(Received for publication, November 8, 1996, and in revised form, May 28, 1997)
From Novartis Pharmaceuticals Corp., Summit, New
Jersey 07901 and the § Department of Medical Biochemistry,
Neurobiotechnology Center and Ohio State Biochemistry Program, Ohio
State University, Columbus, Ohio 43210
In this report we investigate the molecular mechanisms that contribute to tissue damage following ischemia and ischemia coupled with reperfusion (ischemia/reperfusion) in the rat heart and kidney. We observe the activation of three stress-inducible mitogen-activated protein (MAP) kinases in these tissues: p38 MAP kinase and the 46- and 55-kDa isoforms of Jun N-terminal kinase (JNK46 and JNK55). The heart and kidney show distinct time courses in the activation of p38 MAP kinase during ischemia but no activation of either JNK46 or JNK55. These two tissues also respond differently to ischemia/reperfusion. In the heart we observe activation of JNK55 and p38 MAP kinase, whereas in the kidney all three kinases are active. We also examined the expression pattern of two stress-responsive genes, c-Jun and ATF3. Our results indicate that in the heart both genes are induced by ischemia and ischemia/reperfusion. However, in the kidney c-Jun and ATF3 expression is induced only by ischemia/reperfusion. To correlate these molecular events with tissue damage we examined DNA laddering, a common marker of apoptosis. A significant increase in DNA laddering was evident in both heart and kidney following ischemia/reperfusion and correlated with the pattern of kinase activation, supporting a link between stress kinase activation and apoptotic cell death in these tissues.
Ischemia and ischemia coupled with reperfusion (ischemia/reperfusion) in the heart and kidney result in cell death and scar formation in these tissues, which can ultimately lead to congestive heart failure or renal failure and death. Recent studies in heart tissue, both in vitro and in vivo, suggest that cell death upon reperfusion is largely apoptotic in nature (1-4). In vivo studies demonstrated that apoptosis following occlusion of the coronary artery in the rat is the major contributing factor to myocardial damage after the insult. Subsequently, necrosis occurs and contributes to the progressive loss of cardiomyocytes with time after infarction (5). Apoptotic cell death also appears to play a role in hypertrophy of cardiac tissue in response to pressure overload (6, 7). The occurrence of apoptotic cell death in renal tissue has also been demonstrated following brief periods of ischemia and subsequent reperfusion (8).
The molecular mechanisms by which ischemia and ischemia/reperfusion lead to cell death and eventually to tissue damage have not yet been defined. Recent studies suggest that members of the mitogen-activated protein (MAP)1 kinase family, in particular Jun N-terminal kinase (JNK), are activated in the heart and kidney following ischemia and reperfusion of these tissues (9, 10). Therefore, this kinase signaling pathway may be an important molecular component responsible for tissue injury following ischemia and ischemia/reperfusion in the heart and kidney. However, the role of individual isoforms of JNK or the role of p38 MAP kinase has not been addressed.
The MAP kinase family, members of which are characterized as proline-directed serine/threonine-protein kinases, can be divided into three subgroups: the extracellular signal-regulated kinases, JNKs (also referred to as stress-activated protein kinases), and p38 MAP kinases. These kinase pathways are distinguished by activating signals, substrate specificity, and cellular responses (for review, see Refs. 11-13). While the extracellular signal-regulated kinases are predominantly activated by growth factors, the JNKs and p38 MAP kinases are activated by stress signals such as inflammatory cytokines, heat shock, ultraviolet light, and ischemia (9, 10, 14-16). Because JNKs and p38 MAP kinases are generally activated by the same stress signals, they have been collectively referred to as the stress kinases.
