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doi: 10.1152/japplphysiol.00103.2008105:907-914, 2008. First published 26 June 2008;J Appl Physiol

Rozanski and Kaushik P. PatelKeshore R. Bidasee, Hong Zheng, Chun-Hong Shao, Sheeva K. Parbhu, George J.-adrenoceptors

preserves myocardial function: effects on expression ofExercise training initiated after the onset of diabetes

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Exercise training initiated after the onset of diabetes preserves myocardial

function: effects on expression of-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, Universityof Nebraska Medical Center, Omaha, Nebraska

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

Bidasee KR, Zheng H, Shao C-H, Parbhu SK, Rozanski GJ,Patel KP. Exercise training initiated after the onset of diabetespreserves myocardial function: effects on expression of-adreno-ceptors. J Appl Physiol 105: 907914, 2008. First published June26, 2008; doi:10.1152/japplphysiol.00103.2008.The present studywas undertaken to assess cardiac function and characterize -adreno-ceptor subtypes in hearts of diabetic rats that underwent exercisetraining (ExT) after the onset of diabetes. Type 1 diabetes was inducedin male Sprague-Dawley rats using streptozotocin. Four weeks afterinduction, rats were randomly divided into two groups. One groupwas exercised trained for 3 wk while the other group remainedsedentary. At the end of the protocol, cardiac parameters were as-sessed using M-mode echocardiography. A Millar catheter was alsoused to assess left ventricular hemodynamics with and without iso-proterenol stimulation. -Adrenoceptors were assessed using Westernblots and [3H]dihydroalprenolol binding. After 7 wk of diabetes, heartrate decreased by 21%, fractional shortening by 20%, ejection fractionby 9%, and basal and isoproterenol-induced dP/dtby 35%. 1- and2-adrenoceptor proteins were reduced by 60% and 40%, respec-tively, while 3-adrenoceptor protein increased by 125%. Ventricularhomogenates from diabetic rats bound 52% less [3H]dihydroalpreno-lol, consistent with reductions in 1- and 2-adrenoceptors. Threeweeks of ExT initiated 4 wk after the onset of diabetes minimizedcardiac function loss. ExT also blunted loss of 1-adrenoceptor

expression. Interestingly, ExT did not prevent diabetes-induced re-duction in 2-adrenoceptor or the increase of3-adrenoceptor expres-sion. ExT also increased [3H]dihydroalprenolol binding, consistentwith increased 1-adrenoceptor expression. These findings demon-strate for the first time that ExT initiated after the onset of diabetesblunts primarily 1-adrenoceptor expression loss, providing mecha-nistic insights for exercise-induced improvements in cardiac function.

rats; heart; streptozotocin; contractility; isoproterenol

MORE THAN 150 million people worldwide are afflicted withdiabetes mellitus (DM), and this number is expected todouble in the next 25 years (51). The picture is equallydisturbing in the United States. As many as 22 million

Americans have DM, of which1 million are Type 1 (11a).Type 1 diabetes (T1D), like other types of DM, is anestablished risk factor for adverse cardiovascular events,including the development of a diabetic cardiomyopathy(DC) (2, 3). Evidence from both human and animal studiesindicates that DC is a distinct disease entity, independent ofhypertension and macro- and microvascular diseases (18,19). Using experimental T1D animals, Foy and Lucas (21)showed in the mid 1970s that diabetes induces a bradycar-

dia. These workers also found that diabetic animals exhib-ited reduced sensitivities to the pressor effect of norepineph-rine and the positive chronotropic and inotropic effects ofisoproterenol, suggestive of altered expression and/or func-tion of-adrenoceptors. Savarese and Berkowitz (38) laterconfirmed these findings and attributed them to a reductionin density of -adrenoceptors. Since then several groupshave confirmed reductions in expression and function of1-and 2-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 increasedexpression of 3-adrenoceptors in hearts from chronic strepto-zotocin (STZ)-induced diabetic rats (15). Since 3-adrenocep-tor is coupled to Gi, we postulated that the increase in 3-adrenoceptor subtype may be contributing to the bradycardiaand negative inotropy induced during T1D. Agonists andantagonists to 3-adrenoceptors are currently under investiga-tion as antidiabetes agents and as agents to treat heart fail-ure (4, 49).

