Copyright © 2005, European Society of Cardiology
Spontaneous diastolic contractions and phosphorylation of the cardiac ryanodine receptor at serine-2808 in congestive heart failure in rat
aDepartment of Medical Bio-regulation, Division of Cardiovascular Medicine, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan
bDepartments of Medicine, Physiology and Biophysics, Health Sciences Center, University of Calgary, Calgary, Alberta, Canada
* Corresponding author. Department of Physiology/Biophysics, University of Calgary, 3330 Hospital Drive, NW, Calgary, Alberta, Canada, T2N 4N1. Tel.: +1 403 289 7156; fax: +1 403 270 0313. Email address: terkeurs{at}ucalgary.ca
Received 18 February 2005; revised 7 July 2005; accepted 11 July 2005
 |
Abstract |
|---|
 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
|---|
Objective: The role of phosphorylation of the ryanodine receptor at serine-2808 (RyRS2808) in congestive heart failure (CHF) is controversial, and effects of RyRS2808 phosphorylation on contraction are unclear. It has been reported that diastolic sarcomere length (SL) fluctuations accompany propagating contractile waves due to propagating SR Ca2+ release in trabeculae from rats with CHF. Here, we studied the influence of RyR destabilization by FK506 and isoproterenol on twitch force (Ftw) and SL fluctuations in right ventricular (RV) trabeculae. We measured phosphorylation of RyRS2808 in rats with myocardial infarction (MI) with or without β-blockade and in rats during isoproterenol stimulation in order to assess the role of RyRS2808 phosphorylation in SL fluctuations in failing hearts.
Methods: Five groups of male Lewis Brown–Norway rats were studied 3 months after MI: i) Sham; ii) MI with CHF (cMI); iii) MI without CHF; iv) metoprolol-treated MI, with and without CHF. The root mean square (RMSSL) of SL fluctuations in RV trabeculae was calculated.
Results: RMSSL increased strongly both following a short train of stimuli at 2.5 Hz and following catecholamine activation in trabeculae from MI with CHF, resulting in a decrease in Ftw in proportion to RMSSL. RyRS2808 phosphorylation was increased significantly in the left ventricle (LV; 
58%, P<0.05) but not in the RV (n.s.) in MI rats with CHF. FK506 tripled high frequency stimulation-induced RMSSL in nonfailing trabecula but did not further enhance RMSSL in failing trabecula. Isoproterenol increased RMSSL in nonfailing trabeculae only modestly despite a substantial increase in RyRS2808 phosphorylation in the RV (60%, P<0.05). Isoproterenol induced SL fluctuation without an increase in RV-RyRS2808 phosphorylation in failing trabeculae. Chronic β-blockade decreased high frequency and catecholamine stimulation-induced RMSSL while RyRS2808 phosphorylation in the RV was indistinguishable from that in cMI.
Conclusions: Acute RyRS2808 phosphorylation by itself does not cause spontaneous contractile waves owing to RyR2 destabilization. Spontaneous contractile waves in CHF are not caused by RyRS2808 phosphorylation alone, suggesting that factors other than RyRS2808 phosphorylation affect RyR function.
KEYWORDS Ca2+; Contractile function; E–C coupling; Heart failure; Infarction
|
1. Introduction |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
|---|
Myocardial infarction (MI) leads to ventricular dilatation and dysfunction and ultimately to congestive heart failure (CHF). There is considerable evidence that failure of Ca2+ transport in myocytes is a central cause of contractile dysfunction in CHF. A small amount of Ca2+ enters through L-type Ca2+ channel during action potential in normal cardiac muscle and triggers release of large amount of Ca2+ from the sarcoplasmic reticulum (SR) through cardiac ryanodine receptors (RyR2) causing contraction. Spontaneous SR Ca2+ release during the diastolic interval is minimal in right ventricular trabeculae from normal rat and occurs only in the form of Ca2+ sparks without inducing microscopically visible contractile activity [1]. In contrast, noticeable fluctuations of diastolic sarcomere length (SL) accompany propagating contractile waves due to propagating SR Ca2+ release in cells of trabeculae from rat with CHF [2,3]. These SL fluctuations reduce force of the heartbeat and may cause arrhythmias [2]. The underlying spontaneous SR Ca2+ release is dependent on the SR Ca2+ content [4] and the threshold for opening of the RyR2 in response to SR luminal Ca2+ [5] and contributes to a reduction in SR Ca2+ content [6], yet the influence of SL fluctuations on force of cardiac contraction is unclear.
Activation of β1-adrenergic system persists in severe chronic CHF. CHF in dog and human heart is thought to be accompanied by increased phosphorylation of RyR2 by protein kinase A (PKA) causing FK506-binding protein (FKBP)12.6 to dissociate from RyR2, which increases the open probability of RyR2 [7]. It is well known that β-blockers are important tools in the treatment of chronic CHF and it has been reported that beneficial effect of β-blockers is related to normalized PKA phosphorylation of RyR2 at serine-2808 (rat-RyRS2808, corresponding to rabbit-RyRS2809), and a decrease of spontaneous SR Ca2+ release in severe CHF [8]. However, the question whether RyRS2808 is excessively phosphorylated in CHF is debated [8,9]. In addition, the effect of increased phosphorylation of RyRS2808 (RyRS2808 phosphorylation) by PKA on dissociation of FKBP12.6 from RyR2 is controversial [7,8,10], and it has been reported that RyRS2808 phosphorylation by itself does not change the RyR2 activity [11].
