(Circulation. 2001;103:2201.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Increased Myocardial GRP94 Amounts During Sustained Atrial Fibrillation
A Protective Response?
From the CNR Unit for Muscle Physiology and Physiopathology (M.V., L.G.), Department of Biomedical Sciences (L.G.), and Chair of Heart Surgery (A.G., D.C.C.), University of Padova, Padova, Italy, and Departments of Physiology (J.A) and Molecular Cell Biology and Genetics (M.B.), Maastricht University, Maastricht, The Netherlands.
Correspondence to Dr Luisa Gorza, Department of Biomedical Sciences, via G. Colombo 3, 35121, Padova, Italy. E-mail lgorza{at}civ.bio.unipd.it
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Abstract |
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Background—Structural and phenotypic changes of cardiomyocytes characterize atrial fibrillation. We investigated whether changes in the glucose-regulated protein GRP94, which is essential for cell viability, occur in the presence of chronic atrial fibrillation.
Methods and Results—Samples of fibrillating atrial myocardium obtained from both goat and human hearts were analyzed for GRP94 expression by an immunologic approach. In goats, atrial fibrillation was induced and maintained for 2, 4, 8, and 16 weeks. After 16 weeks of atrial fibrillation, cardioversion was applied and followed by 8 weeks of sinus rhythm. GRP94 levels doubled in goat atrial myocytes after 4 to 16 weeks of fibrillation with respect to normal atria and returned to control levels in atrial myocardium of cardioverted goats. Immunohistochemical analyses confirm that GRP94 increase occurred within cardiomyocytes. Significantly, increased levels of GRP94 were also observed in samples from human fibrillating atria. In the absence of signs of myocyte irreversible damage, the GRP94 increase in fibrillating atria is comparable to GRP94 levels observed in perinatal goat myocardium. However, calreticulin, another endoplasmic reticulum protein highly expressed in perinatal hearts, does not increase in fibrillating atria, whereas inducible HSP70, a cytoplasm stress protein that is expressed in perinatal goat hearts at levels comparable to those observed in the adult heart, shows a significant increase in chronic fibrillating atria.
Conclusions—Our data demonstrate a large, reversible increase in GRP94 in fibrillating atrial myocytes, which may be related to the appearance of a protective phenotype.
Key Words: fibrillation • atrium • myocytes • sarcoplasmic reticulum • stress
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Introduction |
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Long-term atrial fibrillation (AF) is accompanied by structural changes within viable cardiomyocytes, characterized by loss of myofibrils, presence of glycogen granules, and organelle aggregates both in the human heart and in experimental chronic AF in goats.1 2 Fibrillating atrial myocytes display a fetal cardiomyocyte phenotype, because
-smooth muscle actin immunoreactivity is
detected and immunostaining for titin and cardiotin is
reduced in goat fibrillating
atria,3 and the ß-myosin
heavy chain isoform is expressed in fibrillating human
auricles.4 On the basis of
this evidence, the phenotypic change that occurs in fibrillating atrial
myocytes has been called
dedifferentiation.2 3 Furthermore, fibrillating atrial myocytes display signs of intracellular calcium overload,5 6 possibly because of rapid atrial depolarization and Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR) stores. A thorough cytochemical analysis of calcium distribution in fibrillating goat atria showed increased sarcolemma-bound and mitochondrial calcium deposits after 1 to 2 weeks of fibrillation.6 The increase in intracellular calcium in AF is associated with or followed by reduced accumulation of transcripts and protein for the sarcolemmal L-type Ca2+ channel and SR Ca2+ pump,7 8 whereas the expression of other SR proteins involved in calcium handling, such as calsequestrin, ryanodine receptor, and phospholamban, appears to be unaffected.7
The glucose-regulated protein GRP94 belongs to a class of stress proteins that localize in the endoplasmic reticulum (ER).9 10 It acts as a molecular chaperone11 and is involved in the maintenance of cell survival because it exerts a specific protection against stresses due to Ca2+ depletion from the ER.12 GRP94 is a low-affinity–high-capacity Ca2+-binding protein, a property it has in common with other ER resident proteins, such as calreticulin and protein disulfide isomerase.13 GRP94 is expressed in the SR of adult rabbit cardiomyocytes.14 Although its precise functional role in cardiac myocytes remains to be determined, it is upregulated after exposure to bacterial lipopolysaccharide and during prenatal development.14 SR Ca2+ depletion and consequent intracellular Ca2+ overload have been recognized as stimuli for GRP94 upregulation.9 12 In the present study, we analyzed how GRP94 expression is affected in the presence of AF, using both an experimental model of chronically induced yet reversible AF in the goat and atrial samples from patients suffering from chronic AF.
