integrales en linea

Journal of Cardiac Failure Vol. 5 No. 4 1999

Noninvasive Evaluation of Cardiac Dysfunction by Echocardiography in Streptozotocin-Induced Diabetic Rats BRIAN D. HOIT, MD,* CESAR CASTRO, BS, * GILBERTO BULTRON, BS, * SELENA KNIGHT, BS, t MOHAMMED A. MATLIB, PhD* Cincinnati, Ohio

ABSTRACT Background: There has not been a noninvasive in vivo longitudinal evaluation of cardiac function in diabetic rats. The objective of this study is to examine the time course of development of cardiac dysfunction in streptozotocin (STZ)-induced diabetic rats. Methods and Results: Cardiac function was evaluated by M-mode and Doppler echocardiography in anesthetized Wistar rats at 2, 4, 5, 6, and 8 weeks after injection with 65 mg of STZ/kg and in age-matched control rats before and after the administration of isoproterenol. Body weight (BW) was significantly less and blood glucose level significantly greater in diabetic rats compared with controls at 2 weeks and remained at these levels at all time points. The calculated left ventricular (LV) mass appeared slightly decreased in diabetic rats. However, LV mass-BW ratios were similar in controls and diabetic rats at 2, 4, and 5 weeks, but were significantly greater in diabetic rats at 6 and 8 weeks. Basal heart rate (HR) was significantly lower in diabetic rats at all time points studied. Basal LV systolic and diastolic dimensions, fractional shortening (FS), velocity of circumferential shortening (Vcf), peak emptying rate (PER), peak filling rate (PFR), and aortic peak velocity (APV) were not significantly different between controls and diabetic rats at 2 and 4 weeks. PER and PFR were significantly less in 5-week diabetic rats. However, Vcf, PER, and PFR were significantly less and FS and APV were similar at 6 and 8 weeks. Administration of isoproterenol increased HR, Vcf, FS, PFR, and PER in controls at all time points, but the increases in diabetic rats at 5, 6, and 8 weeks were less compared with those in controls. The increase in APV was significantly less in diabetic rats at all time points studied. Conclusion: STZ-induced diabetic rats showed bradycardia before contractile dysfunction. Overt and covert contractile dysfunction unmasked by isoproterenol begins at 5 weeks of diabetes. The overt LV systolic and diastolic dysfunction are fully manifested after 6 weeks of diabetes. Key words: diabetes, streptozotocin, cardiomyopathy, heart, contractility, isoproterenol, echocardiography.

From the *Department of Medicine, Division of Cardiology, and ~Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio. Supported in part by grants from the University of Cincinnati Cardiovascular Center, Cincinnati, Ohio (B.D.H., M.A.M.); American Diabetes Association, Alexandria, Virginia (M.A.M.); and grant no. RO1 HL56782 from the National Institutes of Health, Bethesda, Maryland (M.A.M.). Manuscript received February 19, 1999; revised manuscript received June 9, 1999; revised manuscript accepted June 23, 1999. Reprint requests: Mohammed A. MatIib, PhD, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Avenue, PO Box 670575, Cincinnati, OH 45267-0575. Copyright © 1999 by Churchill Livingstone ® 1071-9164/99/0504-0007510.00/0

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Cardiac Dysfunction In Vivo in Diabetic Rats

