(Circulation. 2001;103:1787.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Combined and Individual Mitochondrial HSP60 and HSP10 Expression in Cardiac Myocytes Protects Mitochondrial Function and Prevents Apoptotic Cell Deaths Induced by Simulated Ischemia-Reoxygenation
From the Division of Endocrinology and Metabolism, Department of Medicine (K.M.L., B.L., I.Y.L., W.H.D.), and the Department of Biology (I.E.S.), University of California, San Diego; and the Department of Physiology, Loyola University, Maywood, Ill (R.M.).
Correspondence to Wolfgang H. Dillmann, MD, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0618. E-mail dillmann{at}ucsd.edu
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Abstract |
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Background—The mitochondrial heat-shock proteins HSP60 and HSP10 form a mitochondrial chaperonin complex, and previous studies have shown that their increased expression exerts a protective effect against ischemic injury when cardiac myocytes are submitted to simulated ischemia. The more detailed mechanisms by which such a protective effect occurs are currently unclear. We wanted to determine whether HSP60 and HSP10 could exert a protection against simulated ischemia and reoxygenation (SI/RO)–induced apoptotic cell death and whether such protection results from decreased mitochondrial cytochrome c release and caspase-3 activation and from the preservation of ATP levels by preservation of the electron transport chain complexes. In addition, we explored whether increased expression of HSP60 or HSP10 by itself exerts a protective effect.
Methods and Results—We overexpressed HSP60 and HSP10 together or separately in rat neonatal cardiac myocytes using an adenoviral vector and then subjected the myocytes to SI/RO. Cell death and apoptosis in myocytes were quantified by parameters such as enzyme release, DNA fragmentation, and caspase-3 activation. Overexpression of the combination of HSP60 and HSP10 and of HSP60 or HSP10 individually protected myocytes against apoptosis. This protection is accompanied by decreases in mitochondrial cytochrome c release and in caspase-3 activity and increases in ATP recovery and activities of complex III and IV in mitochondria after SI/RO.
Conclusions—These results suggest that mitochondrial chaperonins HSP60 and HSP10 in combination or individually play an important role in maintaining mitochondrial integrity and capacity for ATP generation, which are the crucial factors in determining survival of cardiac myocytes undergoing ischemia/reperfusion injury.
Key Words: ischemia • apoptosis • reperfusion
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Introduction |
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Myocardial ischemia and the resulting myocardial infarct are a major cause of mortality and morbidity. More recent findings indicate that cardiac myocytes die by distinct mechanisms, necrosis or apoptosis. Necrosis may be the earliest and most prominent mode of cell death in the infarcted tissues. Apoptotic cardiac myocytes are observed later in the areas of infarct and the surrounding tissues. Myocyte cell death, possibly apoptosis, has been related to the cause of subsequent heart failure among patients with coronary artery occlusions.1 2 It has been shown in in vitro cell culture or in vivo animal models of hypoxia/ischemia with or without reoxygenation that apoptosis occurs in cardiac myocytes.3 One of the major mechanisms for the initiation of apoptosis is provided by the release of mitochondrial factors, such as cytochrome c (cytoC) and precursors of caspases, into the cytoplasm.4 As a result, degradation of cellular proteins, cell shrinkage, and genomic DNA fragmentation occur as the characteristics of apoptosis.5
The mitochondrial chaperonins composed of heat-shock protein 60 (HSP60) and HSP10 are the major sites of protein folding in mitochondria. On the basis of the well-studied Escherichia coli model, GroEL (HSP60 from E coli) and GroES (HSP10 from E coli), the structure and details of the folding/unfolding cycle of chaperonins have become clear, although some mammal-specific functions remain unexplored.6 7 8 Two ring-like structures composed of 7 HSP60 subunits capped with 1 complex of 7 HSP10 subunits form a bell-shaped chaperonin unit. The requirement for both HSP60 and HSP10 to complete the tasks of folding proteins has prompted our group to generate an adenoviral vector simultaneously overexpressing HSP60 and HSP10. Overexpressing both HSP60 and HSP10 protected rat neonatal cardiac myocytes and H9C2 cells against simulated induced ischemia injury (SI)9 ; the mechanism of protection by HSP60 and HSP10, however, is unclear. In the present study, we wanted to determine whether individual HSP60 or HSP10 complexes in addition to the combined HSP60/10 complex exert a protection against SI/reoxygenation (RO) in cardiac myocytes. Our results suggest that HSP60 and HSP10 in combination prevent apoptotic cell death induced by SI/RO very effectively. HSP60 or HSP10 by itself, however, also exerts a protection on certain parameters. The chaperonins appear to mediate their protective effects by maintaining mitochondrial function and integrity.
