A unique pathway of cardiac myocyte death caused by hypoxiaacidosis
Department of Molecular and Cellular Pharmacology and the Vascular Biology Institute, University of Miami Medical Center, Miami, FL 33101, USA
* Author for correspondence (e-mail: kwebster{at}chroma.med.miami.edu)
Accepted 20 May 2004
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Summary |
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Key words: cardiac myocyte, heart, apoptosis, ischemia, pH, BNIP3, mitochondria, necrosis
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Ischemic heart disease |
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Ischemia
Numerous reports have documented the extensive cell death and tissue loss
that accompanies myocardial ischemia
(Anversa and Kajstura, 1998;
Narula et al., 1996
;
Chen et al., 1997
; Fiss and
Gattinger, 1996). The contribution of hypoxia to cell death is controversial
and different groups have reported markedly different results
(Tanaka et al., 1994
;
Long et al., 1997
;
Matsui et al., 1999
;
Kang et al., 2000
;
Regula et al., 2002
;
Kubasiak et al., 2002
). The
discrepancies are almost certainly due to differences in the models,
especially related to glucose levels and pH. Our results indicate that it is
the secondary consequences of hypoxia rather than hypoxia per se that
causes cell death (Webster and Bishopric,
1992
; Webster et al.,
1999
). Although hypoxia is an obligatory consequence of ischemia,
it rarely exists alone. Hibernating myocardium may represent a condition of
chronic hypoxia (Heusch and Schulz,
1996
) but in most other conditions of moderate to severe ischemia,
hypoxia is accompanied by or precedes energy depletion, acidosis and/or
reoxygenation. Each of these latter conditions involves cell and tissue loss,
and the pathways of cell death in each case are unique. Reperfusion damage has
been the subject of numerous articles and reviews (Gottlieb et al., 2003;
Kumar and Jugdutt, 2003
;
Valen, 2003
) and will not be
addressed in detail here. The damage caused by reperfusion is widely believed
to be initiated by surges of oxygen free radicals that initiate stress
responses and culminate in cell death. Our studies showed that the exposure of
cardiac myocytes to cycles of hypoxiareoxygenation results in
30%
cell death in each cycle (Webster et al.,
1999
; Dougherty et al.,
2002
). The response of cardiac myocytes to
ischemiareperfusion (I/R) is complex, and the survival/death pathways
have not been fully described. During early ischemia, ATP levels may be
maintained by increased glycolysis but at the expense of the limited reserves
of glucose and glycogen. Glucose utilization increases by >10-fold in
ischemic/hypoxic cardiac myocytes with corresponding lactate accumulation
(Webster et al., 1993
,
1994
,
1999
). Acidosis is further
exacerbated when ATP levels begin to decline
(Allen et al., 1989
;
Allen and Orchard, 1987
;
Neely and Grotyohann, 1984
).
If ATP is depleted during ischemia, necrosis will occur because of the passive
loss of transmembrane ion gradients, followed by cell swelling and loss of
membrane integrity (Buja et al.,
1993
; Hochachka et al.,
1996
; Majno and Joris,
1995
; Reimer and Ideker,
1987
). In the latter condition, apoptosis as well as necrosis
probably occurs, and both may be `programmed' at least in the early stages
(Bishopric et al., 2001
).
Programmed death is an active, energy-consuming process requiring ATP; as ATP
levels fall at late time points, the programmed pathways may fail and be
replaced by a more classical necrotic death
(Kajstura et al., 1996
;
Ohno et al., 1998
;
Buja and Entman, 1998
).
Regulation of pH during ischemia
It has been recognized for some time that proton pumps and pH regulation
may play a role in apoptosis signaling
(Anversa and Kajstura, 1998;
Gottlieb et al., 1996
;
Karwatowska-Prokopczuk et al.,
1998
; Long et al.,
1998
). Ischemic cardiac myocytes generate excess H+
through increased anaerobic metabolism, net hydrolysis of ATP and
CO2 retention (Dennis et al.,
1991
). These protons are extruded from the myoplasm to the
interstitial space by the combined action of three major ion-specific membrane
transporters, including the Na+/H+ exchanger, the
Na+/HCO3 cotransporter and the
vacuolar proton-ATPase
(Karwatowska-Prokopczuk et al.,
1998
; Lagadic-Gossmann et al.,
1992
; Lazdunski et al.,
1985
). Increased activity of the Na+/H+
exchanger can cause Ca2+ overload because the elevated
intracellular Na+ is subsequently exchanged for Ca2+
via the Na+/Ca2+ exchanger
(Pierce and Czubryt, 1995
).
