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Introduction
Our previous study showed that in vivo treatment with insulin reduced postischemic apoptotic death in both
cardiomyocytes and coronary endothelial cells by activating the phosphatidylinositol 3-kinase-Akt-endothelial nitric oxide
synthase (PI3-kinase-Akt-eNOS) pathway, that is, the survival signaling pathway, which might further contribute to the
prolonged improvement of cardiac performance following ischemia/reperfusion
(I/R)[1_3]. In a recent study, we have
demonstrated that insulin exerts direct positive inotropic effect by increasing calcium
(Ca2+) transients in I/R cardiomyocytes. This
insulin-induced improvement of contractile function in I/R myocytes shares the same signaling cascade for protection as
previously described for myocardial
tissue[4]. These observations therefore indicate that this survival pathway is also active
to directly improve cardiac myocyte function recovery following I/R
in vitro experiments that guarantee constant conditions
and exclude other factors, such as stress, hormone secretion, or inflamatory mediators, rendering insulin even more important
as a protective agent applicable to reperfused myocardial tissue. However, the mechanisms underlying the insulin-induced
improvement of I/R myocyte functional recovery through activation of the survival pathway
in vitro remain largely elusive.
Cardiac contraction and relaxation are closely regulated by intrinsic machineries governing sequential rise and fall of
cytosolic Ca2+. Ca2+ enters the cytosolic space through voltage-dependent
Ca2+ channels after membrane depolarization and
triggers release of Ca2+ from sarcoplasmic reticulum (SR), which initiates activation of cardiac contractile proteins and
actin_myosin cross-bridge linking. Termination of contraction and cardiac relaxation are initiated with the removal of cytosolic
Ca2+ mainly through sarcoplasmic reticulum
Ca2+-ATPase (SERCA) and, to a lesser extent,
Na+-Ca2+ exchanger. It was shown that
in rat ventricular myocardium, 92% of calcium removal occurs by SR calcium
uptake[5]. SERCA2a, the isoform of SERCA in
myocardium and slow twitch skeletal muscle, is responsible for not only
Ca2+ extrusion but also ventricular contractility
because of their role in Ca2+ extrusion and SR
Ca2+ loading for the next cardiac
cycle[5,6]. Defects in SERCA2a lead to
decreased peak myocyte con-tractility, reduced intracellular
Ca2+ removal, and prolonged duration of the cardiac cycle. Heart
failure (HF) is a common outcome of myocardial
infarction (MI) and is associated with a poor prognosis. Because of
the loss of contractile function in the infarcted area, MI
results in increased mechanical load on the intact myocardium,
which undergoes molecular, cellular, morphological, and functional
remodeling. Decreased SR
Ca2+ uptake and decreased expression of SERCA2a are key features of cardiac myocyte dysfunction
in both experimental and human
HF[7,8]. Hypertrophic
cardiomyocytes with reduced contractility and relaxation have been found
in the failing rat heart after MI, accompanied by a
reduced peak in Ca2+ transients and prolonged
Ca2+ transients decay[9]. SERCA mRNA and
protein levels decreased with increasing severity of HF after left
coronary artery ligation[7].
Indeed, in cardiomyocytes isolated from the left ventricle of
patients with end-stage HF, SERCA2a overexpression can restore
normal Ca2+
cycling[6]. Previous studies in adult myocytes
isolated from rat hearts 3 weeks after MI demonstrated
abnormal contractility, intracellular
Ca2+ concentration
([Ca2+]i) homeostasis and decreased SERCA2a expression and
activity[10]. However, no data are available so far on the effect of insulin on
SERCA2a activity in I/R cardio-myocytes.
Last, but not least, activation of Akt and its upstream
signal PI3-kinase might dramatically improve
cardiac function and protect against
apoptosis[1,2,4], establishing an important role for Akt in the
maintenance of heart morphology and function.
Transgenic overexpression of protein kinase Akt
enhances myocyte contractility and relaxation through
acceleration of intracellular
Ca2+ transients. The underlying cellular
mechanism(s) include potentiation of L-type
Ca2+ channel function and
upregulation of SERCA2a[11]. However, to date, a direct link between the effects of insulin on SERCA2a activity in I/R
myocardium and the insulin-induced survival pathway and subsequent improvement of I/R myocyte functional recovery has
not been established.
