Effects of diabetes on ryanodine receptor Ca release channel (RyR2) and Ca2+ homeostasis in rat heart.

Abstract

The defects identified in the mechanical activity of the hearts from type 1 diabetic animals include alteration of Ca2+ signaling via changes in critical processes that regulate intracellular Ca2+ concentration. These defects result partially from a dysfunction of cardiac ryanodine receptor calcium release channel (RyR2). The present study was designed to determine whether the properties of the Ca2+ sparks might provide insight into the role of RyR2 in the altered Ca2+ signaling in cardiomyocytes from diabetic animals when they were analyzed together with Ca2+ transients. Basal Ca2+ level as well as Ca2+-spark frequency of cardiomyoctes isolated from 5-week streptozotocin (STZ)-induced diabetic rats significantly increased with respect to aged-matched control rats. Ca2+ transients exhibited significantly reduced amplitude and prolonged time courses as well as depressed Ca2+ loading of sarcoplasmic reticulum in diabetic rats. Spatio-temporal properties of the Ca2+ sparks in cardiomyocytes isolated from diabetic rats were also significantly altered to being almost parallel to the changes of Ca2+ transients. In addition, RyR2 from diabetic rat hearts were hyperphosphorylated and protein levels of both RyR2 and FKBP12.6 depleted. These data show that STZ-induced diabetic rat hearts exhibit altered local Ca2+ signaling with increased basal Ca2+ level.

Full text

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Original Article
Effects of Diabetes on Ryanodine Receptor Ca Release
Channel (RyR2) and Ca2?Homeostasis in Rat Heart
Nazmi Yaras,1Mehmet Ugur,1Semir Ozdemir,1Hakan Gurdal,2Nuhan Purali,3Alain Lacampagne,4
Guy Vassort,4and Belma Turan1
The defects identified in the mechanical activity of the
hearts from type 1 diabetic animals include alteration of
Ca2?signaling via changes in critical processes that regu-
late intracellular Ca2?concentration. These defects result
partially from a dysfunction of cardiac ryanodine receptor
calcium release channel (RyR2). The present study was
designed to determine whether the properties of the Ca2?
sparks might provide insight into the role of RyR2 in the
altered Ca2?signaling in cardiomyocytes from diabetic
animals when they were analyzed together with Ca2?tran-
sients. Basal Ca2?level as well as Ca2?-spark frequency of
cardiomyoctes isolated from 5-week streptozotocin (STZ)-
induced diabetic rats significantly increased with respect
to aged-matched control rats. Ca2?transients exhibited
significantly reducedamplitude
courses as well as depressed Ca2?loading of sarcoplasmic
reticulum in diabetic rats. Spatio-temporal properties of
the Ca2?sparks in cardiomyocytes isolated from diabetic
rats were also significantly altered to being almost parallel
to the changes of Ca2?transients. In addition, RyR2 from
diabetic rat hearts were hyperphosphorylated and protein
levels of both RyR2 and FKBP12.6 depleted. These data
show that STZ-induced diabetic rat hearts exhibit altered
local Ca2?signaling with increased basal Ca2?level.
Diabetes 54:3082–2088, 2005
D
electrical and mechanical properties of the myocardium
from diabetes are significantly impaired (2–5). Ventricular
myocardium from diabetic rats exhibits a reversible de-
crease in the speed of contraction, prolongation of con-
traction, and a delay in relaxation.
Contraction is initiated when a small amount of Ca2?
entering into the cell following membrane depolarization
andprolongedtime
iabetic cardiomyopathy as a distinct entity was
first recognized by Rubler et al. (1) in diabetic
patients with congestive heart failure who had
no evidence of coronary atherosclerosis. Both
triggers a larger release of Ca2?from the sarcoplasmic
reticulum (SR). Spontaneous local Ca2?transients, or
Ca2?sparks, are short-lived Ca2?release events that are
believed to demonstrate the main events of excitation-
contraction in cardiomyocytes (6,7). The global increase in
the amount of free Ca2?in cardiomyocyte during depolar-
ization consists of the summation of these unitary Ca2?
release events in cardiomyocytes (8) and skeletal muscle
(9). On the other hand, it is generally agreed that any
alteration of Ca2?signaling could be a main source of
cardiomyopathy (10). Since the Ca2?flux underlying the
Ca2?sparks reports the summation of ryanodine-sensitive
Ca2?release channel (RyR2) behavior in the spark cluster,
evaluation of the properties of the Ca2?sparks may
provide insight into the role of RyR2 in the altered Ca2?
signaling in cardiomyocytes from diabetic animals (3,4,
11,12).
