Reduced Subendocardial Ryanodine Receptors and
Consequent Effects on Cardiac Function in Conscious Dogs
With Left Ventricular Hypertrophy
Luc Hittinger, Bijan Ghaleh, Jie Chen, John G. Edwards, Raymond K. Kudej, Mitsunori Iwase,
Song-Jung Kim, Stephen F. Vatner, Dorothy E. Vatner
Abstract—The goal of this study was to examine the transmural distribution of ryanodine receptors in left ventricular (LV)
hypertrophy (LVH) and its in vivo consequences. Dogs were chronically instrumented with an LV pressure gauge,
ultrasonic crystals for measurement of LV internal diameter and wall thickness, and a left circumflex coronary blood
flow velocity transducer. Severe LVH was induced by chronic banding of the aorta (1261 months), which resulted in
a 78% increase in LV/body weight. When ryanodine was infused directly into the circumflex coronary artery, it did not
affect LV global function or systemic hemodynamics; however, it reduced LV wall thickening and delayed relaxation
in the posterior wall in control dogs but was relatively ineffective in dogs with LVH. In LV sarcolemmal preparations,
[3H]ryanodine ligand binding revealed a subendocardial/subepicardial gradient in normal dogs. In LVH there was a 45%
decrease in ryanodine receptor binding and a loss in the natural subendocardial/subepicardial gradient, which roughly
correlated inversely with the extent of LVH and directly with regional wall motion. Both mRNA and Western analyses
revealed similar findings, with a reduction of the transmural mRNA levels and a loss in the natural gradient between
subendocardial and subepicardial layers in LVH. Thus, ryanodine receptor message and binding in LVH is reduced
preferentially in the subendocardium with consequent attenuation of the action of ryanodine in vivo. The selectively
altered ryanodine regulation subendocardially in LVH could reconcile some of the controversy in this field and may play
a role in mediating decompensation from stable LVH. (Circ Res. 1999;84:999-1006.)
Key Words: pressure-overload hypertrophy n systole n diastole n Ca21 n sarcoplasmic reticulum
T
he ryanodine receptor plays a critical role in excitationcontraction coupling. Several studies have reported that
administration of ryanodine depresses both systolic and
diastolic cardiac function in the normal heart.1– 6 In left
ventricular (LV) hypertrophy (LVH) and in heart failure, the
regulation of cardiac function by the ryanodine receptor is
controversial. Biochemical studies have suggested that ryanodine receptor density is increased,7 is not changed,8 or is
reduced9 –11 in hypertrophy. Prior studies have reported that
levels of ryanodine receptor mRNA are increased in mild
hypertrophy but decreased in severe hypertrophy.12,13 In mild
hypertrophy, sarcoplasmic reticulum (SR) Ca21 transport is
also increased,14 and in human heart failure, single-channel
recordings are normal.15 In heart failure, which usually
includes a component of hypertrophy, ryanodine receptor
binding may be increased, 16 not changed17,18 or decreased.19,20 Studies examining protein levels of the ryanodine receptor in heart failure have shown no change,16,21,22 but
other studies have shown that mRNA levels are de-
creased.16,19,23 Furthermore, studies have shown selective
decreases in ryanodine receptor mRNA in ischemic cardiomyopathy compared with dilated cardiomyopathy24 or decreases in both ischemia and dilated cardiomyopathy.25 Importantly, prior studies have not directly examined the effects
of ryanodine on global and regional myocardial function in
LVH, and biochemical studies have not focused on regional
aspects but only provide transmural data. This latter aspect is
critical in view of our hypothesis that there is altered
transmural distribution of ryanodine receptors in LVH, potentially related to major transmural differences in blood flow
regulation in LVH.26,27 To reconcile this controversy, it
would be important to obtain in the same study functional
data in vivo in combination with biochemical evidence of
altered transmural ryanodine receptor distribution in the
heart.
To address this question for the first time, a large mammalian model was used that permits this type of investigation.28 The first goal of the current investigation was to
Received August 31, 1998; accepted January 11, 1999.
From the Cardiovascular and Pulmonary Research Institute, Allegheny University of the Health Sciences, Pittsburgh, Pa (B.G., J.C., J.G.E., R.K.K.,
M.I., S-J.K., S.F.V, D.E.V.), and INSERM U400, Faculté de Médecine (L.H.), Créteil, France.
