See discussions, st ats, and author pr ofiles f or this public ation at : https:www .researchgate.ne tpublic ation11963758 [630829]

See discussions, st ats, and author pr ofiles f or this public ation at : https://www .researchgate.ne t/public ation/11963758
IGF-1 Overexpression Inhibits the Development of Diabetic Cardiomyopathy
and Angiotensin II-Mediated Oxidative Stress
Article    in  Diabe tes · July 2001
DOI: 10.2337/ diabe tes.50.6.1414  · Sour ce: PubMed
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IGF-1 Overexpression Inhibits the Development of
Diabetic Cardiomyopathy and Angiotensin II–MediatedOxidative Stress
Jan Kajstura,1Fabio Fiordaliso,1,3Anna Maria Andreoli,1Baosheng Li,1Stefano Chimenti,1
Marvin S. Medow,2Federica Limana,1Bernardo Nadal-Ginard,1Annarosa Leri,1and Piero Anversa1
Stimulation of the local renin-angiotensin system and
apoptosis characterize the diabetic heart. Because IGF-1reduces angiotensin (Ang) II and apoptosis, we testedwhether streptozotocin-induced diabetic cardiomyopa-thy was attenuated in IGF-1 transgenic mice (TGM). Di-abetes progressively depressed ventricular performancein wild-type mice (WTM) but had no hemodynamic effecton TGM. Myocyte apoptosis measured at 7 and 30 daysafter the onset of diabetes was twofold higher in WTMthan in TGM. Myocyte necrosis was apparent only at 30days and was more severe in WTM. Diabetic nontrans-genic mice lost 24% of their ventricular myocytes andshowed a 28% myocyte hypertrophy; both phenomenawere prevented by IGF-1. In diabetic WTM, p53 was in-creased in myocytes, and this activation of p53 was char-acterized by upregulation of Bax, angiotensinogen, Angtype 1 (AT
1) receptors, and Ang II. IGF-1 overexpres –
sion decreased these biochemical responses. In vivo ac-cumulation of the reactive O
2product nitrotyrosine and
the in vitro formation of H2O2-˙OH in myocytes were
higher in diabetic WTM than TGM. Apoptosis in vitro
was detected in myocytes exhibiting high H2O2-˙OH flu –
orescence, and apoptosis in vivo was linked to the pres-ence of nitrotyrosine. H
2O2-˙OH generation and myocyte
apoptosis in vitro were inhibited by the AT1blocker losar –
tan and the O2scavenger Tiron. In conclusion, IGF-1
interferes with the development of diabetic myopathy by
attenuating p53 function and Ang II production and thusAT
1activation. This latter event might be responsible for
the decrease in oxidative stress and myocyte death by
IGF-1. Diabetes 50:1414–1424, 2001Diabetes alters the structure and function of the
human heart, but the mechanisms involved areunknown (1). Type 1 insulin-dependent diabe-tes is characterized experimentally by cardiac
myopathy, in which cell death by apoptosis predominates(2). Hyperglycemia activates the local renin-angiotensinsystem (RAS), resulting in the formation of angiotensin(Ang) II and stimulation of the endogenous cell deathpathway (2). These observations raise the possibility thatIGF-1 may protect the myocardium from the consequencesof diabetes. This growth factor interferes with the myocyteRAS and the synthesis and secretion of Ang II (3). IGF-1enhances the expression of MDM2, which in turn formsMDM2-p53 complexes inhibiting p53 DNA binding (3).Downregulation of p53 function decreases transcription ofangiotensinogen (Aogen), which is the limiting factor inthe synthesis of Ang II in myocytes (4). Therefore, IGF-1may exert a therapeutic effect on ventricular dysfunction(5) and diabetic cardiomyopathy by attenuating the cellu-lar RAS. This hypothesis is supported by the observationthat ACE inhibition reduces cardiovascular events, improv-ing the morbidity and mortality of diabetic patients (6,7).
Diabetes is associated with an exponential increase in
oxidative damage (8). In various cell systems, a direct linkhas been found between Ang II and reactive O
2(9–11).
However, it is unknown whether the generation of reactive
oxygen species (ROS) constitutes the intermediate eventin the transmission of death signals to myocytes by Ang II.Alternatively, cooperation between Ang II and ROS may berequired for cell death to occur in the diabetic heart. In theabsence of ROS-induced DNA damage, Ang II may not beable to execute the death process. We hypothesized thatIGF-1 overexpression may protect the myocardium fromdiabetes by depressing the synthesis of Ang II and thus theformation of ROS and cellular damage. To address theseissues, diabetes was induced in mice homozygous for the
IGF-1 transgene (12). In vivo determinations were per-formed in control and diabetic animals to identify theeffects of IGF-1 on p53 function, p53-dependent genes,activation of the local RAS, accumulation of oxidativestress products, myocyte death, and cardiac hemodynam-ics. In vitro studies analyzed both the impact of diabeteson ROS formation and the efficacy of Ang type 1 (AT
1)
blockade and reactive O2scavenger on oxidative challenge
and myocyte death. Transgenic mice (TGM) with targetedFrom the Departments of1Medicine and2Pediatrics, New York Medical Col –
lege, Valhalla, New York; and the3Istituto Di Ricerche Farmacologiche Mario
Negri, Milan, Italy.