Depression in contraction and relaxation of myocytes
isolated from streptozotocin (STZ)-induced diabetic rats
was found in parallel with reduced rate of rise and decline
of intracellular Ca2?transient elicited by electrical stimu-
lation (5). These effects were mostly attributed to anoma-
lous SR pump activity (11) and SR Ca2?storage (3) and in
part to reduced Na?/Ca2?exchange (13) and RyR2 protein
expression. However, a change in RyR2 expression was
not observed in this same model (14) nor in the diabetic
db/db mouse (15). It rather was proposed that the RyR2
properties are altered. Dysfunction of RyR2 induced by
diabetes could be, in part, due to formation of disulfide
bonds between adjacent sulfhydryl groups (16), while
chronic diabetes increases advanced glycation end prod-
ucts (16,17).
The goal of the present study was to specifically exam-
ine the behavior of the RyR2 receptors in diabetic rats. We
reported here for the first time that local Ca2?release
events (Ca2?sparks) in ventricular cardiomyocytes from
STZ-induced diabetic rats are slower and have higher
frequency with respect to the controls, consistent with
alterations in Ca2?handling and cardiac dysfunction. The
changes in RyR2 opening kinetics could be related to
hyperphosphorylation of RyR2 and FKBP12.6 release.
RESEARCH DESIGN AND METHODS
Diabetes was induced by a single intraperitoneal injection of STZ (50 mg/kg
body wt and dissolved in 0.1 mol/l citrate buffer, pH 4.5) in adult male Wistar
rats (200–250 g body wt). Control rats received citrate buffer alone. One week
after injection of STZ, blood glucose levels were measured, and rats with
blood glucose at least three times higher than the preinjection levels were
used. All rats had free access to standard rat diet and water. All animals were
used 5 weeks after STZ or citrate buffer injections. All experiments were
performed in accordance with Ankara University School of Medicine ethics
guidelines for the care and use of laboratory animals.
From the1Department of Biophysics, School of Medicine, Ankara University,
Ankara, Turkey; the
Ankara University, Ankara, Turkey; the3Department of Biophysics, School of
Medicine, Hacettepe University, Ankara, Turkey; and4INSERM U-637, Phys-
iopathologie Cardiovasculaire, CHU Arnaud de Villeneuve, Montpellier,
France.
Address correspondence and reprint requests to Dr. Belma Turan, Depart-
ment of Biophysics, School of Medicine, Ankara University, Ankara, Turkey.
E-mail: belma.turan@medicine.ankara.edu.tr.
RyR2, ryanodine receptor Ca release channel; SR, sarcoplasmic reticulum;
STZ, streptozotocin.
Received for publication 11 May 2005 and accepted in revised form 4 August
2005.
© 2005 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
2Department of Pharmacology, School of Medicine,
3082DIABETES, VOL. 54, NOVEMBER 2005

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Cell isolation. Hearts were removed rapidly and weighted after rats had been
anesthetized with sodium pentobarbital (30 mg/kg body wt). Hearts were
cannulated on a Langendorff apparatus and perfused retrogradely through the
coronary arteries with a Ca2?-free solution (in mmol/l): 145 NaCl, 5 KCl, 1.2
MgSO4, 1.4 Na2HPO4, 0.4 NaH2PO4, 5 HEPES, and 10 glucose (pH 7.4), bubbled
with O2at 37°C. Hearts were perfused for 3–5 min to clean out the remaining
blood; this was followed by perfusion with the same solution containing 1
mg/ml collagenase (Collagenase A, Boehringer) for 30–35 min. Ventricles
were then removed and minced into small pieces and gently massaged
through a nylon mesh, and dissociated cardiomyocytes were washed with the
collagenase-free solution. The percentage of viable cells was ?70% in either
group. Subsequently, Ca2?was increased in a graded manner to a concentra-
tion of 1 mmol/l. Cells were kept in this solution at 37°C until used in the
experiments. The percentage of Ca2?-tolerant cells was ?60%.