Two members of the JNK family (JNK1 (46 kDa) and JNK2 (55 kDa)) were initially identified as kinases with high affinity for the transcription factor c-Jun. JNKs phosphorylate c-Jun on specific N-terminal serine residues (Ser-63 and Ser-73) and enhance the ability of c-Jun to activate expression of genes containing c-Jun-responsive promoter elements (14, 17-20). Additional substrates for JNKs have been defined since their first identification; the substrates include ATF2 and Elk-1 (21, 22). Recently, at least 10 isoforms of JNK have been identified, which correspond to alternatively spliced variants derived from three genes. These JNK isoforms differ in their interaction with substrate proteins in vitro, suggesting that these isoforms may play specific roles in vivo (23). However, specific activation of individual isoforms in vivo has not been demonstrated to date. Multiple isoforms of p38 MAP kinase have also been identified (24-27). Although p38 MAP kinases and JNKs are activated by similar stress signals, the kinase cascade leading to the activation of p38 MAP kinases is distinct from the kinase cascade leading to the activation of JNKs (for review, see Ref. 28). Furthermore, the substrate specificity of p38 MAP kinase, although not well defined, is distinct from that of JNKs: p38 MAP kinase phosphorylates mitogen-activated protein kinase-activated protein kinase-2 and -3 and CHOP, which are not substrates for JNKs (29-31).
A number of recent studies have indicated an important role for the stress kinases (JNKs and p38 MAP kinase) in stress-induced apoptosis (32-35). Furthermore, one of the major JNK substrates, c-Jun, has been implicated to be important in neuronal apoptosis (36, 37). In light of these observations, we examined the patterns of stress kinase activation in ischemic and ischemic/reperfused heart and kidney. Our results demonstrate specific patterns of stress kinase activation in these injured tissues. Furthermore, we observe a strong correlation between stress kinase activation, increased expression of stress kinase responsive genes, and initiation of apoptotic cell death. These results provide insight into the molecular events leading to tissue injury following ischemia/reperfusion. Their implications with respect to the tissue-specific patterns of injury progression are discussed.
Antibodies specific for phosphorylated p38 MAP kinase (catalog number 9211S) were purchased from New England Biolabs and used at 1 µg/ml for Western blot analysis. Anti-p38 MAP kinase C-terminal antibodies (catalog number sc-535) and anti-JNK antibodies (catalog number sc-571) were purchased from Santa Cruz Biotechnology Inc., and 0.5 µg/ml was used for Western blot analysis. The ECL kit for Western blot analysis was obtained from Amersham Life Science. GST·c-Jun (amino acids 1-223) protein was produced in Escherichia coli and purified using glutathione-Sepharose-4B (Pharmacia Biotech Inc.).
Ischemia and Ischemia/Reperfusion of Isolated Rat HeartAdult male Sprague-Dawley rats were injected with heparin
(5000 IU/kg, body weight intraperitoneally) and then were anesthetized with pentobarbital sodium (75 mg/kg, body weight, intraperitoneally). The hearts were excised and were placed in ice-cold Krebs-Henseleit buffer (containing 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4,
1.2 mM KH2PO4, 25 mM
NaHCO3, 0.5 mM EDTA, and 5 mM
glucose). Each heart was immediately cannulated at the aorta, and
perfusion was initiated with oxygenated (95% O2, 5%
CO2) Krebs-Henseleit buffer (37 °C) at a constant
pressure of 75 mm Hg. Hearts were perfused for 30 min with
Krebs-Henseleit buffer for equilibration. At this point hearts were
subjected to global ischemia for 20 or 45 min and then were reperfused
for a designated period of time. At the end of the experiment, the
hearts were freeze-clamped, and the frozen tissue was pulverized and
was stored at 70 °C.
Purpose-bred mongrel dogs (Barton's West End Farms, Oxford, NJ) were anesthetized (pentobarbital sodium, 30 mg/kg intravenously), intubated, and ventilated with room air. Arterial blood gases and pH were maintained within normal limits by intravenous infusion of bicarbonate and adjustment of the ventilation rate. Rectal temperature was maintained within ±0.5 °C of base-line temperature. Cannulas were inserted into a femoral artery and vein for obtaining arterial blood samples and administering bicarbonate and supplemental doses of pentobarbital, respectively. Arterial pressure was measured with a pressure transducer cannula (Millar Instruments, Inc., Houston, TX) inserted into the aortic arch via the contralateral femoral artery.
