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J Appl Physiol 105: 907-914, 2008. First published June 26, 2008; doi:10.1152/japplphysiol.00103.2008
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Exercise training initiated after the onset of diabetes preserves myocardial function: effects on expression of β-adrenoceptors

Keshore R. Bidasee,1 Hong Zheng,2 Chun-Hong Shao,1 Sheeva K. Parbhu,1 George J. Rozanski,2 and Kaushik P. Patel2

Departments of 1Pharmacology and Experimental Neuroscience and 2Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska

Submitted 30 January 2008 ; accepted in final form 24 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study was undertaken to assess cardiac function and characterize β-adrenoceptor subtypes in hearts of diabetic rats that underwent exercise training (ExT) after the onset of diabetes. Type 1 diabetes was induced in male Sprague-Dawley rats using streptozotocin. Four weeks after induction, rats were randomly divided into two groups. One group was exercised trained for 3 wk while the other group remained sedentary. At the end of the protocol, cardiac parameters were assessed using M-mode echocardiography. A Millar catheter was also used to assess left ventricular hemodynamics with and without isoproterenol stimulation. β-Adrenoceptors were assessed using Western blots and [3H]dihydroalprenolol binding. After 7 wk of diabetes, heart rate decreased by 21%, fractional shortening by 20%, ejection fraction by 9%, and basal and isoproterenol-induced dP/dt by 35%. β1- and β2-adrenoceptor proteins were reduced by 60% and 40%, respectively, while β3-adrenoceptor protein increased by 125%. Ventricular homogenates from diabetic rats bound 52% less [3H]dihydroalprenolol, consistent with reductions in β1- and β2-adrenoceptors. Three weeks of ExT initiated 4 wk after the onset of diabetes minimized cardiac function loss. ExT also blunted loss of β1-adrenoceptor expression. Interestingly, ExT did not prevent diabetes-induced reduction in β2-adrenoceptor or the increase of β3-adrenoceptor expression. ExT also increased [3H]dihydroalprenolol binding, consistent with increased β1-adrenoceptor expression. These findings demonstrate for the first time that ExT initiated after the onset of diabetes blunts primarily β1-adrenoceptor expression loss, providing mechanistic insights for exercise-induced improvements in cardiac function.

rats; heart; streptozotocin; contractility; isoproterenol

MORE THAN 150 million people worldwide are afflicted with diabetes mellitus (DM), and this number is expected to double in the next 25 years (51). The picture is equally disturbing in the United States. As many as 22 million Americans have DM, of which ~1 million are Type 1 (11a). Type 1 diabetes (T1D), like other types of DM, is an established risk factor for adverse cardiovascular events, including the development of a diabetic cardiomyopathy (DC) (2, 3). Evidence from both human and animal studies indicates that DC is a distinct disease entity, independent of hypertension and macro- and microvascular diseases (18, 19). Using experimental T1D animals, Foy and Lucas (21) showed in the mid 1970s that diabetes induces a bradycardia. These workers also found that diabetic animals exhibited reduced sensitivities to the pressor effect of norepinephrine and the positive chronotropic and inotropic effects of isoproterenol, suggestive of altered expression and/or function of β-adrenoceptors. Savarese and Berkowitz (38) later confirmed these findings and attributed them to a reduction in density of β-adrenoceptors. Since then several groups have confirmed reductions in expression and function of β1- and β2-adrenoceptor subtypes in hearts from experimental diabetic animals and explanted cardiac tissues (22, 24, 26, 35, 37, 44). In a more recent study we found increased expression of β3-adrenoceptors in hearts from chronic streptozotocin (STZ)-induced diabetic rats (15). Since β3-adrenoceptor is coupled to Gi, we postulated that the increase in β3-adrenoceptor subtype may be contributing to the bradycardia and negative inotropy induced during T1D. Agonists and antagonists to β3-adrenoceptors are currently under investigation as antidiabetes agents and as agents to treat heart failure (4, 49).

