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Introduction
Sudden cardiac death (SCD) unexpectedly occurs in
patients attributed to cardiomyopathy (CM), heart diseases, or
inherited gene mutations[1_3]. It is a serious medical problem
as the annual incidence of SCD in the general population is
estimated to be as high as 1 in 1000[4]. Etiological factors for
SCD varied, including ischemic heart disease, hypertrophied
and dilated CM, and genetic defect in ion
channels[5].
Patients with congenital mutations of
K+ and Na+ channels (abnormality of ion channels in sarcolemma) manifest long
QT syndrome (LQTS, mainly LQT1, LQT2, and LQT3) to be
at risk for SCD. On the other hand, mutations of the ryanodine
type 2 receptor (RyR2, abnormality of the calcium handling
system in sarcoplasmic reticulum) exhibit catecholaminergic,
polymorphic ventricular tachycardia
(CPVT)[6], which is susceptible to develop ventricular fibrillation (VF). Both
defective RyR2, attributed to the dissociation of FKBP12.6 by
overphosphorylation[7,8] and the disorder of ion channels in
the sarcolemma[5], are implicated in mechanisms underlying
the development of VF. It is interesting that arrhythmias do
not happen in people with disorders of ion channels
(channelopathy) at resting stage. But arrhythmias occurred
when they were under stimulation of stress or other trigger
factors. There are 2 stages of channelopathy in the
myo-cardium, as we proposed
previously[9], involved in the manifestation of VF, of which a resting state of channelopathy in
the myocardium is arrhythmia/VF free, and an activated
(deteriorated) state of channelopathy manifests malignant
arrhythmias or VF[9]. Thus, we hypothesize that changes in
some molecules may abruptly take place within 1-2 min in
the diseased myocardium under stress, therefore, VF
occurred rapidly. To our knowledge, such changes in
molecules responsible for the sudden appearance of VF have
not been explored yet.
To observe the occurrence of VF within a limited time,
ischemia/reperfusion episode is adopted. An incidence of
VF can be observed easily on reperfusion within 10 min.
Additionally, we allowed 10 min for coronary occlusion, which
is shorter than usual practice, leading to a very low to zero
incidence rate of VF on reperfusion in normal rat hearts.
During the ischemic period of thyroxin-induced CM, the
incidence of VF, is substantially high on
reperfusion[10]. The CM caused by
L-thyroxin manifests an enhanced
ICa.L[11,12] and prolonged action potential duration (APD), which
resembles that of inherited LQTS. We also discovered an
enhancement of IKr,
IKs, and
IK1 in isolated myocytes from the
CM[13,14]. These alterations are similar in the findings of
patients suffering from short QT syndrome
(SQTS)[15]. Thus, the CM by thyroxin possesses a strong tendency to
develop exaggerated VF, which is probably related to the
variability of APD in the myocardium. Such an affected heart is
suitable in the investigation of SCD in relation to the abrupt
changes in some biological molecules to ventricle fibrillation
(VF). It is interesting to investigate a possible link at the
molecular level between the rapid occurrence of VF and
abrupt changes in the expression of FKBP12.6, SERCA2a, and the
endothelin (ET) signaling system in the
myocardium[16].
CPU86017, a complex class III antiarrhythmic agent
derived from berberine[17], suppresses
IK (IKr,
IKs) and
ICa.L currents in the sarcolemma. Changes in the expression of
inflammatory factors, the ET system, and FKBP12.6 in the
myocardium were seen in association with the rapid
appearance of VF on reperfusion of the CM and suppressed by
darusentan[16].
We hypothesized that channelopathies may be divided
into 2 stages: the resting stage and the deteriorated stage.
Before and during ischemia, the CM possesses ion
channelo-pathy, but no VF is manifested; this is referred to as the
resting stage of channelopathy. On reperfusion, the
channelopathy is deteriorated, leading to the rapid
appearance of VF which may be triggered by abrupt changes in
some molecular expressions. We intended to search for
molecular changes that are necessary in promoting the
deterioration of channelopathy relevant to the manifestation of
VF within a very short time on reperfusion. These may
include abrupt changes in the expression of FKBP12.6,
SERCA2a, PKA, and the ET system in the myocardium. To
test the hypothesis, the purpose of the rat CM model was to
investigate the transition from ischemia to reperfusion if VF
and further molecular changes rapidly appeared on
reperfu-sion. CPU86017, possessing multi-channels blocking and
antioxidant activity, was applied to verify the theoretical
consideration, and whether it suppresses VF by reversing
these changes on reperfusion of the CM.
