Local insulin-like growth factor I expression induces physiologic, then pathologic, cardiac hypertrophy in transgenic mice

Local insulin-like growth factor I expression induces physiologic, then pathologic, cardiac hypertrophy in transgenic mice M. CRAIG DELAUGHTER,* GEORGE E. TAFFET,†,§ MARTA L. FIOROTTO,‡ MARK L. ENTMAN,§ AND ROBERT J. SCHWARTZ*,1 *Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA; †Huffington Center on Aging, Houston, Texas 77030, USA; ‡USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, USA; and §Department of Cardiovascular Sciences, Baylor College of Medicine, Houston, Texas 77030, USA ABSTRACT In the present study we determined the long-term effects of persistent, local insulin-like growth factor I (IGF-I) expression on cardiac function in the SIS2 transgenic mouse. Cardiac mass/ tibial length was increased in SIS2 mice by 10 wk of age; this cardiac hypertrophy became more pronounced later in life. Peak aortic outflow velocity, a correlate of cardiac output, was increased at 10 wk in SIS2 mice but was decreased at 52 wk. 72 wk SIS2 mouse hearts exhibited wide variability in the extent of cardiac hypertrophy and enlargement of individual cardiac myofibers. Sirius red staining revealed increased fibrosis in 72 wk SIS2 hearts. Persistent local IGF-I expression is sufficient to initially induce an analog of physiological cardiac hypertrophy in which peak aortic outflow velocity is increased relative to controls in the absence of any observed detrimental histological changes. However, this hypertrophy progresses to a pathological condition characterized by decreased systolic performance and increased fibrosis. Our results confirm the shortterm systolic performance benefit of increased IGF-I, but our demonstration that IGF-I ultimately diminishes systolic performance raises doubt about the therapeutic value of chronic IGF-I administration. Considering these findings, limiting temporal exposure to IGF-I seems the most likely means of delivering IGF-I’s potential benefits while avoiding its deleterious side effects.—Delaughter, M. C., Taffet, G. E., Fiorotto, M. L., Entman, M. L., Schwartz, R. J. Local insulin-like growth factor I expression induces physiologic, then pathologic, cardiac hypertrophy in transgenic mice. FASEB J. 13, 1923–1929 (1999)

Key Words: IGF-I z SIS2 transgene z systolic function z hypertension

Cardiac hypertrophy is an adaptive response to pressure or volume overload and a known risk factor for serious pathologies including arrhythmia and 0892-6638/99/0013-1923/$02.25 © FASEB

myocardial infarction. Mild or short-term overload induces physiological hypertrophy, in which the heart enlarges until it establishes a new operational plateau. Severe and/or persistent overload leads to pathological hypertrophy, characterized by myofiber disorganization, increased fibrosis and decreased cardiac output despite greater cardiac mass. Insulin-like growth factor I (IGF-I) is a 70 amino acid peptide hormone produced primarily by the liver in adults in response to growth hormone (GH) secretion by the pituitary gland. IGF-I is the actual mediator of several effects attributed to GH, including increased skeletal muscle mass and bone growth (1). In cultured neonatal ventricular myocytes, the addition of IGF-I induces DNA synthesis (2) (3) and the transcription of several genes associated with hypertrophy and hyperplasia, including myosin light chain-2, troponin and a-skeletal actin (4). The addition of IGF-I to adult cultured cardiomyocytes induces only a hypertrophic response, characterized by increased myofibril production (5). In vivo, IGF-I and its receptor are up-regulated in experimentally infarcted ventricles, possibly followed by DNA replication and mitotic division of a portion of the remaining cardiomyocytes (6, 7). IGF-I protects against apoptosis in cultured (8) and primary cardiomyocytes (9) and in a mouse model of ischemic injury (10). A transgenic mouse with significantly increased IGF-I serum levels exhibits cardiomyocyte hyperplasia but no hypertrophy (11). IGF-I and/or GH have been shown to improve cardiac performance in experimental (12, 13) and human patient cardiac failure (14, 15). Consequently, IGF-I and GH are being seriously considered as potential therapeutic agents for situations in which hypertrophy and/or hyperplasia of cardiomyocytes would be 1 Correspondence: Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA. E-mail: schwartz@ bcm.tmc.edu