In the early 1980s Stein and coworkers (43) advocated for exercise training (ExT) to be incorporated into treatment regimens for T1D. Several studies conducted thereafter have consistently demonstrated that ExT reduces the incidence of cardiovascular morbidity and mortality during diabetes (12, 34, 36) and that these beneficial effects are due in part to normalization of the sympathetic outflow (neurohormonal) and improvement in the responsiveness of the myocardium to autonomic stimulation (5, 17, 40, 52). In addition to reducing circulating levels of catecholamines, ExT also reduces circulating levels of ANG II, aldosterone, vasopressin, neuropeptide Y, atrial natriuretic peptides, and pro-inflammatory mediators (1, 5, 12, 17, 34, 36, 40, 52).

ExT increases cardiac output, a parameter that is dependent on rate and force of ventricular contraction (45). While it is well known that chronotropy and inotropy are regulated in part by β-adrenoceptor complement, the effect of ExT initiated after the onset of diabetes on expression and function of β-adrenoceptors remains incompletely characterized. In the few animal studies conducted to date, ExT was initiated either before or at the onset of diabetes, limiting clinical extrapolation (14, 23, 25, 27, 33, 48). The present study was undertaken to assess cardiac function and characterize β-adrenoceptor subtypes in hearts of diabetic rats that underwent ExT for 3 wk, starting 4 wk after the onset of diabetes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and Drugs

[3H]dihydroalprenolol (specific activity 103.8 Ci/mmol) was purchased from Perkin Elmer Life Sciences (Boston, MA). CGP-20712 was obtained from Tocris Bioscience (Bristol, UK). Antibodies against rat β1-adrenoceptor (sc 568), β2-adrenoceptor (sc 570), and β3-adrenoceptor (sc 1473), β-actin (C-11), and anti-goat and anti-mouse IgG-horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence reagent (ECL reagent) and X-ray Hyperfilms were purchased from Amersham Biosciences (Piscataway, NJ). Ketamine (Ketaset) was obtained from Fort Dodge Animal Health (Fort Dodge, IA) and acepromazine from Boehringer Ingelheim Vetmedica (St. Joseph, MO). All other reagents and solvents used were of the highest grade commercially available.

Induction and Verification of Experimental STZ-Induced T1D

All procedures used for this study were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee and conducted according to the National Institutes of Health's "Guiding Principles for Research Involving Animals." Thirty male Sprague-Dawley rats ({approx}220 g) were purchased from Sasco Breeding Laboratories (Omaha, NE). Animals were housed with a 12:12-h light-dark cycle at ambient temperature 22°C and 30–40% relative humidity. Laboratory chow and tap water were available ad libitum. After acclimatization for 1 wk, rats were assigned randomly to one of two groups: control and STZ-diabetic. STZ-diabetic rats received a single injection of STZ (65 mg/kg ip; Sigma Chemical, St. Louis, MO) in a 2% solution of cold 0.1 M citrate buffer (pH 4.5). Control rats were injected with a similar volume of citrate buffer only. Onset of diabetes occurred rapidly following STZ injection and was identified by polydipsia, polyuria, and blood glucose concentration >250 mg/dl (Accu-chek, Boehringer Mannheim, Indianapolis, IN). Blood sugar levels of >250 mg/dl were maintained throughout the study.

ExT Protocol

Twenty-eight days after injection of STZ (or citrate buffer), control and STZ-diabetic rats were randomly divided into two groups each. One group of control and one group of STZ-diabetic rats underwent ExT for a 3-wk period using a modification of the protocol described by Musch and Terrell (32). During the training period, rats were exercised 10 min/day at an initial treadmill speed of 10 m/min at 0° grade. The treadmill grade and speed were then gradually increased to 5–10% and 20–25 m/min, respectively, and the duration of exercise was increased to 60 min/day. Control and STZ-diabetic rats had the same total workload (5 days/wk for a total of 3 wk). Only animals that ran steadily on the treadmill with very little or no prompting (electrical stimulation) were included in the study. The remaining control and STZ-diabetic rats were handled daily and treated similarly to the ExT rats except for the treadmill running. These animals were referred to as sedentary. In vivo cardiac function measurements were done within 24 h of the last exercise session.