Heart failure is the leading cause of death in patients with diabetes (1). Compared with the general population, the risk for heart failure is approximately 7-fold greater in women with diabetes and 4-fold greater in men with diabetes (2). A considerable amount of evidence in the literature indicates myocardial abnormalities exist in patients with diabetes independent of vascular diseases (3-5). The spectrum of cardiac abnormalities includes left ventricular (LV) covert diastolic and overt systolic and diastolic dysfunctions (3-5). Streptozotocin (STZ) has been extensively used to produce insulin-dependent (type I) diabetes mellitus (for reviews, see [5-8]). Isolated heart preparations of the STZ-induced diabetic rat have been extensively studied, and depressed peak systolic LV pressure and rates of pressure development and decay have been found after long duration (usually 6 to 8 weeks) of diabetes (9-12). Although these approaches provide high-resolution assessment of physiological end points of chamber performance under controlled conditions, they require killing the animal and are devoid of autonomic reflexes and ventricular-vascular coupling. Furthermore, glucose is almost exclusively used as a substrate in the perfusion medium of isolated heart preparations despite the impairment of glucose metabolism in diabetic hearts. Thus, the isolated heart from the diabetic rat may not reflect integrated ventricular performance that may exist in vivo. Echocardiography recently has been shown to provide reliable and repeatable noninvasive measurements of ventricular dimension and ejection phase indices of cardiac performance in rodents in vivo (13-17). The advantage of this approach is the avoidance of the artificial selection of substrate for energy and modification of the autonomic tone that characterizes isolated heart preparations. Furthermore, repeated noninvasive evaluation in a single animal allows not only the monitoring of the progression of the development of cardiac dysfunction, but also examination of the efficacy of potential therapeutic interventions. Indices of cardiac contraction and relaxation are likely to be influenced not only by the duration and severity of diabetes, but also the workload imposed on the heart. However, very few studies have been conducted that critically evaluate either the progression of cardiac dysfunction, particularly at the early stage of development, or the response of the heart to increased workloads at various stages of cardiomyopathy. Identification of the initiation of the contractile dysfunction is critical to the delineation of the underlying mechanism and evaluation of rational therapeutic interventions. The objective of the present study is to noninvasively examine the progression of changes in LV size and contractile function in vivo by echocardiography in anesthetized STZ-induced diabetic and age-matched rats both

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before and after increased workload with intraperitoneal isoproterenol administration.

Methods Development of Diabetes and Management of Animals Seven- to 8-week-old male Wistar rats weighing 195 to 205 g were injected with either STZ (65 mg/kg body weight [BW]; Sigma Chemical Co, St Louis, MO) or the same volume of buffered vehicle (0.1 mol/L of citrate, pH 4.5) into the tail vein. The rats were maintained individually in steel cases on normal rat chow and water ad libitum. Diabetes was determined by measuring the glucose level with a glucose monitor (Lifescan One Touch; Lifescan, Mountain View, CA) in a drop of blood collected from the tails of anesthetized rats at the indicated time of echocardiographic study. Subsequently, we determined blood glucose levels with an enzyme-linked assay kit (no. 510-DA; Sigma Chemical Co) and corrected the value obtained with the Lifescan monitor by multiplying by 1.37. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication no. 85-23, revised 1996).

Experimental Design and Methods Cardiac function was evaluated by echocardiography in anesthetized diabetic rats and age-matched control rats at 2, 4, 6, and 8 weeks after STZ injection. Three series of experiments with 3 control rats and 3 diabetic rats each were studied at each time point. However, 1 diabetic rat died after the 2-week study and was replaced with another diabetic rat (developed at the same time as the deceased) for subsequent studies; 1 control rat died after 4 weeks in another series; and 1 diabetic rat died after 6 weeks in another series. After narrowing down the period to between 4 and 6 weeks as the initiation of overt contractile dysfunction, a group of 5-week diabetic rats were also studied.

Echocardiography Study On the day of the study, the rats were anesthetized with 30 mg of Nembutal (Abbott Labs, Abbott Park, IL)/kg BW and allowed to breathe spontaneously. BW and blood glucose level were measured. The chest was shaved with electric clippers and electrocardiograph leads were attached to each limb with needle electrodes (Grass Instruments, Quincy, MA). The limbs were secured to the examination table with tape. A warming pad

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(Braintree Scientific Inc, Braintree, MA) was used to maintain normothermia. Cardiac ultrasound studies were performed with an Apogee CX-200 ultrasonograph (ATL-Interspec, Amber, PA). A dynamically focused 9-MHz annular array ultrasound transducer with an axial resolution of 0.2 mm was placed on coupling gel over the hemithorax with care so that the transducer head had adequate contact while avoiding excessive pressure on the chest. The heart was imaged in a shallow left lateral decubitus position, and short- and long-axis views of the left ventricle were obtained by slight angulation and rotation of the transducer. Two-dimensionally directed M-mode of the LV minor axis was taken at the papillary muscle level. Color flow-guided pulsed wave Doppler aortic outflow was performed using a 5-MHz transducer with the sample volume just distal to the aortic leaflets. Early and late LV filling waves on transmitral Doppler were fused because of the rapid heart rates (HRs) and could not be consistently measured. The M-mode and Doppler images were recorded on a 0.5-inch VHS videotape at a speed of 100 mm/s.

