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Myocardial depression can be demonstrated following administration of endotoxin. Proposed mechanisms of endotoxin-induced myocardial dysfunction include the release of proinflammatory mediators, focal myocardial ischemia, and the presence of activated leukocytes within the myocardium. Recently, myocardial caspase activation and mitochondria-related apoptotic events (i.e., release of cytochrome c) were demonstrated in the failing septic heart. Here, we tested the hypothesis that immunosuppressors, cyclosporin A and tacrolimus (FK 506), would improve inflammation, heart nuclear apoptosis, and myocardial dysfunction in endotoxin-treated rats. Myocardial contractility was assessed using an isolated heart preparation. Heart leukocyte infiltration was assessed by measurement of heart myeloperoxidase activity. Leukocyte activation was studied using the intravital microscopy of the mesenteric venule. Apoptosis was detected as myocardial DNA fragmentation, downstream caspase activation, and mitochondrial cytochrome c release. Both cyclosporin A and FK 506 reduced heart leukocyte sequestration and venular adhesion in endotoxin-treated rats. Cyclosporin A, which blocks mitochondrial cytochrome c release, was able to reduce endotoxin-induced myocardial end-stage nuclear apoptosis and heart dysfunction, whereas tacrolimus had no such effects. These effects could be related to the unique properties of cyclosporin A to act on mitochondria.
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Keywords: endotoxin; cardiomyocyte; mitochondria; cytochrome c; apoptosis
Human and animal studies have shown that myocardial contractile depression is an early manifestation of sepsis syndrome (1). Myocardial depression can be demonstrated in experimental animal models following administration of endotoxin, a lipopolysaccharide component of the outer membrane of gram-negative
bacteria (2, 4, 5). Although the etiology of myocardial contractile
dysfunction in this condition is incompletely described, potential
mechanisms include the effects of circulating and locally produced "depressant substances" such as proinflammatory cytokines, for example, tumor necrosis factor (TNF)-
and nitric oxide (6), focal myocardial ischemia and necrosis (10), and the
presence of activated leukocytes within the myocardium (11, 12).
In the heart, apoptosis may be activated under conditions
such as cytokine stimulation. Indeed, proinflammatory cytokine
TNF- directly induces apoptosis of cardiomyocytes in vitro
(13). Furthermore, we and other groups have recently shown
that sepsis may also trigger apoptotic pathways in the heart and
that these events may contribute to endotoxin-induced myocardial dysfunction (14). In most cases, the execution phase of
the apoptotic process involves the participation of mitochondria
as caspase activators (17, 18). Mitochondria release cytochrome
c into the cytosol, where it binds to and induces oligomerization
of Apaf-1 (19, 20). Once activated by cytochrome c, oligomerized Apaf-1 then binds procaspase-9, resulting in procaspase-9
proteolytic self-processing and activation of caspase-3, which
participates as a terminal effector of apoptosis by cleaving crucial nuclear substrates (20). In sepsis, myocardial upstream and
downstream caspase activation, as well as mitochondria-related
apoptotic events such as release of cytochrome c, was recently
demonstrated in the failing septic heart (15, 16).
A large body of evidence suggests that inflammation and apoptosis pathway activation contribute to endotoxin-induced myocardial dysfunction. In this context, we hypothetized that immunosuppressive compounds, that is, cyclosporin A (CsA) and tacrolimus (FK 506), would alter heart inflammation and apoptosis and ameliorate myocardial dysfunction in sepsis. Indeed, CsA and FK 506 are calcineurin inhibitors believed to exert their action through binding to cyclophilins and FK binding proteins, also known as immunophilins (21). When complexed with CsA and FK 506, the properties of immunophilins change, leading to the inhibition of the phosphatase calcineurin and subsequent inhibition of T cell activation. In addition, calcineurin inhibitors have been shown to inhibit cell apoptosis in vitro through mechanisms compatible with inhibition of mitochondrial permeability transition related to the opening of a megachannel in the inner mitochondrial membrane, cytochrome c release, and increases in levels of proteins of the Blc-2 family (22, 23). In vivo, analogues of CsA that block the mitochondrial permeability transition in isolated mitochondria are able to reduce reperfusion-induced myocardial dysfunction (22, 24, 25). Although these findings suggest that under certain conditions, calcineurin inhibitors may improve the function of the reperfused myocardium, the effects of CsA and FK 506 on endotoxin-induced myocardial inflammation and apoptosis have not been specifically addressed in the septic heart.