Assessment of Cardiac Function

M-mode echocardiography.   Twenty-four hours after the last bout of ExT, M-mode echocardiography was performed in lightly anesthetized rats (7–8 from each of the 4 groups; 0.3 ml of a cocktail containing 100 mg/ml ketamine and 10 mg/ml acepromazine given ip) with an Acuson Sequoia 512C ultrasound system (Siemens) using an Acuson 15L8 probe. Left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) were measured parameters. Percent fractional shortening (FS) was calculated as FS = [(LVEDD – LVESD)/LVEDD] x 100. Percent ejection fraction (EF) was calculated as EF = [(LVEDV – LVESV)/LVEDV] x100.

In vivo hemodynamics.   Heart rate, left ventricular pressure, left ventricular end-diastolic pressure, and rate of change of left ventricular pressure (±dP/dt) were also evaluated in anesthetized rats to ascertain changes in cardiac function induced by diabetes. For this, rats (7–8 from each of the 4 groups, same animals used for echocardiography) were anesthetized with Inactin (20 mg/kg ip), and a Millar catheter (Millar Instruments, Houston TX) containing a pressure transducer was introduced into the left ventricle via the right carotid artery as previously described (10). Another catheter was inserted via the right femoral vein for administration of isoproterenol. Cardiac hemodynamic parameters were measured in the anesthetized state for the four groups of rats. After assessing basal parameters, a bolus dose of 0.1 µg/kg isoproterenol was administered into the right femoral vein to assess the responsiveness of the heart to β-adrenoceptor stimulation. A Powerlab data-acquisition system (ADInstuments, Colorado Springs, CO) was used for acquiring data. At the end of the study, hemodynamic parameters were extracted, and Microsoft Excel (Microsoft, Seattle WA) and Prism GraphPad (San Diego, CA) were used for analysis of data.

Tissue Collection

At the end of the in vivo measurements, animals were killed (Inactin, 75 mg/kg ip). Chest cavities were opened, and hearts were removed and either quick-frozen by dropping into liquid nitrogen or embedded in crushed dried ice, or placed in Krebs-Henseleit buffer for isolation of myocytes. Soleus muscles from hindlegs were also excised, quick-frozen, and stored at –80°C.

Citrate Synthase Activity

Citrate synthase activity in soleus muscle was measured spectrophotometrically employing methods described by Srere (42), using tissues from all animals from each group. All measurements were performed in duplicate, under the same experimental setting at 20–22°C. Citrate synthase activities were normalized to total protein content and reported as micromoles per milligram protein per minute.

Preparation of Ventricular Membranes

Ventricular tissues (right and left) were homogenized in ice-cold buffer containing 20 mM Tris, pH 8.0, 1 mM dithiothreitol (DTT), with protease inhibitor cocktail for 3 x 10 s using a Polytron at setting 6.5. The homogenates were then centrifuged at 24,000 rpm for 30 min. The pellets were resuspended in buffer containing 20 mM NaPO4, 10 mM MgCl2, 1 mM DTT, pH 7.4, and placed on ice, and protein concentrations were measured.

Relative Density of β1-, β2-, and β3-Adrenoceptors in Ventricular Tissues

Western blot analyses were used to determine relative levels of β1-, β2-, and β3-adrenoceptors (15). Briefly, 30 µg of ventricular homogenates (50 µg for β3-adrenoceptors) were solubilized with gel-dissociation medium and electrophoresed on 4–20% linear gradient polyacrylamide gels (Bio-Rad Laboratories, Burlingame CA) for 2.5 h at 150 V. The proteins were then transferred overnight onto polyvinylidene difluoride membranes. The next day, membranes were blocked (0.01 M Tris·HCl, 0.05 M NaCl, 5% nonfat dry milk, and 0.04% Tween 20, pH 7.4, for 1 h), washed 3x with phosphate-buffered saline, pH 7.4, and incubated for 20 h at 4°C with either anti β1-, β2-, or β3-antibodies. At the end of this time, membranes were again washed 3x with PBS and then incubated for 2 h at room temperature with either anti-goat IgG-horseradish peroxidase (β1 and β2) or anti-mouse IgG-horseradish peroxidase (β3) (Santa Cruz Biotechnology). Membranes were again washed 3x with PBS and then incubated for 1 min with ECL reagent and exposed to X-ray films. Autoradiograms were developed after 2–4 min. Films were then scanned, and relative intensities of signals were measured using Scion Image 1.62c. β-Actin levels were also probed and used as an internal control to correct for sample loading.