This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Dorothy E. Vatner, MD, Cardiovascular and Pulmonary Research Institute, Allegheny University of the Health Sciences, 15th Floor,
South Tower, 320 East North Avenue, Pittsburgh, PA 15212.
© 1999 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
999
1000
Ryanodine in LV Hypertrophy
determine the direct effects of ryanodine on systolic and
diastolic LV function in conscious dogs with severe LVH. To
avoid the effects of LV ryanodine on LV loading conditions,2,6,29 experiments were performed with ryanodine delivered into the coronary artery, where effects on preload and
afterload do not complicate the interpretation of ryanodine
administration. It was particularly important to study conscious dogs, since anesthesia can also affect excitationcontraction coupling.30,31 The second goal of the current
investigation was to determine the density and the affinity of
ryanodine receptors in severely hypertrophied hearts and
whether these changes are affected transmurally in LVH. The
third goal was to determine whether the altered regulation of
ryanodine receptors was posttranslational or due to altered
gene expression.
Materials and Methods
Development of Model
Mongrel puppies of either sex at 8 to 10 weeks of age were
anesthetized with 12.5 mg/kg sodium thiamylal, maintained with
halothane anesthesia (1 to 2 vol %), and ventilated with a respirator
(Harvard Apparatus). A right thoracotomy was performed through
the fourth intercostal space by use of a sterile surgical technique. The
ascending aorta above the coronary arteries was isolated and dissected free of surrounding tissue. A l-cm-wide polytetrafluoroethylene (Teflon) cuff was placed around the aorta and tightened until a
thrill was palpable over the aortic arch; then the chest was closed.
The Teflon band created a fixed supravalvular aortic lesion, which
became relatively more stenotic as the puppies grew. The puppies
were followed for 8 to 19 months.
Implantation of Instrumentation
Two protocols were used in this investigation. Ryanodine was
administered intracoronarily to 6 control and 5 LVH dogs. In vitro
studies depended on an availability of both epicardial and endocardial LV samples and accordingly were performed on a subset of
these dogs and on samples from prior experiments in which
ryanodine had been infused intravenously. After induction with
sodium thiamylal (12.5 mg/kg) and maintenance with halothane
anesthesia (1 to 2 vol %), an incision was made in the fifth left
intercostal space by use of sterile surgical technique. All dogs were
instrumented with Tygon catheters (Norton Elastics and Synthetic
Division) implanted in the descending thoracic aorta and left atrium
and in the LV apex for the aortic-banded dogs. A solid-state
miniature pressure transducer (model P22, Konigsberg Instruments)
was implanted in the left ventricle though the apex to measure LV
pressure in all dogs. LV wall thickness was measured with piezoelectric crystals implanted across the anterior and posterior walls.
The left circumflex coronary artery was isolated 3 to 5 cm from its
origin, and an ultrasonic Doppler blood flow transducer was implanted around the vessel in control dogs and dogs with LVH. An
indwelling silastic catheter was implanted in the left circumflex
coronary artery32 and in the coronary sinus. The thoracotomy
incision was closed in layers, and the animals were allowed to
recover for 2 weeks before the study. The animals used in this study
were maintained in accordance with the Guide for the Care and Use
of Laboratory Animals (National Research Council, revised 1996).
Experimental Measurements
Strain-gauge manometers (model P23 ID, Gould Instruments) were
used to measure aortic, left atrial, and LV pressures. LV pressure was
also measured by use of a solid-state miniature pressure gauge
(Konigsberg Instruments). The position of all catheters and crystals
was confirmed at autopsy. Arterial content and coronary sinus O2
content were measured with an oximeter (model IL 482, Instrumentation Laboratory).
Experimental Protocol
Experiments were performed in a quiet laboratory with the unsedated, conscious dogs resting comfortably in the right lateral position. A stock solution of 5 mg/mL ryanodine (Calbiochem) was
prepared in distilled water and stored in the freezer at –20°C.