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Methods |
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Cell Culture
Neonatal rat cardiac myocytes were cultured and infected with adenoviral vectors expressing HSP60, HSP10, HSP10/60, and inducible HSP70 (HSP70i) as previously described.9 10 The infection protocol was similar to that described previously.9
Lactate Dehydrogenase Release Assay
LDH activity released to the medium or remaining in
the cells was determined with an LDH kit (Sigma Diagnostics).
Cytoplasmic enzyme released was shown as a percentage of LDH activity
in the medium over the total enzyme activity (medium + remaining
activity in the cells). The LDH release by SI/RO is determined as the
difference in LDH release in cells kept as control and in cells after
SI/RO.
Flow Cytometry and TUNEL Assay
A kit using a 2-color staining method for
labeling DNA breaks and total cellular DNA by flow cytometry was used
to study apoptosis (Apo-BRDU kit, Phoenix Flow System). Apoptotic cells
were labeled with bromodeoxyuridine triphosphate nucleotides (Br-dUTP)
followed by detection with fluorescein-labeled anti-BrdU monoclonal
antibody. Total cellular DNA was stained with propidium iodide.
The percentage of BrdU-stained cells in the whole population was then
analyzed by a flow cytometer (Beckman Dickinson) and was designated as
terminal deoxynucleotidyl transferase–mediated dUTP nick
end-labeling (TUNEL)–positive or apoptotic
cells.
Cytoplasmic DNA Fragments Determined by
ELISA
The cytoplasmic DNA fragments in apoptotic myocytes
were detected with a kit (Boehringer Mannheim 1774425) that detects
histone-associated DNA fragments in the cytoplasmic portion of cell
lysates. In short, the cytoplasmic portions of cell lysates were
incubated for 2 hours in a streptavidin-coated microtiter plate with a
monoclonal biotinylated anti-histone antibody and a
peroxidase-conjugated anti-DNA antibody. After unbound antibodies had
been washed off, the amount of DNA fragments was quantified by the
peroxidase retained in the immunocomplex. Peroxidase was determined
photometrically (405/490 nm) with ABTS as
substrate.
Caspase-3 Activity Assay
The caspase-3 activity within the cells was measured
with a caspase-3 colorimetric assay kit (Clontech K2027-2). In short,
the caspase-3–specific substrate DEVD-pNA was incubated with 20 µg
of myocyte lysates at 37°C for 1 hour, and the cleaved product was
measured by a spectrophotometer at 405 nm. The specificity of the
cleavage reaction was verified by including DEVD-CHO (caspase-3
inhibitor) 10 µmol/L in the incubation, and <10% of activity was
detected.
Protein Analysis
In
Figure 1
, rat neonatal cardiac myocytes infected with
AdvSR-, AdvHSP60/10, AdvHSP60, or AdvHSP10 were lysed by RIPA buffer
(Santa Cruz), and proteins were separated by SDS-PAGE with 4% to 20%
gradient gel followed by Western blot. Anti-HSP60 and anti-HSP10
antibodies were from Stressgene. In
Figure 5, mitochondrial proteins and mitochondrion-free
cytoplasmic proteins were separated as described
previously5 with minor
modifications.
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ATP Measurement
The cellular ATP concentration was measured by a
modified luciferase assay protocol. Two million cells were lysed by
addition of 500 µL lysis buffer containing 0.5% Triton X-100. The
average protein concentration in cell lysates from control conditions
was between 0.8 and 1 mg/mL, and no significant difference was observed
among samples with various virus infections. The substrate buffer
contained luciferin (10 mmol/L). Aliquots of 100 µL substrate buffer
were ejected automatically by a luminometer into the assay tube
containing luciferase 5 µL (0.0125 mg/mL) and 10 µL of cell lysate.
The integrated light output was measured for 1 second. Samples with
known ATP concentrations were also prepared and measured to generate
the standard curves for the assay.