Data Analysis M-mode echocardiograms were analyzed from videotape by planimetry of the septal and posterior endocardial echos with a digitizing pad (Summagraphics, Fairfield, CT) interfaced to a dedicated image analysis system (Tomtec, Hamden, CT). LV end-diastolic dimension (LVEDD) was taken as the maximum and LV end-systolic dimension (LVESD) as the minimum LV dimension. The LV fractional shortening (FS) was defined as

Results General Features of the Experimental Animals The BW of the control rats gradually increased from approximately 200 g by 2-fold at 8 weeks after the injection of STZ vehicle (Fig. 1A). In contrast, BW of the STZ-injected rats remained unchanged at approximately 200 g over 8 weeks (Fig. 1A) and was significantly less than that of the age-matched control rats. The difference in BW between control and STZ-injected rats was similar to that observed previously in this laboratory (18). The initial nonfasting blood glucose level in control rats at 2 weeks was approximately 150 mg/dL and was the same up to 8 weeks (Fig. 1B). However, the blood glucose level was 3.5-fold greater in STZ-injected rats at 2 weeks (Fig. 1B) compared with that of age-matched

A 600 ,~ 500

[] STZ

2

4

6

8

2

4

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8

3oo >, 200 O

m 100

100 × ( L V E D D - LVESD)/LVEDD. The peak emptying rate (PER) and peak filling rate (PFR) were computed from the digitized M-mode echocardiograms. Doppler waveforms were analyzed for the aortic peak velocity (APV) and ejection time (ET). The HR-normalized velocity of circumferential FS was calculated from the equation:

[] Control

B --, 700 _1 600 E O

500 400

300

FS/(ET/,~R/R-R-R)

200

where R-R is the interval between 2 beats. The LV mass in vivo was estimated according to Pawlush et al (17).

Statistical Analysis Data are expressed as mean + SE. Sheffe's F test and unpaired t-tests were used to compare data between control and diabetic rats. Paired t-tests were used to compare the changes in parameters before and after isoproterenol administration. A difference between control and diabetic rats was considered significant for P less than .05.

o

100

m

0

0

Weeks Post-STZ Fig. 1. Effect of streptozotocin (STZ) injection on growth and changes in blood glucose level in rats with time. (A) Body weight; (B) blood glucose level. The abscissa represents time after STZ injection in weeks, vertical bars represent SEM, and the numbers inside the bars are number of rats. ([]) Control rats; (ll) STZ-injected rats. *P < .05 v control.

Cardiac Dysfunction In Vivo in Diabetic Rats

control rats. The elevated blood glucose levels remained stable for the duration of diabetes in STZ-injected rats (Fig. 1B). These data indicate the STZ-injected rats were diabetic and remained so for the duration of the study.

Echocardiography M-mode and Doppler echocardiographic images were recorded and stored on videotape. A n example of an M - m o d e image from a control rat and an 8-week diabetic rat are shown in Figure 2. Note the increased HR and decreased cardiac dimensions after isoproterenol administration in the control (Fig. 2A) and diabetic rats (Fig. 2B).

Basal HR and LV Function Basal HR, L V dimensions, and indices o f systolic and diastolic function in both the anesthetized control and diabetic rats are listed in Table 1. H R was significantly less in diabetic rats at all time points studied. L V E D D and L V E S D were similar in control and diabetic rats and did not change significantly over the course of the study, although L V E D D and L V E S D tended to be greater and FS less in diabetic than control rats. The FS was a p p r o x i m a t e l y 50% in control

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rats and r e m a i n e d at this level for the duration o f the study. The FS in diabetic rats was similar to that of control rats at each time point. M o r e o v e r , A P V was similar b e t w e e n control and diabetic rats during the entire duration of diabetes. In contrast, the velocity of circumferential shortening (Vcf) in diabetic rats was similar to that of control rats at 2 and 4 weeks, but was significantly less in diabetic rats at 6 and 8 weeks. PER and P F R were not significantly different b e t w e e n control and diabetic rats at 2 and 4 w e e k s of diabetes. H o w e v e r , PER and P F R were significantly less in the diabetic than control rats at 6 and 8 w e e k s o f diabetes. Thus, in late diabetes (6 and 8 weeks) c o m p a r e d with control, there was a decrease in rate, but not forced e p e n d e n t M - m o d e e c h o c a r d i o g r a p h i c indices of L V function.