Address correspondence and reprint requests to Jan Kajstura, Department
of Medicine, New York Medical College, Vosburgh Pavilion, Room 302A,Valhalla, New York. E-mail: jan_kajstura@nymc.edu.
Received for publication 27 October 2000 and accepted in revised form
15 March 2001.
Ang, angiotensin; Aogen, angiotensinogen; AT
1, Ang receptor type 1; CM-
H2DCFDA, 5-(6)-chloromethyl-2 9,79-dichlorodihydrofluorescein diacetate;
LVEDP, left ventricular end-diastolic pressure; LVSP, left ventricular systolic
pressure; OD, optical density; PI, propidium iodide; RAS, renin-Ang system;ROS, reactive oxygen species; STZ, streptozotocin; TdT, terminal deoxynu-cleotidyl transferase; TGM, transgenic mice; WTM, wild-type mice.
1414 DIABETES, VOL. 50, JUNE 2001

expression of IGF-1 in myocytes were preferred over non-
transgenic animals with chronic administration of growthfactor, because our objective was to characterize the im-pact of IGF-1 on the development of diabetic cardiomyop-athy. Systemic injection of IGF-1 would have influencedother organs, complicating the interpretation of the resultsin the heart.
RESEARCH DESIGN AND METHODS
Diabetes. A total of 31 male wild-type mice (WTM) and 31 homozygous IGF-1
TGM at 5 months of age were injected into the tail vein with streptozotocin(STZ) (200 mg/kg body wt) dissolved in a citrate-saline solution (pH 4.5) (2).Control mice, 23 in each group, were injected with the diluent and fed arestricted diet to match the decrease in body weight with diabetes. This wasdone to avoid the influence of calorie loss (caused by glycosuria) on bodyweight. Mice were killed at 7 and 30 days. Blood glucose was measured at thetime of killing. Serum levels of IGF-1 were obtained at 7 days by NicholsAdvantage chemiluminescence immunoassay. All protocols used were inaccordance with institutional guidelines.Cardiac function. Just before death, animals were anesthetized with tribro-
moethanol (1.2%, 0.2 ml i.p.). The carotid artery was cannulated with amicrotip pressure transducer (SPR-671, Millar Instruments) connected to an
electrostatic chart recorder. The transducer was advanced into the leftventricle for the evaluation of ventricular pressures and the rate of pressurerise ( 1dP/dt) and decay (–dP/dt). Rectal temperature was maintained at
36–38°C (2–4).Tissue fixation and sampling. The heart was arrested in diastole with 0.15
ml cadmium chloride (100 mmol/l), and the myocardium was perfusedthrough the aorta with 10% formalin (13,14). The heart was excised, cardiacweights were recorded, and three slices of the left ventricle were embeddedin paraffin; 48 animals were studied (6 in each group) at 7 and 30 days afterSTZ or diluent injection. Sections were stained with hematoxylin-eosin, and 60fields in each heart were examined at 3400 with a reticle containing 42 points
to yield the volume percentage of myocytes and interstitium. Ventricularvolume was multiplied by the volume fraction of myocytes to compute theaggregate volume of myocytes in the ventricle (13–15).Myocyte death. This terminal analysis was performed in 48 animals. Myocyte
apoptosis was determined by deoxynucleotidyl transferase (TdT) and hairpinprobe with single-base 3 9overhangs, and myocyte necrosis was determined by
hairpin probe with blunt ends. Apoptosis-necrosis was evaluated by TdT andhairpin probe with blunt ends (16). These techniques have been describedpreviously (2,13–18). Myocyte cytoplasm and nuclei were stained by a-sarco-
meric actin antibody and propidium iodide (PI), respectively (2,13–18).
Sections from the base, mid-region, and apical portion of each left ventricle
were examined by confocal microscopy, and the numbers of myocyte nucleithat were labeled by TdT, hairpin probes, and by TdT and hairpin probe withblunt ends were recorded by analyzing a minimum of 13.5 mm
2to a maximum
of 44.8 mm2of tissue. The total number of myocyte nuclei sampled for TdT
at 7 days of diabetes was 78,393, 51,643, 72,255, and 69,486 in control and
diabetic nontransgenic mice and control and diabetic TGM, respectively.Corresponding values for hairpin probe with single-base 3 9overhangs were
77,827, 74,764, 101,469, and 81,027. Values with hairpin probe with blunt endswere 81,252, 77,464, 111,660, and 105,912. In a comparable manner, the totalnumber of myocyte nuclei sampled for TdT at 30 days of diabetes was 81,613,80,849, 77,796, and 81,282 in control and diabetic nontransgenic mice andcontrol and diabetic TGM, respectively. Corresponding values for hairpinprobe with single base 3 9overhangs were 82,994, 86,658, 90,559, and 77,796.
