Cardiomyocyte function characterised through combined analysis of metabolic flux, beat rate and cellular oxygenation and its application in in vitro ischemia reperfusion modelling Ryan McGarrigle, Ph.D. Research Scientist
What we do.. Luxcel Intro may remove Advanced Fluorescence-based technologies Oxygen & ph Sensing Microplate-based Metabolism Assays Cell Metabolism Made Easy www.luxcel.com @LuxcelBio
More than just an ATP factory Mitochondrial reticulum heavily integrated into cellular processes not just an ATP factory Interplay between metabolism and cell signalling
Cell Metabolism Made Easy ph-xtra Glycolysis Assay Cell membrane Glucose ATP ph-xtra Stress Test Glycolysis Pyruvate? Lactate & H + [O 2 ] ATP O 2 H 2 O OxPhos NADH FADH 2 TCA Cycle FAO Glutamate FA MitoXpress -Xtra Oxygen Consumption Assay Mitochondrion Glutamine MitoXpress -Intra MitoXpress Stress Test - Oxygenation Cytosol CO 2 Glutamine Microplate-based cell metabolism assays 2D and 3D compatible Measured on Fluorescence plate reader Multiplexable with conventional assays MitoXpress - FAO Adapted from Nature Reviews Immunology 5, 712-721.
Fluorescent Signal Luxcel Core Technology: O 2 Sensing Family of fluorescent O 2 sensitive probes & dyes O 2 causes a reversible decrease in probe signal Respiration: O 2 = Probe signal [O 2 ] = (Io/I) -1 K D Non-Chemical & Reversible [O 2 ] [O 2 ] [O 2 ] Hypoxic [O 2 ] Normoxic Y. Will, Hynes J. et al. - Nature Protocols, 2007,1: 2563-2572.
MitoXpress-Xtra Oxygen Consumption Assay
Fluorescent Signal Oxygen Consumption: MitoXpress-Xtra Oil (O 2 barrier) Activation Untreated [O 2 ] [O 2 ] Growth media Inhibition Cells Control
CLARIOstar Assay Protocol Buttons
CLARIOstar Assay Protocol Buttons
MitoXpress Xtra: MARS & Data Analysis
µs OCR/h MitoXpress Stress Test: Data Output 35 48 46 44 42 40 38 36 34 32 30 28 0 20 40 60 Time (min) FCCP (2.5 µm) Untreated Oligomycin (1 µm) Antimycin (1 µm) 30 25 20 15 10 5 0 Spare capacity ATP coupled Non-ATP coupled Non ETC Basal Maximal Data by Melanie Schwalfenberg
Assay Platform Integration Beat Rate hips Cardiomyocytes Cor4U (NCARDIA) Impact of isoproterenol on beat rate and metabolism Data generate by Cristina Bertinetti-Lapatki (Roche) Untreated Isoproterenol (1µM) Antimycin (1µM) % O 2 Untreated Isoproterenol (1µM) Courtesy of NCARDIA Time
Glycolytic Flux (ECA): ph-xtra
% Effect Slope (µs/hr) % Effect Cor4U Metabolic Characterisation OxPhos, Glycolysis, FAO 600 500 400 300 O 2 Consumption Glycolytic Flux (ECA) ATP ATP depletion is ameliorated through increased glycolytic flux. 200 100 0 350 300 250 200 DMSO Antimycin A Rotenone FCCP (1 µm) (1 µm) (2.5 µm) O 2 Consumption Glycolytic Flux (ECA) Baseline 12 10 8 LC-FA respiration Non LC-FA respiration Ox Under stressed conditions (FCCP treatment) increased ETC activity is driven mainly by long-chain fatty acid oxidation Facilitates high-throughput metabolic characterisation 150 100 6 4 Non ETC OCR 50 2 0 0 0.5 1 Antimycin (µm) 0 Coupled Uncoupled
Summary 1 Measured mitochondrial function (OxPhos) and Glycolytic flux (ECA) Panel of convenient companion kits to probe metabolism further Simple mix and measure assay procedures Plate-reader based metabolism assays convenient and high throughput No requirement for expensive dedicated instrumentation Can be multiplexed with other relevant parameters (MMP, ROS, Cell viability etc) Flexible - Compatible with any plate type (including 3D plates) Also very well suited to suspensions cells (including bacterial, yeast, fungi etc)
Oxygenation & Ischemia Reperfusion