The thorax was opened at the left fifth intercostal space, and a pericardial cradle was created. Left ventricular pressure and its first derivative (dP/dt) were measured with a Millar catheter inserted into the left ventricular chamber through an apical stab wound. Hemodynamic signals were monitored continuously with a strip chart recorder and a digital data acquisition system (Modular Instruments, Inc., Malvern, PA).
A short segment of the left anterior descending coronary artery was isolated distal to its first major branch. After allowing hemodynamic stabilization for at least 30 min, the experimental protocol was initiated. Regional myocardial ischemia was achieved by occlusion of the left anterior descending coronary artery for 60 min followed by reperfusion for an additional 60 min. The presence of an ischemic insult was verified by a discernible darkening of the myocardial surface circumscribed by the epicardial coronary vasculature distal to the occlusion site.
Transmural myocardial biopsy samples (~10-20 mg) were obtained serially from the core of the ischemic region with a 14-gauge biopsy needle (Tru-Cut, Baxter, Deerfield, IL) at base line (i.e. preischemia), at the end of the ischemic period, and at 2, 5, 10, 11, 22, 26, 30, and 60 min of reperfusion. All samples were obtained from distal to proximal in the perfusion bed to minimize potential confounding effects of localized disruption of blood flow to regions distal to the sampling sites. Additional control tissue specimens were acquired from the nonischemic cardiac region perfused by the left circumflex coronary artery at base line, at the end of ischemia, and at various times during the reperfusion period.
Myocardial tissue samples were removed from the needle, rinsed with
saline, and quickly (within ~10 s) frozen in liquid nitrogen. Frozen
samples were stored at 70 °C until the time of assay.
All
experiments were performed in male Sprague-Dawley rats weighing between
300 and 500 g. Following induction of anesthesia (Inactin, 100 mg/kg intraperitoneally), the left kidney was exposed via a flank
incision, and left renal artery occlusion was performed using an
atraumatic vascular clamp. Reperfusion was achieved by removing the
clamp. The effect of varying the duration of reperfusion was assessed
by occluding the renal artery for 60 min and then reperfusing the
kidney for indicated periods. The right kidney served as a nonischemic
control. Rats were sacrificed upon completion of either ischemia or
ischemia/reperfusion, and kidneys were immediately removed and were
rapidly frozen in liquid nitrogen. All renal tissues were then kept at
70 °C until further assay.
A powder of frozen heart and kidney
tissues was prepared using a mortar and pestle. For homogenization, 100 mg of tissue powder was weighed out and was homogenized in 1 ml of
buffer (20 mM Hepes (pH 7.5), 20 mM
-glycerophosphate, 20 mM sodium pyrophosphate, 0.2 mM sodium vanadate, 2 mM EDTA, 20 mM sodium fluoride, 10 mM benzamidine, 1 mM DTT, 20 µg/ml leupeptin, and 0.2 mM
pefabloc SC) using a Dounce homogenizer. Cell debris was removed by
centrifugation (20,000 × g for 10 min at 4 °C), and
the supernatant was stored in aliquots at
70 °C.
In gel kinase assays were performed as
described previously (38) with some modifications. Proteins (80 µg/sample) extracted from heart and kidney tissues were separated on
a 10% SDS-polyacrylamide gel that had been polymerized in the presence
of 50 µg/ml GST·c-Jun (amino acids 1-223). After electrophoresis,
the gel was washed five times for 10 min with 60 ml of buffer A (20%
isopropyl alcohol in 50 mM Tris-HCl (pH 8.0)) to remove
SDS. Next, the gel was washed five times for 10 min with 60 ml of
buffer B (1 mM DTT, 50 mM Tris-HCl (pH 8.0)).
To denature proteins, the gel was incubated with 60 ml of buffer C (6 M guanidine HCl, 20 mM DTT, 2 mM
EDTA, 50 mM Tris-HCl (pH 8.0)) for 1 h at room
temperature with shaking. Finally, the gel was incubated in 300 ml of
buffer D (1 mM DTT, 2 mM EDTA, 0.05% Tween 20, 50 mM Tris-HCl (pH 8.0)) for 16 h at 4 °C to
renature proteins. For the kinase assay, the gel was first equilibrated
with kinase buffer (20 mM HEPES (pH 7.6), 20 mM
MgCl2, 20 mM -glycerol phosphate, 20 mM p-nitrophenylphosphate, 2 mM DTT,
0.2 mM sodium vanadate) for 1 h at room temperature
with shaking. The kinase assay was initiated by adding 10 ml of kinase buffer containing 20 mM ATP and 10 µCi/ml of
[
-32P]ATP, and the gel was incubated at 30 °C for
60 min with shaking. The gel was washed several times with 5%
trichloroacetic acid and 1% sodium pyrophosphate to remove free
radioactivity, followed by drying and autoradiography.
