5/31/2013. Mechanical-Energetic Coupling in Airway Smooth Muscle. Evolution of Academic Medical Centers

Transcription

1 Mechanical-Energetic Coupling Mayo Clinic One of the World s Largest Medical Centers Gary C. Sieck, Ph.D. Department of Physiology & Biomedical Engineering Mayo Clinic College of Medicine Physiology & BME Founded by Dr. William Worrall Mayo and his sons William J. Mayo and Charles H. Mayo. Today Number of staff physicians & scientists --- ~3, Number of employees --- ~6, Number of Clinic patients --- ~65, Number of hospital admissions --- ~135, Total revenue $9.2 billion Total research funding $55 million ($325 million extramural funding) Evolution of Academic Medical Centers Balance Between Discovery (Basic) Science and Translational/Clinical Research Implicit in the process of natural selection is that inefficient mechanisms will be either improved or discarded. Symmorphosis Hypothesis - In evolution, no structure is formed or maintained unless it satisfies functional demands (Weibel & Taylor, 1981). Biological systems adhere to an economy of design that can be applied to other complex systems. Discovery Science Translational/Clinical Research For academic medical centers, the capacities for basic science discovery, translational research and clinical practice should be matched. Physiology & Biomedical Engineering 1

2 Bedside to Bench Back to Bedside Approach Asthma: Airways Hyperresponsiveness and Remodeling Identify clinical problems Apply research to clinical problems Conduct targeted research Asthma affects 17 million people in the USA (~8% of the population). Characterized by airway smooth muscle hyperresponsiveness and remodeling - triggered by inflammation and mediated by inflammatory cytokines. Dynamic Properties of the Airway Excitation-Contraction Coupling Airway smooth muscle contractile proteins are highly adaptive to intrinsic and extrinsic stresses and are embedded within a malleable cytoskeletal structure that is tethered to the extracellular matrix. The endoplasmic (sarcoplasmic) reticulum (ER/SR) and mitochondria are also highly dynamic motile structures within the cell. Calmodulin (CaM) Elevated [ ] i -CaM -CaM MLC Kinase Cross-Bridge Cycling Cross-Bridge Recruitment MLC 2 Phosphorylation MLC 2 Dephosphorylation / Relaxation MLC Phosphatase 2

3 Excitation-Energy Coupling Excitation mncx ETC TCA ΔΨ m F1Fo- as e [ [ + ] cyt ] mito ADP MCU Mitochondria O 2 PTP leak ANT ADP Inflammatory Cytokines Enhance Intracellular Response Amplitude [ ] (nm) IL-13 Contraction Hydrolysis 2 Response to 1 M ACh Intracellular Regulation Receptor Agonist influx Voltage dependent Store operated Ca2+ efflux Spontaneous Transients (Sparks) G protein PLC PIP 2 IP 3 IP 3 Phosphatase ADPR cyclase CD38 CICR -NAD cadpr CaM FKB12.6 cadpr Hydrolase Reuptake IP 3 R SERCA SR RyR RyR Release 3

4 Sparks Reflect SR Release via RyR Channels In Airway Smooth Muscle Cells Sparks Fuse into Larger Transients Cells Cell #1 Cell #2 2.5 mm Extracellular 2.5 mm Extracellular Incidence (% Total) Zero Extracellular 2.5 mm Extracellular +2 M Ryanodine (nm) sparks are unaffected by altered influx Incidence of sparks increases with an increase in the open probability of RyR channels Anything that increases open probability of RyR channels (e.g., cadpr) increases the incidence of larger transients ACh-Induces Propagating [ ] i Oscillations Cells Localized [ ] i Oscillation ACh-Induced [ ] i Oscillations Blocked by Depletion of SR Stores 1 nm 5 s Larger transients propagate through the cell and recur as [ ] i oscillations within localized regions 1 M ACh 5 mm Caffeine 4

5 Intracellular Regulation Receptor Agonist influx Voltage dependent Store operated Ca2+ efflux Cytokines Decrease SERCA2 Expression and Slow SR Reuptake G protein PLC PIP 2 IP 3 IP 3 Phosphatase ADPR cyclase CD38 CICR -NAD cadpr CaM FKB12.6 cadpr Hydrolase Reuptake IP 3 R SERCA SR RyR RyR Release Store-Operated Entry (SOCE) SOCE Store-Operated Entry (SOCE) Cells TRPC STIM1 IP 3 R SOCE RyR SERCA SR SOCE Key stores in sarcoplasmic reticulum (SR) must be maintained SR depletion triggers store-operated entry SOCE - via transient receptor protein channel TRPC (transient receptor protein channel) Triggering of SOCE involves translocation and aggregation of STIM1(stromal interaction molecule) and interaction with ORAI1 SOCE triggered by depletion of SR stores regardless of underlying cause 5

6 Cytokines Increase Store-Operated Entry TNF Increases Expression of TRPC3 CPA-induced SOCE Store-Operated Entry (% ) Only TRPC3 expression increases in response to TNF- TNF IL13 TRPC3 Knockdown Blunts TNF - Induced Increase in Store-Operated Entry Aggregation of STIM1 Following SR Depletion Influx (% Response) TNF- TRPC3 sirna Untreated TNF- Pre-exposure to nocodazole, an -tubulin inhibitor, prevents STIM1 aggregation following CPA-induced SR depletion Average Puncta Size ( m) Nocodazole CPA Time (min) 6

