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Am J Physiol Heart Circ Physiol 281: H1137-H1147, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 3, H1137-H1147, September 2001

Altered SR protein expression associated with contractile dysfunction in diabetic rat hearts

Yan Zhong, Saadia Ahmed, Ingrid L. Grupp, and Mohammed A. Matlib

Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The goal of this study was to examine whether alteration of sarcoplasmic reticulum (SR) protein levels is associated with early-onset diastolic and late-onset systolic dysfunction in streptozotocin (STZ)-induced diabetic rat hearts. Four-week diabetic rat hearts exhibited slow relaxation, whereas 6-wk diabetic rat hearts exhibited slow and depressed contraction. Total phospholamban level was increased, and phosphorylated level was decreased in 4- and 6-wk diabetic rat hearts. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) protein level was unchanged in 4-wk but decreased in 6-wk diabetic rat hearts. Only the apparent affinity of SR Ca2+ uptake for Ca2+ was decreased in 4-wk diabetic rat hearts, but the apparent affinity and the maximum rate was decreased in 6-wk diabetic rat hearts. Insulin treatment of the diabetic rats normalized SR protein expression and function. It was concluded that an increase in nonphosphorylated phospholamban and a decrease in the apparent affinity of SR Ca2+ pump for Ca2+ are associated with early-onset diastolic dysfunction and decreases in SERCA2 protein level and apparent affinity and maximum velocity of SR Ca2+ pump are associated with late-onset systolic dysfunction in diabetic rats.

phospholamban; Ca2+-ATPase; ryanodine receptor; calsequestrin; diabetic cardiomyopathy; sarcoplasmic reticulum


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HEART FAILURE is the leading cause of death in diabetic patients (29). Cardiomyopathy has been shown to be an important contributing factor of heart failure in diabetic patients independent of atherosclerosis, hypertension, and other complications (26). Cardiomyopathy in diabetic patients is characterized by early diastolic dysfunction, followed by late systolic dysfunction (5). However, the mechanisms underlying the sequential development of cardiac contractile dysfunction and a rational treatment of the disease remain unknown. Cardiac contractile dysfunction has been observed in streptozotocin (STZ)-induced diabetic rats after 6-8 wk of diabetes (5, 7, 24, 31), but the underlying mechanism remains unclear. Furthermore, no study has been conducted in any animal model of diabetes to determine whether the relaxation process is altered at an early stage of diabetes before the contraction process is affected. To examine potential mechanisms underlying the development of cardiomyopathy in diabetes, it is necessary to identify an animal model of diabetes that mimics the sequential development of cardiomyopathy in diabetic patients. Because STZ-induced diabetic rats have been found to develop cardiac contractile dysfunction after a range of duration of diabetes (5, 7, 24, 31), it is logical to examine this animal model to determine whether a slow relaxation develops before slow or depressed contraction. If a sequential development of contractile dysfunction in this animal model of diabetes is established and the underlying mechanisms are understood, the information can be valuable to understand the mechanism of cardiomyopathy in diabetic patients. Therefore, one of the objectives of this study is to examine the hypothesis that cardiac diastolic dysfunction develops before systolic dysfunction in STZ-induced diabetic rats.

Sarcoplasmic reticulum (SR) is one of the critical elements in cardiac contractility. It is the major regulator of cytosolic free Ca2+ concentration ([Ca2+]c) in beat-to-beat contraction and relaxation of cardiac myocytes (18). The ryanodine receptor (RyR)-linked Ca2+ release from sarco(endo)plasmic reticulum contributes about 90% of the free Ca2+ for contraction, and the SR Ca2+-ATPase or Ca2+ pump (SERCA2) sequesters this fraction of Ca2+ during relaxation of rat cardiac myocytes (2). Phospholamban (PLB) is a key regulator of SERCA2 function (12). In its nonphosphorylated form, PLB inhibits SERCA2 function by decreasing its affinity for Ca2+, whereas phosphorylation of PLB enhances SERCA2 function by increasing its affinity for Ca2+. Transgenic ablation of cardiac PLB has been found to increase the affinity of SERCA2 for Ca2+, resulting in a increased rate of Ca2+ sequestration into SR and increased rates of contraction and relaxation (17). On the other hand, transgenic overexpression of cardiac PLB in mice has been shown to decrease the affinity of SERCA2 for Ca2+, resulting in depression of SERCA2 function and contraction and relaxation (11). The importance of SERCA2-PLB interaction in cardiomyopathy and heart failure is reinforced by a recent report (21) demonstrating complete recovery of structure and function with ablation of PLB or disruption of SERCA2-PLB interaction in dilated cardiomyopathy induced by transgenic ablation of muscle-specific LIM protein in mice. These studies demonstrated the importance of SERCA2 and PLB levels in the regulation of SERCA2 function, intracellular Ca2+ cycling, and contraction and relaxation of the heart. Thus it is conceivable that alterations in the expression levels of SR proteins with duration of diabetes may decrease the affinity of SERCA2 for Ca2+ at the early stage and the velocity of Ca2+ sequestration into SR at the late stage of diabetes. Thus a sequential or a gradual change in SR proteins and SERCA2 function can compromise the ability of the SR to regulate [Ca2+]c that may cause slow relaxation at the early stage and depression of cardiac contraction at the late stage of STZ-induced diabetes in rats. This concept of cardiac SR dysfunction underlying sequential development of diastolic and systolic dysfunction has not been examined previously in any diabetic or nondiabetic animal model of cardiomyopathy.

