INTRODUCTION

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OBESITY IS COMMONLY ASSOCIATED with increased cardiac output, elevated intravascular volume, cardiac hypertrophy, and hypertension and is considered as an important cardiovascular risk factor (17, 21). Obesity is often accompanied with hypertension and, when long standing, is associated with left ventricular dysfunction. The reduced ventricular function thus leads to impairment of ejection fraction, fractional shortening, and diastolic compliance (1, 2). Recently, growing efforts have been made to extend the knowledge and efficacious therapeutic remedy of obesity. Several mechanisms, including insulin resistance, salt sensitivity, and sympathetic activation, have been implicated in obesity-related metabolic and cardiovascular disorders (16, 21).

One prominent metabolic feature in obesity is insulin resistance in skeletal and cardiac muscles. Resistance to insulin-stimulated glucose uptake results in hyperglycemia, predisposing the obese subjects to type II diabetes and other cardiovascular complications (21). Evidence has suggested that tissue resistance to the effects of insulin or insulin-like growth factor-I (IGF-I) is a factor linking various metabolic disorders and heart disease (11). IGF-I is synthesized primarily in the liver under the control of growth hormone and mediates most of the biological effects of growth hormone (7). The secretion of growth hormone, however, for undefined reasons, is greatly reduced in obese subjects (15). It has been suggested that such reduction may be due to a negative feedback mechanism of higher serum IGF-I levels, which has been demonstrated in obese subjects (3, 9, 15). Excessive amounts of IGF-I may directly affect cardiac contractile function, growth, and anti-apoptosis (20), which may be related to IGF-I resistance due to prolonged exposure. We recently reported resistance to IGF-I-induced myocardial contractile response in hypertension, a condition that often accompanies obesity (12, 13).

The aim of the present study was to examine the cardiac contractile response of IGF-I in ventricular myocytes from Zucker genetic obese (fa/fa) rats and their lean (fa/?) controls. The Zucker fa/fa rats bear a mutation in the obese gene (ob) product leptin receptor gene (5) and is a well-characterized model of insulin resistance associated with extreme obesity, hyperinsulinemia, and impaired glucose tolerance. We monitored the myocyte shortening by video edge detection, intracellular Ca²⁺ by fura 2 ratiometry, and IGF-I receptor mRNA by ribonuclease protection assay (RPA).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experimental animals. The experimental procedures outlined in this investigation were approved by animal investigation committees from Wayne State University and University of North Dakota. Age-matched female Zucker obese (fa/fa) and lean (fa/?) rats were obtained at 8 wk of age (Harlan Sprague Dawley, Indianapolis, IN) and individually housed in a temperature-controlled room under a 12:12-h light-dark cycle. The rats were allowed access to standard rat chow and tap water ad libitum. The Zucker rat is a well-established obesity model. Although these obese rats do not exhibit hyperglycemia, they develop left ventricular hypertrophy in response to elevation in arterial pressure and total peripheral resistance. Systolic blood pressures, body weights, and plasma glucose levels were measured on a weekly basis by the tail cuff, a balance, and a Yellow Springs Instruments glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH). The animals were killed at 14 wk of age.