Radioligand Binding Assays

Total amount of functional plasma membrane-bound β1- and β2-adrenoceptors was determined using equilibrium binding. For this, 0.4 mg/ml ventricular homogenates were incubated in buffer containing 20 mM NaPO4, 10 mM MgCl2, 1 mM DTT, pH 7.4, for 1 h at 37°C with 6.0 nM [3H]dihydroalprenolol (a nonspecific β-antagonist). After incubation, membranes were rapidly filtered through presoaked glass microfiber filter B filter paper and washed with 3 x 3 ml ice-cold binding buffer. Each filter was then air-dried overnight. The amount of [3H]dihydroalprenolol trapped on the filter paper was determined the next day using liquid scintillation counting. Nonspecific binding was also determined simultaneously by incubating vesicles with 10 µM propranolol (nonselective β-antagonist). Binding experiments were carried out on five separate preparations from each group analyzed in duplicate. Values reported are means ± SE.

Statistical Analysis

Differences among values from each of control, STZ-induced, ExT control, and ExT diabetic rats were evaluated using two-way ANOVA. The data shown are means ± SE. Results were considered significantly different if P < 0.05. Statistical analysis were conducted using Prism 4 (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General Characteristics of Animals

The general characteristics of the animals used in this study are shown in Table 1. Diabetic animals fed normally and moved around in their cages freely, but they did not gain as much weight as nondiabetic animals (P < 0.05). As expected, sedentary control animals had significantly higher body masses than their exercise-trained counterparts (P < 0.05). ExT during diabetes did not significantly alter body mass or blood glucose levels. Citrate synthase activities were significantly greater in skeletal muscles from exercise-trained animals than they were in muscles from sedentary animals (17.2 ± 1.1 µmol·g–1·min–1 for exercise-trained control vs. 10.6 ± 1.2 µmol·g–1·min–1 for sedentary control; and 16.8 ± 1.4 µmol·g–1·min–1 for exercise-trained diabetic vs. 10.4 ± 0.5 µmol·g–1·min–1 for sedentary diabetic, P < 0.05). The increase in citrate synthase activity was similar among exercise-trained groups ({approx}45%), indicative of similar levels of ExT.


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  		Table 1. General characteristics of animals used in the study

 
In Vivo Left Ventricular Function

M-mode echocardiograms.   Compared with nondiabetic controls, sedentary STZ-diabetic rats were bradycardic (Fig. 1). The mean left ventricular end-diastolic diameter in sedentary STZ-diabetic animals was not significantly different from that of sedentary control animals (6.02 ± 0.44 vs. 6.36 ± 0.49 mm, P > 0.05). However, the mean left ventricular end-systolic diameter in sedentary STZ-diabetic animals was significantly larger than in nondiabetic sedentary control animals (2.80 ± 0.23 vs. 2.10 ± 0.23 mm, P < 0.05). Sedentary diabetic animals also exhibited significant reductions in fractional shortening, ejection fraction, stroke volume, and cardiac output compared with sedentary control animals (Fig. 1).


Figure 1
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  		Fig. 1. Representative echocardiograms (top) and cardiac parameters (table at bottom) of sedentary control, sedentary streptozotocin (STZ)-diabetic, exercise-trained (ExT) control, and ExT STZ-diabetic rats. For this, animals were lightly anesthetized with a cocktail containing ketamine (100 mg/ml) and acepromazine (10 mg/ml). Three loops of M-mode were captured for each animal, and data were averaged for 7–8 animals/group. Values shown are means ± SE. LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; bpm, beats/min. *Significantly different from sedentary control (P < 0.05). **Significantly different from sedentary STZ-diabetic group (P < 0.05).

 
ExT did not alter heart rates in both control and diabetic animals (P > 0.05). ExT initiated 4 wk after the onset of diabetes also did not alter percent fractional shortening or left ventricular end-diastolic diameter. However, ExT significantly increased percent ejection fraction and attenuated the increase in left ventricular end-systolic diameter (Fig. 1).

