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Cardiovascular dysfunction and hyperglycemia in STZ diabetesBrazilian Journal of Medical and Biological Research (2004) 37: 1895-1902ISSN 0100-879X

Relationship between cardiovasculardysfunction and hyperglycemia instreptozotocin-induced diabetes in rats

1Instituto de Cardiologia do Rio Grande do Sul,Fundação Universitária de Cardiologia, Porto Alegre, RS, Brasil2Fundação Faculdade Federal de Ciências Médicas de Porto Alegre, Porto Alegre,RS, Brasil3Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre,RS, Brasil4Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brasil5Laboratório de Hipertensão Experimental, Unidade de Hipertensão, Instituto doCoração, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil

B.D. Schaan1,P. Dall’Ago2,C.Y. Maeda3,

E. Ferlin4,T.G. Fernandes3,H. Schmid2 and

M.C. Irigoyen5

Abstract

Streptozotocin (STZ)-induced diabetes in rats is characterized bycardiovascular dysfunction beginning 5 days after STZ injection,which may reflect functional or structural autonomic nervous systemdamage. We investigated cardiovascular and autonomic function, inrats weighing 166 ± 4 g, 5-7, 14, 30, 45, and 90 days after STZinjection (N = 24, 33, 27, 14, and 13, respectively). Arterial pressure(AP), mean AP (MAP) variability (standard deviation of the mean ofMAP, SDMMAP), heart rate (HR), HR variability (standard deviationof the normal pulse intervals, SDNN), and root mean square ofsuccessive difference of pulse intervals (RMSSD) were measured.STZ induced increased glycemia in diabetic rats vs control rats.Diabetes reduced resting HR from 363 ± 12 to 332 ± 5 bpm (P < 0.05)5 to 7 days after STZ and reduced MAP from 121 ± 2 to 104 ± 5 mmHg(P = 0.007) 14 days after STZ. HR and MAP variability were lower indiabetic vs control rats 30-45 days after STZ injection (RMSSDdecreased from 5.6 ± 0.9 to 3.4 ± 0.4 ms, P = 0.04 and SDMMAP from6.6 ± 0.6 to 4.2 ± 0.6 mmHg, P = 0.005). Glycemia was negativelycorrelated with resting AP and HR (r = -0.41 and -0.40, P < 0.001) andwith SDNN and SDMMAP indices (r = -0.34 and -0.49, P < 0.01).Even though STZ-diabetic rats presented bradycardia and hypoten-sion early in the course of diabetes, their autonomic function wasreduced only 30-45 days after STZ injection and these changes werenegatively correlated with plasma glucose, suggesting a metabolicorigin.

CorrespondenceB.D. Schaan

Unidade de Pesquisa

Instituto de Cardiologia do

Rio Grande do Sul

Av. Princesa Isabel, 370

90620-001 Porto Alegre, RS

Brasil

Fax: +55-51-230-3600

E-mail: pesquisa@cardiologia.org.br

Publication supported by FAPESP.

Received August 13, 2003

Accepted August 3, 2004

Key words• Diabetes• Streptozotocin• Heart rate variability• Rats• Autonomic nervous system• Arterial pressure variability

Introduction

Many clinical studies have demonstratedthat chronic diabetic complications occurlate after the onset of the disease, reflectingstructural abnormalities in nerves, kidneys,

retina, and cardiovascular system, stronglyrelating their appearance to the duration ofdiabetes and to glycemic control (1-3). How-ever, some investigators have described theearly appearance of abnormalities whichcould represent a premature development of

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these complications in autonomic and pe-ripheral nerves (4,5). These findings are con-sistent with the hypothesis that poor meta-bolic control is a major determinant of ner-vous damage. Indeed, other studies of type 1diabetic patients have shown that autonomicnervous system abnormalities could be re-versed when better metabolic control wasobtained (6,7).