Polarography to Measure Complex I, Complex III,
and Complex IV Activity
The activity of complex I, complex III, or complex IV
was measured according to a previously described
protocol11 with modification
as noted. Adenovirus-infected myocytes before or after 5 hours of
simulated ischemia were trypsinized and permeabilized by incubation
with digitonin (10 µg/mL for 10 minutes) or until 
95% of the cells
were permeabilized. Cell number and protein concentration in each
sample were counted and measured. Samples with equal amounts of
proteins were divided into aliquots and stored at -80°C. After
ischemia, it took 
3 hours to finish the above procedures, and this
period was considered a period of reoxygenation. These samples (500
µg each) were used later to measure the activity of the complexes.
The complex I, complex III, and complex IV activity was defined as the
rate of oxygen consumption in the presence of the specific substrates
glutamate/malate for complex I, succinate for complex III, and
ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine for
complex IV and was calculated as the fraction that was sensitive to the
inhibitors rotenone for complex I, antimycin for complex III, and
sodium cyanide for complex IV.
Statistics
ANOVA was performed in all comparisons, followed by
post hoc analysis. P values
were determined by Fisher’s protected least significant difference
method. The statistical software used was StatView. All error bars are
SD.
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Results |
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Increased HSP60/10, HSP60, and HSP10 Expression Protects Against SI/RO Damage Determined by LDH Release
One of the aims of our studies was to determine whether HSP60 or HSP10 by itself could exert a protective effect against SI/RO-induced injury. In our protocol, rat neonatal cardiac myocytes were infected with adenovirus constructs expressing equal amounts of HSP60 and HSP10, HSP60 only, and HSP10 only. Figure 1A
demonstrates the level of expression of HSP60 and
HSP10 in myocytes 48 hours after adenoviral infection. Both HSP60 and
HSP10 were increased in myocytes infected with AdvHSP60/10. Only HSP60
was increased by AdvHSP60 infection, and HSP10 was increased by
AdvHSP10 infection. AdvSR- was the control virus without transgene
expression.
After SI/RO, the LDH activity in the medium was 60±12% of
total LDH activity in the cells infected with control virus (AdvSR-),
compared with only 5±2% of total LDH activity found in the medium
with untreated cells for the same period of time (data not shown). As
shown in
Figure 1B, overexpression of HSP60 (AdvHSP60) decreased the
amount of LDH released after SI/RO to a level of only 49% of the
release found in AdvSR--infected cells. Overexpression of HSP10
(AdvHSP10) decreased the LDH release further, to only 23% of that in
AdvSR- after SI/RO. Simultaneous overexpression of both HSP10 and
HSP60 (AdvHSP60/10) resulted in a decrease in LDH release to only 35%
of that in AdvSR-. Judged by the mean values, overexpression of HSP10
seemed to be most effective in reducing the LDH releases after SI/RO;
this protection, however, was not significantly different from that
with overexpression of HSP60 or HSP60/10 together. The protection by
another major HSP, HSP70i, against SI/RO was also examined.
Overexpression of HSP70i was less protective than overexpression of the
mitochondrial HSP60 and HSP10 against SI/RO, because it decreased the
LDH release to only 66% of that with AdvSR-.
Preservation of Respiratory Enzymes by
Overexpression of HSP60 and HSP10
Generation of ATP in mitochondria by ATP synthase
(F1F0-ATPase) is coupled
to the electron transport chain (ETC), in which NADH is oxidized and
oxygen is converted into water. The ETC is composed of NADH-ubiquinone
oxidoreductase (complex I), ubiquinol cytoC oxidoreductase (complex
III), and cytoC oxidase (complex IV). By polarograph, we measured the
activity of complex I, III, and IV in myocytes infected with AdvSR-,
AdvHSP60/10, AdvHSP60, or Adv-HSP10 before and after SI/RO. As
shown in
Figure 2, complex I activity in myocytes was not changed by
overexpression of HSP60 and/or HSP10 before or after SI/RO. Before
SI/RO, complex III activity was significantly higher in myocytes
infected with AdvHSP60/10 and AdvHSP60. Overexpression of HSP10 in
myocytes did not increase the complex III activity. Complex IV is the
complex with the greatest oxidation capacity in the ETC. Overexpression
of HSP60 alone increased the complex IV activity by 50% compared with
AdvSR--infected myocytes, whereas overexpression of both HSP60 and
HSP10 or HSP10 alone did not significantly increase the complex IV
activity
(Figure 2A). After SI/RO, complex III activity was increased
in cells overexpressing HSP60 and HSP10 by 30% and was not changed by
overexpression of HSP60 or HSP10 alone. The complex IV activity was
greater in cells overexpressing HSP60 and HSP10 or HSP60 alone and was
less in cells overexpressing HSP10 alone
(Figure 2B). Taken together, our data suggest that (1)
overexpression of HSP60 alone increases both the complex III and
complex IV activity. Overexpression of HSP10 alone does not have a
beneficial effect on these enzymes; nevertheless, it attenuates the
increases in complex IV activity by HSP60. (2) The beneficial effect of
coexpression of HSP60 and HSP10 on complex III becomes evident after
SI/RO, and the detrimental effect of HSP10 on complex IV is also
amplified after SI/RO.
