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Ann Thorac Surg 2001;72:1650-1656
© 2001 The Society of Thoracic Surgeons

Original article: cardiovascular

Insulin-like growth factor-1 improves postischemic recovery in hypertrophied hearts

Ingeborg Friehs, MDa, Christof Stamm, MDa, Hung Cao-Danh, PhDa, Francis X. McGowan, Jr, MDb, Pedro J. del Nido, MD*a

a Department of Cardiovascular Surgery, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
b Department of Anesthesiology/Critical Care, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

Accepted for publication June 29, 2001.

* Address reprint requests to Dr del Nido, Department of Cardiovascular Surgery, Children’s Hospital, Harvard Medical School, 300 Longwood Ave, Bader 279, Boston, MA 02115, USA
e-mail: pedro.delnido{at}tch.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Severe myocardial hypertrophy is associated with decreased tolerance to ischemia compared with normal hearts. We hypothesized that treatment with insulin-like growth factor-1 (IGF-1) improves postischemic myocardial recovery by increasing glucose uptake during ischemia and early reperfusion.

Methods. Banding of the thoracic aorta in 10-day-old rabbits created pressure-overload hypertrophy. At 5 weeks of age (severe hypertrophy), aortic banded and sham-operated isolated hearts underwent 30 minutes of normothermic ischemia with or without IGF-1 in the preischemic perfusate and cardioplegia followed by 30 minutes of reperfusion.

Results. 2-Deoxyglucose uptake (31P-NMR) and phosphatidylinositol-3-kinase (PI-3-kinase) activity were significantly lower in hypertrophied hearts. Insulin-like growth factor-1 restored glucose uptake and PI-3-kinase activity to control levels in the hypertrophied hearts and both effects were blocked by wortmannin (a PI-3-kinase inhibitor). Postischemic developed pressure was significantly improved in IGF-1-treated hearts compared with untreated or IGF-1+wortmannin-treated hypertrophied hearts.

Conclusions. These data indicate that IGF-1 improves glucose uptake and tolerance to ischemia in hypertrophied hearts. Myocardial IGF-1 effects are likely mediated through a PI-3-kinase-dependent pathway.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Severe myocardial hypertrophy is a known risk factor for mortality and morbidity in cardiac operations [1]. In pressure-overload hypertrophy, adaptive changes include increased sarcomere number, a switch to fetal contractile protein isoforms, and altered calcium regulation for excitation-contraction coupling [2, 3]. Early in the development of hypertrophy, abnormalities of fatty acid metabolism have also been demonstrated, as well as increased glucose utilization for high-energy phosphate production [4]. However, in a rabbit model of severe pressure-overload hypertrophy, we have shown that glucose uptake declines starting at the earliest onset of decompensation and ventricular dilatation, before the development of altered calcium regulation [5, 6]. This finding has also been reported for other models of severe pressure-overload hypertrophy [7]. The decline in glucose uptake is associated with decreased tolerance to ischemia and worsening of contractile function postischemia [6].

Under physiologic conditions, glucose entry into the cell is rate limiting for glycolysis [8]. Glucose transport across the plasma membrane is facilitated by tissue-specific transporter proteins [8]. Ischemia induces multiple changes in myocardial cell metabolism, including a marked increase in glucose uptake because of translocation of insulin-sensitive glucose transporters from intracellular membrane storage vesicles to the sarcolemma [9, 10]. This response is thought to promote glycolysis during ischemia and early reperfusion, thereby facilitating production of high-energy phosphates through anaerobic and aerobic glycolysis. Hypertrophied hearts exhibit an accelerated loss of high-energy phosphates during ischemia, earlier onset of ischemic contracture, and decreased recovery of contractile function after reperfusion compared with nonhypertrophied controls [11, 12]. Based on our earlier findings that impaired glucose uptake in response to insulin was associated with a significantly decreased tolerance to ischemia, we postulated that normalization of the insulin response in hypertrophied myocardium would result in improved postischemic recovery of contractile function [5, 6, 12].

