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Cardiovascular Research 2003 57(3):738-748; doi:10.1016/S0008-6363(02)00788-5
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Copyright © 2003, European Society of Cardiology

High glucose induces cardiac insulin-like growth factor I resistance in ventricular myocytes: role of Akt and ERK activation

Jun Ren*, Jinhong Duan, Kadon K Hintz and Bonnie H Ren

University of Wyoming College of Health Sciences, P.O. Box 3375, Laramie, WY 82071, USA

* Corresponding author. Tel.: +1-307-766-6120; fax: +1-307-766-2953. jren{at}uwyo.edu

Received 20 June 2002; accepted 12 November 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Cardiac resistance to IGF-1 occurs in diabetes and is attributed to cardiac dysfunction in diabetes. However, the mechanism of action responsible for cardiac IGF-1 resistance is still unknown. This study was designed to examine the impact of high glucose on IGF-1-induced contractile response and activation of serine-threonine kinase Akt as well as extracellular signal-regulated kinase (ERK1/2) in cardiac myocytes. Methods: Isolated adult rat ventricular myocytes were cultured for 12–18 h in a serum-free medium containing either normal (NG, 5.5 mM) or high (HG, 25.5 mM) glucose. Mechanical properties were evaluated using an IonOptix MyoCam® system. Myocytes were electrically stimulated at 0.5 Hz and contractile properties analyzed included peak shortening (PS), time-to-PS (TPS) and time-to-90% relengthening (TR90). Intracellular Ca2+-induced Ca2+ release was measured as fura-2 fluorescence intensity change ({Delta}FFI). Protein levels of total and phosphorylated Akt and ERK1/2, indicators of Akt and ERK1/2 activation, IGF-1 receptors (pro-IGF-1R and IGF-1R{alpha}) as well as the glucose transporter GLUT4 were assessed by Western blot. Results: IGF-1 (10–10–10–6 M) elicited a dose-dependent increase in PS and {Delta}FFI in myocytes maintained in NG medium. However, IGF-1 induced a negative response on PS and {Delta}FFI in HG myocytes. The IGF-1-induced responses in NG or HG myocytes were blunted by the IGF-1 receptor antagonist H-1356. Western blot analysis revealed that IGF-1R{alpha} but not pro-IGF-1R was reduced in HG myocytes. While IGF-1 (10–6 M) upregulated total Akt protein levels in both NG and HG myocytes, it only induced a significant activation of Akt in NG but not HG myocytes. IGF-1 elicited comparable ERK1/2 activation in both NG and HG myocytes. Conclusion: These results suggest that the cardiac IGF-1 resistance in diabetes is likely attributed, at least in part, to reduced IGF-1R and attenuated IGF-1-induced Akt phosphorylation under elevated extracellular glucose.

KEYWORDS Calcium (cellular); Cell culture/isolation; Contractile function; Growth factors; Myocytes; Protein kinases


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The physiological performance of the heart depends on a synchronized action between growth and function, the disturbance of which often results in malformation or malfunction of the heart. Insulin-like growth factor I (IGF-1) has been considered a key ‘surviving factor’ synchronizing the interplay between cardiac growth and contractile function [1–3]. IGF-1 has been shown to improve cardiac muscle survival, growth, and differentiation [1,2]. In addition to its antiapoptotic properties, the ability of IGF-1 to directly enhance ventricular contractile function in cardiomyocytes has drawn special attention recently, making IGF-1 a rather unique cardiac hormone compared to other growth factors [1,2,4]. Alteration in the levels of IGF-1 or the IGF-1 signaling pathway may have a significant impact on cardiac growth and function. IGF-1 deficiency or knockout results in cardiac growth retardation and neonatal death [5]. On the other hand, overexpression or exogenous administration of IGF-1 reduces myocyte apoptosis in response to stress or injury and promotes cardiac hypertrophy as well as contractile function [6–8]. The most prominent mediator of IGF-1 signaling is believed to be the phosphoinositol-3 (PI-3) kinase-dependent activation of serine-threonine protein kinase B (PKB), also known as Akt [9]. Although many other growth factors may also activate PI-3-kinase/Akt in the heart, they do not possess the ability to suppress cardiac muscle apoptosis or to enhance cardiac contraction. Activation of Akt has been shown to target on the bcl-2-family member Bad, procaspase-9, the transcription factors nuclear factor-{kappa}B and cAMP response element-binding protein, and thus considered a permissive step in the IGF-1-induced cardiac beneficial effect [9,10]. Disruption of Akt activation has been shown to abolish the antiapoptotic effects of autocrine/paracrine IGF-1 during ischemia/reperfusion injury [10]. Besides Akt, the mitogen-activated protein kinase (MAPK) family is another important signaling pathway for the biological response for IGF-1. Particularly, extracellular signal-regulated kinase (ERK) 1 and ERK 2 have been demonstrated to participate in the IGF-1-induced hypertrophic and antiapoptotic response in the hearts [11–13].