Solutions for intracoronary administration were freshly prepared on
the day of the experiment by dilution of the stock solution with 0.9%
NaCl solution as necessary. The effects of cumulative intracoronary
doses of ryanodine (0.5, 1, 3, and 6 mg) were examined in the 5 dogs
with LVH and the 6 control dogs. Ryanodine was infused directly
into the circumflex coronary at a rate of 1 mL/min. A 5-minute
period for stabilization of the effects of ryanodine was allowed
between each dose.
The data were recorded on a multichannel tape recorder (Honeywell) and played back, and measurements were made using Hem 1.4
(Notocord Systems) on a Vectra XU 5/133C Hewlett Packard
computer. LV end-diastolic wall thickness was measured at the onset
of LV contraction, indicated by the initial increase in LV dP/dt. LV
end systole was defined as the point of maximum negative inflection
of LV dP/dt. To assess initial changes in relaxation during intracoronary administration of ryanodine, the slope of the LV posterior wall
motion during the isovolumic relaxation phase was computed.
Normally, during isovolumic relaxation, the wall begins to thin and
the slope is negative. However, when diastolic relaxation is delayed
or inhibited, the slope become less negative or even positive. LV
systolic function was assessed by examining the extent of systolic
wall thickening.
Membrane Preparation
After the dogs were anesthetized with sodium pentobarbital (40 to 50
mg/kg), their hearts were excised immediately and placed in iced
saline. The left ventricle and septum were weighed, and samples
from the LV free wall were divided in half into subendocardial and
subepicardial layers and trimmed of fat and connective tissue, and a
crude membrane fraction was prepared as previously described.20 It
must be recognized that some ryanodine receptors are lost in the first
centrifugation. This was quantified in heart samples from 3 dogs by
measuring ryanodine receptors in the crude preparation before the
first centrifugation and in the supernatant after the membrane
fraction was prepared. The fraction of ryanodine receptors in
supernatant from subendocardial and subepicardial layers was
similar.
Receptor Binding Studies
To optimize the binding conditions for [3H]ryanodine binding, 12
concentrations of membrane protein (10 to 200 mg) were assayed
with and without unlabeled ryanodine (10 mmol/L) and with 50
nmol/L [3H]ryanodine (61.5 Ci/mmol). Incubation was at 37°C for 2
hours. All assays were performed in triplicate and terminated by
rapid filtration through Whatman glass fiber filters washed with 4
mL of cold buffer (150 mmol/L KCl and 10 mmol/L Tris-HCl, pH
7.4). Filters were vortexed in 5 mL of Ecoscint and counted in a beta
scintillation counter for 5 minutes. Specific binding with 70 mg of
protein was .90%. The optimal time for incubation when the
reaction reached equilibrium was determined by incubating 70 mg of
membrane protein and 8 nmol/L [3H]ryanodine for increasing
amounts of time from 1 to 120 minutes at 37°C. The samples were
filtered and counted. Optimal calcium concentration was determined
by incubating 70 mg of membrane protein with 5 nmol/L [3H]ryanodine in HEPES buffer containing (in mmol/L) HEPES 10, EGTA 1,
KCl 150, and AMP 3 (pH 7.4) with increasing concentrations of
CaCl2 (1 mmol/L to 10 mmol/L). Incubation was carried out at 37°C
for 2 hours.
