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Journal of Diabetes and Its Complications 27 (2013) 1622

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Journal of Diabetes and Its Complications


j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M

Impact of myocardial infarction on cardiac autonomic function in diabetic rats


Bruno Rodrigues a, b,, Cristiano T. Mostarda b, Luciana Jorge b, Catarina A. Barboza a, Camilla F. Grans a, Ktia De Angelis c, Maria Cludia Irigoyen b
a b c

Human Movement Laboratory, So Judas Tadeu University, So Paulo, Brazil Hypertension Unit, Heart Institute (InCor), Medical School of University of So Paulo, So Paulo, Brazil Translational Physiology Laboratory, Nove de Julho University, So Paulo, Brazil

a r t i c l e

i n f o

a b s t r a c t
Aims: We evaluated autonomic and hemodynamic parameters and maximal oxygen consumption (VO2max) as possible determinants of mortality in streptozotocin (STZ) diabetic rats after myocardial infarction (MI). Method: Male Wistar rats were divided into (n=8 of each): control sham (CS), diabetes sham (DS), MI (I), and diabetes+MI (DI). MI was induced 15 days after STZ induction. VO2max was measured at 3 (basal), 30, 60, and 91 days after MI. Hemodynamic and autonomic parameters were evaluated 92 days after MI. Results: MI area was similar in infarcted groups (~44%). Mortality rate increased in the DI (70%) compared with I (53%) group. Cardiopulmonary baroreex, sympathetic (48%) and vagal (33%) tonus, low frequency (LF) band (57%), and LF/high frequency (HF) band ratio (53%) were reduced in DI compared with I animals. Furthermore, cardiac output (CO), peripheral vascular resistance (PVR) impairment, and VO2max reductions were observed in the DI compared with the I group. Conclusions: Our data suggest that the CO and PVR changes as well as VO2max reduction were probably associated with additional cardiac autonomic control impairment, and, consequently, increased mortality rate in diabetic rats after a chronic myocardial infarction. 2013 Elsevier Inc. All rights reserved.

Article history: Received 26 March 2012 Received in revised form 4 July 2012 Accepted 7 August 2012 Available online 6 October 2012 Keywords: Diabetes Myocardial infarction VO2 max Hemodynamic assessments Autonomic dysfunction

1. Introduction The prevalence of diabetes in developed countries is approaching epidemic proportions and has been associated with an increased risk of cardiovascular abnormalities and microvascular complications (Haffner, Lehto, Ronnemaa, Pyorala, & Laakso, 1998). One of the most serious complications of diabetes is cardiovascular autonomic neuropathy, which results in abnormalities in heart rate control and vascular dynamics (Maser, Mitchell, Vinik, & Freeman, 2003) and has been related to the subsequent incidence of a fatal or nonfatal cardiovascular event (Liao, Carnethon, Evans, Cascio, & Heiss, 2002). Diabetic patients have a two-fold higher risk of short-term mortality after MI, even after adjustment for the extent of coronary heart disease (Woodeld et al., 1996). In the thrombolytic era, many diabetic patients have survived cardiac events; however, how diabetes affects the long-term prognosis of these early MI survivors is less certain. Gustafsson et al. (2000) evaluating pre-admission history, presentation, initial treatment, and long-term mortality in patients with MI and diabetes concluded that diabetic compared with

The authors declare that they have no conicts of interest. Corresponding author. Hypertension Unit, Heart Institute (InCor), Av. Dr. Eneas de Carvalho Aguiar, 44-Subsolo, So Paulo, So Paulo, Brazil-05403-000. Tel.: +55 11 2661 5006; fax: +55 11 2661 7887. E-mail address: bruno.rodrigues@incor.usp.br (B. Rodrigues). 1056-8727/$ see front matter 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jdiacomp.2012.08.002