Global cytosolic Ca2?measurement. Intracellular Ca2?transients were
measured by fura-2 fluorescence at room temperature (21 ? 2°C). The cells
were incubated at 37°C for 50 min with 4 ?mol/l fura-2 AM for loading and
then washed with fresh buffer. Fluorescence was recorded using a PTI
Ratiomaster microspectrophotometer and FELIX software (Photon Technol-
ogy International). Cells were excited at 340/380 nm and emission measured
at 510 nm. The fluorescence ratio F340/380of the emitted light on excitation at
340 and 380 nm was calculated and used as an indicator of [Ca2?]i. F340/380
ratio was measured at a frequency of 10 Hz. The composition of the bath
solution used in [Ca2?]imeasurement was as follows (mmol/l): 130 NaCl, 4.8
KCl, 1.2 MgSO4, 1.5 CaCl2, 1.2 KH2PO4, 10 HEPES, and 10 glucose (pH 7.4).
Following monitoring [Ca2?]ifor 20 s at rest, field-stimulation pulses of 20–30
V with 10-ms duration, were applied at 0.2-Hz frequency. Peak amplitude
(difference between basal and peak F340/380ratios), time to peak, and
half-decay time were determined from Ca2?transients evoked by field
stimulation. Background fluorescence measured from a cell-free field was
subtracted from all recordings before calculation of ratios.
Caffeine-induced Ca2?transients were induced by bath application of 10
mmol/l caffeine on cardiomyocytes 30 s after stopping field stimulation of the
cells with electrical pulses (for 2 min at 0.2-Hz stimulation frequency) to
ensure stable SR Ca2?load.
L-Type Ca2?current. Ca2?currents (ICaL) were recorded at room tempera-
ture (22 ? 2°C) using the whole-cell configuration of the patch clamp
technique in the presence of cesium to inhibit K?currents and using voltage
ramp by using a patch-clamp amplifier (Model RF-300; Biologic, Claix, France)
and filtered at 3 kHz. Current traces were digitized at 5 kHz using Digidata
1200 and pClamp 8 software (Axon Instruments, Foster City, CA). The
electrode resistance was 1.2–1.5 mol/l?.
Pipette solution for ICaLcontained (in mmol/l): 120 CsCl, 6.8 MgC12, 5.0
phosphocreatine-Na2, 5.0 ATP-Na2, 11 EGTA, and 20 HEPES (pH 7.2). The
bathing solution for Ca2?currents contained (in mmol/l): 117 NaCl, 1.7 MgCl2,
5.4 CsCl2, 10 HEPES, 1.8 CaCl2, and 11 glucose (pH adjusted to 7.4 with
NaOH). ICaLwas recorded during 250-ms depolarizing pulses to potentials
between ?50 and ?60 mV (with 10-mV steps) applied at a frequency of 0.2 Hz
after a 500-ms ramp to ?45 mV from a holding potential of ?80 mV. Current
amplitude was estimated as the difference between peak inward current and
the current level at the end of the 250-ms pulse.
Ca2?spark measurement. Cardiomyocytes were placed into the recording
chamber, which was set onto the stage of an inverted microscope equipped
with a laser scanning confocal microscope (200 mol/l; LSM-Pascal, Zeiss,
Germany). In the experiments, 40? (NA 1.3) oil immersion objectives were
used for imaging cardiomyocytes located over the cover glass base of the
recording chamber. A 488-nm laser line from an argon laser (25 mW) was used
to excite the Ca2?-sensitive dye, Fluo-3, and emitted fluorescence collected at
505 nm. Changes in [Ca2?]iwere recorded in line-scan mode (spatial [x] vs.
temporal [t], 1.9 ms/line). To prevent photo-bleaching and cell damage, the
laser line was kept at 4–6% of maximal intensity and the confocal pinhole set
to 1–1.5 airy units to achieve the best resolution and emission intensity.
Image analysis was performed using LSM Image Examiner. The F value for
fluorescence intensity of image was calculated by averaging pixels other than
potential spark areas. Then ?F/F image was created by using this F value.
Ca2?sparks were manually detected and converted to temporal lines by
averaging fluorescence intensity of 2–3 pixels aligning the peak of fluores-
cence intensity over time. The signals were filtered using a Butterworth digital
filter. The temporal profiles were then fitted to gamma function to analyze
time to peak, peak amplitude of fluorescence intensity, and decrease time to
half-maximum.
Western blot analysis. Hearts were homogenized with a motor-driven
Teflon-to-glass homogenizer in cold Tris-HCl buffer containing (in mmol/l): 50
Tris-HCl (pH 7.4), 200 NaCl, 20 NaF, 1.0 Na3VO4, 1 dithiothreitol, and protease
inhibitors (complete tablet from Roche) and centrifuged at 1000g for 30 min at
4°C. Supernatant was centrifuged at 30,000g for 30 min, then at 100,000g for
1 h at 4°C. Pellet was suspended with the homogenization buffer and protein
amount measured (18). The samples (100 ?g protein) were subjected to 5%
SDS-PAGE for RyR2 assay and 10% SDS-PAGE for both FKBP12.6 and actin
assays and then transferred electrophoretically to nitrocellulose membrane.