Insulin-like growth factor-1 (IGF-1) has been shown to have both acute and long-term cardiovascular effects. Potential beneficial effects in heart failure include reduction in afterload through vasodilatation, positive inotropy, prevention of apoptosis, increased calcium sensitivity of cardiac myofilaments, and improved recovery of cardiac function after myocardial infarction and during reperfusion after global ischemia in rat models [13–16]. Insulin-like growth factor-1 has also been shown to stimulate glucose uptake in skeletal muscle and adipose tissue [17, 18].

The present study was designed to determine whether acute administration of IGF-1 improves glucose transport and tolerance to ischemia in a rabbit model of pressure-overload hypertrophy. In addition, we sought to determine whether the defect in insulin response in hypertrophied hearts was associated with decreased phosphatidylinositol-3-kinase (PI-3-kinase) activity and whether IGF-1 restored this activity.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Left ventricular hypertrophy model
Pressure-overload hypertrophy was achieved by banding the descending aorta in 10-day-old New Zealand White rabbits as previously described [5, 6, 12]. Implanting a fixed constriction in a young animal and allowing it to grow induced pressure-overload hypertrophy beginning at 3 weeks of age in this model. Using measurements of left ventricular (LV) mass and LV volume with transthoracic echocardiography, we have previously shown that progression of LV hypertrophy reaches a plateau by 5 weeks of age. This early-compensated hypertrophy phase is followed by progressive LV dilation and a fall in the LV mass to LV cavity volume ratio (M/V ratio). A 30% increase in LV M/V ratio was found to indicate the point of onset of uncompensated hypertrophy with ventricular dilatation [5]. In this study all hearts were studied just before the uncompensated stage of hypertrophy.

2-deoxyglucose uptake
In hearts from 5-week-old aortic banded and age-matched control (sham-operated) rabbits, 2-Deoxyglucose (2-DG) uptake was determined using 31P-nuclear magnetic resonance spectroscopy as previously described in detail [5]. Nuclear magnetic resonance spectra were acquired in an 8.45 Tesla vertical bore Bruker spectrometer (Bruker Instruments, Billerica, MA) operating at a proton frequency of 360 MHz and 31P frequency of 145 MHz. Spectra were obtained by signal averaging 120 scans with a 2-second delay. Spectral peak areas were quantified by integration after base line correction with software provided by Bruker. Normalization was carried out by standardizing the 2-Deoxyglucose-6-phosphate (2-DG-6-P) signal integral to an external standard (500 µmol/L dimethylene phosphonic acid) contained in a balloon adjacent to the heart.

After an initial stabilization period, the perfusate was switched to a modified Krebs-Henseleit (KH) solution containing 10 IU/L bovine insulin, 1 mmol/L glucose, and 3 mmol/L 2-DG (Sigma, St. Louis, MO). 2-Deoxyglucose is a competitive inhibitor of glucose that is transported by the sarcolemmal glucose transporter and subsequently phosphorylated by hexokinase to yield 2-DG-6-P. 2-Deoxyglucose-6-phosphate gives a distinct resonance peak and the rise of this peak over time is proportional to 2-DG uptake and phosphorylation by the cell and the clearance of 2-DG-6-P from the heart, which is slow. Thus the rate of 2-DG-6-P accumulation is proportional to glucose transport and phosphorylation [19].

To determine the effects of IGF-1 on 2-DG uptake in hypertrophied hearts, 31P-NMR spectra were obtained over a period of 30 minutes in age-matched, sham-operated, nonhypertrophied control hearts (Control, n = 6); untreated hypertrophied hearts (Hyper, n = 6); a group of hypertrophied hearts (Hyper+IGF, n = 6) receiving IGF-1 (5 x 10-8 mol/L; Research Biochemicals Incorporated, Natick, MA); and an additional group of hypertrophied hearts (Hyper+IGF/wort, n = 6) perfused with IGF-1 (5 x 10-8 mol/L) and wortmannin, a PI-3-kinase inhibitor (1 µmol/L; Sigma, St. Louis, MO) [13]. In all groups, modified KH buffer contained insulin in a concentration of 10 IU/L.