Ryanodine binding sites were quantified with 8 concentrations of
[3H]ryanodine (1 to 40 nmol/L) and 100 mg of membrane protein, in
the presence and absence of 10 mmol/L (unlabeled) ryanodine in a
final volume of 150 mL. The tissue was prepared in HEPES buffer
with 2 mmol/L CaCl2. The triplicate samples were incubated for 2
hours at 37°C, filtered on Whatman glass fiber filters with 4 mL of
wash buffer 3 times for each sample (10 mmol/L Tris-HCl and
Hittinger et al
May 14, 1999
1001
Effects of Intracoronary Ryanodine on Global and Regional Myocardial Function
n
Baseline
Ryanodine
Control (1 mg)
6
11867
11866
LVH (3 mg)
5
22868*
22466*
D Ryanodine
LV systolic pressure, mm Hg
062
2463
LV end-diastolic pressure, mm Hg
Control (1 mg)
6
5.661.2
5.961.3
0.360.3
LVH (3 mg)
5
11.262.4
10.462.3
20.860.4
Control (1 mg)
6
28116287
27976255
214687
LVH (3 mg)
5
32416453
30536396
2188693
Control (1 mg)
6
9268
9769
562
LVH (3 mg)
5
8466
8464
064
Control (1 mg)
6
9665
9965
361
LVH (3 mg)
5
8464
8665
363
Control (1 mg)
6
11.661.5
11.761.5
LVH (3 mg)
5
18.961.3*
18.961.3*
20.060.1
Control (1 mg)
6
25.865.9
21.264.8†
24.661.5
LVH (3 mg)
5
18.263.5
17.963.4
20.360.5*
Control (1 mg)
6
25.263.7
11.965.6†
17.163.7
LVH (3 mg)
5
23.861.2
2.462.1*
Control (1 mg)
6
2864
2964
161
LVH (3 mg)
5
2664
2565
2162
LV dP/dt, mm Hg/s
Heart rate, bpm
Mean arterial pressure, mm Hg
LV end-diastolic wall thickness
0.160.1
LV wall thickening, %
Wall thickness, slope (1023)
6.262.4*
Coronary blood flow velocity, cm/s
Values are mean6SEM.
*P,0.05 LVH different from control.
†P,0.05 effects of ryanodine different for baseline.
150 mmol/L KCl, pH 7.4), and counted in 5 mL of Ecoscint in a beta
scintillation counter with 75% efficiency. Specific binding was 90%.
All assays were standardized by protein content. Protein concentrations were determined using the method of Lowry et al.33 All binding
data were analyzed by the interactive LIGAND computer program of
Munson and Rodbard.34
Western Analysis
The amount of ryanodine receptor protein in cardiac membranes was
measured by immunoblotting and run on precast 4% to 15%
SDS-PAGE (Bio-Rad Laboratories). Proteins were then transferred
to nitrocellulose paper using a wet transblotting apparatus for 4 hours
at 1 A/cm2. The blotting membrane was then incubated in Trisbuffered saline–Tween (TBST; 100 mmol/L NaCl, 100 mmol/L
Tris-HCl, 0.1% Tween-20, and 5% nonfat dry milk) for 1 hour with
shaking at room temperature. This was followed by a 1-hour
incubation at room temperature in TBST, and the paper was then
incubated for 20 minutes at room temperature with sheep anti-mouse
horseradish peroxidase antibody (1:1000 dilution) and washed 4
times with TBST without milk. Autoradiography was performed
using the chemiluminescence system (Amersham Corp) at room
temperature for 1 minute. Autoradiographic densities were determined by densitometry (model PD, Molecular Dynamics).
mRNA Analysis
Changes in cardiac ryanodine receptors were determined by slot-blot
analysis. LV total RNA was prepared and quantified as described
previously.35 A cDNA coding for cardiac ryanodine receptor protein
(cRYR2) was the generous gift of Dr P. Allen).16 Total RNA (10 mg)
was slotted using a manifold (model PR 648, Hoefer, Pharmacia
Biotech). The ryanodine cDNA was radiolabeled by a random prime
method, and blots were normalized for loading using a 28S oligonucleotide (Clonetech) that was radiolabeled using the Prime-a-Gene
Labeling system (Promega). Hybridization conditions were as previously described.35 After washing, the blots were placed under film
at – 80°C or in phosphor image cassettes. Autoradiographs or
phosphor images were quantified using a densitometer or Storm 840
PhosphorImager, respectively (both from Molecular Dynamics).
Statistical Analysis
Statistical analysis was performed using StatView and SuperAnova
software (Abacus Concepts Inc) on a Macintosh computer. The data
are reported as mean6SEM. The comparison of the variations
among groups were performed using the Student t test. The comparisons between groups and between baseline and responses were
analyzed by a 2-way ANOVA for repeated measures, followed if
necessary by a Student t test or a paired t test. Significance was
recorded for probability value of 0.05 or less.