Values with hairpin probe with blunt ends were 92,117, 80,849, 83,687, and81,282. Values for TdT and hairpin probe with blunt ends are not indicatedbecause this simultaneous association was never found. The density ofmyocyte nuclei was obtained by counting (per unit area of tissue) the numberof nuclei in a-sarcomeric actin–positive cells. The number of apoptotic and
necrotic myocytes per 10
6cells was then calculated.
Nitrotyrosine labeling. This analysis was performed in 24 animals (6 in each
group) at 30 days of diabetes. Sections were incubated overnight with rabbitanti-nitrotyrosine antibody (Upstate Biotechnology, Lake Placid, NY) diluted1:40 in phosphate-buffered saline (19). Fluorescein isothiocyanate–labeledgoat anti-rabbit IgG was used as a secondary antibody. Simultaneous presenceof cell death and nitrotyrosine was evaluated with a secondary antibodylabeled with tetramethyl rhodamine isothiocyanate. Sections treated with 10%peroxynitrite were used as a positive control. The percentage of myocytescontaining nitrotyrosine was obtained by confocal microscopy; 300 myocyteprofiles were examined in each animal.Myocyte size and number. Myocyte cell volume was obtained by three-
dimensional section reconstruction of enzymatically dissociated cells. In eachleft ventricle, 150–200 myocytes were measured. The total number of ventric-ular myocytes was computed by dividing the aggregate volume of myocytes bythe average cell volume. This methodology has been described previously(12,19).Myocyte isolation. This was performed in 44 mice, 6 in each group, at 7 days
after diabetes and in 5 at 30 days after diabetes. Left ventricular myocyteswere enzymatically dissociated following a protocol well established in ourlaboratory (3,12–14). Myocyte yields were 2.1 60.4310
6in control WTM,
1.460.53106in diabetic WTM, 2.9 60.83106in control TGM, and 2.6 6
0.73106in diabetic TGM. Contamination from nonmyocytes ranged from 1 to
3% in all cases.
Band shift. Oligonucleotides corresponding to the p53 binding site in the Bax,
Aogen, and AT1promoters were used in mobility shift assays (3,4,17). As a
negative control, nuclear extracts were exposed to a p53 antibody (Pab 240;
Santa Cruz Biotechnology) before the binding assay. Unlabeled Aogen, AT1,
and Bax probes were used as competitors, and unlabeled mutated Aogen, AT1,
and Bax were used as noncompetitors (4,17).
Western blot. A total of 50 mg of myocyte proteins were separated by 12%
SDS-PAGE. Proteins were transferred to nitrocellulose and exposed toanti-human p53 (Pab 240; Santa Cruz Biotechnology), anti-human Bax (P19;Santa Cruz Biotechnology), anti-rat Aogen (Swant), anti-rat renin (Swant),anti-human AT
1(306; Santa Cruz Biotechnology), and anti-human AT2(C-18;
Santa Cruz Biotechnology) antibodies (3,4,17).
Ang II labeling. This was performed in 24 animals (6 in each group) at 7 days
after diabetes. Sections were incubated with Ang II antiserum (2,3). Specificitywas determined by preabsorbtion of antibody with antigen and by stainingwith nonimmune rabbit serum. Ang II–labeled myocytes and Ang II sites permillimeter squared of myocytes were determined (2,3). A total of 200 myocyteprofiles were analyzed in each animal.Reactive O
2.This analysis included five separate myocyte isolations in each
group of mice at 7 days of diabetes, using a total of 20 animals. Isolated myo-
cytes were attached to laminin-coated petri dishes, and cells were loaded with5-(6)-chloromethyl-2 9,79-dichlorodihydrofluorescein diacetate (CM-H
2DCFDA)
(Molecular Probes, Eugene, OR) for 30 min. Fluorescence intensity from
individual cells was measured using an excitation wavelength of 485 nm andan emission wavelength of 530 nm (20,21); 100–140 cells were sampled atrandom in each preparation using an Olympus IX70 inverted microscopeequipped with a digital cooled CCD-IEEE camera (Optronics, Goleta, CA) andImagePro image analysis software (Media Cybernetics, Silver Spring, MD). Toavoid changes in fluorescence intensity, only a single measurement per fieldwas collected. Fluorescence was calibrated with InSpeck microspheres(Molecular Probes). Calibration curves were generated and cell brightnesswas measured. Probe fluorescence was established and this background wassubtracted from all determinations. Preliminary experiments were also per-formed to assess the uptake of CM-H
2DCFDA with time. Maximum fluores –
cence was reached between 20 and 30 min. Therefore, a 30-min interval wasused. The volume of each analyzed myocyte was measured by confocalmicroscopy (9,10), and fluorescence intensity per cell was divided by its vol-ume to correct for differences in cell size. After the completion of the mea-surements of ROS, cells were fixed and stained for apoptosis.Ang II, ROS and apoptosis. The effects of 30 min of exposure to Ang II (10
29
mol/l) on ROS formation were detected as described above. Freshly isolated
myocytes were cultured in laminin-coated petri dishes and serum-free me-dium fo r 1 h before the addition of Ang II. Identical cultures were exposed to
losartan (10
27mol/l) and to losartan plus Ang II, and ROS generation was
measured. Five separate myocyte isolations were used in each condition. For
the evaluation of apoptosis, myocytes were cultured in the presence of Ang IIfor 24 h. Additionally, the effect of losartan or the superoxide anion scavengerTiron (0.1 mmol/l) on Ang II–mediated apoptosis was determined. In theseexperiments, losartan or Tiron were added to the culture 30 min before AngII. Again, six myocyte isolations were used in each protocol.Statistics. All tissue samples were coded, and the code was broken at the end
of the study. Results are presented as the mean 6SD. Statistical significance
between two measurements was determined by the two-tailed unpairedStudent’s ttest, and among groups it was determined by the Bonferroni’s
method (22). Probability values of P,0.05 were considered significant.