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desirable, such as postmyocardial infarction or hypocontracting cardiomyopathies (16, 17). In contrast to these beneficial effects, however, IGF-I has been implicated as a primary factor in the development of cardiac hypertrophy under conditions of pressure overload (18). IGF-I stimulates collagen production in primary cultures of mechanically loaded cardiac fibroblasts (19) and in hypertensive humans (20). Patients with hypersecretion of GH (acromegaly), and consequently IGF-I, often develop a cardiomyopathy characterized by interstitial fibrosis, mononuclear infiltration, and myocyte necrosis (16). Systolic dysfunction is a common late-stage finding in these patients (21). Administration of octreotide, which inhibits GH secretion, can partially relieve these effects (22). Given these conflicting observations, we considered it imperative to determine whether the long-term effects of IGF-I on cardiac function were of a physiological or pathological nature. Here we report that persistent expression of IGF-I in a transgenic model initially induced an analog of physiologic hypertrophy, characterized by increased cardiac mass and improved systolic performance. However, later in life this hypertrophy progressed to a pathological condition characterized by decreased systolic performance and increased interstitial fibrosis.

Pulsed Doppler measurements of systolic performance Our method for pulsed Doppler measurements has been described (24). Immunohistochemistry Hearts from control and SIS2 mice were excised, blotted dry, and flash frozen in 2-methyl-butane chilled with liquid N2. Tissue sections (6 mM) were produced and mounted on polylysine-coated slides. Slides were fixed at 220°C for 20 min in 50 mM glycine in 70% ethanol adjusted to pH 2.0. A 1:20 dilution of conditioned media from a hybridoma expressing anti-rat sarcomeric myosin IgG (hybridoma MF20, University of Iowa Developmental Studies Hybridoma Bank, Iowa City) was applied to the slides for 30 min. Slides were rinsed three times for 5 min with PBS. An FITC-conjugated sheep antimouse IgG secondary antibody (Boehringer Mannheim) was applied to slides for 30 min. Slides were rinsed three times for 5 min with PBS. A 200 ng/ml Hoechst 33258 solution was applied to slides for 5 min to stain nuclei. Fibrosis quantitation After paraffin embedding, 4 mM sections were produced and mounted on polylysine-coated slides. Slides were stained with Sirius red/picric acid. Overlapping partial images of heart cross sections were captured at 403 with a microscopemounted digital camera. Composite images of complete heart cross sections were assembled and quantitation of collagen and noncollagen material was performed. Statistical analysis

MATERIALS AND METHODS

Data sets were compared using Student’s t test, the nonparametric sign test, or analysis of variance, as appropriate. P , 0.05 was considered significant. Results are reported as mean 6 standard error.

Mice The construction of the SIS2 transgene has been described (23) as Sk 733 IGF-I 39Sk. SIS2 transgenics and control mice were of the inbred FVB background strain. Control animals were either littermates lacking the SIS2 transgene or FVB controls purchased from Charles River Laboratories (Wilmington, Mass.). All animals were housed at Baylor College of Medicine in accordance with National Institutes of Health guidelines. Animals were kept on a 10 h light/14 h darkness schedule and were fed autoclaved chow ad libitum. IGF-I radioimmunoassay Rodent and human total IGF-I concentrations in serum and heart homogenates were determined by species-specific radioimmunoassay (Diagnostic Systems Labs, Webster, Tex.) (rodent) or IRMA (human). Serum samples were assayed per manufacturer’s instructions. Heart homogenates were prepared by powdering whole mouse hearts under liquid N2 suspension in 4°C RIPA buffer [phosphate-buffered saline (PBS), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate] plus protease inhibitors [1 mg/ml Pefabloc (Boehringer Mannheim, Indianapolis, Ind.), 0.5 mg/ml EDTA, 10 mg/ml pepstatin, 1 mg/ml aprotinin)] and homogenized. Serum and heart homogenates’ human and rodent IGF-I levels were then assayed per manufacturer’s instructions, except that homogenates were subjected to a single freeze/thaw cycle prior to assay to increase recovery of the hormone. 1924