In this study we hypothesize that the observed SR Ca2+ leak [6–8] occurs in intact cardiac muscle in the form of spontaneous propagating Ca2+ release accompanied by contractile waves which are reflected by SL fluctuations and reduce force of the next contraction [2]. Hence, we studied the effect of SL fluctuations on force development in RV trabeculae from rats after coronary artery ligation leading to MI with CHF (cMI) and without CHF (uMI). We explored the role of RyR2 destabilization, induced by the immunosuppressant FK506 (tacrolimus) and by isoproterenol, in SL fluctuations and force development in trabeculae. We measured RyRS2808 phosphorylation in uMI and cMI with or without β-blockade as well as during isoproterenol stimulation and assessed the role of RyRS2808 phosphorylation in SR Ca2+ release in CHF.
|
2. Methods |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
|---|
2.1 Coronary artery ligation
MI was induced in male Lewis Brown–Norway, F1 rats (250–320 g body weight; Harlan, Indianapolis, IN, USA) [2]. The rats were anesthetized with 40 mg/kg BW of sodium pentobarbital (i.p.), artificially ventilated and after left thoracotomy, the main left coronary artery was ligated using a 6–0 nylon suture. Age-matched sham rats were prepared similarly without coronary artery ligation. The animals received analgesia for 48 hours using 2 mg/kg BW of Butorphanol Tartrate (Wyeth Animal health, Guelph, ON, Canada) (s.c.) and 50 mg/kg/day of Codeine Phosphate (Ratiopharm Inc., Canada) in their drinking water. The procedures were approved by the Animal Care Committee of the University of Calgary and complied with The Guide for the Care and Use of Laboratory Animals (NIH Publication no. 85-23, revised 1996).
We have previously shown that MI rats with lung wet weight/body weight (LW/BW)>[(mean value of LW/BW in sham)+(2 S.D.)] exhibit signs of CHF [2]. The division according to LW/BW leaves no overlap between rats with CHF and those without CHF. Hence, we divided the rats into: MI rats with CHF (cMI) and those without evidence of CHF (uMI). MI rats treated with the β-blocker, metoprolol (βB), were divided into βB-cMI and βB-uMI.
2.2 Echocardiography
Animals were anesthetized with sodium pentobarbital (30 mg/kg BW; i.p.) and echocardiography (12-MHz transducer; Agilent SONOS 5500, Agilent Technologies, Andover, MA, USA) was performed 2, 6 weeks and 3 months after the surgery. Left ventricular (LV) end-diastolic dimension (LVEDD), LV end-diastolic area (LVEDA), and end-systolic area (LVESA) were measured at papillary muscle level. LV-contraction ([LVEDA–LVESA]/LVEDA x 100%), was used as index of LV systolic function.
Diameter of the tricuspid valve annulus (TVD) was measured at aortic valve level in short axis view and used as an index of RV dilation (Fig. 1). Transverse scar length was estimated by planimetric measurement of the LV endocardium that demonstrated a thinned wall and systolic akinesis in the LV short axis view. Animals were excluded after 2 weeks if the echocardiogram revealed a scar of <40% of the LV circumference.
|
significant difference vs. uMI; 
significant difference vs. cMI.Central venous pressure (PRA) and RV systolic pressure (PRV) were measured using a polyethylene catheter (PE50) to estimate RV hemodynamics 3 months after surgery.
The time course of change in heart rate after isoproterenol (200 µg/kg BW i.v.) was measured in control rats as well as the maximal response of heart rate and LV-contraction to isoproterenol (0.01, 0.1, 1, 10, 50, and 200 µg./kg BW i.v.). The response of heart rate of cMI to isoproterenol (10 µg/kg BW i.v.) was also measured.
2.3 β-blocker treatment
Twenty-eight rats were divided into 2 groups and monitored for 3 months. Group 1 received 1g/l metoprolol in their drinking water immediately after recovery from anesthesia. Group 2 received drinking water without metoprolol. There was no difference in water consumption between the groups. We chose 1 g/l metoprolol as the dose proved to inhibit the effect of isoproterenol on LV-contraction by 39% (23.6 ± 4.9% in control (n=5) vs. 14.5 ± 1.0 in control with metoprolol (n=5) 10 min after 1 µg i.v./kg BW; P<0.05).
2.3.1 RV trabeculae
Rats were sacrificed 3 months after surgery. Trabeculae from the RV were isolated and Scar, noninfarcted LV, RV and left atrium appendage were dissected and weighed. The scar area was determined by planimetry. The lungs were removed, blotted, and weighed immediately; then, the lungs were dried and weighed dry.
RV trabeculae were mounted in a perfused chamber on an inverted microscope as described previously [2] and stimulated (2–2.5 ms pulses 1.5 x threshold) at 0.5 Hz. The muscle chamber was perfused at 8 ml/min with a buffer solution with the following composition (in mmol/l): NaCl 137.2, KCl 5, MgCl2 1.2, acetate 2.8, HEPES 10.0, glucose 10, taurine 10, extracellular Ca2+ concentration ([Ca2+]o)=0.4 mmol/l, 26.5 °C; pH 7.4, and equilibrated with O2 for 
40 min.
