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

Luyi Li

Tamara Roach

TimO’Connell

Paul Simpson Bill

Seaman

Melissa Kachura

SusanRicker

The SF VAMCAfCS Lab

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 Flow or Constant Flow

Myocyte Isolation Procedure

Hang. Heart In Situ(Const. Flow)

(50)Const. Pres.

(64)Const. Flow

(38)

Myocytes For Plating (106)% Rod ShapedRod Shaped Myocytes

2.6 ± 0.5 76 ± 10%

2.0 ± 0.5

1.6 ± 0.3 68 ± 8%1.1 ± 0.3

1.7 ± 0.3 67 ± 7%1.1 ± 0.3

# 35 mm Dishes at 50K rods/dish(~62 rods/mm2)

39 22 22

Plating Efficiency@ 1 hr (%)(#attached/#plated)

39%*

In Situ preparation is much easier technicallyIn Situ constant flow preparation is easier than constant pressure

* measured since January, 2002

Myocyte Yields with DifferentIsolation Techniques

0 hr 24 hr 72 hr

Plate for 1 hr on laminin coated dishes in:MEM w/Hanks BSS w/5% BCS10 mM BDMPenicillin

Change Medium to:MEM w/Hanks BSS w/1 g/ml Insulin0.5 g/ml Transferrin0.55 ng/ml Selenium1 mg/ml BSA10 mM BDMPenicillin

Culture for up to 72 hours at 37°C in 2% CO2

Goal: Maintain rod-shaped myocytes that signal for 72 hrs

Myocyte Culture Procedure

MyocyteIsolation

3 hrs

PlatingAssay

SignalingAt 24 hrs

AssaySignalingAt 72 hrs

1 hr 24 hrs 48 hrs

MediumChange

Assays:Gs: cAMP, PLB phosphorylation, myocyte contractionGi: inhibition of cAMPGq: ERK phosphorylation

Myocyte Experimental Timeline

10-10 10-9 10 -8 10 -7 10-6 10-5

20000

8000

12000

16000

4000

0

Isoproterenol (nM)

fmo

l cA

MP

/2

0,0

00

myo

cyt

es

Isoproterenol, a -AR agonist that signal through Gs,increases cAMP in a concentration-dependent manner

EC50 24 nM72 hrs

EC50 28 nM

24 hrs

Activation of Gs Signaling in Myocytes at 24 and 72 Hours

Control Iso

120000

100000

800003500030000

200001500010000

05000

25000

Fsk FskCarb

IsoCarb

Isoproterenol (1 M)Forskolin (100 M)Carbachol (100 M)

fmo

l cA

MP

/2

0,0

00

myo

cyt

es

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

Control PE20 M

ET-1100 nM

PMA100 nM

Phospho-ERK

Total-ERK

Phenylephrine, an 1-AR agonist, and Endothelin-1 which both signal through Gq, increase ERK1/2 phosphorylation

Control PE20 M

ET-1100 nM

PMA100 nM

24 hrs 72 hrs

Activation of Gq Signaling in Myocytes at 24 and 72 Hours

Control Iso1 M

Phospho-PLB

G

Isoproterenol, a -AR agonist that signals through Gs,increases phospholamban phosphorylation

Control Iso1 M

24 hrs 72 hrs

Phospholamban Phosphorylation in Myocytes at 24 and 72 Hours

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QuickTime™ and a decompressor

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Activation of E-C Coupling in Myocytes at 24 Hours

Myocytes contracting under field stimulationMyocytes quiescent for first 5 secondsStimulated at 80V, 1 Hz for 20 seconds

Then increase frequency to 1.5 Hz for 15 seconds

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QuickTime™ and a decompressor

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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

Control 1 M Isoproterenol

0

2

4

6

8

% S

ho

rte

nin

gIsoproterenol induces myocyte contraction

16 16

Myocyte Contraction measured as %Shortening of individual cardiac myocytes

*

* p < 0.05

Activation of E-C Coupling in Myocytes at 72 Hours

270%

Assay Time Points MyocytesPhosphoprotein 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

Requirements for the Ligand Screen

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.