Therefore, in this study, we examined the protective effects
of cyclosporin A and tacrolimus on myocardial dysfunction in heart from endotoxin-treated rats. The results of this study
provide new pieces of information: (1) both CsA and FK 506 were able to reduce heart leukocyte sequestration, vascular
leukocyte activation, and serum concentrations of TNF- and
(2) CsA, but not FK 506, was able to offer protection to endotoxin-induced myocardial cell apoptosis and improve myocardial function.
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Animal Preparation
Care and use of animals were in accordance with our institution's guidelines for the care and use of laboratory animals. Six groups of adult male Sprague-Dawley rats were studied, that is, saline-treated (control animals), endotoxin-treated (Endo. 10 mg/kg of endotoxin from Escherichia coli serotype 055:B5), CsA-treated (10 mg/kg) and FK 506-treated (3 mg/kg) control and endotoxin-treated rats. Four hours after treatment, rats were prepared for either physiological measurements or for in vitro assays.
Physiological Measurements
Isolated and perfused heart preparationMyocardial contractile function was studied using a modified Langendorff isolated heart preparation as we have previously described (26).
Leukocyte adhesion in the mesenteric venulesThe mesenteric microcirculation was observed with the use of an intravital microscope (Eclipse E800 Nikon microscope, Tokyo, Japan). As previously described, single unbranched mesenteric venules were selected for study of leukocyte rolling and adhesion (27, 28).
Biochemical Measurements
Cytokine determination and leukocyte countSerum levels of interleukin (IL)-1
, IL-6, and TNF- were determined by use of commercially available immunoassay kits (enzyme-linked immunosorbent assay [ELISA]). Circulating blood leukocytes and differential counts of leukocytes were performed.
Myeloperoxidase assayMyeloperoxidase (MPO) was extracted from homogenized tissue by suspending the pellets in 0.5% hexadecyl trimethylammonium bromide in 50 mM phosphate buffer and MPO activity was assayed spectrophotometrically as described (29, 30).
DNA fragmentation detectionFor the detection of oligonucleosomes, a cell death detection ELISA plus kit (Boehringer, Mannheim, Germany) was used according to the manufacturer's instructions. For electrophoresis, DNA was extracted from cardiac tissue (LV apex) using a commercially available isolation kit (Genzyme TACS, R&D Systems, MN) (15). The DNA obtained was used in a ligation mediated polymerase chain reaction (LM-PCR) assay according to the manufacturer's instructions (Clontech Laboratories, Palo Alto, CA) (15).
Caspase-3 activity and Western blot analysis for procaspase-3
For measurements of caspase-3 activity, heart proteins were diluted and incubated at 25° C with the colorimetric substrate Ac-DEVD-pNA (Biomol, Plymouth, PA) in 96-well microtiter plates. Cleavage of the p-nitroaniline (p-NA) dye from the peptide substrate was determined by the measure of absorbance of p-NA at 405 nm in a microplate reader Digiscan (Asys Hitech, Cincinnati, OH) (14, 15). Results were calibrated with known concentrations of p-NA and expressed in picomoles of substrate cleaved per minute and per microgram proteins at 25° C.
For procaspase-3 immunoblotting, fresh heart tissue was resuspended in RIPA buffer. Lysates were centrifugated at 14,000 × g and 200 µg of supernatant was loaded on 12.5% polyacrylamide gels, electrophoresed, and transferred as previously described (14). Procaspase-3 was detected using a polyclonal rabbit anti-procaspase-3 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), which recognizes the 32-kD unprocessed procaspase-3.
Isolation of mitochondria and swellingThe mitochondrial fraction was prepared as previoulsy described (31). For determination of swelling, mitochondria were washed and resuspended (1 mg/ml) in sucrose buffer. Mitochondrial swelling was estimated from the decrease in absorbance measured at 520 nm in a spectrophotometer as described (32). Large amplitude swelling was induced by addition of 320 µM calcium at the indicated time.
Western blot analysis for cytochrome c
Mitochondrial protein samples in loading buffer were run on 12% SDS-PAGE gels. The proteins on the gel were electrophoretically transferred to nitrocellulose membranes. After blocking, membranes were treated with mouse monoclonal anticytochrome c antibody (Pharmingen, Becton Dickinson Company). Membranes were then incubated with horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin G (IgG) secondary antibody (Sanofi Diagnostics Pasteur, Bio-Rad), washed, and bound antibodies were detected using chemiluminescence with an ECL Plus kit (Amersham).
Statistical Analysis
For in vitro and in vivo studies, we tested for differences using ANOVA procedures (SPSS 9.0 for Windows, IL). Data are presented as means ± SEM throughout. A value of p < 0.05 was considered significant.