The aims of this study, therefore, were to determine the role of SERCA2a in the insulin-induced improvement of I/R
cardiomyocyte functional recovery, and to further investigate the mechanism involved.
Materials and methods
Preparation of isolated cardiomyocytes The experiments were carried out in adherence with the National Institutes of
Health Guidelines on the Use of Laboratory Animals and were approved by the Fourth Military Medical University
Committee on Animal Care. Calcium-tolerant ventricular myocytes were isolated from adult male Sprague-Dawley rats (body weight,
220_250 g) hearts by a standard enzymatic
technique[4]. Briefly, rats were anesthetized and anticoagulated with heparin
sodium (1000 U/kg, ip). The hearts were rapidly excised and mounted on a Langendorff perfusion apparatus, and immediately
perfused with Ca2+-free Tyrode¡¯s solution containing (in mmol/L) 143.0 NaCl, 5.4 KCl, 0.5
MgCl2, 0.3
NaH2PO4, 5.0 HEPES, 5.0 glucose (pH 7.4,
36 °C, equilibrated with O2, until spontaneous contraction of the heart ceased. The hearts were then
perfused with Ca2+-free Tyrode¡¯s solution containing 0.4 g/L collagenase II (283
U/mg; Worthington Biochemical, Lakewood, USA) and 0.7 g/L bovine serum albumin for approximately 20 min until the heart
became soft. After perfusion with Ca2+-free Tyrode¡¯s solution for 5 min to remove enzymes, the atria and aorta were removed
and the ventricles were minced and incubated with Krebs solution containing (in mmol/L) 70.0
L-glutamic acid, 25.0 KCl, 20.0 Taurine, 10.0
KH2PO4, 3.0 MgCl2, 0.5 EGTA,
10.0 HEPES, 10.0 glucose (pH 7.4) supplemented with 2% bovine serum albumin
before being filtered through a nylon mesh (200 mesh). The cells were subsequently separated by sedimentation for 10 min
twice. Cardiac myocytes were then re-suspended in the
Ca2+-free Tyrode¡¯s solution, and
Ca2+ was slowly added to the cell suspension until
Ca2+ reached a final concentration of 1.8 mmol/L. Approximately 70%_80% rod-shaped cardiomyocytes
were obtained.
Measurement of myocyte contractile and relaxation function
The mechanical contraction of ventricular myocytes was
assessed by a video-based motion edge-detection system (IonOptix, Milton,
USA)[12]. In brief, myocytes were transferred to
a cell chamber on the stage of an inverted microscope (Olympus, Tokyo, Japan) and continuously perfused with Tyrode¡¯s
solution (1 mL/min, 35 °C). Myocyte contraction was induced at a frequency of 0.5 Hz by platinum electrodes connected to
an electrical stimulator. Peak twitch amplitude (PTA), time to peak shortening (TPS), time to 70% relengthening (TR70), and
the maximal velocities of shortening/relengthening
(±dL/dt) were automatically calculated from the cell length data by a
computer. Criteria for choosing myocytes for the experiment included: (1) a rod shape with clear edges; (2) clearly defined
sarcomeric striations; (3) steady contraction in response to electrical stimulation but without spontaneous contractions; and
(4) stable steady-state contraction amplitude for at least 5 min before drugs were given.
Measurement of myocyte Ca2+ transients
Myocytes were loaded with Fura-2/AM (0.5 µmol/L; Alexis Bio-chemicals, San
Diego, USA) for 30 min. The myocytes were excited by light emitted by a 75 W lamp and passed through either a 360 or 380
nm filter, and the Fura emission wavelength (510 nm) was synchronously monitored. Intracellular free
Ca2+ in loaded myocytes was measured as the fluorescence ratio (360/380
nm)[4].
