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Introduction
Heart failure (HF) is one of the leading causes of
morbidity and mortality in our society. The therapeutic prospects
remain poor and this may be attributed to an inadequate
understanding of the multiple and complex mechanisms
underlying HF. In failing hearts, some molecular events which
worsen cardiac performance and are predisposed to
developing life threatening-arrhythmias are likely to be related to
the overactivation of β-adrenergic receptors
(β-AR)[1,2]. However, the mechanisms underlying the consequences of
β-receptor activation in HF remain to be clarified.
Protein kinase A (PKA) phosphorylation as the consequence of
β-AR activation significantly affects proteins of the
intracellular calcium handling
system[3], including ryanodine receptor
type 2 (RyR2), FKBP12.6 (calstabin2), phospholamban (PLB),
and sarcoplasmic reticular Ca2+ ATPase 2a (SERCA2a) in the
sarcoplasmic reticulum (SR). The application of
isoproterenol (ISO) which significantly worsens cardiac performance
is considered to produce PKA
hyperphosphorylation[4]. PKA hyperphosphorylation increases diastolic
[Ca2+]i which seriously disturbs cardiac
function. A calcium leak from the
Ca2+ release channel RyR2 where FKBP12.6 is dissociated by
downregulation and a reduction in the uptake activity of
SERCA2a contribute to elevated diastolic calcium
levels. The normal function of SERCA2a is dependent on its modulating
protein PLB which is markedly affected by PKA
phosphoryla-tion. Thus, the normal expression of FKBP12.6 and PLB is
crucial to maintaining cardiac performance by keeping low
Ca2+ levels at diastole and sufficient calcium stores in the SR
for systolic release under physiological conditions.
In general, depressed SERCA2a and RyR2 and their
modulating proteins PLB and FKBP12.6 are observed in chronic
HF (CHF)[5,6], resulting in an increase in diastolic
[Ca2+]i. The modulating proteins FKBP12.6 and PLB are abnormal in
compromised hearts. Cardiac insufficiency can be induced by a
mutated PLB[7]. Acute cardiac failure caused by
ischemia/reperfusion procedure (I/R) is associated with depressed
Ca2+ uptake and Ca2+ release in association with the
down-regulation of SERCA2a and PLB and the upregulation of
PKA-mediated phosphorylation which are reversed by
β-blockers[8]. The reversal of the abnormal expression of FKBP12.6 has
also been targeted to relieve the molecular disturbance in
HF[9]. Thus, it was of interest to investigate a reversal of the
abnormal expression of FKBP12.6 and PLB other than by
β-blockade to increase our understanding of the molecular
mechanisms underlying HF.
A role for endothelin (ET)-1 has been
implicated. HF and ET blocking agents are considered candidates for
therapeutic uses in the treatment of
HF[10]. The upregulation of the
ET pathway is involved in the pathological process of HF,
and ET receptor antagonism has been successful in treating
acute HF[10,11], but remains controversial in
CHF[12,13]. The application of the dual ET receptor antagonist CPU0213
relieves cardiac dysfunction in diabetic
cardiomyopathy[14] and HF produced by coronary
I/R[15], together with an improvement in the calcium handling system in the
myocardium. Excessive ET may stimulate the myocardium to produce more
reactive oxygen species (ROS); conversely, an increase in
ROS facilitates ET pathway
activation[16,17] to further adversely impact the cardiovascular system.
The downregulation of FKBP12.6 and PLB in association
with compromised cardiac performance in diabetic hearts,
which could be related to the stimulation of β-receptors, is
significantly attenuated by the ET receptor antagonist
CPU0213. Thus, we hypothesized that ISO, which
dramatically downregulates the expression of FKBP12.6 and PLB,
worsens cardiac insufficiency, and may cause activation of
the ET pathway, an intermediate event in the downstream
events following vigorous stimulation of β-AR.
It was of interest to further study the molecular changes relating to
HF by examining whether the downregulation of the
expression of FKBP12.6 and PLB and worsened cardiac
dysfunction by ISO could be reversed by the application of a dual
ETAR/ETBR antagonist CPU0213.
Materials and methods
Animals Male, 12-week old Sprague-Dawley rats,
weighing 220_250 g were purchased from the Animal Center of
Nanjing Medical University (Nanjing, China).