nondiabetic patients treated with oral hypoglycemic agents or insulin, but not those treated with diet alone, have a signicantly increased mortality following acute MI. In post-MI diabetic patients, autonomic function can be affected by diabetes sequelae, MI sequelae, or both, and has been associated with increased risk of morbidity and total mortality (Barthel et al., 2011). In the experimental setting, our group has previously evidenced autonomic dysfunction in streptozotocin diabetic rats (De Angelis et al., 2000; Schaan et al., 2004; Farah et al., 2007; De Angelis, Irigoyen, & Morris, 2009; Mostarda et al., 2009) and MI animals (Lacerda et al., 2007; Flores et al., 2010; Mostarda et al., 2010; Jorge et al., 2011), being normalized by aerobic exercise training. However, the autonomic function when associated with these experimental conditions has been poorly investigated. Exercise intolerance is a common characteristic observed in diabetic (Vinik & Ziegler, 2007) and chronic heart failure patients (Negrao & Middlekauff, 2008) and has been considered an important determinant of mortality in these populations. Autonomic dysfunction may impair exercise tolerance and has been shown to reduce heart rate, blood pressure, and cardiac output responses to exercise in persons with diabetes (Vinik & Ziegler, 2007). Furthermore, it has been proposed that impaired cardiac reserve and peripheral abnormalities, rather than resting cardiac function, may be more directly correlated with impaired exercise ability in heart failure patients (Negrao & Middlekauff, 2008). In this sense, it is

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possible that the set of abnormalities intrinsic to diabetes or MI, or both together, may play a critical role in the abnormal exercise performance in these individuals. We hypothesized that the association between diabetes and chronic MI could be related to additional worsening of cardiac autonomic dysfunction, resulting in hemodynamic and exercise capacity impairments, with consequently increase in mortality rate. Thus, the purpose of this study was to conduct a screening of cardiac autonomic function and hemodynamic measurements, and to evaluate the maximal oxygen consumption (VO2max), and the possible repercussion of these clinical parameters on the mortality rate of diabetic rats after chronic MI. 2. Materials and methods

delimited leading to analysis of the movement of the LV walls. Regions with systolic shortening classied as absent were considered infarcted (Rodrigues, Figueroa, et al., 2011; Rodrigues, Rosa, et al., 2011; Jorge et al., 2011). To conrm the echocardiographic measurement, the MI area was also evaluated after all evaluations by dissecting the brous scar from the remaining LV muscle. Initially, the MI area was conrmed by gross observation of the LV scar. After dissection of the scar area, the outlines of fragments were drawn on graph paper, and their areas were measured by the cross-point method (Flores et al., 2010; De Angelis, Leirner, Irigoyen, & Cestari, 2001; Lindpaintner et al., 1993). The interventricular septum was considered part of the left ventricle.