Immunoblotting was performed using antibodies against RyR2, RyR2-P (kind
gift of Drs. A. Marks and X. Wehrens; dilutions 1/5,000 and 1/3,000, respec-
tively), FKBP12.6, and actin (Santa Cruz), and then enhanced chemolumines-
cence. Briefly, nitrocellulose membranes were incubated 1 h at 4°C in PBS (20
mmol/l NaH2PO4-Na2HPO4, pH 7.6, containing 154 mmol/l NaCl, 3% BSA, and
8% nonfatty dry milk). Blots were washed several times with PBS containing
0.1% Tween and then incubated with antiserum at room temperature for 1–2
h by shaking. Blots were then washed several times with PBS, incubated with
horseradish peroxidase–labeled anti-rabbit IgG (Santa Cruz Biotech, Santa
Cruz, CA) (dilution 1/10,000) for 1 h at room temperature. Blots were washed
several times with PBS and then incubated with ECL Western blotting reagent
(Amersham, Vienna, Austria) for 1 min and exposed to X-ray film for 45–90 s.
RESULTS
General characteristics of STZ-induced diabetic rats.
Diabetic animals had significantly high glucose levels
(458 ? 8 mg/dl) compared with control animals (101 ? 1
mg/dl). They stopped gaining weight (213.6 ? 9.2 vs.
197.8 ? 7.9 g) following STZ injection, while control
animals continued to gain weight (215.0 ? 4.9 vs. 249.2 ?
6.8 g) at the end of the 5-week experimental period. The
heart weight–to–body weight ratio was 4.08 ? 0.58 vs.
4.04 ? 0.38 mg/g in the diabetic versus control groups. To
avoid the possible insensitive heart-weight measurement,
we also measured the capacitance of the isolated cardio-
myocytes and compared the values between the groups.
The mean cell capacitances of the control and diabetic rats
were similar as previously reported (189.9 ? 12.2 and
180.6 ? 13.6 pF, respectively) (19).
Global Ca2?homeostasis in diabetic rat heart. Figure
1A shows original recordings of Ca2?transients elicited in
control and diabetic rat cardiomyocytes. The averaged
peak amplitude of ?F340/380was significantly smaller in
diabetic than in control cells (0.23 ? 0.01 and 0.35 ? 0.02
AU, respectively) (Fig. 1B). The time to peak amplitude of
Ca2?transients of diabetic cells (0.26 ? 0.01 s) was
significantly larger than the control ones (0.18 ? 0.01 s),
and the half-time for recovery, half-decay time was signif-
icantly prolonged with respect to the controls (0.64 ? 0.02
vs. 0.50 ? 0.02 s).
The averaged diastolic values of ?F340/380were 0.49 ?
0.01 and 0.41 ? 0.01AU in diabetic and control groups,
respectively (P ? 0.001). This is indicative that the basal
[Ca2?]ilevel is larger in the diabetic groups (Fig. 1C).
L-Type Ca2?currents. To determine whether a change in
L-type Ca2?currents (ICaL) contributes to the altered Ca2?
sparks activity observed in the diabetic group with respect
to the control group in the present study, ICaLwas exam-
ined with whole-cell patch-clamp technique. The densities
of ICaLof both groups were similar at all voltages from ?50
to ?60 mV. The average peak current densities measured
at 0 mV are given in Fig. 1D. Besides the amplitudes (10.9
? 0.7 and 10.2 ? 0.5 pA/pF in control vs. diabetes,
respectively), the fast and slow activation time constants
of ICaL(8.1 ? 1.2, 52.9 ? 7.1, and 7.6 ? 1.9 ms; 54.9 ? 5.8
ms in control vs. diabetes) obtained by fitting a two-
exponential curve (17) were found to be similar (Fig. 1D,
inset). The current voltage relationships of ICaLof both
groups recorded between ?50 and ?60 mV were similar as
well (data not shown). This result demonstrates that L-type
Ca2?channel function was not significantly different due
to diabetes.
