(The FASEB Journal. 2001;15:393-402.)
© 2001 FASEB
Transgene overexpression of 
B crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion
PARTHA S. RAY*,
JODY L. MARTIN
,
ERIC A. SWANSON,
HAJIME OTANI*,
WOLFGANG H. DILLMANN and
DIPAK K. DAS*1
* Cardiovascular Research Center, Department of Surgery, University of Connecticut Health Center, Farmington, Connecticut 06030-1110, USA; and
Department of Medicine, University of California at San Diego, San Diego, California, USA
1Correspondence: Cardiovascular Division, Department of Surgery, 263 Farmington Ave., Farmington, CT 06030-1110, USA. E-mail : ddas{at}neuron.uchc.edu
 |
ABSTRACT
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We investigated whether enhanced expression of 
B crystallin, a
stress-inducible molecular chaperone of the small heat shock family,
can protect myocardial contractile apparatus against ischemia
reperfusion (I/R) injury. Transgenic mice overexpressing B
crystallin were generated using the 0.76 kb rat B crystallin cDNA
cloned into a pCAGGS plasmid driven by a human cytomegalovirus
expression system. Southern analysis confirmed transgene integration
and Northern and Western blotting characterized expression (3.1-fold
and 6.9-fold elevations in myocardial mRNA and protein levels,
respectively). Extent of functional recovery over a 3 h
reperfusion period following a 20 min ischemic period in transgenic and
wild-type mouse hearts was assessed using an ex vivo
work-performing heart preparation. The transgenic group displayed
significantly higher values of DP at R45 min (29.14±1.9 mm Hg vs.
17.6±0.7 mm Hg), R60 min (31.56±1.7 mm Hg vs. 17.8±0.8 mm Hg), and
R75 min (32.5±2.2 mm Hg vs. 16.9±0.9 mm Hg), and of dLVP/dt at R45
min (1740.2±111.5 mm Hg.s-1 vs. 548.7±82.2 mm
Hg.s-1) and R60 min (1199.8±104.6 mm Hg.s-1
vs. 466.9±61.1 mm Hg.s-1). The transgenic group also
displayed development of less oxidative stress, decreased extent of
infarction, and attenuated cardiomyocyte apoptotic cell death.
Transgene overexpression of B crystallin was therefore successful in
diminishing the independent contributory effects of both necrosis and
apoptosis on I/R-induced cell death.—Ray, P. S., Martin, J. L., Swanson, E. A., Otani, H., Dillmann, W. H., Das, D. K. Transgene overexpression of B crystallin confers simultaneous
protection against cardiomyocyte apoptosis and necrosis during
myocardial ischemia and reperfusion.
Key Words: B crystallin • transgenic • ischemia/reperfusion • oxidative stress
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INTRODUCTION
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EVEN THOUGH TREMENDOUS progress has been made in our
understanding of the pathophysiology of myocardial ischemia reperfusion
(I/R) injury, the precise mechanisms underlying the progression of such
cellular injury to ultimate cell death remain far from clear.
Disturbances in calcium and lipid homeostasis, generation of reactive
oxygen species, loss of high-energy phosphates, and the accumulation of
detrimental catabolites have all been implicated and are still believed
to play a crucial role in the pathogenesis of I/R injury
(1
2
3)
. A significant advance in the field of ischemia
reperfusion research came with recent findings that demonstrated that
necrosis and apoptosis are independent contributors to I/R-induced cell
death (4)
. Oxygen free radicals also play a significant
role in I/R-induced cardiomyocyte apoptosis (5
, 6)
.
The last decade has witnessed an explosive growth in research efforts
to understand why cyclic episodes of short durations of ischemia, each
followed by short durations of reperfusion, can protect the heart from
the otherwise potentially lethal effects of a subsequent more severe
ischemic insult (7
8
9)
. This led to the development of the
concept of ischemic preconditioning (IP). Subsequent research revealed
that it is the myocardial adaptation to stress that comprises the
fundamental basis of IP. Thus, a defined amount of any stress,
including oxidative stress and heat shock, can protect the myocardium
from suffering lethal ischemic injury if administered to the heart in
the form of a preconditioning protocol prior to the ischemic duration
of known lethal effect (10
, 11)
. Several reports now
indicate a role of heat shock protein (HSP) 70 in mediating such
protective effects (12
13
14)
. However, recent studies have
challenged these findings with the observation that expression of HSP
70 may not be directly linked with the heat stress-mediated
cardioprotection (14)
.