In vivo hemodynamics.   Consistent with echocardiography data, mean basal heart rate of Inactin-anesthetized sedentary diabetic animals was significantly lower than that of sedentary control animals (284 ± 10 compared with 370 ± 12 beats/min, P < 0.05). Basal heart rates of ExT animals were not significantly different from those of sedentary animals. Mean peak left ventricular pressures were significantly greater in sedentary control animals than they were in sedentary STZ-diabetic animals (139.7 ± 4.6 vs. 91.5 ± 5.9 mmHg, P < 0.05, Fig. 2). In addition, rates of pressure changes (±dP/dt) were also significantly lower in sedentary diabetic rats than they were in sedentary control rats (6,102 ± 249 vs. 10,215 ± 494 mmHg/s for +dP/dt and 4,120 ± 320 vs. 9,875 ± 620 mmHg/s for –dP/dt, respectively; also see Fig. 3). ExT did not alter peak developed left ventricular pressure in control animals, but it blunted the reduction induced by diabetes (121.6 ± 4.2 mmHg after ExT compared with 91.5 ± 5.9 mmHg for sedentary diabetics with no ExT, Fig. 2B, P < 0.05). Mean left ventricular end-diastolic pressure in sedentary control animals was significantly higher than that of sedentary diabetic animals (8.9 ± 1.8 vs. 1.8 ± 0.3 mmHg, P < 0.05). ExT did not alter left ventricular end-diastolic pressures in either control or diabetic groups.


Figure 2
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  		Fig. 2. A: representative left ventricular (in vivo) pressure recordings of hearts from sedentary control, sedentary STZ-diabetic, ExT control, and ExT STZ-diabetic rats. Animals (7–8 animals/group) were lightly anesthetized with Inactin (20 mg/kg ip), and an F-2 micromanometer-tipped catheter (Millar Instruments, Houston, TX) was inserted via the right carotid artery into the left ventricle. B: mean values of left ventricular (LV) pressure. *Significantly different from sedentary control (P < 0.05). **Significantly different from sedentary STZ-diabetic (P < 0.05).

 

Figure 3
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  		Fig. 3. A: representative recordings of rates of left ventricular pressure changes (±dP/dt) in hearts from sedentary control, sedentary STZ-diabetic, ExT control, and ExT STZ-diabetic rats. Animals (7–8 animals per group) were lightly anesthetized with Inactin (20 mg/kg ip), and an F-2 micromanometer-tipped catheter (Millar Instruments) was inserted via the left carotid artery into the left ventricle. Data were obtained using Powerlab data-acquisition system (ADInstuments, Colorado Springs, CO). B: mean values for ±dP/dt. Iso, isoproterenol. *Significantly different from sedentary controls (P < 0.05). **Significantly different from sedentary STZ-diabetic (P < 0.05).

 
When injected with 0.1 µg/kg isoproterenol, mean developed left ventricular pressure in hearts of sedentary control animals increased to 156.6 ± 5.0 mmHg, while that of sedentary STZ-diabetic animals was 124.6 ± 8.0 mmHg. Although the amplitude of the response to 0.1 µg/kg isoproterenol in diabetic animals was significantly lower than that of control animals, the actual change in pressure was larger in sedentary diabetic animals than in sedentary control animals (33.1 vs. 16.9 mmHg, respectively, P < 0.05). Concomitant with this, rates of developed pressure rise and fall (±dP/dt) in sedentary control animals were also significantly faster than those in sedentary STZ-diabetic animals (Fig. 3). Similar trends were also obtained following injection with 0.5 µg/kg isoproterenol (data not shown). Three weeks of ExT initiated 4 wk after the onset of diabetes enhanced the responsiveness (amplitude as well as ±dP/dt) of both control and diabetic hearts to isoproterenol stimulation, indicative of preservation of β-adrenoceptors (Fig. 3).