RESULTS
Ventricular function. Blood glucose in WTM increased
from 11.4 62.1 to 27.5 63.3 and 36.5 63.6 mmol/l at 7 and
30 days after STZ administration, respectively. Compara-ble changes in blood glucose were detected in TGM: forcontrol, diabetic TGM at 7 days, and diabetic TGM at 30J. KAJSTURA AND ASSOCIATES
DIABETES, VOL. 50, JUNE 2001 1415

days the values were 11.8 61.5, 28.9 64.0, and 37.2 64.5
mmol/l, respectively. As indicated by the small standarddeviations, all animals in each group developed stablehyperglycemia. At 7 days, diabetes in WTM was character-ized by an increase in left ventricular end-diastolic pres-sure (LVEDP) and a decrease in left ventricular systolicpressure (LVSP) and 1dP/dt. Cardiac function was further
depressed at 30 days (Fig. 1). In contrast, diabetes at 7 and30 days did not affect ventricular performance in TGM.Because of pair-feeding, diabetic and nondiabetic mice hadcomparable body and cardiac weights. Heart weight–to–body weight ratios remained constant in control WTM(3.560.2 and 3.6 60.3 mg/g at 7 and 30 days, respectively)
and in diabetic WTM (3.7 60.3 and 3.9 60.5). Similarly,
control TGM (4.4 60.3 and 4.5 60.4) did not differ from
diabetic TGM (4.3 60.5 and 4.7 60.6). Control and dia-
betic TGM had higher heart weight–to–body weight ratios(P,0.01 to P,0.001) than corresponding WTM (12).
It should be noted that the serum level of IGF-1 was ,3
ng/ml in nondiabetic and diabetic WTM. Conversely, IGF-1was 5.7 61.2 ng/ml in nondiabetic and diabetic TGM,
respectively. It cannot be excluded, therefore, that thehigher circulating level of IGF-1 in TGM may have had anadditional effect on the response of the heart to diabetes inthis group.Myocyte loss. Cell death values at 7 and 30 days were
similar in control WTM and control TGM and thus werecombined. Diabetes was associated with an increase inmyocyte apoptosis, which was higher at 7 days than it wasat 30 days in both WTM and TGM (Fig. 2 Aand B).
However, at either interval, cell death was twofold greaterin diabetic WTM than in TGM. The extent of apoptosis didnot differ when measured by TdT or hairpin probe withsingle-base 3 9overhangs. Myocyte necrosis, evaluated by
a hairpin probe with blunt ends, was not increased indiabetic WTM and TGM at 7 days. Conversely, myocytenecrosis with diabetes at 30 days increased 2.9-fold and1.8-fold in diabetic WTM and TGM, respectively (Fig. 2 C).
The 1.7-fold difference between the two groups of diabeticmice was significant. Cells dying by both apoptosis andnecrosis were never found. In comparison with controlWTM, a 24% reduction in the total number of left ventric-ular myocytes was found in diabetic WTM at 30 days (Fig.
