Core D, San Francisco: Laboratory for Development of Signaling Assays
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Transcript of Core D, San Francisco: Laboratory for Development of Signaling Assays
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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
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Luyi Li
Tamara Roach
TimO’Connell
Paul Simpson Bill
Seaman
Melissa Kachura
SusanRicker
The SF VAMCAfCS Lab
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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
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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
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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
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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
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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
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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
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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
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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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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
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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%
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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
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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.