Isolated heart perfusion and contractile function measurements
A separate group of 5-week-old banded and age-matched, sham-operated (control) rabbits were given heparin (500 U/kg intravenous [IV]) and euthanized with a mixture of ketamine (50 to 100 mg/kg IV) and xylazine (2.5 mg/kg IV). The hearts were rapidly excised and placed in cold modified KH solution. After aortic cannulation, the hearts were perfused in the nonworking, nonrecirculating Langendorff mode at a constant perfusion pressure as described previously [6]. After a 30-minute stabilization period, the hypertrophied (Hyper, n = 6) and normal, age-matched control hearts (Control, n = 6) were arrested with a 2-minute infusion of normothermic cardioplegia solution (30 mL) containing modified KH buffer with 10 U/L insulin and with a final KCl concentration of 22.5 mmol/L. Another group of hypertrophied hearts (Hyper+IGF, n = 6) was perfused with modified KH buffer containing (in addition to 10 IU/L insulin) IGF-1 (5 x 10-8 mol/L) for 20 minutes before ischemia and arrested with normothermic cardioplegia containing the same concentration of insulin and IGF-1. An additional group of hypertrophied hearts (Hyper+IGF/wort, n = 6) was perfused with modified KH buffer containing insulin (10 IU/L), IGF-1 (5 x 10-8 mol/L) and the PI-3-kinase inhibitor, wortmannin (1 µmol/L), for 20 minutes before ischemia. The hearts were then arrested with normothermic cardioplegia containing the same concentration of insulin, IGF-1, or wortmannin. The insulin concentration of 10 IU/L was the same in all groups.

Left ventricular pressure measurements were obtained with a latex fluid-filled balloon that was connected to a micromanometry pressure transducer (Millar Instruments Co, Houston, TX). Heart rate, coronary flow, LV-developed pressure (calculated from LV systolic minus end-diastolic pressure) at balloon volumes adjusted to produce an end-diastolic pressure in the range of 5 to 10 mm Hg, were measured at the end of the 30-minute stabilization period for baseline measurements. After induction of ischemia, the intracavitary balloon was emptied and remained empty during the entire ischemic period and for 30 minutes during reperfusion to simulate the beating, nonworking heart. After 30 minutes of reperfusion, the balloon was filled to its preischemic volume and LV pressure measurements were recorded.

Phosphatidylinositol-3-kinase activity
Phosphatidylinositol-3-kinase activity was measured by modification of a previously described method by Folli and colleagues [20] and Ruderman and colleagues [21]. Ventricular tissue from a separate set of hypertrophied (n = 6) and age-matched littermates (n = 6) perfused with KH solution containing 10 IU/L insulin for 15 minutes and hypertrophied animals (n = 6) perfused with insulin (10 IU/L) and IGF-1 (5 x 10-8 mol/L) containing KH buffer was homogenized in ice-cold buffer containing 50 mmol/L HEPES (pH 7.5), 137 mmol/L NaCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 2 mmol/L Na3VO4, 10 mmol/L sodium pyrophosphate, 10 mmol/L sodium fluoride, 2 mmol/L EDTA, 1% Triton X-100, aprotinin (2 µg/mL), antipain (10 µg/mL), leupeptin (5 µg/mL), pepstatin (0.5 µg/mL), benzamidine (1.5 mg/mL), phenylmethylsulfonyl fluoride (34 µg/mL) and centrifuged at 14,000 rpm for 10 minutes. Tissue from nonhypertrophied hearts obtained after addition of wortmannin served as control. The supernatant was stored at -80°C for later analysis. Aliquots of the supernatant containing 0.5 to 2.0 mg protein were immunoprecipitated with antiinsulin-receptor-substrate-1 (anti-IRS-1) followed by protein A/G-PLUS-agarose. The immunoprecipitates were washed thrice with phosphate-buffered saline (PBS) containing 1% Triton X-100 and 100 µmol/L Na3VO4, followed by washing in 100 mmol/L Tris (pH 7.5) with 500 mmol/L LiCl and 100 µmol/L Na3VO4, and subsequently in 10 mmol/L Tris (pH 7.5) with 100 mmol/L NaCl, 1 mmol/L EDTA and 100 µmol/L Na3VO4. The pellets were resuspended in 20 µL of 10 mmol/L Tris (pH 7.5) containing 100 mmol/L NaCl and 1 mmol/L EDTA. Then 10 µL of 100 mmol/L MgCl2 and 10 µL of phosphatidylinositol (2 µg/µL) were added, sonicated in 10 mmol/L Tris (pH 7.5) with 1 mmol/L EGTA. The PI-3-kinase reaction was started by addition of 10 µL of 500 µmol/L ATP containing 10 µCi of [32P]ATP. After 10 to 60 minutes of constant shaking at room temperature, the reaction was stopped by adding 20 µL of 8 N HCl and 160 µL of CHCl3:methanol (1:1; vol/vol). The samples were centrifuged and the lower organic phase was removed and applied to a silica gel thin-layer chromatography (TLC) plate (Merck, Whitehouse Station, NJ) coated with 1% potassium oxalate. Thin-layer chromatography plates were developed in CHCl3:CH3OH:H2O:NH4OH (60:47:11.3:2; vol/vol), dried, and visualized by autoradiography. After exposure on films, quantitative analysis was undertaken by laser densitometry.