Inhibition of Na+/H+ exchange has been shown to protect
against ischemic injury, possibly by preventing this increase in
Ca2+ (Bond et al.,
1993
; Shimada et al.,
1996
). Conversely, inhibition of the vacuolar ATPase promotes
apoptosis, in part by shifting the proton load towards the
Na+/H+ transporter, thereby increasing Ca2+
uptake, and in part by reducing the myocyte capacity to control intracellular
pH (Gottlieb et al., 1996
;
Karwatowska-Prokopczuk et al.,
1998
; Long et al.,
1998
). Acidosis has been shown to correlate with apoptosis in a
number of other systems where it may promote the activation of caspases
(Gottlieb et al., 1995
;
Li and Eastman, 1995
;
Perez-Sala et al., 1998
).
Bcl-2 and BNIP3
The Bcl-2 gene family encodes a group of proteins with the ability to
promote or repress programmed cell death in response to a wide variety of
stimuli (Boise et al., 1995;
Hockenbery, 1995
;
Korsmeyer, 1995
). Additional
genes in this family include those encoding Bcl-Xl, Mcl-1, A1, Bcl-W and
CED-9, which are anti-apoptotic, and Bak, Bax, Bcl-xs, Diva and Mtd/Bok, which
are pro-apoptotic. These proteins are usually associated with the cell
membranes, particularly the mitochondria, endoplasmic reticulum (ER) and
nuclear envelope, where they are anchored by a C-terminal domain. Individual
family members may remain in the cytosol or be loosely membrane bound and
translocate after a death signal is received
(Adams and Cory, 2001
;
Regula et al., 2002
;
Vande Velde et al., 2000
). The
physical location and activity of each Bcl-2 family protein is determined
partly by its binding to other Bcl-2-related proteins in the cell cytosol.
This in turn is determined by the relative concentrations of each protein, and
the balance of pro- and anti-apoptotic members is an important feature of the
regulation. Bcl-2 has been attributed antioxidant and proton translocating
properties (Hockenbery et al.,
1993
; Shimizu et al.,
1998
), and one major function of the family as a whole is to
determine the on/off state of the mitochondrial permeability transition pore
(MPTP; reviewed in Green and Reed,
1998
; Earshaw et al.,
1999
; Crompton,
2000
). In some animal ischemia models of coronary occlusion,
myocardial levels of Bcl-2 proteins have been shown to decline while
pro-apoptotic Bax increases (Kajstura et
al., 1996
; Hockenbery,
1995
). Over-expression of Bcl-2 reduces apoptosis in some models
of neuronal ischemia (Kane et al.,
1993
; Lawrence et al.,
1996
).
BNIP3 is a member of the so-called BH3-only subfamily of Bcl-2 family
proteins that antagonize the activity of pro-survival proteins and promote
apoptosis (Ray et al., 2000;
Vande Velde et al., 2000
).
These proteins do not possess the same protein binding domains (BH1 and BH2)
as the other Bcl-2 family members but instead bind through a common BH3
domain. Related members of this group include Bik, Blk, Hrk, BimL, Bad, Bid
and Nix. BNIP3 was originally identified as a Bcl-XL or E1B 19K-binding
protein (Zhang et al., 2003
).
The BH3 domain of BNIP3 may not be required for the death-promoting functions,
but the C-terminal transmembrane domain is required, indicating membrane
targeting as essential for pro-apoptosis function. BNIP3 is expressed below
detectable levels in most organs including the heart under normal
(non-ischemic) conditions (Bruick,
2000
; Vande Velde et al.,
2000
). Overexpression of BNIP3 protein by transfection of the cDNA
into some cultured cell lines results in membrane translocation and initiation
of cell death (Vande Velde et al.,
2000
). The death pathway has characteristics of both apoptosis and
necrosis, including DNA fragmentation and MPTP opening, but early loss of
plasma membrane integrity and no caspase activation. Expression of the
BNIP3-encoding gene is induced by hypoxia through a 5' promoter
hypoxia-inducible factor-1 (HIF-1)-binding site
(Bruick, 2000
).