In the early 1980s Stein and coworkers (43) advocated forexercise training (ExT) to be incorporated into treatment reg-imens for T1D. Several studies conducted thereafter haveconsistently demonstrated that ExT reduces the incidence ofcardiovascular morbidity and mortality during diabetes (12, 34,36) and that these beneficial effects are due in part to normal-ization of the sympathetic outflow (neurohormonal) and im-provement in the responsiveness of the myocardium to auto-nomic stimulation (5, 17, 40, 52). In addition to reducingcirculating levels of catecholamines, ExT also reduces circu-lating levels of ANG II, aldosterone, vasopressin, neuropeptideY, atrial natriuretic peptides, and pro-inflammatory mediators(1, 5, 12, 17, 34, 36, 40, 52).

ExT increases cardiac output, a parameter that is depen-dent on rate and force of ventricular contraction (45). Whileit is well known that chronotropy and inotropy are regulatedin part by -adrenoceptor complement, the effect of ExT

initiated after the onset of diabetes on expression andfunction of -adrenoceptors remains incompletely charac-terized. In the few animal studies conducted to date, ExTwas initiated either before or at the onset of diabetes,limiting clinical extrapolation (14, 23, 25, 27, 33, 48). Thepresent study was undertaken to assess cardiac function andcharacterize -adrenoceptor subtypes in hearts of diabeticrats that underwent ExT for 3 wk, starting 4 wk after theonset of diabetes.

Address for reprint requests and other correspondence: K. R. Bidasee, Dept.of Pharmacology and Experimental Neuroscience, Univ. of Nebraska MedicalCenter, DRC 3047, Omaha, NE 68198-5800 (e-mail: [email protected]).

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

J Appl Physiol105: 907914, 2008.First published June 26, 2008; doi:10.1152/japplphysiol.00103.2008.

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MATERIALS AND METHODS

Chemicals and Drugs

[3H]dihydroalprenolol (specific activity 103.8 Ci/mmol) was pur-chased from Perkin Elmer Life Sciences (Boston, MA). CGP-20712was obtained from Tocris Bioscience (Bristol, UK). Antibodiesagainst rat 1-adrenoceptor (sc 568), 2-adrenoceptor (sc 570), and

3-adrenoceptor (sc 1473), -actin (C-11), and anti-goat and anti-mouse IgG-horseradish peroxidase were purchased from Santa CruzBiotechnology (Santa Cruz, CA). Enhanced chemiluminescence re-agent (ECL reagent) and X-ray Hyperfilms were purchased fromAmersham Biosciences (Piscataway, NJ). Ketamine (Ketaset) wasobtained from Fort Dodge Animal Health (Fort Dodge, IA) andacepromazine from Boehringer Ingelheim Vetmedica (St. Joseph,MO). All other reagents and solvents used were of the highest gradecommercially available.

Induction and Verification of Experimental STZ-Induced T1D

All procedures used for this study were approved by the Universityof Nebraska Medical Center Institutional Animal Care and UseCommittee and conducted according to the National Institutes ofHealths Guiding Principles for Research Involving Animals. Thirty

male Sprague-Dawley rats (220 g) were purchased from SascoBreeding Laboratories (Omaha, NE). Animals were housed with a12:12-h light-dark cycle at ambient temperature 22C and 3040%relative humidity. Laboratory chow and tap water were available adlibitum. After acclimatization for 1 wk, rats were assigned randomlyto one of two groups: control and STZ-diabetic. STZ-diabetic ratsreceived 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 wasidentified by polydipsia, polyuria, and blood glucose concentration250 mg/dl (Accu-chek, Boehringer Mannheim, Indianapolis, IN).Blood sugar levels of250 mg/dl were maintained throughout thestudy.