The injection of streptozotocin (STZ) inrats leads to the development of a clinicalsyndrome characterized by hyperglycemia,excessive osmotic diuresis and loss of weight,which is similar to human diabetes. More-over, the STZ-diabetic rat develops the usualchronic microvascular complications (ne-phropathy, peripheral and autonomic neuro-pathy) as observed in diabetic patients (8-10). Studies on 5-day STZ-diabetic rats fromour laboratory have shown depressed vagaltone, reduction of vagal effect (11) and im-paired tachycardic response to arterial pres-sure (AP) decreases (12). Fifteen days afterSTZ injection we observed impairment ofthe reflex bradycardia and tachycardia pro-duced by vasopressor and vasodepressoragents, respectively (13,14). Similar cardio-vascular changes were described in this mo-del (15) and there is evidence that some ofthese alterations are reversed by insulin thera-py (16). These findings, associated with theimpairment of baroreflex sensitivity, an ex-cellent gauge of autonomic function, sug-gested the early development of autonomicdysfunction in these animals.

Since the autonomic nervous systemmodulates beat-to-beat fluctuations in heartrate (HR), methods to quantify HR and bloodpressure variability have been evaluated asindicators of sympathetic and parasympa-thetic modulation of the cardiovascular sys-tem in humans (17) and in experimentalmodels (18,19). These methods seemed todetect early autonomic dysfunction at a timewhen other metabolic dysfunctional changeswere not clearly observed. Indeed, the rela-tionship between the abnormalities in auto-

nomic modulation and glycemic control ob-served in humans has not been explored indiabetic animals.

The objective of the present study was toinvestigate cardiovascular and autonomicfunctions, evaluated by HR and blood pres-sure variability, and their relationship to gly-cemic control in STZ-induced diabetic ratsat different times after STZ injection.

Material and Methods

In order to obtain a significant number ofanimals with diabetes of different duration,we grouped data from all cardiovascular ex-periments performed in STZ-diabetic rats inour laboratory from 2000 to 2001, assem-bling data from 111 male Wistar rats. Theexperiments were performed by researcherssimilarly trained in data collection, and con-trol and diabetic rats were always evaluatedsimultaneously in order to minimize pos-sible circadian and seasonal changes. Theanimals, weighing 150-280 g, were obtainedfrom the Animal House of Universidade Fe-deral do Rio Grande do Sul, Porto Alegre,RS, Brazil, and kept in small groups withfree access to tap water and standard ratchow.

Animals were made diabetic (D) by ivinjection of 50-60 mg/kg STZ (Sigma, St.Louis, MO, USA) dissolved in sodium cit-rate buffer, pH 4.5. Controls (C) were in-jected with citrate buffer alone. Rats werefasted overnight before STZ administration.STZ-treated rats were used only when theydeveloped elevated plasma glucose levels(confirmed by qualitative measurements ofblood glucose >300 mg% 48 h after injec-tion). Metabolic control was evaluated onthe basis of plasma glucose and animal weightat the end of the experiments. Rats weregrouped according to the time of hemody-namic evaluation after STZ injection, with24 (13C, 11D), 33 (17C, 16D), 27 (15C,12D), 14 (7C, 7D), and 13 (8C, 5D) ratsbeing evaluated after 5-7, 14, 30, 45, and 90

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days, respectively.Catheters filled with saline were im-

planted under anesthesia into the femoralartery and vein (PE-10) for direct measure-ment of AP and drug administration, respec-tively. One day after catheter placement, thearterial cannula was connected to a strain-gauge transducer (P23Db; Gould-Statham,Oxnard, CA, USA) and blood pressure sig-nals were recorded during a 40-min periodwith a microcomputer equipped with an ana-log-to-digital converter board (CODAS, 2-kHz sampling frequency; Dataq Instruments,Inc., Akron, OH, USA). Rats were consciousand moved freely during the experiments.Recorded data were analyzed on a beat-to-beat basis. To evaluate mean AP (MAP)variability, we used the standard deviationof the mean of MAP (SDMMAP) (20), whileHR variability was evaluated by calculatingthe following indices in the time domain: 1)root mean square of successive differencesof pulse intervals (RMSSD), and 2) standarddeviation of the normal pulse intervals(SDNN) (18). Each method employed toanalyze HR variability reflects different pat-terns of variability: RMSSD reflects high-frequency short-term variations in HR, orvagal activity; SDNN is the square root ofthe variance, which is mathematically equalto the total power of the signal. The totalpower corresponds to the contribution of allharmonic components (short-term and long-

term variation) responsible for the variability.Data are reported as means ± SEM. Sta-

tistical significance was calculated by theStudent t-test to compare unpaired data of Dand C during each period after STZ injec-tion. The relationship between metaboliccontrol, measured by plasma glucose and thedegree of autonomic modulation, measuredby the indices cited above, was evaluated bycalculating the Pearson correlation coeffi-cient. Differences were considered to be sig-nificant at P < 0.05 for all tests.