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RESULTS Expression of the SIS2 transgene Expression of the SIS2 (a-skeletal actin promoter, human IGF-I cDNA, a-skeletal actin 39UTR) transgene had been partially characterized in a previous study (23), which showed transgenic mRNA only in skeletal and cardiac muscle, with skeletal expression being greater than that of cardiac. In the current study, changes in transgene expression with age were examined. Transgenic (i.e., human) and endogenous IGF-I levels in serum and heart homogenates were measured by IRMA or radioimmunoassay at 10, 20, and 32 wk of age (Fig. 1A, B). Human IGF-I (hIGF-I) was detected in transgenic serum at every time point; serum from control mice contained no detectable hIGF-I. The serum level of endogenous IGF-I declined with age in both control and transgenic mice. There was no statistically significant difference in endogenous IGF-I levels between transgenic and control serum samples at any age, implying that the SIS2 transgene was not repressing ex-

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Figure 1. A—D) Statistical significance at individual time points was determined by Student’s t test assuming unequal variance: *P , 0.005, †P , 0.001, ‡P , 0.0001, §P , 0.00001. Boxed P values were determined by ANOVA considering all time points simultaneously. Error bars represent standard error of the mean. Transgenic and endogenous IGF-I levels in serum (A) and heart homogenates (B). Total serum IGF-I levels (human1mouse) were not significantly different in transgenics and controls (A); total heart homogenate IGF-I levels were significantly increased in transgenics at each time point (B). n 5 6. C, D) Cardiac mass and cardiac mass/tibial length were significantly increased in transgenics at each time point. n 5 16.

pression of the endogenous IGF-I gene. Total (human1mouse) serum IGF-I was not significantly different between transgenics and controls at any time point. As with serum, hIGF-I was detected in SIS2 hearts at each time point, but not in control hearts. The level of endogenous IGF-I declined with age in both control and transgenic hearts. There was no statistically significant difference in the endogenous IGF-I level between control and transgenic heart homogenates at any age, suggesting that the transgene’s expression was not repressing that of the endogenous IGF-I gene. However, total heart IGF-I was significantly increased ;threefold by 10 wk of age in transgenic mice compared to controls (29.964.4 vs. 11.863.2 ng/mg, P,0.003). This difference increased to fivefold by 20 wk of age (42.765.5 vs. 8.362.3 ng/mg, P,0.0001). Overall, these data indicate that the SIS2 transgene induces its phenotype primarily through enhanced local, cardiac IGF-I expression. The SIS2 mouse develops cardiac hypertrophy To determine the extent to which the SIS2 transgene is able to induce cardiac hypertrophy, cardiac mass and tibial length were measured in control and SIS2 mice at 10, 20, and 32 wk of age. Cardiac mass (Fig. 1C) was significantly greater in transgenic mice by 10 wk (131.862.2 vs. 112.563.4 mg, P,0.0001), with the increase over controls widening until 20 wk of age (150.362.7 vs. 125.464.0 mg, P,0.00001). Cardiac mass/tibial length (Fig. 1D) was significantly greater in SIS2 mice by 10 wk (7.2260.11 vs. 6.2260.17 mg/mm, P,0.0001), increasing until 20 wk of age (7.9560.15 vs. 6.7260.19 mg/mm, PERSISTENT IGF-I INDUCES PATHOLOGIC CARDIAC HYPERTROPHY

P,0.00001). The SIS2 transgene is able to induce cardiac hypertrophy early in life and stimulates continued enlargement throughout life. IGF-I is initially beneficial, but ultimately detrimental to systolic function Having established that the SIS2 transgene can induce cardiac hypertrophy, we then examined the long-term effect of IGF-I on systolic function (Fig. 2A). Eight SIS2 mice and eight control littermates were subjected to aortic-pulsed Doppler measurements in a longitudinal study. Data were collected at 10, 20, 32, and 52 wk of age. Peak aortic outflow velocity, a measure of systolic performance, was significantly greater (111.966.2 vs. 87.663.8 cm/s; P,0.02) in the transgenics at 10 wk compared to controls. This benefit, however, was not maintained; transgenic peak aortic outflow velocity was not significantly different from controls at 20 or 32 wk and was significantly reduced (95.364.0 vs. 112.164.9 cm/s; P,0.05) by 52 wk compared to controls. Heart rates were not significantly different between control and transgenic animals at any time point (Table 1). Thus, IGF-I expression provided a temporary systolic performance benefit early in life, but continued expression led to compromised performance. SIS2 mice exhibit reduced left ventricular early/ atrial peak filling velocity ratios The accretive gain in cardiac mass and transient increase in peak aortic outflow velocity observed in SIS2 mice compared to controls might be explained by a steady degradation of left ventricular diastolic 1925