Materials and methods
Reagents and instruments CPU86017 (Figure 1) is
synthesized by the Center of the New Drug Research and
Development of the China Pharmaceutical University (Nanjing,
China). The RT-PCR reagents were brought from Promega
(USA); L-thyroxin was from Sigma (St Louis, MO, USA), the
thermal cycler was from Eppendorff (Germany), and Labworks
imaging acquisition and analysis software was from
Ultra-Violet Products (Cambridge, UK).
L-thyroxin induced CM and reperfusion induced
VF The CM was produced as described in previous
research[10]. Briefly, male, adult Sprague-Dawley rats (220±20 g) were
divided into 5 groups (n=6): the normal control, the CM
untreated, CM with coronary artery ligation (CAL),
CM+CAL+reperfusion (RF) group, and CM+CAL+RF+ CPU86017 group. The CM group was produced by 0.4
mg/kg sc L-thyroxin for 10 d; CPU86017 was medicated at 4
mg/kg, sc from d 6 to d 10, respectively. On d 11, the rats were
anaesthetized with 20% urethane (1.5 g/kg, ip) and CAL was
performed for 10 min, followed by reperfusion in rats for 10
min under room temperature at 25 oC. The lead II of the
electrocardiography (ECG) traces was monitored and the
absolute irregular waves in ECG were recognized as VF. The
hearts were rapidly excised and divided into the right and
left ventricle (LV), including the septum, and weighed. The
cardiac weight index was measured by heart weight/body
weight and LV weight/body weight. The mass of the LV was
stored in liquid nitrogen before assay for RT-PCR and
Western blotting.
Calcium transients The cardiomyocytes were isolated
from the CM and those that were rod-shaped and striated
were selected for the calcium transient assay. The assay of
[Ca2+]i was performed as
described[18,19] with some modifica-tions. After being loaded with 10 µmol/L Fluo-3/AM at 37
oC for 30min, unincorporated dye was washed away; the cells,
control cardiomyocytes, CM cardiomyocytes, and the CM
cardiomyocytes treated with CPU86017 1 µmol, were then
transferred to a 300 mL perfusion chamber at room
temperature under an IX71 inverted microscope (Olympus, Japan).
The myocytes were subjected to 0.5 Hz field stimulation with
square wave pulses of 40 V at 5 ms duration. Fluo-3
fluorescence was recorded and converted into absolute
[Ca2+]i by a calibration procedure as follows:
[Ca2+]i=Kd
*(F -
Fmin)/(Fmax -
F)
Where Kd represents the dissociation constant for
Ca2+-bound to Fluo-3, and
F=Fcell -
Fback. Fcell and
Fback is the measured mean fluorescent intensity of myocytes and
background, respectively. Fmax represents the maximum
fluorescence of cells permeable to Ca2+ by adding 5 mmol/L
MnCl2 to 5 mmol/L A23187-treated myocytes and calculated as
follows: Fmax=FMn*5 and
Fmin=Fmax/40. The systolic and
diastolic [Ca2+]i levels can be calculated from this formula.
RT-PCR analysis The total RNA was extracted from a
100 mg, frozen, left ventricular tissue sample by using 1 mL
Trizol and reverse-transcribed into cDNA by AMV reverse
transcriptase. The primers in parallel with GAPDH were
amplified and assayed semiquantitatively. Oligonucleotides
for the primers used are listed in Table 1. The density of the
bands was analyzed by computer-aided Labworks imaging
acquisition and analysis software. The relative densities of
the bands for each mRNA were yielded by dividing the band
with the internal control GAPDH.
Western blotting For the quantitative analysis of total
protein levels of FKBP12.6, phospholamban (PLB), and
SERCA2a in cardiac myocytes, the LV tissue (100_200 mg)
was homogenized in 4 volumes of extraction buffer and
centrifuged at 10 000×g for 10 min, and performed as previous
described[20]. After transferring to the nitrocellulose and
blocking with nonfat milk (5% w/v), followed by incubation
with first antibody (Santa Cruz Biotechnology, Santa Cruz,
CA, USA), sc-6173 for FKBP12s, sc-20511 for PLB and
Affinity Bioreagents, MA3-919 for SERCA2a, 1:500 dilution in 5%
milk-TBS-Tween) for another hour. After 3 washes, the blot
was incubated with horseradish peroxidase-conjugated goat
secondary antibody IgG (1:1000) for an additional 1 h. The
antigen was detected with 0.1% 3, 3'-diamino-benzidine/
0.01% H2O2. A linear relationship between the density of the
blots and the protein load was observed when 20, 40, 60, 80,
and 100 µg membrane protein were used per lane.