The potential cardioprotective role of the small heat shock protein
family has begun to receive attention only recently and came with the
observation that phosphorylation of MAPKAP kinase 2 leading to the
phosphorylation of HSP 27 plays a crucial role in IP (15)
.
This observation is further strengthened by the fact that HSP 27 is
involved in cytoskeletal stabilization (16)
and that
cytoskeletal injury plays a crucial role in the pathogenesis of I/R
injury (17)
. Consistent with these findings is a recent
report demonstrating that enhanced expression of either hsp 27 or B
crystallin in cardiomyocytes results in decreased cytosolic enzyme
release after simulated ischemia (18)
. B crystallin is
a member of the small HSP family that shares many properties with HSP
27. Recent studies have demonstrated that B crystallin is the most
abundantly expressed stress protein in the heart (19)
, and
its production is induced by agents that promote the disassembly of
microtubules (20)
. It has also been demonstrated that B
crystallin helps to preserve microtubular integrity in the face of
simulated ischemia in cardiomyocytes (21)
.
The aim of our study was to assess whether the cardioprotective effects
of enhanced expression of B crystallin against cellular ischemic
injury as suggested by in vitro studies are sufficient to
preserve myocardial contractile function and reduce infarct size when
challenged by an ischemic insult. Furthermore, we also sought to
determine whether B crystallin plays a role in attenuating apoptotic
cell death associated with myocardial I/R injury. In the present study,
transgenic mice overexpressing B crystallin were generated and
characterized. An ex vivo isolated murine working heart
model was used to assess postischemic recovery of contractile function.
Coronary perfusate malonaldehyde levels were measured to assess the
extent of oxidative stress and TUNEL assay to assess the extent of
myocardial apoptotic cell death was performed. Results suggest that
transgene overexpression of B crystallin exerted considerable
cardioprotection against I/R injury as evidenced by its ability to
significantly preserve postischemic contractile function and reduce
associated oxidative stress. In addition, it reduced the extent of
infarction associated with such injury, attenuated cardiomyocyte
apoptotic cell death, and thereby diminished the independent
contributory effects of both necrosis and apoptosis on I/R-induced cell
death.
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MATERIALS AND METHODS
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All animals used in this study received humane care in compliance
with the principles of laboratory animal care formulated by the
National Society for Medical Research and Guide for the Care and Use of
Laboratory Animals prepared by the National Academy of Sciences and
published by the National Institutes of Health (publication no. NIH
85–23, revised 1985).
|
Generation of B crystallin transgenic mice
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For construction of transgenic mice, a rat B crystallin
transgene was cloned into pCAGGS plasmid. Expression of rat B
crystallin cDNA was driven by a human cytomegalovirus immediate-early
enhancer linked to the chicken ß-actin promoter, followed by its
first exon and intron (Fig. 1
). The rat B crystallin with its own stop codon and poly(A) signal is
followed by a rabbit ß-globin poly(A) sequence. The mRNA transcript
of the transgene therefore consists of the first exon of chicken
ß-actin, which is transcribed but untranslated, followed by the B
crystallin transgene. To isolate the expression unit of the B
crystallin transgene, the plasmid was cleaved with SalI and
BglII. Standard techniques were used to generate the
transgenic mice (23)
. In brief, the pronucleus of eggs
from superovulated C3H mice crossed with C57/black 6 male mice were
injected with 1–2 pl of the purified DNA fragment at a concentration
of 2 µg/ml. This amount is equivalent to 200 to 400 copies of the
transgenes. The injected eggs were then transferred into the oviduct of
pseudopregnant BalbC mice. Litters were delivered after 
20 days of
gestation.
To determine transgene integration, genomic DNA was extracted from the
tails of 3-wk-old mice and subjected to Southern blot analysis. The
tail DNA was digested with ApaI and PstI,
resolved on an agarose gel, transferred to a nylon membrane, and
hybridized to a 32P-labeled transgene-specific
probe that corresponds to the first chicken ß-actin intron (Fig. 1)
.
ApaI cuts in the chicken ß-actin intron and
PstI cuts 600 bp into the B crystallin cDNA generating a
1.4 kb DNA fragment. The probe is presented by a 0.75 kb
ApaI/XbaI digest releasing a fragment of the
chicken ß-actin intron that specifically hybridizes to the transgene.