Western immunoblotting of phosphatidylinositol-3-kinase
Immunoprecipitates of ventricular tissue from hypertrophied and age-matched littermates perfused with KH solution containing 10 IU/L insulin for 15 minutes and hypertrophied animals perfused with KH buffer containing insulin (10 IU/L) and IGF-1 (5 x 10-8 mol/L) were used for gel electrophoresis with 10% SDS-PAGE gels. Proteins were electrophoretically transferred to nitrocellulose membranes. After transfer, the membranes were incubated in 5% nonfat dry milk in TBST (10 mmol/L Tris-HCl pH 7.5, 100 mmol/L NaCl, 0.1% Tween 20) for 30 minutes at room temperature to block nonspecific binding sites and then incubated with primary antibody against PI-3-kinase (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:1,500 overnight; this was followed by incubation with horseradish peroxidase-conjugated secondary antibody (Jackson Immuno Research Labs, West Grove, PA) at a dilution of 1:10,000. The bound antibody was detected by the enhanced chemiluminescence method according to the manufacturer’s instruction (Amersham Life Science, Arlington Heights, IL). This method depends on the production of light after the oxidation of luminol by horseradish peroxidase in the presence of H2O2. After exposure on films, quantitative protein analysis was undertaken by laser densitometry.

Statistical analysis
Data were analyzed using SigmaStat software (Jandel Scientific; SPSS Science, Chicago, IL) and are reported as mean ± SEM. Analysis of variance was used for comparison among and between groups, followed by Bonferroni or Dunn post hoc analysis where appropriate. A value of p less than or equal to 0.05 was considered statistically significant.

Animal care
All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication 85-23, revised 1985). The protocol was reviewed and approved by the animal care committee at Children’s Hospital, Boston.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Left ventricle to body weight ratio
Left ventricle to body weight ratio (LV/BW ratio) was 2.2 ± 0.2 g/kg in nonhypertrophied age-matched controls, 3.7 ± 0.6 g/kg in untreated aortic banded animals, 3.4 ± 0.4 g/kg in aortic banded animals perfused with IGF-1, and 3.8 ± 0.5 g/kg in IGF-1+wortmannin (p <= 0.05 for all hypertrophied groups versus control).

2-deoxyglucose uptake
The rate of 2-DG-6-P accumulation over a period of 30 minutes in the aortic banded and age-matched control hearts is shown in Figure 1. The rate of 2-DG-6-P accumulation was lower in untreated hypertrophied hearts compared with normal age-matched control hearts (p <= 0.05) with a slower rate of rise and lower total accumulation after 30 minutes. In IGF-1-treated hypertrophied hearts, 2-DG-6-P accumulation was increased compared with nontreated hypertrophied hearts (p <= 0.05). However, 2-DG-6-P accumulation in IGF-1-perfused nonhypertrophied, control hearts remained unchanged (data not shown). Wortmannin nearly completely inhibited 2-DG-6-P accumulation in hypertrophied hearts perfused with insulin and IGF-1.