The animal handling was in accordance with the Provincial Regulations
of Animal Care and Use in Jiangsu Province (Nanjing, China).
Chemicals ISO was purchased from Shanghai Hefeng
Medicine (Shanghai, China); CPU0213 (0213, dajisentan) and
darusentan (DAR) were from the Department of Medicinal
Chemistry of the China Pharmaceutical University (Nanjing,
China); and Propranolol (PRO) was from Sigma (St Louis,
MO, USA).
Acute HF and ISO The rats were divided into 3 groups
with 10 rats in each: (i) normal; (ii) ISO: administration with 1
mg/kg ISO subcutaneously (sc) on d 1_10; and (iii) CPU0213:
1 mg/kg ISO on d 1_10 and 30 mg/kg CPU0213 (sc) on d
6_10. On d 11, the rats were placed under urethane anesthesia and
the main left coronary artery was occluded after chest
opening. Acute cardiac failure was produced by 10 min
occlusion of the coronary artery (ischemia) followed by
reperfusion for 10 min (I/R). Cardiac function was assessed
by inserting a catheter into the left ventricular chamber to
measure the systolic (LVSP) and diastolic function
(LVEDP). Data were collected for 10 rats in each group.
Semiquantitative determination of FKBP12.6 and PLB
mRNA by RT-PCR The total mRNA was extracted from the
homogenate of the frozen left ventricle using Trizol reagent
and reversely transcribed to cDNA by using AMV reverse
transcriptase (Promega, USA) according to previous
publications and the manufacturer's
instructions[14,18]. The total volume of the PCR reaction was 25 µL: 1 µL cDNA, 2
mmol/L MgCl2, 20 mmol/L each dNTP, 0.2 nmol/L each primer, 2 U
DNA Taq polymerase, and the accompanied
buffer. The cDNA was amplified under the following conditions: initial
denaturation at 94 ºC for 5 min, then cycling and
denaturation at 94 ºC for 40 s, annealing for 40 s extending at 72 ºC for
1 min. The annealing temperature and cycle number of RyR2,
FKBP12.6, PLB, SERCA2a, and GAPDH were 58 ºC, 36 cycles;
61 ºC, 32 cycles; 63 ºC, 30 cycles; 54 ºC, 36 cycles; and 65 ºC,
30 cycles, respectively. It was followed by a final extension
at 72 oC for 10 min. The specific primers for RyR2 (AF130880)
were: sense, 5'-GAATCAGTGAGTTACTGGGCATGG-3' and antisense, 5'-CTGGTCTCTGAGTTCTCCAAAAGC-3'; for
FKBP12.6 (D86642): sense, 5'-GTGAAGGCAGGAAGGAA-3' and antisense, 5'-GCAGCCAACAGAAGATAAG-3'; for
PLB (NM_022707): sense, 5'-TACCTTACTCGCTCGGC-TATC-3' and antisense,
5'-CAGAAGCATCACAATGATG-CAG-3'; for SERCA2a (NM_017290): sense,
5'-CCGTA-TCCGATGACAATG-3' and antisense,
5'-CCAGGCTCCA-GGTAGTTT-3'; and for GAPDH: sense,
5'-GCTGGGGCTCA-CCTGAAGG-3' and antisense, 5'-GGATGACCTTGCC
CA-CAGCC-3', respectively.
The amplification products were separated by agarose
gel electrophoresis (2%), stained with ethidium bromide,
visualized under UV light, and digitally scanned (Syngene,
England). Band density was determined by a gel imaging
analysis system (Genegenus, Syngene, England), and the
relative density of each DNA band was obtained by dividing
that of the GAPDH bands.
Isolation of rat ventricular myocytes The rat ventricular
myocytes were isolated from adult, male Sprague-Dawley
rats as described previously[19].
Briefly, the rats were killed by an intraperitoneal (ip) injection of a lethal dose of
pentobarbital (100 mg/kg), then the chest cavity was opened and
the hearts were excised. The excised hearts were retrogradely
perfused at 7 mL/min through the aorta, first for 5 min with
Ca2+-free Tyrode's solution composed of (in mmol/L): 135.0
NaCl, 5.4 KCl, 2.0 MgSO4, 0.33 NaH
2PO4, 10.0 glucose, and 10.0 HEPES (pH 7.4) at 37 °C, then with
Ca2+-free Tyrode's solution containing collagenase type II (0.33 mg/mL) and
protease (0.16 mg/mL) for 12 min, and finally with Tyrode's
solution containing 0.2 mmol/L CaCl2 for 6
min. The ventricles of the digested heart were then cut into small cubes
that were subjected to gentle agitation to dissociate the
cells. The freshly dissociated cells were stored at room
temperature in Tyrode's solution containing 0.2 mmol/L
CaCl2.