2D). Cell loss in these animals was accompanied by a 28%
myocyte hypertrophy (Fig. 2 E), which resulted in preser-
vation of ventricular mass (Fig. 2 F). The 24% loss of cells
in the ventricle reflected an absolute dropout of 1.1 310
6
myocytes. In contrast, diabetes did not change the num-
ber, size, and aggregate myocyte volume in TGM (Fig.2D–F).
p53 DNA binding and myocyte RAS. p53 Function and
expression of the p53-dependent and regulated genes(Bax, Aogen, and AT
1) and p53-independent genes (renin
and AT2receptor) were measured in myocytes isolated
from control and diabetic WTM and TGM (Fig. 3 A). These
determinations were restricted to 7 days because apopto-sis with diabetes was greater at this interval. p53 Bindingto its cognate DNA sequence on the promoter of Bax(59-AGCTTGCTCACAAGTTAGAGACAAGCCTGGGC-
GTGGCTATATTGA-3 9), Aogen (5 9-AGCCTCTGTACA-
GAGTAGCCTGGGAATAGATCCATCTTC-3 9), and AT
1
(59-GCTGAGCTTGGATCTGGAAGGCGACACTGGG-3 9)
was increased in myocytes of diabetic WTM (Fig. 3 B–D).
The optical density (OD) of the p53-shifted band for eachof these three genes was significantly higher than in dia-betic TGM (OD data not shown). These differences werecoupled with larger quantities of p53 (Fig. 3 E) and Bax
(Fig. 3 F) proteins in WTM myocytes with diabetes. Simi-
larly, Aogen (Fig. 3 G), renin (Fig. 3 I), and AT
1(Fig. 3 H)
increased more in diabetic WTM than in diabetic TGM. AT2
protein did not change in diabetic mice (Fig. 3 J).
The quantitative analysis of Ang II localization in the
myocardium (Fig. 4 A–D) showed labeling in 51 67% (n5
6) and 31 66% ( n56,P,0.001) of the myocytes
in nondiabetic WTM and TGM, respectively. Diabetes in-creased the fraction of Ang II–positive myocytes to 77 6
9% (n56,P,0.001) in WTM and to 50 68% (n56,P,
0.01) in TGM. The number of Ang II sites per millimetersquared of myocytes was twofold ( P,0.001) higher in
nondiabetic WTM (14,000 63,500 per mm
2) than in TGM
(7,000 62,100). With diabetes, these values increased
2.3-fold ( P,0.001) in WTM (32,100 68,300) and 1.7-fold
(P,0.001) in TGM (12,000 63,400). Thus, Ang II sites in
FIG. 1. Effects of diabetes on LVEDP ( A), LVSP
(B), LV 1dP/dt ( C), and LV –dP/dt ( D) in non-
transgenic mice and TGM. Results are means 6
SD. d, Days; C, control; D, diabetic. * P<0.05
vs. C.IGF-1 AND DIABETIC CARDIOMYOPATHY
1416 DIABETES, VOL. 50, JUNE 2001

myocytes were 2.7-fold ( P,0.001) more numerous in
diabetic WTM than in TGM.Nitrotyrosine localization. Nitrotyrosine is formed by
the interaction of peroxynitrite with cytoplasmic proteins(23). This modified amino acid is a product of oxidativestress that can be detected in the myocardium using anitrotyrosine-specific antibody (Fig. 5 AandB). Its associ-
ation with myocyte apoptosis can be identified as well(Fig. 5 CandD). The analysis of nitrotyrosine localization
in myocytes was performed at 30 days of diabetes to allowits accumulation with time. The percentage of myocytesexpressing nitrotyrosine was 28 66% (n56) and 14 64%
(n56;P,0.05) in nondiabetic WTM and TGM, respec-
tively. Diabetes increased the fraction of nitrotyrosine-positive myocytes to 71 616% ( n56,P,0.001) in WTM;
this parameter did not change in diabetic TGM, which hada value of 16 65% (n56, NS). The presence of nitrotyro-
sine does not permit the examination of all nuclei of posi-tive cells; nuclei are often not included in the section plane(Fig. 5 AandB). Moreover, nitrotyrosine labeling exceeds
by several orders of magnitude the extent of cell death. Atotal of 63 apoptotic myocytes were examined in diabeticWTM to detect the potential implication of nitrotyrosine inapoptosis; in all cases, apoptosis was accompanied bynitrotyrosine labeling (Fig. 5 CandD). To confirm the role
of oxidative stress in cell death, 38, 26, and 18 apoptoticmyocytes were analyzed in diabetic TGM and nondiabeticWTM and TGM, respectively; nitrotyrosine was present inevery cell undergoing apoptosis.ROS formation. The increase in nitrotyrosine in myo-
cytes with diabetes suggested that a causative link existedbetween oxidative stress and this disease. Additionally,IGF-1 attenuated the effects of oxidative stress on nitro-tyrosine accumulation in the diabetic heart. To establishwhether a relationship was present between diabetes andAng II formation and between ROS production and celldeath, in vitro studies were performed after 7 days ofdiabetes. This interval was selected because Ang II label-ing of myocytes was obtained at this time and apoptosisin vivo was higher than at 30 days (see above). Myocyteswere isolated from nondiabetic and diabetic WTM, and
TGM and ROS generation was measured using a fluores-cent probe detecting H
2O2andzOH in living cells (Fig. 6 A
andB). In each cell, fluorescence intensity (f) was normal-
ized by the corresponding myocyte cell volume (V). ROSproduction was 44% ( P,0.01) higher in myocytes from
nondiabetic WTM ( n55, 1.34 60.11 f/V) than in cells
from nondiabetic TGM ( n55, 0.93 60.08). Diabetes
increased the formation of H
2O2-zOH in myocytes of WTM
(n55, 2.40 60.29 f/V) and TGM ( n55, 1.23 60.12)
1.8-fold ( P,0.001) and 1.3-fold ( P,0.05), respectively.
