Acute Doxorubicin Cardiotoxicity Involves Cardiomyocyte Apoptosis1

Abstract

Despite well-documented cardiotoxic effects, doxorubicin remains a major anticancer agent. To study the role of myocardial apoptosis follow- ing doxorubicin administration, male Wistar rats were exposed to 1.25, 2.5, and 5 mg/kg of i.p. doxorubicin and terminated on days 1-7 in groups of five. Doxorubicin caused a significant (P < 0.001) and dose-dependent induction of cardiomyocyte apoptosis at 24 - 48 h after the injection. Repeated injections of 2.5 mg/kg given every other day resulted in peaks of apoptosis at 24 h after each injection. However, no additive effect of repeated dosing was noted. In histological samples, alterations in the cytoskeletal apparatus with focal loss of contractile elements were seen after a single injection. Myocyte necrosis was absent. Thus, acute doxo- rubicin-induced cardiotoxicity involves cardiomyocyte apoptosis, a poten- tially preventable form of myocardial tissue loss.

Full text

2000;60:1789-1792. Cancer Res
Olli J. Arola, Antti Saraste, Kari Pulkki, et al.
Apoptosis
Acute Doxorubicin Cardiotoxicity Involves Cardiomyocyte
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[CANCER RESEARCH 60, 1789–1792, April 1, 2000]
Advances in Brief
Acute Doxorubicin Cardiotoxicity Involves Cardiomyocyte Apoptosis
1
Olli J. Arola, Antti Saraste, Kari Pulkki, Markku Kallajoki, Martti Parvinen, and Liisa-Maria Voipio-Pulkki
2
Departments of Medicine [O. J. A., L-M. V-P.], Anatomy [A. S., M. P.], Clinical Chemistry [K. P.], and Pathology [M. K.], University of Turku, FIN-20521 Turku, Finland
Abstract
Despite well-documented cardiotoxic effects, doxorubicin remains a
major anticancer agent. To study the role of myocardial apoptosis follow-
ing doxorubicin administration, male Wistar rats were exposed to 1.25,
2.5, and 5 mg/kg of i.p. doxorubicin and terminated on days 1–7 in groups
of five. Doxorubicin caused a significant (P<0.001) and dose-dependent
induction of cardiomyocyte apoptosis at 24–48 h after the injection.
Repeated injections of 2.5 mg/kg given every other day resulted in peaks
of apoptosis at 24 h after each injection. However, no additive effect of
repeated dosing was noted. In histological samples, alterations in the
cytoskeletal apparatus with focal loss of contractile elements were seen
after a single injection. Myocyte necrosis was absent. Thus, acute doxo-
rubicin-induced cardiotoxicity involves cardiomyocyte apoptosis, a poten-
tially preventable form of myocardial tissue loss.
Introduction
The anthracycline antibiotic DOX
3
is an important antineoplastic
agent because of its high antitumor efficacy in hematological as well
as in solid malignancies. Its use is limited by the not infrequent
induction of dose-dependent chronic cardiomyopathy. The mecha-
nisms of DOX cardiotoxicity include (a) the formation of free reactive
oxygen radicals, (b) direct DNA damage and/or interference with
DNA repair (1, 2), and (c) induction of immune reactions involving
antigen-presenting cells in the heart (3). In addition to nucleic acids
and cellular membranes, the cytotoxic action by anthracyclines in-
volves the cytoskeleton of both tumor cells and cardiomyocytes (4).
Cytoskeletal changes following DOX administration include reduc-
tion in the density of myofibrillar bundles (5), alterations on the Z-disc
structure, and disarray and depolymerization of actin filaments (6, 7).