2.4. Maximal oxygen consumption (VO2max) 2.1. Experimental design Experiments were performed using adult male Wistar rats (230 260 g) obtained from the animal facility at the University of So Paulo (So Paulo, Brazil). The rats were fed standard laboratory chow and water ad libitum. They were housed in collective polycarbonate cages in a temperature-controlled room (22 C) with a 12-h darklight cycle (light 07:0019:00 h). The Institutional Animal Care and Use Committee of the Medical School of the University of So Paulo approved this protocol; this investigation was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). The rats were randomly assigned to 4 groups: control sham (CS, n=10), diabetic sham (DS, n=20), control infarcted (I, n=30), and diabetes+myocardial infarction (DI, n= 30). Experimental diabetes was induced by an intravenous injection of 50-mg/kg streptozotocin (STZ) (Sigma Chemical Co., St. Louis, MO) dissolved in citrate buffer (pH 4.2). The rats were fasted overnight before the STZ injection. The CS and I rats were injected with buffer only (10 mM citrate buffer, pH 4.5). Forty-eight hours after the STZ injection, diabetes was conrmed by the measurement of blood glucose levels above 200 mg/dL in fasted (6 h) rats. 2.2. Myocardial infarction Fifteen days after the STZ injection or administration of the citrate buffer, the rats were anesthetized (80 mg/kg Ketamine and 12 mg/kg Xylazine, administered i.p.) and underwent MI induction by surgical occlusion of the left coronary artery, as described elsewhere (Rodrigues, Figueroa, et al., 2011; Rodrigues, Rosa, et al., 2011; Jorge et al., 2011). Briey, after intubation, animals were positive-pressure ventilated with room air at 2.5 mL, 65 strokes/min with a pressure cycled rodent ventilator (HARVARD Apparatus, Model 683, Holliston, MA). For induction of MI, a 2-cm left lateral thoracotomy was performed in the third intercostal space, and the left anterior descending coronary artery was occluded with a nylon (6.0) suture at approximately 1 mm from its origin below the tip of the left atrium. The chest was closed with a silk suture. The animals were maintained under ventilation until recovery. The sham rats (CS and DS) underwent the same procedures except that myocardial ischemia was not induced. The mortality rate was investigated during the 90 days of the protocol (n=90) beginning after the MI and/or Sham surgery (to exclude the inuence of anesthesia or stress from the surgical procedure). Eight animals from each group were evaluated 90 days after myocardial infarction induction (105 days after diabetes induction) to perform the following experimental protocols. 2.3. Myocardial infarction area determinations The MI area was evaluated by echocardiography 2 days (initial evaluation) and 90 days (nal evaluation) after MI surgery and was VO2max was measured at basal (3 days), 30, 60, and 91 days after MI or Sham surgery. The VO2 max was determined by analyzing expired gas during a progressive exercise ramp protocol, with 3 m/min increments every 3 min and no grade until exhaustion. The experimental animals only started the exercise test when the levels of resting VO2 were near to 305 mL/kg/min. Gas analysis was performed using an oxygen (S-3A/I) analyzer (Ametek, Pittsburgh, PA, USA). The VO2 was calculated using the measured ow through the metabolic chamber, the expired fraction of efuent oxygen, and the fraction of oxygen in room air (Rodrigues et al., 2007; Jorge et al., 2011).

2.5. Hemodynamic measurements One day after the last maximal oxygen consumption evaluation, 2 catheters lled with 0.06 mL of saline were implanted into the femoral artery and femoral vein of the anesthetized rats (80 mg/kg Ketamine and 12 mg/kg Xylazine, i.p.). Twenty-four hours later, the arterial cannula was connected to a strain-gauge transducer (Blood Pressure XDCR; Kent Scientic, Torrington, CT, USA), and arterial pressure (AP) signals were recorded over a 30-min period in conscious animals by a microcomputer equipped with an analog-to-digital converter board (WinDaq, 2 kHz, DATAQ, Springeld, OH, USA). The recorded data were analyzed on a beat-to-beat basis to quantify changes in mean AP (MAP) and heart rate (HR). Pulse interval variability (PIV) in time domain was determined by using the standard deviation of the basal HR recording period for 30 min (Rodrigues, Rosa, et al., 2011; Jorge et al., 2011). Responses to stimulation of cardiopulmonary baroreex sensitivity (CBS) (BezoldJarish reex) were determined in conscious animals. After recording 30 min of resting AP and HR, sequential bolus injections (0.1 mL) of increasing doses of serotonin (2, 4, and 8 g/kg; Sigma Chemical Co. Perth, West Australia) were given to the animals while AP and HR were recorded. For data analysis, maximum reductions in HR and AP for each given dose were used to calculate the CBS. Injections were not repeated until the recorded parameters had returned to basal levels (Lacerda et al., 2007; Flores et al., 2010). After CBS assessment, AP and HR were continuously recorded during the basal state and after methylatropine (3 mg/kg, IV) injection (0.2 mL). Because the HR response to the drug reaches its peak within 3 to 5 min, this time interval was allowed to elapse before HR measurement. Atenolol (8 mg/kg, IV) was injected (0.2 mL) 10 min after methylatropine, and again the response was evaluated after simultaneous blockade with atenolol and methylatropine. The next day, the sequence of injections was inverted (rst atenolol and then methylatropine). The intrinsic heart rate (IHR) was evaluated after simultaneous blockade with atenolol and methylatropine. Sympathetic tonus was determined as the difference between maximum HR after methylatropine injection and IHR. Vagal tonus was obtained by