Local SR Ca2?release in diabetic rat heart. Ca2?
sparks, which represent SR Ca2?release from clusters of
N. YARAS AND ASSOCIATES
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RyR2, were examined in cardiomyocytes isolated from
control and STZ-induced diabetic rat hearts. Representa-
tive line-scan images of the cardiomyocytes from the
control and the diabetic rats (individual Ca2?spark image
x vs. t) are displayed in Fig. 2A. Representative temporal
and averaged spatial profiles of Ca2?sparks recorded in
cells from a control and a diabetic rat are given in Fig. 2B.
The spatial time courses were extracted as the mean of all
recorded spatial or temporal pixels centered at the peak of
the ?F/F of the Ca2?spark. These transients were then
fitted by a gamma distribution function to obtain objective
kinetic and spatial measures of the events (19).
Quantitative data for Ca2?spark characteristics are
summarized in Fig. 2C. Peak amplitude determined as
FIG. 1. Ca2?transients and basal intracel-
lular [Ca2?]iare changed in cardiomyocytes
from diabetic rats. A: Representative Ca2?
transients and fitted curves (dashed lines)
of diabetic (DM) and control (C) rat cardi-
omyocytes field stimulated at 0.2 Hz. B: Bar
graphs representing the changes in peak
amplitude (PA) of the fluorescence [Ca2?]i
transients (?F340/380? F340/380peak ? F340/
380 basal), time to peak (TP), and decay
time to 50% of the peak transient (DT50) of
the diabetic (nrat? 5, ncell? 25) and con-
trol groups (nrat? 5, ncell? 24). C: Change
of basal [Ca2?]iin the diabetic group with
respect to the control group. D: Compari-
son of L-type Ca2?currents (ICaL) recorded
in cardiomyocytes from diabetic (dashed
line) and control rat (continuous line)
hearts. Inset: representive current records,
time shifted for clarity. Currents were re-
corded by applying 250-ms depolarizing
voltage steps at 0.2 Hz from a holding
potential of ?80 to 0 mV. The current
amplitude was estimated as the difference
between peak inward current and the cur-
rent level at the end of the 250-ms pulse
and normalized to cell capacitance. Values
are expressed as means ? SE of 10–12 cells
from at least five rats. *P < 0.001 vs.
control.
FIG. 2. Spatio-temporal properties of Ca2?
sparks in diabetes. A: Representative line-
scan images of isolated cardiomyocytes. C,
control; DM, diabetic. B: Averaged spatial
profiles of Ca2?sparks (continues line: C,
dashed line: diabetic) (right panel) and
temporalprofilestogether
curves (dashed line) of a spark from both
groups (left panel). C: Time to peak ampli-
tude (TP), half decay-time (DT50), and
peak amplitude (PA) of Ca2?sparks mea-
sured in control (C) and diabetic (DM) rat
cardiomyocytes (nrat? 5, ncell? 34, nspark?
152 vs. nrat? 4, ncell? 25, nspark? 145 in
diabetic vs. control). D: Spatial distribu-
tion of the sparks (frequency of Ca2?
sparks width at half their maximal ampli-
tude [FWHM]) (left panel) and comparison
of Ca2?sparks frequency (10 vs. 12 cardi-
omyocytes from control [C] vs. diabetic
[DM] groups) (right panel). *P < 0.0001 vs.
control.
withfitted
Ca2?SPARKS IN DIABETIC CARDIOMYOCYTES
3084DIABETES, VOL. 54, NOVEMBER 2005

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?F/F at the peak of Ca2?spark, frequency of Ca2?sparks
width at half-maximal amplitude, time to peak, and time to
half decrease were calculated from individual gamma distri-
bution function fits. The mean peak amplitude of the diabetic
group (?F/F) was not changed (0.51 ? 0.02 and 0.56 ? 0.02
AU in diabetic and control, respectively), but time to peak
was significantly slower (12.88 ? 0.50 and 9.08 ? 0.30 ms in
diabetic and control groups, respectively) as well as time to
half decrease, which prolonged from 20.78 ? 0.87 to 32.90 ?
1.29 ms with respect to the control group (P ? 0.0001). The
spatial spread (frequency of Ca2?sparks width at half-
maximal amplitude) was not significantly different in the
diabetic group with respect to the control group (2.45 ? 0.85
vs. 2.05 ? 0.92 ?m) (Fig. 2D, left). Spontaneous Ca2?sparks
frequency was higher (P ? 0.0001) in diabetic cardiomyo-
cytes (0.051 ? 0.007 s–1?/m?1) than in the control cells
(0.032 ? 0.003 s–1?/m?1) (Fig. 2D, right).
