© 1998 American Heart Association, Inc.
Original Contributions |
Regulation of Cardiomyocyte Apoptotic Signaling by Insulin-like Growth Factor I
From the Departments of Medicine and Biological Chemistry, Division of Endocrinology, Diabetes, and Metabolism, University of California, Irvine.
Correspondence to Ping H. Wang, MD, University of California, Department of Medicine, Medical Science I, Room C240, Irvine, CA 92697-4086.
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Abstract |
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Abstract—Apoptosis is regulated by specific intracellular signaling pathways. The development of cardiomyopathy involves the apoptosis of cardiomyocytes; however, the details of their apoptotic signaling are not yet known. Insulin-like growth factor I (IGF I) is an important survival growth factor for myocardium and other tissues, but the effects of IGF I on apoptotic signaling remain largely unknown. To study apoptotic signaling pathways in cardiomyocytes and to understand IGF I actions on the apoptotic signaling of cardiac muscle cells, we have defined the effects of IGF I on Bcl-2, Bax, caspase 3, DNA fragmentation, and cell survival in primary cardiomyocytes. Compared with Bax levels, the levels of Bcl-2 were found to be quite low in these cells. Serum withdrawal and doxorubicin reduced cell viability, increased fragmentation of DNA, increased cellular contents of Bax, and activated caspase 3. IGF I enhanced cell viability, suppressed DNA fragmentation, attenuated Bax induction, and suppressed caspase 3 activation. The levels of Bcl-2–associated Bax were increased after serum withdrawal and incubation with doxorubicin and were reduced by IGF I. Thus, cardiomyocyte apoptosis induced by serum withdrawal and doxorubicin likely results, in part, from the induction of Bax and activation of caspase 3, but IGF I may inhibit cardiomyocyte apoptosis by attenuating Bax induction and caspase 3 activation. These findings provide new insight into the mechanisms of cardiomyocytes apoptosis and may help elucidate how IGF I modulates apoptotic signaling in cardiac muscle.
Key Words: apoptosis • insulin-like growth factor I • cardiac muscle • caspase 3 • Bax
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Insulin-like growth factor I (IGF I) can inhibit apoptosis of cardiac muscle cells.1 2 Apoptosis, ie, programmed cell death, is regulated by specific intracellular signaling pathways that ultimately induce cell self-destruction.3 Although these pathways may differ in different tissues,4 they usually converge, indicating common cell death mechanisms.5 The details of these common mechanisms are yet known, but it is widely believed that caspase 3, a cysteine protease that belongs to a family of interleukin-1ß–converting enzymes, plays a critical role in this process.6 Previous studies have shown that activation of caspase 3 leads to nucleosomal fragmentation of DNA,6 a hallmark of apoptosis. The activities of caspase 3 can be regulated by the Bcl-2 protein family. Bcl-2 was the first member of the protein family to be identified; subsequently, several homologous proteins that share common BH domains were cloned.5 Interestingly, some of these proteins, such as Bcl-2, block apoptosis, whereas others, such as Bax, promote cell death. Homodimers of Bcl-2 may stabilize the mitochondrial membrane and prevent the activation of downstream apoptotic signaling.5 Recent data indicate that Bax can neutralize the actions of Bcl-2 by forming heterodimers with Bcl-2.7 Moreover, homodimers of Bax may independently trigger apoptotic signaling further downstream.8
Apoptosis of cardiomyocytes occurs in animal models of cardiomyopathy and human heart failure, and the development of cardiomyopathy and myocardial remodeling may involve increased apoptosis of cardiomyocytes.9 Although apoptosis in cardiac muscle has just now been recognized, how it occurs is not yet clear, and further investigation into its molecular mechanisms is required. IGF I may improve cardiac function in animal models of cardiomyopathy,1 2 10 11 12 13 but the actions of IGF I on apoptotic pathways in cardiac muscle cells are largely unknown. To this end, we have studied the effects of IGF I on Bcl-2, Bax, caspase 3, and DNA fragmentation in primary cardiomyocytes isolated from fetal rats.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
FBS and cell culture medium were purchased from Irvine Scientific. Terminal deoxynucleotidyl transferase was from Pharmacia. Polyvinylidene fluoride membranes and reagents for SDS-PAGE were purchased from Bio-Rad Laboratories. Protein concentrations were analyzed with the Bio-Rad protein assay kit. Anti–Bcl-2 and anti-Bax antibodies were from Santa Cruz Biotechnology or Oncogene Research Products. Recombinant GST–Bcl-2 and GST-Bax fusion proteins were from Santa Cruz Biotechnology. IGF-I was supplied by Genentech. [
-32P]ATP was
from Amersham. Other chemicals were purchased from Sigma Chemical Co
or Fisher.