The procedures for immunoprecipitation and immunoblotting were described previously (39). In short, proteins (500 µg/sample) extracted from heart and kidney tissues were incubated with 2 µg of anti-p38 MAP kinase C-terminal antibodies at 4 °C for 2 h with rotation. Protein A-agarose beads (20 µl/sample) were then added to each sample and incubated for an additional 40 min at 4 °C with rotation. The immune complexes precipitated with protein A-agarose were washed four times with buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM sodium vanadate, 0.2 mM pefabloc SC, 20 µg/ml leupeptin, 150 mM NaCl, 20 mM sodium fluoride, and 0.1% Triton X-100. The immunoprecipitates were immobilized on polyvinylidene difluoride membrane after separation on 10% SDS-PAGE. The membrane was immunoblotted with anti-phospho-p38 MAP kinase antibodies using the ECL method to evaluate tyrosine phosphorylation of p38 MAP kinase. The same membrane was then reblotted with anti-p38 MAP kinase antibodies after stripping to verify that equivalent amounts of p38 MAP kinase protein were loaded in each lane.
In Situ HybridizationIschemic and ischemic/reperfused
hearts were obtained from rats as described previously (40). Ischemic
and ischemic/reperfused kidneys were obtained as described above. The
tissues were frozen in isopentane prechilled with liquid nitrogen and
kept at 70 °C until further assay. In situ
hybridization was carried out using ATF3 or c-Jun antisense RNA probes
as described previously (40).
Total RNA was isolated
using a guanidium isothiocyanate and organic extraction method as
outlined by the manufacturer (5 Prime 3 Prime, Inc., Boulder, CO).
5 µg of total RNA was loaded onto a 1% formaldehyde-agarose gel,
transferred to Hybond membrane (Amersham), UV-cross-linked, hybridized
to 32P-labeled rat c-Jun or rat ATF3 partial cDNAs
using QuikHyb (Stratagene), and washed at high stringency as outlined
by the manufacturer.
The pulverized tissues from rat heart and
kidney were lysed with lysis buffer (50 mM Tris-HCl (pH
8.0), 10 mM EDTA, 0.5% SDS, and 100 mM NaCl)
and digested in lysis buffer containing 1 mg/ml of protease K at
55 °C for 16 h. DNA was then extracted with Isoquik rapid DNA
isolation kit (ORCA Research Inc., Bothell, WA) according to the
manufacturer's instructions, and the concentration was determined
based on absorbance at 260 nm. For visualization of DNA laddering, DNA
(1 µg/sample) was labeled at room temperature for 10-15 min using 5 units of Klenow fragment (NEB 212) in a total volume of 10 µl
containing 0.5 µCi of [-32P]dATP. The labeled DNA
was then subjected to electrophoresis on a 1 or 1.8% agarose gel. The
gels were fixed with 10% acetic acid for 2 h at room temperature
followed by drying, or the DNA was transferred to nylon membranes and
UV-cross-linked followed by baking at 80 °C for 1 h. The dried
gels and membranes were then autoradiographed for periods ranging from
2 h to overnight as indicated in Fig. 8.
Isolated rat hearts were subject to global ischemia for either 20 or 45 min prior to reperfusion. Individual hearts were reperfused for various periods ranging from 1 min to 3 h. Extracts from the hearts were then prepared, and stress kinase activity was measured by either in gel kinase assay using c-Jun (amino acids 1-223) as a substrate, or Western blotting using phospho-specific antibodies.