Histologically, cytoplasmic vacuolization due to dilation of the sar-
cotubules and loss of myofibrils characterize DOX cardiomyopathy
(8). Recently, myocardial apoptosis has been suggested as a common
mechanism of acute and chronic myocyte loss (9–11). DOX induces
apoptosis in several cell lines (12) and, in a rat model, in the kidney
and the intestine (13). Whether DOX-induced cardiotoxicity involves
CA remains unclear. In a rat model, no CA was observed in samples
obtained after a 12-week cumulative DOX treatment (13). In human
heart samples, however, contraction and ring formation of the nuclei
suggestive of apoptosis have been documented within 24 h after DOX
injection (14). Furthermore, apoptosis-type cell morphology with
shrunken focal cardiomyocytes has been observed in connection with
myofibrillar lysis in the classic study by Billingham et al. (7). We
therefore designed an experimental study to investigate whether acute
DOX administration induces CA in vivo, the possible dose-response
of such an acute effect, and the relationship of CA to ensuing histo-
logical myocardial damage typical of anthracycline cardiomyopathy.
Materials and Methods
Experimental Protocol. Adult male Wistar rats weighing 300–470 g (total
n⫽110) were purchased from the Central Animal Laboratory, Turku Uni-
versity, Finland. The rats had free access to standard rodent chow and water.
All procedures for animal care and housing were in compliance with the
contemporary guidelines. The Laboratory Animal Committee and the Social
and Health Department of the Provincial State Office of Western Finland
approved the experimental protocol. DOX was obtained from Pharmacia SPA
(Milan, Italy). DOX cardiomyopathy was induced as described by Iliskovic
and Singal (15). In brief, after rats received s.c. buprenorphin (0.05 mg/kg;
Reckitt & Colman, Hull, England) to provide analgesia, the first 2.5 mg/kg
dose of DOX was injected i.p., and the animals were allowed to recover for
48 h before the procedure was repeated as determined by the subprotocols.
Control animals (n⫽10) were treated with only buprenorphin and i.p. saline.
To first study the occurrence of CA after a single versus multiple DOX
injections, a pilot study with 15 animals was performed. The rats were given
2.5 mg/kg of DOX every other day up to a maximal cumulative dose of 15
mg/kg over 2 weeks. The rats were killed with carbon dioxide on days 1, 3, and
14 after the first injection and on days 1, 7, and 18 after the last DOX injection
(n⫽3–5 in each group).
Being guided by the results from the pilot study, we then studied the early
time course and dose-dependence of apoptosis occurrence. Groups of 15–25
rats received single injections of 1.25, 2.5, or 5 mg/kg of DOX. The rats were
killed in groups of five on days 1, 2, and 3 after the injection. For the 2.5 mg/kg
injections, rats were also killed at 6 h and on day 7. Finally, to study the effects
of consecutive injections, three injections of 2.5 mg/kg each were given every
other day. Ten rats were killed on the day after each injection, and five rats
were killed on days 2 and 3 after the respective injections.
Immediately post mortem, the entire heart was excised. The heart was sliced
transversally, and a midventricular slice was fixed in neutral-buffered 10%
formalin for 24 h, embedded in paraffin, and cut into 5-
␮
m sections for
assessment of apoptosis and histological features.
In Situ Assay for Apoptosis. Apoptotic cardiomyocytes were detected
with the TUNEL assay and previously described methodology (9, 11, 16).
In brief, nuclear DNA strand breaks were end-labeled with digoxigenin-
conjugated dideoxy-UTP by terminal transferase and visualized immuno-
histochemically with digoxigenin antibody conjugated to alkaline phospha-
tase. The assay was carefully standardized using adjacent tissue sections of
each sample, which were pretreated with DNase I (1 unit/ml for 15 min at
37°C; positive control of DNA breaks). The staining of each section was
interrupted when intense positivity in the DNase I-treated section appeared.
This approach provides optimal sensitivity for double-strand DNA breaks
and normalizes the results for differences in tissue permeability of the
reagents. The cardiomyocyte origin of TUNEL-positive nuclei was identi-
fied by the presence of surrounding myofilaments. The validity of this
approach was confirmed by immunofluorescence staining with antimyosin
(cat. no. M-8421; Sigma, St. Louis, MO) of randomly selected TUNEL-
stained sections. To further confirm the specificity of TUNEL staining in
respect to apoptosis, attention was paid to the morphology of positive
nuclei and cells.