Primary Cardiomyocyte Culture
Primary cultures of rat cardiomyocytes were prepared
from the cardiac ventricles of 17- to 19-day (gestational age)
Sprague-Dawley rats as previously described.14
After disaggregation with 0.125% pancreatin, nonmuscle cells were
minimized by differential plating.15 At the time
of experiments, these preparations contained <10% fibroblasts.
Myocytes were cultured at a density of 8x106
cells per T-75 flask overnight in 10 mL DMEM containing 10% FBS. The
cells were trypsinized and plated (
25 000 to 30 000
cells/cm2) in 8-well slides for in situ DNA
fragmentation studies and in 100-mm dishes for other studies. All
experiments in the present study used 60% to 70% confluent cells.
In experiments with IGF I, primary cardiomyocytes were
rinsed 3 times with 0.5% BSA+DMEM, serum-starved for 18 hours in 0.5%
BSA+DMEM, rinsed 3 times with 0.5% BSA+DMEM, and then incubated with
IGF I at the indicated concentrations. To induce apoptosis, the
cells were rinsed 3 times with DMEM and then serum-deprived (DMEM+0.5%
BSA) in the presence or absence of the indicated concentrations of
doxorubicin for 14 to 15 hours. To study the effects of IGF I on
apoptotic signaling, IGF I was added to the culture medium 20
minutes before the addition of doxorubicin for all experiments
involving IGF I.
MTT Assay
Cell viability was analyzed by MTT assay as previously
described.16 For this assay, equal numbers of
contractile cardiomyocytes were plated on 96-well plates
(30 000 cells/well) and maintained in regular growth medium for 2
days. The cells then underwent doxorubicin and IGF I treatment when
indicated, as described earlier. MTT reagents (final concentration,
0.625 mg/mL) were added to each well and incubated at 37°C for 4
hours, and the cells were lysed with acidic isopropanol (0.04N HCl).
After incubation at room temperature for 15 minutes, the plates were
then analyzed with a multiwell ELISA reader at 570 and 650
nmol/L.
DNA Fragmentation
The attached cells were scraped and, together with the detached
cells floating in the medium, were collected by
centrifugation at 2000g for 10 minutes. The
pellets were lysed with a lysis buffer (20 mmol/L EDTA, 50
mmol/L Tris-HCl [pH 8.0], 0.5% SDS, and 100 µg/mL proteinase K)
and incubated at 37°C for 5 hours.17 Nucleic
acids were treated with RNase A (100 µg/mL), and DNA was extracted by
the phenol/chloroform method and precipitated with 60% (vol/vol)
isopropanol. Two micrograms of DNA was labeled with terminal
deoxynucleotidyl transferase (tdt) for 90 minutes
at 37°C.18 The unincorporated
nucleotides were removed, and the labeled DNA was resolved
with 8% acrylamide gel. The gels were dried and exposed
for autoradiography. To quantify fragmentation of DNA,
the radioactivity by autoradiography from 100 bp to 1
kb was determined by laser densitometry.
In Situ TUNEL Assay
In situ labeling of fragmented DNA was performed with tdt UTP
nick end-labeling (TUNEL).19 Monolayers of
cardiomyocytes were grown on slides and fixed with 4%
buffered formalin. TUNEL assay was performed with the commercially
available ApopTag Plus kit (Oncor) according to the manufacturer's
instructions. In brief, nucleosome-sized DNA fragments were tailed with
digoxigenin-nucleotide and then reacted with
fluorescein-conjugated anti-digoxigenin antibodies. The
nucleus was counterstained with DAPI.20 The
apoptotic and nonapoptotic nuclei were visualized with
a Bio-Rad laser scanning confocal fluorescence microscope
equipped with the MRC 1024 UV system. The image was recorded with
LaserSharp software, and the photographs were printed with a Codonic
NP-1600 printer.
