Reduced Ryanodine Receptor to Dihydropyridine Receptor Ratio May Underlie Slowed Contraction in a Rabbit Model of Left Ventricular Cardiac Hypertrophy

J Mol Cell Cardiol 33, 473–485 (2001) doi:10.1006/jmcc.2000.1320, available online at http://www.idealibrary.com on

Reduced Ryanodine Receptor to Dihydropyridine Receptor Ratio May Underlie Slowed Contraction in a Rabbit Model of Left Ventricular Cardiac Hypertrophy James T. Milnes and Kenneth T. MacLeod∗ Dept. Cardiac Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse Street, London, SW3 6LY (Received 21 August 2000, accepted in revised form 8 December 2000, published electronically 18 January 2001) J. T. M  K. T. ML. Reduced Ryanodine Receptor to Dihydropyridine Receptor Ratio May Underlie Slowed Contraction in a Rabbit Model of Left Ventricular Cardiac Hypertrophy. Journal of Molecular and Cellular Cardiology (2001) 33, 473–485. Cardiac hypertrophy is associated with contractile dysfunction, a feature of which is a slowing of the time to reach peak contraction. We have examined the main mechanisms involved in the initiation of contraction and investigated if their functions are changed during cardiac hypertrophy. Cardiac hypertrophy was induced by constriction of the ascending aorta in the rabbit. After 6 weeks left ventricular myocytes were isolated or left ventricular and septal mixed membrane preparations were produced for electrophysiological and radioligand binding studies, respectively. Aortic constriction resulted in a 24% and 23% increase in heart weight to body weight ratio and cell capacitance, respectively. Action potential duration and time-toreach 50% and 90% peak contraction (TTP50 and TTP90, respectively) were significantly prolonged in myocytes from hypertrophied hearts. The prolongation of TTP50 and TTP90 could not be explained by altered peak calcium current density or SR calcium content which were unchanged in hypertrophy. Radioligand binding studies performed on tissue preparations from the same hearts, revealed a 34% reduction in ryanodine receptor (RYR) density with no change in dihydropyridine receptor (DHPR) density. This resulted in a reduction in the ratio of RYR to DHPR from 4.4:1 to 3.3:1 in hypertrophy. Ryanodine receptor Ca2+-sensitivity was unchanged between sham operated and hypertrophied groups. A reduction in the ratio of RYRs to DHPRs may result in a degree of “functional uncoupling” causing defective release of Ca2+ from the SR. These findings may underlie the slowed TTP of myocyte contraction in hypertrophy.  2001 Academic Press K W: Cardiac hypertrophy; Calcium; Contractile function; E–C coupling; Ca channel; Ryanodine receptor; Dihydropyridine receptor; Radioligand binding.

Introduction Myocardial hypertrophy is a compensatory mechanism precipitated by an increased pressure- or volume-workload. It is associated with changes in neural and humoral systems producing altered release of neurotransmitters, endocrine and paracrine

hormones, with direct actions on myocyte growth.1 It also results in decreased active force development, reduced velocity of shortening, increased time-topeak of force development, decreased shortening, decreased rate of shortening and rate of relengthening2,3 of cardiac muscle, but the reasons underlying these contractile changes remain poorly understood.

∗ Please address all correspondence to: Dr Kenneth T. MacLeod. Tel: +44 (0)20 7351 8143; Fax: +44 (0)20 7351 8145; E-mail: [email protected]

0022–2828/01/030473+13 $35.00/0

 2001 Academic Press

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In cardiac muscle, it is widely accepted that contraction is mainly initiated by an action potential depolarisation activating L-type calcium channels or dihydropyridine receptors (DHPRs) causing an influx of Ca2+. This influx elevates the local Ca2+ concentration around the calcium release channels of the junctional sarcoplasmic reticulum (SR) in sufficient amounts to activate them. When a large number of SR calcium release channels or ryanodine receptors (RYRs) are activated, a large amount of Ca2+ is discharged into the cytoplasm of the cell. Together these sources of Ca2+ enter the cytosol and activate contraction.4,5 Efficient coupling of excitation to contraction thus relies on tight functional and spatial coupling of the L-type Ca2+ channels with the SR Ca2+ release channels.6,7 It has been proposed in heart failure that the slowed time-to-peak contraction and reduced Ca2+-transients may be due to a degree of uncoupling between L-type channels and ryanodine receptors and the defect resides in the ability of the ICa to activate Ca2+ release8 and not ICa or RYR density per se. However, it is unknown how excitation–contraction (E–C) coupling is affected in hypertrophy. We have set out to elucidate the changes that occur in the trigger of, and release of, Ca2+ from the sarcoplasmic reticulum in a pressure-overload model of cardiac hypertrophy in the rabbit. We have employed electrophysiological techniques and complemented our functional observations with a parallel radioligand binding study for the DHPR and RYR to investigate changes in hypertrophy at both the cellular and protein level.