LV Mass LV mass and LV mass to B W ratios are shown in F i g u r e 3. LV mass appears decreased in diabetic rats. H o w e v e r , there were no significant differences between control and diabetic rats. H o w e v e r , there were no significant differences b e t w e e n control and diabetic rats in the L V mass to B W ratio at 2 and 4 weeks of diabetes, but at 6 and 8 weeks, the LV mass to B W

A CONTROL RATS Basal

Isoproterenol IVS

Fig. 2. Representative M-mode images of an 8-week (B) diabetic and an age-matched (A) control rat before (basal) and after isoproterenol administration. Time and horizontal dimension scales are the same in all four images. IVS, intraventricular septum; LVPW, left ventricular posterior wall; ECG, electrocardiogram.

LVPW

ECG

B Basal

8W DIABETIC RATS Isoproterenol

T

1 cm

$ (

1 second

>

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Journal of Cardiac Failure Vol. 5 No. 4 December 1999 Table 1. Basal Contractile Function in Control and Streptozotocin-Induced Diabetic Rats 2 Weeks

Parameters HR (beats/min) LVEDD (cm) LVESD (cm) FS(%) Vcf (cm/s) PER (circ/s) PFR (circ/s) APV (cm/s)

4 Weeks

C O N ( n = 9)

D I A ( n = 9)

CON(n = 9)

425 ± 13 0.69 -+ 0.02 0.32 _+ 0.03 53+-4 9.35 _+ 0.68 7.44 _+ 0.80 7.15 _+ 0.76 0.80 + 0.05

342 _+ 15" 0.65 -- 0.03 0.30 + 0.02 54±4 9.21 _+ 0.75 6.05 ± 0.75 6.63 ± 1.10 0.71 _+ 0.05

367 ± 14 0.67 +- 0.03 0.31 ± 0.04 61--7 8.60 ± 0.85 6.07 _+ 0.61 6.12 ± 0.73 0.58 + 0.I0

6 Weeks

D I A ( n = 9) 268 0.72 0.35 51 7.71 5.18 5.19 0.61

± 14" -+ 0.04 _+ 0.03 +2 ± 0.46 ± 0.41 ± 0.35 ± 0.11

8 Weeks

C O N ( n = 8)

D I A ( n = 9)

CON(n = 9 )

D I A ( n = 8)

401 _+ 11 0.68 -+ 0.05 0.31 _+ 0.05 56-+6 9.82 _+ 0.86 6.85 -+ 0.61 7.10 +_ 0.62 0.76 + 0.03

291 _+ 17" 0.70 ± 0.04 0.35 _+ 0.03 50-+4 7.06 _+ 0.48* 4.42 _+ 0.6i* 5.05 _+ 0.65* 0.83 _+ 0.04

346 ± 12 0.69 -+ 0.02 0.27 _+ 0.04 60-+6 10.19 + 0.54 7.81 _+ 1.00 7.11 _+ 1.06 0.58 _+ 0.03

241 _+ 16" 0.76 + 0.04 0.36 ± 0.02 51_+2 7.59 +_ 0.26* 4.97 _+ 0.29* 4.70 +_ 0.64* 0.63 _+ 0.01

Number in parentheses indicates number of rats studied from which measurable data were obtained. CON, controls; DIA, diabetic rats; HR, heart rate; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; FS, fractional shortening; Vcf, velocity of circumferential shortening; PER, peak emptying rate; circ/s, circumferential dimension/second; PFR, peak filling rate; APV, aortic peak velocity. * P < .05 v controls.

ratio was significantly greater in diabetic than control rats.

A Cardiac LV Function After Isoproterenol Administration

2 "-, o')

1.5

== =E >

1

"

0.5

2

4

6

8

2

4

6

8

B 8 7 X

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3 2 1 0

Weeks Post-STZ Fig. 3. Effect o f streptozotocin (STZ)-induced diabetes on

cardiac left ventricular (LV) mass and body weight (BW) ratio of control ([]) and diabetic (il) rats. (A) Absolute LV mass; (B) LV mass-body weight ratios. The LV mass in vivo was calculated from M-mode echocardiographic images and the corresponding body weight of control and diabetic rats were from the data in Fig. 1. The numbers inside the bars represent number of rats studied in that category. *P < .05 v control.