Experimental protocols Ischemia conditions were simulated by chemical anoxia solution according to the method
described by Esumi et al[13], which contained (in mmol/L): 137.0 NaCl, 15.8 KCl, 0.49
MgCl2, 0.9
CaCl2·2H2O, 4.0
HEPES, 10.0 deoxyglucose, 0.75 sodium dithionate and 20.0 lactate, pH 6.5. Ventricular myocytes from rats were perfused with
Tyrode¡¯s solution and field stimulated at a frequency of 0.5 Hz, 5 ms duration. After 10_15 min of equilibration, the myocytes
were exposed to one of the following treatments (15_20 myocytes from 6_8 rats/group): (1) control (Con), myocytes were
perfused with Tyrode¡¯s solution for 45 min; (2) control plus insulin (Con+Ins), myocytes were perfused with
10-7 mol/L insulin for 45 min; (3) (I/R), after ischemia was simulated by perfusion of myocytes with chemical anoxia solution for 15 min, the cells
were reperfused with Tyrode¡¯s solution for 30 min; (4) I/R plus insulin
(I/R+Ins), after undergoing the same ischemia procedure as the I/R group, the cardiomyocytes were reperfused with insulin
for 30 min; (5) I/R plus insulin plus Akt inhibitor
(I/R+Ins+AI), the isolated myocytes were subjected to the same I/R procedure as the I/R plus insulin group, and pre-treated
with a selective Akt inhibitor, AI (1L-6-Hydroxy-
methyl-chiro-inositol
2-(R)-2-O-methyl-3-O-octadecyl-carbonate, 5.0 µmol/L; Calbiochem, Darmstadt,
Germany)[14,15] for 2 h before the I/R procedure and were treated with AI in the same concentration throughout the whole I/R duration. Myocyte
shortening and intracellular Ca2+ transients were assessed as described above.
Western blot analysis To determine the mechanism of the protective effect of insulin on I/R cardiomyocytes, an
additional experiment was carried out to examine Akt expression and activation by Western blot analysis as
described[1]. Isolated cells were randomly divided into 5 groups to receive the treatments according to the experimental protocols. Ventricular
myocytes were washed with phosphate-buffered saline and centrifuged at
1000×g. Cells were collected, washed and
homogenized in lysis buffer containing (in mmol/L) 20.0 Tris, 150.0 NaCl, 1.0 EDTA, 1.0 EGTA, 1% Triton X-100, 2.5 sodium
pyrophosphate, 1.0 b-glycerolphosphate, 1.0
Na3VO4, 1.0 g/L leupeptin, and 1.0 phenylmethylsulphonyl fluoride (pH 7.5).
After sonication, the lysate was centrifuged at 4 °C (12
000×g, 10 min). The protein of the lysate was quantified and separated
by sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride-plus membrane.
After being blocked with 5% milk, the immunoblots were probed with anti-pAkt (1:1000) antibodies overnight at 4 °C,
followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The blots were visualized
with ECL-plus reagent (Amersham Pharmacia Biotech, USA). pAkt immunoblots were then stripped with stripping buffer at
50 °C for 30 min and reblotted for total Akt (1:1000). Anti-Akt and anti-pAkt antibodies (#9272 and #9271, respectively) were
obtained from Cell Signaling Technology (Beverly, USA).
Preparation of SR from rat heart SR was prepared according to the methods of Jones as modified by Kodavanti
et al[16_18]. The myocytes were put in homogenizing medium, containing (in mmol/L) 50.0
Na2HPO4, 10.0 Na2
EDTA, and
25.0 NaF (pH 7.4). The minced ventricle tissue was placed in 10 mL of ice-cold homogenizing medium and homogenated 3
times. An additional 5 mL of homogenizing medium was added and the homogenate was sedimented twice for 20 min at 14
000×g at 4 °C. The supernatant was recentrifuged at
45 000×g for 30 min. The pellet obtained after this centri-fugation, consisting of crude membrane vesicles (SR), was susper
milliliter, preincubated for 10 min at 37 °C and the reaction was initiated by the addition of ATP. The ATP hydrolysis that
occurred in the absence of Ca2+ (1 mmol/L EGTA) was subtracted to determine the activity of
Ca2+-stimulated ATPase. Ouabain was added fresh to a final concentration of 1 mmol/L in the media, which remained unchanged throughout the
incubation. Mitochondrial contamination was assessed by determining the activity of azide-sensitive ATPase, that is, that
activity inhibited by 5 mmol/L sodium
azide[19].
Statistical analysis All values in text, tables and figures are presented as mean±SEM. Differences were compared by
Student¡¯s t-test or anova, where appropriate. Probabilities of <0.05 were considered to be statistically significant. All of the
statistical tests were carried out with GraphPad Prism software version 4.0 (GraphPad Software, San Diego, USA).