and unused portion of the cable tie was removed and the thorax closed using Ethibond Excel 2-0 suture. Muscle layers and skin were reapposed and sutured using Dexon II 2-0 reabsorbable suture. Post-operative analgesia was provided by 0.01–0.05 mg/kg buprenorphine i.m. (Vetgesic, Alstoe Ltd., Melton Mowbray, UK) and/or 3 mg/kg carprofen s.c. (Rimadyl, Pfizer Animal Health, Sandwich, UK) for 48 h. Sham-operated animals underwent the same procedure with the exception of placing a cable tie around the aorta. Animals were kept for 6 weeks and allowed chow and water ad libitum. All surgical procedures were carried out in sterile conditions and adhered to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996) and with the U.K. Animals (Scientific Procedures) Act of 1986. After 6 weeks, animals were heparinized (500 U/kg via the marginal ear vein), killed by an injection of sodium pentobarbitone (400 mg/kg also administered via marginal ear vein; Euthanal, Rhoˆne Me´rieux, Harlow, UK), their hearts excised and placed into icecold normal Tyrode’s solution (NT) containing 2 m Ca2+ (see solutions for composition). Hearts were then used either for cellular or biochemical studies. End stage body, lung, liver, heart and left ventricular weights were recorded.

Isolation of left ventricular myocytes Left ventricular cardiac myocytes were isolated using a modification of methods described in detail elsewhere (Naqvi et al.9).

Materials and Methods

Electrophysiological and contractility studies

Animal pressure-overload model

The experimental set up and cell-length detection system were identical to those published previously.9,10 All experiments were carried out at 20–22°C. Electrophysiological studies were undertaken using an Axoclamp 2B amplifier (Axon Instruments, CA, USA). Sharp, high resistance microelectrodes (18–30 M
) filled with a KCl-based solution (containing in m: KCl, 2000; EGTA, 0.1; HEPES, 5: adjusted to pH 7.2 with 1  KOH) were used to avoid dialysis of the intracellular milieu. Current and voltage-clamp protocols were controlled, and data acquired and analysed using pClamp 6.0.3 software (Axon Instruments). Rapid solution changes were evoked through a system of solenoids driven from pClamp software. Action

New Zealand White rabbits (Charles River, Margate, UK), 2.5–3.0 kg, were subjected to a constriction of the ascending aorta. Animals were anaesthetized with 0.3 ml/kg i.m. Hypnorm (Janssen-Cilag, High Wycombe, UK) and 2.5 mg/kg i.p. Hypnovel (Roche, Welwyn Garden City, UK). Tracheal intubation was carried out and the animals were kept on a positive pressure ventilator for the whole surgical procedure. A left thoracotomy, between the 2nd and 3rd ribs, was performed to allow access to the heart and great vessels. Obtrusive fatty tissue was removed to allow a sterilized cable tie to be placed around the aorta. This was tightened, reducing the diameter of the aorta by approximately one third. The free

Ca Channel Stoichiometry in LV Hypertrophy

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potentials were initiated using 10 ms pulses of current, 50% above threshold applied at 0.5 Hz. Cell capacitance was measured as the integral of the transient capacitive current following a 10 mV hyperpolarizing step from a holding potential of −40 mV. To measure the L-type calcium current (ICa) cells were depolarized with a series of 100 ms pulses from a holding potential of −40 mV to 0 at 0.5 Hz. The peak ICa was taken as the peak inward current sensitive to 2 m Cd2+. The continuous presence of 10
 niflumic acid11 assured that the measurements of ICa was not contaminated by a Ca2+activated Cl− conductance.12 At this concentration, niflumic acid does not affect ICa.13 Sarcoplasmic reticulum Ca2+ content was assessed by integration of the caffeine-induced transient Na+/Ca2+ exchanger current.14,15 To induce this current, myocytes were voltage clamped at their resting membrane potential and the superfusate rapidly changed to NT containing 10 m caffeine via a glass pipette positioned in close proximity to the cell. For comparisons between the two groups currents are normalized to cell capacitance.