Width of the trabeculae (in mm) was: 0.24 ± 0.06, 0.24 ± 0.10, 0.27 ± 0.09, 0.30 ± 0.10 and 0.33 ± 0.14, in Sham, uMI, βB-uMI, βB-cMI, and cMI, respectively; thickness (in mm) was: 0.12 ± 0.03, 0.11 ± 0.02, 0.11 ± 0.01, 0.14 ± 0.04 and 0.18 ± 0.05, in Sham, uMI, βB-uMI, βB-cMI and cMI, respectively. Trabeculae from cMI were thicker than from Sham and uMI but there was no significant difference between cMI and βB-cMI.
2.3.2 Force and SL measurements
Force was measured using a strain gauge (AE 801, Sensor One Technologies Corp, Sausalito, CA, USA). Twitch force (Ftw) was normalized to cross-sectional area. Ftw was measured at SL=2.10 ± 0.02 µm. SL was measured by laser diffraction as described previously [2,12]. A region of the muscle with minimal translation during contraction was selected for SL measurement and was monitored using a CCD camera (SONY SSC-M370, SONY Co., Japan). The position of the median of the 1st order of the diffraction pattern was converted electronically to a voltage proportional to SL which was calibrated using test gratings. The resolution of SL in quiescent muscle was 2 nm [13]. Ftw and SL were sampled at 1 kHz by a 12-bit A/D board (National Instruments Corporation, Austin, TX, USA).
2.3.3 Frequency potentiation
Stimulation at 2.5 Hz (30 stimuli) led to twitch potentiation (F1, F2, etc.) upon return to stimulation at 0.5 Hz (Fig. 2). F1 after a 2.5-Hz train equaled maximal developed Ftw (Fmax) at optimal [Ca2+]o [12]; hence, we measured Fmax using frequency potentiation. Ftw of potentiated beats returned exponentially to the steady state, obeying:
|
|
|
where Fn represents Ftw of the nth contraction after frequency potentiation and Fn+1 that of the subsequent beat (Fig. 3A(a)). D has been interpreted to reflect the fraction of Ca2+ re-circulating to the SR [12].
|
2.3.4 Analysis of diastolic SL fluctuations
We have described that SL fluctuations occur together with microscopically visible contractile waves (lasting
300 ms and propagating at 100 µm/s) in cells of the trabeculae at elevated [Ca2+]o from rats with CHF [2]. The waves caused multiple sub-peaks in the 1st order of the diffraction pattern, which were converted to fluctuations of the median of the SL distribution. We calculated the root mean square value of SL fluctuations (RMSSL) around the average SL (SLmean): |
|
RMSSL (in nm) correlated with the incidence of contractile waves (i.e. 3 nm/(% of cells that generate waves/sec)) [2].
When, in control experiments, the bath temperature was raised to 35 °C, Ftw in cMI decreased as has been shown before (by 34% at 0.5 Hz [14]) and RMSSL was at the detection limit of the system (1.3 nm). RMSSL increased substantially in cMI during the first twitches (0.5 Hz) after frequency potentiation at 35°C and significantly more than in uMI (6.97 ± 3.54 nm (cMI n=4) vs. 1.70 ± 0.27 (uMI n=3), P<0.05). Raising [Ca2+]o at 35 °C to 1.25 mmol/l also increased RMSSL substantially in cMI at 0.5 Hz but not in uMI (6.67 ± 2.81 nm (cMI n=4) vs. 1.83 ± 0.12 (uMI n=3), P<0.05). These data show that the phenomenon of spontaneous contractile activity occurs both at body temperature and at 26.5 °C. Trabeculae are stable for a longer time at 26.5 °C; hence, we explored the effect of RyR2 destabilization, induced by FK506 and isoproterenol, on RMSSL at 26.5 °C. Furthermore, trabeculae from cMI are more sensitive [Ca2+]o than normal trabeculae [2] and SL fluctuations lead to muscle deterioration [2,13]; hence we performed these studies at [Ca2+]o=0.4 mmol/l.
2.4 Effect of FK506 and isoproterenol on Ftw and SL fluctuations
After frequency potentiation, FK506 (5 µmol/l; generously provided by Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan) was added to 100 ml of circulating solution; Ftw and RMSSL at 0.5 Hz and after frequency potentiation were measured 60 min later. We also measured the response of Ftw and RMSSL in trabeculae at 0.5 Hz at 8–10 min after exposure of isoproterenol (100 nmol/l) using 35 rats. Finally, we tested the effects of isoproterenol on 4 hearts in vivo and on 4 hearts in vitro in a standard Langendorff setup.
2.5 Phosphorylation of RyRS2808
Phosphorylation of RyRS2808 was measured in triplicate in homogenates of the LV and RV from uMI and cMI, as well as in RV homogenates from in vivo rat hearts after i.v. injection of isoproterenol (1, 10, and 200 µg/kg BW) and in Langendorff-perfused hearts exposed to isoproterenol (100 nmol/l; at [Ca2+]o=0.4 mmol/l) for 10 min. Furthermore, RyRS2808 phosphorylation was measured in RV homogenates from cMI at 6 min after i.v. injection of isoproterenol (10 µg/kg BW).