Quantification of Apoptotic Cells. The number of apoptotic cardiomyo-
cytes in TUNEL-stained sections was counted by use of light microscopy with
an ocular grid (⫻250 magnification; area of the field, 0.25 mm
2
). An average
of 184 and 210 microscopic fields were analyzed per DOX-treated and control
animal, respectively. The average number of cardiomyocytes per field was
Received 11/1/99; accepted 2/15/00.
The costs of publication of this article were defrayed in part by the payment of page
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18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported financially by the Aarno Koskelo Foundation, Turku
University Foundation, and Finnish Cultural Foundation.
2
To whom requests for reprints should be addressed, at Department of Medicine,
Turku University Central Hospital, P.O. Box 52, 20521 Turku, Finland. E-mail:
olli.arola@utu.fi.
3
The abbreviations used are: DOX, doxorubicin; CA, cardiomyocyte apoptosis;
TUNEL, terminal transferase-mediated DNA nick end labeling.
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287 ⫾42 (mean ⫾SD) in DOX-treated animals and 320 ⫾45 in controls. The
results are expressed as the percentage of apoptotic nuclei of the total number
of labeled cardiomyocyte nuclei, which were counted in the corresponding
DNase I-treated section. No apoptotic nuclei in the interstitial space or in the
endothelial lining of blood vessels were found. The correlation between the
results of repeated procedures with this method is 0.88 (P⬍0.01; Ref. 11).
Histological Analysis. Formalin-fixed, paraffin-embedded heart tissue sec-
tions were stained with van Gieson to demonstrate general histological fea-
tures. To visualize the cytoskeletal elements, formalin-fixed, paraffin-embed-
ded sections were stained for the intermediate filament protein desmin.
Sections were first deparaffinized with xylol, and then boiled twice for 5 min
in a microwave oven in 10 mMcitrate buffer (pH 6.0). The primary antibody
mouse antidesmin (Zymed Laboratories Inc., San Francisco, CA) was diluted
1:30, and the bound antibody was detected with a biotin-avidin-peroxidase
complex (Vectastain ABC kit; Vector Laboratories Inc., Burlingame, CA). The
sections were counterstained with hematoxylin.
Statistical Analysis. Quantitative results are expressed as means ⫾SD.
Percentages of apoptotic cardiomyocytes between DOX-treated and control
rats were compared using one-way ANOVA followed by Dunnett’s two-sided
ttest. In the case of multiple comparisons (one dose versus multiple doses,
different dosages, or consecutive injections) the least significant difference
post hoc test with Bonferroni correction was used. The software used was
Fig. 1. van Gieson-stained sections of rat myocardium from a control animal (A) and 24 h after a single injection of 2.5 mg/kg DOX (B). Note the vacuolization of cardiomyocytes
(arrows) and edema (ⴱ)inB.C, dark brown-colored TUNEL-positive nucleus in a sample obtained 24 h after injection of 2.5 mg/kg DOX. Note the condensation of the nucleus and
shrunken appearance of the cell. D, double staining with antimyosin shows the same nucleus located within the cardiomyocyte. E–G, immmunohistochemical staining for desmin. E,
control rat myocardium; F, myocardium after a single injection of 2.5 mg/kg DOX; G, myocardium after the cumulative DOX dose of 15 mg/kg and a follow-up for 7 days. Note the
gradual decrease in the intensity of staining and disorganization of cross-striated staining pattern (ⴱ). Magnification: A,B,E–G,⫻200; bar ⫽50
␮
m; Cand D,⫻1000; bar ⫽10
␮
m.
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SPSS 8.0 for Windows (SPSS Inc, Chicago, IL). Differences were considered
significant at P⬍0.05.
Results
General Effects and Histological Analysis. After the total cumu-
lative DOX dose of l5 mg/kg over 2 weeks (pilot study), attenuation
in weight gain and signs of clinical DOX-induced cardiomyopathy
(ascites, hepatic enlargement, and some degree of general lethargy)
were observed as originally described by Iliskovic and Singal (15).
