The application of C-TAB for excitation propagation photocontrol in cardiac tissue

1.

The application of C-TAB for excitation propagation
photocontrol in cardiac tissue.
Gorbunov V., Erofeev I., Agladze K.
Moscow Institute of Physics and Technology
Introduction
Photocontrol allows to precisely
affect excitation propagation by
light with help of special
photosensitive molecules.
Recently a novel photosensitive
substance C-TAB was developed.
The aim of the present work was
to investigate the action of C-TAB
on the excitability of cardiac tissue.
N+
+Azo-TAB
+C-TAB
>30 uM
+C-TAB
<30 uM
C-TAB
O
O
UV
N+
UV
UV
UV
O
Wash out
Wash out
Wash out
O
9
8
7
6
5
4
3
2
1
0
100%
Relative propagation speed
Propagation speed (sm/s)
Fig. 1 Photocontrol diagram for Azo-TAB and C-TAB, photoisomerisation scheme
0
5
10
15
20
25
30
UV power 5 mW/sm2
80%
5uM
60%
10uM
40%
15uM
20%
35
0%
0
30
60
C-TAB concentration (uM)
1000%
Cell survival
800%
600%
400%
200%
0%
10
100%
100
C-TAB concentration (uM)
Sell survival
80%
60%
40%
20%
0%
1
10
100
Azo-TAB concentration (uM)
Conclusions
Integral signal intence
Fig. 3 Relative toxicity
of C-TAB and Azo-TAB
by MTT test on 3T3
fibroblasts for 1 hour
of substance presence.
Cells were incubated
overnight in 96-well
1000
plate in DMEM 10%
FBS, 37°C 5% CO2.
After substance wash
out and MTT test
optical density of
particular wells was
related to control
1000 (substance free) wells
180
210
Fig. 4 The effect of C-tab cis-form on the excitation
propagation speed (UV exposure power 5 mW/sm²;
trans- form is present in media). For 15 uM propagation
stopped after 1 minute of exposure, for 10 uM after 3
minutes, for 5 uM after more than 10 minutes.
40 minutes after wash out
120%
shadow zone
100%
UV exposed zone
80%
60%
40%
20%
Time (s)
0%
0
Integral signal intence
1200%
120
UV exposure time (s)
Fig. 2 Dose dependence of excitation propagation speed
on the concentration of C-tab trans-form (no UV).
monitored by optical mapping of Ca2+ waves. In rat
neonatal cardiac cell monolayer propagation speed
decreases until the complete stop at 30 uM.
Toxicity
90
2
4
6
8
10
12
14
16
18
48 hours after wash out
110%
shadow zone
100%
UV exposed area
90%
80%
70%
60%
50%
0
2
4
6
8
10
12
14
16
18
Time (s)
Fig. 5 Restoration of excitation propagation after 48
hours. Fluorescence intensity diagrams of Ca2+ sensitive
dye show every propagation cycle as a peak. The half of
the culture with 50 uM C-TAB in media was exposed by 5
mW/sm ² UV for 1 minute than examined for excitation
propagation in next 48 hours. Exposed zone stayed
inactive for at least 20 hours and partly restored
propagation in 48 hours.
The citotoxicity of C-TAB was discovered to be 10 times less than for Azo-TAB. The blocking concentration
for trans-C-TAB was estimated as 30 uM and for lower concentration than that the possibility of
photocontrol before wash out was demonstrated. The photo ablation process with wash out was
introduced for concentrations higher than 30 uM.