Results

Body weight and plasma glucose

Body weights were similar in all experi-mental groups at baseline. Five to 7 daysafter STZ injection, the weight of diabeticrats was similar to that of controls, althoughtending to be lower. The weight of the dia-betic animals was lower for all other diabeticgroups compared to their controls after 14,30, 45, and 90 days of diabetes. Plasmaglucose levels measured after STZ injectionwere higher than after citrate buffer injectionat all times studied (Table 1).

Arterial pressure and heart rate

Groups studied 5-7 and 14, 30, 45, and90 days after the induction of diabetes pre-

Table 1. Characterization of diabetic (D) and control (C) rats 5-7, 14, 30, 45, and 90 days after STZ injection.

5-7 days 14 days 30 days 45 days 90 days

D (11) C (13) D (16) C (17) D (12) C (15) D (7) C (7) D (5) C (8)

Initial weight (g) 164 ± 5 167 ± 6 206 ± 13 199 ± 14 203 ± 17 157 ± 4 237 ± 10 251 ± 12 225 ± 11 241 ± 9Final weight (g) 170 ± 4 195 ± 9 189 ± 12* 235 ± 15 223 ± 13* 264 ± 5 183 ± 8* 297 ± 11 207 ± 11* 301 ± 10PG (mg/dl) 332 ± 6* 119 ± 7 466 ± 42* 133 ± 8 486 ± 40* 131 ± 21 409 ± 47* 132 ± 18 258 ± 48* 89 ± 13MAP (mmHg) 108 ± 1 112 ± 2 104 ± 5* 121 ± 2 100 ± 4* 114 ± 3 100 ± 6* 115 ± 3 109 ± 0.2 114 ± 2HR (bpm) 332 ± 5 363 ± 12 334 ± 16* 369 ± 9 317 ± 12 347 ± 11 285 ± 7* 379 ± 18 323 ± 30 337 ± 9

Data are reported as means ± SEM for the number of animals indicated in parentheses. PG = plasma glucose; MAP = mean arterial pressure; HR= heart rate.*P < 0.05 vs C rats (Student t-test).

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Figure 1. Standard deviation ofthe normal pulse intervals(SDNN, panel A), root meansquare of successive differenceof pulse intervals (RMSSD,panel B) and standard deviationof the mean of MAP (SDMMAP,panel C) in diabetic (D) and con-trol (C) animals 5-7 (13C, 11D),14 (17C, 16D), 30 (15C, 12D), 45(7C, 7D), and 90 (8D, 5C) daysafter STZ injection. *P < 0.05compared to the respective con-trol (Student t-test).

Figure 2. Scattergrams of meanarterial pressure (MAP, N = 85,panel A), and heart rate (HR, N =87, panel B) versus plasma glu-cose in diabetic and control rats.Correlation was evaluated by thePearson test.

SD

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5-7 14 30 45 90Days after STZ

Diabetic Control

*

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s)S

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(bpm

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sented a significantly lower resting MAPcompared to control at each time point of thestudy. HR was also lower in the D groupcompared to the C group at all times studied,although the difference was not statisticallysignificant for all periods of diabetes evalu-ated (Table 1).

Autonomic function

The evaluation of autonomic functionusing indices of HR variability (SDNN andRMSSD) did not identify differences be-tween the groups at 5-7, 14, 30, 45, and 90days after STZ or citrate injection. The onlyexception was the RMSSD of the 45-day Danimals, which was lower than their C group(P = 0.04) (Figure 1). Also, MAP variabilityevaluated by the SDMMAP was significant-ly lower in the D vs C groups at 30 and 45days after induction (4.2 ± 0.6 and 4.9 ± 0.5vs 6.6 ± 0.6 and 6.8 ± 0.4 mmHg for D and C,respectively; Table 1).

Relationship between cardiovascular changesand plasma glucose

By plotting the results obtained for allanimals, D and C, it was possible to identifya negative relationship between plasma glu-cose levels and MAP (r = -0.41, P < 0.001),HR (r = -0.40, P < 0.001; Figure 2A and B,respectively), SDNN (r = -0.34, P < 0.001)and SDMMAP (r = -0.49, P < 0.001; Figure3A and B, respectively). There was no rela-tionship between RMSSD and plasma glu-cose levels (r = -0.14, P = 0.15).