Initially, the generation of isoproterenol dose-response curves was attempted in control and diabetic rats. However, a high mortality in diabetic rats associated with the m a x i m u m dose of isoproterenol forced us to use a submaximal bolus dose of 0.06/xg/kg intraperitoneally. The results are listed in Table 2 as changes in absolute values and shown in Figure 4 as percentage of increase from pretreatment values. Administration of this dose of isoproterenol increased HR to the same extent (approximately 25%) at all time points, but the difference between control and diabetic rats was preserved. In both control and diabetic rats, L V E D D remained similar at 2, 4, and 6 weeks, but was significantly greater in diabetic rats at 8 weeks of diabetes. The L V E S D was similar between control and diabetic rats at 2 and 4 weeks of diabetes; however, the L V E S D in diabetic rats was significantly greater at 6 and 8 weeks of diabetes. Thus, although the FS of control and diabetic rats at 2 and 4 weeks was not different, the FS in diabetic rats were significantly less at 6 and 8 weeks. The Vcf was significantly less at 2, 6, and 8 weeks, but not at 4 weeks. Compared with control rats, P F R and PER in diabetic rats were similar at 2 and 4 weeks, but significantly less at 6 and 8 weeks of diabetes. The A P V was significantly less in diabetic rats at 2, 6, and 8 weeks. Although the A P V in absolute values was not significantly different at 4 weeks, the percentage of increase with isoproterenol was significantly less in diabetic rats compared with that in control rats.

Cardiac Dysfunction In Vivo in Diabetic Rats

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Table 2. Contractile Function in Control and Streptozotocin-Induced Diabetic Rats After Isoproterenol Administration

2 Weeks Parameters HR (beats/min) LVEDD (cm) LVESD (cm) FS (%) Vcf (cm/s) PER (circ/s) PFR (circ/s) APV (cm/s)

CON (n = 9)

DIA (n = 9)

533 _+9 40l _+24* 0.58 -+ 0.02 0.64 ± 0.02 0.16 + 0.01 0.19 + 0.02 73-+3 70-+3 14.76 -+ 1.04 11.75 -+ 0.85* 8.99 + 0.89 8.68 ± 0.98 8.95 -+ 0.92 8.75 ± 1.00 1.05 ± 0.08 0.78 ± 0.03*

4 Weeks CON (n = 9)

6 Weeks

DIA (n = 9)

CON (n = 8)

DIA (n = 9)

8 Weeks CON (n = 9)

DIA (n = 8)

461 ± 25 346 ± 21" 518 _+6 299 + 25* 458 _+ 18 291 ± 23* 0.65 ± 0.03 0.64± 0.03 0.57 + 0.03 0.64_+0.03 0.61 _+0.04 0.72 + 0.02* 0.19 -+ 0.04 0.17-+ 0.02 0.10 ± 0.02 0.27 +- 0.04* 0.16 -+ 0.03 0.29 ± 0.04* 70 +5 74 +3 82±2 60-+4" 74±4 60-+4" 12.69 2 1.23 11.46 + 1.04 15.53± 1.55 9.36 -+ 1.29" 16.62± 1.17 10.54_+ 1.78" 8.61 ± 0.75 8.14+ 0.66 9.12 ± 0.49 5.02 + 0.57* 9.11 -+ 0.84 5.96 ± 0.69* 8.48 ± 0.88 8.59+ 0.70 9.43 ± 0.36 5.80 -+ 0.57* 9.38 + 1.02 5.98 +- 0.65* 0.87 + 0.17 0.68 + 0.12~ 1.27 -+ 0.14 0.90 -- 0.05* 1.28 -+ 0.11 0.71 ± 0.02*

Numbers in parentheses indicate number of rats studied from which measurabledata were obtained. CON, controls;DIA, diabeticrats; HR, heart rate; LVEDD, left ventricularend-diastolicdiameter; LVESD, left ventricularend-systolicdiameter; FS, fractionalshortening;Vcf, velocityof circumferentialshortening;PER, peak emptyingrate; circ/s, circumferentialdimension/second;PFR, peak filling rate; APV, aortic peak velocity. * P < .05 v controls. P < .05 v controls on the basis of percentage increase with isoproterenol.