Recent evidence has indicated that the overt cardiac contractile dysfunction seen in diabetes is associated with alterations in the levels of IGF-1 and IGF-1 receptor [14–18]. Although the precise mechanism behind the interplay between diabetic cardiac dysfunction and changes in IGF-1 system is largely unknown, supplementation and overexpression of IGF-1 have convincingly antagonized the diabetes-induced cardiac dysfunction [7,8]. This cardiac protective effect of IGF-1 is consistent with the notion that IGF-1 may be used, either alone or with insulin, to improve systemic glucose tolerance and insulin resistance in diabetes. However, the precise mechanism behind the attenuated cardiac IGF-1 resistance under diabetes is largely unknown. The aim of the present investigation was to examine if this elevated extracellular glucose level itself alters IGF-1-induced cardiac contractile function, IGF-1 receptor and post-receptor signaling such as Akt and ERK1/2. We took advantage of a ‘diabetic-like’ myocyte culture system whose diabetic cardiac dysfunction may be reproduced in myocytes [19].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell isolation procedures
The experimental procedure was approved by the University of North Dakota Animal Care and Use Committee (Grand Forks, ND). Ventricular myocytes were isolated enzymatically from the hearts of adult male Sprague–Dawley rats (200–250 g; Harlan, Indianapolis, IN) by coronary perfusion and prepared for primary culture as previously described [20]. In brief, ventricular myocytes were dissociated by collagenase (176 U/ml) and hyaluronidase (0.1 mg/ml), and further digested by trypsin (0.02 mg/ml) during trituration (5 min) after the tissue was removed from the perfusion apparatus and minced. Isolated myocytes were maintained in a defined medium consisting of Medium 199 with Earle's salts containing HEPES (25 mM) and NaHCO3 (25 mM), supplemented with albumin (2 mg/ml), L-carnitine (2 mM), creatine (5 mM), taurine (5 mM), insulin (100 nM), penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin (5 µg/ml). This medium also contained either normal glucose (NG, 5.5 mM) or high glucose (HG, 25.5 mM). The high glucose is comparable to serum glucose levels in diabetic rats [21]. A subset of myocytes was maintained in normal glucose medium supplemented with 20 mM D-mannitol (to which the cell is impermeable) in order to examine if the high glucose-induced effects were due to changes in extracellular osmotic pressure. The cells were maintained at 37 °C in an incubator with 100% humidity and 5% CO2 for 12–18 h.

2.2 Cell shortening/relengthening
Mechanical properties of ventricular myocytes were assessed using an IonOptix MyoCam® system (IonOptix, Milton, MA) [20]. In brief, cells were placed in a Warner chamber mounted on the stage of an inverted microscope (Olympus, IX-70) and superfused (~1 ml/min at 30 °C) with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, at pH 7.4. Cells were field stimulated with suprathreshold voltage and at a frequency of 0.5 Hz. The myocyte being studied was displayed on the computer monitor using an IonOptix MyoCam camera. SoftEdge software (IonOptix) was used to capture changes in cell length during shortening and relengthening.