Cell isolation procedures. Single ventricular myocytes were isolated as described previously (14). Briefly, animals were killed, and the hearts were rapidly removed and perfused (at 37°C) with oxygenated (5% CO₂-95% O₂) Krebs-Henseleit bicarbonate (KHB) buffer (in mM: 118 NaCl, 4.7 KCl, 1.25 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 10 HEPES, and 11.1 glucose, pH 7.4). Hearts were subsequently perfused with a nominally Ca²⁺-free KHB buffer for 2-3 min until spontaneous contractions ceased followed by a 20-min perfusion with Ca²⁺-free KHB containing 223 U/ml collagenase (Worthington Biochemical, Freehold, NJ) and 0.1 mg/ml hyaluronidase (Sigma Chemical, St. Louis, MO). After perfusion, the left ventricle was removed and minced, under sterile conditions, and incubated with the above enzymatic solution for 3-5 min. The cells were further digested with 0.02 mg/ml trypsin (Sigma) before being filtered through a nylon mesh (300 µm) and subsequently separated from the enzymatic solution by centrifugation (60 g for 30 s). Myocytes were resuspended in a sterile filtered Ca²⁺-free KHB buffer containing (mM) 131 NaCl, 4 KCl, 1 MgCl₂, 10 HEPES, and 10 glucose, supplemented with 2% BSA with a pH of 7.4 at 37°C. Cells were initially washed with Ca²⁺-free KHB buffer to remove remnant enzyme, and extracellular Ca²⁺ was added back incrementally to 1.25 mM. Isolated myocytes were maintained for 12-24 h in a serum-free medium consisting of medium 199 (Sigma). Mechanical properties remained relatively stable in myocytes maintained for 12-24 h in the serum-free medium. Cells were not used if they had any obvious sarcolemmal blebs or spontaneous contractions.

Cell shortening/relengthening measurements. Mechanical properties of ventricular myocytes were assessed using a video-based edge-detection system (Crescent Electronics, Sandy, UT) as described (14). In brief, coverslips with cells attached were placed in a chamber mounted on the stage of an inverted microscope (Olympus X-70) and superfused (~2 ml/min at 37°C) with a buffer containing (in mM) 131 NaCl, 4 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES, at pH 7.4. The cells were field stimulated at frequency of 0.5 Hz, 3-ms in duration. A video-based edge-detector was used to capture and convert changes in cell length (CL) during shortening and relengthening into an analog voltage signal (IonOptix Corp, Milton, MA). The myocyte being studied was displayed on a Sony monitor using a Pulnix camera, which rapidly scans the image area at 120 Hz to ensure good fidelity of signal. Cell shortening and relengthening were assessed using the following indexes: peak twitch amplitude (PS), time-to-90% PS (TPS), time-to-90% relengthening (TR₉₀), and maximal velocities of shortening (+dL/dt) and relengthening (dL/dt), respectively.

Intracellular Ca²⁺ transient measurement. For these experiments, myocytes were loaded with fura 2-AM (0.5 µM) for 10 min at 30°C, and fluorescence measurements were recorded with a dual-excitation single-emission fluorescence photomultiplier system (IonOptix). Myocytes were placed on an inverted microscope and imaged through an Olympus Fluor ×40 oil objective. Myocytes were exposed to light emitted by a 75-W halogen lamp through either a 360- or 380-nm filter while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after initial illumination at 360 nm for 0.5 s and then at 380 nm for the duration of the recording protocol. The 360-nm excitation scan was repeated at the end of the protocol and qualitative changes in intracellular Ca2+ concentration ([Ca²⁺]_i) was inferred from the ratio of the fura-fluorescence intensity (FFI) at both wavelengths. Fluorescence decay time (FDT) was also measured as an indication of the intracellular Ca²⁺ clearing rate (14).