The ventricles were crushed by an aluminum clamp precooled in liquid N2 and stored at –80 °C. Frozen samples were pulverized in liquid N2 and homogenized immediately (Brinkmann Polytron PT 15; setting 8; 4 x 15 s) in 6 volumes of buffer ((in mmol/l): 30 KH2PO4 (pH 7.0), 40 NaF, 5 EDTA, 300 sucrose, and protease inhibitors). Aliquots of homogenates were solubilized in 50 mmol/l Tris–HCl, pH 7.5, and 3% SDS for 1 h at room temperature, and insoluble materials were removed by centrifugation (16,000 x g for 10 min). Equal portions of the supernatant were subjected to 6% SDS–PAGE; then, the resolved proteins were transferred to nitrocellulose membranes in the presence of 0.01% SDS (45 V for 18–20 h at 4 °C). The membranes were blocked with PBS containing 0.5% Tween-20 and 5% skim milk powder for 30 min. Two parallel membranes were loaded with equal amounts of protein. One membrane was, then, incubated for 3 hours at room temperature with anti-RyR2 antibody (1:1000; a polyclonal rabbit antibody kindly provided by Dr. Anthony Lai at Cardiff University, UK); the other membrane was incubated simultaneously with anti-S2808(PO3) antibody (1:5000) [9,10].
Alternatively, a membrane was first probed with anti-RyR2 antibody (1:1000) and, then, stripped of RyR2 antibody by incubation in a buffer containing 50 mM Tris–HCl, 2% SDS, and 0.1 M β-mercapto-ethanol (pH 6.8, 55 °C, 30 min). After the membrane had been washed with PBS (3 x; three times for 5 min each) and re-probed with the anti-S2808(PO3) antibody (1:5000), it was washed (15 min x 3) with PBS containing 0.5% Tween-20 and incubated with secondary goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:20,000; 30 min) and washed again.
RyRS2808 phosphorylation was determined by densitometry of luminescence of the Western blots and expressed as anti-serine-2808 luminescence/anti-RyR2 luminescence (S2808P/RyR2).
2.6 Statistics
Results are expressed as mean ± S.D. Comparison between groups was performed by ANOVA followed by Scheffe's post hoc test or Student's unpaired t-test. Intra-group comparison was performed using Student's paired t-test. Linear regression analysis was used to describe the decay of Ftw-potentiation. Differences were considered significant at P<.05.
|
3. Results |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
|---|
3.1 Echocardiography and pathology after coronary artery ligation
cMI was accompanied by a larger scar size and RV hypertrophy compared to uMI (Table 1). RV weight in cMI was twice that of Sham, similar to previous reports [15]. Ventricular dilatation in cMI was echocardiographically significantly more severe than in uMI at 2 weeks and 3 months after surgery (LVEDD: 6.7 ± 0.2, 9.7 ± 0.6, and 11.5 ± 0.4 mm in Sham, uMI, and cMI, respectively; LVEDA: 31.2 ± 2.9, 71.9 ± 7.3, and 95.1 ± 9.4 mm2 in Sham, uMI, and cMI, respectively). The RV was dilated (TVD>5.0 mm) in all cMI. TVD correlated with systolic RV pressure in Sham, uMI and cMI (r2=0.75, P<0.0001, n=14). Hence, we used TVD to diagnose CHF in MI rat in vivo (Fig. 1).
|
3.2 Ftw and SL fluctuations in RV trabeculae in CHF
Trains of 30 stimuli at 2.5 Hz potentiated Ftw after return to 0.5 Hz; the potentiated Ftw decreased exponentially to the steady state Ftw at 0.5 Hz in Sham trabeculae (Fig. 2A), giving rise to a linear relationship between the amplitude of subsequent contractions (Fig. 3A(a) slope=0.83, r2=0.999, P<0.0001) [12,16]. In contrast, the time-course of Ftw after frequency potentiation in cMI trabeculae showed reduced force of the first twitches after return to 0.5 Hz. We calculated the apparent deficit in Ftw (Ftw-deficit) as follows. First, we fitted the decay of force in 4th to 15th twitches after frequency potentiation by Eq. (1) when SL fluctuations had disappeared (slope=0.80, r2=0.999, P<0.0001 in sham trabeculae; slope=0.77, r2=1, P<0.0001 in cMI trabecula). Second, Ftw-deficit was calculated from the difference between Ftw predicted by Eq. (1) and measured Ftw of the first three twitches after frequency potentiation. The Ftw-deficit appeared proportional to RMSSL (slope=1.9% measured Ftw/nm, r2=0.64, P<0.0001, n=148) in Sham, uMI, and cMI (Fig. 3B).