2.3 Intracellular fluorescence measurement
A separate cohort of myocytes was loaded with fura-2/AM (0.5 µM) for 10 min and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (IonOptix) as described [20]. Myocytes were placed in a chamber on an Olympus IX-70 inverted microscope (30 °C) and imaged through a Fluor 40x oil objective. Cells were exposed to light emitted by a 75 W lamp and passed through either a 360 or a 380 nm filter (bandwidths were±15 nm), while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after first illuminating cells at 360 nm for 0.5 s then at 380 nm for the duration of the recording protocol (333 Hz sampling rate). The 360 nm excitation scan was repeated at the end of the protocol and qualitative changes in intracellular Ca2+ concentration ([Ca2+]i) were inferred from the ratio of the fluorescence intensity at two wavelengths.

2.4 Western analysis of total and phosphorylated Akt and ERK1/2, IGF-1Receptor, β-actin and GLUT4
Membrane proteins from NG or HG-cultured myocytes with or without a 15-min IGF-1 (10–6 M) treatment were extracted as described [22]. Myocytes were collected, sonicated and the supernatants were centrifuged at 7000xg for 30 min at 4 °C. The pellets were cellular membrane fractions and were used for immunoblotting of Akt, phosphorylated Akt (pAkt), ERK1/2, phosphorylated ERK1/2 (p-ERK1/2), IGF-1R{alpha}, β-actin and GLUT4. We confirmed that these membrane fractions did not contain any detectable collagens. Membrane proteins (50 µg/lane) were separated on 7% (Akt and pAkt), 10% (IGF-1R{alpha}) or 15% (GLUT4, β-actin) or 20% (ERK1/2 and p-ERK1/2) SDS–polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad) and transferred to polyvinylidene difluoride membranes. The membranes were blocked (4% Block Ace, Dainippon Pharmaceutical, Osaka, Japan) and then incubated with anti-Akt (1:1000), anti-pAkt (1:1000), anti-ERK1/2 (1:500), anti-p-ERK1/2 (1:500), anti-IGF-1R{alpha} (1:1000), anti-β-actin (1:8000) and anti-GLUT4 (1:4000) antibodies. Anti-Akt and Anti-pAkt antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-ERK1/2 polyclonal and anti-p-ERK1/2 monoclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-IGF-1 receptor {alpha} polyclonal antibody was from Cell Signaling Technology (Beverly, MA). Anti-β-actin monoclonal antibody was from Novus Biologicals (Littleton, CO). GLUT4 polyclonal antibody was purchased from Chemicon International (Temecula, CA, USA). The antigens were detected by the luminescence method (ECL Western blotting detection kit, Amersham) with peroxidase-linked anti-rabbit (pAkt, ERK1/2, IGF-1R{alpha} and GLUT4) or anti-sheep (Akt), anti-mouse (p-ERK1/2 and β-actin) IgG (1:5000 dilution). After immunoblotting, the film was scanned and the intensity of immuoblot bands was detected with a Bio-Rad Calibrated Densitometer (Model GS-800).

2.5 Statistical analyses
For each experimental series, data are presented as Mean±S.E.M. Statistical significance (P<0.05) for each variable was estimated by analysis of variance (ANOVA) or t-test, where appropriate (SYSTAT, Evanston, IL, USA).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effect of IGF-1 on myocyte peak cell shortening in normal and high glucose-cultured myocytes
The average resting cell length (CL) of ventricular myocytes used in this study was 133±3 and 138±4 µm in the normal glucose and high glucose groups, respectively (38–40 cells/group). The peak shortening (PS, normalized to CL) in response to electrical stimuli in myocytes from normal glucose and high glucose medium was 5.2±0.5% and 5.7±0.6%, respectively (n=38–40 cells/group, P0.05). Consistent with previously published results using the same myocyte culturing system [23,24], myocytes maintained in high glucose medium exhibited significantly prolonged duration of relengthening (TR90: 489±37 ms, n=40) compared to cells maintained in normal glucose medium (399±23 ms, n=38, P<0.05). The maximal velocity of shortening and relengthening (±dL/dt) was not significantly different between the normal glucose group (+dL/dt=64±8 µm/s; –dL/dt = –43±6 µm/s, n=38) and the high glucose group (+dL/dt=70±7 µm/s; –dL/dt = –61±9 µm/s, n=40). Interestingly, the duration of cell shortening (TPS) was significantly shorter in myocytes maintained in high glucose medium (176±7 ms, n=40) compared to the cells maintained in normal glucose medium (210±12 ms, n=38, P<0.05). Fig. 1A shows representative traces depicting the typical effect of IGF-1 (10–7 M) on cell shortening in myocytes maintained in normal or high glucose medium. A 10-min exposure of this concentration of IGF-1 disparately affected cell shortening amplitude (PS) in myocytes from normal glucose and high glucose groups, with little effect on duration of shortening or relengthening. IGF-1 (10–10–10–6 M) caused a concentration-dependent increase or decrease in PS in the normal glucose and high glucose groups, respectively. The threshold of IGF-1 effect was between 10–9 and 10–8 M (Fig. 1B). The effect of IGF-1 on cell shortening reached maximal at about 6 min and was partially reversible upon washout (data not shown). Furthermore, IGF-1 did not affect TPS or TR90 in either group (Table 1).


