrial respirometry in single oocytes and embryos. The biosensor was embedded in a PMMA (polymethyl methacrylate) micro-chamber to allow investigation of single oocytes/embryos immersed in up to 100 μl of respiration buffer. The micro-chamber was sealed to avoid oxygen exchange between the inside of the chamber and the atmosphere, while being maintained at a temperature of 38.5 °C to preserve cell viability. Using amperometry, the oxygen consumption of cells inside the micro-chamber was measured. The measurements from the sensor system revealed basal cell respiration supported by endogenous substrates, respiration associated with proton leak induced by inhibition of the adenosine triphosphate (ATP) synthase (complex V) with oligomycin, and the maximal non-coupled respiratory capacity revealed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) titration. Potential applications of this oxygen sensor system include evaluating effects of metabolic therapies on oocyte bioenergetics and monitoring mitochondrial function throughout oocyte maturation and blastocyst development to predict embryo viability to compliment assisted reproductive technologies.

Scanning Microwave Microscopy of Vital Mitochondria in Respiration Buffer.

We demonstrate imaging using scanning microwave microscopy (SMM) of vital mitochondria in respiration buffer. The mitochondria are isolated from cultured HeLa cells and tethered to a solid graphene support. The mitochondria are kept vital (alive) using a respiration buffer, which provides nutrients to sustain the Krebs cycle. We verify that the mitochondria are "alive" by measuring the membrane potential using a voltage sensitive fluorescent dye (TMRE). The organelles are measured capacitively at 7 GHz. Several technical advances are demonstrated which enable this work: 1) The SMM operates in an electrophysiologically relevant liquid (hence conducting) environment; 2) The SMM operates in tapping mode, averaging the microwave reflection measurement over many tapping periods; 3) A tuned reflectometer enables increased sensitivity; 4) Variable frequencies up to 18 GHz are used; 5) In contrast with traditional matching/resonant methods that exhibit high quality factor that fail in the presence of liquids, interferometric/tuned reflectometer gives the possibility to adjust the quality factor or sensitivity even in the presence of the liquid.

Effects of Buffer Composition and Substrates on Calcium‐stimulated Mitochondrial Respiration and Free Radical Emission

Mitochondrial ATP synthesis is tightly coupled to oxygen consumption in a highly regulated process; however, unavoidable byproducts in the form of reactive oxygen species (ROS) are formed. An important regulator of this process is calcium, but the precise relationship between calcium and free radical production is obscure. Therefore, we measured ROS production under a variety of experimental conditions that mimic the pathological environmental setting. The Oroborous oxygraph system was used to simultaneously measure ROS emission and the oxygen consumption rate from isolated cardiac guinea pig mitochondria energized with different substrates and challenged with different calcium concentrations. The Amplex Red assay was used to quantify ROS emission rates. The substrates used utilized a mixture of the carbohydrate and fatty acid dependent metabolic pathways. Two respiration buffers, one lactobionate‐based and the other potassium chloride‐based, were used to quantify the differences in respiration and ROS emission rates. Relative to the potassium chloride‐based respiration buffer, the lactobionate‐based buffer resulted in higher ADP‐stimulated respiration rates and higher ROS emission rates for each substrate combination. On average, the presence of calcium depressed ROS emission rates during the leak respiration, sodium/calcium cycling, and ADP‐stimulated states. The lower respiration rates in the potassium chloride‐based buffer is likely due to the presence of inhibitory levels of chloride. The high chloride levels also appear to depress ROS emission. Overall, these data refute the hypothesis that calcium stimulates ROS emission from isolated mitochondria. Studies are ongoing to further characterize ROS emission from cardiac guinea pig mitochondria and elucidate the relationship between calcium and ROS emission.Support or Funding InformationR00‐HL121160This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The effect of respiration buffer composition on mitochondrial metabolism and function.

Functional studies on isolated mitochondria critically rely on the right choice of respiration buffer. Differences in buffer composition can lead to dramatically different respiration rates leading to difficulties in comparing prior studies. The ideal buffer facilities high ADP-stimulated respiratory rates and minimizes substrate transport effects so that the ability to distinguish between various treatments and conditions is maximal. In this study, we analyzed a variety of respiration buffers and substrate combinations to determine the optimal conditions to support mitochondrial function through ADP-stimulated respiration and uncoupled respiration using FCCP. The buffers consisted of a standard KCl based buffer (B1) and three modified buffers with chloride replaced by the K-lactobionate, sucrose, and the antioxidant taurine (B2) or K-gluconate (B3). The fourth buffer (B4) was identical to B2 except that K-lactobionate was replaced with K-gluconate. The substrate combinations consisted of metabolites that utilize different pathways of mitochondrial metabolism. To test mitochondrial function, we used isolated cardiac guinea pig mitochondria and measured oxygen consumption for three respiratory states using an Oroboros Oxygraph-2k. These states were the leak state (energized mitochondria in the absence of adenylates), ADP-stimulated state (energized mitochondria in the presence of saturating ADP concentrations), and uncoupled state (energized mitochondria in the presence of FCCP). On average across all substrate combinations, buffers B2, B3, and B4 had an increase of 16%, 26%, and 35% for the leak state, ADP-simulated state, and uncoupled state, respectively, relative to rates using B1. The common feature distinguishing these buffers from B1 is the notable lack of high chloride concentrations. Based on the respiratory rate metrics obtained with the substrate combinations, we conclude that the adenine nucleotide translocase, the dicarboxylate carrier, and the alpha-ketoglutarate exchanger are partially inhibited by chloride. Therefore, when the goal is to maximize ADP-stimulated respiration, buffers containing K-lactobionate or K-gluconate are superior choices compared to the standard KCl-based buffers.