The clinical cardiomyopathy syndrome became gradually more evi-
dent during the 18-day observation time following the last injection;
in particular, greater ascites formation was noted. The pilot study
revealed the presence of TUNEL positivity in myocardial samples
obtained early during the course of DOX treatment. However, in
samples obtained 1–2 weeks after the cumulative treatment, the num-
ber of TUNEL-positive cells declined and was not significantly dif-
ferent from controls (data not shown).
In the van Gieson-stained light microscopic sections, alterations
consistent with DOX-induced toxicity were seen: cardiomyocyte vac-
uolization (Fig. 1, Aand B) was present after a single injection of 2.5
mg/kg DOX. Notably, necrotic cardiomyocytes were not seen. Neither
polymorphonuclear inflammatory cell infiltration nor lymphocyte in-
filtration was present.
Immunohistochemical staining of desmin, which is an intermediate
filament protein important in the anchorage of the contractile elements
in cardiac myocytes (17), was profoundly altered after a single injec-
tion of DOX. The uniformly intense staining for desmin vanished, and
the normal cross-striation of the cardiomyocytes (Fig. 1E) disappeared
in a focal manner (Fig. 1F). After the cumulative 15 mg/kg of DOX
and follow-up for 7 days, the desmin staining showed even more
striking changes: the staining of cardiomyocytes appeared patchy; and
the sarcomeres and intercalated discs were hardly visible. These
heavily damaged cells were, however, often adjacent to cells that
appeared normal.
Single DOX Injection: Time Course and Dose-dependent In-
duction of TUNEL Positivity. Microscopic examination of the
TUNEL-stained sections showed the presence of sparse single posi-
tive nuclei scattered across the ventricular wall. These were sur-
rounded by cardiomyocyte myofilaments (Fig. 1C) as visualized by
double staining with antimyosin (Fig. 1D). In general, TUNEL-
positive cardiomyocytes showed condensation of nuclei and,
sometimes, shrinking of the cytoplasm consistent with apoptotic mor-
phology (Fig. 1, Cand D). Only very few TUNEL-positive inflam-
matory and other non-myocyte cells were observed equally in all
hearts, including the control samples.
TUNEL-positive CA was a very rare event in control animals
[0.0065 ⫾0.0022% (mean ⫾SD)]. A significant induction of CA to
0.033 ⫾0.012% (P⬍0.001) of total cardiomyocytes was seen at 24 h
after a single 2.5 mg/kg injection of DOX. The percentage of CA
peaked on the first day after the injection and declined thereafter (Fig.
2A). The onset of TUNEL positivity was very rapid: at 6 h, the mean
percentage was increased to 0.034 ⫾0.027% (P⬍0.05). The gradual
decline of TUNEL positivity continued at day 7, confirming prelim-
inary observations from the pilot study.
In response to 1.25, 2.5, and 5 mg/kg DOX, a dose-dependent
induction of CA was observed. As shown in Fig. 2B, the highest
relative percentages of apoptotic cardiomyocytes were observed on
day 1 after the 1.25 and 2.5 mg/kg doses and on day 2 after 5.0 mg/kg.
Compared with sham-injected controls (day 0; 0.0065 ⫾0.0022%),
the peak amount of TUNEL-positive nuclei was statistically signifi-
cantly increased after the 2.5 mg/kg (0.033 ⫾0.012%; P⬍0.001) and
Fig. 2. A, time course of apoptosis occurrence after a single injection of 2.5 mg/kg DOX. B, dose-dependent induction of apoptosis following single injections of 1.25, 2.5, and 5.0
mg/kg DOX.
Fig. 3. Three consecutive injections of 2.5 mg/kg DOX administered 1 day apart. Note
that the increase of apoptosis is cumulative but not additive. Arrows indicate injections.
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5.0 mg/kg (0.066 ⫾0.020%; P⬍0.001). A gradual decline of
TUNEL positivity was observed on day 3, but the percentages still
remained above control values.
Consecutive Injections. Fig. 3 shows the amount of CA after three
consecutive injections of 2.5 mg/kg DOX administered 1 day apart.
After each injection, a new increase in the mean amount of CA was
observed (0.033, 0.042, and 0.049% on days 1, 3, and 5, respectively;
Fig. 3). Notably, the effect of each new DOX dose was attenuated so
that the relative increase was 7-fold after the first injection, 33% after
the second injection, but only 16% after the third injection.