ryanodine from the receptor.18 Non-specific binding was assessed using a 1000-fold excess of cold ryanodine (Sigma, Poole, UK). Assays were incubated at 37 °C for 1 h. The reaction was terminated with 5 ml ice-cold basic wash buffer (see solutions for composition), and samples were filtered using a negative pressure sampling manifold (Millipore, Watford, UK) through Whatman GF/B filters presoaked in basic wash buffer containing 2% w/v “Polymin P” (Merck, Poole, UK). Filters were then placed in 10 ml scintillant (Ultima Gold MV, Packard Bioscience, Groningen, Netherlands) and analysed in a Wallac 1219 Betarack counter. Specific binding was determined as mean total binding minus nonspecific binding at each concentration. Non-specific binding was <25–30% at the greatest concentration of [3H] ryanodine. Results were normalized to total protein determined by a Bradford assay. Binding data from each heart were pooled and fitted by nonlinear regression to a single high affinity binding site model using Prism ver. 3.0 (Graphpad software), and the equilibrium dissociation constants (Kd) and maximum binding site densities (Bmax) were calculated.

Mixed membrane preparation for biochemical studies

Ca2+-dependent [3H] ryanodine binding

The atria and right ventricle were cut free and discarded and the left ventricle and septum were placed in ice-cold homogenate buffer containing a protease inhibitor (see solutions for composition). The tissue was diced using scissors then homogenized using an Ultra-Turrax T25 for nine 15 s bursts, at maximum speed. The homogenate was then centrifuged at 1000×g for 15 min at 4°C. The pellet was discarded and the supernatant aliquoted, snap frozen in liquid nitrogen and stored at −80°C until use.

Ca2+-dependent [3H] ryanodine binding was performed to study RYR Ca2+-sensitivity. Solutions containing a range of target sub-maximally activating free [Ca2+] calculated using MaxChelator (see solutions for composition) were made up in bulk, and frozen at −20°C until used, allowing one set of Ca2+ solutions to be used for all assays. Due to the pH-dependence of the apparent Ca2+ association constant of HEDTA, pH was well buffered with 25 m HEPES and adjusted to 7.200±0.005 while concurrent free [Ca2+] measurements were made using a Ca2+-sensitive electrode (Orion Research) at 37.0±0.2°C. Measured free [Ca2+] were used in subsequent calculations as opposed to expected calculated values.

[3H] ryanodine binding Preliminary experiments and the methods and findings of others determined conditions for maximal binding.16–18 Binding studies were conducted in a final volume of 1 ml buffer (see solutions for composition) containing 0.25–20 n [3H] ryanodine (NEN, Hounslow, UK). High affinity ryanodine binding occurs preferentially to the open state of the channel thus conditions were optimized to maximize open channel probability, and hence maximum specific binding. This was achieved by a high ionic strength buffer (>1 ) containing 100
 CaCl2, 1 m ATP, and BSA to slow the dissociation of

[3H] PN200-110 binding Preliminary experiments determined optimum conditions for the assay based on the methods of Stiffel and Bers.17 Assays were conducted in a final volume of 1 ml incubation buffer (see solutions for composition) containing 12–555 p [3H] PN200-110 (Amersham, Little Chalfont, UK) for 1 h at 37°C. Binding was carried out on the same tissue preparations as those used for ryanodine binding to

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allow more quantitative assessment of channel stoichiometry. Non-specific binding was determined by addition of 1000-fold excess of nifedipine. Specific binding was normalized to total protein, and nonspecific binding accounted for <20% of total binding at the greatest concentration of [3H] PN200-110. All solutions containing dihydropyridines were protected from light by wrapping vessels in foil and prepared under red-light illumination with the incubations being conducted in total darkness.

Solutions Normal Tyrode’s (NT) contained (m): NaCl, 140; KCl, 6; MgCl2, 1; CaCl2, 2; glucose, 10; N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid (HEPES), 10: pH adjusted to 7.4 with 2  NaOH. Homogenization buffer contained (m): KCl, 500; HEPES, 25; phenylmethylsulfonyl fluoride (PMSF), 5
: adjusted to pH 7.2 with 1  KOH at 20°C. Ryanodine binding incubation buffer contained (m): KCl, 1000; sucrose, 300; HEPES, 25; adenosine triphosphate disodium salt, 1; bovine serum albumen (BSA), 0.1 mg/ml; CaCl2, 0.1; PMSF, 5
: adjusted to pH 7.2 with 1  KOH at 20°C. Ca2+-dependent ryanodine binding solutions contained (m): KCl, 1000; HEPES, 25; N-hydroxyethylethylenediminetriacetic acid (HEDTA), 1; PMSF, 5
; [pCa 6.3–4.5] or no chelator [pCa 4.3–4.0]; CaCl2, added to give desired free [Ca2+] as calculated using MaxChelator software: adjusted to pH 7.200±0.005 at 37.0±0.2°C with 1  KOH. Basic wash buffer for ryanodine binding contained (m): KCl, 1000; HEPES, 25: adjusted to pH 7.2 with 1  KOH at 20°C. PN200-110 binding incubation buffer contained (m): Tris, 25; Na-HEPES, 10; ethylenediaminetetra-acetic acid disodium salt (EDTA disodium), 1; MgCl2, 1.1; PMSF, 5
: adjusted to pH 7.4 with HCl at 20°C. Basic wash buffer for PN200-110 binding contained (m): Na-HEPES, 10: adjusted to pH 7.4 at 20°C. In radioligand binding experiments PMSF was added from a 200 m stock in methanol. Ryanodine (Sigma) was added from a 1 m stock solution in reagent grade water. Nifedipine (Sigma) was added from a 10 m stock in ethanol. In electrophysiological experiments niflumic acid was made as a 100 m stock in ethanol, stored at −20°C and added fresh on the day of experimentation.