The objective of this study is to determine whether there is a sequential or a gradual alteration of expression and function of SR proteins associated with sequential development of diastolic dysfunction preceding systolic dysfunction in STZ-induced, insulin-deficient diabetic rat hearts.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Development and characterization of diabetic rats. Male 6-wk-old Wistar rats weighing 150 ± 10 g were purchased from Harlan Industries (Indianapolis, IN) and housed in the University of Cincinnati animal facility. After 2 days of acclimation, the rats were anesthetized with an intraperitoneal injection of pentobarbital sodium 30 mg/kg body wt. Each rat was then injected with a single dose of streptozotocin (STZ, 65 mg/kg body wt, Sigma; St. Louis, MO) into the tail vein. Because STZ was dissolved in 100 mM sodium citrate (pH 4.5), the control rats received the same volume (0.1 ml) of sodium citrate buffer. The rats were housed individually in steel cages and were provided with normal rat chow and water ad libitum until they were anesthetized with an intraperitoneal injection of pentobarbital sodium 60 mg/kg body wt. The heart was excised and washed in ice-cold 0.9% NaCl solution, and the atria and aorta were cut off. The ventricles were freeze-clamped at liquid N2 temperature between a pair of stainless steel plates and stored at -80°C. The ventricles were freeze-clamped within 40 s after excision of the heart. Before cardiac excision, the blood glucose level was measured in a drop of blood collected from the tail by using glucose test strips (Bayer Glucometer, Bayer; Elkhart, IN). After cardiac excision, blood was immediately collected from the thoracic cavity, and serum was separated from blood cells by centrifugation and frozen for insulin assay.

The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Publication No. 85-23, Revised 1996) and was performed according to a protocol approved by the Institutional Animal Care and Use Committee.

Insulin treatment of diabetic rats. Rats were injected with either STZ or citrate buffer as described above. After 4 wk, all animals were weighed and had their blood glucose measured using the Bayer Glucometer. The diabetic rats were randomly divided into two groups. One group of diabetic rats were injected subcutaneously with 5 IU of insulin (PZI, Blue Ridge Pharmaceuticals; Greensboro, NC) between 16:30 and 17:00 h for the first 3 days, followed by 4 IU at the same time of the day for 11 days. The other group of diabetic rats and the control rats did not receive any insulin. The rats were weighed and their blood glucose level determined every day before insulin injection. On the 15th day (~09:00 h), all rats were anesthetized with 60 mg/kg of pentobarbital sodium and killed. The ventricles were collected and frozen as described above. Blood glucose was determined and the serum was frozen at -20°C for later insulin assay.

Determination of serum insulin level. Serum insulin level was determined by radioimmunoassay (Amersham Life Sciences, Little Chalfont; Buckinghamshire, UK).

Measurements of cardiac contractility in isolated Langendorff-perfused heart preparation. Contractile function of isolated hearts from 4- and 6-wk diabetic rats and age-matched control rats was evaluated by the Langendorff procedure as described previously (9). Before anesthesia, a rat was injected intraperitoneally with heparin 5,000 IU/kg body wt. The rat was anesthetized with an intraperitoneal injection of pentobarbital sodium 45 mg/kg body wt and placed on a ventilator via a tracheal tube to ensure sufficient oxygenation for the heart. The heart was quickly excised through a midline thoracotomy, and the aorta was cannulated immediately and retrograde perfusion was started with warm oxygenated Kreb-Henseleit buffer containing (in mM) 118 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 0.5 NaEDTA, and 5.5 glucose. The perfusion buffer was continuously gassed with 95% O2-5% CO2 and maintained at 37°C for a final pH of 7.4. A catheter was inserted through a pulmonary vein and advanced through the mitral valve into the left ventricular wall to the apex. It was pierced through the apex, and the posterior end was secured to remain fixed and opened to the inside of the left ventricle chamber as described previously (9). The anterior free end of the catheter was connected to a Cobe pressure transducer (Argon-Maxim; Athens, TX) to record heart rate, intraventricular pressure (IVP), the rate of pressure development (+dP/dt), and the rate of decline of developed pressure (-dP/dt). Aortic pressure was fixed at 55 mmHg. These parameters were recorded simultaneously on a polygraph (model P7, Grass Instruments; Quincy, MA) interfaced to a computer for data collection and storage. Coronary effluent from the heart was collected continuously in a container resting on a digital scale. Coronary flow rate was determined by converting the weight of the fluid to volume. Coronary resistance was calculated by dividing aortic pressure with coronary flow (in ml · min-1 · g heart-1).

Determination of PLB, SERCA2, calsequestrin, and alpha -actin protein levels by quantitative immunoblotting. The frozen ventricles in liquid nitrogen of control and diabetic rats were separately powdered in a stainless steel mortar tightly fitted with a pestle cooled with liquid nitrogen. The frozen tissue was powdered by pressing the pestle into the mortar with a hammer. The powdered tissue was resuspended in the proportion of 100 mg of tissue to 1.5 ml of ice-cold medium containing 10 mM imidazole-HCl buffer (pH 7.0), 300 mM sucrose, 0.3 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium metabisulfite, and 1 mM dl-dithiothreitol (DTT). The suspension was homogenized at 0-4°C using a motor-driven glass/Teflon homogenizer (size A, Thomas Scientific). Protein concentration was determined by the method of Lowry et al. (15), using bovine serum albumin for standard curve.

The homogenates were diluted to 0.1 mg/ml with a medium containing 62.5 mM Tris · HCl buffer (pH 6.8), 10% glycerol, 2% SDS, 5% beta -mercaptoethanol, and 2.5% bromophenol blue. Aliquots containing 0.25, 0.5, 0.75, and 1.0 µg of protein for PLB, calsequestrin (CSQ), or alpha -actin and 8, 16, 32, and 64 µg for SERCA2 were loaded on the SDS-polyacrylamide gel and separated at 4°C by electrophoresis in 4% acrylamide stacking gel and 12% acrylamide separating gel (13) initially at 120 V for 15 min and then at 180 V for 30 min. In each gel, identical protein concentration range of control and diabetic rat heart homogenate proteins were loaded on the gel as described above. The separated proteins were transferred electrophoretically from the gel onto nitrocellulose membranes (0.2 µm pore size, Bio-Rad; Hercules, CA) at 200 mA at 4°C for 90 min (30) in a buffer containing 25 mM Tris base, 192 mM glycine, and 20% methanol and by using the Bio-Rad Trans-Blot electrophoretic transfer system (Bio-Rad). The nitrocellulose membranes were washed for 5 min with 100 mM Tris · HCl buffer (pH 7.4) containing 0.9% NaCl solution (TBS) and blocked with 5% Carnation instant milk in TBS for 1 h at room temperature.