From 44 founder animals, 4 transgene positive mice were identified. Two
of the founders were bred into lines B1 and B2. The B1 mice
had the highest expression levels and were used primarily for the
reported studies.
|
Northern and Western blot analysis
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Isolation of RNA was performed as described by Chomezynski et al.
(22)
. For electrophoresis, Northern transfer, and
hybridization, standard protocols were used (23)
. As a
loading standard, GAPDH mRNA was used. The Northern blots were
densitometrically scanned and the ratio of B crystallin over GAPDH
mRNA was expressed. For Western blot analysis, protein extracts were
fractionated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and electrotransferred onto nitrocellulose using a
semidry electrophoresis apparatus (Bio-Rad, Hercules, Calif.). The
nitrocellulose membranes were reacted with polyclonal antibodies
directed against B crystallin or rodent HSP25 (StressGen, Victoria,
B.C., Canada). Protein loading was normalized by using monoclonal
antibodies to constitutive HSP C70 (StressGen) and sarcomeric actin
(Sigma, St. Louis, Mo.). Blots were then processed using the Enhanced
Chemiluminescent kit (Amersham, Arlington Heights, Ill.). Protein and
RNA results were quantitated by scanning the images. The images were
then digitized and analyzed with the NIH image public domain image
analysis software program.
|
Murine ex vivo isolated work-performing heart model
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Wild-type or transgenic B6C3 female mice (n=15)
weighing 25–30 g were anesthetized with sodium pentobarbital (200
mg/kg b.w. intraperitoneal (i.p.) injection, Abbott Laboratories, North
Chicago, Ill.) and anticoagulated with heparin sodium (500 U/kg b.w.
i.p. injection, Elkins-Sinn Inc., Cherry Hill, N.J.). After ensuring
adequate depth of anesthesia, a thoracotomy was performed, and the
heart was excised and immediately immersed in ice-cold perfusion
buffer. The aortic arch was quickly isolated and incised. The aorta was
cannulated and retrograde perfusion in the Langendorff mode through the
aortic cannula was initiated at a perfusion pressure of 60 mm Hg. The
perfusion buffer used in this study consisted of a modified
Krebs-Henseleit bicarbonate buffer [KHB: composed of (in mM) 118 NaCl,
4.7 KCl, 1.2 MgSO4, 25
NaHCO3, 10 glucose, and 1.7
CaCl2, gassed with 95% O2:5%
CO2, filtered through a 5 µm filter to remove
any particulate contaminants, pH 7.4] that was maintained at a
constant temperature of 37°C and gassed continuously for the duration
of the experiment.
Next, the pulmonary venous opening was located. The beveled sharp end
of a PE-50 catheter was inserted through this opening and passed
through the left-atrium, mitral valve and pushed out the apex so that
its fluted end remained in the left ventricular lumen. This catheter
was connected to a pressure transducer to monitor left ventricular
pressure. The pulmonary venous opening was then cannulated with a short
piece of PE-50 tubing and connected to the left atrial inflow line.
Side arms of the aortic and left atrial cannulas were connected to
pressure transducers to permit continuous monitoring of the respective
chamber pressures. At this time, perfusion was switched to the working
heart mode by stopping retrograde perfusion through the aortic cannula,
opening the aortic outflow line and initiating antegrade perfusion
through the left atrial inflow line, in that order. A bubble
trap/pressure chamber containing an air cushion of 1.5 ml was located
in the aortic output line to allow for adequate elastic recoil. The
method followed was essentially the same as that described previously
(24
, 25)
except for a slight modification in that for our
model we used a fixed preload of 15 mm Hg and a fixed afterload of 50
mm Hg maintained by hydrostatic columns. Left ventricular, aortic, and
left atrial pressures (LVP, AOP, and LAP, respectively) were monitored,
analyzed, and recorded in real time using the Digimed data acquisition
and analysis system (Micromed, Louisville, Ky.). Heart rate (HR), left
ventricular (DP) defined as the difference of the maximum, and minimum
left ventricular pressures and maximum positive dLVP/dt were all
derived or calculated from the continuously obtained left ventricular
pressure signal.
|
Perfusion protocol for global ischemia reperfusion
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The preparation was allowed to attain steady-state values of
functional parameters. After a 30 min stabilization period, baseline
functional data were recorded and perfusate samples were collected. The
hearts were then subjected to 20 min of global ischemia by arresting
buffer flow through the left atrial cannula. Reperfusion for the first
10 min began in the retrograde Langendorff mode to allow for
postischemic stabilization. Thereafter, perfusion was switched to the
antegrade working heart mode as described above to allow for monitoring
of functional parameters, which were recorded at 15, 30, 45, 60, 75,
90, 120, 150, and 180 min of reperfusion. Perfusate samples were also
collected at the above time points and at 1, 3, 7, and 10 min of
reperfusion to allow for assessment of oxidative stress in the
immediate-early reperfusion period, when typically it is at its
greatest.