Statistical analysis Data are shown as mean±... where n is number of cells. Exceptions to this are body and organ weights, and radioligand binding studies in which n refers to the number of rabbit hearts. Statistical comparisons were made using a two-tailed Student’s t-test and statistical significance was deemed to be P<0.05. Non-significance is denoted by “n.s.”.

Results Characterization of cardiac hypertrophy At 6 weeks post-operation there was no significant difference between the end body, lung or liver weights and no animals in this study showed overt signs of heart failure such as ascites, cyanosis or breathlessness. Heart weight and left ventricle weight to body weight ratio (HW:BW and LVW: BW, respectively) were used as an index of cardiac hypertrophy and were 24% and 38% greater in the banded group compared with the sham-operated animals (P<0.05) (see Table 1). Cell capacitance was increased by 23% in myocytes isolated from the hearts of banded animals compared with those isolated from sham animals (Table 1). This clearly demonstrates that banding the ascending aorta of the rabbit produces left ventricular cardiac hypertrophy at both the organ and cellular level.

Functional electrophysiological and contraction studies Figure 1A shows representative action potential records from left ventricular myocytes. Action potential durations measured at both 50% and 90% repolarization (APD50 and APD90, respectively) are significantly prolonged in cells isolated from the banded group of animals compared with those isolated from sham animals. APD50 increased from 273±33 ms (n=18) in the sham group to 413±27 ms (n=24) in the hypertrophied group (P<0.05), and APD90 increased from 439±40 ms (n=18) in the sham group to 570±29 ms (n= 24) in the hypertrophied group (P<0.05). This prolongation was not associated with changes in myocyte resting membrane potential (Em) between the two groups [−74±2 mV (n=15) and −73±1 mV (n=24), n.s., in sham and hypertrophy, respectively]. Figure 1B shows the corresponding cell shortening traces normalized to the peak of contraction elicited from the action

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Ca Channel Stoichiometry in LV Hypertrophy Table 1 Model validation

Start BW (kg) End BW (kg)  BW (kg) HW:BW (×100) LVW (g) LVW:BW Liver weight (g) Lung weight (g) Cm (pF)

Sham-operated

Aortic-banded

Statistical significance

2.92±0.09, n=10 3.57±0.15, n=10 0.65±0.12, n=10 0.229±0.01, n=10 4.0±0.3, n=5 1.12±0.04, n=5 103.1±8.3, n=10 12.0±0.7, n=10 130±7, n=32

2.84±0.10, n=14 3.47±0.13, n=14 0.63±0.10, n=14 0.284±0.02, n=14 5.7±0.5, n=6 1.54±0.13, n=6 105.5±6.1, n=13 14.0±2.7, n=13 160±5, n=51

n.s. n.s. n.s. P<0.05 P<0.05 P<0.05 n.s. n.s. P<0.05

Body weight (BW); heart weight (HW); left ventricular and septal weight (LVW); cell capacitance (Cm). Data on left ventricular and septal weight collected from hearts used in radioligand binding studies only.

Figure 1 Electrophysiological and contractile changes in hypertrophy. Panel A shows representative action potential records from isolated left ventricular myocytes superfused with NT containing 2 m Ca2+. Action potentials were elicited in current clamp mode at a frequency of 0.5 Hz. Action potentials have been superimposed and clearly show increased duration in cells isolated from hypertrophied hearts compared with sham hearts. Panel B shows cell shortening traces corresponding to the action potentials in Panel A (note different time base). Cell shortening traces are shown superimposed and normalized to peak contraction. It can clearly be seen that the time-to-reach peak contraction is increased in hypertrophy compared with myocytes isolated from the sham group.