Basal Contractile Function and Changes With Isoproterenol Administration in 5 - W e e k Diabetic Rats As described above, we determined that overt cardiac contractile dysfunction develops between 4 and 6 weeks of diabetes. Therefore, 5-week diabetic rats were studied to determine whether the changes begin to develop at this time. These rats were made diabetic and maintained under identical conditions, as previously described. B W and blood glucose levels of the 5-week diabetic and age-matched control rats (data not shown) were not significantly different from those of 4- and 6-week diabetic and age-matched control rats, shown in Figure I. Basal HRs of the 5-week diabetic and age-matched control rats were similar to those of 4- and 6-week diabetic rats (Table 3). However, an abnormally low HR of a diabetic rat resulted in a larger SE for this group and a P greater than .05 in comparison to those in control rats. The basal PER and PFR in 5-week diabetic rats were significantly less than those of control rats without other significant changes. The administration of isoproterenol resulted in increases in HR, FS, Vcf, PER, PFR, and APV and a decrease in LVESD. However, diabetic rats showed a significantly reduced response in all parameters except LVEDD and PFR. There was no significant increase in LV m a s s - B W ratio in 5-week diabetic rats. These data indicate overt cardiac contractile dysfunction and abnormal response to isoproterenol begin at 5 weeks of STZinduced diabetes in rats.

Discussion To our knowledge, this is the first study to examine noninvasively cardiac function in vivo over time in STZinduced diabetic rats and to identify the precise time

point at which overt contractile dysfunction develops. Abnormalities of cardiac contraction and relaxation are influenced by the duration of diabetes, suggesting the immediate change in the metabolism that occurs after the development of diabetes may not underlie cardiac contractile dysfunction. Rather, changes caused by metabolic shifts in either the activity or expression of certain gene(s)/protein(s) to a critical level may be responsible. To identify such protein(s), it is important to define the period during which covert contractile dysfunction (for example, evident only after isoproterenol challenge) or overt basal changes develop after the induction of diabetes. Our study indicates overt cardiac contractile dysfunction begins to develop at 5 weeks and is fully manifested after 6 weeks of STZ-induced diabetes in the rat. Our study indicates contractile dysfunction unmasked by isoproterenol also develops at 5 weeks of diabetes in this model. The rats made diabetic with STZ in this study have the usual characteristics of decreased BW and significantly elevated blood glucose level, as previously observed (9-12,18). However, in some studies of isolated hearts or cardiac muscle strips, severe contractile dysfunction was observed 4 weeks after STZ-induced diabetes in the rat. Because glucose was used as substrate in these studies, it is possible that abnormal metabolism of glucose and energy deprivation may have produced an exaggeration of the contractile dysfunction. Severity of diabetes may also be responsible for severity of cardiac contractile dysfunction. In the present in vivo study of diabetic rats in which the blood glucose level was approximately 500 mg/dL, basal LV systolic and diastolic functions in diabetic rats were not significantly different from control rats during the first 4 weeks. The basal contractile dysfunction began at 5 weeks and was fully manifested after 6 weeks of diabetes. In some studies in which the blood

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Journal of Cardiac Failure Vol. 5 No. 4 December 1999

A Control

120 100

80 Fig. 4. Changes in cardiac function in (A) control and (B) diabetic rats after isoproterenol administration. The data from Table 2 are presented as percentage of non-isoproterenol-treated rats. Note that overall increases at 6 and 8 weeks are less in diabetic compared with control rats. HR, heart rate; LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-systolic dimension; FS, fractional shortening; Vcf, velocity of circumferential shortening; PER, peak emptying rate; PFR, peak filling rate; APV, aortic peak velocity. *P < .05 v control.

i 40 20 0

2

4

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6

8

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Diabetes m O ¢-

2 o. O

120

• LVEDD

100

[] LVESD [] FS

80

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60

[] PER [] PFR

~- 40

Ir'IAPV

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u

20 i

2

4

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6

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W e e k s of Diabetes

glucose level of diabetic rats was greater (600 to 700 mg/dL), basal contractile dysfunction in isolated heart or cardiac muscle strips was observed at earlier times after STZ injection (19-21). The results of these studies indicate severity of diabetes may also influence the time course of development of cardiac dysfunction. Overall, the contractile dysfunction in vivo observed by echocardiography in the present study appears to be less severe than that observed ex vivo (9-12,18). We have previously shown a decreased rate of LV relaxation with increased work load induced by a maximum effective dose of isoproterenol in isolated working heart preparations from 4-week diabetic rats (18). In contrast, the present in vivo study showed neither cardiac systolic nor diastolic dysfunction with isoproterenol after

4 weeks of diabetes. The reasons for the discrepancy could be caused by (1) the use of a submaximal dose of isoproterenol in the in vivo study, and (2) the use of glucose as substrate in isolated heart preparations as opposed to glucose, fatty acids, ketones, and amino acids available in intact animals. It is also possible that if the maximum effective dose could be used, it could not only unmask contractile dysfunction at 4 weeks, but also produce more severe dysfunction at 6 and 8 weeks of diabetes. Nevertheless, a submaximal dose of isoproterenol increased HR and LV systolic and diastolic functions in both control and diabetic rats at all the time points studied, but the increases of 6- and 8-week diabetic rats were significantly less than those of the control rats.