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Preservation of ATP Levels by HSP60/10
Overexpression
Oxidative phosphorylation and the formation of ATP are
essential functions of mitochondria. We therefore determined potential
protective effects of HSP60 and HSP10 on ATP levels in myocytes
subjected to 8 hours of simulated ischemia followed by 4 and 8 hours of
reoxygenation. It is interesting to note that control myocytes not
subjected to SI but infected with AdvHSP60/10 versus myocytes infected
with control adenovirus (AdvSR-) showed a 30% increase in ATP
levels. Myocytes infected with adenovirus expressing only HSP60 or
HSP10 had ATP levels not significantly different from those of the
AdvSR- group
(Figure 3). Myocytes exposed to an 8-hour period of ischemia
showed an 80% drop in ATP levels. Increased expression of HSP60/10
or HSP10 did not influence the ischemia-mediated decline in ATP. In
HSP60-overexpressing cells, ATP levels were slightly but significantly
higher than those in other groups. After 4 hours of reoxygenation, ATP
levels showed minimal but significant recovery in AdvHSP60/10-infected
myocytes but not in other groups. A reoxygenation period of 8 hours
showed a significant increase in ATP levels primarily in the
HSP60/10-overexpressed cells.
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Apoptosis of Cardiac Myocytes Induced by SI/RO
Is Decreased by HSP60 and HSP10 Expression
To determine apoptosis in cardiac myocytes after SI/RO,
an ELISA-based assay for cytoplasmic DNA fragments was used. Ischemia
for 8 hours followed by 4-hour reoxygenation resulted in a >10-fold
increase in ELISA signal for cytoplasmic DNA fragments compared with
the untreated condition in AdvSR--infected cardiac myocytes (data not
shown). As shown in
Figure 4A, after the same SI/RO episodes, overexpression of
HSP60 and HSP10 together, HSP60 by itself, and HSP10 by itself resulted
in 23%, 44%, and 35%, respectively, of the amount of cytoplasmic DNA
fragments seen in cells infected with the control virus.
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To confirm the protection against apoptosis by simultaneous
expression of HSP60 and HSP10, a flow cytometry–based TUNEL assay was
used to measure myocyte apoptosis. In myocytes infected with AdvSR-,
the percentage of myocytes that stained positive in TUNEL assay
increased from 3% under control conditions to 24% of total cells
after simulated ischemia followed by 24 hours of reoxygenation
(Figure 4B). In myocytes infected with AdvHSP60/10, 16% of
cells became TUNEL-positive after SI/RO, which represented a decrease
of 40% in apoptosis by AdvHSP60/10 compared with AdvSR-infected
myocytes
(Figure 4B).
Decrease in cytoC Release and Caspase-3
Activation by Overexpression of HSP60 and HSP10 in Myocytes Under
SI/RO
The release of mitochondrial cytoC into the cytosol has
been shown to be involved in triggering the apoptotic program. We
therefore determined the release of cytoC by Western blotting of the
mitochondrion-free cytosolic proteins collected from myocytes with
altered chaperonin expression submitted to SI/RO.