Neither JNK46 nor JNK55 activity, measured by
in gel kinase assay, was detectable in control hearts or in hearts
subjected to ischemia for either 20 min (data not shown) or 45 min
(Fig. 1A). However,
JNK55 activity was detectable within 1 min following
reperfusion; it continued to increase, reaching maximal activity at
approximately 1 h postreperfusion. Significant levels of
JNK55 activity were still present at 3 h
postreperfusion. Interestingly, no significant levels of
JNK46 activity were detectable upon reperfusion of the
heart until 1 h postreperfusion, when some weak JNK46
activity was evident. The lack of significant JNK46
activation in the heart was not a consequence of the absence of
JNK46 expression, because JNK46 protein was
readily detectable by Western analysis using an antibody recognizing
both isoforms (Fig. 1B). Furthermore, the Western analysis
confirmed that similar levels of JNK were present in all samples. This
result indicates that there is a preferential activation of
JNK55 in the isolated heart following reperfusion. The
preferential activation of JNK55 was confirmed in
vivo by examining JNK activation in dog heart tissue following
ischemia/reperfusion (Fig. 1C). Small biopsies of cardiac
tissue were removed from the exposed dog heart in vivo prior
to ischemia and at the indicated time points following ischemia and
reperfusion. The profile of JNK activation is essentially identical to
that observed in the isolated rat heart. However, the peak in
JNK55 activity appears to be reached sooner in
vivo. Preferential activation of a JNK isoform in tissues has not
been reported previously, and these observations may suggest a specific
role for a JNK55 in the heart.
p38 MAP kinase activity was measured by Western analysis using
anti-phospho-p38 antibodies (Fig.
2A). In a parallel experiment, p38 MAP kinase protein levels were measured by Western analysis using
an anti-p38 antibody to ensure that similar levels of p38 MAP kinase
protein were present in all samples (Fig. 2B). No detectable p38 MAP kinase activity was present in control hearts. However, consistent with the recent report by Bogoyevitch et al.
(41), p38 MAP kinase was activated in the heart subjected to 20 min of
ischemia (Fig. 2B, right part). This activation
was transient, because p38 MAP kinase activity was not detectable after
45 min of ischemia (Fig. 2A, left part).
Significantly, upon reperfusion following 45 min of ischemia, p38 MAP
kinase was rapidly reactivated: the kinase activity was detectable
within 1 min postreperfusion, reached a maximal level at approximately
15 min, and returned to a low level within 3 h. We conclude that
in the ischemic heart, p38 MAP kinase is transiently activated.
Independent of the time of ischemia, p38 MAP kinase is active upon
reperfusion.
Pattern of Stress Kinase Activation in Ischemic and Ischemic/Reperfused Rat Kidney
Rat kidneys, in vivo, were made ischemic by ligation of the renal artery for 1 h. Kidneys were reperfused for various periods ranging from 5 min to 3 h. Extracts were then prepared from the treated kidney or control (i.e. contralateral) kidney.
JNK activity was measured by in gel kinase analysis. As observed in the
heart, no JNK activity was detectable in the control kidneys or in the
ischemic kidney. Upon reperfusion, however, both JNK46 and
JNK55 were activated within 10 min, unlike in the heart
where JNK55 was preferentially activated. The activities of
both kinases were high for approximately 90 min and returned to near
basal level by 2 h (Fig.
3A). Therefore, the pattern of
JNK isoform activation differs significantly in the kidney and heart.
As a control, we showed that similar levels of JNK46
protein were present in all samples (Fig. 3B).
Interestingly, the levels of JNK55 appeared to be lower in
the kidney than in the heart, because the JNK antibody, which
cross-reacts with both JNK46 and JNK55, failed
to detect JNK55 in the kidney (compare Figs. 1B
and 3B). In each case, the same amount of total cellular
protein was loaded on the gels for Western analysis.
p38 MAP kinase activity in the kidney was measured by Western analysis
using anti-phospho-p38 MAP kinase antibodies. As observed in the heart,
no p38 MAP kinase activity was detectable in control kidneys. However,
in contrast to the heart, where p38 MAP kinase was transiently
activated by short periods of ischemia (20 min), p38 MAP kinase was not
activated by short periods of ischemia (15 min or 30 min) in the kidney
(data not shown). Rather, p38 MAP kinase was activated only after
prolonged ischemia for 60 min (Fig.