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  		Fig 1. Glucose uptake measured as accumulation of 2-Deoxyglucose-6-phosphate (2-DG-6-P) by 31P-NMR spectroscopy over a period of 30 minutes in hypertrophied hearts (Hyper), age-matched nonhypertrophied control hearts (Control), insulin-like growth factor-1-perfused hypertrophied hearts (Hyper+IGF), and IGF-1+wortmannin-perfused hypertrophied hearts (Hyper+IGF/wort). n = 6/group, *p <= 0.05 versus Hyper+IGF and Control. All solutions contained a constant amount of insulin (10 IU/L).

 
Coronary flow and contractile function
Preischemic heart rate, coronary flow, diastolic pressure, and developed pressure did not differ between hypertrophied and nonhypertrophied, control hearts. Addition of IGF-1 to the perfusate did not influence preischemic cardiac contractile function in either hypertrophied or nonhypertrophied control hearts (Table 1). Heart rate remained unchanged in all groups postischemia. Coronary flow was decreased during reperfusion in nonhypertrophied control (34 ± 1 mL/min), untreated hypertrophied (31 ± 3 mL/min) and IGF-1+wortmannin-treated hypertrophied hearts (27 ± 4 mL/min) compared with IGF-1-treated hypertrophied hearts (37 ± 2 mL/min; p <= 0.05).


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  		Table 1. Preischemic and Postischemic Values

 
Postischemic developed pressure (measured after 30 minutes of reperfusion) was higher in nonhypertrophied control hearts compared with untreated hypertrophied hearts (p <= 0.05, Fig 2). Addition of IGF-1 to the perfusate and cardioplegia improved recovery of contractile function in hypertrophied hearts, comparable to control levels. The beneficial effects of IGF-1 were blocked by addition of wortmannin. (Note that IGF-1 ± wortmannin was not present in the perfusate during reperfusion).



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  		Fig 2. Developed pressure measured 30 minutes after reperfusion (expressed as percent of preischemic values) in hypertrophied hearts (Hyper) with and without insulin-like growth factor-1 (Hyper + IGF-1) perfusion, with IGF-1+wortmannin (Hyper+IGF/wort) containing perfusate, and age-matched nonhypertrophied control hearts. n = 6/group, *p <= 0.05 versus IGF-1 perfused hypertrophied hearts and age-matched control hearts (Control).

 
Phosphatidylinositol-3-kinase activity
Figure 3A shows a representative autoradiograph of TLC separation of the reaction products from PI-3-kinase assays. Quantitation was undertaken by laser densitometry (Fig 3B). No PI-3-kinase activity was present in the anti-IRS-1 immunoprecipitates from nonhypertrophied control hearts treated with wortmannin. Lower levels of PI-3-kinase activity were found in hypertrophied hearts perfused with insulin (10 IU/L) containing KH buffer compared with nonhypertrophied, control hearts after perfusion with insulin containing KH buffer (p <= 0.05). The PI-3-kinase activity in anti-IRS-1 immunoprecipitates was higher in LV tissue from hypertrophied hearts perfused with insulin+IGF-1 compared with those perfused with insulin alone (p <= 0.05).



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  		Fig 3. (A) Representative blot of phosphatidylinositol-3-kinase (PI-3-kinase) activity in antiinsulin-receptor-substrate-1 immunoprecipitates from left ventricular muscle extracts of hypertrophied hearts perfused with Krebs-Henseleit (KH) solution containing insulin only or KH containing insulin+insulin-like growth factor-1 (IGF-1) compared with hearts from age-matched control animals perfused with KH containing insulin or insulin/wortmannin. The radioactivity at the origin is caused by [32P] ATP residues and other 32P-labeled materials. PIP indicates the position of migration of PI-3-P standard. (B) Summary of densitometry values expressed as arbitrary densitometry units for the three groups. *p <= 0.05; versus IGF-1-perfused hypertrophied hearts and age-matched control.

