Biomedical Engineering Reference
In-Depth Information
RT-PCR. The expression of
I
K1
channels increases developmentally and the adult cur-
rent density is achieved at postnatal days nine [32]. The basis of the developmental
increase has been variously ascribed to increased channel number, open probability or
unitary conductance [34]. We found that the CMG cells expressed Kir2.1 before dif-
ferentiation, and that their expression was maintained (Fig. 2a). Kir2.2 was not ex-
pressed until 6 weeks, when they showed ventricular myocyte-like action
potentials.
We next investigated expression of the transient outward K
+
I
to
) channel subunits
K
V
1.2, K
V
1.4, K
V
1.5, K
V
.2.1, K
V
4.2 and K
V
4.3. Wickenden reported that during car-
diomyocyte development, the predominant K
+
channel mRNA species switches from
K
V
1.4 to K
V
4.2 and K
V
4.3 [35]. Undifferentiated CMG cells expressed none of the
subunits that compose
I
to
(Fig. 2b). Interestingly, the K
V
1.4 subunit was expressed
from 2 weeks, and the K
V
2.1 subunit was expressed from 4 weeks. Expression of
K
V
4.2 was detected 6 weeks after differentiation, when the CMG cells showed ven-
tricular myocyte-like potentials. However, neither K
V
1.2, K
V
1.5 nor K
V
4.3 were in-
duced in differentiated CMG cells. These expression patterns could explain how
CMG cells developed ventricular cardiomyocyte-like action potentials at 4 or 6
weeks. We propose that an increase in the density of I
to
, along with the previously
reported K
V
1.4 to K
V
4.2/K
V
4.3 isoform switch, produced the shortening of the action
potential duration and the notchshaped transient repolarization following the spike
(phase 1 of action potential).
Next, we investigated the expression of the ERG K
+
channels that underlie native
rapidly activating delayed rectifier K
+
currents (
I
kr
), and the K
V
LQT1 and KCNE1
channels that underlie the native slowly activating delayed rectifier K
+
current (
I
Ks
).
Mouse ERG (MERG) was expressed both before and after differentiation (Fig. 2c),
whereas K
V
LQT1 and KCNE1 were not expressed either before or after differentia-
tion (Fig. 2d).
The K
+
channel rapidly activated by application of acetylcholine (
I
K,Ach
channel) is en-
coded by the combination of GIRK1, GIRK2B and GIRK4. We previously reported
that bone marrow-derived cardio-myocytes express functional Ach receptors [13]. RT-
PCR revealed that none of these subunits were expressed before cardiomyocyte induc-
tion (Fig. 3a). In contrast, the differentiated CMG cells expressed GIRK1 and GIRK2B
after 6 weeks, when the cells showed ventricular myocyte-like action potentials.
ATP-sensitive inwardly rectifying K
+
(
I
K,ATP
) channels are expressed in a variety of
tissues including heart. The
I
K,ATP
channel has been reconstituted in functional form
by coexpression of SUR, the sulfonylurea-binding protein, and the inwardly rectifying
K
+
channel subunit, Kir6.2 [7]. CMG cells did not express these subunits before dif-
ferentiation, but the differentiated cells expressed both Kir6.2 and SUR2A from 4
weeks onwards (Fig. 3b).
3.3 Expression of Pacemaker and Ca
2+
Channels in CMG Cells
The hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channel gene
family is known to contribute significantly to cardiac pacemaker current (
I
f
). The pre-
sent study demonstrated that the HCN family was not expressed before cardiomyocyte