IGF-I receptor mRNA RPA. The RNA probe construction for IGF-I receptor has been described previously (23). A 265-bp EcoR I-Rsa I fragment was isolated from one of the rat IGF-I receptor cDNA clones and subcloned into the plasmid vector pGEM-3 that had been digested with EcoR I and Sma I. The resulting construct was linearized with EcoR I, gel-purified, and used to generate a ³²P-labeled rat IGF-I receptor antisense RNA by using SP6 RNA polymerase and [-³²P]UTP (Amersham). The antisense transcript was consisted of 40 bases of vector sequence and 265 bases complementary to 15 bases of 5'-untranslated sequence as well as to the region encoding the signal peptide and the first 53 amino acids of the a subunit. The IGF-I receptor mRNA RPA assay was based on protocol from Ambion (Austin, TX). In brief, 20 µg of total RNA extracted from the left ventricle was coprecipitated with 1 ng of IGF-I receptor RNA probe and 1 ng of human cyclophilin RNA probe in a solution containing LiCl (142 mM) and ethanol (75%). The RNA/probe pellets were resuspended in 30 µl hybridization solution [75% of formamide, 40 mM of piperazine-N,N'-bis(2-ethanesulfonic acid), 400 mM NaCl, and 1 mM EDTA, pH 8.0], followed by hybridization at 60°C overnight. Ribonuclease T1 (1,400 U/ml) in T1 buffer solution (in mM: 300 NaCl, 10 Tris, pH 7.5, and 5 EDTA, pH 8.0) was used to digest single-strand RNA. The protected double-stranded RNAs were precipitated in ethanol, resuspended in 8 µl of loading buffer, and subject to electrophoresis through 6% acrylamide gel containing 8 M urea. The hybridized RNAs were transferred to a positively charged nylon membrane, followed by cross-linking under ultraviolet light. The signal of biotin-labeled RNA was detected using CDP-Star (1 µl streptavidin-alkaline phosphatase conjugate) (Ambion). The signals were visualized by exposure to a film and quantified by scanning laser densitometry. The ratio of IGF-I receptor intensity to cyclophilin intensity was used to correct for differential loading.

Data analysis. Data are presented as means ± SE. Differences between and within groups were evaluated by two-way ANOVA with repeated measures (SYSTAT). A Tukey test was used as a follow-up for the multiple comparisons. To determine significant differences in the repeated measures (concentrations of IGF-I), the "within subjects" MSerror and dferror terms from the parent ANOVA were used. To determine significant differences between strains at a given concentration of IGF-I, the "between subjects" MSerror and dferror terms from the parent ANOVA were used. Statistical significance was considered to be P < 0.05. On average, four to eight cells were studied per rat heart for each given data point.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

General features of lean and obese Zucker rats. After 14 wk of genetic obesity, we found the body weight of the obese Zucker rats were significantly heavier than their age-matched lean counterparts. Obesity was also associated with overt elevation of systolic pressure and enlargement of heart, liver, and kidneys, although the relative size of heart normalized to body weight was reduced due to an increased body weight. No hyperglycemia was seen in obese Zucker rats compared with their lean controls (Table 1).

Baseline mechanical and fluorescent properties of lean and obese myocytes. As expected, obesity leads to cardiac hypertrophy. The average resting CL of ventricular myocytes isolated from lean and obese Zucker rat hearts was 101 ± 3 and 134 ± 4 µm, respectively (P < 0.05, 64 cells/group). The peak shortening (PS) was significantly depressed in obese myocytes compared with lean ones (5.0 ± 0.4 vs. 6.1 ± 0.3% CL, respectively, P < 0.05). The maximal velocities of shortening and relengthening (±dL/dt) were comparable in myocytes from both groups (Table 2). However, obese myocytes exhibited a prolonged duration of TR₉₀ compared with their lean counterparts, although the TPS was comparable.

Effect of insulin and IGF-I on myocyte PS. Acute IGF-I and insulin exposure did not affect resting myocyte CL over the range of concentrations tested. Representative traces depicting the typical effect of IGF-I (100 ng/ml) on lean or obese myocyte shortening are shown in Fig. 1. At the end of a 5-min exposure to this concentration of IGF-I, PS was increased by ~15% in the lean group, whereas no effect was observed in the obese group. IGF-I exhibited little effect on TPS or TR₉₀. Both IGF-I (1-500 ng/ml) and insulin (1-500 nM) caused a concentration-dependent enhancement of PS in lean myocytes, with maximal increases of 29.0 and 12.7%, respectively. Interestingly, the positive response on cell shortening was blunted by obesity and reversed to inhibition for both IGF-I and insulin in myocytes isolated from obese rat heart. The concentrations at which IGF-I and insulin elicited EC₅₀ were 13.9 ng/ml and 15.7 nM, respectively, in lean myocytes (Figs. 1 and 2). The stimulatory effects of IGF-I and insulin on cell shortening were maximal within 4 min of exposure and were partially reversible on washout. Finally, IGF-I exerted little action on the duration of either TPS or TR₉₀ (Table 2). Similarly, insulin did not affect TPS or TR₉₀ (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   A and B: typical experiments showing the effect of insulin-like growth factor-I (IGF-I; 100 ng/ml) on myocyte peak shortening (PS) in a lean (A) and an obese (B) myocyte. Myocyte shortening and relengthening (twitch) were recorded with a high-resolution (120 Hz) video edge-detection system at 37°C before and 5 min after IGF-I administration. C: concentration-dependent response of IGF-I (1-500 ng/ml) on PS in both lean and obese myocytes. PS is expressed as the percent change of respective baseline value. Means ± SE, * P < 0.05 vs. baseline value, # P < 0.05 vs. lean.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent response of insulin (1-500 nM) on PS in both lean and obese myocytes. PS is expressed as the percent change of respective baseline value. Means ± SE, * P < 0.05 vs. baseline value, # P < 0.05 vs. lean.