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Cyclosporin A Prevented Endotoxin-induced Myocardial Dysfunction
As shown in Table 1, left ventricle (LV) developed pressure (LVDP) and its first derivatives (i.e., dP/dtmax and dP/dtmin) were significantly decreased 4 h after administration of endotoxin as compared with control animals. Coinjection of CsA (10 mg/kg) largely prevented LV systolic function alterations of endotoxin-treated hearts (n = 7 in each group). FK 506 (3 mg/kg) had no detectable effects on myocardial function in endotoxin-treated animals (n = 7 in each group) (Table 1). Animals cotreated with endotoxin and CsA exhibited LVDP-preload relationships that were shifted upward compared with animals treated with endotoxin alone, suggesting improved systolic myocardial performance (Figure 1). Animals cotreated with endotoxin and FK 506 exhibited LVDP-preload relationships that were similar to animals treated with endotoxin alone (Figure 1). CsA and FK 506 treatment had no effects on myocardial function in control animals (Table 1).
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5 to +20 mm Hg in order
to construct LV developed
pressure (LVDP)-preload relationship curves. Compared
with control, the LVDP-
preload relationships were
shifted downward (in the
direction of LV systolic performance decrease) in endotoxin-treated rats and in
endotoxin + FK 506-treated rats. Endotoxin + CsA-treated rat hearts exhibited a shift of LVDP-preload relationships upward compared with
endotoxin-treated rat hearts (in the direction of LV systolic performance increase). Endotoxin + FK 506-treated rat hearts exhibited
LVDP-preload relationships similar to endotoxin-treated rat hearts.
CsA and FK 506 had no effects on LV function in control rats (data not
shown). *p < 0.01 compared with control rats.
Cyclosporin A and Tacrolimus Reduced Endotoxin-induced Inflammation
CsA and FK 506 reduced serum TNF- levels in endotoxin-treated rats but had no effects on IL-1 and IL-6 levels (Figure 2A). Compared with control rats, heart MPO activity, an
index of heart leukocyte infiltration, was increased in endotoxin-treated rats. CsA and FK 506 reduced heart MPO activities
in endotoxin-treated rats (Figure 2B). Consistently, leukocyte
adhesion on the mesenteric venule, a surrogate of vascular inflammation, was increased in endotoxin-treated rats compared
with control rats. In endotoxin-treated rats, CsA and FK 506 administration provided significant reduction of adhesive interactions between leukocytes and venular endothelium (Table 2). Compared with endotoxin-treated rats, measured hemodynamic variables (i.e., mean arterial pressure and shear
rate) were unchanged (Table 2) in CsA and FK 506 endotoxin-treated rats, which suggest that these compounds did not interfere with the physical forces that modulate leukocyte behavior within the microvasculature.
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p < 0.01 compared with endotoxin-treated rats). (B) Compared with control rats,
heart myeloperoxidase (MPO) activity, an index of heart leukocyte infiltration, increased in endotoxin-treated rats. CsA and FK 506 prevented heart MPO activities in endotoxin-treated rats. Results are displayed as mean ± SEM. (*p < 0.01 compared with control rats; n = 5 in each group;
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Cyclosporin A Prevented Endotoxin-induced Myocardial Cell Nuclear Apoptosis
As shown in Figure 3A, significant nuclear DNA fragmentation detected by agarose gel electrophoresis was evident 4 h after endotoxin injection in the myocardium. Oligonucleosomal DNA fragmentation confirmed nuclear apoptosis 4 h after endotoxin injection (Figures 3A and 3B). CsA protected myocardial cells from DNA fragmentation induced by endotoxin, whereas FK 506 had no effects (Figure 3).
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Effects of Cyclosporin A and Tacrolimus on Endotoxin-induced Caspase-3 Activation
Four hours after endotoxin administration, caspase-3 activity assessed by measuring hydrolysis of the preferential substrate Ac-DEVD-pNA, increased in rat heart (n = 8 rats in each group) (Figure 4A). Results of immunoblotting (n = 4 in each group) indicated that in contrast to control hearts, the proteolytic activation of procaspase-3 was significant in endotoxin-treated myocardium (Figure 4B). Neither CsA nor FK 506 prevented proteolytic activation of procaspase-3 and caspase-3 activity increases in endotoxin-treated rat hearts.
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Cyclosporin A Prevented Mitochondrial Cytochrome c Release in the Septic Heart
Results of immunoblotting indicated that cytochrome c, in contrast to normal hearts, was substantially reduced in the mitochondrial fraction of endotoxin-treated rat hearts (Figure 5). The reduction in mitochondrial cytochrome c observed in endotoxin-treated rats was prevented by CsA, but not by FK 506 (Figure 5).