4A). After reperfusion, p38
MAP kinase activity was maintained for 5 min and then returned to basal
level within 2 h postreperfusion (Fig. 4A). Similar
levels of p38 MAP kinase protein were present in all samples as
determined by Western analysis (Fig. 4B). Taken together,
our results on the activation of stress kinases in the heart and kidney
demonstrate very distinct tissue-specific patterns of stress kinase
activation by ischemia and ischemia/reperfusion. These distinct
patterns of kinase activation are likely to have important implications
in the progression of tissue injury, as discussed below.
Increased Expression of Stress Kinase-responsive Genes in Ischemic and Ischemic/Reperfused Tissues
Stress stimuli, which result in stress kinase activation, have been shown to lead to increased expression of several immediate early genes, including c-Jun and ATF3 (40, 42). We have examined the expression levels of these immediate early genes in response to ischemia and ischemia/reperfusion in both the heart and kidney.
In the heart, c-Jun expression was measured by in situ
hybridization. In the control hearts, the antisense c-Jun RNA probe failed to detect a signal, indicating undetectable levels of c-Jun expression (Fig. 5, left
panels). Ischemia alone, induced by ligation of the left anterior
descending coronary artery, resulted in localized expression of c-Jun
on the endocardium side of the ventricle wall (Fig. 5, upper
right panel). As noted above, we observed transient activation of
p38 MAP kinase early in ischemia (Fig. 2A). It is likely
that induced c-Jun expression in the endocardium following ischemia is
mediated by ATF2, a transcription factor whose activity is enhanced by
p38 MAP kinase phosphorylation and has been shown to regulate c-Jun
transcription (43). Following reperfusion, c-Jun expression was induced
throughout the endocardium and epicardium (Fig. 5, lower right
panel). The broad pattern of c-Jun expression upon reperfusion is
most likely mediated by the phosphorylation and activation of both
c-Jun and ATF2 transcription factors by JNK and p38 MAP kinase. The
role of the stress kinases in mediating induced c-Jun expression
through activation of both c-Jun and ATF2 transcription factors is
supported by the expression pattern of ATF3 following ischemia and
ischemia/reperfusion. Previously, we examined the expression of ATF3 by
in situ hybridization following ischemia and
ischemia/reperfusion in the heart (40). The expression pattern of ATF3
is very similar to that of c-Jun. Like c-Jun transcription, ATF3
transcription is regulated by both c-Jun and ATF2 transcription factors
(44). Activation of these transcription factors by both p38 MAP kinase
and JNK most likely induces ATF3 expression in a pattern similar to
that of c-Jun. Because the endocardium is more severely damaged than
the epicardium in the ischemic heart, we speculate that the expression
of both c-Jun and ATF3 in the endocardium following ischemia is linked
to cellular damage in this region. Expression of c-Jun and ATF3
throughout the ventricular wall upon reperfusion may play a role in
more widespread tissue damage.
The expression levels of c-Jun and ATF3 in the kidney were examined by
Northern analysis (Fig. 6). No detectable
levels of either c-Jun or ATF3 mRNA were observed in control
kidneys or kidneys subjected to ischemia for 1 h. This correlates
with our inability to detect p38 MAP kinase activity in the ischemic
kidney until 1 h after ischemia. However, within 20 min following
reperfusion, both c-Jun and ATF3 mRNA levels increased
significantly, reached maximal levels by 1.5 h, were maintained at
a high level for at least 3 h, and returned to the basal level by
24 h. We conclude that ischemia followed by reperfusion leads to a
rapid and transient expression of these stress-responsive genes in the
kidney as a consequence of stress kinase activation.
To examine the localization of c-Jun and ATF3 expression in the kidney,
we performed in situ hybridization (Fig.
7). Confirming the results of the
Northern analysis, no detectable levels of either c-Jun or ATF3
expression were observed in the control (data not shown) or ischemic
kidneys (Fig. 7A). Upon reperfusion of the kidneys for 90 min, high levels of both c-Jun and ATF3 expression were observed (Fig.