At the end of the 3 h reperfusion period, hearts were removed from
the apparatus and the atrial tissue was dissected away. The ventricles
were either fixed in 10% buffered formalin or immersed in 1%
triphenyl tetrazolium chloride (TTC) solution in phosphate buffer
(Na2HPO4 88 mM,
NaH2PO4 1.8 mM) at 37°C
for 10 min. Hearts kept in formalin were later embedded in paraffin
following standard procedures, and 3 µM thick transverse ventricular
sections were obtained to perform TUNEL assays for the detection of
apoptosis. Hearts immersed in TTC were stored at -70°C for later
processing in infarct size calculations.
Six hearts from the transgenic group and seven hearts from the control
group were excluded from the study on account of low baseline cardiac
contractile performance after the 30 min stabilization period. Three
hearts from the transgenic group and two hearts from the control group
were also excluded as they did not maintain recordable contractile
function until the end of the 3 h reperfusion period.
|
Measurement of malonaldehyde for assessment of oxidative stress
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Malonaldehyde (MDA) was assayed as described (26)
to
monitor the development of oxidative stress during ischemia
reperfusion. Coronary perfusates were collected at the time of
recording baseline functional parameters and thereafter at 1, 3, 5, 7,
15, 30, 60, 90, 120, 150, and 180 min into reperfusion for the
measurement of MDA. MDA in the collected coronary perfusate samples was
derivatized using 2,4-dinitrophenylhydrazine (DNPH); 1 ml of perfusate
was added to 0.1 ml of DNPH reagent (310 mg DNPH in 100 ml 2N HCl, 1.56
mmol DNPH) in a 20 ml TeflonTM lined screw-capped test tube, contents
were vortexed, and 10 ml of pentane was added prior to intermittent
rocking for 30 min. The aqueous phase was extracted three times with
pentane, blown down with N2, and reconstituted in
200 µl of acetonitrile. Aliquots of 25 µl in acetonitrile were
injected onto a Beckman Ultrasphere C18 (3 mm)
column in a Waters HPLC (Waters Corp., Milford, Mass.). The products
were eluted isocratically with a mobile phase containing
acetonitrile-H2 0-CH3COOH
(34:66:0.1, v/v/v) and detected at three different wavelengths of 307
nm, 325 nm, and 356 nm. The peak for malonaldehyde was identified by
co-chromatography with a DNPH derivative of the authentic standard,
peak addition, comparison of the UV patterns of absorption at the three
wavelengths, and gas chromatography-mass spectrometry. The amount of
MDA was quantitated by performing peak area analysis using the Maxima
software program (Waters) and expressed in pmol/ml.
|
TUNEL assay for assessment of apoptotic cell death
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Immunohistochemical detection of apoptotic cells was carried out
using TUNEL in which residues of digoxigenin-labeled dUTP are
catalytically incorporated into the DNA by terminal deoxynucleotidyl
transferase11, an enzyme that catalyzes a template-independent addition
of nucleotide triphosphate to the 3'-OH ends of double- or
single-stranded DNA. The incorporated nucleotide was incubated with a
sheep polyclonal anti-digoxigenin antibody, followed by a
FITC-conjugated rabbit anti-sheep IgG as a secondary antibody as
described by the manufacturer (Apop Tag Plus, Oncor Inc., Gaithersburg,
Md). The sections (n=3) were washed in phosphate-buffered
saline (PBS) three times, blocked with normal rabbit serum, and
incubated with mouse monoclonal antibody recognizing cardiac myosin
heavy chain (Biogenesis Ltd., Bournemouth, U.K.), followed by staining
with TRIRC-conjugated rabbit anti-mouse IgG (200:1 dilution, Dako
Japan; Tokyo, Japan). The fluorescence staining was viewed with a
confocal laser microscope (Olympus Co., Tokyo, Japan).