Figure 1
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  		Fig. 1 (A) Typical experiments showing the effect of IGF-1 (10–7 M) on cell shortening in myocytes maintained for 12–18 h in normal glucose (5.5 mM, left) or high glucose (25.5 mM, right) medium; solid and dotted traces depict cell twitches before and 10 min after IGF-1 addition. (B) Concentration-dependent response of IGF-1 (10–10 to 10–6 M) on cell shortening in ventricular myocytes from normal glucose or high glucose medium. Data are presented as percent change from the respective baseline (0 IGF-1) value. The number of myocytes is given in parentheses. Mean±S.E.M. *P<0.05 vs. baseline.

 

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  		Table 1 Effect of IGF-1 on duration of myocyte shortening (TPS) and relengthening (TR90) in ventricular myocytes maintained in normal and high glucose medium

 
3.2 Effect of IGF-1 on myocyte shortening in the presence of H-1356 or high D-mannitol
To determine the role of IGF-1 receptor in IGF-1-induced cardiac contractile response, myocyte shortening in response to IGF-1 was re-examined in the presence of IGF-1 analogue, H-1356 (Bachem Bioscience, King of Prussia, PA). This peptide inhibits the autophosphorylation of the IGF-1 receptor by IGF-1, thereby decreasing its activity [25]. To block the action of IGF-1, H-1356 (20 µg/ml) was pre-incubated with the cardiomyocytes for 4 h and was presented throughout the experimental protocol. Not surprisingly, the IGF-1 (10–7 and 10–6 M)-induced positive/negative contractile response in cell shortening in normal and high glucose groups was completely blunted by H-1356 (Fig. 2). These results indicate that the IGF-1 receptor is likely to mediate the IGF-1-induced disparate response in both normal and high glucose environments. To determine whether the high glucose-induced reduction in the contractile response to IGF-1 was due to changes in extracellular osmotic pressure, we examined the cardiac contractile response to IGF-1 in myocytes cultured in normal glucose supplemented with 20 mM D-mannitol for 12–18 h. Mannitol itself had no effect on the mechanical indices examined (data not shown), consistent with our earlier observation [24]. Similar to its action in normal glucose-cultured myocytes, IGF-1 (10–7 and 10–6 M) elicited a positive contractile response in myocytes cultured with high mannitol, suggesting that the high glucose-induced cardiac IGF-1 resistance is unlikely due to a change in extracellular osmotic pressure.


Figure 2
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  		Fig. 2 (A) Effect of the IGF-1 receptor antagonist H-1356 (20 µg/ml) or high D-mannitol (20 mM) on IGF-1 (10–7 and 10–6 M)-induced cell contractile response in myocytes maintained in normal glucose medium. (B) Effect of the IGF-1 receptor antagonist H-1356 (20 µg/ml) on IGF-1 (10–7 and 10–6 M)-induced cell contractile response in myocytes maintained in high glucose medium. Data are presented as percent change from the respective baseline (0 IGF-1) value. The number of cells is given in parentheses. Mean±S.E.M. *P<0.05 vs. baseline.

