Electrocardiographic Abnormalities in Hypertension Models

Background: Hypertensive condition can lead to abnormalities in heart structure and electrical activity. The electrocardiogram (ECG) is a recording of the electrical activity of the heart and widely used to diagnose and detect heart problem. Objective: We conducted a comparative ECG analysis between two hypertension models (L-NAME and SHR) and their controls (Wistar and Wistar-Kyoto) at six and 15 th week of age. Methods: Blood pressure was measured at the end of the 15 th week, and electrocardiography was performed at six and 15 weeks of age in anaesthetized rats. Data normality was confirmed by Kolmogorov-Smirnov test followed by unpaired Student’s t-test and the Mann-Whitney for parametric and non-parametric data, respectively. Results are expressed as mean ± SD. The accepted level of significance was set at p < 0.05. Results: L-NAME exhibited prolongation of JT and QT intervals and SHR showed a decrease in heart rate when compared to Wistar-Kyoto and L-NAME. Wistar-Kyoto exhibited short PR interval with increased QRS complex, and only QT prolongation at 15 weeks compared to Wistar. Conclusions: All the hypertension models used in this study featured an increase in blood pressure. However, while SHR showed cardiac dysfunction, L-NAME exhibited changes in ventricular performance. These results may guide future studies on different types and models of hypertension. (Int J Cardiovasc Sci. 2019; [online]. ahead print, PP.0-0)


Introduction
2][3] This imbalance can result in several types of diseases, including cardiovascular diseases, which are the leading cause of death in the world. 4][10] Classically, there are two well established models of sympathetic overactivity: the NG-monomethyl-L-arginine methyl ester (L-NAME)-induced hypertension and the spontaneously hypertensive rats (SHR). 10pertensive condition can lead to abnormalities in cardiac structure, and consequent dysfunction in cardiac electrical activity. 11,12The electrocardiogram (ECG) is a recording of the electrical activity of the heart and widely used to diagnose and detect heart problems. 13his test shows typical upward and downward 1 deflections (waves) that reflect the alternate contraction of the atria and the ventricles. 14The first wave, P, is due to atrial contraction and its prolongation has been associated with hypertension caused by endothelial dysfunction and interatrial conduction delay. 12,15The Q, R, S complex indicates ventricular depolarization. 12he duration of the QRS interval and the amplitude of the waves separately are related to mortality in hypertension. 16,17The final wave, named T, represents the repolarization of the ventricles and its correlation with the QRS complex (QT-interval).It is an important marker of ventricular activity and has been shown to have clinical utility. 12,18In the present study, we aimed to conduct a comparative ECG analysis between two hypertension models (L-NAME and SHR) and their controls (Wistar and Wistar-Kyoto).

Animals
Studies were conducted with adult, male, six and 15-week-old Wistar (HanUnib:WH), Wistar-Kyoto (NTacUnib:WKY) and SHR (SHR/NTacUnib) rats (Rattus norvegicus).All animals were provided by the Multidisciplinary Center for Biological Research (CEMIB -UNICAMP).The rats were housed in collective cages (3 rats per cage) at 22 o C on a 12 h light-dark cycle (lights on at 06:30 a.m.) with ad libitum access to standard chow (Labina Purina ® ) and filtered water.All animal housing, animal care and experimental procedures, and sample size were approved by the Ethics Committee on Animal Experimentation (CEUA) of the Institute of Biology of Unicamp in Campinas, Brazil (no.2615-1), in accordance with the NIH guidelines.The animals were divided into four groups: control Wistar (WIS, n = 6), induced hypertension (L-NAME, n = 6), control Wistar Kyoto (WKY, n = 6), and genetic hypertension (SHR, n = 6).For the L-NAME group, we inhibited nitric oxide synthesis by administration of L-NAME (Enzo Life Sciences International, Inc.5120 Butler Pike, Plymouth Meeting, PA 19462) 40 mg/kg/day, for 5 weeks in the drinking water, started at the 10 th week of life of WIS. 19ater was changed three times a week, with correction in dose/weight.The rats were anesthetized with tiletamine 29 mg kg -1 and zolazepam 29 mg kg -1 , i.p. (Zoletil 50 ® -Virbac Laboratories, Carros, France); and xylazine 12.88 mg kg -1 , i.p. (Anasedan ® -Sespo Ind. e Com.Ltda, Paulínia/SP, Brazil).