F0.5 Hz and F0.5 Hz/Fmax and half relaxation time (RT50) were increased in cMI compared to sham and uMI (Fig. 4A). Although RMSSL was small (
2 nm) in all groups at 0.5 Hz, RMSSL at 0.5 Hz after frequency potentiation in cMI increased significantly compared to other groups (Fig. 4B).
|
3.3 Effects of FK-506 on Ftw and SL fluctuations
FK506 (5 µmol/l) neither changed Ftw nor increased RMSSL at a stimulus rate of 0.5 Hz. However, FK506 tripled RMSSL after 2.5 Hz stimulation and decreased potentiation of Ftw in Sham and uMI (Table 2). Ftw-deficit was again proportional to RMSSL (slope=2.8% measured Ftw/nm, r2=0.57, P<0.0001 in sham (n=32), slope=4.3, r2=0.75, P<0.0001 in uMI (n=36)). FK506 increased neither the already high RMSSL nor the deficit of Ftw in cMI.
|
3.4 Effects isoproterenol on Ftw, SL fluctuations, and cardiac muscle function
Isoproterenol (100 nmol/l) enhanced RMSSL three-fold in cMI trabeculae stimulated at 0.5 Hz, which contrasted a modest increase observed in all other groups (Fig. 5). Furthermore, the substantial increase in Ftw due to isoproterenol found in all other groups was absent in cMI, although isoproterenol accelerated relaxation in cMI in the same way as in the other groups.
|
Isoproterenol injection in vivo increased the heart rate to a maximum (16.2 ± 3.9%) at 4 min after 200 µg/kg BW i.v. followed by a decrease to near basal level in the next 30 min (17.8 ± 4.2% at 10 min, and 4.2 ± 0.6% at 30 min (Fig. 6)). The responses of functional and biochemical cardiac parameters in vivo and in vitro were nearly identical (Fig. 7). Heart rate and LV-contraction at 4 min increased with the isoproterenol dose and saturated at 10 µg/kg BW (i.v.) (Fig. 7). Maximum acceleration of the heart rate (16.8 ± 3.0%) by isoproterenol (10 µg /kg BW i.v., at 4 min, n=3), in vivo, was similar to the steady state effect of isoproterenol (100 nmol/l for 10 min) on heart rate of spontaneously beating Langendorff perfused hearts (15.6 ± 6.9%, n=4). Importantly, isoproterenol (10 µg/kg BW i.v., at 4 min) increased heart rate in cMI considerably less (36 ± 14 bpm) than in control (69 ± 12 bpm; P<0.05).
|
|
HR, 3.5 Phosphorylation of RyRS2808
Baseline RyRS2808 phosphorylation (S2808P/RyR2) was substantial in the RV taken directly from the rat and similar to that in the RV from hearts that had been perfused with HEPES solution for 20 ± 3 min. S2808P/RyR2 in the LV of cMI, but not of uMI, was increased compared to Sham (P<0.0004). S2808P/RyR2 in the RV of cMI was not significantly higher than in Sham (P=0.15; Fig. 8).
|
Isoproterenol (10 µg/kg BW i.v.), in vivo, increased S2808P/RyR2 substantially (P<0.003) in the RV of control hearts and as much as in the isolated heart (100 nmol/l for 10 min, P<0.02; Fig. 7), but failed to increase S2808P/RyR2 in the RV of cMI (1.06 ± 0.043 (n=3) vs. 1.00 ± 0.089 in drug-free cMI (n=4), P=0.17).
3.6 Effects of chronic β-blockade
Metoprolol did not reduce LV size of cMI (LVEDA (in mm2) 95.1 ± 9.4 in cMI (n=16) vs. 92.6 ± 8.3 in βB-cMI (n=9), P=0.97) but clearly reduced RV size (TVD) and RV hypertrophy observed 3 months after surgery (Fig. 1 and Table 1). Metoprolol also reduced RMSSL and Ftw-deficit after frequency potentiation (Fig. 4) significantly, both in uMI and in cMI. Furthermore, chronic treatment with metoprolol reduced S2808P/RyR2 in the LV of cMI significantly compared to cMI (P<0.04; Fig. 8). S2808P/RyR2 in the RV of cMI with metoprolol remained indistinguishable from Sham-operated animals.
|
4. Discussion |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
|---|
We show here that i) In RV trabeculae from rats, which exhibit all criteria of CHF (cMI), substantial SL fluctuations develop following increased frequency of stimulation and catecholamine activation, resulting in reduced Ftw in proportion to RMSSL. ii) RyRS2808 phosphorylation was significantly increased in the LV but not in the RV of cMI. iii) FK506 increased RMSSL, induced by high frequency stimulation, 3-fold only in nonfailing trabeculae, but iv) isoproterenol at a saturating concentration increased RMSSL only modestly in nonfailing trabeculae in spite of a substantial increase in RyRS2808 phosphorylation in the RV (P<0.05). On the other hand: v) isoproterenol induces SL fluctuations in failing trabeculae without significant increase in RV RyRS2808 phosphorylation. vi) Chronic β-blockade decreased SL fluctuations induced by high frequency stimulation and catecholamines in trabeculae of cMI while RyRS2808 phosphorylation in the RV remained indistinguishable from that in sham-operated animals and cMI.
4.1 Ftw in RV trabeculae in CHF
Coronary artery ligation causing a scar in excess of 35% of the LV surface caused severe LV dilatation, pulmonary congestion, pulmonary hypertension, RV hypertrophy and RV dilation[2]. F0.5 Hz increased and relaxation prolonged in RV trabeculae from the hearts of these cMI animals compared to uMI and Sham, probably due to an increase in [Ca2+]i transient amplitude due to action potential prolongation [17]. These findings are consistent with those observed in severe pressure-induced RVH in a transitional stage to heart failure [18].