Results
Chronic pressure overload induced by aortic banding increased LV weight/body weight ratio by 78% (P,0.01) in
1002
Ryanodine in LV Hypertrophy
Figure 1. Representative recordings of LV global function and
regional contraction in a control dog before (baseline, left panel)
and after intracoronary administration of 1 mg (middle panel)
and 6 mg (right panel) ryanodine. Ryanodine markedly altered LV
posterior regional contraction in the territory in which ryanodine
was perfused. Note that the slope of posterior wall thickness
during relaxation (top panels, dotted line between marks) was
negative before ryanodine infusion but was delayed and became
positive after ryanodine infusion.
dogs with LV hypertrophy compared with control dogs. Right
ventricular weights were similar in both groups.
Baseline Hemodynamics
Baseline hemodynamics are included in the Table. LV
systolic pressure was doubled in dogs with LVH, but LV
end-diastolic pressure, LV dP/dt, heart rate, and mean arterial
pressure were similar in dogs with LVH and control dogs. LV
end-diastolic wall thickness was 66% greater than in control
dogs, reflecting the hypertrophy of the myocardial wall
(Table).
Effects of Intracoronary Ryanodine
A typical example of the effects of intracoronary ryanodine in
one of the control dogs is shown in Figure 1. Intracoronary
ryanodine did not alter LV global function, systemic hemodynamics, or coronary blood flow velocity (Table). At a dose
of 1 mg IC, ryanodine decreased LV posterior wall thickening
(–18.162.5%, P,0.05) in control dogs (Figure 1 middle
panel) but did not affect wall thickening in dogs with LVH
(Figure 2, right panel). A clear dose-response relationship
was observed in control dogs, whereas dogs with LVH
demonstrated less depression of contraction in response to
intracoronary ryanodine at any dose studied (Figure 2). At the
highest dose of ryanodine studied, 6 mg, posterior LV wall
thickening fell slightly (– 8.962.6%) in dogs with LVH but
much more (P,0.05) in control dogs (– 42.065.6%). Intracoronary ryanodine delayed relaxation of the posterior LV
wall more in the control dogs than in dogs with LVH.
Intracoronary ryanodine replaced the normal wall thinning
during isovolumic relaxation with a prolonged thickening, as
illustrated in Figure 1. To account for potential dilutional
effects in the larger hearts with dogs with LVH, the effects of
3 times the dose of ryanodine was compared in dogs with
LVH (3 mg IC) versus control dogs (1 mg) in the Table. At 1
mg of ryanodine, changes in LV wall thickening and in the
Figure 2. Graphs showing effects of intracoronary ryanodine
(0.5 to 6 mg) on LV anterior (E) and posterior wall contraction
(F) in control dogs (left panel) and in dogs with LVH (right panel). In control dogs, intracoronary ryanodine reduced LV posterior wall contraction dose dependently. In contrast, in dogs with
LVH, intracoronary ryanodine did not alter LV posterior wall
contraction.
slope of the motion of the posterior LV wall thickness during
isovolumic relaxation after intracoronary ryanodine were
greater (P,0.05) in control dogs than with 3 mg in dogs with
LVH, suggesting that ryanodine impaired local contraction
and relaxation more in control dogs than in dogs with LVH.
Neither coronary blood flow velocity (Table) nor coronary
sinus oxygen content (data not shown) changed in both
groups.
Ryanodine Receptors
Ryanodine binding studies for control and LVH hearts are
shown in Figure 3. Figure 3 illustrates Scatchard analyses of
compiled data from 13 animals, including 5 controls and 8
LVH hearts. There was a significant (P,0.01) transmural
decrease in the amount of ryanodine receptor binding in LVH
Figure 3. Combined Scatchard analyses of [3H]ryanodine binding in the subendocardial (Endo) and subepicardial layers (Epi)
in control (n55) and LV hypertrophied (n58) hearts. There was a
significant decrease in ryanodine binding in LVH hearts and a
loss in the natural subendocardial/subepicardial gradient. The x
intercept indicates the amount bound. Bar graph shows transmural [3H]ryanodine binding density in control hearts (open bars)
compared with LVH hearts (filled bars). There was a significant
decline (*P,0.01) in receptor density (Bmax) in LVH hearts.
Hittinger et al
May 14, 1999
1003
Figure 6. Representative slot-blot analysis of mRNA coding for
cRYR2 in the subendocardium and the subepicardium of 3 control and 3 LVH dogs.