Histological findings in SIS2 mice

Figure 2. A, B) Statistical significance at individual time points was determined by Student’s t test assuming unequal variance: *P , 0.05, †P , 0.02. Boxed P value was determined by ANOVA considering all time points simultaneously. Error bars represent standard error of the mean. A) Peak aortic outflow velocity, a measure of systolic performance, was significantly increased in transgenic mice over controls at 10 wk of age (111.9617.5 vs. 87.6610.8 cm/s; P,0.02), declining with age until the transgenics showed significantly decreased values by 52 wk (95.369.4 vs. 112.1610.9 cm/s; P,0.05); n56. B) Early/atrial peak filling velocity ratios were lower in transgenic mice at all time points. Considering all time points by ANOVA testing revealed a significant trend of reduced E/A peak velocity ratios in SIS2 mice compared to controls (P,0.03).

function. To test this hypothesis, early/atrial peak filling velocity ratios measured by transmitral pulsed Doppler were examined (Fig. 2B). E/A peak velocity ratios were consistently lower in SIS2 mice than in controls. When all time points were considered simultaneously, SIS2 mice demonstrated a significant reduction in early/atrial peak filling velocities (P,0.03). Heart rates were not different between groups at any time point. Thus, it is likely that the long-term effect of a loss of left ventricular compliance or impairment of relaxation is at least partially responsible for the systolic performance changes present in the SIS2 mouse. 1926

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Having observed that the SIS2 transgene provides a short-term systolic performance benefit but a longterm detriment, we searched for structural differences between transgenic and control mice that could provide a rationale for the functional effect. Hearts from 52-wk-old transgenic and littermate control mice were sectioned and immunostained for sarcomeric myosin (Fig. 3). Control heart sections displayed uniform myosin expression. A few punctate regions lacking myosin expression were determined to be nuclei on counterstaining with Hoechst 33258. These findings contrasted sharply with those of the transgenic heart sections. In these, myosin expression was intermittent; large areas were devoid of myosin protein. Most of these nonmyosin regions were not nuclei, as they failed to stain with Hoechst 33258. Sections from transgenic hearts also revealed dramatic myofiber hypertrophy; myofiber disorganization was evident in some transgenic sections. Myofiber hypertrophy and disorganization are common elements of pathological cardiac hypertrophy. Regions of the transgenic sections that failed to stain with sarcomeric myosin antibody or Hoechsht 33258 might be explained by yet another feature of pathological hypertrophy, interstitial fibrosis. To test the hypothesis that these regions contained collagen, all mice from the longitudinal study were killed at 72 wk of age and their hearts were fixed for histological examination. Upon dissection, transgenics displayed wide variability in cardiac mass compared to controls (188.4622.0 mg vs. 151.7614.4 mg, P,0.05). Some 72 wk transgenic mouse hearts exhibited overt organ pathology (Fig. 4A). Individual myofibers from some transgenic heart sections were hypertrophied severalfold compared to controls (Fig. 4B), but this varied with the degree of cardiac hypertrophy in each mouse. To determine the extent of interstitial fibrosis, sections from each longitudinal study heart were stained with Sirius red, which binds collagen quantitatively. Transgenic TABLE 1. Heart rate of control and transgenic mice at 10, 20, 32, and 52 wk of agea

Age (wk)

Control heart rate

SIS2 heart rate

Statistical significance (P)

10 20 32 52 Overall

265 6 24 297 6 38 353 6 22 329 6 15 307 6 15

271 6 33 300 6 36 347 6 47 300 6 16 304 6 17

0.889 0.945 0.911 0.217 0.416

a Statistical significance of individual time points was determined by Student’s t test assuming unequal variance. Overall significance was determined by ANOVA. Heart rates are expressed in beats/min as mean 6 standard error. At no time was there a significant difference between control and transgenic mouse heart rates.