potentials shown in Figure 1A. Time-to-reach 50% and 90% peak shortening (TTP50, TTP90, respectively) are significantly increased in the banded group compared with sham. TTP50 in sham animals was 184±13 ms (n=9) and 240±14 ms (n=20) in cells from the banded group (P<0.05). TTP90 in sham animals was 319±22 (n=9) compared with 419±21 (n=20) in cells from the banded group (P<0.05). It is apparent that this model of cardiac hypertrophy is associated with contractile and electrophysiological changes at the cellular level. We have previously shown that the increased TTP of contraction is associated with a slowing of the TTP of the Ca2+ transient monitored using Indo-1.9 This suggests that this phenomenon results from alterations to intracellular Ca handling and is not a consequence of altered myofilament Ca sensitivity. Several factors may explain the prolongation of the time-to-peak of shortening observed in hypertrophied cells. First, the size of the trigger of CICR (ICa) could be changed; second, the efficacy with which ICa triggers Ca2+ release from the SR may be reduced; or third, the process of Ca2+ release from the SR could be altered. We first investigated if Ca2+ influx via ICa was reduced. Figure 2A and B show representative normalized Ca2+ current traces. Since the cells were not being dialysed with K+ channel blockers, contamination of the current record by outward K+ conductances (mainly the transient outward current, Ito) is likely. We therefore measured ICa as a cadmium-sensitive current. The peak Cd2+-sensitive current density (ICa) was not statistically different between the two groups [2.8±0.3 pA/pF (n=8) in the sham group compared with 2.5±0.1 pA/pF (n=15) in the hypertrophied group (n.s.) (see Fig. 2A and B]. These results suggest that in hypertrophy the size of the trigger of CICR is unchanged.

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Figure 2 The effect of hypertrophy on peak L-type calcium current. Panels A and B show representative calcium current traces recorded from myocytes isolated from sham and hypertrophied hearts, respectively. Voltage-clamped myocytes were held at −40 mV and stepped to 0 for 100 ms at a frequency of 0.5 Hz to steady-state. ICa was measured as a Cd2+-sensitive current. The upper panels show the current record before and after rapid application of NT containing 2 m Cd2+. The lower panel shows the respective Cd2+-sensitive subtraction currents (ICa). Normalization of ICa to cell capacitance revealed no difference between the two groups. Panel C shows saturation binding curves for the dihydropyridine PN200-110 to a left ventricle and septal crude mixed membrane preparation from sham (Ε) and hypertrophied (Μ) hearts. Calculated Bmax and Kd were not significantly different between sham and hypertrophied hearts.

PN200-110 binding assays We employed radioligand binding to estimate the numbers of Ca2+ channels in a cardiac mixed membrane preparation from sham and banded animals. Figure 2C shows pooled data from saturation binding experiments using [3H] PN200-110, a dihydropyridine, to assess the maximum density of dihydropyridine receptors. Saturation binding

curves were best fitted assuming a single binding site in a similar manner to other studies.17,19,20 There was no difference in the maximum number of sites when normalized to total protein (Bmax) between the sham and hypertrophied groups (88.9±6.5 fmol/mg protein, n=5, and 78.5±8.1 fmol/mg protein, n=5, respectively; n.s.). The apparent dissociation constant was also not statistically different between sham and hyper-

Ca Channel Stoichiometry in LV Hypertrophy

Figure 3 [3H] ryanodine binding studies. Panel A shows pooled data from equilibrium [3H] ryanodine binding experiments to a cardiac mixed membrane preparation. Binding experiments were performed on the same tissue samples as those in Figure 2C. Calculated Bmax is reduced by 34% in hypertrophy (Μ) compared with sham (Ε). There was no difference in Kd between the two groups (see Table 2). Panel B shows representative Ca2+-dependent binding curves fitted with a sigmoidal dose–response curve. Calculation of the half maximally activating calcium concentration (pCa50) revealed no difference between hypertrophied (Μ) and sham (Ε) hearts (see Table 2).

trophied groups (134.9±23.9 p, n=5, and 131.0±31.8 p, n=5, respectively). These binding data support the findings of an unchanged peak ICa density.

Ryanodine binding assays The experiments described above suggest that the slow TTP is not caused by a reduced trigger of CICR. Therefore, we examined the site of Ca2+ release from the SR, namely the ryanodine receptor. Figure 3A shows pooled data from a parallel series of [3H] ryanodine binding experiments. Saturation