Results
Insulin improved cardiomyocyte contractile functional recovery in simulated
I/R A simulated I/R model of myocytes was developed according to Esumi
et al[13], and the changes in cell contractile activity and intracellular
Ca2+ transients during I/R were determined. Ventricular myocytes were perfused with Tyrode¡¯s solution for 10_15 min while the field was stimulated
at a frequency of 0.5 Hz, 5 ms duration. Following this equilibration period the myocytes were exposed to chemical anoxia
solution for 15 min. PTA of myocytes undergoing ischemia was gradually decreased to 38%±8% of the pre-ischemic level
(P<0.01, n=20 myocytes from 8 hearts). On commencement of reperfusion, PTA rapidly returned to near or transiently above
pre-ischemia, then gradually declined to 87%±10% of the pre-ischemic level
(P>0.05) with continued reperfusion (Figure 1A).
Consistent with the contractile amplitude,
±dL/dt also had similar changes under conditions of simulated I/R (Figure 1B).
Insulin (10-7 mol/L, the concentration used in most
in vitro studies)[20,21] exerted significant inotropic action in normal
myocytes (21.8%±0.5% of Con+Ins group
vs 17.9%±
0.8% of Con group; P<0.05, n=20 myocytes from 8 hearts) (Table 1). Although the contractile function of simulated I/R
myocytes (underdoing ischemia for 15 min and reperfusion with Tyrode¡¯s solution for 30 min) could not completely recover
to the level of the control group, in which the cells were perfused with Tyrode¡¯s solution for 45 min, there was no significant
difference in contraction of the cells in the two groups (17.9%±0.8% of Con group
vs 14.5%±0.7% of I/R group; P>0.05,
n=20 myocytes from 8 hearts). However, consistent with our previous results, treatment with insulin at the beginning of reperfusion
increased myocyte shortening in a concentration-dependent
manner[4]. As summarized in Table 1, insulin
(10-7 mol/L) significantly increased PTA (% baseline) and
±dL/dt, and markedly reduced time to peak shortening (TPS) and time to 70%
relengthening (TR70) (20 myocytes from 7 rats,
P<0.05 vs I/R group). The insulin-induced effect on myocyte shortening
appeared rapidly with the maximal response, which occurred within 5 min of exposure to insulin, and cells recovered almost
completely after washout. These data showed that insulin improved cardiomyocyte contractile functional recovery in simulated
I/R.
Insulin increased intracellular calcium transients in simulated I/R
cardiomyocytes The time-course of the fluorescence signal augment [time to peak
Ca2+ (TTPCa)] and decay [time to 50% diastolic
Ca2+ (T50Dca)] were evaluated to assess
the rate of intracellular Ca2+ release and clearing, respectively. Simulated ischemia by perfusion with chemical anoxia solution
for 15 min significantly decreased calcium transients amplitude
(DFFI, shown as twitch of fluorescence 360/380 ratio) (34%±9%
of pre-ischemic level, P<0.01, n=20 myocytes from 8 hearts). Reperfusion with vehicle (Tyrode¡¯s solution) for 30 min almost
recovered the twitch of fluorescent ratio to the pre-ischemic level, although not completely (90%±8% of pre-ischemic level,
P>0.05) (Figure 2).
Consistent with our previous results, insulin significantly increased the calcium transients amplitude in normal myocytes
(0.39±0.03 of Con+Ins group vs 0.35±0.04 of Con group,
P<0.05, n=20 myocytes from 8 hearts) (Table 2).
Although vehicle reperfusion had no effect on the intracellular
Ca2+ transients, it was observed that insulin treatment at the
onset of reperfusion significantly enhanced the DFFI from 0.32±0.03 of the I/R group to 0.36±0.04
(P<0.05) 5 min after reperfusion with insulin. Significantly shorter TTPCa and
T50DCa were seen after exposure to insulin in I/R myo-cytes (both
P<0.05), whereas the diastolic
Ca2+ level (resting FFI) had no significant change compared with the Con or
I/R groups (Table 2). These data suggested that there was enhanced
Ca2+ handling elicited by insulin in I/R myocytes.