7 STIM1 sirna Knockdown Reduces Store-Operated Entry STIM1 Aggregates Towards TRPC3 Following SR Depletion Influx (% Response) Nonsense STIM1 sirna FRET Energy Transfer Efficiency (% ) HBSS + CPA Cy3-tagged STIM1 (donor fluorophore) and Cy5-tagged TRPC3 (acceptor fluorophore). Acceptor photobleaching increases FRET efficiency when molecules are in close proximity Mitochondrial Regulation and SR Repletion SOCE TRPC STIM1 IP 3 R RyR Mitochondria SERCA SR Mitochondria are juxtaposed to plasma membrane and SR Mitochondria contain channels uniporter, Na + / exchanger and /H + exchanger Mitochondrial production is dependent and essential for SR reuptake (i.e., SERCA) Proximity of SR, Mitochondria and Plasma Membrane Is Important in the Regulation of Intracellular SOCE SOCE Proximity Buffering STIM1 SERCA SR IP 3 R Ca2+ SERCA Reuptake SR RyR STIM1 Regulation IP 3 R RyR 7

8 Mitochondria Move Away from Plasma Membrane during Agonist Stimulation Mitochondria Move Toward SR during Agonist Stimulation and with SOCE Mitochondrial - PM Distance ( m) Phalloidin Time after ACh (s) Mitochondrial - SR Distance ( m) ACh CPA Time (min) Mitochondria and SR are Functionally Coupled Dynamic Coupling of Mitochondria and SR is Mediated by MNF2 488 nm Laser Excitation <1 nm distance ER-GFP Donor 488 nm excitation nm emission MFN2-YFP Acceptor 514 nm excitation nm emission Spectral Unmixing Resting ER-GFP FRET Resting ASM FRETSignal 1 MACh 8

9 Hot Spot Theory for Mitochondrial Regulation Cytosolic and Mitochondrial Responses are Coupled Cells Cytosol Mitochondria ~1 s lag 1 MACh Dorn and Maack. J Mol Cell Cardiol 55: 42-49, 213. Inflammatory Cytokines Reduce MFN2 Expression MFN2 Protein Expression (Relative to GAPDH) MFN2 GAPDH IL-13 IL-13 Inflammatory Cytokines Uncouple Cytosolic and Mitochondrial Responses Amplitude [ ] (nm) Cytoplasmic IL-13 Mitochondrial 9

10 Inflammatory Cytokines Trigger SR-Mitochondrial Uncoupling Mitochondrial Network Fusion/Fission 8 Mitochondria red SR - green 1 m % of SR in Proximity with Mitochondria Koopman. Mitochondrial Dynamics. Nijmegen Center for Mitochondrial Disorders Inflammatory Cytokines Trigger Mitochondrial Fragmentation Inflammatory Cytokines Trigger Mitochondrial Fragmentation 24 h exposure ASM cells 1 m Form Factor (FF): Metric of mitochondrial length and degree of branching 2 perimeter FF 4π Area -exposed ASM cells 1 m Aspect Ratio (AR) : Metric of mitochondrial length major axis length AR minor axis length 1

11 Role of DRP1 (Fusion) and MFN2 (Fission) in Mitochondrial Dynamics Effect of Inflammatory Cytokines on Excitation-Energy Coupling Excitation-Energy Coupling Inflammatory Cytokines Increase Force Generation Excitation O 2 ETC ΔΨ m F1Fo- as e TCA mncx [ [ + ] cyt ] mito ADP MCU Mitochondria PTP leak ANT ADP Force (mn/g protein) Contraction Hydrolysis TNFa IL13 11

12 Consumption Increases with Force Generation A. B. Force (N cm -2 ) hydrolysis rate (nmol cm -3 s -1 ) Tension cost (nmol N -1 cm -1 s -1 ) Phalloidin Phalloidin Time (min) 8 Phalloidin Time (min) 1 8 Phalloidin Time (min) A Force (N cm -2 ) Inflammatory Cytokines Increase Tension Cost Cyto D +Cyto D B Hydrolysis Rate (nm cm -3 s -1 ) Cyto D +Cyto D C Tension Cost (nm N -1 cm -1 s -1 ) Cyto D +Cyto D Inflammatory Cytokines Increase Mitochondrial Respiration /ADP Ratio (F49/F435) Mitochondrial Membrane Potential ( Ψm) TMRM Fluorescence (AU) ACh FCCP Time (s) Δ/ADP Ratio (1mMACh Stimulation) Time (s). 12

13 Inflammatory Cytokines Increase Mitochondrial Respiration Mitochondrial Fragmentation Leads to Mitogenesis 1 ASM cells 1 m Mitochondrial Volume Density (%) exposed ASM cells 1 m Inflammatory Cytokines Increase Reactive Oxidant Species (ROS)Generation Excitation-Energy Coupling Excitation O 2 ROS Generation Fluorescence (AU) CM-H 2 DCFDA MitoSOX ETC ΔΨ m F1Fo- as e TCA mncx [ [ + ] cyt ] mito ADP MCU Mitochondria PTP leak ANT ADP TNFa IL-13 Contraction Hydrolysis 13

14 Inflammatory Cytokines Trigger and Unfolded Protein Response (ER/SR Stress) P P Inflammation/ROS PERK SR(ER) Stress Marker Proteins ATF6 Splicing of XBP1 mrna XBP1 protein SR stress target genes Chaperones, MFN2? IRE1α P P PERK Protein Expression (Relative to GAPDH) Spliced XBP1 mrna Expression (Relative to GAPDH) IL-13 IL-13 Inflammatory Cytokines Trigger ER/SR and Mitochondrial Stress There is no such thing as a new idea. It is impossible. We simply take a lot of old ideas and put them into a sort of mental kaleidoscope. We give them a turn and they make new and curious combinations. Cellular Imaging and Physiology Laboratory Department of Physiology & Biomedical Engineering Mark Twain Research supported by grants from the NIH HL9675, HL7439, HL3768, GM56686, & AR