4.2 SL fluctuations
We assume that microscopically visible propagating contractile waves, underlying SL fluctuations, reflect the response of sarcomeres to propagating waves of SR Ca2+ release. This assumption is supported by: i) increase of [Ca2+]i in the presence of SL fluctuations [13] and ii) their suppression by ryanodine [2,19]. The assumption predicts that increased SR Ca2+ load–such as due to high frequency stimulation [6]–will bring RyR2 closer to the threshold for spontaneous Ca2+ release [5] and cause SL fluctuations. This happened in fact in cMI trabeculae, but not in uMI or Sham, suggesting that the threshold of the RyR2 for spontaneous Ca2+ release is reduced in CHF. Excessive SL fluctuations were found in cMI at both 26.5 °C and 35 °C, showing that they reflect genuine properties of RV cardiac muscle in CHF.
Spontaneous SR Ca2+ release, underlying SL fluctuations, is expected to render the SR temporarily unable to respond fully to an action potential. The latter would cause non-uniform Ca2+ release and reduced force development by partially–along with fully activated–sarcomeres. Our data show that SL fluctuations (at RMSSL=12 nm) indeed are accompanied by a substantially reduced twitch force (i.e. up to 25% of maximal Ftw), and that this reduction is tightly coupled to the intensity of SL fluctuations immediately preceding the twitch.
Furthermore, recurrent spontaneous SR Ca2+ release is expected to cause a chronic leak of Ca2+ from the SR. We estimated the Ca2+ leak from the SR owing to SL fluctuations in cMI (at RMSSL=12 nm) to be 5%/s of the SR Ca2+ content [2]); this value is similar both to the reported SR Ca2+ leak in rabbit HF myocytes (7–10%/s; [6]) and to Ca2+ leak estimated from isolated canine cardiac SR vesicles in which FKBP12.6 dissociation had been induced by FK506 (2–4% of Ca2+-uptake into SR/s; [20]).
FK506 tripled stimulation-induced SL fluctuations and reduced potentiation of Ftw in nonfailing (Sham and uMI) trabeculae, consistent with reports that FK506 dissociates FKBP12.6 from RyR and causes spontaneous SR Ca2+ release [20,21]. FK506 did not further increase stimulation-induced SL fluctuations in trabeculae from cMI, suggesting that the mechanism causing SL fluctuations in failing rat trabeculae was saturated. Taken together, these observations suggest that CHF induces instability of the RyR2 SR Ca2+ release channels causing SL fluctuations that lead to reduced twitch force development.
4.3 Phosphorylation of serine-2808 – the effect of isoproterenol
Studies in failing dog heart and in explanted human failing heart have suggested that RyRS2808 phosphorylation is increased in CHF [7,8]. In the present study, RyRS2808 phosphorylation increased significantly in the LV but not in the LV of uMI suggesting that LV RyRS2808 phosphorylation depends on the extent of ventricular remodeling. However, RyRS2808 phosphorylation was not increased in the RV of MI rats with CHF nor in uMI.
We studied the functional effects of adrenergic stimulation in RyRS2808 phosphorylation further by exposing intact anesthetized animals and both isolated heart and trabeculae to isoproterenol. In vivo i.v. injection of isoproterenol increased heart rate and LV contraction in a dose-dependent manner. Using the volume of distribution and half-life of isoproterenol in vivo (216 ml/kg BW and 4.2 min, respectively; [22]), we converted the isoproterenol dose to the concentration of isoproterenol in the extracellular space ([isoproterenol]). The EC50 of isoproterenol in vivo appeared to be near identical to the EC50 of [isoproterenol] for force in trabeculae (Fig. 7; [23]). Furthermore the maximal increase of heart rate after isoproterenol in vivo was the same as that in Langendorff heart perfused with a saturating [isoproterenol], while the maximal increase of LV contraction after isoproterenol in vivo was similar to that of force in trabeculae in vitro (Fig. 7) [23]. Fig. 7 also shows that RyRS2808 phosphorylation increases steeply with isoproterenol at a dose that is near the EC50 for the functional parameters and shows that saturation occurs both in vivo and in vitro at 100 nmol/l. These data suggest that the cardiac response to isoproterenol in vivo and in vitro are similar and suggest that RyRS2808 phosphorylation contributes to the observed functional changes.
Isoproterenol accelerated relaxation and induced SL fluctuations in all groups. However, isoproterenol induced threefold larger SL fluctuations in cMI than in the other groups and, unlike uMI and Sham muscles, no increase of Ftw was detected in cMI trabeculae. The modest increase of SL fluctuations induced by 100 nmol/l isoproterenol in nonfailing trabeculae is consistent with the report that increase of the amplitude of Ca2+ sparks by PKA is due to increase in SR Ca2+ content rather than destabilization of RyR2 [24]. This observation suggests that acute RyRS2808 phosphorylation does not destabilize RyR2 enough to induce spontaneous Ca2+ waves, consistent with the assumption that complete RyRS2808 phosphorylation by PKA does not dissociate FKBP12.6 from normal RyR2 [9].