The membranes were then washed three times with TBS and were incubated at 4°C overnight with mouse PLB monoclonal antibody (Affinity Bioreagents; Golden, CO) or mouse alpha -actin monoclonal antibody (Sigma) or rabbit CSQ monoclonal antibody (a gift from Dr. Larry R. Jones, Indiana University School of Medicine, Indianapolis, IN). The primary antibody dilution was 1:1,000 for PLB, 1:2,000 for alpha -actin, and 1:5,000 for CSQ in 2% Carnation instant milk-TBS. The solution was decanted, the membranes were washed in TBS for 30 min at room temperature with agitation, and the TBS was changed every 10 min. The membranes were then incubated for 5 h with a secondary antibody conjugated to horseradish peroxidase after dilution in 2% Carnation instant milk-TBS. The secondary antibodies and the dilutions were donkey anti-mouse at 1:500 for PLB, goat anti-mouse at 1:5,000 for alpha -actin, and goat anti-rabbit at 1:5,000 for CSQ. The secondary antibody solution was decanted, and the membranes were washed for 30 min with TBS alone.

For SERCA2 protein, the membranes were washed three times with TBS plus 0.05% Tween 20 and incubated at room temperature for 2 h with a goat polyclonal anti-rat SERCA2 (Santa Cruz Biotechnology; Santa Cruz, CA) as a primary antibody at 1:400 dilution in 2% Carnation instant milk-TBS-Tween 20. The solution was decanted and TBS-Tween 20 solution was added. The membranes were washed for 30 min with agitation, and the TBS-Tween 20 solution was changed every 10 min. The membranes were incubated for 2 h with a secondary anti-goat antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) at a 1:4,000 dilution in 2% Carnation instant milk-TBS-Tween 20. The solution was decanted and washed for 30 min with TBS-Tween 20 solution.

Quantitative immunoblotting of ryanodine receptor. The preparation of cardiac homogenate proteins for RyR immunoblot was the same as that described above. Aliquots of SDS-digested homogenate proteins were loaded on the SDS-polyacrylamide gel at 4, 8, 16, and 32 µg of protein and separated by electrophoresis (4% acrylamide stacking gel and 6% acrylamide separating gel) at 120 V for 15 min and then at 180 V for 30 min. In each gel, homogenates from an age-matched control and a diabetic rat in identical protein concentration range as described above were loaded. The separated proteins were electrophoretically transferred onto polyvinylidene difluoride membranes (0.2 µm pore size, soaked in methanol just before use, Bio-Rad) in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol and using the Bio-Rad Trans-Blot electrophoretic transfer system at 250 mA at 4°C for 3 h, and then at 50 V for about 12 h. The membranes were treated with 5% Carnation instant milk in TBS with 0.2% Tween 20 for 1 h at room temperature and then for 24 h with anti-ryanodine receptor (Affinity Bioreagents), at 1:700 dilution in 0.5% Carnation instant milk/TBS with 0.2% Tween 20. The primary antibody solution was decanted, and the membranes were washed three times with TBS-0.2% Tween 20 solution each time for 10 min. The blots were then incubated for 2 h with a secondary anti-mouse antibody conjugated to horseradish peroxidase (Affinity Bioreagents) at 1:500 dilution in 0.5% Carnation instant milk-TBS with 0.2% Tween 20. The secondary antibody solution was decanted, and the membranes were washed for 30 min with TBS-0.2% Tween 20.

Determination of phosphorylated PLB by immunoblotting. Preparation of cardiac homogenate and immunoblotting were the same as described for total PLB level. For measurements of phosphorylation at serine-16 and threonine-17, site-specific polyclonal antibodies (Fluorescience; Leeds, UK) were used as described previously (17). The dilution of the antibodies used in this study was 1:5,000.

Analysis of immunoblots. The bands representing the pentameric PLB (25 kDa), SERCA2 (110 kDa), RyR (565 kDa), CSQ (65 kDa), and alpha -actin (42 kDa) proteins were visualized using ECL system (Amersham Pharmacia Biotech; Piscataway, NJ). The protein levels were determined by using linear regression analysis with the linear lines of the number of pixels versus the amount of homogenate protein. The slope (r2 > 0.90) of the lines (pixels/µg) of a control and a diabetic rat separated in the same gel was compared. The levels of each of the proteins in diabetic rat hearts were expressed as a percentage of those of the control rat hearts separated in the same gel. This procedure eliminates false results due to errors during loading and separation of proteins in the gel and transfer of protein bands to membrane and experiment-to-experiment variations in measurement of density of the bands.

Determination of Ca2+ uptake into SR. Frozen ventricles were powdered by stainless steel mortar and pestle cooled with liquid N2. The powdered tissue was suspended in a medium containing 300 mM sucrose, 50 mM K+-phosphate buffer (pH 7.0), 10 mM NaF to inhibit phosphatases, 0.3 mM PMSF to inhibit proteases, and 0.5 mM DTT to prevent oxidation and breakdown of proteins containing a sulfur-sulfur bond. The suspension was homogenized four times each time with 10 passes with a Teflon pestle in a glass Potter-Elvehjelm tissue homogenizer attached to a drill driven at an output of 50 W (120 V). During homogenization, the temperature was maintained at 2-4°C by submerging the homogenizer in a plastic bottle packed with saline-soaked crushed ice. The homogenate was centrifuged at 35,000 g for 30 min, and the supernatant was discarded. The pellet was resuspended in the homogenization buffer in a ratio of 1 g tissue to 15 ml of buffer. The homogenate for Ca2+ uptake study was used within 2 h after preparation.