Discussion

The present results demonstrate that STZ-diabetic rats present changes in resting HRand AP very early in the course of diabetes.Nevertheless, autonomic function evaluatedby time-domain indices of HR and MAPvariability was reduced by diabetes only 30to 45 days after STZ injection. Interestingly,

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all of these changes showed a negative cor-relation with plasma glucose.

Bradycardia in this animal model of dia-betes has already been described by us andothers (11-13,18,21). Autonomic nervoussystem dysfunction, indicated by an increasein vagal tone or a decline in sympathetic toneto the heart may reduce HR, as suggested byJackson and Carrier in 1983 (15), but previ-ous data from our laboratory showed that 5-day diabetic rats had in fact reduced vagalfunction, whereas their sympathetic tone wasnot significantly affected (12). Perhaps theenhanced bradycardia induced by electricalvagal stimulation or methacholine adminis-tration could account in part for the finalresult of lower HR (22). This bradycardiamay also be caused by a change in the elec-trophysiological properties of the sinoatrialnode, since intrinsic HR is reduced (12).Previous results reported by our group (23)demonstrated that diabetes-induced brady-cardia was attenuated by training, a changethat was positively correlated with intrinsicHR. The metabolic improvement character-istic of exercise training may contribute tothese changes.

Although some investigators have de-scribed a so-called STZ hypertension, thisprobably reflects discrepancies between thedirect and indirect blood pressure measure-ments (24), since we and others, using the firstmethod, consistently found hypotension in thisanimal model independently of the duration ofdiabetes (11-13,18). Osmotic diuresis causinghypovolemia or myocardial dysfunction re-ducing contractile force are putative mechan-isms of hypotension in STZ-diabetic rats(23,25). Interestingly, both mechanisms re-flect metabolic derangement. Moreover, anexaggerated pressor response to chronic hy-perinsulinemia has been reported to occur inspontaneously hypertensive diabetic rats (26),an observation that allows us to expect theopposite change in blood pressure in responseto hypoinsulinemia, which is characteristic ofthe STZ-diabetic rat.

In humans, the earliest detectable featureof diabetic cardiac autonomic neuropathy isa defective parasympathetic control, repre-sented by persistent resting tachycardia andloss of beat-to-beat variation during deepbreathing (27), functional changes related tostructural changes, as confirmed by post-mortem studies (28). Fazan et al. (19) sug-gested that impairment of cardiac parasym-pathetic nerve function is present early in thecourse of diabetes in this animal model, asindicated by decreased HR variability. How-ever, other investigators doubt its occurrencebefore diabetes has been present for sometime (29). Indeed, previous results from ourgroup, evaluating MAP variability by thethree-dimensional return map, showed thatrats with short-term STZ diabetes even pres-ent a normal autonomic control of HR andMAP (18). The present results did not dem-onstrate any change in SDNN, the sameindex used by Fazan et al. (19) to assessparasympathetic autonomic neuropathy from5 to 90 days of diabetes duration. Anotherstudy, although evaluating spontaneouslyhypertensive diabetic rats, analyzed the al-terations in AP and HR variability usingspectral analysis approaches and found a

Figure 3. Scattergrams of thestandard deviations of the nor-mal pulse intervals (SDNN, N =85, panel A) and of the mean ofMAP (SDMMAP, N = 86, panelB) versus plasma glucose in dia-betic and control rats. AU = arbi-trary units. Correlation wasevaluated by the Pearson test.

SD

NN

(A

U)

30

252015

10

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r = -0.34P < 0.001

0 500 1000

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0 200 1000

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reduced variability of AP without changes inHR variability in diabetic animals (30). Thisdisagreement may be accounted for by thedifferences in the sensitivity of the methodsused to evaluate autonomic dysfunction, i.e.,time domain or frequency domain indices.The differences between the current resultsand previous ones obtained by our groupcould also be accounted for by differentmethodologies. Disturbances in autonomicfunction were observed in 5-day diabeticrats when evaluated by baroreflex sensitivityand after autonomic blockade with propran-olol and atropine (11,12), which could bemore sensitive than the indices used in thepresent study.

