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Core D, San Francisco: Laboratory for Development of Signaling Assays

Core D, San Francisco: Laboratory for Development of Signaling Assays. B Lymphocytes Initiate ligand screen, 1st publication (with Core C, Dallas) Long term culture Myocytes. The SF VAMC AfCS Lab. Tim O’Connell. Paul Simpson. Luyi Li. Bill Seaman. Tamara Roach. Melissa

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Core D, San Francisco: Laboratory for Development of Signaling Assays

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  1. Core D, San Francisco: Laboratory for Development of Signaling Assays • B Lymphocytes • Initiate ligand screen, 1st publication (with Core C, Dallas) • Long term culture • Myocytes

  2. The SF VAMC AfCS Lab Tim O’Connell Paul Simpson Luyi Li Bill Seaman Tamara Roach Melissa Kachura Susan Ricker

  3. Myocyte Isolation Procedure Hanging Heart In Situ Perfusion Steps in both: • Ca++ wash out • Collagenase digestion ( 50 mM Ca++) • Mechanical disaggregation • Collagenase inhibition (BCS) • Ca++ reintroduction • Wash & count Constant Flow Constant Pressure or Constant Flow pump pump • • needle inserted in LV apex in situ • • drain atrium & clamp aorta • • constant pressure (~75 mmHg, 125 cm) • or • constant flow (4 ml/mim) • dissect heart • cannulate aorta • constant flow (4 ml/min)

  4. Myocyte Yields with DifferentIsolation Techniques * measured since January, 2002 In Situ preparation is much easier technically In Situ constant flow preparation is easier than constant pressure

  5. Myocyte Culture Procedure Goal: Maintain rod-shaped myocytes that signal for 72 hrs Plate for 1 hr on laminin coated dishes in: MEM w/Hanks BSS w/ 5% BCS 10 mM BDM Penicillin Change Medium to: MEM w/Hanks BSS w/ 1 mg/ml Insulin 0.5 mg/ml Transferrin 0.55 ng/ml Selenium 1 mg/ml BSA 10 mM BDM Penicillin Culture for up to 72 hours at 37°C in 2% CO2 0 hr 24 hr 72 hr

  6. Myocyte Experimental Timeline Plating Assay Signaling At 24 hrs Assay Signaling At 72 hrs Myocyte Isolation Medium Change 3 hrs 1 hr 24 hrs 48 hrs Assays: Gs: cAMP, PLB phosphorylation, myocyte contraction Gi:inhibition of cAMP Gq:ERK phosphorylation

  7. 20000 16000 12000 8000 4000 0 1 0 - 1 0 1 0 - 9 1 0 - 8 1 0 - 7 1 0 - 6 1 0 - 5 Activation of Gs Signaling in Myocytes at 24 and 72 Hours Isoproterenol, a b-AR agonist that signal through Gs, increases cAMP in a concentration-dependent manner 24 hrs EC50 28 nM fmol cAMP/ 20,000 myocytes 72 hrs EC50 24 nM Isoproterenol (nM)

  8. 120000 100000 80000 35000 30000 25000 20000 15000 10000 5000 0 Activation of Gi Signaling in Myocytes at 24 and 72 Hours Carbachol, a muscarinic agonist that signals through Gi, reduces isoproterenol- and forskolin- induced cAMP accumulation Isoproterenol (1 mM) Forskolin (100 mM) Carbachol (100 mM) fmol cAMP/ 20,000 myocytes 24 hrs 72 hrs Fsk Iso Carb Fsk Carb Control Iso

  9. Activation of Gq Signaling in Myocytes at 24 and 72 Hours Phenylephrine, an a1-AR agonist, and Endothelin-1 which both signal through Gq, increase ERK1/2 phosphorylation 24 hrs 72 hrs Control PE 20 mM ET-1 100 nM PMA 100 nM Control PE 20 mM ET-1 100 nM PMA 100 nM Phospho-ERK Total-ERK

  10. Phospholamban Phosphorylation in Myocytes at 24 and 72 Hours Isoproterenol, a b-AR agonist that signals through Gs, increases phospholamban phosphorylation 24 hrs 72 hrs Control Iso 1 mM Control Iso 1 mM Gb Phospho-PLB

  11. Activation of E-C Coupling in Myocytes at 24 Hours Myocytes contracting under field stimulation Myocytes quiescent for first 5 seconds Stimulated at 80V, 1 Hz for 20 seconds Then increase frequency to 1.5 Hz for 15 seconds

  12. Activation of E-C Coupling in Myocytes at 24 Hours Isoproterenol, a b-AR agonist that signals through Gs and increases phospholamban phosphorylation, induces myocyte contraction

  13. 8 6 4 2 0 Activation of E-C Coupling in Myocytes at 72 Hours Isoproterenol induces myocyte contraction Myocyte Contraction measured as %Shortening of individual cardiac myocytes * % Shortening 270% 16 16 Control 1 mM Isoproterenol * p < 0.05

  14. Requirements for the Ligand Screen Assay Time Points Myocytes Phosphoprotein 0, 2, 5, 12, 30 min 5 x 35 mm dish (250,000 myocytes) RNA Array 0, 30, 120, 240 min 16 x 60 mm dish (2,400,000 myocytes) cAMP 0, 2, 5, 12, 30 min 5 x 35 mm dish (250,000 myocytes) Calcium in development Total 2.9 x106 myocytes Project that we need 3 hearts/ligand

  15. Summary: Myocytes • Criteria • Acute signaling: cAMP, phosphorylation, contraction. • Suitable for mutation (RNAi, antisense, transfection, etc). • Reproducible within and between labs. • Convenient, sufficient throughput. • Mouse, normal, adult.

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