 
Western immunoblotting of phosphatidylinositol-3-kinase
Phosphatidylinositol-3-kinase content determined in anti-IRS-1 immunoprecipitates from nonhypertrophied control hearts, hypertrophied hearts with insulin perfusion, and hypertrophied hearts treated with IGF-1 showed no difference in PI-3-kinase protein between any of the groups. A representative immunoblot is shown in Figure 4A. Figure 4B summarizes the densitometry results. These data indicate that the lower PI-3-kinase activity in hypertrophied hearts is not a result of lower expression but rather posttranscriptional regulation. Similarly, IGF-1 resulted in higher PI-3-kinase activity independent of PI-3-kinase protein content in myocardium.



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  		Fig 4. (A) Representative Western immunoblot of phosphatidylinositol-3-kinase (PI-3-kinase) protein of antiinsulin-receptor-substrate-1 immunoprecipitates from left ventricular muscle extracts from hypertrophied hearts perfused with Krebs-Henseleit (KH) solution containing insulin only or KH containing insulin and insulin-like growth factor-1 (IGF-1) compared with hearts from age-matched control animals perfused with KH containing insulin. (B) Summary of densitometry values expressed as arbitrary densitometry units for the three groups. There is no significant difference of PI-3-kinase protein content in any group.

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
Our results indicate that IGF-1 acutely improves and normalizes the 2-DG-6-P accumulation indicative of glucose uptake in hypertrophied hearts, in association with improved recovery of postischemic contractile function. The beneficial effect of IGF-1 on functional recovery and glucose uptake are associated with higher PI-3-kinase activity. These effects are blocked by wortmannin, an inhibitor of the PI-3-kinase reaction. These data indicate that IGF-1 improves ischemic tolerance and postischemic myocardial function in hypertrophied hearts most likely through a PI-3-kinase-dependent increase in glucose uptake and oxidation.

During ischemia, anaerobic glycolysis is the primary source for ATP production, since lack of oxygen and accumulation of NADH in the mitochondria rapidly inhibit oxidative phosphorylation. Glycolysis has been shown to be especially important for postischemic recovery of contractile function and preservation of calcium homeostasis during ischemia and early reperfusion [22, 23]. Transport of glucose across the plasma membrane is the initial step in myocardial glucose metabolism, and the amount and activity of the facilitative glucose transporter protein present in the sarcolemma determine, in great part, the rate of glucose uptake. The predominant transporter in adult myocardium is the insulin-sensitive transporter GLUT-4. GLUT-4 can rapidly increase glucose uptake in response to insulin. Under basal conditions, GLUT-4 protein is stored in intracellular vesicles and, on insulin stimulation, is translocated to the plasma membrane [24]. Ischemia also causes substantial translocation of this insulin-responsive glucose transporter [10].

We have previously shown that glucose uptake, determined by 2-DG uptake and by tritiated water production from [2-3H] glucose, is significantly impaired in pressure-overload hypertrophied hearts. The impairment in glucose uptake is detectable at the earliest onset of decompensated hypertrophy, ie, onset of LV dilatation [5]. The slower glucose uptake in the hypertrophied hearts may in part explain the accelerated loss of high-energy phosphates during ischemia and worse recovery of postischemic contractile function [6, 11, 12]. Myocardial insulin insensitivity, as seen in noninsulin-dependent diabetes mellitus, has been shown to be associated with impaired glucose uptake during ischemia [25]. Therefore it is reasonable to speculate that failure of insulin-regulated recruitment of the glucose transporter, GLUT-4, to the plasma membrane is responsible for impaired glucose transport in hypertrophied myocardium.