ExT Protocol

Twenty-eight days after injection of STZ (or citrate buffer), controland STZ-diabetic rats were randomly divided into two groups each.One group of control and one group of STZ-diabetic rats underwentExT for a 3-wk period using a modification of the protocol describedby Musch and Terrell (32). During the training period, rats wereexercised 10 min/day at an initial treadmill speed of 10 m/min at 0grade. The treadmill grade and speed were then gradually increased to510% and 2025 m/min, respectively, and the duration of exercisewas increased to 60 min/day. Control and STZ-diabetic rats had thesame total workload (5 days/wk for a total of 3 wk). Only animals thatran steadily on the treadmill with very little or no prompting (electri-cal stimulation) were included in the study. The remaining control andSTZ-diabetic rats were handled daily and treated similarly to the ExT

rats except for the treadmill running. These animals were referred toas sedentary. In vivo cardiac function measurements were done within24 h of the last exercise session.

Assessment of Cardiac Function

M-mode echocardiography.Twenty-four hours after the last boutof ExT, M-mode echocardiography was performed in lightly anesthe-tized rats (78 from each of the 4 groups; 0.3 ml of a cocktailcontaining 100 mg/ml ketamine and 10 mg/ml acepromazine given ip)with an Acuson Sequoia 512C ultrasound system (Siemens) using anAcuson 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] 100.Percent ejection fraction (EF) was calculated as EF [(LVEDV LVESV)/LVEDV] 100.

In vivo hemodynamics. Heart rate, left ventricular pressure, leftventricular end-diastolic pressure, and rate of change of left ventric-ular pressure (dP/dt) were also evaluated in anesthetized rats toascertain changes in cardiac function induced by diabetes. For this,rats (78 from each of the 4 groups, same animals used for echocar-

diography) were anesthetized with Inactin (20 mg/kg ip), and a Millarcatheter (Millar Instruments, Houston TX) containing a pressuretransducer was introduced into the left ventricle via the right carotidartery as previously described (10). Another catheter was inserted viathe right femoral vein for administration of isoproterenol. Cardiachemodynamic parameters were measured in the anesthetized state forthe four groups of rats. After assessing basal parameters, a bolus doseof 0.1 g/kg isoproterenol was administered into the right femoralvein to assess the responsiveness of the heart to -adrenoceptorstimulation. A Powerlab data-acquisition system (ADInstuments, Col-orado Springs, CO) was used for acquiring data. At the end of thestudy, hemodynamic parameters were extracted, and Microsoft Excel(Microsoft, Seattle WA) and Prism GraphPad (San Diego, CA) wereused 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 wereremoved and either quick-frozen by dropping into liquid nitrogen orembedded in crushed dried ice, or placed in Krebs-Henseleit buffer forisolation of myocytes. Soleus muscles from hindlegs were also ex-cised, quick-frozen, and stored at 80C.

Citrate Synthase Activity

Citrate synthase activity in soleus muscle was measured spectro-photometrically employing methods described by Srere (42), usingtissues from all animals from each group. All measurements wereperformed in duplicate, under the same experimental setting at 2022C. 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-coldbuffer containing 20 mM Tris, pH 8.0, 1 mM dithiothreitol (DTT),with protease inhibitor cocktail for 3 10 s using a Polytron at setting6.5. The homogenates were then centrifuged at 24,000 rpm for 30min. The pellets were resuspended in buffer containing 20 mMNaPO4, 10 mM MgCl2, 1 mM DTT, pH 7.4, and placed on ice, andprotein concentrations were measured.