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hearts (72-wk-old) demonstrated a significantly greater percentage of fibrosis per complete cross section (12.062.0 vs. 3.360.5%, P,0.01) compared to controls (Fig. 4C). Thus, it is likely that the nonmyosin, nonnuclear regions in sections from 52-wk-old transgenic also contained collagen. The significantly decreased systolic performance seen in aged SIS2 mice corresponded with their increased cardiac interstitial fibrosis. We also examined young SIS2 mice to determine whether the converse held true. Sirius red-stained heart sections from 15-wk-old transgenic mice trended toward increased fibrosis compared to controls, but did not achieve statistical significance (7.861.2 vs. 4.460.5%, P,0.08). Myofiber disorganization was not evident and myofiber hypertrophy was marginal in 15-wk-old transgenic mice. Although a cause-and-effect relationship cannot be proved, erosion of the temporary benefit in systolic performance conferred by the SIS2 transgene closely paralleled the development of interstitial fibrosis.

Figure 4. A) Control and SIS2 longitudinal study hearts at 72 wk of age. B) Left ventricle cross sections from panel A stained with Sirius red/picric acid to reveal interstitial fibrosis. Dramatic myofiber hypertrophy is evident in the SIS2 heart. C) Statistical significance at individual time points was determined by Student’s t test assuming unequal variance: *P , 0.01. Fibrosis as a percentage of total cross sectional area in transgenic and control hearts. Transgenics present increased (but not yet significant) fibrosis by 15 wk of age (7.861.2 vs. 4.460.5%, P,0.08). Significantly increased fibrosis has developed by 72 wk (12.065.4 vs. 3.361.1%, P,0.01); n 5 5.

DISCUSSION

Figure 3. Control and SIS2 left ventricle cross sections at 52 wk of age. FITC (green) indicates the presence of sarcomeric myosin; Hoechst 33258 stains nuclei blue. SIS2 sections display dramatic myofiber hypertrophy compared to controls. Note that in control sections, virtually every region lacking myosin antibody corresponds to a nucleus. In the transgenic heart sections, several nonmyosin, nonnuclear regions can be seen (black areas). PERSISTENT IGF-I INDUCES PATHOLOGIC CARDIAC HYPERTROPHY

The development of pressure overload-induced cardiac hypertrophy has been shown to be concomitant with increased IGF-I expression by the heart (18). We present evidence that cardiac expression of IGF-I in the absence of significantly increased serum IGF-I is sufficient to initiate and promote an analog of physiologic cardiac hypertrophy, which eventually progresses to a pathological state. The phenotypic progression of the SIS2 mouse closely parallels that of the mammalian heart in hypertension. In the early stages of mammalian hypertension, individual myocytes increase the number of myofibers they contain in an attempt to normalize the increased systolic pressure applied to them. Grossly, the heart begins to develop a concentric hypertrophy of the left ventricle. A microscopic examination of the heart at 1927