Relative Levels of β-Adrenoceptors

Western blot analyses.   To characterize β-adrenoceptor isoforms involved in enhanced isoproterenol response following ExT, Western blot analyses were conducted. As shown in Fig. 4A, membrane homogenates from sedentary STZ-diabetic hearts contained 59.6 ± 5.2% less β1-adrenoceptor protein compared with homogenates from sedentary control animals (P < 0.05). Homogenates from sedentary STZ-diabetic rat hearts also contained 28.4 ± 8.1% less β2-adrenoceptors than that in sedentary control (P < 0.05, Fig. 4B). Consistent with earlier studies we also found a 125.8 ± 10.2% increase in steady state levels of β3-adrenoceptor in homogenates from STZ-diabetic rat hearts when compared with sedentary controls (15). Three weeks of ExT did not significantly alter expression of β1-, β2-, or β3-adrenoceptors in hearts of control animals. However, in STZ-diabetic rats, ExT blunted the loss of expression of β1-adrenoceptor and potentiated the increased expression of β3-adrenoceptor induced by diabetes but had no effect on expression of β2-adrenoceptor (Fig. 4B).


Figure 4
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  		Fig. 4. Representative Western blots showing steady state levels of β1- (A), β2- (B), and β3-adrenoceptors (AR) (C) and corresponding β-actin in left ventricular tissues from sedentary control, sedentary STZ-diabetic, ExT control, and ExT STZ-diabetic rat hearts. For this, 30 or 50 µg of membranes was used. Values n graphs represent the average data obtained from >5 separate preparations from each group analyzed in duplicate. *Significantly different from sedentary control (P < 0.05). **Significantly different from sedentary diabetic (P < 0.05).

 
[3H]dihydroalprenolol binding.   Receptor binding was also used to confirm increased levels of β-adrenoceptor protein. As shown in Fig. 5A, membranes from STZ-diabetic rat hearts exhibited a 53.1 ± 4.6% reduction in [3H]dihydroalprenolol binding compared with membranes from sedentary control animals. ExT during diabetes blunted [3H]dihydroalprenolol binding loss to 20.1 ± 5.6% of control. Since ExT enhanced steady-state levels of β1-adrenoceptor, but not β2-adrenoceptors, the increased [3H]dihydroalprenolol binding has been attributed to the increase in steady-state levels of β1-adrenoceptors. Using the selective β1-adrenoceptor antagonist CGP-20712, we found that β1-adrenoceptors accounted for 50.1 ± 8.4% of the total [3H]dihydroalprenolol binding in homogenates from sedentary controls (Fig. 5B) and ExT control rat hearts, consistent with previous studies (Ref. 2 and references within). In sedentary STZ-diabetic rat hearts β1-adrenoceptors accounted for 30.1 ± 10.1% of the total [3H]dihydroalprenolol binding. However, in ExT STZ-diabetic rats β1-adrenoceptors accounted for 39.2 ± 4.7% of the total [3H]dihydroalprenolol binding (P < 0.05).


Figure 5
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  		Fig. 5. A: shows the ability of membranes prepared from sedentary control, sedentary STZ-diabetic, ExT control, and ExT STZ-diabetic rat hearts to bind [3H]dihydroalprenolol. Values in graphs represent the average data from >5 separate preparations from each group analyzed in duplicate. *Significantly different from sedentary control (P < 0.05). **Significantly different from sedentary diabetic (P < 0.05). B: displacement [3H]dihydroalprenolol binding assays were used to determine relative levels of β1- and β2-adrenoceptors in rat ventricular tissues using the β1-adrenoceptor selective agonist CGP-20712. Graph represents the average of >5 separate experiments done in duplicate (membranes were prepared from sedentary control ventricular tissues).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Clinical studies have demonstrated that ExT initiated after the onset of diabetes slows and/or delays myocardial contractility loss and improves the responsiveness of the heart to adrenergic stimulation. However, the effect of ExT on β-adrenoceptor complement remains incompletely characterized. In the present study a multifaceted approach was used to demonstrate for the first time that the increased responsiveness of diabetic hearts to adrenergic stimulation following ExT stems principally from preservation of β1- but not β2-adrenoceptors or a reduction in expression of β3-adrenoceptors. Using echocardiography and in vivo hemodynamics we establish that ExT initiated 4 wk after the onset of diabetes (chronic) slows myocardial contractility loss and increases the responsiveness of hearts to isoproterenol (catecholamine) stimulation. Using Western blot, we found that hearts from ExT diabetic animals that exhibit increased responsiveness to adrenergic stimulation expressed elevated levels of β1-adrenoceptor but not β2- or β3-adrenoceptors. These hearts also bound more [3H]dihydroalprenolol, consistent with elevated levels of β1-adrenoceptor. The present study is also unique in that ExT was initiated 4 wk after the onset of sedentary diabetes (chronic) and persisted in a graded manner for 3 wk, making it relevant to patients who may be administered ExT as a therapeutic modality after diagnosis of diabetes. Prior experimental animals studies initiated ExT protocols either before or at the onset of hyperglycemia and measured only one of the above parameters (14, 23, 25, 27, 33, 48).