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Methods |
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Goat Model of Chronic AF
As previously described,15 an Itrel pacemaker (Medtronic) was implanted in the neck of adult female goats under general anesthesia (1% to 2% halothane and N2O). A bipolar screw-in electrode was inserted through the jugular vein and fixed to the right atrial appendage. One week after surgery, the pacemaker was switched on, and AF was induced and maintained by bursts of stimuli (50 Hz, 2 seconds, 4 times diastolic threshold). Because in subsequent days the duration of paroxysms of AF became longer, the interburst period was gradually prolonged.15 Thirty-six goats were included in the present study. They were divided into the following groups: sinus rhythm (n=6); AF of 2, 4, 8, and 16 weeks’ duration (n=6 each); and 8 weeks postcardioversion after 16 weeks of AF (n=6). Animal handling was carried out according to the Dutch law on animal experimentation and the European Directive for Protection of Vertebrate Animals.
At the end of each protocol, the goats were anesthetized, and the left and right atrial appendages were excised. Left ventricular samples and biopsy samples from limb skeletal muscles were taken from the control group. Additionally, atrial and ventricular samples were obtained from 2 prenatal goats (1 to 2 weeks before birth) and 5 neonatal goats (3 days after birth). The samples were frozen immediately after excision in isopentane precooled with liquid nitrogen.
Human Heart Samples
Samples from the left atrial appendage were obtained
from 13 human hearts. Control samples were excised at the time of heart
explantation from 5 healthy individuals who died because of
subarachnoid hemorrhage (mean age±SE 38±2.9 years).
Eight fibrillating atrial samples were provided during cardiac surgery
for mitral valve replacement. Patients (mean age±SE 45±9 years)
displayed sustained AF for >6 months and were undergoing digitalis
treatment. Five of them received additional
calcium-antagonist medication. Each sample was collected
after procedures in accordance with institutional guidelines from the
Heart Surgery Center of the University of
Padova.4 16
Antibodies
Monoclonal antibody 3C4 reacted specifically with
rabbit GRP9414 ; monoclonal
antibody BN-59 reacted with both cardiac and skeletal troponin T of
several different species.17
Monoclonal anti-HSP70 inducible form (SPA810) and polyclonal
anti-calreticulin antibodies (SPA-600) were from Stressgen
Biotechnologies Corp. Anti-desmin antibody (clone DE-B-5) was from
Chemicon. Peroxidase-conjugated antibodies were from BioRad and Dako.
Fluorescein-conjugated antibody was from
Cappel.
Western Blotting
Cryostat sections from tissue samples were
homogenized in electrophoresis sample buffer in the absence
of blue bromophenol. After heating for 5 minutes and
centrifugation at
15 000g for 15 minutes at
4°C, protein concentration was determined as described by Lowry et
al18 with bovine serum
albumin used as
standard.19 Equal amounts of
samples were run either in 6% to 12% gradient or in 10% linear
SDS-polyacrylamide gel together with molecular weight standards
(BioRad) at 5 mA, transferred to nitrocellulose, saturated with
ovalbumin, and incubated with primary antibodies. After being
washed with TBST (10 mmol/L Tris-HCl, pH 8.00, 150 mmol/L
NaCl, 0.05% Tween 20), filters were incubated with
anti-immunoglobulins conjugated with peroxidase and revealed by
diaminobenzidine14 or
chemiluminescence19 when the
same blot was stained for both GRP94 and desmin.
For troponin T cross-linking studies, homogenization was preceded by incubation of sections of control goats with propionate buffer for 30 minutes at 37°C in the presence or absence of 10 mmol/L CaCl2, as described previously.17 20 Additional experiments were performed in the presence of the calpain inhibitor calpeptin (Z-Leu-Nle-OH, Novabiochem).
Quantitative and Statistical
Analysis
Quantitative densitometry of Western blots was
achieved with a Shimadzu chromatoscanner CS-930, as described
previously.14 19
Variability among different experiments was compensated for by use of
the same control sample as internal reference. Sample values were also
normalized to the corresponding densitometric value of desmin
immunoreactivity for control and 4- and 16-week-AF samples. Statistical
analysis was performed with the unpaired Student’s
t test.
Immunocytochemistry
Ten-micrometer cryosections were fixed
for 10 minutes with 4% freshly prepared buffered
paraformaldehyde, rinsed with PBS (10 mmol/L Na
phosphate, 150 mmol/L NaCl, pH 7.4), permeabilized
with 0.1% Triton X-100, and incubated with 3C4 monoclonal
antibody for 1 hour. After being washed in PBS and incubated
with secondary antibodies, peroxidase was revealed with
diaminobenzidine.14 Controls
were performed with nonimmune mouse immunoglobulins. For confocal
microscopy analysis (BioRad MRC 1024ES), a
fluorescein-conjugated secondary antibody was used, with
the addition of 1 µmol/L propidium
iodide.