Statistical analysis GraphPad Instat version 3.05
(GraphPad Software, San Diego, CA, USA) was used to
analyze the results. Data were presented as mean±SEM.
Statistical analyses were performed by ANOVA, followed by a
Bonferroni t-test. Fisher's exact test (one-tailed) was used
to test the incidence of VF. A value of P<0.05 was
considered statistically significant.
Results
Ventricular hypertrophy and an incidence in VF on
reperfusion The cardiac weight index and heart rate (HR)
significantly increased in the CM relative to the control. An
increased HR was attributed to the stimulation of the
beta-adrenergic receptors by chronic thyroxin medication (Table
2). There was no VF observed in the CM before and during
CAL. Within 1_2 min following reperfusion, an increased
incidence of VF was seen relative to the control and CM
groups (P<0.01, Fisher's exact test, one-tailed). Following
intervention with CPU86017, VF was markedly suppressed
(P<0.05, Fisher's exact test, one-tailed) in association with a
regression in cardiac hypertrophy (Figure 2).
Exaggerated Ca2+ transients and elevated diastolic
Ca2+ To test whether an abnormal calcium homeostasis
contributes to augmented VF in CM, cardiomyocytes freshly
isolated from the CM were tested for calcium transients. A huge
peak level (Ca2+ in systole) of intracellular calcium was found
in CM (1360±120 nmol/L), relative to the control (460±40
nmol/L, P<0.05, Figure 3A) and provided evidence of
dysfunction of the intracellular calcium handling system in the
CM. An increase in the trough of
[Ca2+]i levels
(Ca2+ in diastole, 180±32 nmol/L, Figure 3B) which increases
significantly (P<0.05) relative to the control (107±11 nmol/L),
indicated that an elevated diastolic calcium served as a risk
factor to an increased incidence of VF. Treatment with
CPU86017 significantly reduced systolic (480±30 nmol/L) and
diastolic (112±5 nmol/L)
[Ca2+]i, P<0.05) compared with the
untreated CM, respectively.
Abnormal expression of the calcium handling system
and PKA For further investigation of the abnormality of the
calcium handling system, the mRNA expression of the
system was conducted. The upregulation of the RyR2 mRNA
level was markedly increased by +82% in the CM and
CM+CAL groups (P<0.05, vs control), respectively. These
were not further changed following reperfusion (Figure 4A).
FKBP12.6 mRNA was significantly was downregulated in
the CM and unchanged during 10 min CAL; however,
further downregulation was found on reperfusion,
(P<0.05 vs CM+CAL+RF, Figure 4B). The mRNA of SERCA2a and PLB
was downregulated (P<0.05) in the CM group, relative to the
control, respectively, and no further changes were found by
ischemia and reperfusion (Figure 4C, 4D). The mRNA
expression of PKA was significantly upregulated
(P<0.01, vs control) in association with changes in the mRNA of the
calcium handling system. Further upregulation of the PKA
mRNA expression was found on reperfusion against the
untreated CM (P<0.05, Figure 4E). This abnormal
expression of the calcium handling system and PKA were
completely reversed by CPU86017 treatment
(P<0.01).
During CAL and RF, the protein expression of
FKBP12.6 was downregulated significantly relative to the CM
(P<
0.05); however, the expression of FKBP12.6 before ischemia
in the CM was upregulated relative to the control
(P<0.01, Figure 5A). The protein levels of SERCA2a significantly
decreased in the CM group (P<0.01
vs control) and further downregulation was found on reperfusion
(P<0.01 vs control, Figure 5B). The change in the PLB protein level was mild and
no difference was found among the groups (Figure 5C).
CPU86017 significantly regressed the downregulation of the
protein expression of FKBP12.6 and SERCA2a, respectively
(P<0.01 vs CM+CAL+RF).