Cardiac Dysfunction In Vivo in Diabetic Rats

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Table 3. Basal Contractile Function and Changes With Isoproterenol Administration in 5-Week Streptozotocin-Induced Diabetic and Age-Matched Control Rats Basal Parameters HR (beats/min) LVEDD (cm) LVESD (cm) FS (%) Vcf (cm/s) PER (circ/s) PFR (circ/s) APV (crrds) LV mass/BW ratio X 10 3

After Isoproterenol Treatment

CON (n = 5)

DIA (n = 5)

CON (n = 5)

DIA (n = 5)

384 _+ 10 0.80 ± 0.03 0.33 _+ 0.03 58 ± 3.8 8.9 +- 0.7 10.1 _+0.6 10.1 ± 0.6 0.67 ± 0.05 1.42 ± 0.11

292 ± 62 0.74 -+ 0.03 0.32 ± 0.03 56 +- 4.0 8.5 -+ 0.7 6.3 -+ 0.8* 7.3 ± 1.0t 0.62 +- 0.04 1.60 ± 0.09

501 ± 37 0.70 -+ 0.04 0.16 ± 0.01 77 -+ 1.5 16.7 ± 1.0 14.4 ± 0.5 13.0 ± 1.2 1.05 ± 0.04 --

369 + 57 0.74 +_0.03 0.24 ± 0.02* 66 + 4.0* 10.9 ± 1.4" 9.2 + 1.5" 11.0 ± 1.8' 0.70 ± 0.09* --

CON, controls; DIA, diabetic rats; HR, heart rate; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; FS, fractional shortening; Vcf, velocity of circumferential shortening; circ/s, circumferential dimension/second;PER, peak emptying rate; PFR, peak filling rate; APV, aortic peak velocity; LV, left ventricular; BW, body weight. * P < .05 v control isoproterenol-treated. -I-p < .05 v control basal.

The decreased response of A P V to isoproterenol was the earliest abnormality observed in diabetic rats. It is possible A P V may be a more sensitive indicator of the abnormal response to isoproterenol in STZ-induced diabetic rats. However, we cannot exclude an effect owing to a variable intercept angle between blood flow and ultrasound beam and the relation between LV stroke volume and the dimension of the aortic root, neither of which were measured in the present study. The basal HR in anesthetized diabetic rats was significantly lower compared with that of the control rats at all time points studied. However, the decrease in basal HR observed previously ex vivo in isolated working heart preparations from 4-week diabetic rats was less severe and was not statistically different from controls (18). This discrepancy may be caused by the sympathetic tone in vivo and/or washout of the circulating catecholamines ex vivo in the isolated heart preparations. This may be true because the ex vivo HR in the control rats in that study was lower (18) compared with that observed in vivo. Significantly lower heart rates have also been observed in conscious STZ-induced diabetic rats (19). Thus, bradycardia in vivo is characteristic of the STZinduced diabetic rat. Hypothyroidism (9,22) and/or decreased glucose metabolism (23) may be responsible for the bradycardia in STZ-induced diabetic rats. One limitation of this study is that the depressed velocity-dependent indices of LV function (eg, Vcf, PFR, PER) observed in diabetic compared with control rats could be caused by the slower HRs in the former. However, despite similar differences in HR between control and diabetic groups of rats over 8 weeks, a clear and statistically significant disparity in functional indices, particularly P F R and PER, are evident at 5, 6, and 8 weeks and not earlier despite the lower HR. Furthermore, there was a similar response to HR, but not LV function,