Using 0.2-f/V increments, the frequency distribution offluorescence intensity of myocytes was plotted (Fig. 6 C–
F). In comparison with WTM, IGF-1 overexpression in
nondiabetic and diabetic hearts was characterized by ashift to the left of the reactive O
2signal. In nondiabetic
WTM and TGM and diabetic WTM and TGM, respectively,
a total of 563, 585, 601, and 643 myocytes were examinedand 1, 0, 7, and 2 apoptotic cells were found. All dyingmyocytes had high levels of H
2O2-zOH; f/V values ranged
from 3 to 5. The frequency of apoptotic myocytes found invitro were higher than in vivo. This is a consistent phe-
FIG. 2. Effects of diabetes on myocyte apoptosis evaluated with TdT
(A) and Hairpin probe ( B), and the effects of diabetes on necrosis ( C),
myocyte number ( D), myocyte cell volume ( E), and aggregate myocyte
mass in the left ventricle ( F). See legend to Fig. 1 for symbols. * P<
0.05 vs. C; ** P<0.05 vs . D 7 days; and † P<0.05 vs. nontransgenic
mice. A–C: C 7–30 days, n512; D 7 days, n56; D 30 days, n56.
D–F:n55.J. KAJSTURA AND ASSOCIATES
DIABETES, VOL. 50, JUNE 2001 1417

FIG. 3IGF-1 AND DIABETIC CARDIOMYOPATHY
1418 DIABETES, VOL. 50, JUNE 2001

nomenon associated with cell isolation and culture condi-
tions (4,17).
To identify whether the cellular release of Ang II was
responsible for the induction of H2O2-zOH at baseline and
with diabetes or whether oxidative stress was independentfrom AT
1receptor activation, myocytes were exposed for
30 min, respectively, to the AT1antagonist losartan (1027
mol/l) or the intracellular reactive O2scavenger Tiron (0.1
mmol/l). H2O2-zOH fluorescence was reduced by losartan
(Fig. 6 G–J) in myocytes obtained from diabetic WTM (from
2.4060.29 to 1.53 60.13 f/V; 582 cells, two apoptosis) and
TGM (0.91 60.14; 610, one). Values in losartan-treated
nondiabetic WTM and TGM were, respectively, 1.26 60.18
f/V (541 cells, one apoptosis) and 0.85 60.09 f/V (572 cells,
no apoptosis). Similarly, Tiron-attenuated oxidative stressmimicked the effects of losartan at a more distal level:WTM 51.1260.17 f/V (551 cells, no apoptosis), TGM 5
0.7860.11 (539, none), diabetic WTM 51.2960.21 (680,
one), and diabetic TGM 50.9160.14 (554, none).
Ang II, ROS formation and apoptosis. Myocytes from
control WTM were exposed to Ang II, and the H
2O2-zOH
signal was measured (Fig. 7 A). Fluorescence intensity per
cell nearly doubled with Ang II. In contrast, pretreatmentof cells with losartan prevented the effects of Ang II onROS production. Myocyte apoptosis markedly increased24 h after the addition of Ang II. Importantly, Tiron orlosartan inhibited the apoptotic signal transmitted by AngII (Fig. 7 B). Thus, Ang II activated apoptosis via the AT
1
receptor by enhancing oxidative stress.
DISCUSSION
IGF-1 overexpression protects from diabetic cardio-
myopathy. Whether diabetes per se—in the absence of
coronary artery disease and hypertension—leads to car-diac myopathy in humans has been a controversial ques-tion (1). Animal models of diabetes have not resolved thisissue. Biochemical, mechanical, structural, and electro-physiological alterations have been identified in combina-tion with modest abnormalities in the diastolic propertiesof the heart (2,24,25). However, indexes of severe ventric-ular dysfunction and failure have not been observed (1,2).Results presented here document for the first time that in amouse model of type 1 insulin-dependent diabetes, cardiacperformance is impaired soon after the onset of the dis-ease and deteriorates chronically. Myocyte loss and hyper-trophy of the remaining cells characterize the diabeticdecompensated heart, mimicking cardiac myopathies inhumans and animals (26).