ISO stimulates cardiomyocytes The ventricular
myo-cytes were isolated as described earlier, plated onto
laminin-coated coverslip chambers, and cultured at 37 °C in
MEM/EBSS serum-free medium. Four hours later, drugs were added
into chambers to assess the change of PLB expression as
follows: (i) control (0.2 µL dissolvent); (ii) ISO
(1×10-6 mol/L ISO+0.2 µL dissolvent); (iii) PRO-5 and PRO-6
(1×10-6 mol/L
ISO+1×10-5 mol/L or
1×10-6 mol/L PRO); (iv) DAR-5 and
DAR-6 (1×10-6 mol/L
ISO+1×10-5 mol/L or
1×10-6 mol/L DAR); and (v) 0213-5 and 0213-6
(1×10-6 mol/L
ISO+1×10-5 mol/L or
1×10-6 mol/L CPU0213). In a separate experiment, changes in the
FKBP12.6 expression in cardiomyocytes were investigated
with PRO and dajisentan (CPU0213) at
1×10-5 mol/L and
1×10-6 mol/L added to the medium and ISO at
1×10-6 mol/L.
Immunocytochemistry assay of PLB Twenty-four hours
after drug incubation, the ventricular myocytes were fixed
using 4% paraformaldehyde for 15 min at 37
°C. The fixed cells were rinsed 3 times in phosphate-buffered saline (PBS)
and then incubated for 30 min with 2% bovine serum
albumin in PBS to reduce non-specific binding.
After overnight incubation (at 4 °C) with the primary antibody against PLB
(Santa Cruz Biotechnology, Santa Cruz, CA, USA), the cells
were rinsed 4 times with PBS, thereafter, the secondary
antibody conjugated with fluorescein isothiocyanate (FITC)
(Santa Cruz Biotechnology, USA) was added for 2 h at room
temperature. Following a further 4 washes, the stained cells
were imaged under fluorescence microscopy.
The negative control, which received identical treatment with the
exception of the primary antibody treatment, was conducted to
exclude non-specific staining.
For the convenience of data analyses, the color images
were converted to red- and blue-filtered, gray-scale images
by ImagePro 5.0 software. In the gray-scale images the
"green" staining of PLB was highlighted above a uniform
background. This step resulted in the narrow and peaked
gray density distribution of pixels in the
cardiomyo-cytes. The gray density represented positive-stained
pixels. The darkest pixel (black) was set to gray value 0 and the brightest
pixel (white) was set to gray value 255, thus, the pixels were
set to gray images between 20_120.
Western blot analysis of PLB and FKBP12.6
After 24 h incubation, the ventricular myocytes were then washed twice
with cold PBS (137 mmol/L NaCl, 1.47 mmol/L
KH2PO4, and
8.9 mmol/L Na2HPO4, pH 7.4) and put into 500 µL lysing buffer
containing 50 mmol/L Tris-HCl, 1% Triton X-100, 150 mm
NaCl, 1 mmol/L EDTA, 0.5% SDS, and 1 mmol/L
phenyl-methylsulfonyl fluoride. The homogenates were centrifuged
for 10 min at 10 000×g at 4
oC and the supernatants were collected. The cell lysates (40 mg) were analyzed by 10%
SDS-PAGE at 100 V for 2 h. After transfer, the nitrocellulose
membranes were incubated in a blocking buffer (50 mmol/L
Tris-HCl, 200 mmol/L NaCl, 0.2% Tween 20, and 5% non-fat
dried milk) for 1 h at room temperature, followed by 2 h in the
same buffer containing 1% non-fat dried milk with a polyclonal
antibody raised against PLB and FKBP12.6 (Santa Cruz
Biotechnology, USA). The membranes were washed with
the same buffer without milk and then incubated for 1 h with
the rabbit antigoat antibody (Wuhan Boster Biological
Technology, Wuhan, China). After washing 3 times for 15
min each time, the immunoreactive bands were visualized by
enhanced chemiluminescence detection reagent (Wuhan
Boster Biological Technology, China) and quantified by
densitometry as described earlier.