Immunoblotting and Immunoprecipitation
Cell monolayers were incubated with defined medium in the
presence or absence of doxorubicin and IGF I. The cells were then
rinsed once with 1x PBS and solubilized with a buffer containing
50 mmol/L Tris (pH 7.4), 100 mmol/L sodium chloride, 10
µg/mL aprotinin, 5 mmol/L EDTA, 1 mmol/L
phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, and 1%
Triton X-100 on ice. The homogenates were
centrifuged at 10 000g for 10 minutes. Protein
concentrations in the supernatants were determined by the Bradford
method,21 and equal amounts of supernatants (20
to 400 µg protein/lane) were separated by 12% SDS-PAGE. The resolved
proteins were electronically transferred to polyvinylidene
fluoride filters in a transfer buffer (192 mmol/L glycine,
15% methanol [vol/vol], and 25 mmol/L Tris-HCl). To reduce
nonspecific binding, the filters were incubated in a blocking buffer
(20 mmol/L Tris [pH 7.6], 137 mmol/L NaCl, 5% nonfat milk,
and 0.05% Tween 20) at 25°C for 1 hour. The filters were incubated
with anti–Bcl-2 or anti-Bax antibodies overnight at 4°C and washed
with washing buffer (1x TBS [pH 7.6] and 0.05% Tween 20) 6 times
for 5 minutes each. The results of immunoblotting were
visualized with enhanced chemiluminescence.22 In
selective experiments, immunoprecipitation was carried out by
incubating cell lysates with agarose-conjugated anti– Bcl-2 antibodies
overnight at 4°C. The agarose beads were collected by brief
centrifugation and washed 4 times. The protein
complexes were then eluted from the beads and applied to SDS-PAGE for
subsequent immunoblotting with anti-Bax antibodies.
Caspase 3 Activities
The activities of caspase 3 were determined with the CPP32 assay
kit (Clontech) by the detection of chromophore
p-nitroanilide after cleavage from the labeled substrate
Asp-Glu-Val-Asp (DEVD)-p-nitroanilide as previously
described.5 In brief,
2x106 cells were solubilized, and equal amounts
of protein lysates were reacted with 50 µmol/L
DEVD-p-nitroanilide at 37°C for 1 hour. The activity was
read in a spectrophotometer at 405 nmol/L, and the results were
calibrated with known concentrations of p-nitroanilide. The
units of protease activity were defined as the amount of caspase 3
required to produce 1 pmol of p-nitroanilide at 25°C.
Nucleosome ELISA
The quantities of mononucleosomes and oligonucleosomes generated
in the apoptotic cardiomyocytes were determined
with a quantitative nucleosome ELISA by affinity capturing of free
nucleosomes with precoated DNA binding proteins. Anti-histone 3
biotin– labeled antibodies were used to detect the levels of
nucleosomes with a microplate ELISA reader. This assay was carried out
with a commercially available kit from Calbiochem.
Statistical Analysis
All autoradiogram were analyzed by laser
densitometry. The data were presented as mean±SE, and the
statistical significance was tested by the Student t
test.
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Results |
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IGF I Suppressed Apoptosis of Cardiomyocytes and Enhanced Cell Survival
DNA Fragmentation and Cell Survival
The purpose of this series of experiments was to determine whether we could induce apoptosis in vitro in cultured primary cardiomyocytes and to assess whether IGF I might suppress the apoptosis of cultured cardiomyocytes. Serum withdrawal has been shown to induce apoptosis in cells. To assess whether serum withdrawal may induce apoptosis in cardiomyocytes, we performed pilot studies and found that overnight serum withdrawal causes DNA fragmentation. However, fragmentation of DNA under serum withdrawal was not as dramatic as previously reported in other types of cells. Since doxorubicin has been shown to induce apoptosis in other cells and doxorubicin may cause cardiomyopathy in humans, we added doxorubicin to the cells incubated with serum-free medium and found that DNA fragmentation was significantly increased. As shown in Figure 1
, moderate DNA fragmentation was found
in the cells grown overnight in serum-free medium, and the addition of
doxorubicin further increased DNA fragmentation in these cells. To test
whether IGF I can suppress apoptosis of
cardiomyocytes, the cells were incubated with serum-free
medium containing IGF I for 20 minutes before the addition of
doxorubicin. IGF I partially suppressed the DNA fragmentation induced
by serum withdrawal and doxorubicin, indicating that apoptosis
of cardiomyocytes can be attenuated with IGF I treatment.