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binding was performed on the same tissue preparations as used for PN200-110 binding. Data from saturation binding experiments were bestfitted assuming a single binding site. The calculated mean Bmax is reduced by 34% in the banded group compared with sham, with no apparent difference in Kd (see Fig. 3 and Table 2). The protein yield of the mixed membrane preparations used in the binding studies was not statistically different between the two groups (60.8±5.7 mg/g wet weight tissue (n=5) and 55.3±7.6 mg/g wet weight tissue (n=5) in sham and hypertrophy, respectively) and thus cannot provide an explanation for the reduced RYR density. The use of radioligand binding in the same tissue samples enables quantitative assessment of the ratio of RYR to DHPR. The ratio of RYR to DHPR decreases from 4.4:1 to 3.3:1 in hypertrophy. Because ryanodine binding occurs preferentially to the open state channel, it can be used as a conformational and thus functional probe for the ryanodine receptor. The affinity of the channel for calcium was examined more closely. A series of solutions containing a range of free [Ca2+] (pCa 6.55–4.03) were used to study Ca2+-dependent binding. Representative Ca2+-dependent ryanodine binding curves are shown in Figure 3B. Calculation of the free [Ca2+], at which half maximal binding (pCa50) occurred, revealed no statistical difference between the two groups (see Fig. 3B and Table 2). These results suggest that reduced RYR density and not channel Ca2+-sensitivity may be a key factor in slowing release of Ca2+ from the SR and thus slowing of the TTP contraction.

Quantification of SR Ca2+ content SR Ca2+ content has been shown to effect the kinetics of Ca2+ release and is thus a possible factor contributing to slowed TTP of contraction. Myocytes superfused with NT containing 2 m Ca2+ and 10
 niflumic acid were subjected to a conditioning train of 20 pulses at 0.5 Hz in current clamp mode. This enabled loading of the SR to a steady-state using the cells own action potentials. Following repolarization of the 20th action potential, the cell was voltage-clamped at its resting membrane potential and the superfusate was changed to NT containing 10 m caffeine. The integral of the elicited caffeine-induced Na+/Ca2+ exchanger current was used to calculate SR Ca2+ content (Fig. 4). Values were adjusted for accessible cell volume (a.c.v.) using a conversion factor of 4.58 for cell surface area to cell volume in rabbit

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Figure 4 Effect of hypertrophy on SR Ca2+ load. Panels A and B show inward Na+/Ca2+ exchanger currents (upper trace) induced by rapid application of NT containing 10 m caffeine, in voltage-clamped myocytes from sham and hypertrophied hearts, respectively. The integral of the current is shown in the lower trace. Calculated SR Ca2+ load when normalized to cell capacitance was no different between the two groups.

Table 2 Pooled [3H] ryanodine binding data

RYR Bmax (fmol/mg protein) RYR Kd (n) pCa50 Ratio RYR:DHPR

Sham-operated

Aortic-banded

Statistical significance

393.4±17.1, n=5 1.2±0.2, n=5 5.41±0.04, n=5

258.9±20.7, n=5 0.99±0.3, n=5 5.41±0.03, n=5

P<0.05 n.s. n.s.

4.4:1

3.3:1

Maximum RYR density (Bmax); dissociation constant (Kd); half-maximal activating Ca2+ concentration (pCa50).

myocytes21 and mitochondrial occupancy of 25%. Calculated SR content was not statistically different between the sham and hypertrophied groups [28±2
mol/l a.c.v. (n=7) and 23±2
mol/l (n= 15), respectively; n.s.] and these values were not statistically different from measured SR Ca2+ content in control un-operated rabbit cells [23±2
mol/l a.c.v. (n=21)]. The results suggest there are no differences in SR content in sham or hypertrophied hearts and so differences in content

cannot explain altered TTP of contraction in hypertrophy.

Discussion In this study we have investigated some of the possible changes underlying the slowed time-topeak of cardiac myocyte shortening observed in cardiac hypertrophy. We have focused on the two