7B). Interestingly, c-Jun and ATF3 were expressed at high
levels within the medulla of the kidney, the region of the kidney most
susceptible to damage following ischemia/reperfusion (45). This result
is reminiscent of the observation that, in the ischemic heart, c-Jun
and ATF3 were initially induced in the endocardium, the region of the
heart most susceptible to damage. This observation further supports the
hypothesis that the high level of both c-Jun and ATF3 expression may be
linked to tissue damage.
Apoptotic Cell Death in Cardiac and Renal Tissue following Ischemia and Ischemia/Reperfusion
A number of recent studies have demonstrated a link between stress kinase activation and initiation of apoptotic cell death (32-35). To examine if there was evidence of apoptotic cell death in the rat heart and kidney following stress kinase activation by ischemia/reperfusion, we examined these tissues for DNA laddering, a marker of apoptotic cell death (46).
Only trace amounts of DNA laddering were observed in control heart and heart subjected to 45 min of ischemia, indicating a very low level of apoptotic cell death in these tissues. However, DNA laddering was detectable within 30 min of reperfusion, and the maximal amount of DNA laddering was evident 1 h following reperfusion (Fig. 8A), indicating a rapid increase in apoptotic cell death following reperfusion and stress kinase activation in the heart.
Similar to the heart, in the kidney no obvious amount or only a trace amount of DNA laddering was observed in the control kidney and kidney subjected to ischemia alone. Upon reperfusion an increase in DNA laddering was observed, but it was not detectable until 2-3 h following reperfusion. The degree of DNA laddering observed at 24 h following reperfusion was significantly above that observed at 3 h following reperfusion (Fig. 8B). This observation suggests that the initiation of apoptotic cell death in the kidney, as measured by DNA laddering, occurs at a slower rate than that observed in the heart. These results, combined with the pattern of stress kinase activation described above, indicate that there is a strong correlation between the time course in the initiation of apoptotic cell death and the activation of the stress kinases in the heart and kidney following ischemia/reperfusion and lend support to the hypothesis that stress kinase activation is linked to apoptotic cell death in these organs.
In this study we have demonstrated both tissue-specific and isoform-specific patterns of stress kinase activation following ischemia/reperfusion in the heart and kidney. This stress kinase activation leads to an increase in the expression levels of both c-Jun and ATF3, which are believed to play a role in cell death (36, 37, 40). The pattern of increased expression of these transcription factors corresponds to regions within the heart and kidney most susceptible to damage following ischemia/reperfusion. The proposal that stress kinase activation is linked to apoptotic cell death is further supported by our observation of a strong correlation between the time course of stress kinase activation and the increase in DNA laddering observed in ischemic/reperfused heart and kidney tissue. These results provide insight into the potential molecular mechanisms that lead to damage of both cardiac and renal tissue following ischemia/reperfusion.
One of the most interesting observations in our study is the transient activation of p38 MAP kinase following a short period of ischemia (15 min) in the heart, whereas prolonged ischemia (60 min) was required for p38 MAP kinase activation in the kidney. It is interesting to note that the length of time of ischemia required to give severe loss of function differs significantly between the rat heart and kidney. In the rat heart, ischemia for longer than 20 min results in severe loss of function (47). However, in the rat kidney, ischemia for 60-90 min is required to observe severe loss of function (48). We propose that activation of p38 MAP kinase during ischemia may initiate events that ultimately contribute to cell death and tissue damage. A role for p38 MAP kinase in apoptotic cell death is supported by several recent studies (34, 49). The activation of p38 MAP kinase early during ischemia in the heart may, at least in part, be the mechanism responsible for the more rapid loss of function in the heart compared with the kidney, where p38 MAP kinase is activated only after prolonged ischemia. The studies performed to measure p38 MAP kinase activity do not distinguish between the multiple isoforms of p38 MAP kinase identified to date (24-27). It is possible that individual isoforms of p38 MAP kinase are activated by specific conditions in the heart and kidney, leading to distinct patterns in the progression of tissue injury.