Relative Density of1-, 2-, and3-Adrenoceptorsin Ventricular Tissues

Western blot analyses were used to determine relative levels of1-,

2-, and 3-adrenoceptors (15). Briefly, 30 g of ventricular homog-enates (50 g for 3-adrenoceptors) were solubilized with gel-disso-ciation medium and electrophoresed on 420% linear gradient poly-acrylamide gels (Bio-Rad Laboratories, Burlingame CA) for 2.5 h at150 V. The proteins were then transferred overnight onto polyvinyli-dene 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 3 with phosphate-bufferedsaline, pH 7.4, and incubated for 20 h at 4C with either anti 1-, 2-,or 3-antibodies. At the end of this time, membranes were againwashed 3 with PBS and then incubated for 2 h at room temperaturewith either anti-goat IgG-horseradish peroxidase (1 and 2) oranti-mouse IgG-horseradish peroxidase (3) (Santa Cruz Biotechnol-ogy). Membranes were again washed 3 with PBS and then incu-

908 EXERCISE DURING DIABETES PRESERVES -ADRENOCEPTOR FUNCTION

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bated for 1 min with ECL reagent and exposed to X-ray films.Autoradiograms were developed after 24 min. Films were thenscanned, and relative intensities of signals were measured using ScionImage 1.62c. -Actin levels were also probed and used as an internalcontrol to correct for sample loading.

Radioligand Binding Assays

Total amount of functional plasma membrane-bound 1- and 2-adrenoceptors was determined using equilibrium binding. For this, 0.4mg/ml ventricular homogenates were incubated in buffer containing20 mM NaPO4, 10 mM MgCl2, 1 mM DTT, pH 7.4, for 1 h at 37Cwith 6.0 nM [3H]dihydroalprenolol (a nonspecific -antagonist). Afterincubation, membranes were rapidly filtered through presoaked glassmicrofiber filter B filter paper and washed with 3 3 ml ice-coldbinding buffer. Each filter was then air-dried overnight. The amountof [3H]dihydroalprenolol trapped on the filter paper was determinedthe next day using liquid scintillation counting. Nonspecific bindingwas also determined simultaneously by incubating vesicles with 10M propranolol (nonselective -antagonist). Binding experimentswere carried out on five separate preparations from each groupanalyzed in duplicate. Values reported are means SE.

Statistical Analysis

Differences among values from each of control, STZ-induced, ExTcontrol, and ExT diabetic rats were evaluated using two-wayANOVA. The data shown are means SE. Results were consideredsignificantly different ifP 0.05. Statistical analysis were conductedusing Prism 4 (GraphPad Software, San Diego, CA).

RESULTS

General Characteristics of Animals

The general characteristics of the animals used in this study areshown in Table 1. Diabetic animals fed normally and movedaround in their cages freely, but they did not gain as much weightas 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 notsignificantly alter body mass or blood glucose levels. Citratesynthase activities were significantly greater in skeletal musclesfrom exercise-trained animals than they were in muscles fromsedentary animals (17.2 1.1 molg1min1 for exercise-trained control vs. 10.6 1.2 molg1min1 for sedentarycontrol; and 16.8 1.4 molg1min1 for exercise-traineddiabetic vs. 10.4 0.5 molg1min1 for sedentary diabetic,P 0.05). The increase in citrate synthase activity was similaramong exercise-trained groups (45%), indicative of similarlevels of ExT.

In Vivo Left Ventricular Function

M-mode echocardiograms.Compared with nondiabetic con-trols, sedentary STZ-diabetic rats were bradycardic (Fig. 1).The mean left ventricular end-diastolic diameter in sedentarySTZ-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-systolicdiameter in sedentary STZ-diabetic animals was significantlylarger than in nondiabetic sedentary control animals (2.80 0.23 vs. 2.10 0.23 mm, P 0.05). Sedentary diabeticanimals also exhibited significant reductions in fractionalshortening, ejection fraction, stroke volume, and cardiac output

compared with sedentary control animals (Fig. 1).ExT did not alter heart rates in both control and diabetic

animals (P 0.05). ExT initiated 4 wk after the onset ofdiabetes also did not alter percent fractional shortening or leftventricular end-diastolic diameter. However, ExT significantlyincreased percent ejection fraction and attenuated the increasein left ventricular end-systolic diameter (Fig. 1).