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Hypoxia and cardiac death |
---|
As discussed above, the role of hypoxia in ischemia-mediated cell death is
controversial. We previously reported that rapidly contracting cardiac
myocytes remained fully viable and contractile during culture under severe
hypoxia for up to 5 days (Webster and
Bishopric, 1992). Glycolysis was induced by 10-fold within 1 h,
and there was no evidence of major cell loss; ATP remained high and the only
significant change was a reduced level of contractility that correlated with
lower intracellular cAMP in the hypoxic cultures. These results contrast with
other reports of significant cardiac myocyte cell loss by apoptosis during
2472 h of exposure to an equivalent degree of hypoxia
(Tanaka et al., 1994
;
Long et al., 1997
;
Matsui et al., 1999
;
Regula et al., 2002
). To
resolve this apparent controversy, we subjected cardiac myocytes to severe
hypoxia for a week under conditions where the glucose and extracellular pH
([pH]o) were constantly monitored and maintained within the physiological
range, and we measured both caspase activity and cell death at intervals. As
indicated in Fig. 1A, there was
no significant change in the number of apoptotic nuclei at either 3 days or 7
days compared with aerobic cultures. Under the same conditions of hypoxia,
endothelial cells died within 3 days (data not shown). Caspase activities are
shown in Fig. 1B; there were no
significant changes in the activities of caspases 3, 8 or 9 at any time during
this time course (staurosporine-treated plates incubated in parallel generated
a maximal 2.7-fold activation of caspase 3 and 1.8-fold activation of caspase
9; not shown). These results confirm our previous reports that hypoxia alone
does not activate programmed cell death in neonatal cardiac myocytes. This
conclusion cannot necessarily be extrapolated to other models or to the intact
heart. To remain viable under hypoxia, the cells must be able to maintain
glycolysis at a level that is sufficient to sustain ATP, a condition that
requires a continuous supply of glucose. In addition, the cell must be able to
clear excess acid produced by anaerobic glycolysis. If these conditions can
not be fully accommodated, the cell will die. Todor et al.
(2002
) recently reported that
cardiac myocytes from failing hearts, but not those from normal hearts, were
susceptible to hypoxia-mediated death. It is possible that the failing
myocytes were compromised in their ability to activate adaptations to hypoxia.
It should also be noted that oxygen tension may be an important determinant in
the cellular response including the mechanism of oxygen sensing. Budinger et
al. (1998
) described a model of
myocardial ischemia where myocytes were subjected to 3% O2. There
was a small decrease in contractility under hypoxia coincident with a similar
small loss of ATP but no loss of cell viability. The authors concluded that
oxygen sensing, and the positive adaptation, was probably initiated by
reactive oxygen species (ROS) generated in the mitochondria under these
conditions because of partial inhibition of cytochrome oxidase. Under some
conditions, mitochondrial ROS may play a driving role in cell death during
hypoxia. Yermolaieva et al.
(2004
) described a stroke
model where death of PC12 cells subjected to a brief period of hypoxia
followed by reoxygenation correlated with ROS. In our studies, oxygen tension
is maintained at less than 0.5%, mitochondrial functions are severely impaired
but we did not detect any increase of ROS as measured by the lucigenin reagent
(N-methyl-acridinium nitrate;
Dougherty et al., 2004
).
|
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Hypoxiaacidosis and cardiac death |
---|
|
In Fig. 2DF, the
effects of coincident hypoxia and acidosis on myocyte structure and integrity
are shown by staining with Hoechst 33342 and anti--MHC antibody (see
Dougherty et al., 2002
;
Webster et al., 1999
). In
cultures grown aerobically, abundant myofilaments with clear cross-striations
are evident with the
-MHC stain
(Fig. 2D). The white arrows
indicate smooth, oval-shaped nuclei with sparse evidence of condensation or
internal fragmentation. In this field, 29 nuclei were scored normal and one
was condensed. At 72 h, thehypoxicacidotic cultures
(Fig. 2E) still stained
strongly with
-MHC antibody but there was clear deterioration of the
myofilaments, and cross-striations were no longer clearly visible. In the
field shown, 22 nuclei were scored condensed (examples are indicated by the
arrows), and 14 were normal. By contrast, hypoxicneutral cells still
exhibited myofilaments with intact cross-striations after 72 h
(Fig. 2F, see arrow at far
right), and most of the nuclei were normal. Control aerobic cultures contained
57% apoptotic cells; this increased to 44% after 48 h of hypoxia with
metabolite build-up and to 60% after 72 h of hypoxia.
These results demonstrate that cardiac myocyte death under hypoxia occurs only if the media is not frequently changed, indicating either that proapoptotic factors accumulate in the media during hypoxia or vital components are depleted. To test these possibilities, medium from 48 h hypoxic cultures that were just beginning to show signs of DNA laddering was transferred to fresh cardiac myocytes and the cells were incubated for an additional 2448 hunder either hypoxic or aerobic conditions. Apoptosis was again monitored by DNA fragmentation. Control plates received medium from hypoxic cells that underwent daily medium replacement. Results are shown in Fig. 3. Significant apoptosis was apparent in both aerobic and hypoxic cultures 24 h after exposure to the spent medium, but significantly more DNA fragmentation appeared in the sample from the 24 h hypoxic plate correlating with the lower [pH]o. These results implicate a death factor(s) associated with deteriorating pH control.
|
As a second approach to identify the death factors, we collected the spent
medium from 48 h hypoxic cultures and readjusted the pH to 7.6 before
transferring to fresh cardiac myocytes. The effect of neutralized spent medium
is shown in Fig. 4. Acidic
spent medium again caused extensive apoptosis of fresh myocytes but the
neutralized medium caused minimal DNA fragmentation
(Fig. 4A). This suggests that
acidosis is necessary to induce apoptosis of hypoxic cultures. To confirm
this, parallel cardiac myocyte cultures were again exposed to hypoxia; in one
set, acid accumulated in the medium and in the other the acid was neutralized
every 12 h to maintain [pH]o above 7.0 during hypoxic incubation. These
results are shown in Fig. 4B.