Monitoring Oxygenation MitoXpress -Intra Intracellular Oxygen Assay Unique cell-penetrating O 2 sensitive nanoparticle probe Long-term stability Simple loading procedure Multiplexable Plate-reader compatible Robust O 2 scale via ratiometic TRF Measurements
Ischemia Reperfusion Ischaemia-reperfusion (IR) injury: Occurs when tissue blood supply is restricted and subsequently restored (myocardial infarction, renal failure and stroke) Reperfusion associated with ROS-mediated damage - Triggers inflammatory - cell death. Rational development of targeted therapeutic interventions requires good in vitro model Current In vitro IR models ATP depletion Oil overlays to induce autohypoxia glucose oxidase addition to cause sample deoxygenation N 2 purged hypoxia chambers to induce a slow reduction in dissolved O 2 They do not replicate conditions of IR injury in vitro Need rapid, controlled, transient, ischemic shock and rapid controlled reperfusion Need real-time cellular oxygenation monitoring to allow accurate IR characterisation Need parallel measurements of critical IR injury parameters such as reactive oxygen species (ROS) and mitochondrial membrane potential (MMP).
Ischemia Reperfusion Cor.4U cells MitoXpress -Intra CLARIOstar with ACU
MitoXpress-Intra Signal (µs) MitoXpress -Intra Intracellular Oxygen Assay 70 65 60 55 50 45 40 35 30 25 0 5 10 15 20 % O 2 Real-time monitoring of cellular oxygenation Data by Conn Carey & Melanie Schwalfenberg
MitoXpress -Intra Intracellular Oxygen Assay Real-time monitoring of cellular oxygenation allows us to question key assumptions Assumption 1: To maintain cells at desired [O 2 ], simply set chamber to that value
MitoXpress -Intra Intracellular Oxygen Assay Instrument O 2 Antimycin Treated (ETC inhibition) Untreated (Metabolically Active) Real-time monitoring of cellular oxygenation allows us to question key assumptions Assumption 1: To maintain cells at desired [O 2 ], simply set chamber to that value
Cellular Oxygenation is Dynamic Basal metabolism has reduced O 2 concentrations to ~14% Treatment with the ETC inhibitor Antimycin ETC & [O 2 ] returns to ambient levels β-adrenoreceptor agonist isoproterenol: beat rate - O 2 consumption - [O 2 ] ~6% Reduction in intracellular O 2 driven by Cor4U respiration % O 2 Compound treatment Antimycin (1µM) Assumption 2: Respiratory deoxygenation is fixed, just requires a simple correction Invalid Untreated Further O 2 depletion caused by Isoproterenol treatment Isoproterenol (1µM) Time (min) Data by Conn Carey Cor4U cardiomyocytes from Ncardia
DHE Intensity JC-1 Ratio Multiparametric Analysis (Cor4U) %O 2 20 18 16 14 12 10 8 6 4 2 0 0 50 100 150 Time (min) Data by Conn Carey & Melanie Schwalfenberg ACU Non Respiring Respiring MMP ROS 1 0.8 0.6 0.4 0.2 0 40000 35000 30000 25000 20000 15000 10000 5000 0 Untreated Untreated Cor4U cells Ncardia (www.ncardia.com) Antimycin Antimycin
Summary 2 Cells in vivo are typically cultured at hyperglycaemic and hyperoxic conditions This can have significant biological consequences Technology exits control cellular oxygen (e.g. BMG CLARIOstar & ACU) Monitoring is the critical aspect MitoXpress -Intra THE ONLY AVAILABLE SOLUTION Not correct that cells experience the O 2 set value (Respiration V Diffusion) Not correct that respiratory contribution to hypoxia is fixed Awareness of these factors can improve physiological relevance of in vitro models Multiparametric analysis - delineate relationship between O 2 & metabolism/ signalling Providing new insights into Tumour metabolism & ischemia reperfusion
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