In vivo hemodynamics. Consistent with echocardiographydata, mean basal heart rate of Inactin-anesthetized sedentarydiabetic animals was significantly lower than that of sedentarycontrol animals (284 10 compared with 370 12 beats/min,P 0.05). Basal heart rates of ExT animals were not signifi-cantly different from those of sedentary animals. Mean peakleft ventricular pressures were significantly greater in sedentarycontrol animals than they were in sedentary STZ-diabeticanimals (139.7 4.6 vs. 91.5 5.9 mmHg,P 0.05, Fig. 2).In addition, rates of pressure changes (dP/dt) were alsosignificantly lower in sedentary diabetic rats than they were insedentary control rats (6,102 249 vs. 10,215 494 mmHg/sfor dP/dtand 4,120 320 vs. 9,875 620 mmHg/s for dP/dt,respectively; also see Fig. 3). ExT did not alter peak developedleft ventricular pressure in control animals, but it blunted thereduction induced by diabetes (121.6 4.2 mmHg after ExTcompared with 91.5 5.9 mmHg for sedentary diabetics withno ExT, Fig. 2B,P 0.05). Mean left ventricular end-diastolicpressure in sedentary control animals was significantly higherthan 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.

When injected with 0.1 g/kg isoproterenol, mean devel-oped left ventricular pressure in hearts of sedentary controlanimals increased to 156.6 5.0 mmHg, while that of seden-tary STZ-diabetic animals was 124.6 8.0 mmHg. Althoughthe amplitude of the response to 0.1 g/kg isoproterenol indiabetic animals was significantly lower than that of controlanimals, the actual change in pressure was larger in sedentarydiabetic 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 sedentarycontrol animals were also significantly faster than those insedentary STZ-diabetic animals (Fig. 3). Similar trends werealso obtained following injection with 0.5 g/kg isoproterenol(data not shown). Three weeks of ExT initiated 4 wk after theonset of diabetes enhanced the responsiveness (amplitude aswell as dP/dt) of both control and diabetic hearts to isopro-

Table 1. General characteristics of animals used in the study

Sedentary Control(n 8)

Sedentary STZ-Diabetic(n 7)

Exercise-Trained Control(n 8)

Exercise-Trained STZ-Diabetic(n 7)

Body mass, g 4207 27710* 38215* 2628*Blood glucose, mg/dl 1077 55919* 1067 53226*

Values are means SE. STZ, streptozotocin. *Significantly different from sedentary control.

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terenol stimulation, indicative of preservation of-adrenocep-tors (Fig. 3).

Relative Levels of-Adrenoceptors

Western blot analyses. To characterize -adrenoceptor iso-forms involved in enhanced isoproterenol response followingExT, Western blot analyses were conducted. As shown in Fig. 4A,membrane homogenates from sedentary STZ-diabetic heartscontained 59.6 5.2% less 1-adrenoceptor protein comparedwith homogenates from sedentary control animals (P 0.05).Homogenates from sedentary STZ-diabetic rat hearts also con-

tained 28.4 8.1% less 2-adrenoceptors than that in seden-tary control (P 0.05, Fig. 4B). Consistent with earlier studieswe also found a 125.8 10.2% increase in steady state levelsof 3-adrenoceptor in homogenates from STZ-diabetic rat

hearts when compared with sedentary controls (15). Threeweeks of ExT did not significantly alter expression of1-, 2-,or 3-adrenoceptors in hearts of control animals. However, inSTZ-diabetic rats, ExT blunted the loss of expression of1-adrenoceptor and potentiated the increased expression of3-adrenoceptor induced by diabetes but had no effect onexpression of2-adrenoceptor (Fig. 4B).