The response to isoproterenol (10 µg/kg BW i.v.) on heart rate in cMI in this study was substantially reduced compared to control, consistent with desensitization of the β1-adrenergic signaling pathway in cMI owing to chronic activation of β1-adrenergic system in CHF [25]. This dose of isoproterenol induced SL fluctuations without a significant increase in RyRS2808 phosphorylation in RV of cMI. Acceleration of Ca2+ uptake stimulated by isoproterenol may explain this finding because the resultant increase of the SR Ca2+ content may have exceeded the threshold for opening of the RyR2 in cMI. On the other hand, it cannot be ruled out that chronic CHF has altered a property of the SR Ca2+ release channel in the RV other than by RyRS2808 phosphorylation such that it has become vulnerable to adrenergic drive. One possible–untested–alternative mechanism could be nitric oxide (NO) mediated; e.g. increased S-nitrosylation of thiol-groups on RyR2 may have contributed to RyR2 destabilization and spontaneous SR Ca2+ release [26] in CHF [27].
4.4 The effects of chronic β-blockade
Chronic β-blockade suppressed the increase of RyRS2808 phosphorylation in the LV of cMI corroborating the assumption that RyRS2808 phosphorylation is mediated by chronic adrenergic effects on the heart in CHF. Chronic β-blockade clearly reduced SL fluctuations induced by high frequency stimulation and exposure of isoproterenol. However, the lack of a significant increase of RyRS2808 phosphorylation in the RV suggests that other factors are involved in the development of SL fluctuations in CHF.
4.5 Conclusions
SL fluctuations are increased following stimulation at high stimulus rate and during catecholamine stimulation in CHF and are accompanied by a decrease in Ftw in proportion to the degree of SL fluctuations measured by the RMSSL. Our data suggest that spontaneous SR Ca2+ release causes the inability of failing cardiac muscle to increase its force in response to increased heart rate and sympathetic stimulation. These data also show that acute RyRS2808 phosphorylation by itself does not cause spontaneous SR Ca2+ release owing to RyR2 destabilization. The lack of a significant increase of RyRS2808 phosphorylation in the RV in chronic CHF makes it unlikely that RyR2 destabilization is caused by RyRS2808 phosphorylation alone. Further studies are required to reveal the other factors that are involved in RyR2 destabilization in chronic CHF.
|
Acknowledgments |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
|---|
This study was supported by grants to H.E.D.J. ter Keurs and S.R.W. Chen from CIHR. H.E.D.J. ter Keurs is supported as AHFMR Medical Scientist. S.R.W. Chen is a senior scholar of AHFMR.
|
Notes |
|---|
Time for primary review 29 days
|
References |
|---|
|
TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
|---|
- Wier W.G., ter Keurs H.E., Marban E., Gao W.D., Balke C.W. Ca2+ sparks and waves in intact ventricular muscle resolved by confocal imaging. Circ Res (1997) 81:462–469.
[Abstract/Free Full Text] - Davidoff A.W., Boyden P.A., Schwartz K., Michel J.B., Zhang Y.M., Obayashi M., et al. Congestive heart failure after myocardial infarction in the rat: cardiac force and spontaneous sarcomere activity. Ann NY Acad Sci (2004) 1015:84–95.[CrossRef][Web of Science][Medline]
- Miura M., Boyden P.A., ter Keurs H. Ca2+ waves during triggered propagated contractions in intact trabeculae: determinants of the velocity of propagation. Circ Res (1999) 84:1459–1468.
[Abstract/Free Full Text] - Diaz M.E., Trafford A.W., O'Neill S.C., Eisner D.A. Measurement of sarcoplasmic reticulum Ca2+ content and sarcolemmal Ca2+ fluxes in isolated rat ventricular myocytes during spontaneous Ca2+ release. J Physiol (1997) 501:3–16.
[Abstract/Free Full Text] - Jiang D., Xiao B., Yang D., Wang R., Choi P., Zhang L., et al. RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR). Proc Natl Acad Sci U S A (2004) 101:13062–13067.
[Abstract/Free Full Text] - Shannon T.R., Pogwizd S.M., Bers D.M. Elevated sarcoplasmic reticulum Ca2+ leak in intact ventricular myocytes from rabbits in heart failure. Circ Res (2003) 93:592–594.
[Abstract/Free Full Text] - Marx S.O., Reiken S., Hisamatsu Y., Jayaraman T., Burkhoff D., Rosemblit N., et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell (2000) 101:365–376.[CrossRef][Web of Science][Medline]
- Reiken S., Wehrens X.H.T., John V.A., Barbone A., Klotz S., Mancini D., et al. β-blockers restore calcium release channel function and improve cardiac muscle performance in human heart failure. Circulation (2003) 107:2459–2466.
[Abstract/Free Full Text] - Xiao B., Jiang M.T., Zhao M., Yang D., Sutherland S., Lai A., et al. Characterization of a novel protein kinase A phosphorylation site, serine-2030, reveals no hyperphosphorylation of the cardiac ryanodine receptor in the heart failure. Circ Res (2005) 96:847–855.
[Abstract/Free Full Text] - Xiao B., Sutherland C., Walsh M.P., Chen S.R. Protein kinase A phosphorylation at serine-2808 of the cardiac Ca2+ release channel (ryanodine receptor) does not dissociate 12.6-kDa FK 506-binding protein (FKBP12.6). Circ Res (2004) 94:487–495.
[Abstract/Free Full Text] - Stange M., Xu L., Balshaw D., Yamaguchi N., Meissner G. Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor. J Biol Chem (2003) 278:51693–51702.