Consistent with earlier studies (15, 16, 29), in the present study we found that β1- and β2-adrenoceptors were significantly reduced in hearts from 7-wk sedentary STZ-diabetic rats. Although the exact mechanism(s) responsible for downregulation remains poorly defined, chronic activation of the sympathetic nervous system and diabetes-induced hypothyroidism are contributing factors (8). We also found that ExT selectively preserved expression of β1-adrenoceptor over β2-adrenoceptor. To date, the reason for this selectivity remains unclear since ExT is known to reduce circulating levels of catecholamines, which agonize both β1- and β2-adrenoceptors. What we do know, however, is that this selectivity is not due to ExT-induced increase in thyroid hormones, which serve as positive regulator of β1-adrenoceptor gene (Ref. 28 and reference within). Studies have demonstrated that while ExT enhances thyroid levels in healthy subjects, it does not enhance T3/T4 levels in Type 1 diabetic patients and or experimental T1D animals (8, 11, 14; Shao C-H and Bidasee KR, unpublished data). β1- and to a lesser extent β2-adrenoceptors are involved in cardiac contractility (20). Since ExT enhanced steady-state levels of β1-adrenoceptor protein but not β2-adrenoceptor protein, we concluded that the increased responsiveness of diabetic hearts to isoproterenol stimulation stems primarily from preservation of β1-adrenoceptor protein. This conclusion is consistent with the notion that β1-adrenoceptor is the principal regulator of chronotropy and inotropy in rat hearts (2, 31).

Consistent with earlier results (15), in this study we also found that β3-adrenoceptor is upregulated in hearts of STZ-diabetic rats. Fève et al. (20) also showed that exposure of mouse 3T3-F442A adipocytes to insulin for 4 days decreased β3-adrenoceptor expression 3.5-fold. Thus it is tempting to speculate that the upregulation of β3-adrenoceptor is also due in part to STZ-induced reduction in circulating insulin levels. In this study we found that ExT during diabetes also potentiated expression of β3-adrenoceptor subtype. The reason for this increase is unclear, but these data support the idea that β3-adrenoceptor antagonists might be useful in the treatment of heart failure (49).

Recently Barbier et al. (6) found that after 8 wk of ExT, hearts of healthy, nondiabetic female Wistar rats expressed 20% less β1-adrenoceptor protein and 39% more β3-adrenoceptor protein. These data could help explain the bradycardia induced by ExT in healthy individuals. Comparing their data with that of our control animals (sedentary control and ExT control), we did not see a significant change in β1- or β3-adrenoceptor subtypes after 3 wk of ExT. These differences could be accounted for by substantial differences in the experimental paradigm, including the duration and intensity of ExT protocol, strain of rat, and sex of rat.

From echocardiographic studies, there was a trend toward increased basal heart rates in ExT diabetic animals compared with sedentary STZ-diabetic animals; however, the data did not reach statistical significance. Similarly, in in vivo hemodynamic studies, basal heart rates of sedentary animals were not significantly different from those of ExT animals. While ExT should induce a bradycardia in control animals, we anticipated ExT to increase heart rate in diabetic animals; however, this was not the case in this study. Studies with the duration of ExT increased to ≥4 wk and with ExT initiated earlier during the diabetes (after 3 wk of diabetes) remain to be done.