SR Ca2?content of cardiomyocytes. Caffeine applica-
tion caused a sudden and transient increase in intracellu-
lar Ca2?of cardiomyocytes due to Ca2?release from SR.
The size of the caffeine-induced Ca2?transient has been
used to assess the SR Ca2?load of control and diabetic
cardiomyocytes. To assure stable SR Ca load, cells were
first stimulated and caffeine (10 mmol/l) rapidly applied
30 s after cessation of electrical stimulation. The caffeine-
induced Ca2?transient recorded in cardiomyocytes from
diabetic rats was smaller than the corresponding control
one (Fig. 3A). The averaged caffeine responses (?F/F)
were 0.42 ? 0.02 vs. 0.36 ? 0.02 AU in the control versus
diabetic cells, and the difference between these two
groups is statistically significant (Fig. 3B).
Effects of insulin on Ca2?sparks. To clarify whether
the altered parameters of Ca2?sparks arise due to STZ-
induced diabetes (type 1, insulin-dependent diabetes) or a
result of the direct toxic effect of STZ on cardiomyocytes,
cells from diabetic rats were incubated in the presence or
not of insulin (100 nmol/l) for 4–5 h at 37°C. Then, we
recorded Ca2?sparks and calculated the time to peak and
the time to half decrease of the sparks. The mean peak
amplitudes of the sparks (?F/F) from these two groups of
cells were not affected by insulin incubation, but the time to
peak of the insulin incubated group of cells (58 sparks in five
cells from two rats) was significantly recovered (10.06 ? 0.30
vs. 13.03 ? 0.52 ms) compared with untreated cells (45
sparks in four cells from two rats). Also, time to half increase
was recovered from 30.04 ? 1.81 to 25.06 ? 1.19 ms (P ?
0.001). If we compare these parameters with those from
normal controls (time to peak 9.08 ? 0.30 ms, time to half
decrease 20.78 ? 0.87 ms), we can see significant recoveries
with insulin incubation of the cardiomyocytes from STZ-
induced diabetic rats (Fig. 3C).
Biochemical analysis of RyR2 in diabetes. The mecha-
nisms underlying the dysfunction of RyR2 in diabetes
remain undefined. We hypothesized that the alterations in
Ca2?release behavior seen at the global and local spark
level in STZ-induced diabetic rat cardiomyocytes could be
due to a defect in RyR2 function following its phosphory-
lation and release of FKBP12.6, an accessory protein of
RyR2 macromolecular complex, as documented in heart
failure (20). The phosphorylation level of RyR2 in diabetic
and control rat heart tissue was evaluated using specific
antibodies directed against RyR2 and phosphorylated
RyR2 (Fig. 4A). Total RyR2 in the diabetic groups was
?44% less than in the control group as estimated from the
Western-blot bands (Fig. 4B). There is strong evidence of
phosphorylation of RyR2 in diabetic rat heart but no
appearance of it in the control rat hearts. The amount of
FKBP12.6 was decreased by 41% in the diabetic rat heart
compared with the control rat heart (Fig. 4B). There was
no significant difference in the protein levels of actin in
diabetic and control groups (Fig. 4B).
DISCUSSION
This study on STZ-induced type 1 diabetes in rats reports
significant alterations in Ca2?homeostasis that can ac-
FIG. 3. Effect of diabetes on caffeine re-
sponse. A: Representative tracings of caf-
feine-induced peak
transients elicited in cardiomyocytes iso-
lated from control (C) and diabetic (DM)
animals. Traces have been shifted for sake
of clarity. B: ?F340/380values of the two
groups. Values are expressed as means ?
SE of 12–17 cells from at least five animals.
*P < 0.01 vs. control. C: Recovery of al-
tered parameters of Ca2?sparks following
100-nmol/l insulin incubation for 4–5 h at
37°C. Values are expressed as means ? SE
of 58 cells (insulin incubated, two diabetic
rats) vs. 45 cardiomyocytes (nonincubated,
two diabetic rats). *P < 0.001 vs. nonincu-
bated.
intracellularCa2?