Statistical analysis The fluorescence images were
analyzed by ImagePro 5.0 software. Data are presented as
mean±SD or mean±SEM. The homogeneity of the data was
tested by one-way ANOVA and the differences between
specific means were tested for significance by
Bonferroni's multiple comparison tests. A difference between 2 means
was considered statistically significant when
P<0.05.
Results
Worsening cardiac dysfunction and ventricular
fibrillation (VF) rate by ISO Cardiac dysfunction was evaluated
by measuring intracardiac pressure following I/) episodes in
normal and ISO-treated rat hearts. A decrease in systolic
(LVSP, Figure 1A) and diastolic (LVEDP, Figure 1B)
performance was found by I/R procedure relative to the control.
Acute HF during I/R was worsened in the ISO-treated rats
with sharply depressed systolic and diastolic function
relative to I/R in the normal rats (P<0.05). Thus, a worsened HF
model was established attributed to ISO medication which
strongly stimulates b-receptors. In the CPU0213 group the
impaired cardiac performance was attenuated relative to the
ISO group, indicating that the adverse impact by ISO on the
cardiac function was significantly reversed
(P<0.05) by antagonism of ET receptors by CPU0213.
Downregulation of mRNA of the calcium handling
system by ISO The exacerbation of cardiac dysfunction was
produced by ISO subsequent to I/R. The downregulation of
RyR2, FKBP12.6, PLB, and SERCA2a (Figure 2A_2D) was
established by RT-PCR to be significant (P<0.01) relative to
the control, respectively. CPU0213 intervention significantly
reversed the downregulation of the expression of the
calcium handling system compared to the ISO group
(P<0.01).
Downregulation of PLB by ISO in vitro
The incubation of the adult rat ventricular myocytes with ISO at
1×10-6 mol/L was conducted to establish an in vitro of downregulation of
PLB. After incubation of the second antibody conjugated
with FITC, the PLB proteins in the cardiac myocytes were
flamed with green fluorescence (Figure 3). Following 24 h
incubation with ISO, the green fluorescence was reduced
significantly and the mean gray value of the cardiomyocytes
was reduced to 31.7±7.1, from 57.6±6.0 for the control group
(P<0.01; Figure 4). ISO suppressed the peak of gray density
distribution in the cardiomyocytes relative to the control
(Figure 4A, 4B).
Reversal of PLB downregulation by PRO in
vitro It was of interest to identify whether the downregulation of PLB by
ISO was due to the activation of β-AR. The altered gray
images of PLB in isolated cardiomyocytes were completely
reversed by PRO at either 1×10-5 mol/L (51.45±1.8,
P<0.01) or 1×10-6 mol/L (47.98±7.5,
P<0.05), respectively. In this cellular
model, the downregulation of PLB by ISO was subsequent
to an overactivation of the β-AR in vitro (Figure 4C, 4D).
Reversal of PLB downregulation by ET receptor
antagonism in vitro To further investigate whether ET receptor
antagonism could reverse the downregulation of PLB by ISO,
the effects were compared with PRO in
vitro. A selective ETA blocker DAR at
1×10-5 mol/L and
1×10-6 mol/L significantly elevated the mean gray values in ventricular myocytes to
59.49×1.0 (P<0.01) and 47.38×7.5
(P<0.05), respectively (Figure 5A, 5B), relative to the ISO group, and the pattern of
the images was restored towards the control levels.
It was also interesting to determine whether the reversal
of the downregulation of PLB could also be achieved by a
dual ETA/ETB blocker CPU0213
in vitro. Incubation with CPU0213 at
1×10-5 mol/L and
1×10-6 mol/L dramatically upregulated the PLB protein levels, as an increase in
immuno-staining levels (54.61±3.2, P<0.01 and 47.10±3.5,
P<0.05) relative to the ISO group (Figure 5C, 5D). The protection of
cardiomyocytes from the downregulation of PLB in the
presence of ISO was significant by either DAR or CPU0213
in vitro (Figure 5E, 5F).