Using echocardiography, we found that ExT during diabetes enhanced ejection fraction, consistent with the notion that ExT is preserving cardiac function during diabetes. However, other parameters such as fractional shortening, stroke volume and cardiac output were not significantly changed, although there was a trend toward improvement. The short duration and later onset of ExT may be contributing factors. Using high-resolution magnetic resonance imaging, Loganathan et al. (27) recently showed improvement in cardiac function after 9 wk of ExT. It should also be pointed out that in the study by Loganathan et al. (27), ExT was initiated before the onset of diabetes. Using echocardiography, we also found that ExT significantly reduced left ventricular end-systolic diameters but did not change left ventricular end-diastolic diameters. These data suggest that ExT during diabetes has minimal effect on left ventricular chamber size but instead is increasing the extent of ventricular contraction. In an earlier study we found that ExT initiated at the onset of diabetes blunted vascular endothelial dysfunction induced by diabetes (30). To date, it is not clear if ExT initiated after chronic diabetes is also able to blunt endothelial dysfunction. It should also be mentioned that none of the diabetic animals that underwent ExT during this study died, suggesting that postexercise hypoglycemia or delayed onset hypoglycemia was not an issue.

In hemodynamic studies, we found that ExT blunted the decrease in basal left ventricular pressure and increased rates of pressure rise and fall (±dP/dt). The responsiveness of hearts to isoproterenol stimulation (increase in left ventricular pressure and ±dP/dt) was also enhanced with ExT. These data are in agreement with clinical data indicating that ExT during diabetes slows the progression of DC.

As indicated earlier, β1-adrenoceptor is the dominant isoform in the heart and regulates cardiac function primarily by Gs-mediated activation of adenylyl cyclase and activation of protein kinase A (PKA). PKA then phosphorylates several proteins, including the L-type calcium channel, ryanodine receptor calcium-release channel, phospholamban, and troponin I (9). β2-Adrenoceptor subtype also mediates positive inotropic effects via Gs-mediated activation of adenylyl cyclase and activation of PKA, but to a lesser extent (31). More recent studies also suggest that persistent activation of PKA reduces the affinities of β1- and β2-adrenoceptor for Gs and increase their affinities for Gi (13, 46). In addition to activation of PKA, persistent stimulation of β1-adrenoceptors and resultant elevation in cAMP, as is the case with diabetes, can also activate the guanine nucleotide exchange factor activated by cAMP (Epac), which maybe able to activate CaMKII via a Rap/PLC{varepsilon} pathway (41). In earlier studies, our laboratory and others showed that diabetes enhances phosphorylation of RyR2 at the PKA site Serine 2809 (39, 50). Whether diabetes also increases phosphorylation of RyR2 at CaMKII site (Serine 2815) and whether changes observed (if any) are attenuated with ExT both remain to be determined. Studies that investigate whether Gs to Gi switching also occurs following persistent activation of β1- and β2-adrenoceptors by catecholamines during chronic diabetes remain to be done.

In healthy cardiac myocytes, β3-adrenoceptor is present in low levels and produces negative inotropic effects by coupling to Gi and nitric oxide synthase pathways (47). The absence of phosphorylation sites for cAMP-dependent PKA or G protein-coupled receptor kinase in the short COOH terminus of β3-adrenoceptor is also noteworthy as this would impart resistance to agonist-induced downregulation (47) and may serve a protective role to combat against high levels of circulating catecholamine levels.

In conclusion, we have shown for the first time in a comprehensive way that ExT initiated after a period of chronic diabetes enhances cardiac contractility and the responsiveness of hearts to isoproterenol (catecholamine) stimulation and that these effects are due principally from ExT-induced increase in β1-adrenoceptor protein.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported in part by grants from the Edna Ittner Research Foundation and Dean's Indirect Cost (K. R. Bidasee), American Diabetes Association (1-06-RA-11, K. R. Bidasee), and National Institutes of Health (HL-085061, K. R. Bidasee; NS-39751, K. P. Patel; and HL-66446, G. J. Rozanski).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. R. Bidasee, Dept. of Pharmacology and Experimental Neuroscience, Univ. of Nebraska Medical Center, DRC 3047, Omaha, NE 68198-5800 (e-mail: kbidasee{at}unmc.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.


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