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

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

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

5. 0 hr24 hr72 hr 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  g/ml Insulin 0.5  g/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% CO 2 Goal: Maintain rod-shaped myocytes that signal for 72 hrs Myocyte Culture Procedure

6. Myocyte Isolation 3 hrs Plating Assay Signaling At 24 hrs Assay Signaling At 72 hrs 1 hr24 hrs48 hrs Medium Change Assays: Gs:cAMP, PLB phosphorylation, myocyte contraction Gi:inhibition of cAMP Gq:ERK phosphorylation Myocyte Experimental Timeline

7. 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 20000 8000 12000 16000 4000 0 Isoproterenol (nM) fmol cAMP/ 20,000 myocytes Isoproterenol, a  -AR agonist that signal through Gs, increases cAMP in a concentration-dependent manner EC 50 24 nM 72 hrs EC 50 28 nM 24 hrs Activation of Gs Signaling in Myocytes at 24 and 72 Hours

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

9. ControlPE 20  M ET-1 100 nM PMA 100 nM Phospho-ERK Total-ERK Phenylephrine, an  1-AR agonist, and Endothelin-1 which both signal through Gq, increase ERK1/2 phosphorylation ControlPE 20  M ET-1 100 nM PMA 100 nM 24 hrs72 hrs Activation of Gq Signaling in Myocytes at 24 and 72 Hours

10. ControlIso 1  M Phospho-PLB GG Isoproterenol, a  -AR agonist that signals through Gs, increases phospholamban phosphorylation ControlIso 1  M 24 hrs72 hrs Phospholamban Phosphorylation in Myocytes at 24 and 72 Hours

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

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

14. AssayTime PointsMyocytes Phosphoprotein 0, 2, 5, 12, 30 min5 x 35 mm dish (250,000 myocytes) RNA Array 0, 30, 120, 240 min16 x 60 mm dish (2,400,000 myocytes) cAMP 0, 2, 5, 12, 30 min 5 x 35 mm dish (250,000 myocytes) Calciumin development Total2.9 x10 6 myocytes Project that we need 3 hearts/ligand Requirements for the Ligand Screen

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.