Reversal of PLB and FKBP12.6 protein by Western
blotting Data from Western blots present further proof of the
downregulation of PLB protein levels by ISO in
vitro. After incubation of
1×10-6 mol/L ISO for 24 h, there was a 39%
depression of the PLB protein (P<0.01) relative to the control,
and the downregulation of PLB was effectively reversed by
either PRO or CPU0213 at 1×10-5 mol/L
(P<0.01) and 1×10-6 mol/L
(P<0.05), respectively (Figure 6A).
A dramatic downregulation of FKBP12.6 was also found
in the cardiac myocytes stimulated by ISO in
vitro. The protein level of FKBP12.6 decreased by 48%
(P<0.01) compared to the control, and the downregulation of the
FKBP12.6 protein was restored by PRO at
1×10-5 mol/L (P<0.01) and
1×10-6 mol/L (P<0.05). The ET receptor antagonism of
CPU0213 significantly elevated the FKBP12.6 protein at both
1×10-5 mol/L (P<0.01) and
1×10-6 mol/L (P<0.05), respectively,
relative to the ISO group. Both the β-blocker and ET
receptor antagonist can reverse the downregulation of FKBP12.6
by ISO (Figure 6B).
Discussion
Despite extensive investigations, the molecular events
and the mechanisms underlying the progression of HF
remain unclear. In failing hearts, the cardiac adrenergic
receptor signaling pathway is activated, and the most striking
target for the treatment of HF is directed to the downstream
events of β-AR activation[20]. Molecular aspects of HF have
been focused at the depressed expression of
FKBP12.6[9] and
SERCA2a[6,21] which are potential therapeutic targets.
Changes in the downstream signaling pathways (RyR2,
FKBP12.6, SERCA2a, PLB, etc) in response to
β-adrenergic stimulation are also considered to be important targets for
future treatments of HF[22]. In the present study, cardiac
failure produced by I/R procedure is exacerbated by ISO
treatment which also produces significant downregulation
in the mRNA expression of RyR2, FKBP12.6, PLB, and
SERCA2a in the myocardium in vivo. These changes are
attenuated by CPU0213 which is a dual ET receptor
antagonist[15].
The overactivation of the sympathetic nervous system
and the β-AR are found in congestive HF where the
expression of PLB and SERCA2a is
downregulated[23]. The worsened cardiac dysfunction following ISO treatment correlates
well with a marked reduction in the expression of PLB and
SERCA2a, possibly resulting from hyperphosphorylation by
PKA in response to the overstimulation of β-AR. PKA
phosphorylation of PLB under normal conditions releases its
sustained suppression of SERCA2a, resulting in elevated
activity of SERCA2a in response to an accelerated heart rate in a
shortened cardiac cycle. In the presence of excess of ISO,
the marked downregulation of both SERCA2a and PLB is
produced in the myocardium, which is in agreement with
previous studies[24].
The RyR2/calcium release channel in the SR comprises a
macromolecular complex in association with FKBP12.6
(calstabin2). FKBP12.6, an 11.8 kDa cis-trans peptidyl-prolyl
isomerase (apparent molecular mass 12.6 kDa), stabilizes the
closed state of the RyR2 channel at diastole, and FKBP12.6
can be targeted for improving cardiac
performance[25]. However, the underlying mechanism of this regulation has
not been fully clarified. It remains to be established whether
the expression of RyR2, FKBP12.6, SERCA2a, and PLB is
mediated by the activation of ET receptors in the process
following the stimulation of β-receptors.
Cardiac dysfunction exaggerated by ISO in association
with the downregulation of the calcium handling system is
alleviated by ET receptor blockade in vivo. The beneficial
effects may be explained by an indirect compensatory
response via a reflex or some humoral factors rather than a
direct involvement in the downstream events of the
stimulated β-receptors. An in vitro assessment of this possibility
has been carried out. ISO incubated with
cardiomyocytes in vitro produces overactivity of
β-receptors and the down-regulation of PLB and FKBP12.6 proteins. The present study
of in vitro mechanisms provided the support that the ET
pathway is directly linked to the downregulation of PLB by
the activation of β-AR. Interestingly, when ET receptors are
blocked by either the ETAR blocker DAR or
ETAR/ETBR blocker CPU0213, a reversal of the downregulated protein
expression of PLB and FKBP12.6 is equally significant. This
suggests that the ETAR is involved mainly in the response to
β-receptor stimulation. Additionally, the protein expression
of PLB and FKBP12.6 is downregulated by ISO in cultured
myocytes in vitro and the downregulation of PLB and
FKBP12.6 in the presence of ISO is blocked by CPU0213, the
effects of which are comparable to PRO. The present study
provides evidence to support the thesis that the
downregula-tion of the calcium handling system in the presence of
β-receptor stimulation is at least partly mediated by the
activation of the ET pathway. Thus, ET receptor antagonism is
effective in treating HF[16,27] and is of potential therapeutic
significance in preventing patients from sudden cardiac
death[26,28]. The present study may offer more information in
the molecular events relating to the downregulation of RyR2
and FKBP12.6 in HF[26].