Figure 4. Graphs showing correlation between [3H]ryanodine
receptor density, averaged from subepicardial and subendocardial samples, and LV weight/body weight ratio (left panel) and
baseline LV wall thickening (right panel). Baseline LV contraction
(wall thickening) was correlated closely with ryanodine binding.
The extent of hypertrophy was correlated inversely with ryanodine binding.
hearts, with no significant change in the dissociation constant
(KD). Figure 4 shows that ryanodine receptor density for each
animal studied was correlated with LV weight/body weight
(r50.860) (inverse) and LV wall thickening (r50.720) (direct). Regional distribution analysis demonstrated a gradient
of ryanodine receptor density between the subendocardial and
subepicardial layers in control dogs. This was not observed in
dogs with LVH (Figures 3 and 5, left panel). Western analysis
confirmed this finding by also showing a loss in the natural
subendocardial/subepicardial gradient in LVH.
mRNA Analysis
By Northern analysis, preliminary experiments determined
that the cRYR2 probe produced only a single band of '16 kb
(data not shown). Because of the size of the ryanodine
mRNA, efficient transfer did not always appear quantitative,
Figure 5. Bar graphs showing regional distribution of ryanodine
receptor density (left) and normalized ryanodine mRNA levels
(right) in subendocardial (o) and subepicardial layers (M) in control (left) and LVH dogs (right). In control dogs, ryanodine receptor density and message was greater in the subendocardial
compared with the subepicardial layers (*P,0.05). In LVH dogs
the ryanodine receptor density and message gradient were lost.
and therefore slot-blot analysis was used to make comparisons between control and experimental animals. Compared
with control animals (n55), transmural LV cardiac ryanodine
receptor mRNA levels were significantly decreased in LVH
(n58). When regional analysis was performed, a gradient in
mRNA levels of ryanodine was observed between the subendocardial and subepicardial layers in control dogs. This
gradient was not observed in LVH dogs (Figures 5, right
panel, and 6).
Discussion
The effects of LV hypertrophy and heart failure on ryanodine
regulation remains controversial. It is unlikely that this
amount of controversy can be due to one factor. Prior studies
have found both decreased9 –11 and unchanged11 ryanodine
receptor density, whereas Limas et al14 found enhanced
calcium transport by the SR. This discrepancy may be
explained by (1) methodological considerations (ie, measurement of ryanodine receptor binding9 –11 versus mRNA level
measurements)12; (2) species differences, with the downregulation of ryanodine receptors appearing less marked in rats
than in guinea pig or ferret11; and (3) potentially, the duration
or extent of LV hypertrophy, with upregulation of the
ryanodine receptor being observed in mild hypertrophy or
during the development of hypertrophy12,13 and downregulation occurring after prolonged overload in severe hypertrophy.9 –11 One other factor has not been considered, but may
help reconcile this controversy, ie, the transmural distribution
of changes. For example, if changes in ryanodine regulation
occur selectively in one part of the heart, prior studies
sampling nonselectively could have concluded no change if
this was not taken into account and samples were diluted with
tissues from regions where ryanodine regulation was not
altered. The present study performed in dogs with severe
LVH after prolonged pressure overload demonstrated clearly
that ryanodine receptors are downregulated, but this downregulation is selective for the subendocardium. Furthermore,
the decreased density of the ryanodine receptors correlates
with the extent of hypertrophy and the baseline LV wall
thickening (Figure 4).
Importantly, the current study demonstrated for the first
time that there is a normal transmural gradient of ryanodine
receptors, which is lost in LVH. The selective loss of
ryanodine receptor regulation subendocardially may help to
reconcile some of the controversy in this area. For example,
prior negative studies may have been affected by subepicar-
1004
Ryanodine in LV Hypertrophy
dial sampling, which does not show marked changes. The
normal gradient from the endocardium to the epicardium for
ryanodine receptors may be required to provide calcium for
contraction in subendocardial layers, characterized by larger
compressive forces. With the development of severe LVH,
there is a loss of this natural transmural gradient of the
ryanodine receptors between the subendocardial and subepicardial layers. Although the reason for the natural transmural
gradient in the normal situation or its modification in LVH is
not known, it is interesting to speculate that impairment in
subendocardial ryanodine receptors is due to the subendocardial ischemia that develops on stress in LVH because of
reduced subendocardial reserve.28,36 Interestingly, the deficit
only becomes apparent with severe LVH, when restricted
subendocardial coronary reserve is expressed. Furthermore,
in patients with ischemic cardiomyopathy, downregulation of
ryanodine receptor mRNA is observed.24 In addition, several
studies have shown that acute myocardial ischemia can also
reduce ryanodine receptors.37–39
A natural question is whether the downregulation of
ryanodine receptors observed in LVH has to do with altered
gene expression for the receptors or some other posttranslational event. It appears that the former alternative is responsible, as reflected by decreased levels of mRNA in the
subendocardium in LVH. Interestingly, these experiments
also demonstrated a transmural gradient for ryanodine mRNA
in normal dogs and a loss of this transmural gradient in LVH.