N. YARAS AND ASSOCIATES
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count for the well-known contractile dysfunction in such
pathological conditions. At first we confirmed the defec-
tive intracellular Ca2?signaling with both lower amplitude
and slower kinetics of Ca2?transients as well as the
decreased SR Ca2?load (5). Furthermore, we clearly
established that these defects could be attributed to anom-
alous RyR2 behavior, as revealed by the spatio-temporal
properties of Ca2?sparks that especially exhibited slower
kinetics. The reduced amount of RyR2 and FKBP12.6
levels and the protein kinase A–dependent phosphoryla-
tion of RyR2 could be responsible for most of these
observations. A lower SR Ca2?load reinforced these
defects in Ca2?release channel properties.
The diabetic cardiomyopathy, starting with asymptom-
atic left ventricular diastolic dysfunction, progresses to
compromised systolic function that leads to increased
incidence in morbidity and mortality. Coincident with the
alteration of cardiac function are a variety of metabolic
and biochemical abnormalities that include change in the
predominance of myosin isoenzymes (21), an activity of
the SR Ca2?-ATPase (3,5,11,22,23). In addition, diabetes
has been associated with a reversible increase in the
duration of the action potential attributable to a reduced
transient outward K?current (IK1), while the L-type Ca2?
current (ICaL) was unaffected (5,24–26). Thus, diabetes
exhibits some similarities with heart failure in which it has
been reported that IK1is reduced as a consequence of
elevated diastolic Ca2?following altered RyR2 properties
(27).
Despite the fact that ICaLis not modified by diabetes, the
cardiomycytes from diabetic rats demonstrate significant
alterations in Ca2?homeostasis. These include increases
in rise time and half-decay time of Ca2?transient together
with decrease in Ca2?transient amplitude and SR Ca2?
load as well as increase in diastolic Ca2?. All these
changes could be consequent to the alterations in RyR2
behavior that we describe here. The decrease in SR Ca2?
load has been reported in several previous studies (5) and
was already suggested by rapid cooling contracture exper-
iments (28). This is attributable to reduced sarcoplasmic
reticulum Ca-ATPase and increased phospholamban ex-
pression (29,30) as well as to the anomalous RyR2 activity.
These factors could also be responsible for the larger
diastolic Ca2?level seen here in diabetic cells (31–33),
although others reported no change (34,35) or even a
decrease (36,37). However, this latter work (37) was
performed using a low-Ca (0.5 mmol/l)–containing extra-
cellular solution. It needs to be emphasized that the
current controversy in resting Ca2?levels in both tissue
and isolated myocytes in diabetic hearts is under consid-
eration and needs to be clarified with additional data.
Our data also show that insulin incubation of the
cardiomyocytes from diabetic rats for 4 h significantly
restored the altered parameters of Ca2?sparks and thus
provides support for the origin of these alterations. It is
well known that STZ can induce cardiac dysfunction
depending on insulin deficiency (type 1 diabetes), while
insulin treatment of these animals/cells could recover
almost all of these parameters (25,28,38–41). Besides
these published data, it has been shown that altered SR
Ca2?content and RYR2 binding, altered SR protein expres-
sion, as well as altered RyR2 mRNA levels and RyR2
protein levels (5,14,17) were normalized following insulin
treatment of the STZ-induced diabetic animals in vivo
and/or in vitro.
It is now well documented that an increase in [Ca2?]iis
FIG. 4. Amount and phosphorylated level of
RyR2. A: Representative Western blots of
RyR2, RyR2-P, FKBP12.6, and actin in con-
trol (C) and diabetic (DM) rats. B: The
averagechanges of
FKBP12.6 are 44 and 41%, respectively, vs.
control, while there was no significant
change in actin level (at least two animals
with double assays in each sample from
each group for each type of measurement).
*P < 0.05 vs. control.
bothRyR2 and
Ca2?SPARKS IN DIABETIC CARDIOMYOCYTES
3086DIABETES, VOL. 54, NOVEMBER 2005

Page 6

one of the major steps in the sequences of events leading
to contraction of cardiac muscle. These sequences of
events are demonstrated in part by Lopez-Lopez et al. (7),
in which we have shown that parallel defects in contrac-
tion and [Ca2?]itransients in our diabetes model clearly
indicate that defective intracellular Ca2?signaling contrib-
utes to contractile dysfunction in diabetes. In our diabetes
model, although there is significant prolongation in action
potential duration as well as delayed rise time and pro-
longed decay in contractile activity, the density of ICaLwas
similar at all voltages from ?50 to ?60 mV, and L-type Ca2?
channel function did not show any difference due to diabetes
(5,26). Therefore, it is concluded that ICaLdid not contribute
to the altered activity of [Ca2?]itransient observed in the
diabetic group; rather, the gain of function of the excitation-
contraction coupling is markedly decreased.