Mg(2+) differentially regulates two modes of mitochondrial Ca(2+) uptake in isolated cardiac mitochondria: implications for mitochondrial Ca(2+) sequestration.

The manner in which mitochondria take up and store Ca(2+) remains highly debated. Recent experimental and computational evidence has suggested the presence of at least two modes of Ca(2+) uptake and a complex Ca(2+) sequestration mechanism in mitochondria. But how Mg(2+) regulates these different modes of Ca(2+) uptake as well as mitochondrial Ca(2+) sequestration is not known. In this study, we investigated two different ways by which mitochondria take up and sequester Ca(2+) by using two different protocols. Isolated guinea pig cardiac mitochondria were exposed to varying concentrations of CaCl2 in the presence or absence of MgCl2. In the first protocol, A, CaCl2 was added to the respiration buffer containing isolated mitochondria, whereas in the second protocol, B, mitochondria were added to the respiration buffer with CaCl2 already present. Protocol A resulted first in a fast transitory uptake followed by a slow gradual uptake. In contrast, protocol B only revealed a slow and gradual Ca(2+) uptake, which was approximately 40% of the slow uptake rate observed in protocol A. These two types of Ca(2+) uptake modes were differentially modulated by extra-matrix Mg(2+). That is, Mg(2+) markedly inhibited the slow mode of Ca(2+) uptake in both protocols in a concentration-dependent manner, but not the fast mode of uptake exhibited in protocol A. Mg(2+) also inhibited Na(+)-dependent Ca(2+) extrusion. The general Ca(2+) binding properties of the mitochondrial Ca(2+) sequestration system were reaffirmed and shown to be independent of the mode of Ca(2+) uptake, i.e. through the fast or slow mode of uptake. In addition, extra-matrix Mg(2+) hindered Ca(2+) sequestration. Our results indicate that mitochondria exhibit different modes of Ca(2+) uptake depending on the nature of exposure to extra-matrix Ca(2+), which are differentially sensitive to Mg(2+). The implications of these findings in cardiomyocytes are discussed.

The Potential for Another Calcium Uptake Mode in Cardiac Mitochondria

Cytosolic Ca2+ levels are tightly regulated in cardiomyoctes. In many instances mitochondria play a direct role in this regulation. They take up Ca2+ via the Ca2+ uniporter, buffer Ca2+ by calcium-phosphate sequestration, and release Ca2+ primarily through the electrogenic Na+/Ca2+ exchanger. However, the manner in which Ca2+ is taken up is still in dispute. In this study we used energized mitochondria isolated from guinea pig hearts to explore the potential for two modes of Ca2+ uptake. In addition, we sought to demonstrate the manner in which mitochondria take up, sequester, and release Ca2+, and how Ca2+ transport is differentially modulated by Mg2+. To carry out this study, we monitored extra-matrix and matrix [Ca2+] during Ca2+ loading and unloading experiments using Fura-4F PP and Fura-4F AM fluorescence to quantify extra-matrix and matrix [Ca2+], respectively. Two loading protocols were used: 1) a bolus of CaCl2 was added to a suspension of mitochondria in respiration buffer and 2) mitochondria were added to respiration buffer already containing CaCl2. In all experiments, ruthenium red was later added to stop Ca2+ uptake and NaCl was added to initiate Ca2+ efflux. Also, each protocol was executed in the presence and absence of extra-matrix MgCl2. Depending on the protocol, two distinct profiles of Ca2+ uptake were observed, whereby using protocol 1 resulted in a faster mode of Ca2+ uptake and protocol 2 in a slower mode. Furthermore, Ca2+ uptake and efflux were inhibited by MgCl2. We found that Mg2+ reduced the ability of mitochondria to sequester Ca2+ independent of the protocol. In summary, these observations derived from our experiments show the potential for at least two modes of Ca2+ uptake and provide us with a better understanding of how matrix Ca2+ dynamics change under physiological and pathophysiological conditions.