Ca Channel Stoichiometry in LV Hypertrophy

major proteins involved in the release of Ca2+ from the SR and additional factors that influence Ca2+ release. We demonstrate a reduction in RYR density in hypertrophy and reduced channel stoichiometry between the RYR and DHPR. The increased TTP of contraction in hypertrophy can be attributed, in part, to these observations and not to reduced Ca2+ current or SR Ca2+ content which are not statistically different in hypertrophy. Six weeks of aortic constriction resulted in a 24% increase in HW:BW, a 38% increase in LVW:BW and a 23% increase in cell membrane capacitance indicating left ventricular cardiac hypertrophy at the organ and cellular level. Due to the absence of overt signs of acute heart failure we conclude the model to be one of a compensated stage of cardiac hypertrophy. These findings are similar to those of Naqvi et al.9 who also report a 23% increase in cell capacitance in this model, although a larger increase in HW:BW. Contractile data clearly demonstrate a prolongation in the time to reach both 50% and 90% of peak contraction in hypertrophied myocytes when stimulated to contract using their own action potential. The increase in TTP is associated with an increase in action potential duration at both 50% and 90% repolarization. These findings are similar to those found in other pressure overload models of hypertrophy. Mann et al.22 were the first to show that contractile changes in cardiac hypertrophy are intrinsic to the myocyte itself, and they reported increased TTP of cell shortening. We have demonstrated increased TTP of contraction in a Goldblatt model of hypertrophy in the guinea pig10 and that increased TTP of contraction in this rabbit model of hypertrophy is associated with an increased time-to-peak of the intracellular Ca2+ transient measured using Indo-1.9 Other groups using pressure-overload models, while not measuring TTP of contraction per se, have reported reduced rates of myocyte shortening,2 sarcomere shortening23 and increased time-to-peak of the [Ca2+]i transient.3 Nagata et al.24 found TTP of contraction to be unchanged in a compensated stage of cardiac hypertrophy in Dahl-salt sensitive rats, but prolonged in myocytes from animals that had developed heart failure. Together these observations suggest that the prolongation of TTP of contraction appears to be a consistent finding in both hypertrophy and heart failure,25 although there may be species differences in when these changes become apparent.24 Prolongation of the action potential is a consistently well-documented characteristic of myocytes from models of cardiac hypertrophy26–28 and heart failure.29,30 These demonstrable changes in

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electrophysiological and contractile function in a compensated stage of hypertrophy are not new, but the mechanisms that underlie these changes remain poorly understood.

Ca2+ current in hypertrophy The lack of effect of hypertrophy on peak ICa density provides convincing evidence that the trigger for Ca2+ release is unchanged in hypertrophy. This is consistent with many other studies of ICa in hypertrophy where no change in peak ICa density was seen in pressure-overload induced cardiac hypertrophy in the cat26 or rat.31 This is also the finding in other models of compensated cardiac hypertrophy.8,10 Reports of altered ICa may stem from models of severe hypertrophy or heart failure.28,32 In many of these studies the patch-clamp technique was employed. This technique dialyses the cell, changing the intracellular environment, and is often associated with a time-dependent run down of currents. In this study we have favoured the use of switch-clamping using sharp microelectrodes to prevent dialysis of the intracellular milieu. The intracellular environment is retained and is therefore more physiologically and pathophysiologically relevant than if patch pipettes are used. However, the technique has its limitations. The major difficulty is in the dissection of individual currents. This can be achieved more effectively using wide-bore pipettes and an appropriate dialysate. However, the Cd2+-sensitive subtraction current allows assessment of peak ICa without interference from outward currents. The present findings are consistent with other sharp electrode studies that show an unchanged peak ICa density, in this model,9 the guinea pig10 and in the spontaneously-hypertensive rat.27 Reports by Ryder et al.28 of increased ICa density in guinea pigs following 20 weeks aortic-coarctation may, as stated previously, be a consequence of severe and sustained hypertrophy. Overall, the results suggest that increased TTP of contraction cannot be readily explained by a reduced trigger of CICR.

Radioligand binding [3H] PN200-110 and [3H] ryanodine binding activities were measured in the same tissue preparations to investigate possible changes in numbers of DHPR and RYR, respectively, and to allow quantitative comparisons between the two receptor populations and their stoichiometry. Crude muscle homogenates have been used in preference to purified membrane fractions17,33 due to reports

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of differential membrane extraction between normal and failing hearts34 and differential recovery of sarcolemmal and SR membranes.35 However, there are two disadvantages of using crude muscle homogenate in binding studies. First, a high non-specific binding17 produces a low signal-to-noise ratio. This can be problematic with a low receptor density as in the case of DHPR in cardiac tissue. To address this a high ionic strength homogenate buffer was used to solubilize the contractile proteins and lowspeed centrifugation was employed to remove large cellular elements and debris, while retaining a crude mixed membrane supernatant. In this way nonspecific binding was minimized as was the loss of binding sites through the use of a nominal purification protocol producing an essentially crude preparation. Second, the use of a crude preparation can require longer incubation times of the radioligand leading to increased protein degradation. This was addressed by the use of high protein concentration samples and protease inhibitors. Incubation times were selected considering maximum total binding and sample degradation, and both binding assays were incubated at the same temperature for the same time. The tightness of the data in each group and similar protein yields lend weight to consistency of our preparations.