[3H]dihydroalprenolol binding.Receptor binding was alsoused to confirm increased levels of-adrenoceptor protein. Asshown in Fig. 5A, membranes from STZ-diabetic rat heartsexhibited a 53.1 4.6% reduction in [3H]dihydroalprenolol

binding compared with membranes from sedentary controlanimals. ExT during diabetes blunted [3H]dihydroalprenololbinding loss to 20.1 5.6% of control. Since ExT enhancedsteady-state levels of1-adrenoceptor, but not 2-adrenocep-

Fig. 1. Representative echocardiograms (top) andcardiac parameters (table at bottom) of sedentarycontrol, sedentary streptozotocin (STZ)-diabetic,

exercise-trained (ExT) control, and ExT STZ-di-abetic rats. For this, animals were lightly anesthe-tized with a cocktail containing ketamine (100mg/ml) and acepromazine (10 mg/ml). Threeloops of M-mode were captured for each animal,and data were averaged for 78 animals/group.Values shown are means SE. LVEDD, leftventricular end-diastolic diameter; LVESD, leftventricular end-systolic diameter; bpm, beats/min.*Significantly different from sedentary control(P 0.05). **Significantly different from seden-tary STZ-diabetic group (P 0.05).

Fig. 2. A: representative left ventricular (in vivo) pressure recordings of hearts from sedentary control, sedentary STZ-diabetic, ExT control, and ExTSTZ-diabetic rats. Animals (78 animals/group) were lightly anesthetized with Inactin (20 mg/kg ip), and an F-2 micromanometer-tipped catheter (MillarInstruments, Houston, TX) was inserted via the right carotid artery into the left ventricle. B: mean values of left ventricular (LV) pressure. *Significantly differentfrom sedentary control (P 0.05). **Significantly different from sedentary STZ-diabetic (P 0.05).




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tors, the increased [3H]dihydroalprenolol binding has beenattributed to the increase in steady-state levels of1-adreno-ceptors. Using the selective 1-adrenoceptor antagonist CGP-20712, we found that 1-adrenoceptors accounted for 50.1 8.4% of the total [3H]dihydroalprenolol binding in homoge-nates from sedentary controls (Fig. 5B) and ExT control rathearts, consistent with previous studies (Ref. 2 and referenceswithin). In sedentary STZ-diabetic rat hearts 1-adrenoceptorsaccounted for 30.1 10.1% of the total [3H]dihydroalprenolol

binding. However, in ExT STZ-diabetic rats 1-adrenoceptorsaccounted for 39.2 4.7% of the total [3H]dihydroalprenololbinding (P 0.05).

DISCUSSION

Clinical studies have demonstrated that ExT initiated afterthe onset of diabetes slows and/or delays myocardial contrac-tility loss and improves the responsiveness of the heart toadrenergic stimulation. However, the effect of ExT on -ad-renoceptor complement remains incompletely characterized. Inthe present study a multifaceted approach was used to demon-strate for the first time that the increased responsiveness ofdiabetic hearts to adrenergic stimulation following ExT stems

principally from preservation of1- but not 2-adrenoceptorsor a reduction in expression of3-adrenoceptors. Using echo-cardiography and in vivo hemodynamics we establish that ExTinitiated 4 wk after the onset of diabetes (chronic) slowsmyocardial contractility loss and increases the responsivenessof hearts to isoproterenol (catecholamine) stimulation. UsingWestern blot, we found that hearts from ExT diabetic animalsthat exhibit increased responsiveness to adrenergic stimulationexpressed elevated levels of 1-adrenoceptor but not 2- or

3-adrenoceptors. These hearts also bound more [3

H]dihydro-alprenolol, consistent with elevated levels of1-adrenoceptor.The present study is also unique in that ExT was initiated 4 wkafter the onset of sedentary diabetes (chronic) and persisted ina graded manner for 3 wk, making it relevant to patients whomay be administered ExT as a therapeutic modality afterdiagnosis of diabetes. Prior experimental animals studies ini-tiated ExT protocols either before or at the onset of hypergly-cemia and measured only one of the above parameters (14, 23,25, 27, 33, 48).

Consistent with earlier studies (15, 16, 29), in the presentstudy we found that 1- and 2-adrenoceptors were signifi-cantly reduced in hearts from 7-wk sedentary STZ-diabetic

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 (78 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 fromsedentary STZ-diabetic (P 0.05).