 
3.3 Effect of IGF-1 on intracellular Ca2+ ([Ca2+]i) transients
To determine whether the differential response of IGF-1 in normal glucose and high glucose cultured cardiac myocytes was due to changes in intracellular Ca2+ concentration, we used the fluorescent dye fura-2 to estimate intracellular Ca2+ transient properties in myocytes from both groups. The difference between the peak and resting intracellular Ca2+ level was used to estimate the electrically stimulated Ca2+-induced Ca2+ release (CICR). The time constant of fluorescence signal decay ({tau}) was calculated to assess the intracellular Ca2+ clearing rate. Myocytes from both normal and high glucose groups exhibited similar clearing rates and resting intracellular Ca2+ levels. Acute IGF-1 exposure (10–10–10–6 M) caused a concentration-dependent increase of CICR in myocytes maintained in normal glucose medium whereas elicited a negative response in cells maintained in high glucose medium (Fig. 3). The threshold of IGF-1-induced response was between 10–9 and 10–8 M in the normal glucose group and between 10–10 and 10–9 M in the high glucose group. The intracellular Ca2+ response to IGF-1 achieved steady state at 6 min and recovered partially following washout. Neither resting intracellular Ca2+ levels nor {tau} were affected by IGF-1 (Table 2).


Figure 3
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  		Fig. 3 Effect of IGF-1 on electrically stimulated Ca2+-induced Ca2+ release in ventricular myocytes from normal glucose or high glucose cultured myocytes. (A) Typical traces showing the effect of IGF-1 (10–7 M) on Ca2+-induced Ca2+ release before and 10 min after IGF-1 addition in myocytes maintained in normal or high glucose medium. (B) Concentration-dependent response of IGF-1 (10–10–10–6 M) on Ca2+-induced Ca2+ release. Mean±S.E.M., *P<0.05 vs. baseline (0 IGF-1). Cell number is given in parentheses.

 

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  		Table 2 Effect of IGF-1 on resting intracellular Ca2+ transient level (360/380 ratio) and fluorescence decay time ({tau}) in ventricular myocytes maintained in normal and high glucose medium

 
3.4 Western blotting of IGF-1 receptor, Akt, ERK1/2 and GLUT4
It has been demonstrated that the IGF-1-induced cardiac response is mediated through its membrane receptor and post-receptor signaling pathways such as PI-3 kinase, the serine-threonine kinase Akt and ERK1/2 [9,11–13,20]. It has also been reported that the diabetes-associated defects may be due to alteration of membrane glucose transport protein GLUT4 [26,27]. To examine the role of membrane IGF-1 receptor and post-receptor signaling including Akt, ERK1/2 and GLUT4 in the disparate IGF-1 response under normal and high glucose conditions, the protein levels of IGF-1 receptor (both IGF-1R{alpha} and pro-IGF-1R), Akt and ERK1/2 (both total and phosphorylated), and GLUT4 were measured in myocytes maintained under normal or high glucose environment. Our immunostaining analysis revealed that while pro-IGF-1R was similar between the two groups, the levels of IGF-1R{alpha} were significantly reduced in high glucose cells compared to the normal glucose group (Fig. 4). Furthermore, total Akt protein levels may be upregulated by a 15-min IGF-1 (10–6 M) exposure in both normal glucose and high glucose cultured myocytes. Not surprisingly, IGF-1 stimulated the activation of Akt shown as enhanced phosphorylated Akt (pAkt) in normal glucose-cultured myocytes. However, the IGF-1-stimulated activation of Akt was blunted by the high glucose myocytes. The pAkt/total Akt ratio was significantly less in high glucose myocytes compared to normal glucose myocytes (Fig. 5), suggesting reduced IGF-1-induced Akt activation under high glucose condition. The total ERK1/2 levels were not different between the two groups, and were not affected by IGF-1 incubation. IGF-1 treatment elicited a comparable degree of ERK1/2 activation in both normal glucose and high glucose myocytes (Fig. 6). Finally, total GLUT4 protein levels were unchanged between the two groups and were not altered by the short-duration of IGF-1 treatment (Fig. 7). In each set of experiments, an equal amount of protein was used for gel blotting analysis, as evidenced by β-actin abundance.