Mechanisms involved in improvement of contractile function
Defects in SERCA2a lead to decreased peak myocyte
contractility, reduced intracellular
Ca2+ removal, and prolonged duration of the cardiac
cycle[10]. To study whether SERCA2a is also involved in the insulin-induced improvement of contractile function following I/R, the present study examined
Ca2+-ATPase activity by an optical assay in crude SR
extracted from control and simulated I/R myocytes. As shown in Figure 3,
insulin increased the SR
Ca2+-ATPase activity in normal myocytes. It was seen that the SR
Ca2+-ATPase activity was significantly decreased at the end of ischemia, whereas the vehicle reperfusion almost recovered the SR
Ca2+-ATPase activity in I/R myocytes compared with that of control group. Interestingly, treatment with insulin at the onset of reperfusion
significantly increased the SR
Ca2+-ATPase activity in I/R myocytes (7.8±0.4
mmol Pi·mg protein-1·
h-1 vs 6.8± 0.6 mmol Pi·mg
protein-1·h-1 of I/R group,
n=8, P<
0.05), suggesting that increased SERCA2a activity might be involved in the insulin-induced improvement of I/R myocyte
contraction.
A previous study demonstrated that transgenic over-
expression of Akt increases myocyte contractility through
acceleration of intracellular
Ca2+ transients[11]. To study the role of
Akt in the insulin-induced positive inotropic effects on I/R myocytes, an additional experiment was carried out to determine
the insulin-induced Akt activation in isolated cardiomyocytes by Western blot. As shown in Figure 4, insulin markedly
activated Akt activity in normal myocytes. Although the I/R procedure made no change in Akt activation, treatment with
insulin resulted in a 2.9-fold increase in Akt phosphorylation
(n=5, P<0.01 vs I/R group). There was no difference in total Akt
among the groups studied (Figure 4). These results showed that the insulin-induced inotropic effect on I/R myocytes might
be, at least in part, Akt-dependent.
To further establish a cause-effect relationship between increased SERCA2a activity and the improved contractile
response to insulin in simulated I/R myocytes through Akt activation, we pre-treated the myocytes with a selective AI (5.0
µmol/L) before insulin stimulation and assessed the changes in SERCA2a activity and myocyte contractile function.
Pre-treatment with AI not only markedly decreased the Akt phosphorylation by insulin
(P<0.01 vs I/R plus insulin group, Figure
4), but also inhibited the augmentation of SERCA2a activity in the insulin-treated I/R myocytes
(n=8, P<0.05, Figure 3). In addition, AI treatment also almost completely abolished the contractile effect and the augmented intracellular calcium
transients induced by insulin synchronously, whereas the same concentration of AI alone had no effect on either myocyte
contraction or calcium transients (Tables 1,2). Previous studies and our preliminary
experiment showed that AI, at the concentration of 5.0
µmol/L, selectively blocked Akt phosphorylation induced by insulin but did not affect PI-3 kinase
activity[22,23]. These results provided direct evidence demonstrating that increased SERCA2a activity, which is at least partly by
enhanced Akt activation, plays a critical role in cardiac contractile response to insulin in I/R myocytes.
Discussion
In the present study we have demonstrated, for the first time, that insulin increases the SERCA2a activity and
subsequently improves functional recovery in simulated I/R myocytes. This enhancement of SERCA2a activity by insulin is at
least partly due to Akt activation. This finding suggests that insulin plays a more important role in the cardioprotection of
ischemic/reperfused myocardium by modulating the intracellular
Ca2+ concentration homeostasis.
Increasing evidence indicates that, in addition to its inimitable function in glucose metabolism, insulin plays critical roles
in a variety of other physiological and pathological modulations, such as regulation of inflammatory response and nitric oxide
production[1,24,25]. We have also previously demonstrated that insulin improves myocardial functions following I/R in an
in vivo model[3]. However, the mechanism underlying these insulin-induced effects is unclear. In a recent study, we provided
evidence that insulin exerts direct positive inotropic action on
cardiomyocytes[4]. To further determine the effect of insulin on
myocardium in I/R conditions in vitro, in our present study, ventricular myocytes isolated from adult rat were subjected to 15
min ischemia and subsequent 30 min reperfusion, during which the cells were field-stimulated and myocyte
shortening/relengthening and intracellular
Ca2+ transients were simultaneously observed.