|
Measurement of infarct size
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Hearts to be used for infarct size calculations (n=3)
were taken upon termination of the experiment, immersed in 1%
triphenyl tetrazolium solution in PBS
(Na2HPO4 88 mM,
NaH2PO4 1.8 mM) for 10 min
at 37°C, and stored at -70°C for later processing. Frozen hearts
(including only ventricular tissue) were sliced transversely in a plane
perpendicular to the apico-basal axis into 0.5 mm thick sections,
blotted dry, placed between microscope slides, and scanned on a
Hewlett-Packard Scanjet 5p single-pass flat bed scanner
(Hewlett-Packard, Palo Alto, Calif.). Using the NIH Image 1.6.1 image
processing software, each digitized image was subjected to equivalent
degrees of background subtraction, brightness, and contrast enhancement
for improved clarity and distinctness. Risk as well as infarct zones of
each slice were traced and the respective areas were calculated in
terms of pixels. The weight of each slice was then recorded to
facilitate the expression of total and infarct masses of each slice in
grams in order to remove the introduction of any errors due to
nonuniformity of heart slice thickness. The individual risk masses and
infarct masses of each slice were summed to obtain the risk and infarct
masses for the whole heart. Infarct size was expressed as a percentage
of the area at risk for any one heart.
|
Statistical analysis
|
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The values for myocardial functional parameters, MDA, risk, and
infarct volumes, and infarct sizes were all expressed as the mean ± standard error of mean (SE). Differences between data
were analyzed for significance by performing a Student’s t
test. The results were considered significant if P<0.05.
|
RESULTS
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Characterization of transgenic mice
Southern blot analysis of mouse tail DNA with a transgene specific
probe identified members of the B 1 line as shown in Fig. 2
. In the hearts of heterozygous B1 mice, B crystallin mRNA levels
were 3.1-fold elevated above the level in control mice. There was a
6.9-fold increase at the protein level. Endogenous B crystallin
expression in the rodent heart is relatively high, with published
protein concentration estimates ranging up to 0.5% of total protein
(27)
. The Western blot of the mouse heart extracts
(Fig. 3
) demonstrates a basal level of closer to 0.1% of total protein in our
studies. The level of transgene expression in the heart was quite high,
especially in lane 1. In addition, significant protein expression
occurred in skeletal muscle: the increase in protein levels was even
more dramatic than that observed in the heart. A 4.8-fold increase was
seen at the RNA level and a 7.9-fold increase at the protein level
(Fig. 4
). Significant increases in protein expression also occurred in the
brain. In other organs like liver, kidney, and spleen, no B
crystallin derived transgene expression could be identified. The level
of HSP25 mRNA or protein was not significantly altered in the B
crystallin transgenic mice in the heart or in the other organs where
transgene expression occurred.
Recovery of myocardial contractile performance
There were no statistically significant differences between the
control and transgenic groups with regard to baseline cardiac function.
Evidence of improved postischemic recovery of myocardial contractile
recovery, however, was clearly apparent in the transgenic group as
early as R15, although statistically significant differences could only
be reported from R30 onward.
The peak LVP (maximum systolic left ventricular pressure) recovered
most dramatically in the transgenic group (Fig. 5
, top), being most prominent at R30 (57.22±3.99 mm Hg vs. 32.68±2.04
mm Hg in control group P<0.05), R45 (69.44±2.67 mm Hg vs.
29.45±2.36 mm in control group, P<0.001), and R60
(60.3±3.19 mm Hg vs. 26.0±1.17 mm Hg in control group
P<0.05). This represented the percent of recoveries of
66.92%, 81.22%, and 70.53% at time points of R30, R45, and R60
compared to baseline peak LVP in the transgenic group whereas in the
control group the corresponding percent recoveries were 41.88%,
37.74%, and 33.32% of baseline peak LVP at the same time points. The
transgenic group continued to have consistently higher values of peak
LVP until termination of the experiment, being significantly different
when compared to values of the control group even at R180. Mean aortic
pressure (Fig. 6
, bottom) closely followed the above trend, being significantly higher
in the transgenic group than in the control group at time points of
R30, R60, and R75 (29.48±1.54 mm Hg vs. 19.73±1.65 mm Hg, 41.78±2.67
mm Hg vs. 17.38±1.32 mm Hg, and 36.48±3.44 mm Hg vs. 16.43±1.18 mm
Hg, respectively). Heart rate (Fig. 7
, top) was preserved closer to baseline values in the transgenic group
for the entire experiment. This was especially apparent at R60
(306.6±11.71 beats/min vs. 203.83±4.60 beats/min in the control
group).