this stage reveals hypertrophy of well-organized myocytes, but fibrosis and myofiber disorganization are not yet evident. From a functional standpoint, the heart effectively compensates for its high-pressure operating environment, as there is no loss in cardiac output. In the present study, the SIS2 mouse developed statistically significant increases in cardiac mass and cardiac mass/tibial length by 10 wk of age, but neither myofiber disorganization nor statistically significant fibrosis had developed by 15 wk. The hypertrophy in 10 wk SIS2 mice actually provided an increase in systolic performance when compared to controls. Thus, young SIS2 mice present an analog of physiologic cardiac hypertrophy. Were pressure overload actually present, these findings might well be classified as compensated pathologic hypertrophy, and it is this early phenotypic stage of hypertensive heart disease for which the young SIS2 mouse may be an effective model. With severe or even mild but persistent hypertension, the mammalian heart will undergo further hypertrophy until the phenotype progresses to a more pathological state. Grossly, this is characterized by pronounced concentric hypertrophy of the left ventricle. Other chambers may reveal pathological anatomic changes as well. Microscopically, the pathologically hypertrophied heart reveals prominent hypertrophy of individual myocytes, disorganization of myofibers, and increased interstitial fibrosis. Despite the increased heart mass and consequent increase in oxygen and metabolite consumption, cardiac output actually declines. Often this is due to a loss of ventricular compliance. In effect, the thicker ventricular wall and its increased collagen content lead to less efficient diastolic filling. In a similar fashion, the SIS2 mouse exhibits a progressive development of pathological features as the animal matures. The statistically significant increases in cardiac mass and cardiac mass/tibial length that SIS2 mice exhibit by 10 wk of age become more pronounced with age. Histological examination of 72-wk-old SIS2 mice revealed hypertrophy of individual myocytes and significantly increased interstitial fibrosis when compared to controls. Despite the increased cardiac hypertrophy, the SIS2 transgenic mouse’s systolic function steadily deteriorates from 10 wk of age (if not earlier) until it is significantly worse than that of control mice (at 52 wk). Although no relative decrease over time was evident, early/atrial peak filling velocity ratios were also significantly reduced in SIS2 transgenic mice compared to controls, indicating that diastolic function may also be compromised in this animal model. The IGF-I expression pattern and the phenotype progression observed in the SIS2 mouse may make this animal an informative model for pressure overload-induced cardiac hypertrophy. No model is perfect, but we believe the limitations 1928

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inherent in the SIS2 mouse model are outweighed by its potential utility. Given the increase in skeletal muscle mass seen in the SIS2 mouse (23), it could be argued that increased metabolic demand by peripheral tissues is the primary factor driving the development of cardiac hypertrophy. However, the presence or absence of the SIS2 transgene was the only statistically significant factor in determining cardiac mass after normalizing cardiac mass to lean body mass, a metric dominated by skeletal muscle mass (data not shown). Combined with the statistically significant increase in hIGF-I detected in the SIS2 mouse heart, these data suggest that the transgene exerts a direct hypertrophic effect on cardiac tissue. If present, changes in afterload resistance might account for the majority of this study’s reported differences in systolic performance between SIS2 and control mice. We did not measure systemic aortic blood pressure on a longitudinal basis in this study, as this would have required repeated catheterization. The possible presence of vascular hypertrophy was examined as part of the original characterization of the SIS2 mouse, but no evidence of this phenomenon was found. This information is corroborated by the lack of transgene mRNA expression in smooth muscle tissue (23) and by the lack of significant circulating hIGF-I in the current study. Peak aortic outflow velocity is also sensitive to changes in preload and heart rate. Heart rate was not significantly different among transgenic and control animals at any time point in this study (Table 1). This study does present evidence of decreased early/ atrial peak filling velocity ratios in SIS2 mice compared to controls, suggesting that diastolic function is compromised in the transgenic animals. However, it is important to note that the greatest deficit in transgenic E/A peak velocity ratios compared to control values occurs at 10 wk of age, when the transgenic animals’ peak aortic outflow velocity is 128% of the control value. At 52 wk of age, peak aortic outflow velocity of SIS2 mice is only 85% of that of control mice, but the relative difference between transgenic and control E/A peak velocity ratios is essentially unchanged from the 10 wk time point. This lack of correlation between the relative decline in peak aortic outflow velocity and the relative E/A peak velocity ratio strongly suggests that preload changes alone are unlikely to account for the erosion of systolic performance in the SIS2 transgenic mouse. The well-documented ability of IGF-I to improve cardiac performance, at least temporarily, in clinical (15) and experimental (12, 25) cardiac pathologies has sparked much interest in the use of this hormone as a novel therapy in the failing heart. Here we examined the cardiac effect of local IGF-I expression throughout the majority of an animal model’s life