Overactivated ET system and upregulated
iNOS The ET-converting enzyme (ECE), prepro-ET-1 (pp-ET-1),
endothelin A receptor (ETAR), and iNOS mRNA expression
was significantly upregulated in the CM, CM+CAL and
CM+CAL+RF groups (P<0.05 vs control, Figure 6), but no
difference was found between ischemia and reperfusion,
except that the mRNA of ECE was further upregulated on
reperfusion (P<0.05, Figure 6A). CPU86017 completely
reversed these abnormalities in the CM.
Discussion
Sudden augmented VF has been found on reperfusion
(CM+CAL+RF) relative to the ischemia (CAL+CM) group.
The expression of some molecules changed rapidly on
reperfusion of the CM, relative to the CM or CAL; among
them, the protein expression of FKBP12.6 and SERCA2a
downregulated significantly relative to the CM
(P<0.05). In contrast, no change in RyR2 and PLB was found. These
changes in the calcium handling system are associated with
an increased rate of VF on reperfusion. A suppression on
abnormal expression by CPU86017 results in the
disappearance of VF during the 10 min of reperfusion.
Characteristics of dysfunction of the RyR2 in
cardio-myocytes freshly isolated from the CM reveals a great
increment in systolic
[Ca2+]i. A significant peak of calcium is
attributed to an increased release by RyR2, and is partly
attributed to an increase in Ca2+ influx via
ICa.L by L-thyroxin
treatment[11,12]. The abnormality of RyR2 is likely to develop
a calcium leak which elevates diastolic
[Ca2+]i and delays the repolarization in the myocardium so that it is favorable to
arrhythmogenesis[21]. Besides the unstable
RyR2[8], an
insufficient calcium pump attributable to the downregulation
of SERCA2a may be implicated[7].
On reperfusion, a dramatic further downregulation of
FKBP12.6 and SERCA2a deteriorates the calcium homeostasis in the diastole to retard the
repolarization process, thus, a non-reentrant mechanism is
triggered to help a process of the delayed after
depolarization (DAD), torsades de pointes, and finally
VF[8,22].
An upregulation of RyR2 mRNA in this study is in
agreement with findings of previous
reports[16,23]. However, an upregulation of mRNA of RyR2 in the CM is at odds with
those of the post-myocardial infarcted rat heart stimulated
by isoproterenol[24], in a failing
heart[8], diabetic CM[20,25],
and CM by chronic anthracycline
administration[26] where the downregulation of RyR2 mRNA is remarkable. The
upregulation of RyR2 and FKBP12.6 in the CM before
reperfusion may be explained by increased transcription in
the nucleus by L-thyroxin. Another possible reason of an
upregulation of FKBP12.6 in the CM may be related to the
use of the polyclonal antibody which conjugates the total
protein of FKBP12, including the subunit
FKBP12.6[37].
An activated endothelin signaling pathway is implicated
in cardiovascular dysfunction and
disorders[28] and presumably exerts a role in deteriorating channelopathy of the
SERCA2a and RyR2. The upregulation of the mRNA of
pp-ET-1, ECE, and ETAR is found in the CM before and
during ischemia, manifesting no VF; however, an abrupt
upregulation of ECE mRNA is associated with VF on reperfusion indicating that an activated ET signaling system
may play an active part in the rapid manifestation of VF,
coinciding with findings in a previous
report[16]. The upregulation of the iNOS gene expression is related to the
genesis of the reactive oxygen system (ROS) which
combines an activated ET pathway to form an ET-ROS signaling
pathway[29]. The ROS injures the
Ca2+ handling function and gene expression on
reperfusion[30] and promotes the formation of an activated
NFκB and TNFα, both of which facilitate the transcript process in the nucleus responsible for an
upregulation of the ET system, and then an increase in the
susceptibility of the CM to arrhythmogenesis. An excess of
ROS is suppressed when an activated ET pathway is
antagonized[31]. Oxidative stress is augmented by thyroxin
treatment and suppressed by
CPU86017[32], thus, antioxida-tive activity of CPU86017 does supplement a relief of the
deteriorated channelopathy on reperfusion.
Increased calcium in the diastole mediates triggered
activity to initiate DAD, and eventually malignant
tachyven-tricular arrhythmias[24]. Compound JTV-519, a derivative of
diltiazem, possesses a blockade on calcium and potassium
channels[33] and has been discovered to correct deficiencies
of RyR2 and FKBP12.6 effectively[34]. Its profile resembles
those of CPU86017. A calcium antagonism has been found
clinically to improve the lifespan of patients who suffer from
CPVT[35].