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the difference between the lowest HR after atenolol injection and IHR (Mostarda et al., 2009). 2.6. Cardiac output and peripheral vascular resistance determinations After cardiovascular assessments, the left ventricle (LV) was catheterized in anesthetized rats (pentobarbital sodium, 40 mg/kg). One catheter of PE-50 was inserted into the right carotid artery and advanced into the LV. Yellow (150,000) 15-mm Dye-Trak microspheres (Triton Technology, San Diego, CA, USA) were infused for cardiac output (CO) and peripheral vascular resistance (PVR) determinations. Microsphere infusion and processing were performed as described previously (Jorge et al., 2011). 2.7. Cardiac autonomic modulation The overall variability of PIV was assessed in the time and frequency domains by spectral estimation. Fluctuations in PI were further assessed in the frequency domain by autoregressive spectral estimation. The theoretical and analytical procedures for autoregressive modelling of oscillatory components have been described previously (Malliani & Pagani, 1991; Mostarda et al., 2010). Briey, the PI series derived from each recording was divided into 300 beat segments with a 50% overlap. The spectra of each segment were calculated via the LevinsonDurbin recursion and the order of the model chosen according to Akaike's criterion, with the oscillatory components quantied in low frequency (LF; 0.20.6 Hz) and highfrequency (HF; 0.63.0 Hz) ranges. The normalized units were obtained by calculating the power of LF and HF correlating each to the total power, after subtracting the power of the very LF component (frequencies b 0.2 Hz). 2.8. Statistical analysis The data are reported as meansSEM. After conrming that all continuous variables were normally distributed using the Kolmogorov Smirnov test, statistical differences between the groups were identied using two-way analysis of variance (ANOVA) or repeated-measures ANOVA followed by the StudentNewmanKeuls post-test. Pearson's correlation was used to study the association between the variables. A survival curve was constructed using the KaplanMeier method, and the survival curves of the experimental groups were compared using the log-rank test. All of the tests were 2-sided, and the signicance level was established at P b .05. Calculations were performed with Statistical Package for Social Sciences (SPSS) software, version 12.0.

3. Results 3.1. Animals Body weight was similar among study groups at the beginning of the study (~23015 g). At the end, STZ-diabetic rats exhibited a reduction in body weight (DS: 21510 and DI: 21010 g) compared with that in normoglycemic rats (CS: 5122 and I: 4628 g). Glycemia was higher in STZ-diabetic rats (DS: 40425 and DI: 376 32 mg/dL) than in normoglycemic rats (CS: 921 and I: 954 mg/dL). MI area measured by echocardiography was similar between infarcted groups at the initial (I: 442 and DI: 432% of LV wall) and nal (I: 463 and DI: 425% of LV wall) evaluations. We also evaluated the MI area by millimeter graph paper stamps. Corroborating the echocardiographic data, MI groups had a similar MI area at the end (I: 502 and DI: 484%) and displayed a positive correlation (r=0.87, P=.001) with the MI area evaluated by echocardiography. 3.2. Mortality rate Total mortality rate evaluation (KaplanMeier survival curve) showed that the I (16 deaths among 30 animals, 53% mortality) and DI (21 deaths among 30 animals, 70% mortality) groups had a higher mortality rate than the C (no deaths) and D groups (8 deaths among 20 animals, 40% mortality) (P=.001). However, it is important to highlight that the DI compared with I group had an increased mortality rate. 3.3. Maximal oxygen consumption (VO2max) Values of VO2max at basal, 30, 60, and 91 days after MI are presented in Fig. 1. The DS group displayed a VO2max reduction in all evaluated times compared with the CS group. As expected, MI induced a reduction in VO2max in I and DI compared with CS and DS animals, in all experimental evaluated periods. Furthermore, the DI group had an additional reduction in VO2 max at 60 and 91 days compared with the I group. 3.4. Hemodynamic and autonomic evaluations Hemodynamic measurements are presented in Table 1. STZdiabetic groups (DS and DI) had an additional reduction in systolic arterial pressure (SAP) compared with CS and I rats. Furthermore, MI induced a reduction in systolic arterial pressure in the I compared with the CS group. HR was reduced in DS and DI groups compared