In the absence of additional buffer, [pH]o dropped to 5.7 at 48 h, and there
was extensive apoptosis. In the pH-neutral cultures, there was no visible DNA
fragmentation. Hoechst 33342 and anti--MHC antibody stains again
revealed >60% apoptosis of acidotic cardiac myocytes and <10% when the
pH was neutralized.
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BNIP3 and the hypoxiaacidosis death pathway |
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Activation of BNIP3 by acidosis |
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Hypoxiaacidosis: an atypical programmed death pathway |
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Integration and significance of the BNIP3 pathway |
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The extreme effect of acidosis on survival of myocytes under hypoxia may
explain why other groups have reported that hypoxia alone promotes apoptosis
(Tanaka et al., 1994;
Long et al., 1997
;
Matsui et al., 1999
;
Kang et al., 2000
;
Regula et al., 2002
). BNIP3 is
induced under all conditions of hypoxia and may undergo low-level activation
even at neutral [pH]o. Although our results show that acid build-up drives
newly synthesized BNIP3 into membranes, it is likely that prolonged hypoxia
will promote a continuous low-level BNIP3 activation because of the increased
effective proton activity. Hypoxic cells produce >10-fold more acid than
aerobic cells with a corresponding increase of proton flux. This condition
must lead to the establishment of a new ionic equilibrium involving a net
increase of intra- and extracellular H+
(Webster and Bishopric, 1992
;
Webster et al., 1994
). This
change may be sufficient to stimulate low-level BNIP3 translocation in some
cells and a consequent low-level activation of the death program. Consistent
with this model, proton pumps have been shown to play a role in apoptosis
signaling (Anversa and Kajstura,
1998
; Karwatowska-Prokopczuk
et al., 1998
), and acidosis has been shown to correlate with
apoptosis in a number of other systems
(Gottlieb et al., 1995
;
Perez-Sala et al., 1998
).
Loss of cardiac myocytes is a central feature of heart disease of both
ischemic and non-ischemic origin (reviewed in
Anversa and Kajstura, 1998;
Kajstura et al., 1998
;
Narula et al., 1996
). It has
been described in multiple regions of the myocardium during infarction,
hibernation (Heusch and Schulz,
1996
) and during both ischemia and subsequent reperfusion
(Chen et al., 1997
;
Fliss and Gattinger, 1996
).
Death pathways involving necrosis, apoptosis and oncosis have been described
(reviewed in Kajstura et al.,
1996
; Ohno et al.,
1998
). The probability that hypoxia and acidosis coexist in
diseased and/or infarcted myocardial tissue is high because of the disrupted
vasculature and elevated lactic acid production in ischemic tissue
(Chen et al., 1997
;
Krayenbuehl and Hess, 1992
).
Direct measurements of myocardial tissue pH indicate that it drops by
12 units within 10 min of ischemia
(Marzouk et al., 2002
).
Therefore, BNIP3 may be expected to play a significant role in cell loss
during ischemic heart disease. Combined hypoxia and acidosis reflects a
greater hemodynamic disruption than acidosis or hypoxia alone, and the dual
signal may provide a selective advantage by activating the death pathway only
as a last resort. Acidosis may also activate BNIP3 in skeletal muscle to allow
myocyte drop-out under conditions of severe ischemia or hypoxia where the
probability of irreversible damage is higher.
Further work is required to characterize this pathway of cardiac myocyte
death. We need to determine the contribution of BNIP3 to tissue loss in the
intact heart during ischemic heart disease and during infarction. To what
extent does this pathway contribute to acute and chronic cell loss during
coronary artery disease and congestive heart failure? If this is substantial
then it will be important to develop methods to selectively block the pathway.
Previous work from this and another laboratory
(Kubasiak et al., 2002;
Regula et al., 2002
) indicates
that N-terminal deletions of BNIP3 that lack the transmembrane domain are
protective. It may be possible to develop cell-permeable peptides or other
small organic mimics that can block BNIP activation very specifically.
Finally, the requirement of this death pathway for MPTP opening but absence of
caspase activation remains a paradox.
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Acknowledgments |
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