Spontaneous fluctuations in MAP andHR have been used to detect autonomicdysfunction in humans (17). The reducedMAP variability, evaluated by SDMMAP,observed in the diabetic groups in the pres-ent study may indicate early functional auto-nomic cardiovascular dysfunction (31). Theindices used to evaluate MAP and HR varia-bility were negatively correlated with plasmaglucose, suggesting that MAP and HR couldbe altered by diabetic metabolic decompen-sation. Accordingly, in spontaneously hy-pertensive diabetic rats, reflex bradycardicindex and the power of low frequency oscil-lations of systolic arterial pressure are in-versely related to blood glucose, reinforcingthe crucial role of metabolic changes in thecardiovascular disorder in this model (30).Hicks et al. (21), in 1998, observed the sametendencies as observed here: a decline in themagnitude of the circadian variation anddecreased sympathetic and parasympatheticnervous tone to the heart after STZ injection,changes which were reversed by insulin treat-ment.

It is unlikely that bradycardia and hypo-tension could be the result of autonomicneural structural injury; instead, they couldrepresent autonomic neural dysfunctioncaused by metabolic changes induced bySTZ diabetes. This is supported by the nega-

tive correlation observed between MAP andHR with plasma glucose. Indeed, other in-vestigators have demonstrated that brady-cardia and hypotension induced by experi-mental diabetes can be prevented by insulintreatment (16,30). Our group observed re-duced intrinsic HR in short-term STZ diabe-tes, which could be the result of metabolicinjury to pacemaker cells (11). Reducedmotor nerve conduction velocity is also nor-malized by insulin treatment in this animalmodel (32). These data suggest that theseearly phenomena constitute a stage of neuro-logical dysfunction distinct from “true” neu-ropathy (28). In fact, other investigators didnot find structural abnormalities in the vagusnerve, which consists of parasympatheticpreganglionic fibers, or enteric ganglia, whichconsist of parasympathetic neurons in STZ-diabetic rats with a 6- to 12-month durationof diabetes (33,34). Conversely, relevantstructural changes characterized by axonaldystrophy did occur in the sympathetic pre-ganglionic nerve fibers, but these changeswere observed 12 months after STZ injec-tion (34).

Several mechanisms have been consid-ered to be involved in the pathogenesis ofdiabetic neuropathy in the STZ-diabetic rat,but hyperglycemia is always implicated.Structural changes occurring in peripheralnerves resemble those of human diabeticneuropathy and are preceded by hyperglyce-mia-induced biochemical abnormalities.Recent evidence has emphasized the impor-tance of vascular dysfunction, driven bymetabolic insults to the nerves, as a cause ofdiabetic neuropathy. Non-enzymatic glyco-sylation of myelin components, reduction ofendoneural blood flow, increased oxygenfree radical activity, or production and dep-rivation of the nerve growth factor are alsoinvolved (32,35).

In agreement with our results, Burger etal. (36) described the reversibility of earlycardiac autonomic neuropathy assessed bypower spectral analysis of HR variability by

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glycemic control in type 1 diabetic patients,and comparable results were also publishedby Muhr-Becker et al. (37) in a study evalu-ating cardiac sympathetic dysinnervationscintigraphically in a similar group of pa-tients.

A direct effect of hyperglycemia on vas-cular and myocardial cells should also beconsidered as a cause for cardiovascular dys-function in this model. The protein kinase C(PKC) pathway, which is activated by hy-perglycemia, has been recently recognizedas an important mechanism in the develop-ment of diabetic complications including car-diomyopathy and angiopathy (38). Up-regu-lation of PKCß2 was demonstrated in theheart and aorta at both the transcriptionaland translational levels during the early stagesof experimental diabetes, suggesting its role

in the diabetic injury to the cardiovascularsystem (39).

Our results demonstrate some well-knowncardiovascular alterations presented by acommonly used diabetic model, the STZ-diabetic rat, such as hypotension and brady-cardia, and transitory autonomic index de-rangements. Considering data reported byother investigators who demonstrated thatautonomic nerve structural lesions do notappear as early as these functional changes,we question whether these changes could beassigned to early development of autonomicneuropathy in this model, but we accept thepresence of reversible neurological dysfunc-tion. The negative correlation observed be-tween cardiovascular dysfunction and plasmaglucose is in accordance with these observa-tions.

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