Effect of insulin and IGF-I on intracellular Ca²⁺ transients. To determine whether IGF-I and insulin-induced increase of myocyte shortening was due to enhanced availability of [Ca²⁺]_i, we used fura 2 to estimate changes in [Ca²⁺]_i in response to both hormones. The time course of FDT was evaluated to assess the rate of intracellular Ca²⁺ clearing. Results from the fluorescent measurements indicated that obese myocytes possessed a slight decrease, although not significantly, in resting intracellular Ca²⁺ levels (lean 0.764 ± 0.008 vs. obese 0.751 ± 0.012) and prolonged FDT (lean 564 ± 44 vs. obese 887 ± 60 ms) compared with the lean group. Representative traces of intracellular Ca²⁺ transients shown in Fig. 3, B and C, depict that 100 ng/ml IGF-I increased FFI by ~11% in lean myocytes, whereas it had no effects in obese myocytes. Both IGF-I and insulin elicited concentration-dependent increase of FFI in both lean and obese myocytes (Fig. 3). The stimulation of FFI suggests that an increase in intracellular free Ca²⁺ is likely to be responsible for IGF-I and insulin-induced positive actions on myocyte shortening. Neither resting FFI (representing resting Ca²⁺ level) nor FDT was affected by IGF-I or insulin (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent response of IGF-I (A, 1-500 ng/ml) and insulin (D, 1-500 nM) on intracellular Ca2+ transient changes (FFI) in myocytes from lean and obese rat hearts. B and C: typical experiments showing the effect of IGF-I (100 ng/ml) on intracellular Ca2+ transients in a lean (B) and an obese (C) myocyte. Solid and dashed traces show Ca2+ transients recorded from fura 2-loaded myocytes before and 5 min after IGF-I exposure. FFI is expressed as the percent change of respective baseline value. Means ± SE, * P < 0.05 vs. baseline value, # P < 0.05 vs. lean.

Quantitation of IGF-I receptor mRNA levels in Zucker lean and obese rat hearts. To explore the potential mechanism(s) of action related to the disparate response of IGF-I in lean and obese heart, IGF-I receptor mRNA was measured using RPA. Figure 4 shows that the level of IGF-I receptor mRNA was significantly reduced in hearts from obese rats, indicating that depressed IGF-I-related cardiac action may be associated with diminished IGF-I receptor level. Additionally, the cardiac IGF-I mRNA levels were indifferent between the Zucker lean and obese rats.