Clinical and experimental studies aiming at the identifi-
cation of a therapeutic role for exogenously administratedIGF-1 or growth hormone in pathologic states of the hearthave not provided a consistent answer. Positive observa-tions (5,27) have been contrasted by negative results (28,29). IGF-1 overexpression in TGM has previously been
shown to inhibit both myocyte apoptosis in the survivingmyocardium after infarction (13) and myocyte necrosisafter nonocclusive coronary artery constriction (14). Inter-ference with cell death improved cardiac anatomy anddecreased diastolic wall stress in both situations. How-ever, these beneficial effects were not accompanied by acorresponding amelioration in ventricular hemodynamics,possibly due to the presence of large infarcts and restric-
tion in coronary perfusion, respectively. Therefore, thetherapeutic impact of IGF-1 on the diseased heart remainsunclear. The current findings demonstrate unequivocallythat IGF-1 overexpression affected the level of activationof myocyte death with diabetes and preserved ventricularperformance. Myocyte death has been questioned as anetiological factor capable of inducing functional alter-ations. Cell death has been claimed to be an epiphenom-enon that has little influence on the onset and evolution ofcardiac failure (30). Current data in diabetic nontransgenicmice and TGM challenge this contention.IGF-1 overexpression inhibits Ang II synthesis andROS formation. Recent observations in diabetic patients
(7) and in rats after STZ administration (2) have demon-strated that the systemic and local RAS are activated withdiabetes, exerting a detrimental effect on the course of thedisease. Formation of Ang II in the myocardium andstimulation of AT
1receptors cause myocyte apoptosis and
cardiac remodeling (2). The in vivo results obtained here
are consistent with the concept that upregulation of p53leads to enhanced expression of the p53-regulated gene(Aogen) responsible for the increased levels of Ang II inmyocytes with diabetes. Aogen is the limiting factor in theformation of Ang II in cardiac muscle cells (4,17,31), andinactivation of p53 inhibits generation of the octapeptide(4). IGF-1 attenuates p53 transcriptional activity andthereby downregulates Aogen and the synthesis of Ang II.
This negative modulation of RAS by IGF-1 is mediated byMDM2 and the generation of MDM2-p53 inactive com-plexes (3,32). Ang II leads to oxidative stress in several cellsystems through NADH/NADPH oxidase (9). This enzymeis the major source of superoxide; p22
phoxis critical for the
transfer of electrons from NADH or NADPH to O2and the
production of reactive O2(33).
Ang II increases the formation of ROS in neonatal
myocytes (34), endothelial cells (11), and smooth musclecells (9) in vitro by activating AT
1receptors (11). How –
ever, a link between ROS and apoptosis has not beenestablished. It is technically impossible to measure thegeneration of ROS in cardiac myocytes in vivo. Therefore,the localization of nitrotyrosine in myocytes was evalu-ated. Superoxide anion interacts with nitric oxide, formingperoxynitrite (ONOO
2) (23). ONOO2induces oxidative
damage to proteins, leading to the production of a modi-
FIG. 3. Left ventricular myocytes from a diabetic nontransgenic mouse at 7 days ( A);a-sarcomeric actin staining of the cytoplasm (red
fluorescence and PI labeling of nuclei (yellow fluorescence), 3300. B–D: Gel mobility assays of p53 binding to its consensus sequence in the
promoter of bax ( B), Aogen ( C), and AT1(D). Bax, Ao, and AT1; probes in the absence of nuclear extracts. Co, competition with an excess of
unlabeled self-oligonucleotide; Ab, competition with monoclonal p53 antibody; Mut, preincubation with unlabeled mutated form of oligonucle-
otides; SV-T2, nuclear extracts from SV-T2 cells; C, control; D, diabetic; W, nontransgenics; T, transgenics. OD values of the shifted bands are notlisted. E–J: Western blots of p53 ( E), Bax ( F), Aogen ( G), AT
1(H), renin ( I), and AT2(J). ODs for p53: WC 51.960.31, WD 56.761.5 ( P<
0.001); TC 50.56 60.16, TD 52.160.48 ( P<0.05). ODs for Bax, WC 51.460.26, WD 55.760.81 ( P<0.001); TC 50.50 60.15, TD 50.78 6
0.25 ( P<0.05). ODs for Aogen: WC 56.261.3, WD 51964(P<0.001); TC 51.360.44, TD 52.460.64 ( P<0.01). ODs for AT1:W C 53.76
1.0, WD 51363(P<0.001); TC 50.99 60.29, TD 51.860.54 ( P<0.01). ODs for renin: WC 50.88 60.29, WD 52.160.64 ( P<0.001); TC 5
0.71 60.19, TD 51.160.28 (NS). ODs for AT2:W C 50.93 60.33, WD 50.91 60.24 (NS); TC 50.85 60.26, TD 50.82 60.27 (NS). n56i n
all cases.J. KAJSTURA AND ASSOCIATES
DIABETES, VOL. 50, JUNE 2001 1419

FIG. 4. Myocardium of nondiabetic ( AandB) and diabetic ( CandD) WTM. Green fluorescence ( AandC) and yellow fluorescence ( BandD)