Considering the cardiac myocyte is composed of bundles of myofibrils that contain sarcomeres representing the basic
contractile units of the myocyte and related to the intracellular free
Ca2+ concentration
([Ca2+]i), we reflected the cardiomyocyte
contractile function with the change of the myocyte sarcomere length. It was seen that simulated ischemia by chemical anoxia
solution decreased the amplitude of contraction to approximately 40% of the pre-ischemic level, which was restored, though
not completely, to the pre-ischemic level by reperfusion with Tyrode¡¯s solution. Interestingly, treatment with insulin
(10-7 mol/L) at the onset of reperfusion significantly enhanced the recovery of contractile function as evidenced by increased PTA
and ±dL/dt and shortened TPS and TR70 in I/R cardiomyocytes, compared with those in cells reperfused with vehicle (Table
1). Concomitantly, the intracellular
Ca2+ transients (Ca2+ fluorescence ratio,
DFFI) induced by field-stimulation was restored to the pre-ischemic level by reperfusion with vehicle in simulated I/R myocytes. As the amplitude of
Ca2+ transients was increased, and TTPCa and
T50DCa were overtly reduced by insulin treatment in I/R myocytes (Table 2), the alteration of
myocyte Ca2+ handling is largely responsible for the enhanced contractile response of myocytes to insulin. Taken together,
these data indicate that insulin exerts direct positive inotropic effects on myocytes in I/R conditions by modulating
intracellular Ca2+.
The Ca2+-ATPase of SR catalyzes the most important step in relaxation by coupling cleavage of ATP to transport two
Ca2+ into the SR
lumen[26,27]. A dysfunction of SERCA2a has been proposed as a contributing factor to the development of
cardiovascular diseases in which cytokines are involved, such as genetic hypertension, I/R injury, myocardial stunning, and
heart failure[28]. However, SERCA2a overexpression in cardiomyocytes from the left ventricle of patients with HF can restore
normal Ca2+
cycling[6]. In the present study, to determine the role of SERCA2a in the insulin-induced alteration of
Ca2+ transients, we measured SERCA2a activity in myocytes. No remarkable change was observed in myocytes undergoing
simulated I/R procedure, which is different from some previous data that SERCA2a expression and activity decreased with
increasing severity of HF after MI[10]. The difference might be due to the shorter time-course of I/R
in vitro in this study. In contrast, we found that treatment with insulin significantly increased SERCA2a activity compared with vehicle reperfusion,
suggesting that increased SERCA2a activity might contribute to the improvement of intracellular
Ca2+ transients and subsequent contraction by insulin in I/R myocytes.
We have demonstrated that insulin exerts anti-apoptotic effects in both cardiomyocytes and coronary endothelial cells
following I/R by Akt-eNOS signal in vivo, that is, the survival signaling pathway, which might further contribute to the
prolonged improvement of cardiac function after
reperfusion[1_3]. In addition, it was also shown in our previous study that
insulin has a direct positive inotropic effect on isolated cardiomyocytes by activating Akt. This is consistent with the notion
that transgenic overexpression of Akt enhances myocyte contractility and relaxation through acceleration of intracellular
Ca2+ transients in mice[11]. However, the direct link between Akt activation and improvement of myocyte contraction by
insulin remains largely unclear. In the present study, we found that insulin resulted in a 2.9-fold increase in phosphorylation
of Akt in I/R myocytes, which was inhibited by pretreatment of the cells with a specific AI. Most importantly, inhibition of Akt
activity with AI also significantly abolished increased SERCA activity,
Ca2+ transients and contractile activity by insulin
simultaneously in I/R myocytes. These data suggest that Akt is probably involved in the effects of insulin on SERCA2a,
intracellular Ca2+ handling and subsequent contractile function in I/R cardiomyocytes.
In summary, the present study demonstrated that insulin improves the recovery of contractile function in simulated I/R
cardiomyocytes in an Akt-dependent and SERCA2a-mediated fashion. This finding suggests that the insulin-activated Akt
survival signaling not only contributes to the previously observed cardiac protective effects, but also plays a causative role
in the direct inotropic action induced by insulin in I/R myocardium.
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