Blood Pressure
Blood pressure monitoring was performed at the end of the 15 th week under anaesthesia.Blood catheterization was performed by insertion of a cannula (PE 50) into the right carotid artery of anesthetized animals, attached to a straingauge pressure transducer that was connected to a MLS370 amplifier/7 blood pressure Module (ADInstruments -Australia), and to the data acquisition system PowerLab 8/30.For analysis of the results, we used the Software LabChart Pro (ADInstruments -Australia). 20

Electrocardiography
Electrocardiography was performed in anesthetized rats in the supine position during spontaneous breathing.Recordings were performed at 6 and 15 weeks of age, using hypodermic needle electrodes, with computerized electrocardiography (MLS360/7 ECG Analysis Module, ADInstruments, Australia), for five minutes. 21

Statistical Methods
Data are presented as mean ± SD.Normality of data distribution was confirmed by Kolmogorov-Smirnov test and then we performed unpaired Student's t-test for parametric and the Mann-Whitney test for nonparametric data.All statistical analysis was performed with Graph Pad Prism version 7.00 for Windows (Graph Pad Software, San Diego, California, USA).The accepted level of significance was p < 0.05.

Blood pressure
L-NAME and SHR 15-week-old rats exhibited increased systolic blood pressure, diastolic blood pressure, mean diastolic pressure and mean pressure when compared to their respective controls, WIS and WKY rats.There were no differences in systolic blood pressure, diastolic blood pressure, mean diastolic pressure and mean pressure between hypertensive rats (L-NAME vs. SHR) and control groups (WIS vs. WKY).Peak time was higher in SHR when compared with WKY but was not different when compared with L-NAME.No difference was observed in this parameter in control rats.There were no differences in pulse pressure, ejection and non-ejection time and cycle duration between the study groups (Table 1).

Electrocardiographic (ECG) analysis:
The six-week-old L -N A M E rats showed no ECG changes compared with WIS.However, the 15-weekold L-NAME rats exhibited increase in QT, QTc and JT intervals when compared to their controls, suggesting impaired ventricular conduction after the intervention (Figure 1, Table 2).SHR at six weeks of age showed a decrease in heart rate (HR) and an increase in the RR interval when compared with WKY.At this age, SHR also exhibited changes in atrial electrical conduction, such as increased PR interval and P-wave duration.Changes in ventricular function were also observed, with a decrease of QT and QTc intervals, increase in Q wave amplitude and decrease in R and S waves' amplitudes.By visual analysis, the ECG revealed a delay in atrial conduction and shortening in ventricular conduction compared with six-week-old WKY (Figure 1, Table 2).
The 15-week-old SHR continued to exhibit lower HR and increased RR and PR interval than WKY, indicating an impairment of atrial conduction with increase in P-wave duration.These rats failed to show a decrease in QT, QTc, JT and P wave amplitude when compared to WKY, probably due to a failure in ventricular conduction at 15 weeks of life, and a decrease in S wave amplitude at six weeks.Ventricular extrasystole followed by compensatory pauses may characterize impaired ventricular conduction in the SHR when compared with WKY (Figure 1, Table 2).
Regarding the differences between hypertension models used in this study, our results showed that sixweek-old SHR exhibited several changes when compared to L-NAME rats, such as a decrease in HR, increase in RR and PR intervals and duration, increased P wave amplitude, as well as a reduction of QT interval, QTc and JT.At 15 weeks of age, SHR exhibited higher RR interval and P amplitude than L-NAME, an increase in P wave amplitude and shorter JT interval when compared to L-NAME (Figure 1, Table 2).
Analysis between the control rats demonstrated that six-week-old WKY had a reduction in PR interval and an increase in QRS interval when compared with WIS.At 15 weeks of age, WKY showed an increase in the QT interval when compared with the WIS (Figure 1, Table 2).

Discussion
In both hypertension models, the animals showed higher systolic, diastolic and mean pressure values at 15 weeks.Ribeiro et al., 22 identified progressive increase in blood pressure in L-NAME rats after 4-6 weeks of life, reaching 164 ± 6 mmHg when compared with 108 ± 3 mmHg in controls.SHR rats developed hypertension at 4-6 weeks of age without any type of intervention and