The activation of the ET pathway may initiate oxidative
stress, which in turn releases more ET-1 to affect the
cardiovascular system; a positive therapeutic outcome of an
antagonism of the ET receptor also results from antioxidative
activities[14,16]. Additionally, a relief of cardiac failure by ET
receptor antagonists also corresponds to an improvement
of mitochondrial respiratory complex activities in the
myocardium[27].
The findings of this study both in vivo and
vitro is that β-AR activation, which induces the downregulation of the
calcium modulating system, is at least partly relayed by an
activated ET pathway. The blockade of ET receptors
reverses the abnormal expression of the calcium handling
system in the SR and the worsened cardiac function
produced by ISO. This study offers direct evidence that ET-1 is
actively involved in the downregulation of PLB and
FKBP12.6 through β-AR overactivation and that this mode of action is
consistent with an attenuation of heart failure by ET
antagonism.
Acknowledgement
We are most grateful to Prof David J TRIGGLE from the
State University of New York at Buffalo for assistance in
revising the English of the manuscript.
References
1 Feldman DS, Carnes CA, Abraham WT, Bristow
MR. Mechanisms of disease: beta-adrenergic receptors — alterations in
signal transduction and pharmacogenomics in heart
failure. Nat Clin Pract Cardiovasc Med 2005; 2: 475_83.
2 Schwinger RH. Treatment of heart failure with
beta-receptor-blockers. Dtsch Med Wochenschr 2002; 127: 682_8.
3 Olson EN. A decade of discoveries in cardiac
biology. Nat Med 2004; 10: 467_74.
4 Reiken S, Gaburjakova M, Guatimosim S, Gomez AM, D'Armiento
J, Burkhoff D, et al. Protein kinase A phosphorylation of the
cardiac calcium release channel (ryanodine receptor) in normal
and failing hearts. Role of phosphatases and response to
isoproterenol. J Biol Chem 2003; 278: 444_53.
5 Gupta RC, Mishra S, Rastogi S, Sharov VG, Sabbah HN.
Improvement of cardiac sarcoplasmic reticulum calcium cycling in dogs
with heart failure following long-term therapy with the Acorn
Cardiac Support Device. Heart Fail Rev 2005; 10: 149_55.
6 Yano M, Ikeda Y, Matsuzaki M. Altered intracellular
Ca2+ handling in heart failure. J Clin Invest 2005; 115: 556_64.
7 Schmitt JP, Kamisago M, Asahi M, Li GH, Ahmad F, Mende U,
et al. Dilated cardiomyopathy and heart failure caused by a
mutation in phospholamban. Science 2003; 299: 1410_3.
8 Temsah RM, Dyck C, Netticadan T, Chapman D, Elimban V,
Dhalla NS. Effect of beta-adrenoceptor blockers on
sarcoplasmic reticular function and gene expression in the
ischemic-reperfused heart. J Pharmacol Exp Ther 2000; 293: 15_23.
9 Wehrens XH, Marks AR. Novel therapeutic approaches for heart
failure by normalizing calcium cycling. Nat Rev Drug Disc 2004;
3: 565_73.
10 Ertl G, Bauersachs J. Endothelin receptor antagonists in heart
failure: current status and future directions. Drugs 2004; 64:
1029_40.
11 Teerlink JR, McMurray JJ, Bourge RC, Cleland JG, Cotter G,
Jondeau G, et al. Tezosentan in patients with acute heart failure:
design of the Value of Endothelin Receptor Inhibition with
Tezosentan in Acute heart failure Study (VERITAS). Am Heart J
2005; 150: 46_53.
12 Anand I, McMurray J, Cohn JN, Konstam MA, Notter T, Quitzau
K, et al. Long-term effects of darusentan on left-ventricular
remodelling and clinical outcomes in the Endothelin A Receptor
Antagonist Trial in Heart Failure (EARTH): randomised,
double-blind, placebo-controlled trial. Lancet 2004; 364: 347_54.