In parallel with these studies, the viability of the cells was
analyzed with MTT assay as shown in Figure 2. The cells underwent serum withdrawal,
doxorubicin, and/or IGF I treatment as described above. Serum
withdrawal overnight induced 23% reduction in MTT activities, and the
addition of doxorubicin further reduced MTT activities to 53% of the
control cells. Consistent with the effects of IGF I on DNA
fragmentation, IGF I partially enhanced the viability of
cardiomyocytes in the presence of serum-free medium and
doxorubicin.
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In Situ TUNEL Assay
To assess the occurrence of apoptosis in situ, broken DNA
in the nucleus was labeled with TUNEL assay and visualized by
fluorescence microscopy. Figure 3
shows photomicrographs of a typical TUNEL assay; the apoptotic
nuclei were stained green. All nuclei, apoptotic and
nonapoptotic, were counterstained with DAPI, which appeared
blue. Compared with the control cells, more apoptotic nuclei
were identified in the cells grown in serum-free medium and doxorubicin
(Figure 4). The results of multiple TUNEL
assays show that serum withdrawal increased the number of
apoptotic nuclei by 6-fold and that the addition of doxorubicin
further increased apoptotic nuclei by 13-fold. IGF I treatment
significantly decreased the number of apoptotic nuclei after
serum withdrawal and doxorubicin incubation. These data are
consistent with the results of DNA fragmentation experiments
described above and confirm that apoptosis of
cardiomyocytes can be induced with serum withdrawal and
doxorubicin and that IGF I can attenuate the apoptosis of
cardiomyocytes induced by serum withdrawal and
doxorubicin.
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IGF I Regulated the Contents of Bax Protein and Modulated
Bcl-2–Bax Interactions
The execution of apoptosis is mediated by specific
apoptotic signaling molecules. To understand how serum
withdrawal and doxorubicin induced apoptosis and how IGF I
attenuated cardiomyocyte apoptosis, we have
determined the abundance of Bcl-2 and Bax by immunoblot
analysis (Figure 5). The levels
of Bcl-2 were low in these cells. Although Bcl-2 protein can be easily
detected in cardiac fibroblasts, the band representing
Bcl-2 was very faint in cardiomyocytes, and in many
experiments we were not able to detect a clear Bcl-2 band with the use
of 3 different anti–Bcl-2 antibodies that react with rat Bcl-2. In
contrast, Bax protein can be readily detected with antibodies that
interact with rat Bax. Bax was induced by serum withdrawal and
doxorubicin and partially attenuated by IGF I. In contrast, the
abundance of tubulin was not significantly altered by serum withdrawal,
doxorubicin, or IGF I. Bax protein was visualized by the enhanced
chemiluminescence technique after <20 seconds of exposure. However, we
had to load abundant cell lysates (100 to 400 µg protein/lane) on
SDS-PAGE and expose the autoradiograph for a considerable period of
time (15 to 30 minutes) to visualize a faint band, if there is any, of
Bcl-2. To more accurately compare the abundance of Bax and Bcl-2
proteins, their levels were compared with known dilutions of
recombinant GST–Bcl-2 and GST-Bax fusion proteins (Figure 6). The results shows that Bax levels
were much higher than Bcl-2 levels in these cells.
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Compared with the control condition, the abundance of Bax was increased
by 80% after serum deprivation and by 3-fold after the
addition of doxorubicin (Figure 7),
suggesting that Bax is involved in the apoptotic signaling
induced by serum withdrawal and doxorubicin treatment. When
cardiomyocytes were pretreated with IGF I before serum
withdrawal and incubation with doxorubicin, induction of Bax protein
was reduced. Since increased Bax expression is associated with
increased apoptosis, these results suggest that IGF I may
attenuate the apoptotic effects of serum withdrawal and
doxorubicin partly by inhibiting Bax induction.
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Increased expression of Bax may lead to the formation of Bax–Bcl-2
heterodimers. To determine whether serum withdrawal and doxorubicin
result in increased formation of Bax–Bcl-2 complexes, cell lysates
were first immunoprecipitated with anti–Bcl-2 antibodies and then
immunoblotted with anti-Bax antibodies (Figure 8). Since the level of Bcl-2 protein is
quite low in these cells, considerably large amount of cell lysates (1
mg protein/sample) were used for this experiment. The results show that
Bcl-2–associated Bax was significantly increased after serum
withdrawal and doxorubicin treatment and that IGF I treatment reduced
the abundance of Bcl-2–associated Bax in cardiomyocytes.