A number of recent studies have also suggested an important role for JNK in apoptosis induced by various stress stimuli in vitro (32-35). Our results provide evidence to support a role for JNK in apoptosis in vivo by demonstrating a strong correlation between JNK activation and apoptosis in both the ischemic/reperfused heart and kidney. Reperfusion in both the heart and kidney results in rapid activation of JNK. The activation of JNK is followed by the occurrence of apoptotic cell death, as measured by DNA laddering. In the heart apoptosis is evident within 30 min, whereas it is not until 2-3 h after reperfusion that apoptosis is evident in the kidney. We suggest that the early activation of p38 MAP kinase during ischemia in the heart may initiate events that allow for a more rapid acceleration of apoptosis upon JNK activation in the heart compared with the kidney. This hypothesis is supported by the recent observations of Fliss and Gattinger (50), published while this manuscript was under review. They observed that apoptosis was evident in rat heart tissue after 2.25 h of continuous ischemia. We speculate that this apoptotic cell death may be initiated by p38 MAP kinase activation. They also noted that the degree of apoptosis was accelerated by reperfusion after 45 min of ischemia. This, we believe, is likely to be mediated by JNK activation. Another recent observation is that prolonged activation of JNK is required to induce apoptosis in T-cells, while transient JNK activation (<1 h) led to T-cell activation (51). We observe prolonged JNK activity following reperfusion in both the heart and kidney, again supporting a link between JNK activity and apoptosis.
Of particular interest in this study is the preferential activation of the JNK55 isoform in the heart. It has been shown in vitro that JNK isoforms have different affinities for their substrates and hence are likely to have different activities with regard to substrate phosphorylation (19, 20, 23). A recent study demonstrated stronger activation of JNK46 compared with JNK55 following cytokine stimulation of chondrocytes, and it was suggested that the JNK46 isoform may play a more important role in chondrocytes (52). However, to date there has been no evidence demonstrating specific activation or a specific role for JNK isoforms in vivo. Our results demonstrate that there is preferential activation of a JNK55 isoform in the heart, both in vitro and in vivo, following ischemia/reperfusion. Therefore, a JNK55 isoform may play a specific role in the processes leading to damage of heart tissue following ischemia/reperfusion. While this manuscript was in preparation, Bogoyevitch et al. (41) reported the activation of both JNK46 and JNK55 isoforms following ischemia/reperfusion in the isolated rat heart, which is inconsistent with our results. The reason for the discrepancy remains to be determined. However, we have observed the preferential activation of JNK55 in the heart upon reperfusion both in vitro and in vivo in two species, rat and dog.
It has been proposed that stress kinase activation has a positive rather than a negative effect following ischemia/reperfusion, being involved in ischemic "preconditioning" rather than apoptosis (9, 41). Preconditioning is associated with induction of proteins thought to be cardioprotective, including heat shock proteins. Heat shock proteins are substrates for the kinase mitogen-activated protein kinase-activated protein kinase-2, which is activated by p38 MAP kinase (53). However, for the reasons discussed above, we currently favor a role for the stress kinases in tissue damage rather than protection following ischemia/reperfusion. Confirmation of the role of stress kinase activation in either apoptosis or cardioprotection awaits the availability of specific kinase inhibitors.
We have provided evidence for the occurrence of apoptotic cell death in both heart and kidney following ischemia/reperfusion, the progression of which appears to be linked to the tissue-specific pattern of stress kinase activation. The importance of apoptotic cell death in the in vivo evolution of infarct size has been demonstrated in a number of species from rat to human (2, 5, 54). A central role for apoptosis in tissue injury following ischemia/reperfusion suggests that inhibition of the apoptotic pathway may be a novel treatment for both acute myocardial infarction and acute renal damage. Because the activation of stress kinases may be an early event in the pathway leading to apoptosis, an ability to inhibit stress kinase activation may reduce apoptotic cell death and presumably the consequent tissue damage.
We thank Nancy Yao and Dongming Sun for assistance in preparation of kidney and heart samples, respectively. We are grateful to Dr. Karin Przyklenk for advice on obtaining the needle biopsy samples. We also thank Dr. Rodney Lappe for support of this work and Dr. Benjamin Bowen for critical reading of the manuscript.