Figure 5A summarizes the results from 4 experiments. By
taking the cytoC from the cytosolic fraction of AdvSR--infected cells
after SI/RO as 100%, the cytoC was found minimally in the cytosolic
fractions of all 4 virus-infected myocytes before SI/RO. After ischemia
for 8 hours, the cytoC was greatly increased in all virus-infected
groups. Only sole expression of HSP60 lowered the ischemia-initiated
cytoC release, compared with that from the AdvSR--infected group.
Reoxygenation for 4 hours after ischemia almost doubled the initial
cytoC release in AdvSR--infected myocytes. Overexpression of HSP60
and HSP10 together or HSP10 alone decreased the cytoC release after
reoxygenation by 20%, which suggests a specific protection against
reoxygenation-induced injury. Although after SI/RO the cytoC release in
myocytes with expression of HSP60 alone was significantly less than
that in the AdvSR--infected group, HSP60 overexpression appeared not
to be effective in lowering the cytoC release induced by reoxygenation.
Expression of only HSP10 in myocytes seemed most effective in reducing
cytoC release after reoxygenation.
Activation of caspase-3 has been found in many models of
apoptosis and plays an important role as a downstream event of cytoC
release. We therefore determined the caspase-3 activity in myocytes by
an in vitro substrate-cleaving reaction. After 8-hour ischemia, there
was a 2.8-fold increase of caspase-3 activity from control conditions
in myocytes infected with AdvSR-. Overexpression of HSP60 and HSP10
together or separately did not significantly reduce the
ischemia-induced caspase-3 activation
(Figure 5B). The subsequent reoxygenation period after
ischemia further increased the caspase-3 activity. After 4-hour
reoxygenation, the increases in caspase-3 activity were 6.5-fold in
AdvSR--infected, 3.9-fold in AdvHSP60/10-infected, 2.9-fold in
AdvHSP60-infected, and 4.2-fold in AdvHSP10-infected myocytes.
Caspase-3 activation occurs primarily during reoxygenation, and HSP60
or HSP10 by itself exerts an inhibitory effect on caspase-3 activity
equal to that seen with the HSP60/10
combination.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Cardiac myocytes are specifically rich in mitochondria to generate the high amount of ATP needed for normal cardiac contractile function. To maintain the protein members of the oxidative phosphorylation cascade in a normal folding state, a specific set of mitochondrial HSPs, especially HSP60 and HSP10, is used to form the chaperonin complex for efficient protein folding. The majority of mitochondrial proteins are nucleus-encoded and need to be refolded after mitochondrial import by use of the chaperonin complex made by HSP60 and HSP10. The role that HSP60 and HSP10 play in helping myocytes to recover from SI/RO injury is only incompletely explored. A general protection of combined expression of HSP60 and HSP10 in cardiac myocytes against ischemic injury has been demonstrated.9 It is currently unclear, however, whether increased expression of HSP60 and HSP10 by themselves exerts a protection, whether this protection results in better preservation or recovery of ATP levels after an ischemia-reperfusion episode, and whether the protection includes prevention of apoptotic cell death. Our results provide answers to these questions with the demonstration that HSP60 and HSP10 by themselves have a protective effect in addition to the HSP60/10 combination. Part of the protective mechanism is due to better preservation and accelerated recovery of ATP levels after SI/RO. In addition, protection against apoptotic cell death occurs that may be mediated by decreased release of cytoC from mitochondria. The beneficial effect that HSP60/10 or HSP60 and HSP10 individually exert on different cellular events linked to SI/RO injury will be discussed in more detail below.