Discussion
Our data demonstrate a significant induction of CA in the early
phase following DOX administration. The percentage of TUNEL-
positive cells peaked within 24–48 h after a single injection, followed
by a gradual decline to baseline levels by day 7 after the moderate 2.5
mg/kg dose. The number of TUNEL-positive cardiomyocytes also
declined rapidly during the follow-up after a cumulative high-dose
DOX treatment. Thus, our findings are not inconsistent with those
obtained by Zhang et al. (13), who could not demonstrate CA after
prolonged (42–84 days) DOX administration and follow-up.
The fact that DOX is toxic to and interferes with DNA raises
concern regarding the specificity of the TUNEL assay in assessing
apoptosis as a mechanism of DOX cardiotoxicity. The validity of our
method has been discussed previously in detail (9, 11, 16). In brief,
the standardization procedure using DNase I-treated control sections
results in optimization of the TUNEL assay for apoptosis-specific
double-strand DNA breaks. During microscopic examination, typical
nuclear and cellular morphological features of apoptosis were found
in association with TUNEL positivity. Recently, induction of signif-
icant CA by DOX in a rat ventricular cell line was reported by Delpy
et al. (18). A protective effect by thermal preconditioning in this in
vitro model was also described by Ito et al. (19). In addition to the
TUNEL assay, these researchers used also agarose DNA gel electro-
phoresis (DNA ladder assay). In our experience, DNA ladders are
only demonstrable in the TUNEL-positive areas when the amount of
positive cells exceeds ⬃0.04% (9).
A theoretical basis for reduced toxicity by fractional DOX dosing is
provided by the observation that the single high dose of 5 mg/kg
increased TUNEL positivity more (0.066 ⫾0.020%) than did two 2.5
mg/kg injections (0.051 ⫾0.018%). The induction of TUNEL posi-
tivity was cumulative after repeated injections, but the relative in-
crease was blunted. This further supports dose fractionation and
suggests induction of protective mechanisms. Because the number of
apoptotic cardiomyocytes was reduced to nonsignificant levels 3 days
after the cumulative dose was achieved, induction of apoptosis by
DOX seems to be an acute effect in DOX cardiotoxicity. Cardio-
myocyte necrosis is characterized by swelling of the cell, which
eventually leads to leakage of cellular components, causing an inflam-
matory response in adjacent areas. In principle, cells may proceed to
or transform to a necrotic type of cell death. However, immediate or
delayed histological signs of necrosis did not develop in our model
even after a dose considered to be pharmacological or after three
repeated injections.
Myocardial apoptosis is not, however, the only mechanism of
deteriorating contractile force because the remaining myocardium is
also dysfunctional. In addition to the apparent loss of cellular ele-
ments, our data show that the very first injection of DOX induces
changes in the cytoskeleton and in the contractile element of the
cardiomyocyte. The rapid damage observed in the cytoskeleton is
likely to cause myocardial dysfunction and contribute to clinical heart
failure syndrome (20). Whether this effect is due to direct DOX
toxicity or a failure in protein synthesis reflecting DNA damage
remains unclear. These cytoskeletal interferences are likely to favor
cell shrinkage and affect tension development (20). This effect can
modify or even speed up apoptosis.
Attenuation of apoptotic cell death by a caspase inhibitor has been
demonstrated in a myocardial reperfusion injury model. The caspase
inhibitor pretreatment effect was achieved in cells receiving a signal
that usually promotes apoptosis (21). In cultured cardiomyocytes,
exposition to thermal preconditioning before DOX administration
attenuates apoptosis occurrence (19). Thus, myocardial apoptosis is a
potentially modifiable and preventable form of myocardial tissue loss.
The acute cardiotoxicity of DOX is multifactorial. The very first
injection of DOX alters the organization of the cardiomyocyte; in
addition, an early induction of apoptosis is observed. This potentially
novel mechanism is transient, but it may be of key importance to the
ensuing heart failure. Inhibition and modification of this mechanism
warrants further study.
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