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rats. Although the exact mechanism(s) responsible for down-regulation remains poorly defined, chronic activation of thesympathetic nervous system and diabetes-induced hypothy-roidism are contributing factors (8). We also found that ExTselectively preserved expression of1-adrenoceptor over 2-

adrenoceptor. To date, the reason for this selectivity remainsunclear since ExT is known to reduce circulating levels ofcatecholamines, which agonize both 1- and 2-adrenoceptors.What we do know, however, is that this selectivity is not dueto ExT-induced increase in thyroid hormones, which serve aspositive regulator of1-adrenoceptor gene (Ref. 28 and refer-ence within). Studies have demonstrated that while ExT enhances

thyroid levels in healthy subjects, it does not enhance T3/T4 levelsin Type 1 diabetic patients and or experimental T1D animals (8,11, 14; Shao C-H and Bidasee KR, unpublished data). 1- and toa lesser extent 2-adrenoceptors are involved in cardiac contrac-tility (20). Since ExT enhanced steady-state levels of1-adreno-ceptor protein but not 2-adrenoceptor protein, we concluded thatthe increased responsiveness of diabetic hearts to isoproterenolstimulation stems primarily from preservation of1-adrenoceptorprotein. This conclusion is consistent with the notion that 1-adrenoceptor is the principal regulator of chronotropy and inot-ropy in rat hearts (2, 31).

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 5separate preparations from each group analyzed in duplicate. *Significantlydifferent from sedentary control (P 0.05). **Significantly different fromsedentary diabetic (P 0.05).B: displacement [3H]dihydroalprenolol bindingassays were used to determine relative levels of1- and 2-adrenoceptors in ratventricular tissues using the 1-adrenoceptor selective agonist CGP-20712.Graph represents the average of5 separate experiments done in duplicate(membranes were prepared from sedentary control ventricular tissues).

Fig. 4. Representative Western blots showing steady state levels of1- (A),2- (B), and 3-adrenoceptors (AR) (C) and corresponding -actin in leftventricular tissues from sedentary control, sedentary STZ-diabetic, ExT con-trol, and ExT STZ-diabetic rat hearts. For this, 30 or 50 g of membranes wasused. Values n graphs represent the average data obtained from 5 separatepreparations from each group analyzed in duplicate. *Significantly differentfrom sedentary control (P 0.05). **Significantly different from sedentarydiabetic (P 0.05).




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Consistent with earlier results (15), in this study we alsofound that 3-adrenoceptor is upregulated in hearts of STZ-diabetic rats. Feve et al. (20) also showed that exposure ofmouse 3T3-F442A adipocytes to insulin for 4 days decreased3-adrenoceptor expression 3.5-fold. Thus it is tempting tospeculate that the upregulation of3-adrenoceptor is also duein part to STZ-induced reduction in circulating insulin levels.

In this study we found that ExT during diabetes also potenti-ated expression of 3-adrenoceptor subtype. The reason forthis increase is unclear, but these data support the idea that3-adrenoceptor antagonists might be useful in the treatment ofheart failure (49).

Recently Barbier et al. (6) found that after 8 wk of ExT,hearts of healthy, nondiabetic female Wistar rats expressed20% less 1-adrenoceptor protein and 39% more 3-adreno-ceptor protein. These data could help explain the bradycardiainduced by ExT in healthy individuals. Comparing their datawith that of our control animals (sedentary control and ExTcontrol), we did not see a significant change in 1- or 3-adrenoceptor subtypes after 3 wk of ExT. These differencescould be accounted for by substantial differences in the exper-imental paradigm, including the duration and intensity of ExTprotocol, strain of rat, and sex of rat.

From echocardiographic studies, there was a trend towardincreased basal heart rates in ExT diabetic animals comparedwith sedentary STZ-diabetic animals; however, the data did notreach statistical significance. Similarly, in in vivo hemody-namic studies, basal heart rates of sedentary animals were notsignificantly different from those of ExT animals. While ExTshould induce a bradycardia in control animals, we anticipatedExT to increase heart rate in diabetic animals; however, thiswas not the case in this study. Studies with the duration of ExTincreased to 4 wk and with ExT initiated earlier during thediabetes (after 3 wk of diabetes) remain to be done.