These experiments suggest that the key role of subendocardial reserve and subendocardial ischemia, which is so critical
in mediating the transition from LVH to failure, may be part
of the mechanism for reduced ryanodine receptors. This
further links deficits in ryanodine binding to the development
of heart failure, particularly ischemic cardiomyopathy, as
already noted.19,20,24
It is also known that remodeling occurs with severe LVH,
and there is increased fibrosis in subendocardial layers,27
which could decrease ryanodine message and receptors.
However, in this model, the increase in fibrosis accounts for
,2% of the subendocardial mass.27 Thus, a dilutional effect
due to fibrosis was not responsible for the findings.
Although the observation that there is a downregulation of
ryanodine receptors in severe LVH is important, little is
known regarding the impact of this and the functional
consequences. A major part of the current investigation was
designed to examine the effects of ryanodine on LV function
in LVH. The effects of ryanodine have been studied repeatedly in normal hearts and have consistently shown a dosedependent decrease in LV systolic function.1–5 At the nanomolar concentration range, a range of concentrations
produced by the doses of ryanodine given here,6 ryanodine
binds to a high-affinity site, locking the channel in the
semiopen state, resulting in calcium efflux and SR calcium
depletion and finally a prolongation of the time course of LV
contraction and relaxation.6 Since, in conscious dogs, ryanodine intravenously administered increases heart rate and
changes the loading conditions2 and therefore alters systolic
and diastolic function indices, in the current investigation
ryanodine was delivered intracoronarily. Under these conditions, ryanodine did not elicit a major effect on global LV or
systemic hemodynamics and coronary blood flow velocity.
However, intracoronary ryanodine decreased systolic wall
thickening more at each dose studied in control dogs than in
dogs with LVH.
It is also well known that the calcium-release channel also
regulates diastolic cardiac function. Several prior studies both
in vitro5,40 and in vivo2,3 have demonstrated dose-dependent
decreases in LV systolic and diastolic function with ryanodine administered systemically. This was confirmed in the
present study in control dogs. However, we were surprised to
find that the action of intravenous ryanodine was attenuated
strikingly in LVH (data not shown). It is important to keep in
mind, however, that changes in loading conditions alter the
interpretation of diastolic functional indices.41 With ryanodine administered intracoronarily, in the current study, effects
due to altered loading conditions were minimized, but the
most frequently used indices of diastolic LV function (eg,
tau) could not be used, because diastolic function is usually
assessed for global LV function. Accordingly, we examined
the pattern of relaxation selectively in the posterior wall in
response to intracoronary ryanodine. The wall thinning normally observed during isovolumic relaxation of the left
ventricle was replaced by either no change or a prolonged
thickening in both groups of dogs after ryanodine administration. However, the changes in the slope of the posterior
wall motion in the territory perfused with ryanodine were
greater in control dogs than in dogs with LVH, suggesting a
more potent impairment of relaxation in control dogs than in
dogs with LVH. Therefore, ryanodine exerts less of a negative lusitropic and less of a negative inotropic effect on the
hypertrophied myocardium of conscious dogs. Interestingly,
in failing human myocardium, which also generally involves
hypertrophy, there was a diminished stimulation of Ca21
accumulation by ryanodine18; this is consistent with the
physiological data presented in the current study.
A recent study in young spontaneously hypertensive rats,
characterized by enhanced cardiac function at baseline, found
increased responsiveness to ryanodine.42 These results, although apparently inconsistent with our results, can actually
be reconciled readily. First, Mill et al42 did not measure
ryanodine receptors, but if they did, downregulation would
not be likely, since this is not observed in mild LVH.