reflect Ang II labeling (arrows) and laminin staining of the interstitium. Red fluorescence shows a-sarcomeric actin staining of the cytoplasm ( B
andD), and blue fluorescence shows PI labeling of nuclei ( BandD). Magnification: 31,200.IGF-1 AND DIABETIC CARDIOMYOPATHY
1420 DIABETES, VOL. 50, JUNE 2001

FIG. 5. Nitrotyrosine localization in the myocardium of a diabetic WTM at 30 days ( Aand B), shown by blue fluorescence ( A) and pink
fluorescence ( B).a-Sarcomeric actin staining of the cytoplasm is shown by red fluorescence ( B) and PI-labeling of nuclei (green-yellow
fluorescence, AandB).CandDfrom the same heart show an apoptotic nucleus (green-yellow fluorescence, arrow) detected by hairpin probe with
single-base 3 *overhangs. Stainings for nitrotyrosine and myocyte cytoplasm are the same as those in AandB. Magnification: 31,200.J. KAJSTURA AND ASSOCIATES
DIABETES, VOL. 50, JUNE 2001 1421

fied amino acid (nitrotyrosine). The increased frequency of
nitrotyrosine-positive myocytes in diabetic nontransgenicmice pointed to an oxidative challenge in vivo. Thiscellular response was prevented in diabetic IGF-1 TGM,correlating with the lower level of Ang II in the myocar-dium. However, these in vivo results did not prove whetherAng II was the trigger for the induction of ROS or whetherIGF-1 reduced oxidative stress by interfering only with thesynthesis of Ang II. IGF-1 could have affected the activityof NADH/NADPH oxidase, limiting superoxide formation(23). Importantly, AT
1blockade in myocytes from diabetic
WTM and TGM decreased the H2O2-zOH signals. Although
the levels of H2O2-zOH varied in the presence and absence
of IGF-1 overexpression, inhibition of Ang II binding m ark-
edly depressed the generation of ROS in either myocytepopulation. Similar results were obtained with the reactiveO
2scavenger Tiron. Thus, Ang II was the mediator of re –
active O2, and IGF-1 attenuated oxidative stress by reduc -ing the local synthesis of Ang II in the myocardium with
diabetes.IGF-1 overexpression attenuates myocyte death withdiabetes. Myocyte apoptosis and necrosis are both in-
volved in the development of diabetic cardiomyopathy.Necrosis temporally follows apoptosis, contributing to thechronic loss of ventricular myocytes with diabetes. Thestrict association between nitrotyrosine and apoptosissuggests that oxidative damage is causally implicated inthe activation of this form of cell death. Although a similarlink was not investigated for myocyte necrosis, differentlevels of reactive O
2trigger apoptosis or necrosis; high
quantities induce necrosis and low amounts promote apo-
ptosis (35). IGF-1 attenuated necrosis and apoptosis butdid not prevent cell death completely. However, myocyteapoptosis and necrosis in TGM did not affect the aggregatenumber of ventricular myocytes 1 month after STZ admin-istration. This apparent inconsistency may be explained bymyocyte regeneration, which could have occurred withIGF-1 overexpression (12).
In conclusion, the positive correlation between the ex-
tent of oxidative challenge and myocyte apoptosis in vitro
FIG. 6. Fluorescence of the H2O2-zOH probe in myocytes isolated from
a control ( A) and diabetic ( B) WTM. Magnification: 3150. C–J: Fre-
quency distribution of H2O2-zOH signals in myocytes from nondiabetic
and diabetic mice in the absence ( C–F) and presence of losartan ( G–J).
Fluorescence increments of 0.2 f/V were used to construct these curves.Regression lines were fitted using Weibull’s equation. Solid circlescorrespond to groups of myocytes within 0.2 f/V. Solid triangles corre-spond to apoptosis of individual myocytes.
IGF-1 AND DIABETIC CARDIOMYOPATHY
1422 DIABETES, VOL. 50, JUNE 2001

and between nitrotyrosine localization and myocyte apo-
ptosis in vivo strongly suggest that oxygen toxicity mayconstitute an important cell death signal responsible forcell dropout in the diabetic heart. Diabetic cardiomyopa-thy may be viewed as a ROS-dependent myopathy in whichcell loss initially produces moderate ventricular dysfunc-tion and chronically leads to a severely decompensatedheart. IGF-1 protects the myocardium from the detrimen-tal effects of diabetes by attenuating Ang II synthesis andthus reactive O
2damage and, ultimately, cell death. Wheth –
er the protective role of IGF-1 in diabetes persists over longtime periods remains an important unanswered question.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Healthgrants HL-38132, HL-39902, HL-43023, AG-15756, HL-66923,AG-17042, and HL-65577 and by JDF grant 1-2000-62. Theinitial helpful suggestions of Dr. Ashwani Malhotra areacknowledged.
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