Initial rate of Ca2+ uptake into SR as a function of time and free Ca2+ in the assay medium was measured using 45Ca2+ as a tracer by Millipore filtration technique (16). The rate of Ca2+ uptake was measured in a 1.5-ml reaction medium containing (in mM) 40 imidazole-HCl buffer (pH 7.0), 95 KCl, 0.5 EGTA, 5 potassium oxalate, 5 MgCl2, 5 NaN3, and 0.001 ruthenium red, and 120-150 µg of protein and free Ca2+ concentration varying from 0.001 to 10 µM achieved by adding varying volume of 10 mM CaCl2 with 45CaCl2 tracer according to an established titration program (25). After preincubation for 5 min at 37°C in ATP free assay buffer, Ca2+ uptake was initiated by the addition of 5 mM ATP (final concentration). During the estimation of free Ca2+ concentration, the 5 mM ATP concentration was also taken into consideration. A 300-µl aliquot was removed at 30, 60, and 90 s after addition of ATP and within 2 s filtered through Millipore filters (0.45 µm pore size) and washed quickly three times with 3.5 ml of solution containing 20 mM Tris · HCl buffer (pH 7.0) and 2 mM EGTA (pH 7.0 with KOH). The filters were dried and counted for radioactivity in 15 ml of Budget-Solve (Research Products International; Mount Prospect, IL). The radioactivity in the filter in the absence of ATP was subtracted from that in the presence of ATP. To verify that Ca2+ uptake into SR vesicles was measured, the uptake was also determined in the presence of SR Ca2+-pump inhibitor thapsigargin (10). Thapsigargin (2.5 µM) inhibited Ca2+ uptake by 93% (9.35 ± 0.32 vs. 0.61 ± 0.16 nmol · min-1 · mg protein-1 in 3 untreated vs. 3 treated samples) in control hearts and similar extent of inhibition in diabetic rat hearts. This test indicated that ATP-dependent Ca2+ uptake into SR was indeed measured in this study.

Statistical analysis. Data were calculated for means ± SE of each group of rats. The statistical analysis for difference between mean were analyzed by unpaired Student's t-test. The P value of < 0.05 was considered as the statistically significant change between control and diabetic rats or diabetic and insulin-treated diabetic rats.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

General characteristics of experimental rats. The food and water intake, the body weight (BW), the blood glucose level, the serum insulin level, the ventricular weight (VW), the VW-to-BW ratio of the control, and STZ-injected rats are presented in Table 1. The STZ-injected rats had significantly (P < 0.05) lower mean body weight compared with that of the age-matched control rats. However, the decreased body weight of STZ-injected rats was not due to starvation or anorexia, because food and water intake of these rats were higher. Arrested growth was a likely cause for decreased body weight in STZ-injected rats compared with that of age-matched control rats. The blood glucose level was increased to about 400% (P < 0.05), and the serum insulin level was decreased to about 85% (P < 0.05) in STZ-injected rats compared with age-matched control rats. The ventricular weight of the diabetic rats was significantly lower (P < 0.05) compared with control rats. The VW-to-BW ratio in diabetic rats was significantly higher at 4 and 6 wk (P < 0.05). The results demonstrate that STZ-induced diabetic rats are hyperglycemic and insulin deficient, which are characteristics of type 1 diabetes.

                              
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  		Table 1.   Characteristics of STZ-induced diabetic rats

Contractile function. Cardiac function was evaluated in isolated heart preparations, and the results are presented in Table 2. The intrinsic heart rate (HR) of the diabetic rat hearts was significantly (P < 0.05) lower compared with that of age-matched control rat hearts. The left ventricular +dP/dt, time to peak pressure (TPP), and intraventricular peak pressure (IVP) were not significantly different in 4-wk diabetic rat hearts and age-matched control rat hearts. However, the +dP/dt, TPP, and IVP were significantly (P < 0.05) decreased in 6-wk diabetic rat hearts compared with those of age-matched control rat hearts. The rate of relaxation as assessed by the -dP/dt was significantly (P < 0.05) decreased, and both the time to 50% relaxation (RT50) and 90% relaxation (RT90) were significantly (P < 0.05) prolonged in 4- and 6-wk diabetic rat hearts compared with those of age-matched control rat hearts. The contractile dysfunction in diabetic rat hearts is not due to decreased HR because similar changes in diabetic rat hearts were observed when the HR of control and diabetic rats was equalized to 300 beats/min by electrical pacing (data not shown here). Coronary resistance in 4-wk diabetic rat hearts was increased by about 20% compared with that of control rat hearts, but it was decreased by about 20% in 6-wk diabetic rat hearts. The reasons for this pattern of change in coronary resistance could not be determined in this study.

                              
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  		Table 2.   Hemodynamics and contractile function of isolated hearts

To determine whether replacement of insulin in diabetic rats prevents the development of delayed relaxation and depressed contraction in diabetic rat hearts, 4-wk diabetic rats were treated with insulin for 2 wk and examined at 6 wk after STZ injection. Daily insulin treatment of diabetic rats for 2 wk normalized the blood glucose and serum insulin levels (Table 3). The slow relaxation and slow and depressed contraction in diabetic rat hearts observed after 6 wk of diabetes were also normalized to the levels of the control rat hearts (Table 2).

                              
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  		Table 3.   Characteristics of insulin-treated diabetic rats

The results of cardiac function study demonstrate slow rate of relaxation in 4-wk diabetic rat hearts and slow rates of contraction and relaxation and depressed magnitude of contraction in 6-wk diabetic rat hearts and that these changes are reversible or preventable with insulin replacement.

SR Ca2+-cycling protein expression. The immunoblots indicated the presence of pentameric form (mol mass 25 kDa) of PLB illustrated with a typical experiment in Fig. 1A. No detectable trace of the monomeric (mol mass 5 kDa) form of the PLB in the immunoblots of the control or diabetic rat heart homogenates was observed (not illustrated here). Homogenates from both control and diabetic rat hearts were run in the same gel. The pentameric PLB level in the diabetic rat hearts was determined relative to the density of the 25-kDa bands in the age-matched control rat hearts. Cumulative data revealed a significant increase in PLB level in diabetic rat hearts by 31% at 4 wk and 60% at 6 wk of diabetes compared with age-matched control rat hearts (Fig. 1A, bar graphs).