Figure 4
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  		Fig. 4 Effect of high glucose culture on IGF-1 receptor expression. Inset, representative gels depicting immunostaining using anti-IGF-1R antibodies in ventricular myocytes maintained in normal glucose or high glucose medium. Bar graph shows summarized data from five isolations each. Mean±S.E.M., *P<0.05 vs. normal glucose group.

 

Figure 5
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  		Fig. 5 (A) Representative gels depicting immunostaining using anti-Akt and anti-pAkt antibodies. (B–D) Western blot of total non-phosphorylated Akt (B), phosphorylated Akt (C) and ratio of pAkt/Akt (D) in ventricular myocytes maintained in normal glucose or high glucose medium, with or without a 15-min IGF-1 (10–7 M) stimulation. Mean±S.E.M., n=6 cell isolations, *P<0.05 vs. baseline (0 IGF-1), #P<0.05 vs. respective normal glucose group.

 

Figure 6
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  		Fig. 6 (A) Representative gels depicting immunostaining using anti-ERK1/2 and anti-p-ERK1/2 antibodies. (B–D) Western blot of total non-phosphorylated ERK1/2 (B), phosphorylated ERK1/2, or p-ERK1/2 (C) and ratio of p-ERK1/2:ERK1/2 (D) in ventricular myocytes maintained in normal glucose or high glucose medium, with or without a 15-min IGF-1 (10–7 M) stimulation. Mean±S.E.M., n=4 cell isolations, *P<0.05 vs. baseline (0 IGF-1).

 

Figure 7
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  		Fig. 7 Western blot analysis of GLUT4 protein levels in ventricular myocytes maintained in normal glucose or high glucose medium, with or without a 15-min IGF-1 (10–7 M) treatment. Inset, representative gels depicting immunostaining using anti-GLUT4 antibody. Mean±S.E.M., n=4.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
IGF-1 promotes cardiac growth and contractility, facilitates glucose metabolism, increases insulin sensitivity, and improves lipid profile, suggesting its physiological role and therapeutic potential [1]. However, IGF-1 and its binding proteins may be considered as markers for the presence of certain cardiac abnormalities since altered levels and function of the IGF-1 system have been reported in several cardiovascular co-morbidities such as diabetes and hypertension [16,28]. Data from the present study suggest that elevated glucose, a hallmark in all forms of diabetes, may directly contribute to the altered cardiac contractile function of IGF-1 in individual ventricular myocytes. Furthermore, our data provide evidence, for the first time, that depressed cardiac mechanical responsiveness to IGF-1 under elevated glucose environment is associated with reduced IGF-1 receptor abundance and diminished activation of Akt but not that of ERK1/2 in response to IGF-1 stimulation.

IGF-1 has been shown to enhance myocardial contractility [13]. Several mechanisms have been postulated for this including IGF-1-induced increase in intracellular Ca2+ concentration [29] and intracellular Ca2+ sensitivity [20,30]. The cardiac response of IGF-1 is featured by rapid onset, long lasting and modest magnitude compared with other endogenous substances, suggesting its role as an ‘intrinsic’ regulator of myocardial contractility. In the present study, the IGF-1-induced response in myocyte shortening was paralleled by a similar pattern of response in intracellular Ca2+ transients under either normal or high glucose cultured myocytes. These results indicate that the disparate response to IGF-1 in cell shortening under normal or high glucose conditions is likely due to a similar pattern of response in intracellular Ca2+ level. Our result does not favor the disparate IGF-1 contractile response under normal or high glucose to be related to any acute effect on the glucose transporter GLUT4 or ERK1/2. The fact that IGF-1-induced cardiac contractile effect was blunted by the IGF-1 receptor antagonist H-1356 suggests a selective IGF-1 receptor-mediated mechanism. Our earlier work showed that H-1356 itself depresses cardiac contraction [20], suggesting a role for endogenous IGF-1 in the maintenance of myocardial contractile function. Our Western blot analysis clearly revealed reduced IGF-1R{alpha} abundance (although not the pro-IGF-1R fraction) in high glucose myocytes, indicating potential contribution of IGF-1R in high glucose-induced alteration in cardiac contractile response to IGF-1.