Myocardial ischemia is frequently followed by reperfusion
because of therapeutic interventions leading to the opening of occluded
cardiac arteries. Reperfusion and the resultant reoxygenation lead to
the generation of oxygen radicals that can by themselves cause
myocardial damage called reperfusion injury. We used a myocyte culture
model of SI/RO to mimic the in vivo events. It is interesting to note
that LDH release as a marker of general myocyte injury was markedly
reduced in myocytes undergoing SI/RO, with increased expression of the
combination of HSP60 and HSP10, but also in myocytes expressing only
increased amounts of HSP60 or HSP10 alone
(Figure 1). Previous studies in yeast have shown that HSP60
or HSP10 by itself can influence the folding state of a certain subset
of proteins.12 13
It has also been postulated that the sequestered space formed by a
ring-like structure composed of only HSP60 could serve as a protective
holding reservoir for proteins during noxious cell
stress.7
In this report, we specifically identified protection of
increased expression of HSP60/10 or HSP60 as well as HSP10 by itself
against apoptotic cell death. This protective effect can be linked to
decreased release of cytoC from mitochondria to the cytoplasm
(Figure 5). CytoC has a well-established role of combining
with Apaf1, leading to caspase-9 activation and thus triggering the
apoptotic program.14 In our
study, we found decreased cytoC release, decreased caspase-3 activity,
and decreased DNA fragmentation measured by TUNEL and ELISA by
overexpressing HSP60 and HSP10. The cell death program is complicated
and can be elicited by different
pathways14 ; cell death
induced by SI/RO is an even more complex event. Our data from rescuing
myocytes by overexpressing chaperonins against SI/RO serve as an
example of this complexity. For some parameters, eg, LDH release and
cytoC release, sole HSP10 overexpression appeared most protective, but
for other parameters, such as caspase-3 activity and DNA fragmentation,
HSP10 protection was not better, if not worse, than combined
HSP60/HSP10 expression. Generally, expression of HSP60 alone is most
protective against ischemia in most parameters, and expression of HSP60
and HSP10 in combination protects better after a period of
reoxygenation.
Several mechanisms can be involved by which increased expression of the mitochondrial chaperonins leads to decreases in cytoC release. Higher ATP levels occur when cardiac myocytes with increased expression of HSP60/10 are submitted to SI/RO. In such HSP60/10-overexpressing myocytes, we also observed a specific protective effect on individual complexes of the ETC. The activity of complex I that contains the largest amount of individual proteins was not influenced by increased mitochondrial chaperonin expression. In contrast, the activities of complex III and complex IV were significantly higher in HSP60- or HSP60/10-overexpressed myocytes, and the protection of complex III and IV was also evident in these cells after SI/RO. Whether better preservation of ETC and ATP synthesis leads to a smaller amount of cytoC leaving the mitochondrial intermembrane space is still unclear. Our data suggest a relation between mitochondrial function (ETC) and integrity (cytoC release). HSP60 and HSP10 are localized in the mitochondrial matrix, whereas cytoC is positioned at the intermembrane space outside the mitochondrial matrix. A direct physical contact between the HSP60/10 ring-like structure and cytoC therefore appears unlikely, and intermediate molecules, such as those that are part of the ETC, will be necessary to lead to less cytoC release after SI/RO.
It should also be noted that a recent article demonstrated that overexpression of HSP60 results in an increase in apoptosis in Jurkat cells.15 Our present results obtained from myocytes seem at odds with the previous finding. The effect of overexpression of mitochondrial chaperonins (HSP60) may be different in myocytes from that seen in T cells, such as Jurkat cells, which are prone to go into apoptosis. In contrast, cardiac myocytes are particularly rich in mitochondria but relatively resistant to apoptosis. Thus, it is possible that HSP60 and HSP60/10 may be particularly protective in myocytes against apoptosis induced by SI/RO while being proapoptotic in cells already destined to apoptosis.
In summary, our results show that overexpression of HSP60 and HSP10 individually in addition to combined expression of HSP60 and HSP10 results in protection of cardiac myocytes against apoptotic cell death induced by SI/RO. Preservation of oxidative phosphorylation, accelerated ATP recovery after SI/RO, decreased cytoC release, and caspase-3 activation indicating a better preservation of mitochondria by mitochondrial chaperonins contributed to this beneficial effect.
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Acknowledgments |
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This work was supported by NIH grant HL-49434. Dr Lin is the recipient of an NIH postdoctoral fellowship. We would like to thank Denis Rogers for secretarial assistance.
Received August 9, 2000; revision received October 19, 2000; accepted October 19, 2000.