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  		Fig. 1.   Quantitative immunoblots demonstrating increased level of PLB protein (A) in 4 wk (4W) and 6 wk (6W) diabetic rat hearts and decreased levels of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) (B), and ryanodine receptor (RyR) (C) proteins in 6 W diabetic rat hearts compared with age-matched control rat hearts. Bands are quantitative immunoblots of a representative control (C) and diabetic (D) rat heart run in the same gel. Slope of linear regression line (r2 > 0.90) of control rat heart was taken as 100% to determine the change in diabetic rat hearts. Bars represent cumulative data of means ± SE of 4 hearts in each group. *Significantly different from control rat hearts (P < 0.05).

The immunoblot of the SERCA2 protein is also illustrated in Fig. 1B. SERCA2 protein level as determined by the density of the 110-kDa bands relative to that of age-matched control rat heart homogenates was not significantly altered in 4-wk diabetic rats (Fig. 1B, bar graphs). However, SERCA2 was significantly decreased by 36% in 6-wk diabetic rat hearts compared with the age-matched control rat hearts. The PLB-to-SERCA2 ratio was significantly increased (P < 0.05) in 4-wk (1.42 ± 0.2) and in 6-wk (2.5 ± 0.2) diabetic rat hearts compared with 1.00 ± 0.04 in 4-wk and 1.01 ± 0.19 in 6-wk control rat hearts.

The immunoblot of the RyR protein is also illustrated in Fig. 1C. The RyR protein level in 4-wk diabetic rat hearts as determined by the density of the 565-kDa bands relative to that of the age-matched control rat hearts was not significantly changed in diabetic rat hearts (Fig. 1C, bar graphs). However, RyR was significantly decreased by 31% in 6-wk diabetic rat hearts compared with the age-matched control rat hearts.

CSQ is a SR luminal Ca2+-buffering protein (19). To determine whether CSQ was altered in diabetic rat hearts, immunoblots of homogenates of 4- and 6-wk diabetic rat hearts and age-matched control rat hearts were performed. The results presented in Fig. 2A demonstrate no significant change in CSQ protein level in 4- or 6-wk diabetic rat hearts.


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  		Fig. 2.   Quantitative immunoblots demonstrating no significant change in the levels of calsequestrin (CSQ; A) and alpha -actin proteins (B) in 4W and 6W diabetic rat hearts compared with age-matched control rat hearts. Same hearts as in Fig. 1 were studied. Format of data presentation is the same as in Fig. 1.

alpha -Actin is a myofilament protein (28). To determine whether alpha -actin was altered in diabetic rat hearts, immunoblots of the cardiac homogenates of control and diabetic rat hearts were performed. The results presented in Fig. 2B demonstrate no change in alpha -actin level in 4- or 6-wk diabetic rat hearts relative to that of the age-matched control rat hearts.

The results of SR Ca2+-cycling protein expression study demonstrate a sequential alteration of SR Ca2+-cycling proteins as indicated by increase in PLB level in 4-wk diabetic rat hearts and decrease in SERCA2 and RyR levels along with increased PLB level in 6-wk diabetic rat hearts without any change in SR luminal Ca2+-buffering protein CSQ or myofilament protein alpha -actin.

Basal phosphorylated levels and reversibility with insulin treatment. Phosphorylation level of PLB determines its ability to inhibit SERCA2 by decreasing its affinity for Ca2+. Therefore, phosphorylated PLB level was determined in diabetic and age-matched control rat hearts. The basal phosphorylation level at the serine-16 site of the PLB (P-Ser16) was significantly decreased in 4-wk and 6-wk diabetic rat hearts compared with age-matched control rat hearts (Fig. 3A). The basal phosphorylation at the threonine-17 site of the PLB (P-Thr17) was also significantly decreased in 4-wk and 6-wk diabetic rat hearts (Fig. 3B). Insulin treatment of the diabetic rats completely prevented the decreased levels of P-Ser16 (Fig. 3A) and P-Thr17 (Fig. 3B). These results indicate that the increased PLB in diabetic rat hearts is predominantly unphosphorylated form.


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  		Fig. 3.   Immunoblots demonstrating basal levels of phosphorylated phospholamban (PLB) protein at serine-16 (A) and threonine-17 (B) in 4W and 6W diabetic rat hearts and age-matched control rat hearts. Bars represent cumulative data of mean ± SE of 4 hearts in each group. ID, insulin-treated diabetic rat hearts. *Significantly different from control rat hearts (P < 0.05). #Significantly different from diabetic rat hearts (P < 0.05).

SR Ca2+ uptake. The initial rate of Ca2+ uptake into SR was increased with increasing free Ca2+ concentration from 100 nM and above, approaching the maximum rate at about 1 µM Ca2+ of 4- and 6-wk diabetic and age-matched control rat hearts (Fig. 4A). The concentration of free Ca2+ that produced half of the maximum rate (EC50) of Ca2+ uptake into the SR of control rat heart membrane homogenates was 0.195 ± 0.02 µM and did not significantly change with the increasing age of the rats (Fig. 4B). However, the EC50 of Ca2+ uptake into SR of 4- and 6-wk diabetic rat hearts was significantly increased, respectively, by 31% and 56% compared with that of age-matched control rat hearts (Fig. 4B). The data indicate a decrease in the affinity of SR Ca2+ pump for Ca2+ in diabetic rat hearts, consistent with the increase in PLB level (see Fig. 1A). The maximum velocity (Vmax) of Ca2+ uptake into SR of 4- and 6-wk control rats was 11.75 ± 0.50 and 11.67 ± 0.72 nmol · min-1 · mg protein-1, respectively, indicating no significant change in Vmax with increasing age of the control rats (Fig. 4C). The Vmax of Ca2+ uptake into SR of 4-wk diabetic rat hearts was not significantly different from that of age-matched control rat hearts. However, it was significantly decreased by about 27% in 6-wk diabetic rat hearts (Fig. 4C) consistent with the decrease in SERCA2 protein level (see Fig. 1B).