A) WIS at six weeks; B) WIS at 15 weeks; C) L-NAME at six weeks; D) L-NAME at 15 weeks; E) WKY at six weeks; F) WKY at 15 weeks; G) SHR at six weeks; H) SHR at 15 weeks. WIS: Wistar; WKY: Wistar-Kyoto; L-NAME: NG-monomethyl-L-arginine methyl ester (L-NAME)-induced hypertension; SHR: spontaneously hypertensive rats.
exhibited an increased cardiac output with normal total peripheral resistance. 9The development of hypertension in SHR progressively promoted structural changes in the heart, which were associated with progressive cardiac hypertrophy. 9Our results corroborate those reported by Anishchenko et al., 23 showing an increase in systolic and diastolic blood pressure in SHR compared with WKY at six weeks of life. 23There are no data comparing ECG measures between WIS and WKY rats in the literature.
SHR showed a decrease in HR when compared to WKY and L-NAME.A possible interpretation of this data is the involvement of cyclic adenosine monophosphate (cAMP), which may be less available due to increased depletion induced by phosphodiesterase 3A (PDE3A).5][26] Increased expression of PDE3A results in reduced availability of cAMP and changes in the cyclic nucleotide balance, with a direct relationship with many diseases, including hypertension. 27The availability of cAMP is also linked to hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, where beta-adrenergic stimulation activates adenylate cyclase, resulting in an increase in cAMP synthesis. 28Increased cAMP raises the membrane potential, leading to higher depolarization rate, and subsequent increase in heart rate, acting as a second messenger in the modulation of HCN channels. 29This could be a possible explanation for the cardiac abnormalities found in WKY.However, studies in the literature that explain these effects are scarce and technical limitations avoid us to validate this hypothesis, so further studies are needed to confirm it.
El-Mosallamy et al., 30 demonstrated that L-NAME rats exhibited increase in RR interval, longer duration of the P wave and ST-segment elevation.However, in our results, the treatment with L-NAME induced an increase in QT, QTc and JT intervals at 15 weeks.Prolongation of QT and JT intervals are considered an indication of ventricular arrhythmia, the major cause of sudden death in hypertension. 12,31The RR interval may change with dysregulation of the atrial electrical activity, and ceases to be constant in irregular heartbeats. 32SHR showed increased RR interval, resulting in bradycardia.This change may be related to the ventricular extrasystole observed in this strain. 33e PR interval corresponds to the period that electrical signals are delayed at the atrioventricular (AV) node, before it travels through the ventricular branches to induce cardiac depolarization and may be prolonged during AV nodal dysfunction. 34Therefore, increased PR interval is also linked to bradycardia. 32We found a failure in atrial conduction in SHR rats, which are consistent with the studies by Hazari et al., 35,36 that showed prolongation of PR interval in SHR compared with WKY.
In addition to the atrial conduction delay, SHR also showed impaired ventricular conduction, with a decrease in QT and QTc, and shortening of QT at six weeks of life.These results are commonly related to electrolyte disorders such as hyperkalemia. 37azari et al., 35 described that 12-week-old SHR have prolongation of the interval QT, JT and QTc.9][40] The ECG confirmed that SHR, beyond the electrical conduction failure, show T-wave inversion, which may lead to ischemic heart failure with advanced hypertension.Animal restraining, restraintstress and difficulties with placing the electrodes in the same position in different rats are significant limitations of the method.

Conclusion
The present study shows that cardiac function is different in SHR compared with L-NAME rats.While SHR showed cardiac dysfunction, L-NAME exhibited changes in ventricular performance.Although decreased levels of PDE3A may have contributed to the changes observed in WKY, it was not sufficient to cause hypertension in this strain.Thus, all the hypertension models used in this study featured an increase in blood pressure, but each with its distinct adaptive mechanism.These results can serve as a basis for future studies on different types and models of hypertension.

Potential Conflict of Interest
No potential conflict of interest relevant to this article was reported.

Sources of Funding
This study was funded by CAPES, Faepex-PRP, SAE-Unicamp and FAPESP.

Study Association
This article is part of the thesis master submitted by Ana Gabriela Conceição-Vertamatti, from Universidade de Campinas.

Table 1 -Evaluation of blood pressure in hypertensive (L-NAME and SHR) and normotensive (WIS and WKY) rats at 15 weeks of age
*Blood pressure parameters of Wistar (WIS), Wistar-Kyoto (WKY), NG-monomethyl-L-arginine methyl ester (L-NAME)-induced hypertension, and spontaneously hypertensive rats (SHR) at six and 15 weeks of age.Data are presented as mean ± SD. *p < 0.05 compared to controls (WIS vs. L-NAME; WKY vs. SHR), n = 6.