Nanochannel Trap Arrays for Monitoring Single Mitochondrion Behavior

We present a nanofluidic device for trapping and fluorescence imaging of individual isolated mitochondria. The device consists of an array of 10 parallel nanochannels fabricated out of PDMS each with a cross section of 500 nm x 2 µm. Mitochondria are isolated from a human cervical cancer cell line (HeLa). Respiration buffer containing the mitochondria is pumped into the device using a syringe pump. Due to the small height of the trap channels individual mitochondria get immobilized one by one along the channels. Time lapsed fluorescence microscopy of JC-1 and TMRM stained mitochondria reveals that trapped mitochondria maintain their membrane potential. The effect of adding substrates and calcium on the membrane potential is studied and the results indicate that mitochondria remain vital and functional in this trapped state. Flickering of membrane potential in some substrate fed mitochondria is observed.

Nanofluidic Platform for Single Mitochondria Analysis Using Fluorescence Microscopy

Using nanofluidic channels in PDMS of cross section 500 nm × 2 μm, we demonstrate the trapping and interrogation of individual, isolated mitochondria. Fluorescence labeling demonstrates the immobilization of mitochondria at discrete locations along the channel. Interrogation of mitochondrial membrane potential with different potential sensitive dyes (JC-1 and TMRM) indicates the trapped mitochondria are vital in the respiration buffer. Fluctuations of the membrane potential can be observed at the single mitochondrial level. A variety of chemical challenges can be delivered to each individual mitochondrion in the nanofluidic system. As sample demonstrations, increases in the membrane potential are seen upon introduction of OXPHOS substrates into the nanofluidic channel. Introduction of Ca(2+) into the nanochannels induces mitochondrial membrane permeabilization (MMP), leading to depolarization, observed at the single mitochondrial level. A variety of applications in cancer biology, stem cell biology, apoptosis studies, and high throughput functional metabolomics studies can be envisioned using this technology.

Mitochondrial DNA Variant for Complex I Reveals a Role in Diabetic Cardiac Remodeling

Myocardial remodeling and dysfunction are serious complications of type 2 diabetes mellitus (T2DM). Factors controlling their development are not well established. To specifically address the role of the mitochondrial genome, we developed novel conplastic rat strains, i.e. strains with the same nuclear genome but a different mitochondrial genome. The new animals were named T2DN(mtFHH) and T2DN(mtWistar), where the acronym T2DN denotes their common nuclear genome (type 2 diabetic nephropathy (T2DN) rats) and mtFHH or mtWistar the origin of their mitochondria, Fawn Hooded Hypertensive (FHH) or Wistar rats, respectively. The T2DN(mtFHH) and T2DN(mtWistar) showed a similar progression of diabetes as determined by HbA1c, cholesterol, and triglycerides with normal blood pressure, thus enabling investigation of the specific role of the mitochondrial genome in cardiac function without the confounding effects of obesity or hypertension found in other models of diabetes. Echocardiographic analysis of 12-week-old animals showed no abnormalities, but at 12 months of age the T2DN(mtFHH) showed left ventricular remodeling that was verified by histology. Decreased complex I and complex IV but not complex II activity within the electron transport chain was found only in T2DN(mtFHH), which was not explained by differences in protein content. Decreased cardiac ATP levels in T2DN(mtFHH) were in agreement with a lower ATP synthetic capacity by isolated mitochondria. Together, our data provide experimental evidence that mtDNA sequence variations have an additional role in energetic heart deficiency. The mitochondrial DNA background may explain the increased susceptibility of certain T2DM patients to develop myocardial dysfunction.

Mitochondrial Dysfunction Impairs Tumor Suppressor p53 Expression/Function

Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying mechanisms by which mitochondria suppress tumorigenesis are far from being clear. In this study, we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53 using a set of respiration-deficient (Res(-)) mammalian cell mutants with impaired assembly of the oxidative phosphorylation machinery. Our data suggest that normal mitochondrial function is required for γ-irradiation (γIR)-induced cell death, which is mainly a p53-dependent process. The Res(-) cells are protected against γIR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in the absence of the MWFE subunit reduces the steady-state level of the p53 protein, although there is no effect on the p53 protein level in the absence of the ESSS subunit that is also essential for complex I assembly. The p53 protein level was also reduced to undetectable levels in Res(-) cells with severely impaired mitochondrial protein synthesis. This suggests that p53 protein expression is differentially regulated depending upon the type of electron transport chain/respiratory chain deficiency. Moreover, irrespective of the differences in the p53 protein expression profile, γIR-induced p53 activity is compromised in all Res(-) cells. Using two different conditional systems for complex I assembly, we also show that the effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings will have major implications in the understanding of cancer development and therapy.