| Summary: | 碩士 === 國防醫學院 === 生理學研究所 === 93 === Introduction
The hereditary cardiomyopathic strain of the Syrain hamster (Bio
14.6) had been extensively used as an experimental model for the study
of cardiomyopathy and heart failure (HF). It was assumed that, because
of the inherited defects in the cytosolic calcium (Ca2+
i) reuptake process
in the sarcoplasmic reticulum (SR), the myopathic myocytes would prone
to develop Ca2+
i overload and the subsequent triggered arrhythmia.
However, paradoxically, part of our previous experiments revealed that
the myopathic hamsters had a lower incidence of reentrant atrial
tachyarrhythmias and a smaller transient inward current (Iti) after
prolonged depolarizing pulses (＞1000 ms) in concomitant with a longer
APD and a reduced transient outward k+ current (Ito) in ventricular
myocytes.
Aims
The aims of the present experiment were:
(1) to study the ventricular muscles and cardiomyocytes from healthy
and cardiomyopathic hamsters under conditions of intracellular
calcium overload (perfusion with high [Ca]o-low [K]o solution or
after repeated electrical pacing);
(2) to examine the genesis of ventricular arrhythmias in the
absence or presence of insulin, a polypeptide known to modulate
intracellular calcium ([Ca2+]i) and increase contractile function of
myopathic hamsters.
Material and Methods
1. Preparation of hamster ventricular tissues :
Animals (35-40 week-old) were anesthetized with an intreperitoneal
injection of sodium pentobarbital (50 mg/kg). Papillary muscle
obtained from left ventricle and perfused in vitro at 37℃. Steady-state
(2 Hz) and post-rest (20s) action potentials were recorded by
microelectrode techniques and twitch force by a transducer.
2. Isolation of ventricular myocytes:
Male myopathic Syrian hamsters (Bio 14.6, 52-60 week-old) and
healthy hamsters were anesthetized with pentobarbital and the heart
quickly removed and immersed in HEPES-Tyrode solution. The hearts
were perfused in a retrograde manner via polyethylene tube connected
through the aorta and left ventricule into the atrium. The free end of the
polyethylene tubing was connected to a Langendorff perfusion column
for perfusion with oxygenated HEPES-Tyrode solution at 37℃. The
perfusate was replaced with oxygenated Ca2+-free HEPES-Tyrode
solution, then was replaced with containing collagenase (1 mg/ml) and
protease (0.01 mg/ml) finally. The piece of tissue was cut into fine
pieces and gently shaken in 20 ml of high-K storage solution until
single cardiomyocytes were obtained.
3. Electrophysiology study:
The isolated cells were placed in a 1-ml chamber mounted on the stage
of
an inverted microscope and superfused (at 3 ml/min) with
extra-cellular solution appropriate to each patch-clamp experiment.
Results
1. Ventricular papillary muscle obtained from 35-40 week-old
myopathic hamsters (vs. those from healthy hamsters) are prone to
develop signs of calcium overload: delayed afterdepolarization (DAD)
and post-rest triggered arrhythmias in the presence of high-[Ca2+]o
l o w - [ K + ] o p e r f u s a t e .
2. In ventricular myocytes isolated from 52-60 week-old myopathic
hamsters, all preparations (n=6) generate transient inward currents (Iti)
after repeated (up to 20 times) depolarizing pulses (from -40 to +40
mV for 500 ms every sec). In healthy preparations, only 2 of 7
m y o c y t e s
develop Iti and the Iti are smaller in size.
3. Insulin (1 µM), a polypeptide known to enhance Ca2+ influx and induce
positive inotropic response in myocardial cells, exaggerates DAD and
Iti but does not increase inward rectifier K+ currents (IK1) in
preparations from healthy hamsters. In myopathic ventricular muscles,
insulin increases (to a lesser extent) the DAD but may increase or
decrease Iti in myopathic myocytes due to difference in the age of
animals and individual variation.
Conclusions
1. With the experimental protocols adopted in the present study (repeated
depolarizing pulses, post-rest potentiation of triggered actitivity, etc.),
it has been demonstrated that preparations from myopathic hamsters
are prone to develop triggered arrhythmias as compared to the healthy
control.
2. Further experiments are required to clarify the discrepancies in results
between ventricular tissues and cardiomyocytes.
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