View larger version (47K): [in this window] [in a new window]   Fig. 4.   Ribonuclease protection assay (RPA) quantitation of IGF-I receptor mRNA levels in Zucker lean and obese rat hearts. Top: representative autoradiography showing RPA using IGF-I receptor and IGF-I RNA probes. The size of the IGF-I receptor mRNA-protected band is 265 bp. The IGF-I RNA probe gave 2 protected bands, corresponding to IGF-Ia mRNA (224 bp) and IGF-Ib mRNA (376 bp). Only the 224-base protected band, which constitutes >90% of the total IGF-I mRNA, is shown. Bottom: summary of the RPA quantitation of IGF-I receptor mRNA from 12 experiments. * P < 0.05 vs. lean.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, we characterized altered contractile functions manifested as reduced PS, prolonged TR₉₀, and slowed intracellular Ca²⁺ clearing rate in left ventricular myocytes from Zucker obese rats compared with age-matched lean controls. Alteration of cardiac contractile functions has been reported in pressure-overload conditions of hypertrophy, including spontaneous hypertension (12, 13) and obesity (1, 2). The findings are somewhat consistent between the two hypertrophic models in the prolongation of relaxation and depression of cardiac contractility. Therefore, the obesity-induced cardiac dysfunction may be deduced, at least in part, from the presence of hypertension in these obese Zucker rats. However, caution must be taken because obesity is also associated with other vascular or metabolic abnormalities.

Compromised cardiac systolic function has been reported in Zucker obese rats (19), supporting our observation of reduced PS in myocytes from obese rats. Decrease in adrenergic responsiveness, including altered receptor density and/or postreceptor signaling mechanism, has been speculated to play a role in the obesity-related contractile dysfunction (4, 22). Our data indicated prolongation in myocyte relengthening in obesity, which was not associated with alterations of TPS and ±dL/dt. Decreased diastolic compliance was observed in the obese rabbit heart (2). This prolongation may be related to ventricular hypertrophy-induced reduction of sarcoplasmic Ca²⁺ uptake (2). In addition, the fura 2 recording exhibited a slowed rate of intracellular Ca²⁺ removal in obese myocytes, corresponding to prolonged myocardial relaxation. However, whether obesity directly impairs the intracellular clearing mechanisms such as sarcoplasmic Ca²⁺- ATPase and Na/Ca exchanger remains to be explored.

Most importantly, this study demonstrated, for the first time, resistance to IGF-I-induced cardiac contractile response in obesity. Growth hormone-IGF-I axis has been one of the center issues in the knowledge and therapy of obesity. As we mentioned earlier, growth hormone secretion is greatly reduced in obesity and can progressively increase with reduction of body weight. Growth hormone is mainly controlled by the hypothalamus through growth hormone-releasing hormone (stimulatory) and somatostatin (inhibitory), which is driven by pituitary hormone. This loop of growth hormone function is modulated by a number of central (such as neuropeptides) and peripheral factors (such as IGF-I). In obesity, it is believed that free IGF-I levels are elevated and then provide the negative feedback inhibition of growth hormone release. Reduction of IGF binding proteins has been reported to be responsible for the increase of free IGF-I available (15). Little up-to-date information is available regarding the relationship between IGF-I receptor levels and obesity. This study observed a reduced IGF-I receptor mRNA level in obese rat heart, which is in accordance with the attenuated IGF-I response in obesity. The mechanism of the reduction in IGF-I receptor mRNA is not clear but may be associated with high circulating IGF-I-induced receptor downregulation.

In the present study, IGF-I exerted similar action to that of insulin in both lean and obese groups. Several studies have shown that IGF-I reduces blood glucose and serum insulin in patients with insulin resistance (6). Although the mechanisms of action of IGF-I under insulin resistance are unclear, it seems that IGF-I acts through mechanisms similar to, but distinct from, those of insulin itself, possibly exclusively through IGF-I receptor. This is supported by the observation of normal IGF-I response in patients with mutation in the insulin-receptor gene or postreceptor defects (10, 18).

In conclusion, the present study demonstrated, for the first time, resistance to IGF-I-induced cardiac contractile response in insulin-resistant Zucker obese rats at the cellular level. Although these data provide some interesting information regarding the pathogenesis of obesity-related cardiac contractile dysfunction, the underneath cellular mechanisms remain to be determined. In addition, whether severe insulin resistance may cause or predispose acquired IGF-I resistance through alteration of IGF-I bioavailability or postreceptor signaling, also warrants further investigation.