Biomedical Engineering Reference
In-Depth Information
resistance 15-30 Mȍ). Membrane potentials were measured in current clamp mode
(MEZ-8300 amplifier; Nihon Kohden, Tokyo, Japan) with the built-in 4-pole Bessel
filter set at 1 kHz.
2.3 RNA Extraction and Reverse Transcription Polymerase Chain Reaction
(RT-PCR) Analysis
Total RNA was extracted from cells isolated at 0, 2, 4 and 6 weeks following cardio-
myocyte induction, using Trizol Reagent (GIBCO) as described previously [13]. RT-
PCR was performed to detect Kir2.1, Kir2.2, K
V
1.2, K
V
1.4, K
V
1.5, K
V
2.1, K
V
4.2,
K
V
4.3, MERG, K
V
LQT1, KCNE1, HCN2, HCN4, Ca
V
1.2, GIRK1, GIRK2B, GIRK4,
Kir6.2, SUR2A, and GAPDH mRNA. The expression of
ȕ
1
and
ȕ
2
-adrenergic receptor
mRNAs was also analyzed. PCR was performed for 30-35 cycles, with each cycle
consisting of denaturation at 94°C, annealing at 54-60°C, and amplification at 72°C
(each for 1 min). The primers and PCR cycles used are shown in Table 1. Primary
cultured cardiomyocytes were used as positive controls. Prior to the quantitative
analysis, the linear range of the PCR cycles was measured for each mRNA, and the
appropriate number of PCR cycles was determined. GAPDH was used as an internal
control for each sample.
2.4 cAMP Accumulation Assays
Cells were incubated in serumfree medium with 10
-4
mol/l of 3-isobutyl-1-
methylxanthine (Sigma-Aldrich, St Louis, MO, USA) for 30 min, and stimulated with
isoproterenol (Sigma-Aldrich) for 10 min. In some experiments, cells were preincu-
bated with propranolol (Sigma-Aldrich) for 20 min. The medium was aspirated rap-
idly, and incubation was terminated by addition of 1 ml of ice-cold 0.1 N HCl. The
lysates were centrifuged at 3,000 rpm for 10 min, and the supernatants were used as
samples. The cAMP levels were counted by radioimmunoassay using an assay kit
(YAMASA, Chiba, Japan).
2.5 Videotape Recording
The cultured cells were observed through an inverted phase contrast video microscope
(IX70; Olympus, Tokyo, Japan) equipped with a 4×quartz objective lens and a
1×relay lens. The culture dishes were maintained at 33°C using a thermoplate. The
cell images were obtained using an intensified charged couple device camera
(CS220; Tokyo Denshi, Japan) and digitally videotaped with a VHS recorder (wv-
DR9; Sony, Tpan). Beating rate was then measured using the recorded video images.
2.6 Construction and Transfection of Myosin Light Chain 2v-Promoted
Enhanced Green fluorescent Protein (EGFP) Plasmid
An expression vector, pMLC2v-EGFP, was constructed by cloning a 2.7 kb
Hind
III-
Eco
RI fragment of the rat myosin light chain-2v (MLC-2v) promoter region [15, 26]
into the
Hind
III-
Eco
RI site of pEGFP-1 (Clontech, Palo Alto, CA, USA), so that