Using echocardiography, we found that ExT during diabetesenhanced ejection fraction, consistent with the notion that ExTis preserving cardiac function during diabetes. However, otherparameters such as fractional shortening, stroke volume andcardiac output were not significantly changed, although therewas a trend toward improvement. The short duration and lateronset of ExT may be contributing factors. Using high-resolu-tion magnetic resonance imaging, Loganathan et al. (27) re-cently showed improvement in cardiac function after 9 wk ofExT. It should also be pointed out that in the study byLoganathan et al. (27), ExT was initiated before the onset ofdiabetes. Using echocardiography, we also found that ExTsignificantly reduced left ventricular end-systolic diameters butdid not change left ventricular end-diastolic diameters. These

data suggest that ExT during diabetes has minimal effect onleft ventricular chamber size but instead is increasing the extentof ventricular contraction. In an earlier study we found thatExT initiated at the onset of diabetes blunted vascular endo-thelial dysfunction induced by diabetes (30). To date, it is notclear if ExT initiated after chronic diabetes is also able to bluntendothelial dysfunction. It should also be mentioned that noneof the diabetic animals that underwent ExT during this studydied, suggesting that postexercise hypoglycemia or delayedonset hypoglycemia was not an issue.

In hemodynamic studies, we found that ExT blunted thedecrease in basal left ventricular pressure and increased rates ofpressure rise and fall (dP/dt). The responsiveness of hearts to

isoproterenol stimulation (increase in left ventricular pressureand dP/dt) was also enhanced with ExT. These data are inagreement with clinical data indicating that ExT during diabe-tes slows the progression of DC.

As indicated earlier, 1-adrenoceptor is the dominant iso-form in the heart and regulates cardiac function primarily byGs-mediated activation of adenylyl cyclase and activation of

protein kinase A (PKA). PKA then phosphorylates severalproteins, including the L-type calcium channel, ryanodine recep-tor calcium-release channel, phospholamban, and troponin I (9).2-Adrenoceptor subtype also mediates positive inotropic ef-fects via Gs-mediated activation of adenylyl cyclase and acti-vation of PKA, but to a lesser extent (31). More recent studiesalso suggest that persistent activation of PKA reduces theaffinities of1- and 2-adrenoceptor for Gsand increase theiraffinities for Gi (13, 46). In addition to activation of PKA,persistent stimulation of1-adrenoceptors and resultant eleva-tion in cAMP, as is the case with diabetes, can also activate theguanine nucleotide exchange factor activated by cAMP (Epac),which maybe able to activate CaMKII via a Rap/PLC path-way (41). In earlier studies, our laboratory and others showedthat diabetes enhances phosphorylation of RyR2 at the PKAsite Serine 2809 (39, 50). Whether diabetes also increasesphosphorylation of RyR2 at CaMKII site (Serine 2815) andwhether changes observed (if any) are attenuated with ExTboth remain to be determined. Studies that investigate whetherGs to Gi switching also occurs following persistent activationof1- and 2-adrenoceptors by catecholamines during chronicdiabetes remain to be done.

In healthy cardiac myocytes, 3-adrenoceptor is present inlow levels and produces negative inotropic effects by couplingto Gi and nitric oxide synthase pathways (47). The absence ofphosphorylation sites for cAMP-dependent PKA or G protein-coupled receptor kinase in the short COOH terminus of3-

adrenoceptor is also noteworthy as this would impart resistanceto agonist-induced downregulation (47) and may serve a pro-tective role to combat against high levels of circulating cate-cholamine levels.

In conclusion, we have shown for the first time in a com-prehensive way that ExT initiated after a period of chronicdiabetes enhances cardiac contractility and the responsivenessof hearts to isoproterenol (catecholamine) stimulation and thatthese effects are due principally from ExT-induced increase in1-adrenoceptor protein.

GRANTS

This work was supported in part by grants from the Edna Ittner ResearchFoundation and Deans Indirect Cost (K. R. Bidasee), American DiabetesAssociation (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).

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