Fig. 1. Maximal oxygen consumption (VO2max) at basal, 30, 60, and 91 days after myocardial infarction from control (CS), diabetes (DS), myocardial infarction (I), and diabetes+myocardial infarction (DI) rats (n=8 for each group). # P b .05 vs. initial evaluation; * P b .05 vs. C; P b .05 vs. D; P b .05 vs. I.

B. Rodrigues et al. / Journal of Diabetes and Its Complications 27 (2013) 1622 Table 1 Hemodynamic measurements in control (CS), diabetes (DS), myocardial infarction (I), and diabetes+myocardial infarction (DI) groups. Measurement/Group SAP (mm Hg) DAP (mm Hg) MAP (mm Hg) HR (bpm) CO (mL/min) PVR (mmHg/mL/min) CS 1254 913 1083 3365 1114 0.950.08 DS 1042* 852 944* 2813* 555* 1.650.09* I 1142* 863 1002 3496 646* 1.450.08* DI 1032* 832 932* 28210* 454* 1.750.09*

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Table 2 Autonomic evaluations in control (CS), diabetes (DS), myocardial infarction (I), and diabetes+myocardial infarction (DI) groups. Measurement/Group Autonomic modulation SD-PI (bpm) VAR-PI (ms2) LF (ms2) LF (nu) HF (ms2) HF (nu) LF/HF Autonomic tonus ST (bpm) VT (bpm) IHR (bpm) CS 304 230.8 3.10.5 21.91.5 11.01.4 78.11.5 0.290.03 491 663 3735 DS 192* 151.9* 1.00.2* 13.21.0* 6.81.2* 86.81.0* 0.150.01* 385 354* 2904* I 213* 131.4* 1.20.2* 15.51.2* 6.71.1* 84.81.2* 0.170.02* 916* 273* 35015 DI 101* 101.2* 0.40.1* 6.70.9* 5.61.0* 93.30.9* 0.080.01* 477 182* 2854*

Values are expressed as meanSEM. Systolic arterial pressure (SAP); Diastolic arterial pressure (DAP); Mean arterial pressure (MAP); Heart rate (HR); Cardiac output (CO); Peripheral vascular resistance (PVR). * P b .05 vs. CS; P b .05 vs. DS; P b .05 vs. I.

with that in CS and I groups. Cardiac output was reduced, and peripheral vascular resistance was increased in all experimental groups compared with the CS group, with additional impairment of these hemodynamic parameters in the DI group compared with the I group. CBS evaluated by bradycardic responses to serotonin was reduced in DS, I, and DI groups compared with the CS group in the 4 and 8 g/kg doses (Fig. 2A). However, STZ-diabetic groups (DS and DI) had additional impairment compared with the I group in all injected doses. Hypotensive responses to serotonin were reduced in DS and DI groups compared with that in CS and I groups. Moreover, in the 8-g/kg dose, the I group had a reduced hypotensive response compared to the CS group (Fig. 2B). Autonomic evaluations are represented in Table 2. The PIV, evaluated in the time domain by standard deviation of the pulse interval (SD-PI) and by total variance (VAR-PI) was reduced in all experimental groups compared with that in the CS group, with additional impairment in the DI compared with DS and I (only to SDPI) groups (Table 1). In the frequency domain, the DS, I, and DI groups had a reduction in the low frequency (LF) and high frequency (HF) bands, in the absolute and normalized values, compared with the CS group. The DI rats had an accentuated reduction in the LF and HF normalized values in relation to DS and I rats. Taken together, as a consequence of the two frequency band reductions, the LF/HF ratio was also reduced in DI compared with that in CS and I groups, suggesting an additional impairment in autonomic balance in the association of diabetes and MI. Sympathetic tonus was increased in the I group compared with CS, D, and DI groups. Vagal tonus declined in all experimental groups compared to that in CS animals. Moreover, the DI group had additional vagal tonus impairment in relation to the D group. A