The trends observed with respect to left ventricular developed pressure
(Fig. 5
, bottom) and positive dLVP/dt (Fig. 6
, top) were, however,
different. The transgenic group did display considerable postischemic
recovery of these parameters up until the R60 time point, but was
unable to maintain such recovery thereafter. Significantly different
values for DP were observed at R45 (29.14±1.90 mm Hg transgenic vs.
17.6±0.66 mm Hg control), R60 (31.56±1.65 mm Hg transgenic vs.
17.75±0.84 mm Hg control), and R75 (32.5±2.21 mm Hg transgenic vs.
16.95±0.88 mm Hg control). Significant differences in values of
positive dLVP/dt were demonstrable at R45 (1740.18±111.51 mm Hg/s
transgenic vs. 548.73±82.23 mm Hg/s control) and at R60
(1199.82±104.57 mm Hg/s transgenic vs. 466.85±61.11 mm Hg/s control).
MDA formation
Production of MDA is an index of the occurrence of lipid
peroxidation and the development of oxidative stress. Coronary
perfusate MDA levels were found to be significantly reduced in the
transgenic group as compared to the control group (Fig. 7
, bottom), the
difference being especially apparent at time points of R1 (45.57±7.05
pmol/ml vs. 72.79±8.96 pmol/ml), R7 (43.39±4.17 pmol/ml vs.
161.67±23.11 pmol/ml), and R10 (39.03±4.56 pmol/ml vs. 84.36±5.82
pmol/ml).
Infarct size
Infarct size (percent infarct of total area at risk) was
noticeably reduced in the transgenic group as compared to the control
group (35.41±2.95% vs. 18.59±0.89%) as shown in Fig. 8
.
Apoptotic cell death
Control hearts displayed massive apoptosis as detected with TUNEL
in both cardiomyocytes and noncardiomyocytes. As stated in Materials
and Methods, we used double antibody to specifically stain
cardiomyocytes. Thus, in conjunction with TUNEL, we used monoclonal
antibody recognizing cardiac myosin heavy chain, followed by staining
with TRIRC-conjugated rabbit anti-mouse IgG. The results shown in
Fig. 9A
, C
demonstrate increased apoptotic cardiomyocytes in the
wild-type ischemic reperfused hearts. Apoptotic cardiomyocytes were
rarely observed in B crystallin transgenic mouse hearts (Fig. 9B
, D
).
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|
Figure 9. Double immunofluorescent staining used to demonstrate cardiac myosin
heavy chain (red) and apoptotic nuclei (yellow) using TUNEL in mouse
hearts subjected to 20 min of global ischemia and 180 min of
reperfusion. A, C) Images of control hearts; B,
D) images of B crystallin transgenic hearts. Original
magnification 600x.
|
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|
DISCUSSION
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The results of our study documented that transgenic mice
overexpressing B crystallin are resistant to myocardial ischemic
reperfusion injury. Using the isolated working mouse heart preparation,
we were able to show that the mouse hearts overexpressing B
crystallin had improved postischemic recovery of left ventricular
function compared to wild-type mouse hearts. These observations were
further supported by results that revealed that transgenic mouse hearts
had reduced infarct size and lower incidence of cardiomyocyte apoptosis
compared to wild-type hearts.
The crystallins (A and B) are predominantly present in the
vertebrate lens and are members of the small heat shock protein family
(24)
. Whereas A crystallin is predominantly expressed
in lens, B crystallin mRNA and protein have been found to occur in
high levels in heart, lung, skeletal muscle, and kidney
(28)
. In heart, B crystallin is bound to myocardial
cytoskeletal proteins (29)
, suggesting its role in
cytoskeletal stabilization. Although the actual physiological function
of B crystallin remains unclear, this small heat shock protein
(25–27 kDa family) is known to function as a molecular chaperone in
protein biosynthesis to facilitate protein folding and translocation
(30)
, and undergoes posttranslational modifications
(autokinase activity) (31)
. B crystallin also has a
role in development and differentiation of striated muscle lineages
(32)
.