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  		Fig. 4.   Ca2+ uptake into sarcoplasmic reticulum (SR) demonstrating decreased affinity (increased EC50) for Ca2+ in 4 W diabetic rat hearts and decreased maximum velocity (Vmax) in 6 W diabetic rat hearts compared with age-matched control rat hearts. Same hearts as in Fig. 1 were studied. Initial linear rate (r2 > 0.90) of Ca2+ uptake into SR of diabetic rat hearts was compared with age-matched control rat hearts. A: initial rate of Ca2+ uptake with increasing free Ca2+ concentration; B: EC50; C: Vmax of Ca2+ uptake into SR in membrane homogenate prepared from each heart were determined with a nonlinear curve-fitting (PRIZM) program. Bar graphs represent means ± SE of 4 D and 4 C rat hearts. *Significantly different from control rat hearts (P < 0.05).

The results of Ca2+ uptake into the SR study demonstrate decreased apparent affinity of SR Ca2+-pump for Ca2+ in 4-wk diabetic rat hearts and decreased apparent affinity for Ca2+ and maximum velocity of Ca2+ uptake into the SR in 6-wk diabetic rat hearts. Thus a sequential change in Ca2+ uptake into the SR of diabetic rat hearts was also observed.

Reversal of change in PLB and prevention of the changes in SERCA2 and RyR protein levels and SR Ca2+-pump activity with insulin treatment. To determine whether changes in the SR protein expression and function were preventable or reversible with insulin replacement, rats diabetic for 4 wk were treated with insulin for 2 wk and SR protein expression and Ca2+ uptake into SR were examined. Four-week diabetic rats were selected because PLB level was increased but SERCA2 and RyR levels were unchanged at this stage. This time point was selected because treatment of diabetic patients with insulin begins shortly after diagnosis of the disease, most likely after some cellular changes have already occurred and before long-term changes have begun. The goal of the treatment is to reverse any changes that may have occurred and prevent further changes and complications. Thus the objective of our study was to determine whether the early onset increase in PLB is reversible and the decreases in SERCA2 and RyR levels are preventable. Insulin treatment of the diabetic rats completely normalized blood glucose and serum insulin levels (Table 3). The insulin-treated rats gained body weight significantly but not to the level of that of control rats. The VW-to-BW ratio was not decreased to that of control rats. Instead it was significantly increased in the insulin-treated rats.

Insulin treatment of diabetic rats resulted in complete normalization of PLB, SERCA2, and RyR protein levels to that of control rat hearts (Fig. 5). The results of Ca2+ uptake into the SR of 6-wk diabetic rat hearts, insulin-treated diabetic rat hearts, and control rat hearts are presented in Fig. 6. The EC50 of the Ca2+ uptake into the SR was normalized to that of control rat hearts, and the Vmax was normalized almost to the level of control rat hearts. The results demonstrate that changes in SR protein expression and dysfunction are associated with insulin deficiency in STZ-induced diabetic rat hearts.


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  		Fig. 5.   Quantitative immunoblots of SR proteins demonstrating reversal of the increase in PLB protein level (A), and prevention of the decrease in SERCA2 (B) and RyR (C) proteins levels after 2 wk of insulin treatment of 4W diabetic rats. For simplicity, typical immunoblots of a fixed amount protein (1 µg for PLB, 64 µg for SERCA2, and 32 µg for RyR) of homogenate of a C, D, and an ID rat heart are presented. Bars represent cumulative data of means ± SE of 4 hearts in each group. *Significantly different from control rat hearts (P < 0.05).



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  		Fig. 6.   Ca2+ uptake (A) into SR demonstrating reversal of the decrease in apparent affinity (increased EC50, (B)) of Ca2+ and prevention of the decrease in Vmax (C) after 2 wk of insulin treatment of 4W rats. Same hearts as in Fig. 5 were studied. Same experimental conditions and data analysis were used as in Fig. 4. *Significantly different from control rat hearts (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first report demonstrating an increase in PLB protein level associated with slow rate of relaxation in an animal model of cardiomyopathy. Although depression of SR Ca2+-pump and Ca2+-ATPase activity in STZ-induced diabetic rat hearts have been reported (8, 14, 23), this is the first report of a sequential alteration of expression and function of SR proteins associated with early-onset slow rate of relaxation and late onset slow rates of contraction and relaxation and depressed magnitude of contraction in this model of diabetic cardiomyopathy. The sequential and antithetic changes in expression of the SR Ca2+-cycling proteins accompanying sequential alteration of contractile function of diabetic rat hearts, to our knowledge, have not been observed in any other animal models of cardiomyopathy. In this respect, our discovery is novel, indicating that diabetic cardiomyopathy may be a unique type of cardiomyopathy.

Early-onset diastolic dysfunction preceding systolic dysfunction in diabetic patients has been observed (for a review, see Ref. 6), but the underlying mechanism is currently unknown. The observation in the present study of sequential alteration of expression and function of SR proteins associated with sequential alteration of contractile function in STZ-induced diabetic rats underscores the potential role of a similar mechanism in human diabetic patients. However, the levels of SR Ca2+-cycling proteins and Ca2+-pump activity in diabetic human heart muscle have not yet been determined. Thus it remains to be seen whether a sequential alteration of expression of SR proteins underlies early-onset diastolic dysfunction and late-onset systolic dysfunction in diabetic human hearts. Alteration of PLB level in idiopathic failing human hearts has been controversial; decreased or unchanged level of this protein has been reported (for a review see Ref. 12), but increased PLB level has not been reported. Decreased SERCA2 and RyR levels have been observed in failing human hearts and in nondiabetic animal models of cardiomyopathy and heart failure (1, 20), but sequential change in the expression or function of SR proteins has not been observed. Thus it also remains to be seen whether diabetic cardiomyopathy is different from idiopathic cardiomyopathy.