Thus, serum withdrawal and doxorubicin lead to increased formation of
Bcl-2–Bax complexes, and IGF I reduces Bcl-2–Bax formation.
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IGF I Attenuated Activation of Caspase 3 by Serum Withdrawal
and Doxorubicin
Increased abundance of Bax and formation of Bcl-2–Bax
heterodimers may result in activation of caspase 3, an important
component of the final pathway leading to the occurrence of cell death.
To this end, the activities of caspase 3 were determined, and the
results are shown in Figure 9. Compared
with caspase 3 activity in the control condition, caspase 3 activities
increased by 80% 14 hours after serum withdrawal and by 4.5-fold when
0.5 µmol/L doxorubicin was added. In parallel to its effects on
DNA fragmentation and Bax, IGF I partially inhibited the activation of
caspase 3 by 43%. In cardiomyocytes treated with
serum-free medium and doxorubicin, the addition of a specific caspase 3
inhibitor, DEVD-CHO, in vivo to the medium or in vitro to
the reaction buffer suppressed the activities of caspase 3 to the
control level, indicating that the activities of caspase 3 that we
measured were quite specific. These data show that apoptosis of
cardiomyocytes may involve caspase 3 and that the
antiapoptotic action of IGF I on cardiomyocytes can
be partially explained by the inhibition of caspase 3 activation.
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Dose-Response Effects of IGF I on Apoptotic Signaling
The dose-response effects of IGF I on apoptotic signaling
were defined with nucleosome ELISA and caspase 3 activation
(Table). The generation of nucleosomal DNA
represents a hallmark of apoptosis; nucleosome ELISA
allowed us to acutely quantify the presence of mononucleosomes and
oligonucleosomes. The results show that half-maximal effects of IGF I
occurred below 10-9 mol/L in primary
cardiomyocytes. These data indicate that
antiapoptotic actions can occur at
physiological plasma concentrations of IGF I,
suggesting that endogenous IGF I may exert its
antiapoptotic effects on cardiac muscle in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cardiomyocytes are highly differentiated cells and rarely replicate after birth. In adult mammals, loss of cardiomyocytes will result in a permanent reduction of the number of functioning units in the myocardium. For decades it has been assumed that cardiomyocytes die only by necrosis, such as in the case of reduced oxygen supply. However, with improved assays and technology, recent studies show that another form of cell death, apoptosis, also occurs in cardiomyocytes and contributes to the development of heart failure.23 24 25 Necrosis of the myocardium rarely occurs in nonischemic forms of cardiomyopathy.26 Instead, patchy loss of cardiomyocytes through apoptosis contributes to the progressive deterioration of myocardial function. This emerging concept of cardiomyocyte apoptosis has important implications regarding myocardial function, because loss of cardiomyocytes could be a fundamental part of the myocardial process that initiates or aggravates heart failure in cardiomyopathy.26
The in vitro antiapoptotic effect of IGF I on cardiomyocytes that we have observed is consistent with the results of recent in vivo studies. In a murine model of myocardial ischemia/reperfusion, IGF I administration resulted in a decreased incidence of myocardial apoptosis.1 When the coronary artery was ligated to create an experimental myocardial infarction in mice, transgenic mice overexpressing IGF I in myocardium showed decreased cell death and less ventricular dilatation and wall stress.2 IGF I is capable of augmenting cardiac function in experimental cardiomyopathy.10 11 12 13 Although the contribution of its antiapoptotic actions to the overall effects of IGF I on the heart still awaits to be determined, IGF I may conceivably attenuate the loss of functioning myocardial units by suppressing apoptosis in cardiomyocytes.
Apoptotic signaling pathways in cardiomyocytes remain largely unknown. We have begun to study apoptotic signaling pathways in primary cardiomyocytes. These cells were harvested from near-term fetuses and became nonproliferating contractile cardiomyocytes several days after isolation. The content of antiapoptotic Bcl-2 protein is much lower than proapoptotic Bax in fetal cardiomyocytes. Thus, the ratio of Bcl-2 to Bax is quite low in primary cardiomyocytes. After the induction of apoptosis with serum withdrawal and doxorubicin, the levels of Bax protein increased, and the formation of Bax–Bcl-2 complexes also increased. These changes may play a role in the subsequent activation of caspase 3 and the progress of apoptosis. It is believed that once the caspase cascades are activated, the cell death process cannot be reversed.