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Results |
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Goat Fibrillating Atria Display Increased GRP94 Levels
Western blotting showed reactivity of 3C4 antibody with a single polypeptide with the apparent molecular weight of 99 kDa, corresponding to goat GRP94, in both atrial and ventricular homogenates (Figure 1A
). Because we demonstrated that GRP94 is highly
expressed in the heart conduction
system,14 we restricted our
quantitative analysis to regions such as atrial appendages and
left ventricular free wall, where working
myocardium predominates. GRP94 amount did not differ
significantly between atrial and ventricular samples of
control goats
(Table 1). Analysis was extended to left and
right goat atrial appendages obtained at 2, 4, 8, and 16 weeks after
onset of AF. No significant increase in GRP94 amount was observed in
2-week-AF samples, whereas a 2- to 3-fold increase was detected after
4, 8, and 16 weeks of AF
(Figure 1B;
Table 1). Comparable results were obtained when GRP94
content in control and 4- and 16-week AF was normalized for desmin (not
shown). Conversely, samples obtained from goat hearts that were in
normal sinus rhythm for 8 weeks after 16 weeks of AF showed normal
GRP94 levels
(Figure 1C;
Table 1).
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GRP94 is expressed in every heart
cell.14 Immunohistochemistry
revealed that the GRP94 increase detected in 4-, 8-, and 16-week-AF
samples was due to the stronger GRP94 immunoreactivity within
cardiomyocytes
(Figure 2). Conversely, reactivity of cardioverted myocytes
was similar to that of controls
(Figure 2D). Confocal microscopy
(Figure 3) showed that the increase in GRP94 immunoreactivity
was characterized by the presence of a diffuse dotted staining, which
almost masked the striped pattern at the Z-line level detectable in
control
cardiomyocytes.14
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GRP94 Increase, Myocyte Damage, and
Dedifferentiation
Irreversible myocyte damage can determine increased
GRP94 expression. We previously showed that myocyte death due to
intracellular calcium overload or apoptosis is accompanied by
troponin T proteolysis due to calpain and subsequent cross-linking to
myofibrillar proteins by
transglutaminase.17 20
Consequently, Western blot displays high-molecular-weight polypeptides
that are immunoreactive with anti-troponin T antibodies corresponding
to cross-linked troponin T. In vitro experiments, performed by exposing
control goat atrium homogenates to millimolar amounts of
calcium, showed the appearance of 66- and 80-kDa molecular weight
polypeptides that were reactive with anti-troponin T antibodies
(Figure 4A). Coincubation with calpeptin, a calpain
inhibitor, confirmed the specificity of the process (not
shown). Fibrillating goat atrial samples were negative for the presence
of high-molecular-weight, troponin T–immunoreactive polypeptides,
indicating the absence of irreversible damage
(Figure 4B).
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66 and 80 kDa) were observed only in control sample exposed to calcium. Migration of protein Mr standard (97 400; 66 200; 45 000) is indicated.
Reexpression of fetal cardiac proteins appears to be a
feature of fibrillating atrial myocytes. GRP94 expression in goat
newborn atrial and ventricular myocardium is
significantly higher than in the adult myocardium
(Figure 5 and
Table 1
) and comparable to the levels detected in
fibrillating atria. However, when we analyzed calreticulin
expression, we found that it was present in newborn samples,
whereas it was barely detectable in both adult control and fibrillating
samples
(Figure 5).
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We then investigated whether the expression of another
stress protein, inducible HSP70, changes during AF. The amount of
inducible HSP70 increases significantly in fibrillating atria and
returns to control levels in cardioverted atria
(Figure 5 and
Table 2). However, the amount of inducible HSP70 in the
newborn heart did not differ significantly from that observed in the
adult heart
(Figure 5 and
Table 2).
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GRP94 and HSP70 Expression in Human
Fibrillating Atria
We extended the analysis for GRP94 and
inducible HSP70 expression to samples obtained from patients suffering
chronic AF to ascertain whether this condition also affected the
expression of these proteins in the human heart. GRP94 level was
significantly increased in fibrillating atria with respect to controls
(Figure 6 and
Table 1), whereas the amount of inducible HSP70 appeared
reduced
(Figure 6 and
Table 2).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study shows that myocytes from chronic fibrillating atria display approximately a 2-fold increase in GRP94, which returns to the control level after cardioversion. The increase in GRP94 that occurs within cardiomyocytes was observed in an experimental model of AF in the goat and in human samples obtained from patients with chronic AF. The consistent findings outline the relevance of obtaining and characterizing an experimental model of sustained yet electrically reversible atrial fibrillation.