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 A. Y. W. Chang, J. Y. H. Chan, J. L. J. Chou, F. C. H. Li, K.-Y. Dai, and S. H. H. Chan Heat shock protein 60 in rostral ventrolateral medulla reduces cardiovascular fatality during endotoxaemia in the rat J. Physiol., July 15, 2006; 574(2): 547 - 564. [Abstract] [Full Text] [PDF]
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N. C Chi and J. S Karliner Molecular determinants of responses to myocardial ischemia/reperfusion injury: focus on hypoxia-inducible and heat shock factors Cardiovasc Res, February 15, 2004; 61(3): 437 - 447. [Abstract] [Full Text] [PDF]
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 A. Ramachandran, E. Ceaser, and V. M. Darley-Usmar Chronic exposure to nitric oxide alters the free iron pool in endothelial cells: Role of mitochondrial respiratory complexes and heat shock proteins PNAS, January 6, 2004; 101(1): 384 - 389. [Abstract] [Full Text] [PDF]
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L. Argaud, A.-F. Prigent, L. Chalabreysse, J. Loufouat, M. Lagarde, and M. Ovize Ceramide in the antiapoptotic effect of ischemic preconditioning Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H246 - H251. [Abstract] [Full Text] [PDF]
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Y.-x. Shan, T.-L. Yang, R. Mestril, and P. H. Wang Hsp10 and Hsp60 Suppress Ubiquitination of Insulin-like Growth Factor-1 Receptor and Augment Insulin-like Growth Factor-1 Receptor Signaling in Cardiac Muscle: IMPLICATIONS ON DECREASED MYOCARDIAL PROTECTION IN DIABETIC CARDIOMYOPATHY J. Biol. Chem., November 14, 2003; 278(46): 45492 - 45498. [Abstract] [Full Text] [PDF]
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 S. Hirono, E. Dibrov, C. Hurtado, A. Kostenuk, R. Ducas, and G. N. Pierce Chlamydia pneumoniae Stimulates Proliferation of Vascular Smooth Muscle Cells Through Induction of Endogenous Heat Shock Protein 60 Circ. Res., October 17, 2003; 93(8): 710 - 716. [Abstract] [Full Text] [PDF]
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C. Depre, L. Wang, J. E. Tomlinson, V. Gaussin, M. Abdellatif, J. N. Topper, and S. F. Vatner Characterization of pDJA1, a cardiac-specific chaperone found by genomic profiling of the post-ischemic swine heart Cardiovasc Res, April 1, 2003; 58(1): 126 - 135. [Abstract] [Full Text] [PDF]
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K. J Ashton, K. Holmgren, J. Peart, A. R Lankford, G Paul Matherne, S. Grimmond, and J. P Headrick Effects of A1 adenosine receptor overexpression on normoxic and post-ischemic gene expression Cardiovasc Res, March 1, 2003; 57(3): 715 - 726. [Abstract] [Full Text] [PDF]
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E. Cabiscol, G. Belli, J. Tamarit, P. Echave, E. Herrero, and J. Ros Mitochondrial Hsp60, Resistance to Oxidative Stress, and the Labile Iron Pool Are Closely Connected in Saccharomyces cerevisiae J. Biol. Chem., November 8, 2002; 277(46): 44531 - 44538. [Abstract] [Full Text] [PDF]
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 O. Hori, F. Ichinoda, T. Tamatani, A. Yamaguchi, N. Sato, K. Ozawa, Y. Kitao, M. Miyazaki, H. P. Harding, D. Ron, et al. Transmission of cell stress from endoplasmic reticulum to mitochondria: enhanced expression of Lon protease J. Cell Biol., June 24, 2002; 157(7): 1151 - 1160. [Abstract] [Full Text] [PDF]
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S.R. Kirchhoff, S. Gupta, and A.A. Knowlton Cytosolic Heat Shock Protein 60, Apoptosis, and Myocardial Injury Circulation, June 18, 2002; 105(24): 2899 - 2904. [Abstract] [Full Text] [PDF]
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 C. GILL, R. MESTRIL, and A. SAMALI Losing heart: the role of apoptosis in heart disease--a novel therapeutic target? FASEB J, February 1, 2002; 16(2): 135 - 146. [Abstract] [Full Text] [PDF]
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J. Jayakumar, K. Suzuki, I. A. Sammut, R. T. Smolenski, M. Khan, N. Latif, H. Abunasra, B. Murtuza, M. Amrani, and M. H. Yacoub Heat Shock Protein 70 Gene Transfection Protects Mitochondrial and Ventricular Function Against Ischemia-Reperfusion Injury Circulation, September 18, 2001; 104(90001): I-303 - 307. [Abstract] [Full Text] [PDF]
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R. S. Vander Heide Increased expression of HSP27 protects canine myocytes from simulated ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H935 - H941. [Abstract] [Full Text] [PDF]
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