Insulin-like growth factor-1, an anabolic polypeptide, has been shown to have therapeutic benefits when administered exogenously [14]. Beneficial effects of IGF-1 on cardiac contractility have been demonstrated, including improved cardiac function in patients with congestive heart failure [15]. In a clinical study, acute administration of IGF-1 for treatment of chronic heart failure significantly improved cardiac performance by afterload reduction and possibly positive inotropic effects, whereas glucose and electrolyte blood levels remained unchanged [15]. Other beneficial effects may include reduction in blood glucose and triglyceride levels by increasing glucose uptake rate in adipose tissue and skeletal muscle of patients with type II (noninsulin-dependent) diabetes mellitus, a hyperglycemic disorder characterized by insulin resistance [26].

Studies on isolated cardiomyocytes and isolated perfused hearts have indicated that IGF-1 has direct effects on cardiac function. Acute administration of IGF-1 increased contractility in cultured neonatal rat cardiomyocytes and elicited a positive inotropic response in isolated perfused rat hearts, probably through a mechanism involving increased calcium sensitivity of myofilaments [14, 27]. To differentiate the direct inotropic effects of IGF-1 from the effects on glucose transport into myocytes, in our study, we administered IGF-1 only before and at the onset of ischemia. Thus, during reperfusion the hearts were perfused with only insulin containing perfusate. Therefore, the differences observed in postischemic contractile function are likely due to the cardioprotective effects of IGF-1. In addition, the IGF-1-stimulated increase in glucose uptake rate was seen only in hypertrophied hearts whereas control hearts showed similar levels of glucose uptake with insulin only and after the addition of IGF-1. Insulin-like growth factor-1 did not have a significant impact on preischemic contractile function in normal, nonhypertrophied hearts in the dosage used in this study.

Insulin-like growth factor-1 shows structural similarities to insulin but interacts with its own specific receptor. Myocardial tissue has been shown to contain receptors for IGF-1 and insulin. Both receptors are structurally and functionally homologous, but their binding domains are different, resulting in a 10-fold higher affinity for its respective hormone [28]. Binding of IGF-1 to its receptor induces autophosphorylation of the ß-subunit and subsequent activation of a tyrosine kinase. The signal transducing tyrosine kinase domains of the insulin and IGF-1 receptors, however, are similar and activate common intracellular pathways including PI-3-kinase [29, 30]. Therefore, the difference in the physiologic effect of insulin and IGF-1 in myocytes is probably a result of intrinsic differences early in the signaling pathways at their receptors, because both pathways converge at PI-3-kinase [29]. To determine whether IGF-1 can stimulate glucose uptake in the myocardium through a PI-3-kinase sensitive pathway, we measured PI-3-kinase activity in IGF-1-treated and nontreated, hypertrophied hearts with or without wortmannin. Wortmannin has been shown to block PI-3-kinase activity and glucose uptake (at concentrations similar to the ones we used) in skeletal muscle and heart [29]. The results obtained with wortmannin are supported by the measurements of PI-3-kinase activity in LV tissue extracts from hypertrophied animals. Addition of IGF-1 increased the PI-3-kinase activity in hypertrophied hearts significantly, whereas wortmannin completely blocked PI-3-kinase activity. Differences in total protein content of PI-3-kinase in myocardium were excluded by Western immunoblotting using a PI-3-kinase-specific antibody.

In summary, the results of this study demonstrate that acute administration of IGF-1 just before and during early ischemia can improve recovery of postischemic contractile function in hypertrophied hearts to levels observed in control, nonhypertrophied hearts. Importantly, this study provides evidence to support the conclusion that IGF-1 elicits its effect by normalizing glucose uptake in hypertrophied hearts. The improvement in glucose uptake is most likely through a mechanism that bypasses the early steps in the insulin–insulin receptor signaling cascade. Insulin-like growth factor-1 effects are likely mediated through a PI-3-kinase-sensitive pathway associated with its own receptor mediated signaling cascade. Clinically, IGF-1 has already been successfully tested for treatment of chronic heart failure, thus our findings may have additional implications in designing better cardioprotective strategies for patients with severe ventricular hypertrophy who are at increased risk during cardiac surgical procedures.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
This work was supported in part by grants from NIH: HL 63095 (Pedro J. del Nido) and HL 52589 (Francis X. McGowan, Jr).


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

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