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In Vitro Effects of Cyclosporin A and Tacrolimus on Heart Mitochondria Isolated from Control and Endotoxin-treated Rats
In these experiments, we have tested the capacity of CsA to inhibit mitochondria permeability transition (MPT) in our model of sepsis. Heart mitochondria were purified from control (Figure 6A) and endotoxin-treated rats (Figure 6B). Then, MPT was determined as large amplitude swelling after addition of a high dose of calcium, a standard protocol for the induction of permeability transition. As shown in Figure 5, no difference could be detected in the MPT-dependent colloidosmotic swelling induced by calcium of isolated mitochondria from control (Figure 5A) and endotoxin-treated rats (Figure 5B). As expected, preincubation of mitochondria with CsA fully inhibited calcium-induced PT (Figure 6). The MPT inhibitory effect of CsA was observed to the same extent on mitochondria from control and also on mitochondria from endotoxin-treated rats. In contrast, FK 506 was unable to protect mitochondria from MPT (Figure 6) even at high concentrations (data not shown).
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In this rat model of endotoxin-induced myocardial dysfunction,
we found evidence of inflammation and end-stage apoptosis that may involve downstream caspase activation and mitochondria-related pathway events such as cytochrome c release. Pharmacological manipulation with immunosuppressors reduced endotoxin-induced inflammation and myocardial apoptosis. Both
CsA and FK 506 reduced serum TNF- levels, heart leukocyte
sequestration, and venular endothelial adhesion in endotoxemic rats. CsA, but not FK 506, was able to offer protection
from endotoxin-induced myocardial dysfunction and apoptosis. We speculated that the differential effects of the calcineurin inhibitors on myocardial dysfunction may be related to the unique effects of CsA on the mitochondrial function.
Proinflammatory mediators and leukocytes activated by
proinflammatory cytokines contribute to the development of
cardiovascular dysfunction associated with endotoxemia (2, 4,
7, 11, 12). The relevance of cytokine release and leukocyte
adhesion in mediating cardiovascular dysfunction is illustrated
by the finding that reduction of circulating cytokines and
leukocyte activation attenuates endotoxin-induced cardiovascular dysfunction in shock models (33). In our model, endotoxin induced increases in TNF- serum levels, heart MPO,
and mesenteric endothelial-leukocyte interactions, which were
mainly prevented by the administration of calcineurin inhibitors
CsA and FK 506. These results are consistent with the finding
that CsA and FK 506 modulate cytokine and adhesion molecule gene expression (36, 37) and attenuate cardiovascular failure by interfering with the processes of leukocyte adhesion and
proinflammatory cytokine production in experimental models (37, 38).
Indeed, the identification of calcineurin as a target for the
immunosuppressive drugs cyclosporin and FK 506 suggests a
critical role for this phosphatase in the regulation of T cell reactivity and cytokine gene expression (22, 23). Cyclosporin
and FK 506 inhibit calcineurin catalytic activity through complexes with cyclophilins and FKBP12, respectively. The mechanism whereby calcineurin promotes T-cell activation and cytokine activation has been largely attributed to the family of
transcriptional regulators referred to as nuclear factor of activated T cells (NFAT). Dephosphorylation of NFAT and its
translocation to the nucleus are followed by induction of several cytokines, including IL-2, IL-4, and TNF- (39). Whereas
NFAT factors are important downstream effectors, calcineurin also regulates activity of the transcriptional regulatory factors nuclear-
B, Elk-1, and myocyte enhancer factor-2 (39, 40). In
addition, recent findings suggest that calcineurin promotes nitric oxide synthase, c-jun N-terminal kinase, extracellular signal-regulated kinase, and protein kinase C and 
activation (40). Overall, these findings suggest a critical role for calcineurin in the regulation of several gene expressions (22, 23,
40) involved in the adaptative myocardial response to stress.
Hence, inhibition of calcineurin activity may reduce endotoxin-induced cardiovascular inflammation and myocardial
dysfunction. However, in our model of sepsis, CsA, but not
FK 506, prevented myocardial dysfunction, suggesting that cyclosporin could improve myocardial function by interfering
with different pathways. Indeed, in addition to their immunosuppressive effects, calcineurin inhibitors regulate processes
of cellular apoptosis, suggesting that calcineurin is critical in
death signaling (21, 41).