The increase of PLB protein level observed in diabetic rat hearts is moderate compared with the twofold transgenic overexpression of PLB in mouse hearts, which was accompanied with about 80% decrease in apparent affinity (increase in EC50) of SR Ca2+ pump for Ca2+ (11). Twofold increase of PLB in mice was found to accompany depression of both contraction and relaxation of the heart (11). The increase in PLB in diabetic rat hearts was predominantly a nonphosphorylated form indicating that it was interacting with SERCA2 to decrease its apparent affinity for Ca2+. In 4-wk diabetic rats, we observed a 30% increase in PLB level accompanied with a 30% increase in EC50 of Ca2+ uptake into the SR. Thus the moderate increase in nonphosphorylated PLB and reduction in apparent affinity of SR Ca2+ pump for Ca2+ may slow the rate of Ca2+ sequestration into the SR and delay relaxation without affecting contraction of the heart at the early stages of diabetes. The rate of decrease in Ca2+ sequestration at the early stages of diabetes may not be to the extent that decreases the SR Ca2+ store and the rate of Ca2+ release from SR to depress the rate and magnitude of contraction of the heart. This could be the underlying cause for slow relaxation without any change in contraction of the heart observed at the early stage of diabetes. On the other hand, during increased cardiac workload even this moderate increase in PLB may more drastically depress SR function and thus cause both systolic and diastolic dysfunction. The decreased SERCA2 protein level at the late stage was accompanied by a 27% decrease in maximum velocity of Ca2+ uptake into SR. The decrease in SERCA2 and RyR protein levels along with a further increase (to ~60%) in PLB protein level at the late stage of diabetes may have further a decreased the SR function resulting in a decreased SR Ca2+ store and rate of Ca2+ release from the SR. These changes not only can slow relaxation but also can slow contraction of the heart. Our data demonstrate that 1) moderate increase in PLB protein level and decrease in apparent affinity of SR Ca2+ pump for Ca2+ are associated with slow relaxation, and 2) decreases in SERCA2 and RyR protein levels further increase in PLB protein level, and decrease in maximum velocity and apparent affinity for Ca2+ of SR Ca2+ pump are associated with slow contraction and relaxation in diabetic rat hearts. The results are consistent with the indications that abnormal cellular calcium handling is probably linked to abnormal mechanical function in diabetes (27). However, contribution of other processes, such as isoform shift in myosin heavy or light chain proteins, slow repolarization of membrane potential, or altered energy metabolism on early-onset slow relaxation or late-onset slow and depressed contraction cannot be excluded. Nevertheless, the results of this study strongly implicate altered SR Ca2+ cycling as an important contributing factor underlying the sequential development of contractile dysfunction in diabetes.

The mechanism of alteration of SR protein expression in STZ-induced diabetic rat hearts is unclear at present. The data presented in this study demonstrate that PLB overexpression and decreased SR Ca2+-pump affinity for Ca2+ were completely reversed, and SERCA2 and RyR proteins underexpression and decreased SR Ca2+-pump maximum velocity were prevented with insulin replacement. The results indicate that alteration of cardiac SR protein expression and depression of the SR Ca2+ pump are associated with insulin deficiency in STZ-induced diabetic rats. The acute effect of insulin receptor signaling in muscle, adipose cells, and liver is to mobilize glucose transporter (Glut-4) from a cytosolic compartment to cell membrane and regulate substrate metabolism in the cell (33). However, insulin receptor signaling also chronically regulates gene transcription and translation (22). Insulin receptor and its signaling process have been demonstrated in cardiac myocytes (32). Insulin has been shown to regulate protein synthesis in cardiac myocytes (4). Therefore, it is likely that insulin receptor signaling could be involved in SR gene or protein expression. Nevertheless, it remains to be seen whether insulin deficiency and downregulation of its signaling or indirect effects of insulin deficiency, such as alteration of growth hormones or hyperglycemic stress, underlie alteration of transcription or translation of SR proteins in insulin-deficient (type 1) diabetic rat hearts. It is possible that alteration of SR protein expression may also underlie cardiac contractile dysfunction in type 2 diabetes because insulin signaling is downregulated in insulin-resistant (type 2) diabetes.

In conclusion, this study demonstrates a novel increase in PLB protein at the early stage and decrease in SERCA2 and RyR proteins at a later stage of STZ-induced diabetic rats hearts. The early increase in nonphosphorylated PLB level is accompanied with decrease in the apparent affinity of SR Ca2+ pump for Ca2+ and slow rate of relaxation of the heart. The decrease in SERCA2 and RyR protein levels and the further increase in PLB protein level are accompanied with decreased maximum velocity of SR Ca2+ pump and slow rates of contraction and relaxation and depressed magnitude of contraction of the heart. The results of the study strongly indicate a contribution of a sequential alteration of SR Ca2+ protein expression and function underlying development of early onset slow relaxation and late onset slow and depression of contraction in diabetic cardiomyopathy.


    ACKNOWLEDGEMENTS

We thank Gilbert Newman and Jianhua Zhang for technical assistance and Dr. Ronald Millard and Kin Man Choi for reading the manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant RO1 HL-56782 and by the American Diabetes Association.

Address for reprint requests and other correspondence: M. A. Matlib, Dept. of Pharmacology and Cell Biophysics, Univ. of Cincinnati College of Medicine, 231 Albert B. Sabin Way, PO Box 670575, Cincinnati, OH 45267-0575 (E-mail: matlibma{at}uc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 February 2001; accepted in final form 30 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arai, M, Matsui H, and Periasami M. Sarcoplasmic reticulum gene expression in cardiac hypertrophy and heart failure. Circ Res 74: 555-564, 1994[Free Full Text].

2.   Bassani, JW, Bassani RA, and Bers DM. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol (Lond) 476: 279-293, 1994[Abstract/Free Full Text].

3.   Chu, G, Dorn GW, Luo W, Harrer JM, Kadambi VJ, Walsh RA, and Kranais EG. Monomeric phospholamban overexpression in transgenic mouse hearts. Circ Res 81: 485-492, 1997[Abstract/Free Full Text].

4.   Decker, RS, Cook MG, Behnke-Barclay M, and Decker ML. Some growth factors stimulate cultured adult rabbit ventricular myocyte hypertrophy in the absence of mechanical loading. Circ Res 77: 544-555, 1995[Abstract/Free Full Text].

5.   Fein, FS, and Sonnenblick EH. Diabetic cardiomyopathy. Cardiovasc Drugs Ther 8: 65-73, 1994[ISI][Medline].

6.   Fein, FS, Kornstein LB, Strobeck JE, Capasso JM, and Sonnenblick EH. Altered myocardial mechanics in diabetic rats. Circ Res 47: 922-933, 1980[Abstract/Free Full Text].