Exactly how caspases are activated is not yet clear, but it appears that the Bcl-2 gene family is involved in this process. The formation of homodimers and heterodimers between the antiapoptotic family members and the proapoptotic family members has been shown to provide a potential mechanism of apoptosis execution.5 Homodimers of Bcl-2 associate with mitochondrial membrane, stabilize membrane permeability, and prevent the efflux of cytochrome C and the subsequent activation of caspase 3.4 The stabilizing effect of Bcl-2 on mitochondria permeability disappears when Bcl-2 homodimers are sequestered by the formation of the Bcl-2–Bax heterodimer.3 4 Furthermore, Bax homodimers can be associated with mitochondria and directly activate caspase 3 and other potential cell death pathways.4 How IGF I regulates apoptotic signaling is not well understood, but our study suggesting that the inhibitory actions of IGF I on caspase 3 activation may represent a critical step through which IGF I modulates apoptotic signaling in cardiomyocytes. The effects of IGF I on the Bcl-2 family were not the same in different types of cells. IGF I restored the contents of Bcl-2 and/or Bcl-xL in myeloid cells and PC-12 cells.27 28 But the expression of Bax was not modulated by IGF I in myeloid cells.28 In neuronal cells, activation of the IGF I receptor is accompanied by the induction of Bcl-2 and Bcl-xL and the suppression of caspase 3.29 However, IGF I inhibits the activation of interleukin-1ß–converting enzyme without changing the expression of Bcl-2, Bcl-x, or Bax in COS cells.30 Although differential IGF I regulation of the Bcl-2 family may be in part due to different apoptosis inducers, these studies and our results show that IGF I usually suppresses caspases regardless of the changes in the Bcl-2 family. Cardiomyopathy resulting from right ventricular hyperplasia is associated with increased expression of caspase 3 in the myocardium.31 These patients have progressive loss of cardiomyocytes from their myocardium; thus, suppression of caspase 3 may offer a potential target of therapeutic manipulation to prevent cardiomyocyte loss in cardiomyopathy.
Clinical use of doxorubicin is associated with increased risk of heart failure.32 Although the molecular mechanisms underlying doxorubicin cardiomyopathy are not yet fully understood, the formation of free radicals was increased when myocardium was exposed to doxorubicin in vivo.33 Since free radical formation may induce the occurrence of apoptosis,34 it is possible that doxorubicin-induced cardiomyopathy may involve apoptosis in cardiomyocytes. To test the effects of IGF I in the present study, apoptosis was induced under serum-free conditions. Apoptosis in cardiomyocytes also occurred when doxorubicin was added to the regular growth medium that contained 10% serum. However, higher doses of doxorubicin used in subsequent studies (2 to 4 times higher than the dose used for the present study) were required to produce comparable cytotoxic effects (authors' unpublished data, 1998). This is probably because growth factors in serum, including IGF I, provided protective effects against apoptotic induction.
In summary, apoptosis of cardiomyocytes is an intriguing paradigm that may have significant physiological and pathophysiological implications regarding normal and diseased myocardium. We have shown that in primary cardiomyocytes, apoptosis induced by serum withdrawal and doxorubicin is associated with the induction of Bax and activation of caspase 3. IGF I treatment resulted in attenuated Bax induction and caspase 3 activation and inhibited DNA fragmentation. The formation of Bcl-2–Bax complexes was also increased after the induction of apoptosis and was reduced after IGF I treatment. These data suggest that Bax and caspase 3 are key elements of the signaling pathway through which IGF I inhibits the induction of apoptosis in cardiomyocytes. Our findings may provide new insight into how doxorubicin and IGF I modulate myocardial structure and function.
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Acknowledgments |
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This study was supported in part by grants from the National Institutes of Health, Heart, Lung, and Blood Institute (HL-55533 to Dr P.H. Wang), from the American Heart Association (96010960 to Dr P.H. Wang), and from the Optical Biology Shared Resource of the Cancer Center Support Grant (CA62203). The authors wish to thank Genentech (South San Francisco, Calif) for kindly providing recombinant human IGF I.
Received March 3, 1998; accepted June 16, 1998.
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