[Abstract/Free Full Text] - Banijamali H.S., Gao W.D., Macintosh B.R., ter Keurs H.E.D.J. Force-interval relation of twitches and cold contractures in rat cardiac trabeculae: effect of ryanodine. Circ Res (1991) 69:937–948.
[Abstract/Free Full Text] - Stuyvers B.D.M., Miura M., ter Keurs H.E.D.J. Dynamics of viscoelastic properties of rat cardiac sarcomeres during the diastolic interval: involvement of Ca2+. J Physiol (1997) 502:661–677.
[Abstract/Free Full Text] - de Tombe P., ter Keurs H.E. Force and velocity of sarcomere shortening in trabecula from rat heart; effects of temperatures. Circ Res (1990) 66:1239–1254.
[Abstract/Free Full Text] - Fletcher P.J., Pfeffer J.M., Pfeffer M.A., Braunwald E. Left ventricular diastolic pressure–volume relations in rats with healed myocardial infarction. Circ Res (1981) 49:618–626.
[Abstract/Free Full Text] - ter Keurs H.E. Post-extrasystolic potentiation and its decay. The interval–force relationship: Bowditch revisited. (1992) Cambridge University Press.
- Kaprielian R., Wickenden A.D., Kassiri Z., Parker T.G., Liu P., Backx P.H. Relationship between K+ channel down-regulation and [Ca]i in rat ventricular myocytes following myocardial infarction. J Physiol (1999) 517:229–245.
[Abstract/Free Full Text] - Kogler H., Hartmann O., Leineweber K., Nguyen P., Schott P., Brodde O.E., et al. Mechanical load-dependent regulation of gene expression in monocrotane-induced right ventricular hypertrophy in the rat. Circ Res (2003) 93:230–237.
[Abstract/Free Full Text] - A. Davidoff, Cardiac contraction in failing rat heart. PhD thesis. University of Calgary 2002; Chap. 4–5:55–51.
- Yano M., Kobayashi S., Kohno M., Doi M., Tokuhisa T., Okuda S., et al. FKBP12.6-mediated stabilization of calcium release channel (ryanodine) as a novel therapeutic strategy against heart failure. Circulation (2003) 107:477–484.
[Abstract/Free Full Text] - Gomez A.M., Schuster I., Fauconnier J., Prestle J., Hasenfuss G., Richard S. FKBP12.6 overexpression decreased Ca2+ sparks amplitude but enhances [Ca2+]i transient in rat cardiac myocytes. Am J Physiol (2004) 287:H1987–H1993.[Web of Science]
- Reyes G., Schwartz P.H., Newth C.J.L., Eldadah M.K. The pharmacokinetics of isoproterenol in critically ill pediatric patients. J Clin Pharmacol (1993) 33:29–34.[Abstract]
- de Tombe P., ter Keurs H.E.D.J. Lack of effect of isoproterenol and unloaded velocity of sarcomere shortening in rat cardiac trabeculae. Circ Res (1991) 68:382–391.
[Abstract/Free Full Text] - Li Y., Kranias E.G., Mignery G.A., Bers D.M. Protein kinase A phosphorylation of the ryanodine receptor does not affect calcium sparks in mouse ventricular myocytes. Circ Res (2002) 90:309–316.
[Abstract/Free Full Text] - Kompa A.R., Gu X.H., Evans B.A., Summers R.J. Desensitization of cardiac β-adrenoceptor signaling with heart failure produced by myocardial infarction in the rat. Evidence for the role of Gi but not Gs or phosphorylating proteins. J Mol Cell Cardiol (1999) 31:1185–1201.[CrossRef][Web of Science][Medline]
- Xu L., Eu J.P., Meissner G., Stamler J.S. Activation of the cardiac calcium release channel (ryanodine receptor) by Poly-S-Nitrosylation. Science (1998) 279:234–237.
[Abstract/Free Full Text] - Hare J.M., Givertz M.M., Creager M.A., Colucci W.S. Increased sensitivity to nitric oxide synthase inhibition in patients with heart failure: potentiation of β-adrenergic inotropic responsiveness. Circulation (1998) 97:161–166.
[Abstract/Free Full Text]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  P. A. Boyden The failing ventricle: what initiates the complex ventricular arrhythmias? Am J Physiol Heart Circ Physiol, October 1, 2009; 297(4): H1198 - H1199. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lehnart and A. R. Marks Regulation of Ryanodine Receptors in the Heart Circ. Res., October 12, 2007; 101(8): 746 - 749. [Full Text] [PDF]
|
|
|
|
|
|
Y. Chen-Izu, C. W. Ward, W. Stark Jr., T. Banyasz, M. P. Sumandea, C. W. Balke, L. T. Izu, and X. H. T. Wehrens Phosphorylation of RyR2 and shortening of RyR2 cluster spacing in spontaneously hypertensive rat with heart failure Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2409 - H2417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. E. D. J. ter Keurs and P. A. Boyden Calcium and Arrhythmogenesis Physiol Rev, April 1, 2007; 87(2): 457 - 506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Belin, M. P. Sumandea, T. Kobayashi, L. A. Walker, V. L. Rundell, D. Urboniene, M. Yuzhakova, S. H. Ruch, D. L. Geenen, R. J. Solaro, et al. Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2344 - H2353. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||