13 Kelland NF, Webb DJ. Clinical trials of endothelin antagonists in
heart failure: a question of dose? Exp Biol Med (Maywood)
2006; 231: 696_9.
14 Qi MY, Xia HJ, Dai DZ, Dai Y. A novel endothelin receptor
antagonist CPU0213 improves diabetic cardiac insufficiency
attributed to up-regulation of the expression of FKBP12.6,
SERCA2a, and PLB in rats. J Cardiovasc Pharmacol 2006; 47:
729_35.
15 Dai DZ, Ji M, Huang M, Liu LG. Endothelin receptor antagonist
activity and selective blocking the ETA and
ETB of Compound 0213. J China Pharm Univ 2004; 35: 552_7.
16 Li L, Chu Y, Fink GD, Engelhardt JF, Heistad DD, Chen AF.
Endothelin-1 stimulates arterial VCAM-1 expression via NADPH
oxidase-derived superoxide in mineralocorticoid hypertension.
Hypertension 2003; 42: 997_1003.
17 Xu FP, Chen MS, Wang YZ, Yi Q, Lin SB, Chen AF,
et al. Leptin induces hypertrophy via endothelin-1-reactive oxygen species
pathway in cultured neonatal rat cardiomyocytes. Circulation
2004; 110: 1269_75.
18 Zhang TT, Cui B, Dai DZ. Downregulation of Kv4.2 and Kv4.3
channel gene expression in right ventricular hypertrophy
induced by monocrotaline in rat. Acta Pharmacol Sin 2004; 25:
226_30.
19 Ma YP, Hu HJ, Hao XM, Zhou PA, Wu CH, Dai DZ. Reduced
sodium currents in isolated mammalian myocytes treated with
chronic L-thyroxine. Drug Dev Res 2003; 58: 111_5.
20 Weil J, Schunkert H. Pathophysiology of chronic heart failure.
Clin Res Cardiol 2006; 95 (Suppl 4): 1_17.
21 Armoundas AA, Rose J, Aggarwal R, Stuyvers BD, O'rourke B,
Kass DA, et al. Cellular and molecular determinants of altered
Ca2+ handling in the failing rabbit heart: primary defects in SR
Ca2+ uptake and release mechanisms. Am J Physiol Heart Circ
Physiol 2007; 292: H1607_18.
22 Sucharov CC. Beta-adrenergic pathways in human heart failure.
Expert Rev Cardiovasc Ther 2007; 5: 119_24.
23 Armoundas AA, Rose J, Aggarwal R, Stuyvers B, O'Rourke B,
Kass DA, et al. Cellular and molecular determinants of altered
Ca2+ handling in the failing rabbit heart: Primary defects in SR
Ca2+ uptake and release mechanisms. Am J Physiol Heart Circ
Physiol 2007; 292: H1607_18.
24 Saliaris AP, Amado LC, Minhas KM, Schuleri KH, Lehrke S, St
John M, et al. Chronic allopurinol administration ameliorates
maladaptive alterations in Ca2+ cycling proteins and
beta-adrenergic hyporesponsiveness in heart failure. Am J Physiol Heart
Circ Physiol 2007; 292: H1328_35.
25 Huang F, Shan J, Reiken S, Wehrens XH, Marks AR. Analysis of
calstabin2 (FKBP12.6)-ryanodine receptor interactions: rescue
of heart failure by calstabin2 in mice. Proc Natl Acad Sci USA
2006; 103: 3456_61.
26 Phrommintikul A, Chattipakorn N. Roles of cardiac ryanodine
receptor in heart failure and sudden cardiac death. Int J Cardiol
2006; 112: 142_52.
27 Marin-Garcia J, Goldenthal MJ, Moe GW. Selective endothelin
receptor blockade reverses mitochondrial dysfunction in canine
heart failure. J Card Fail 2002; 8: 326_32.
28 Xia HJ, Dai DZ, Dai Y. Up-regulated inflammatory factors
endothelin, NFκB, TNF-α and iNOS involved in exaggerated
cardiac arrhythmias in L-thyroxine-induced cardiomyopathy are
suppressed by darusentan in rats. Life Sci 2006; 79: 1812_9.
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