with isoproterenol in diabetic rats at 2 and 4 weeks. Finally, we corrected Vcf for HR; therefore, we believe the differences in LV function that we observed were not entirely caused by differences in HR. Interestingly, a similar disparity between force-dependent and velocitydependent indices (eg, FS v Vcf, respectively) has been shown in primates with pressure-overload hypertrophy (24). It may also be argued that the B W differences are potentially responsible for the echocardiographic changes observed in diabetic rats. Although significant B W differences existed between control mad diabetic rats at 2 and 4 weeks, there were no significant changes in the echocardiographic parameters in diabetic rats. Therefore, it is unlikely differences in B W are entirely responsible for the changes in echocardiographic parameters in diabetic rats at 5, 6, and 8 weeks. Similarly, high glucose or hyperosmolarity per se cannot be responsible for the observed changes because 2- and 4-week diabetic rats were hyperglycemic a n d hyperosmolar and yet no significant difference was observed in echocardiographic parameters. However, these changes may gradually alter cardiac function by affecting some yet unknown process. Another limitation is that differences in blood pressure may potentially account for the differences in echocardiographic parameters in 5-, 6-, and 8-week diabetic rats. However, in separate groups of control and diabetic rats in parallel with the echocardiographic study groups, we monitored systolic blood pressure by tail-cuff method with a pneumatic pulse transducer over 8 weeks. The rats of that study were identical to those used in the current study with respect to B W and blood glucose levels. The systolic blood pressure of control and diabetic rats was within normal range ( < 1 2 0 m m Hg), and there were no significant differences except in the 8-week diabetic rats, in which there was a slight increase (data not shown).

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Therefore, despite the limitation of the tail-cuff technique (8,25), systolic blood pressure cannot be entirely responsible for the changes in echocardiographic parameters in diabetic rats. The underlying mechanism for the contractile dysfunction is not clear. Decreased circulating catecholamine levels appear not to be responsible because HR remained decreased even after isoproterenol administration. Also, increased serum and cardiac norepinephrine levels have been observed in STZ-induced diabetic rats (26). Involvement of abnormal /3-adrenergic receptor function in STZ-induced diabetic rats is controversial. /3-adrenergic receptor numbers and signal transduction pathways have been found to be both preserved (27,28) and decreased in this diabetic rat model (29-33). The reason for this disparity in results is unknown. Now that we have defined the time of beginning of contractile dysfunction that can be unmasked with isoproterenol, the issue of whether /3-adrenergic receptor and its signal downregulation is responsible for the contractile dysfunction can be resolved. Furthermore, the question of whether the alteration of expression of any specific gene and protein involved in the regulation of cardiac contraction or relaxation processes and/or energy deprivation underlie the initiation of contractile dysfunction in diabetes can be examined and accurately evaluated. Although there was no significant difference in LV mass-BW ratio between control and diabetic rats after 2, 4, and 5 weeks, the LV mass-BW ratio was significantly increased at 6 and 8 weeks of diabetes. Weight loss may be the predominant mechanism of this increased ratio, but we cannot exclude the development of LV hypertrophy. Measurements of LV weight-tibial length ratio and morphometry of cardiac myocytes are required to determine whether cardiac hypertrophy is responsible for the apparent increase in LV mass-BW ratio in 6- and 8-week diabetic rats. We observed no significant difference in systolic blood pressure between control and STZ-induced diabetic rats (data not shown). Therefore, the apparent increase in calculated relative LV mass could not be attributed to systolic pressure overload. There appears to be no sign of heart failure up to 8 weeks of diabetes because there was no significant decrease in the LV basal FS. Conversely, in the presence of isoproterenol, there was a significant decrease in LV FS. Therefore, increased workload may hasten heart failure in this model of diabetes.

Conclusion In conclusion, this study shows covert and overt cardiac contractile dysfunction in vivo begin at 5 weeks of STZ-induced diabetes. The decreased systolic and diastolic functions were fully manifested at 6 weeks of

STZ-induced diabetes. Isoproterenol failed to enhance cardiac contractile function of 5 weeks or longer duration of diabetic rats to the level of that of age-matched control rats. The temporal resolution of the initiation of cardiomyopathy in vivo in diabetic rats in the present study will allow examination of potential mechanisms, such as alteration of the expression of specific genes and expression and activity of specific proteins that may underlie the development of cardiac contractile dysfunction. This study also establishes echocardiography as a feasible noninvasive approach for in vivo longitudinal evaluation of the development of cardiac contractile dysfunction in diabetic and other rodent models. This approach may also be used for the longitudinal evaluation of efficacy of chronic metabolic or pharmacological interventions to prevent or treat diabetic cardiomyopathy in vivo.

Acknowledgment The authors acknowledge the secretarial support of Rita Eveleigh and Norma Burns.

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