In the heart, apoptosis has been implicated as a major process in diseases including dilated cardiomyopathy, myocarditis, and ischemic reperfusion injury (42, 43). In cardiomyocytes, apoptosis pathways can also be activated under conditions such as cytokine and endotoxin stimulation, in vitro (13). Recently, we (14, 15) and other groups (16) have shown that in vivo, endotoxin induces myocardial apoptosis. In these studies, however, the observed small extent of cardiomyocyte nuclear apoptosis was likely insufficient to directly account for myocardial dysfunction. Therefore, it has been suggested that apoptotic pathways may be linked to myocardial dysfunction in other ways, involving multiple caspase activation and altered mitochondrial signaling. In the present study, we confirmed that endotoxin induces heart nuclear apoptosis as evidenced by detection of oligonucleosomes and electrophoresis DNA laddering (44). Consistent with previous results (13, 14, 16), we documented activation of apoptotic pathways in endotoxin-treated heart by measuring cytosolic activation of downstream caspase-3 and mitochondria cytochrome c release.
Growing evidence suggests that calcineurin participates in
pathways of apoptotic death. Calcineurin inhibitors regulate
apoptosis in cells stimulated by TNF- and nitric oxide (23,
45). For example, cyclosporin and FK 506 potently inhibit cytotoxicity induced by TNF- in rat hepatoma cells. Cyclosporin
and FK 506 may oppose programmed cell death by inhibiting
calcineurin activity, downstream caspase activaty, and mitochondrial signaling such as the mitochondrial pore transition
and expression of Bcl-2. Consistently, in endotoxin-treated
heart, CsA reduced heart internucleosomal DNA fragmentation. We found that neither cyclosporin nor FK 506 prevented
increases in downstream caspase-3 activity, which was assessed by procaspase-3 protein level and DEVDase activity.
However, cyclosporin, but not FK 506, prevented mitochondrial cytochrome c release in vivo and mitochondrial pore
transition in vitro. These results suggest that in our model, cyclosporin may oppose cell death by inhibiting mitochondrial
signaling (23, 41).
The differential effects of CsA and FK 506 on myocardial dysfunction could be related to their known opposite effects on mitochondrial permeability transition and cytochrome c release (21, 41). The mitochondrial permeability transition is caused by the opening of a nonspecific pore in the inner mitochondrial membrane under conditions of mitochondrial calcium overload, oxidative stress, and adenine nucleotide depletion (21, 32). CsA, but not FK 506, prevents in vitro mitochondrial permeability transition and cytochrome c release by blocking translocation of the matrix-specific cyclosporin D to the inner membrane of the mitochondria, thereby decreasing the mitochondrial megachannel sensibility to succinate and calcium ions and reducing mitochondrial swelling (21, 22). Indeed, we confirmed that CsA, as opposed to FK 506, prevented mitochondrial cytochrome c release in vivo in septic hearts and mitochondrial swelling in energized isolated mitochondria from control and septic hearts in vitro. However, in endotoxemic rats, CsA and FK 506 in vivo treatment effects on mitochondrial swelling were not tested because no differences between control and septic energized mitochondria were observed in our experimental setting. Overall, our results are consistent with previous studies showing that analogues of CsA inhibit the permeability transition pore and are able to provide myocardial protection (22, 24). Indeed, CsA provide protective effects in reoxygenated cardiac myocytes and reperfused heart (22, 24). The mechanisms of cyclosporin-induced myocardial protection remain speculative. It could be hypothetized, however, that beneficial effects of CsA on endotoxin-induced dysfunction may be related to independent mitochondria permeability transition effects (46), such as regulatory roles of immunophilins at the ryanodine receptor and antiapoptotic gene product bcl-2 (23, 39) and changes in nitric oxide production (50, 51).
In conclusion, these observations strongly suggest that (1) mitochondrial cytochrome c release may play an important role in the onset of end-stage nuclear apoptosis in endotoxemic heart and (2) CsA, which blocks cytochrome c release, was able to reduce endotoxin-induced heart end-stage nuclear apoptosis and myocardial dysfunction, which was not observed with FK 506. The precise mechanisms of the beneficial effects of cyclosporin warrant further investigations.
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Correspondence and requests for reprints should be addressed to Dr. Rémi Nevière, M.D., Ph.D., Département de Physiologie, Faculté de Médecine, 1, place de Verdun, Lille Cedex 59045, France. E-mail: rneviere{at}univ-lille2.fr
(Received in original form May 17, 2001 and accepted in revised form November 26, 2001).
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.orgAcknowledgments: This work was supported by IFR22, INSERM, Université de Lille 2, and by grants from MENRT and INSERM: Biologie et pathologie des régulations cellulaires. U459 and EA 2689 belong to IFR 22 (CHU, COL, INSERM, IRCL, Université Lille 2).
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