In addition to the changes in [Ca2?]itransients, here we
report alterations in the properties of spontaneous Ca2?
sparks in cardiomyocytes from diabetic rats. Populations
of Ca2?sparks from diabetic rat cardiomyocytes have
significantly slower temporal kinetics. It has been demon-
strated in skeletal muscle that the rising phase of the Ca2?
spark fluorescence represents the underlying open time of
RyR Ca2?channels (9). This similar conclusion could be
made in cardiomyocytes. Alternatively, the decay phase of
spark fluorescence is characterized by the diffusion of
Ca2?away from the SR and is thought to be independent
of SERCA activity. Thus, the spark decay may be influ-
enced by intrinsic Ca2?buffering processes (42).
The Western blot analysis supports altered activity of
RyR2 in diabetes. There are, however, controversies about
the RyR2 properties in diabetes. Similar data were pub-
lished by Guatimosim et al. (12) and Bidasee et al. (14).
They have mentioned the importance of evaluation of the
properties of the Ca2?sparks as insight into the role of
RyR2 in the altered Ca2?signaling in diabetic cardiomyo-
cytes. Their data have also shown that although expres-
sion did not change significantly after 6 weeks of diabetes,
the ability of RyR2 to bind the specific ligand [3H]ryano-
dine was significantly lowered (14). These authors also
demonstrated that chronic diabetes (8 weeks) induced a
decrease in cardiac contractility in part due to a decrease
in expression and an alteration in function of RyR2 (16).
Another origin of altered parameters of Ca2?signaling
might be the deficiency in SR Ca2?load. Indeed, a similar
application of caffeine released a significantly less amount
of Ca2?from SR, in agreement with previous results by
Choi et al. (5) in a similar diabetes model. These studies,
therefore, identify important alterations in SR function
due to either a decreased level and altered function of
RyR2 and/or a depressed level of SERCA.
RyR2 receptors are regulated by a variety of proteins,
including FKBP12.6. Due to its importance in the coupled
gating of RyR2, its deficit and alteration has been involved
in heart failure. However, there are controversies related
to its effects on Ca2?spark parameters (20,43,44). Our
data showed that kinetic parameters of Ca2?sparks are
slower in diabetic compared with control rats. These
alterations are due to defects in RyR2 receptor behavior.
According to the RyR2-coupled gating hypothesis (45),
FKBP12.6 allows all RyR2 clusters to open and close
simultaneously. Therefore, any type of alteration in this
protein, associated here with a decreased amount of RyR2,
will cause a depression in both time to peak and time to
half decrease of Ca2?sparks. The question thus arises as
to how less RyR2 and FKBP12.6 and phosphorylated RyR2
can cause no significant change in the amplitude of Ca2?
transient together with significant prolongations in both
time to peak and time to half decrease. This effect is may
be due to leaky SR in diabetes. This hypothesis is consis-
tent with several studies and also supported by our data on
both increased basal Ca2?level and Ca2?spark frequency
in diabetes (5,15,46). A further study on quantitative
assessment of RyR2 and FKBP12.6 mRNA levels of hearts
from control and diabetic groups is needed to fully clarify
their roles in excitation-contraction coupling and its alter-
ation during diabetes.
These alterations in Ca2?homeostasis might be respon-
sible for heart remodeling that occurs without significant
cell growth. Like most others (25,47), we observed no
change in membrane capacitance, while after longer STZ-
treatment of younger rats, Choi et al. (5) observed a
decrease in membrane capacitance. This was associated
with a lack of increase in body weight during the 5 weeks
of STZ treatment that indicates a relative cell hypertrophy
in diabetic rats. This is in line with the increase in heart
weight–to–body weight ratio previously reported (23,48).
In summary, we have demonstrated that cardiac con-
tractile dysfunction is closely related to altered Ca2?
signaling as well as altered electrical activity. Therefore, it
has been demonstrated for the first time that altered
parameters of both Ca2?transients and Ca2?sparks are
responsible for the altered Ca2?signaling in diabetes, and
these alterations may be in part associated with not only
less Ca2?loading of SR, but also phosphorylation of RyR2
in diabetes.
ACKNOWLEDGMENTS
This work has been supported by grants from Ankara
University Biotechnology Institute (project no. 2001K120-
240) and the Turkish Scientific and Technical Research
Council (project no. 104S591).
We thank Drs. A.M. Marks and X. Wehrens for the
generous gift of RyR2 and RyR2-P antibodies.
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