Values are expressed as meanSEM. Standard deviation of pulse interval (SD-PI); Total variance of pulse interval (VAR-PI); Low frequency band (LF); High frequency band (HF); LF/HF balance; Sympathetic tonus (ST); Vagal tonus (VT); Intrinsic heart rate (IHR); normalized units (nu). * P b .05 vs. C; P b .05 vs. D; P b .05 vs. I.

reduction in intrinsic heart rate was observed in DS and DI groups compared with the CS and I groups (Table 2). 3.5. Correlations We additionally observed some associations between the presented data, as depicted in Fig. 3. A negative correlation between peripheral vascular resistance and VO2max (Fig. 3A), and a positive correlation between vagal tonus and SD-PI (Fig. 3B) were observed in all experimental animals. 4. Discussion We screened autonomic, functional, metabolic, and hemodynamic markers in diabetic rats undergoing MI. We found an additional autonomic impairment represented by reduced CBS, vagal tonus, and cardiac modulation in DI rats. These cardiac autonomic changes were probably associated with exacerbated hemodynamic (cardiac output and PVR) and cardiorespiratory capacity (VO2max) dysfunctions, as well as increased mortality rate in DI animals compared to I animals. Patients with diabetes mellitus experience a high rate of cardiovascular events, and cardiac death is their leading cause of mortality. Several studies reported that diabetes was associated with a worse cardiovascular prognosis at 6-month to 2-year follow-up

A
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5 HT
2 g/Kg 4 g/Kg 8 g/Kg

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-100 -150 -200 -250 -300

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-15 -20 -25 -30 -35 -40

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Fig. 2. Cardiopulmonary baroreex assessment evaluated by injection of 2, 4, and 8 g/kg serotonin doses. HR (A) and AP (B) responses in the control (CS), diabetes (DS), myocardial infarction (I), and diabetes+myocardial infarction (DI) rats (n=8 for each group). * P b .05 vs. C; P b .05 vs. D; P b .05 vs. I. 5 HT, serotonin; HR, heart rate; AP, arterial pressure.

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90 80 VO2m max (ml/Kg/min-1) 70 60 50 40 30 20 10 0 0.0 0.5 1.0 1.5 2.0 2.5 PVR (mmHg/mL/min) r = - 0.56 P = 0.008

4.1. Autonomic impairment Impairment in autonomic function has classically been observed in diabetic subjects (Vinik & Ziegler, 2007), MI patients (La Rovere, Bigger, Marcus, Mortara, & Schwartz, 1998), and in both (Barthel et al., 2011). Either pathology, i.e., MI and diabetes-related autonomopathy, has been separately linked to an increased mortality risk (La Rovere et al., 1998; Maser et al., 2003). However, a substantial fraction of diabetic post-MI patients presenting with severe autonomic failure might suffer from a combination of both (Barthel et al., 2011). In the present study, we observed that DI animals displayed additional autonomic dysfunction compared to I animals, observed by reduced cardiopulmonary baroreex sensitivity, PIV, LF band, and LF/ HF ratio. These data suggest that STZ-diabetes represents an additional role in the impairment of autonomic cardiovascular control after MI. In fact, Fazan, Ballejo, Salgado, Moraes, and Salgado (1997) reported reduced PIV in STZ diabetic rats, associated with reductions in the LF and HF components. Furthermore, studies from our group showed a reduction in both PIV and blood pressure variability in STZ diabetic rats (Schaan et al., 2004; Farah et al., 2007). These autonomic changes were correlated with plasma glucose, suggesting that increased glycemia was associated with neural dysfunction. To conrm our spectral analysis data, we evaluated the autonomic tonus through the infusion of atenolol and methylatropine. As expected, sympathetic tonus was increased and vagal tonus was reduced in the I group. It is likely that activation of cardiac sympathetic afferent bers increases sympathetic outow during the phase of diastolic dysfunction after MI. The later development of ventricular dysfunction is also associated with further increments in neurohumoral excitation, due to arterial and cardiopulmonary baroreceptor abnormalities (Malliani & Montano, 2002). On the other hand, STZ-diabetic groups (DS and DI) displayed maintenance of sympathetic tonus and reduction of vagal tonus in comparison with CS and I groups. However, the DI group had an additional reduction in vagal tonus in relation to all the experimental groups. These data corroborate previous ndings of our group that have shown a reduction in vagal tonus and maintenance of sympathetic tonus to the heart (De Angelis et al., 2000), suggesting the presence of cardiac vagal neuropathy that was accentuated by the MI. Furthermore, a positive correlation was observed between vagal tonus and standard deviation of pulse interval, suggesting that the reduction of the cardiac parasympathetic component may have been the main determinant of pulse interval variability impairment in diabetic and MI animals. Taken together, in the present investigation the association between diabetic neuropathy and autonomic dysfunction triggered by MI may explain the additional worse cardiac autonomic function and cardiopulmonary reex observed in the DI group.