7.   Flarsheim, CE, Grupp IL, and Matlib MA. Mitochondrial dysfunction accompanies diastolic dysfunction in diabetic rat heart. Am J Physiol Heart Circ Physiol 271: H192-H202, 1996[Abstract/Free Full Text].

8.   Ganguly, PK, Pierce GN, Dhalla KS, and Dhalla NS. Defective sarcoplasmic reticular calcium transport in diabetic cardiomyopathy. Am J Physiol Endocrinol Metab 244: E528-E535, 1983[Abstract/Free Full Text].

9.   Grupp, IL, and Grupp G. Isolated heart preparation perfused or superfused with balanced salt solutions. Methods Pharmacol 5: 111-128, 1984.

10.   Inesi, G, and Sagara Y. Specific inhibitors of intracellular Ca2+ transport ATPases. J Membr Biol 141: 1-6, 1994[ISI][Medline].

11.   Kadambi, VJ, Ponniah S, Harrer JM, Hoit BD, Dorn GW, Walsh RA, and Kranias EG. Cardiac specific overexpression of phospholamban alters calcium kinetics and resultant cardiomyocyte mechanic in transgenic mice. J Clin Invest 97: 533-539, 1996[ISI][Medline].

12.   Koss, KL, and Kranias EG. Phospholamban: a prominent regulator of myocardial contractility. Circ Res 79: 1059-1063, 1996[Free Full Text].

13.   Laemmli, UK. Cleavage of structural proteins during assambly of the ead of bacteriophage T4. Nature 227: 680-683, 1970[Medline].

14.   Lopaschuk, GD, Tahilini AG, Vadlamudi RVSV, Katz S, and McNeill JH. Cardiac sarcoplasmic-reticulum function in insulin-treated or carnitine-treated diabetic rats. Am J Physiol Heart Circ Physiol 245: H969-H976, 1983.

15.   Lowry, OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurements with Folin phenol reagent. J Biol Chem 193: 265-275, 1951[Free Full Text].

16.   Luo, W, Grupp IL, Harrer J, Ponniah S, Grupp G, Duffy JJ, Doetschman T, and Kranias EG. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta -agonist stimulation. Circ Res 75: 401-409, 1994[Abstract/Free Full Text].

17.   Luo, W, Chu G, Sato Y, Zhou Z, Kadambi VJ, and Kranias EG. Transgenic approaches to define the functional role of dual site phospholamban phosphorylation. J Biol Chem 273: 4734-4739, 1998[Abstract/Free Full Text].

18.   Lytton, J, and MacLennan DH. Sarcoplasmic reticulum. In: The Heart and the Cardiovascular System (2nd ed), edited by Fozzard HA, Haber E, Jennings RB, Katz AM, and Morgan HE.. New York: Raven, 1991, p. 1203-1222.

19.   Mahoney, L, and Jones LR. Developmental changes in cardiac sarcoplasmic reticulum in sheep. J Biol Chem 261: 15257-15265, 1986[Abstract/Free Full Text].

20.   Marks, AR. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol Heart Circ Physiol 272: H597-H605, 1997[Abstract/Free Full Text].

21.   Minamisawa, S, Hoshijima M, Chu G, Ward CA, Frank K, Gu Y, Martone ME, Wang Y, Ross J, Kranias EG, Giles WR, and Chien KR. Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell 99: 313-322, 1999[ISI][Medline].

22.   Molue, SK, and Denton RM. Multiple signaling pathways involved in the metabolic effects of insulin. Am J Cardiol 80: 41A-49A, 1997[Medline].

23.   Penpargkul, S, Fein F, Sonnenblick EH, and Scheuer J. Depressed cardiac sarcoplasmic reticular function from diabetic rats. J Mol Cell Cardiol 13: 303-309, 1981[ISI][Medline].

24.   Penpargkul, S, Schaible T, Yipnistsoi T, and Scheuer J. The effects of diabetes on performance and metabolism of rat hearts. Circ Res 47: 911-921, 1980[Abstract/Free Full Text].

25.   Robertson, S, and Potter JD. The regulation of free Ca2+ ion concentration by metal chelators. Methods Pharmacol 5: 63-75, 1984.

26.   Rubler, S, Dlugash J, Yuceoglu YZ, Kumral T, Branwood AW, and Grishman A. New type of cardiomyopathy associated with diabetic glomerosclerosis. Am J Cardiol 30: 595-602, 1972[ISI][Medline].

27.   Schaffer, SW, and Mozaffari M. Abnormal mechanical function in diabetes: relation to myocardial calcium handling. Coron Artery Dis 7: 109-115, 1996[ISI][Medline].

28.   Spudich, J, and Watt S. The regulation of rabbit skeletal muscle contraction. I Biochemical studies of the interaction of the tropomyosin-tropnin complex with actin and the proteolytic fragments of myosin. J Biol Chem 246: 4866-4871, 1971[Abstract/Free Full Text].

29.   Stamler, J, Vaccaro O, Neaton JD, and Wentworth D. Diabetes, other risk-factors, and 12-yr cardiovascular mortality for men screened in the multiple risk factor intervention trial. Diabetes Care 16: 34-44, 1993.

30.   Towbin, H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354, 1979[Abstract/Free Full Text].

31.   Vadlamudi, RVSV, Rodgers RL, and McNeill JH. Effect of experimental diabetes on isolated rat heart responsiveness to isoproterenol. Can J Physiol Pharmacol 60: 902-911, 1982[ISI][Medline].

32.   Velloso, LA, Carvalho CRO, Rojas FA, Folli F, and Saad JA. Insulin signaling in heart involves insulin receptor substrates-1 and -2, activation of phosphatidylinositol 3-kinase and the JAK 2-growth related pathway. Cardiovasc Res 40: 96-102, 1998[Abstract/Free Full Text].

33.   While, MF, and Kahn CR. The insulin signaling system. J Biol Chem 269: 1-4, 1994[Free Full Text].

Am J Physiol Heart Circ Physiol 281(3):H1137-H1147
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