Secondly, the late phase of severe LVH is characterized by
depressed cardiac function, as was observed in the current
investigation (Figure 4). Importantly, Bers’ laboratory has
shown that there are marked differences in ryanodine receptor
density among different species and that the depressant
effects of ryanodine correlate with the receptor density.43,44
We observed a similar relationship between ryanodine receptor density (higher in normal than in LVH hearts) and
depressant effects of ryanodine (greater in normal than in
LVH hearts). Because in severe LVH there was also a
diminished response to ryanodine, it appears that both downregulation of ryanodine receptors and reduced responsiveness
to ryanodine presage the decrease in LV function with
increasing severity of LVH.
Before concluding that the differences in LV function in
response to intracoronary ryanodine were due to the downregulation of the ryanodine receptors, it was important to
Hittinger et al
eliminate the possibility of a dilutional effect. It is unlikely
that less drug was delivered to the heart because of differences in coronary blood flow. In this model of LVH,
myocardial blood flow per gram of tissue is normal under
baseline conditions.26,28 Furthermore, mean coronary blood
flow velocity did not differ between control dogs and dogs
with LVH in the current study. However, to account for this
possible source of dilutional error with intracoronary drug
delivery, one analysis used a comparison of a 3-fold increase
in dose to the LVH dogs compared with control dogs (Table).
Even under these conditions, intracoronary ryanodine exerted
significantly greater effects on both systolic and diastolic
regional function in control dogs (Table). Finally, in a
subgroup of dogs, when norepinephrine was directly infused
into the circumflex coronary artery and norepinephrine
plasma levels were measured in the coronary sinus, no
differences were observed between control and hypertrophied
hearts, eliminating the possibility of a dilutional effect (data
not shown).
One other source of error for the physiological experiments
must be addressed. Although some calcium entry blockers
(eg, dihydropyridine compounds) elicit marked coronary
vasodilation45 and could affect drug delivery to the heart, in
the present study ryanodine did not exert a major effect on
coronary blood flow velocity in control dogs. Similar findings
were observed in dogs with LVH, indicating a relative lack of
effect of ryanodine on coronary vasoactivity. Prior in vitro
studies on the effects of ryanodine on coronary vessels also
demonstrated that ryanodine affected coronary vasoactivity
only modestly.5,40,45 Therefore, drug delivery was similar in
both groups of dogs. Apparently, cardiac muscle is more
sensitive to ryanodine than coronary vascular tissue.
Finally, it is important to recognize that the changes in
transmural ryanodine receptor distribution may not be the
only cause of the functional alterations noted, but rather an
important but parallel event. Potentially equally as important
are the total decrease in ryanodine receptors and changes in
SERCA, phospholamban, and other SR proteins. More definitive proof of the causal role of altered ryanodine receptor
distribution in LVH and LV failure awaits experiments with
effective blockers or genetic alterations in mice, as well as
documentation of changes in calcium regulatory proteins.
In summary, normally there is a transmural gradient of
ryanodine receptors and message from subendocardium to
subepicardium. This normal gradient is lost in LVH, as
ryanodine receptors are reduced preferentially in the subendocardium in severe LVH. This is accompanied by attenuation of the action of ryanodine to depress both systolic and
diastolic LV function when it is administered either intravenously or intracoronarily. In view of the correlation between
ryanodine receptors and the degree of LVH, the more
important correlation with the function of the hypertrophied
heart, and the knowledge that the development of heart failure
in this model is preceded by a decline in subendocardial LV
function as well as a decrease in subendocardial coronary
reserve,27,28 it is interesting to speculate that the decrease in
ryanodine receptors and decreased responsiveness to ryanodine presage the decompensation from stable LVH to LV
failure.
May 14, 1999
1005
Acknowledgments
This work was supported in part by US Public Health Service Grants
HL59139, HL33107, HL33065, HL37404, and HL591417. B.G. was
a recipient of the Association Française pour la Recherche Thérapeutique. The cDNA coding for cRYR2 was provided by Dr P. Allen
through National Institutes of Health Grant AR 413140.
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