B
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r = 0.57 P = 0.004

0 0 10 20 30 40 50 60 70 80

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Fig. 3. Correlation obtained by linear regression in all of the studied animals between (A) peripheral vascular resistance (PVR) and maximal oxygen consumption (VO2max); and (B) vagal tonus and standard deviation of pulse interval (SD-PI).

(Zuanetti et al., 1993; Mak et al., 1997; Mukamal et al., 2001; Murcia et al., 2004). Community-based studies, including that reported by Orlander et al. (1994), showed that after adjustment for age, sex, and ethnicity, the risks of death were 37.4% among patients with diabetes and 23.3% among those without after 44 months. Recently, Bot, Pouwer, Zuidersma, van Melle, and de Jonge (2012) reported that the coexistence of diabetes and depression after MI is related to increased mortality, beyond the effect on mortality of diabetes and depression alone in MI patients. These data reinforce the importance of the psychosocial aspects that may impair the time course of diabetes and MI. In the experimental setting, Greer, Ware, and Lefer (2006) using an excellent model of type 2 diabetes, the db/db mice, observed that diabetic animals also experienced signicantly greater mortality after both 30 and 45min of myocardial ischemia compared with either sham operated diabetic or nondiabetic mice. The authors also showed that this pathological response was generally consistent with the development of heart failure in db/db mice, revealing a novel model of heart failure in the diabetic myocardium that allows for the investigation of the pathophysiology of heart failure in Type 2 diabetes. Our group previously demonstrated that, although the increased left ventricular contractility, STZ-diabetic animals after 90days of MI also displayed an increased mortality rate compared with nondiabetics (Rodrigues, Rosa, et al., 2011). A possible explanation observed in this study was that the diabetic MI animals had worse autonomic function, as assessed only by arterial baroreex sensitivity (Rodrigues, Rosa, et al., 2011). Thus, to further investigate the changes in autonomic function and its possible hemodynamic and functional complications in diabetic animals after chronic MI, some important clinical parameters were assessed in these pathological conditions.

4.2. Hemodynamic changes and exercise capacity Regarding hemodynamic data, the diabetic groups displayed reduced arterial pressure compared with nondiabetic groups. Jackson and Carrier (1983) have suggested that the decrease in arterial pressure in STZ-induced diabetic rats at rest may be the result of decreased cardiac output due to hypovolemia caused by hyperglycemic osmotic diuresis. In fact, in the present study, the reduction in HR in diabetic rats, associated with cardiac contractility evoked by MI may have been the main cause of additional CO reduction, and consequently arterial pressure decrease in DI animals. The reduction of HR in diabetic animals at rest has been attributed to changes in the sinoatrial node (De Angelis et al., 2009), although functional alterations in the cholinergic mechanism cannot be excluded as a causal factor. In this sense, an additional reduction of IHR was observed in diabetic groups (DS and DI) compared with nondiabetic