B crystallin expression can be induced in response to stress stimuli
such as heat shock and oxidative stress (33
, 34)
. Evidence
is rapidly accumulating to support the protective effects of B
crystallin against noxious stresses like hyperthermia and environmental
stress. For example, ectopic expression of B crystallin has been
found to render NIH 3T3 cells thermoresistant (35)
as well
as glioma cells through stabilization of cytoskeletal structure
(36)
. In ischemic rats, B crystallin was found to bind
with the myofibrils, suggesting its cardioprotective role
(29)
. Recovery from exercise was associated with specific
transient changes in the expression of B crystallin, which suggests
that this heat shock protein may have specific roles in the remodeling
process evoked by repeated bouts of contractile activity
(37)
. B crystallin and HSP 25 have been found to be
associated with sarcomeric structures directly after heat shock,
indicating a cytoskeletal protective function (38)
.
Recent studies point to the involvement of B crystallin in mediating
cellular protection against myocardial ischemia reperfusion injury.
Ischemia was found to cause a rapid redistribution of B crystallin
from the cytosolic pool to intercalated disks and Z lines of the
myofibrils, which suggests its role in the protection of myocardial
contractile apparatus against ischemia reperfusion injury
(39)
. This study showed that ischemia-induced
phosphorylation of cardiac B crystallin is accompanied by a complete
shift of this protein from the soluble to insoluble fraction, followed
by its translocation to intercalated disks and myofibrillar Z lines,
two cytoskeletal structures known to become destabilized during
prolonged ischemia. An increased expression of B crystallin through
an adenovirus vector system protected the adult cardiomyocytes against
injury mediated by simulated ischemia (18)
. In another
related study, the same investigators found that microtubular integrity
was significantly better preserved after ischemia in cells
overexpressing B crystallin (40)
. The results of the
present study demonstrating improved postischemic ventricular
contractile function are consistent with these previous reports.
Our study further documented that hearts of the transgenic mice
overexpressing B crystallin had significantly reduced infarct size.
Recent studies indicate that necrosis and apoptosis are two independent
contributors of myocardial infarction (4)
. Reperfusion of
ischemic myocardium has been found to contain a significant number of
apoptotic cardiomyocytes (41)
. However, after 180 min of
reperfusion it is unlikely that apoptotic cells will be TTC positive
(red stain indicating viability). Therefore, it is a realistic
contention that TTC will not stain either necrotic or apoptotic cells
at 180 min reperfusion. Therefore, since there are two contributors to
TTC nonstaining, infarct size (TTC nonstained area) should be greater
in the control group. If an experimental intervention reduces only
necrotic cell death, infarct size in the experimental group should be
less than that observed in the control group, but not by as much as in
a group where the experimental intervention reduces both contributors
to TTC nonstaining: necrosis and apoptosis. This explains why there was
a large difference in the observed infarct sizes between the two groups
in our study and supports our contention that overexpression of B
crystalline confers simultaneous protection against both cardiomyocyte
apoptosis and necrosis.
The ex vivo isolated work-performing heart model was used
primarily to detect differences in cardiac contractile function and
cannot be expected to reveal hemodynamic data comparable to that which
might be obtained from an in vivo model. The main advantage
of the ex vivo model is that it affords the investigator a
means to study effects on cardiac contractile function independent of
innate neurohumoral protective/compensatory mechanisms that stay intact
in an in vivo system. Only after studies using an ex
vivo model can one conclude that cardioprotective effects, if any,
are primarily due to the experimental intervention under consideration.
If only the in vivo system is used to assess such
cardioprotection (even if enhanced postischemic ventricular contractile
function is observed in such a study), it cannot reasonably be
concluded that such cardioprotection is due solely to the experimental
intervention and has no contribution from the aforementioned innate
protective/compensatory mechanisms. For this reason we used the
ex vivo model. Furthermore, there is deterioration over time
in the contractile function of such an ex vivo heart
preparation (regardless of whether we use mouse or rat heart) even
under control conditions in the absence of an ischemic insult. It is
therefore the accepted standard that cardioprotection in the form of
enhanced contractile function, if any, can be reliably detected only in
the early to midreperfusion times when using such a model.
This is why, in the late reperfusion period in an ex vivo
isolated working heart model, the functional parameters measured do not
necessarily parallel myocyte cell viability at 180 min reperfusion. We
do not know whether there is greater apoptotic cell death occurring in
the control hearts above and beyond the 40% infarction in control.