GRP94 level increases significantly in goat samples only
after 
4 weeks of AF, and this occurs in the absence of extensive
irreversible myocyte damage, indicating that the upregulation of the
GRP94 gene is not part of a generic stress response. At present, we
cannot exclude that synthesis of stress proteins may occur very early
after the initiation of AF by burst pacing; nevertheless, the amount of
GRP94 after 2 weeks of AF is not different from the value observed in
control atria. Furthermore, we could rule out death of fibrillating
myocytes as an additional stimulus for a stress response;
consistent with previous observations that did not detect
degenerative changes in fibrillating
myocytes,2 we obtained no
evidence for troponin T cross-linking, which has been characterized as
an early marker for irreversible myocyte damage due to intracellular
calcium
overload.17 20
Dedifferentiation of fibrillating myocytes, namely, the switch to a fetal-like phenotype,3 is a possible explanation for GRP94 increase in AF. Indeed, newborn goat atrial and ventricular myocardium displays higher GRP94 levels than the adult heart, as do fibrillating atrial samples. However, a similar upregulation does not occur for calreticulin, another ER calcium-binding protein that is highly expressed in developing cardiomyocytes.21 Such a finding not only indicates that the dedifferentiation process occurs only partially in fibrillating atrial myocytes but reveals GRP94 as the only SR calcium-binding protein that is increased during chronic AF, because no change was reported for calsequestrin.7 Mechanisms responsible for the GRP94 increase in fibrillating atria remain speculative. Perturbations of ER/SR are followed by increased synthesis of GRP94,9 10 and SR disorganization has been described within myolytic areas of fibrillating goat atrial myocytes2 ; interestingly, a significant increase of myolysis occurred from 4 weeks of AF onward.6 Using confocal microscopy, we now demonstrate that the distribution of GRP94 within the SR/ER is greatly modified in the presence of chronic AF, in conjunction with the appearance of SR disorganization.
In this context, the increased accumulation of GRP94 might have important consequences: it may prevent SR protein aggregation through its chaperoning function9 10 22 and/or may cooperate to restore calcium homeostasis through its calcium binding sites. Fibrillation is accompanied by intracellular calcium overload,23 and consistent ultrastructural changes have been described.5 6 Interestingly, sarcolemmal and mitochondrial calcium deposits, which increased in 1- to 2-week-AF goat atria, slowly returned to control levels in 4- to 8-week-AF samples,6 ie, when the GRP94 amount increased. We presently do not know whether a comparable time course in the recovery of calcium deposits also occurs in human fibrillating atria. Although calcium-blocker treatment may variably affect atrial electrical remodeling,24 25 we report significantly increased GRP94 levels irrespective of the type of therapy. Thus, we speculate that GRP94 may improve survival of fibrillating atrial myocytes due both to chaperoning and calcium-binding properties. To date, GRP94 has been implied in the maintenance of cell viability after exposure to ER calcium depletion or other ER stresses.12 Goat fibrillating atrial myocytes, but not human fibrillating samples, also display significantly higher levels of inducible HSP70, a cytoplasm stress protein that improves myocyte survival after ischemic stress26 27 ; nevertheless, it has been shown that cell viability is maintained even in the absence of HSP70 upregulation if ER stress proteins are increased.28 Although the present study does not provide evidence for the functional consequence of GRP94 increase in AF, preliminary observations reveal that muscle cell lines overexpressing GRP9419 show increased survival with respect to cells with normal levels of GRP94 when exposed to intracellular calcium overload (M. Vitadello and L. Gorza, unpublished data, 2000).
Taken together, our findings suggest that sustained AF induces the appearance of a protective phenotype in myocytes. The increase in GRP94 expression that occurs in both experimental and human chronic AF and the return to control levels after cardioversion in the goat may be part of a cell protective program that can be switched on and off. Such a hypothesis is not in contrast with the dedifferentiated phenotype proposed thus far: embryonic and neonatal hearts not only display higher resistance to ischemic injury with respect to adult hearts29 but are also more calcium tolerant.30
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Acknowledgments |
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This study was supported by grants from MURST (COFIN98 "Lesioni molecolari e metaboliche indotte nel miocardio dalla riperfusione post-ischemica e meccanismi di protezione" to Dr Gorza), by CNR grant No. 98-03045-CT04 to Dr Gorza, and by Giunta Regione Veneto-Ricerca sanitaria finalizzata (grant No. 758/01/97 to Dr Gorza), the Netherlands Heart Foundation (grant No. 96-155 to Dr Ausma), and the Netherlands Organization for Scientific Research (NWO N94-170 to Dr Ausma).
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Footnotes |
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1 Drs Vitadello and Ausma contributed equally to this work.
Received October 24, 2000; revision received November 30, 2000; accepted December 14, 2000.
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