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groups (CS and I), showing possible alterations in the cardiac automatism in these animals. Reduction in the VO2max has been classically reported in diabetic (Yki-Jrvinen, DeFronzo, & Koivisto, 1984) and ischemic heart failure patients (Weber, Janicki, & McElroy, 1987) and in patients who are both diabetic and have ischemic heart failure (Miche et al., 2006) due to cardiac and peripheral damage. In the present investigation, DS and I groups had a reduction in VO2max compared with that in the CS group in all experimental evaluations. These data are in accordance with the previous ndings of our group that demonstrated a reduction in the VO2max in STZ-diabetic rats (Rodrigues et al., 2007) and after chronic MI (Jorge et al., 2011). Furthermore, when the two pathological conditions occurred together, an additional impairment in cardiopulmonary capacity was observed, as demonstrated by DI group data. It is possible that the additional reduction of CO combined with the increase of PVR have compromised the delivery of O2 and removal of CO2, culminating in the VO2max reduction in DI animals. In fact, a negative correlation was observed between PVR and VO2max in all experimental animals, suggesting that the additional increase of PVR in DI rats may be directly inuencing the accentuated VO2max reduction observed in this group. In addition, we cannot ignore that the reduced body weight in diabetic animals, observed in our study, probably associated with skeletal and cardiac muscle loss, may also have inuenced the CO and VO2max reduction in these animals. In rats with diabetes induced by STZ, in addition to insulin, the secretion of hormones, such as the growth hormone, glucagon, pancreatic polypeptide and, consequently, growth factor similar to that of insulin, is altered and affects the growth (Gomes et al., 2009). It is also known that diabetes induces increased use of fatty acids and accelerates protein catabolism (Howarth et al., 2008). 4.3. Study limitations Some possible limitations of the present investigation deserve comment. First, we understand that our model of diabetes by STZ does not provide the ideal condition for studying diabetes pathophysiology, such as the genetically modied ob/ob or db/db mouse models. However, STZ-diabetes is widespread in the literature and consistently used by our group to better understand the functional and autonomic changes of diabetes over recent years. Second, the hemodynamic and autonomic parameters were evaluated at the end of the protocol, and comparisons were made by including a control group in the experimental design. Indeed, the direct method of recording blood pressure depends on the catheterization of arterial vessels that are functional during a small time period. Consequently, the biological signals were recorded only at the end of the experimental period, leading to the lack of baseline values in the same animals at the start of the study. Third, since the experimental animals started the protocol with similar values of MI area, glycemia, and VO2max, the prediction of survival (or mortality) based on these parameters becomes difcult and is limited to methods used in this study. 4.4. Conclusions This study is the rst to provide a range of functional, hemodynamic, and autonomic assessments to better understand the pathophysiological evolution of myocardial infarction in diabetes, a clinical condition usually observed in these patients. Our data suggest that the additional impairment of cardiac autonomic function was probably related to subsequent cardiac output, peripheral vascular resistance and cardiopulmonary capacity dysfunctions, and these parameters might have been the main determinants of mortality in diabetic rats after a chronic myocardial infarction. Furthermore, these results highlight the importance of these clinical parameters that should be taken into consider-

ation when therapeutic approaches are being sought for the management of diabetic patients after an ischemic event. Acknowledgments This work was supported by the Fundao de Amparo a Pesquisa do Estado de So Paulo (FAPESP-07/58942-0), Conselho Nacional de Pesquisa e Desenvolvimento (CNPq-482520/2009-4; 306011/20107). BR has doctorate and postdoctorate scholarships from the Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES) and Conselho Nacional de Pesquisa e Desenvolvimento (CNPq), respectively. MCI and KDA had nancial support from Conselho Nacional de Pesquisa e Desenvolvimento (CNPq-BPQ). References
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