Even if it is occurring in control hearts and is being prevented in the
B crystallin transgenic hearts, this increased cell viability cannot
be expected to be reflected in functional measurements so late in the
reperfusion period in an ex vivo isolated working heart
model due to the deficiencies in the model outlined above. The only way
to confirm this is in an in vivo model, which can
potentially constitute the subject of future studies.
Cardiomyocyte apoptosis appears to occur in a variety of mammalian
species including mouse, rat, rabbit, and pig (42
43
44)
.
Reactive oxygen species appear to play a crucial role in cardiomyocytes
apoptosis, because antioxidants such as superoxide dismutase or ebselen
can reduce the number of apoptotic cells significantly
(45)
. In another related study, when subjected to
transgenic mice overexpressing GSHPx-1 were found contain minimal
number of apoptotic cardiomyocytes whereas the knockout mice devoid of
any copy of GSHPx-1 contained significantly higher number of apoptotic
cells compared to the wild-type mouse hearts (46)
. The
results of the present study showed very few apoptotic cardiomyocytes
and nonmyocyte cells in transgenic mouse hearts subjected to
ischemia/reperfusion compared with the wild-type mouse hearts.
Although molecular characterization of the B crystallin transgenic
mice suggested expression primarily in parenchymal tissues such as
cardiac myocytes and not diffusely in other tissues, the findings of
the TUNEL assay suggested that such overexpression was successful in
preventing apoptosis in both cardiac myocytes and nonmyocytes. Possible
reasons might include the general reduction in reactive oxygen
species/oxidative stress as evidenced by the reduced formation of MDA
(thereby reducing the occurrence of apoptosis in both myocytes and
nonmyocytes equally) or the reduced secretion from myocytes of a
potential paracrine proapoptotic mediator. Although the reduction in
oxidative is probably potentially responsible for the observation of
reduced apoptotic cell death, the possibility of reducing the release
of a potential paracrine proapoptotic mediator from myocytes cannot be
ruled out. Nevertheless, we used double antibody to specifically stain
cardiomyocytes. Thus, in conjunction with TUNEL, we used monoclonal
antibody recognizing cardiac myosin heavy chain, followed by staining
with TRIRC-conjugated rabbit anti-mouse IgG. Thus, our results
correctly reflect increase in apoptotic cardiomyocytes in the control
ischemic reperfused hearts. We have certainly identified apoptotic
cardiomyocytes and nonmyocytes in the control ischemic reperfused
myocardium, but our interpretation is based on the results of apoptotic
myocytes.
In concert, reduced amount of MDA formation was found in the transgenic
mouse hearts overexpressing B crystallin subjected to ischemia and
reperfusion. A large number of studies support that reactive oxygen
species are generated during ischemia and reperfusion leading to the
development of oxidative stress (47)
. Our HR demonstrated
that B crystallin overexpression in hearts reduces the formation of
oxygen-derived free radicals during ischemia and reperfusion as
evidenced by reduced MDA formation, a presumptive marker for oxidative
stress (48)
.
MDA levels were elevated in the hearts of wild-type mice subjected to
ischemia reperfusion, the elevation being most marked in the early
reperfusion period, which is consistent with what is generally observed
in the working heart model (for both mouse and rat heart). Our data
clearly demonstrate that MDA levels in the hearts of wild-type mice
stay significantly elevated above baseline levels even after 180 min of
reperfusion. Thus, the MDA findings suggest more than just short term
protection/stunning of the myocardium since, in the B crystallin
group, MDA levels were maintained at or near baseline levels throughout
the course of the experiment.
In summary, our results demonstrated that transgenic mouse hearts
overexpressing B crystallin are resistant to ischemic reperfusion
injury compared to those from wild-type mice as evidenced by improved
postischemic ventricular contractile function and reduced infarct size.
Out results also showed for the first time that B crystallin
possesses an antiapoptotic role because overexpression of this protein
in the hearts prevented both myocytes and nonmyocytes apoptosis.
Reduction of oxidative stress in the ischemic reperfused myocardium
suggests that B crystallin may reduce cardiomyocyte apoptosis by
virtue of its ability to reduce oxidative stress.
|
ACKNOWLEDGMENTS
|
|---|
This study was supported in part by NIH HL 34360, HL 22559, HL
56803, and HL 49434.
Received for publication March 27, 2000.
Revision received July 14, 2000.
|
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TOP
ABSTRACT
INTRODUCTION
and 
indicate performance in control and 
and