APD, or the process of repolarization, depends on the
balance of total inward and outward ion currents and an
increment in IKr or
IKs which has been discovered in the CM,
facilitating the repolarization process to shorten
APD[13,14]. SQTS from mutated genes exhibits an increase in
IKr, IKs, or
IK1 by "gain of function" mutation, which predisposes
patients to life-threatening
arrhythmias[36]. Thus, the CM may possess a reentry mechanism resembling SQTS.
Additionally, an enhanced ICa.L current in the
CM[11,12] produces a prolonged APD indicating that a non-reentrant
mechanism is likely to be involved. Thus, the variability of
repolarization in the CM serves as a basis for predisposition
to severe cardiac arrhythmias in stress[37,38]
. An abnormality of the expression of the calcium handling system may be
associated with the variability of repolarization, eventually
contributing to the appearance of VF in stress.
An exacerbated VF incidence is rapidly found on
reperfu-sion against a state of VF free of the CM and during 10 min
ischemia. Reperfusion acts as stress to produce molecular
changes for the exacerbation and appearance of VF.
Theore-tically, 2 stages of
channelopathy[9] or multi-hit
concept[3,39] have been proposed to explain the complicated processes
underlying mechanisms in SCD. A patient with mutated genes
does not exhibit cardiac arrhythmias, but is potentially at
risk to develop severe cardiac
arrhythmias[40]. The frequency of genetic mutations of individual channels as
SCN5A or CPVT is much higher, up to 10%_20% of the population,
than those to manifest LQTS or the CPVT. This
phenomenon supports the concept that the manifestation of LQTS
and CPVT needs molecular activation of ion channelopathy.
It is necessary to make such a conversion in CM from the
resting state to the deteriorated stage. The deterioration of
channelopathies in molecular aspects contributing to the
SCD or malignant arrhythmias, or the manifestation of LQTS,
can be triggered by either physical and mental stress or drug
insults[25,40,41].
SCD is always abrupt, unpredictable, and unpreventable;
however, an awareness of molecular activation at the
upstream to channelopathy responsible for the sudden
appearance of malignant arrhythmias hopefully provides a
way to prevent patients from the SCD. Thus, the upstream
in the signaling pathways and transcription process in the
nucleus has been the center of focus, rather than the
limitation to the ion channels, and these targets are helpful in
searching for effective agents to prevent the SCD. Based on
the findings of this study, we suggested that reperfusion
produces oxidative stress which causes biosynthesis and
the release of more ET, therefore, the facilitation of PKA
upregulation and abnormality of the calcium handling
system and the ion channels in sarcolemma is the result. An
augmented ICa.L channel is attributed to PKA (presumably
including PKC) phosphorylation relating to an activated ET
system[42] and
arrhythmogenesis[24].
In the CM there may be a link (which may be an
upregula-tion of PKA or PKC) between augmented
IKr, IKs, and
ICa.L currents and abnormal expression of FKBP12.6, RyR2,
SERCA2a, and PLB. PKA and PKC are both responsible for
the predisposition of the hearts to malignant arrhy-thmias.
Reperfusion facilitates molecular events which include an
activation of NF-κB, mitogen activated protein kinase
(MAPK), and AP-1[16,43] to increase the transcription
process for the upregulation of these genes encoding ECE and
PKA. Reperfusion also causes variability of the
repolarization process in the myocardium, thus, a blockade by
CPU86017 on IKr and
IKs suppresses the shortening of APD
in the CM (data not shown); the
ICa.L blocking effect of CPU86017 inhibits a prolongation of APD in the
myocard-ium[17]. Eventually, as a result, the susceptibility of the CM
to VF is dramatically relieved by CPU86017.
In conclusion, abrupt molecular changes contributing to
the sudden occurrence of VF within 1_2 min of reperfusion
has been discussed. A swift downregulation of FKBP12.6
and SERCA2a, and the upregulation of PKA and the ET
signaling pathway, are the main events relevant to manifest VF.
We also demonstrated a conversion of a silent resting state
into an activated channelopathy contributing to the
occurrence of VF. The CM by thyroxin is applied to induce basic
channelopathy (the resting state), but VF free and the
sudden manifestation of VF on reperfusion is triggered by
dramatic worsening in channelopathies. With a blockade on
multi-channels and an anti-oxidative effect, abrupt
molecular changes on reperfusion are markedly suppressed by
CPU86017, resulting in the prevention of sudden
appearance of VF.
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