occurs with the functional channel, not to individual monomers,39 and correlates with open channel probability (Po).16 Unlike PN200-110 binding, ryanodine binding is a functional assay dependent upon both Po and the number of binding sites. Since our conditions are identical and optimized, a reduction in Bmax in hypertrophy implies reduced numbers of receptors. The Ca2+-sensitivity of RYR in both sham and hypertrophied groups was similar, so we conclude our conditions were suitable to stimulate Po maximally in both groups, and thus our Bmax values represent the true maximum number of binding sites. It can be concluded that increased TTP may be attributed to reduced RYR density and may not be explained by reduced sensitivity of the RYR to calcium. However, it should be noted that the measurements of the Ca2+ sensitivity of the RYR were carried out in conditions optimized for the binding assay. The reduction in RYR density may slow or alter the way Ca2+ is released from the SR in hypertrophy. Although there are some exceptions,8,40 these findings are in agreement with other pressure-overload,20,41 and volume-overload3 models of compensated and decompensated hypertrophy,20,42,43 or heart failure.34,35,37 This may be a key factor in compensated hypertrophy and its progression into a decompensated stage.

PN200-110 binding

Calcium channel stoichiometry

Dihydropyridine binding revealed a single population of DHPR with no change in Bmax or Kd. The DHPR is synonymous with the L-type Ca2+ channel and Lew et al.19 provide good evidence that the density of DHPR is almost identical to “functional” Ca2+ channels in the rabbit. The similar receptor density and ligand-receptor complex binding affinity, considered together with the similar peak current density, provide complimentary evidence that DHPRs are functional L-type channels and are unchanged in hypertrophy. This is in agreement with the work of a number of other groups working with models of cardiac hypertrophy20,36 and heart failure,35,37 and in human heart failure.38

It must be considered that results demonstrating reduced RYR density, when normalized to total protein, could be artifactual due to an increase in myocyte and non-myocyte derived protein. This would cause an increase in total protein but RYR numbers may remain unchanged. However, protein yield from the two groups was not significantly different and this trend was not mirrored when DHPR density was normalized to total protein. The calculation of the channel ratio eliminates total protein as the common denominator. The ratio of RYR to DHPR in sham animals was 4.4:1 similar to the value of 4:1 found by Stiffel and Bers in rabbit ventricular homogenate.17 This finding adds further support to our assumption that there was minimal, non-preferential loss of binding sites during the tissue preparation. This ratio was found to be reduced in hypertrophy to 3.3:1. Reduced numbers of RYR may lead to poorer coupling within the functional release units such that the same calcium influx may be unable to trigger SR Ca2+ release as effectively. Reduced RYR density alone may also affect the rate of Ca2+ release from the

Ryanodine binding The SR Ca2+ release channel is a co-operatively coupled homotetrameric structure possessing a single high-affinity ryanodine binding site and three low-affinity sites.39 High-affinity ryanodine binding

Ca Channel Stoichiometry in LV Hypertrophy

SR producing slowed contraction. Similar findings of altered calcium channel ratio in microsomal fractions from hypertrophied hearts20 and in membrane fractions from failing hearts35,37 have been reported.

SR Ca2+ content SR Ca2+ content has been shown to modulate Ca2+ release at the level of the calcium spark, the unitary release event thought to underlie the global intracellular Ca2+ transient.44 Changes in spark amplitude assessed by noise analysis45 occur with varying SR Ca2+ load. Bilayer studies also show good evidence for luminal Ca2+ modulating RYR channel gating.46 In the whole cell, SR Ca2+ content can modulate fractional Ca2+ release from the SR for a given trigger,47 the TTP and magnitude of cell shortening and of the cytosolic Ca2+ transient15,48 and the efficacy with which a given trigger can evoke CICR.48 Thus a decrease in SR Ca2+ load is a potential cause of increased TTP of contraction. Our data suggest that SR load is unchanged in hypertrophy and neither group (sham or hypertrophy) is significantly different from control unoperated animals. Others have also been unable to detect a change in SR load in hypertrophy9,31 or in heart failure in the rabbit.29 Values shown for SR load are smaller than those reported by others29,49 in the rabbit, but this may be due to the use of patch pipettes that will dialyse the cytoplasm. We have presented data showing that in hypertrophy there are decreased numbers of functional RYR with no increase in SR load. The well documented increase in the TTP contraction in compensated hypertrophy may be due to such a reduction in RYR density. The slowed release of Ca2+ cannot be explained by changed RYR Ca2+sensitivity or a change in SR Ca2+ content but rather a reduced ability of the SR to release Ca2+ stemming from an alteration to calcium channel stoichiometry. This may affect the efficacy with which the L-type Ca2+ channel can recruit clusters of Ca2+ release channels in the SR.

Acknowledgements We thank Peter O’Gara for preparation of cardiac myocytes. We also thank Prof. Alan J. Williams and the members of his laboratory, namely Toby ScottWard, Andrew Griffin and Dr Duncan West for their advice and assistance with the radioligand binding

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studies. This work was funded by the British Heart Foundation.

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