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span. This long-term perspective has proved to be of critical importance. We confirmed the short-term benefit in systolic performance After IGF-I administration reported in other studies. However, we have determined that this benefit is transient, and that with continued exposure to IGF-I, the heart develops pathological features including reduced systolic performance and marked interstitial fibrosis. Our demonstration that IGF-I ultimately compromises cardiac function and induces undesirable cellular changes in the heart may limit the therapeutic potential of chronic IGF-I administration. Consequently, the potential cardiovascular benefits of IGF-I must be carefully balanced against the potential dangers inherent in stimulation by this growth factor. Considering our findings, limiting temporal exposure to IGF-I seems the most efficacious means of delivering the potential benefits of IGF-I while avoiding its deleterious side effects. The authors acknowledge Thuy Pham for her pulsed Doppler technical expertise, Alida Evans and Stephanie Butcher for their enthusiastic histological work, Jennifer Pocius for her excellent mouse surgery skills, and Drs. Said Akli and Ruxandra Draghia-Akli for their insight and helpful discussion. This study was supported by USPH 5 T32 AG000183, NIDDK 2 T32 DK07696, NIH HL42550, NIH HL53982, and NIH AG13251. It was funded in part by the U.S. Department of Agriculture, Agricultural Research Service under Cooperative Agreement 58 – 6250-6001. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does the mention of trade names, commercial products, or organization imply endorsement by the U.S. Government.

9. 10.

11.

12.

13. 14.

15.

16.

17. 18.

19.

REFERENCES 1. 2. 3.

4.

5.

6.

7.

8.

Cohick, W. S., and Clemmons, D. R. (1993) The insulin-like growth factors. Annu. Rev. Physiol. 55, 131–53, 131–153 Kardami, E. (1990) Stimulation and inhibition of cardiac myocyte proliferation in vitro. Mol. Cell Biochem. 92, 129 –135 Kajstura, J., Cheng, W., Reiss, K., and Anversa, P. (1994) The IGF-1-IGF-1 receptor system modulates myocyte proliferation but not myocyte cellular hypertrophy in vitro. Exp.Cell Res. 215, 273–283 Ito, H., Hiroe, M., Hirata, Y., Tsujino, M., Adachi, S., Shichiri, M., Koike, A., Nogami, A., and Marumo, F. (1993) Insulin-like growth factor-I induces hypertrophy with enhanced expression of muscle specific genes in cultured rat cardiomyocytes. Circulation 87, 1715–1721 Donath, M. Y., Zapf, J., Eppenberger-Eberhardt, M., Froesch, E. R., and Eppenberger, H. M. (1994) Insulin-like growth factor I stimulates myofibril development and decreases smooth muscle alpha-actin of adult cardiomyocytes. Proc. Natl. Acad. Sci. USA 91, 1686 –1690 Reiss, K., Kajstura, J., Zhang, X., Li, P., Szoke, E., Olivetti, G., and Anversa, P. (1994) Acute myocardial infarction leads to upregulation of the IGF-1 autocrine system, DNA replication, and nuclear mitotic division in the remaining viable cardiac myocytes. Exp.Cell Res. 213, 463– 472 Reiss, K., Meggs, L. G., Li, P., Olivetti, G., Capasso, J. M., and Anversa, P. (1994) Upregulation of IGF1, IGF1-receptor, and late growth related genes in ventricular myocytes acutely after infarction in rats. J. Cell Physiol. 158, 160 –168 Wang, L., Ma, W., Markovich, R., Lee, W. L., and Wang, P. H. (1998) Insulin-like growth factor I modulates induction of

PERSISTENT IGF-I INDUCES PATHOLOGIC CARDIAC HYPERTROPHY

20.

21.

22.

23.

24.