The high glucose-induced attenuation of cardiac contractile response to IGF-1 is consistent with the data reported in streptozotocin-induced diabetes [16]. The reduced response of PS and {Delta}FFI to IGF-1 in HG myocytes may implicate that the elevated glucose level is likely to be responsible for the altered IGF-1 system in diabetes. Although the precise mechanism underscoring the loss of IGF-1 action under a high glucose environment is still not clear, several speculations may be made at this time besides the possible contribution from reduced IGF-1R due to high glucose mentioned above. The cardiac regulating property of IGF-1 is likely mediated through post-IGF-1 receptor activation of PI-3 kinase, which produces phosphoinositides acting on a number of downstream targets, including the Akt proto-oncogene, a serine-threonine protein kinase [31]. Activation of Akt promotes survival of several cell types, including cardiac myocytes, neurons and lymphocytes [31,32], and plays a central role in anti-apoptosis by modulating the Bcl-2 family proteins, caspase 9, and Fas ligand [33,34]. This PI-3-Akt signaling mechanism is believed to be responsible for IGF-1-induced preservation of cardiac morphology and function. The involvement of PI-3 kinase signaling in IGF-1-induced cardiac contractile response was confirmed by our earlier finding that the PI-3 kinase inhibitor wortmannin blunted IGF-1-induced cardiac response [20]. The results of the present study suggest a possible involvement of Akt signaling in the mediation of the cardiac contractile action elicited by IGF-1. However, linkage of PI-3 kinase and reduction of Akt activation to the altered cardiac contractile response to IGF-1 under high glucose environment is still not clear. Direct evidence is not yet available for cardiac contractile response of Akt in ventricular myocytes. Studies from two independent groups have provided compelling evidence for the functional role of Akt. Enhanced myocardial contraction in conjunction with increased Ca2+ release from ryanodine-receptor Ca2+-release channels, Ca2+ sparks and electrically stimulated Ca2+ transient was reported to be paralleled with an augmented PI-3 kinase-dependent phosphorylation of Akt [35]. In vivo gene transfer of constitutively active Akt mutant in a rat model of cardiac ischemia–reperfusion injury has led to dramatically improved cardiac function [36]. Further study revealed that this positive cardiac contractile effect does not simply reflect infarct reduction but is rather due to direct functional benefit of Akt. In contrast, dominant negative Akt, which blocks Akt activation, accelerated hypoxia-induced cardiomyocyte dysfunction and death [36]. The potential cardiac contractile effect of Akt may also be evidenced by the cardiac contractile response induced by PI-3 kinase and its downstream signaling phospholipase C [35,37]. The antiapoptotic signals induced by IGF-1 have been shown to be mediated independently by either the PI-3 kinase/Akt or the MAPK pathways in cardiomyocytes, since pharmacological inhibition of either pathway antagonizes the antiapoptotic effect of IGF-1 in an additive manner [11]. However, our present study did not observe any difference in the IGF-1-stimulated phosphorylation of ERK1/2, the most significant members of the MAPK family involved in IGF-1-mediated cell growth and survival [11–13], between the normal and high glucose myocytes. This result did not favor any involvement of ERK1/2 in high glucose-induced alteration of cardiac response to IGF-1. ERK1/2 has been speculated to be stimulated by IGF-1 in a manner independent of PI-3 kinase/Akt pathway. The ERK1/2 activation in response to IGF-1 has been reported to differ from that of Akt in the time course of peaking and dependence on PI-3 kinase [11]. Considering the complexity of the IGF-1 signaling cascade, future research is needed to clarify the role of the Akt and possibly MAPK or ERK1/2 pathways in the regulation of cardiac contractile function under normal and diabetic conditions. It may be speculated that Akt may represent an important control point determining not only cardiomyocyte survival but function as well. This should provide invaluable information regarding new drug development for heart diseases.

Time for primary review 22 days.


   Acknowledgements
 
This work was supported in part by the American Diabetes Association and the American Heart Association–Northland Affiliate. IGF-1 was obtained from National Hormone and Peptide Program, National Institutes of Diabetes and Digestive Kidney Diseases (NIDDK) and Dr. Parlow. The authors acknowledge the generous help from Miss Glenda I. Scott and Dr. Xiaochun Zhang.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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