25.

apoptotic signaling in H9C2 cardiac muscle cells. Endocrinology 139, 1354 –1360 Wang, L., Ma, W., Markovich, R., Chen, J. W., and Wang, P. H. (1998) Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor I. Circ. Res. 83, 516 –522 Buerke, M., Murohara, T., Skurk, C., Nuss, C., Tomaselli, K., and Lefer, A. M. (1995) Cardioprotective effect of insulin-like growth factor I in myocardial ischemia followed by reperfusion. Proc. Natl. Acad. Sci. USA 92, 8031– 8035 Reiss, K., Cheng, W., Ferber, A., Kajstura, J., Li, P., Li, B., Olivetti, G., Homcy, C. J., Baserga, R., and Anversa, P. (1996) Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice. Proc. Natl. Acad. Sci. USA 93, 8630 – 8635 Duerr, R. L., Huang, S., Miraliakbar, H. R., Clark, R., Chien, K. R., and Ross, J. J. (1995) Insulin-like growth factor-1 enhances ventricular hypertrophy and function during the onset of experimental cardiac failure. J. Clin. Invest.. 95, 619 – 627 Yang, R., Bunting, S., Gillett, N., Clark, R., and Jin, H. (1995) Growth hormone improves cardiac performance in experimental heart failure. Circulation 92, 262–267 Fazio, S., Sabatini, D., Capaldo, B., Vigorito, C., Giordano, A., Guida, R., Pardo, F., Biondi, B., and Sacca, L. (1996) A preliminary study of growth hormone in the treatment of dilated cardiomyopathy [see comments]. N. Engl. J. Med. 334, 809 – 814 Donath, M. Y., Sutsch, G., Yan, X. W., Piva, B., Brunner, H. P., Glatz, Y., Zapf, J., Follath, F., Froesch, E. R., and Kiowski, W. (1998) Acute cardiovascular effects of insulin-like growth factor I in patients with chronic heart failure. J. Clin. Endocrinol. Metab. 83, 3177–3183 Lombardi, G., Colao, A., Cuocolo, A., Longobardi, S., Di Somma, C., Orio, F., Merola, B., Nicolai, E., and Salvatore, M. (1997) Cardiological aspects of growth hormone and insulinlike growth factor- I. J. Pediatr. Endocrinol. Metab. 10, 553–560 Sacca, L. (1997) Growth hormone: a newcomer in cardiovascular medicine. Cardiovasc. Res. 36, 3–9 Donohue, T. J., Dworkin, L. D., Lango, M. N., Fliegner, K., Lango, R. P., Benstein, J. A., Slater, W. R., and Catanese, V. M. (1994) Induction of myocardial insulin-like growth factor-I gene expression in left ventricular hypertrophy. Circulation 89, 799 – 809 Butt, R. P., and Bishop, J. E. (1997) Mechanical load enhances the stimulatory effect of serum growth factors on cardiac fibroblast procollagen synthesis. J. Mol. Cell Cardiol. 29, 1141– 1151 Diez, J., and Laviades, C. (1994) Insulin-like growth factor I and collagen type III synthesis in patients with essential hypertension and left ventricular hypertrophy. J. Hum. Hypertens. 8 (Suppl. 1), S21–S25 Fazio, S., Cittadini, A., Cuocolo, A., Merola, B., Sabatini, D., Colao, A., Biondi, B., Lombardi, G., and Sacca, L. (1994) Impaired cardiac performance is a distinct feature of uncomplicated acromegaly. J. Clin. Endocrinol. Metab. 79, 441– 446 Chanson, P., Timsit, J., Masquet, C., Warnet, A., Guillausseau, P. J., Birman, P., Harris, A. G., and Lubetzki, J. (1990) Cardiovascular effects of the somatostatin analog octreotide in acromegaly. Ann. Intern. Med. 113, 921–925 Coleman, M. E., DeMayo, F., Yin, K. C., Lee, H. M., Geske, R., Montgomery, C., and Schwartz, R. J. (1995) Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J. Biol. Chem. 270, 12109 –12116 Taffet, G. E., Hartley, C. J., Wen, X., Pham, T., Michael, L. H., and Entman, M. L. (1996) Noninvasive indexes of cardiac systolic and diastolic function in hyperthyroid and senescent mouse. Am. J. Physiol. 270, H2204 –H2209 Stromer, H., Cittadini, A., Douglas, P. S., and Morgan, J. P. (1996) Exogenously administered growth hormone and insulinlike growth factor- I alter intracellular Ca21 handling and enhance cardiac performance. In vitro evaluation in the isolated isovolumic buffer-perfused rat heart. Circ. Res. 79, 227–236 Received for publication February 15, 1999. Revised for publication July 19, 1999.

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