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Experimental Physiology 90.4 pp 621-626
DOI: 10.1113/expphysiol.2004.029785
© The Physiological Society 2005
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Depolarization pattern of ventricular epicardium in two-kidney one-clip hypertensive rats

Sergey N Kharin1, Valeria V Krandycheva1 and Dmitry N Shmakov1

1 Laboratory of Cardiac Physiology, Institute of Physiology of the Komi Science Centre of the Russian Academy of Sciences, 50 Pervomayskaya st., GSP-2, Syktyvkar, 167982, Komi Republic, Russia


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The present study is the first attempt to examine the effect of left ventricular hypertrophy (LVH) on the excitation pattern of the ventricular epicardium in experimental hypertensive rats. The left renal artery was clipped in Wistar rats (n = 8; 6–8 months old; weight, 174–295 g) to produce two-kidney one-clip (2K1C) hypertension. After 4 weeks, blood pressure was measured, and epicardial potential mapping was performed under sinus rhythm from 64 unipolar electrodes regularly distributed over the ventricular epicardium. Systolic blood pressure was ~40% higher in the rats with a clipped renal artery (162 ± 14 mmHg, mean ± S.D.) than in the normotensive rats (115 ± 3 mmHg). LVH (~23% increase in the ratio of the left ventricular weight to the body weight, P < 0.05) was observed in the 2K1C hypertensive rats. The depolarization pattern of the ventricular epicardium in the normotensive rats was similar to that in the rats with 2K1C hypertensive LVH. The duration of ventricular epicardial activation was shown to increase (~35%, P < 0.05) in the hypertensive rats as compared to the normotensive animals. This study provides an explanation for alterations of the body surface potential distribution in hypertensive patients with LVH.

(Received 29 December 2004; accepted after revision 14 April 2005; first published online 15 April 2005)
Corresponding author S. N. Kharin: Institute of Physiology of the Komi Science Centre of the Russian Academy of Sciences, 50 Pervomayskaya st., GSP-2, Syktyvkar, 167982, Komi Republic, Russia. Email: s.kharin{at}physiol.komisc.ru


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The results of a number of investigations have suggested that the body surface potential distribution is important for determination of myocardial hypertrophy in humans (Igarashi et al. 1987; Yamaki et al. 1989; Kornreich et al. 1989, 1990; Moroshkin et al. 1995; Moroshkin & Gusarov, 1997). However, little is known about the mechanisms underlying alterations of the body surface potential distribution during the development of left ventricular hypertrophy (LVH) in humans, because investigations of myocardial activation of the human heart in vivo are uncommon and limited by ethical principles. Thus a number of problems in cardiology may be resolved with experimental animal models of pathological processes.

Rats are suitable animals for arterial hypertension and myocardial hypertrophy modelling (Doggrell & Brown, 1998; Pinto et al. 1998). Some studies have been conducted on normotensive rats to investigate the activation pattern of the ventricular epicardium (Suzuki et al. 1992; Roshchevskaya et al. 1999). It was shown that the earliest epicardial breakthroughs occur on the right ventricular epicardium and on the apex of the left ventricle. The right ventricular breakthrough tends to occur earlier than the left ventricular one. The basal epicardium of the left ventricle is the last to be depolarized. This results in a general apex-to-base direction of ventricular epicardial excitation.

However, experimental investigations of the body surface potential distribution in rats with myocardial hypertrophy (Roshchevsky et al. 1988; Bernadic & Zlatos, 1996) are very few and do not provide exhaustive information on ventricular excitation under LVH, in spite of the fact that myocardial excitation sequence plays the major role in the body surface potential distribution formation. Thus, the aim of the present study is to investigate the depolarization pattern of the ventricular epicardium in rats with two-kidney one-clip (2K1C) hypertensive LVH.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals

Sixteen 6- to 8-month-old Wistar rats weighing between 174 and 295 g were used. During the experiment the animals were kept in unisexual groups of two individuals, in a room with the light provided from 08.00 h until 20.00 h and the temperature maintained at 20–24°C. The bedding of the cages (40 cm x 30 cm x 20 cm) consisted of wood shavings. All rats had a free access to unlimited food and water. The body weight of animals did not change during the experiment. The study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85–23, revised 1996).

Surgical procedure

Hypertension was produced by clipping the left renal artery, as previously described (Kharin & Krandycheva, 2004). Briefly, a loop of the left renal artery was pulled into a plastic tube (i.d., 0.5 mm; length, 2 mm) through the left flank incision in rats (n = 8) anaesthetized with ether. Wistar rats (n = 8) of matching body weight, sex and age were used as a control.

Four weeks after the surgery the rats were anaesthetized with ether and their blood pressure was measured. A catheter (o.d., 0.5 mm; i.d., 0.3 mm) filled with heparinized 0.85% NaCl solution was inserted into the abdominal aorta through a midline abdominal incision. The catheter was connected to a blood pressure transducer SensoNor 840 (50 µV V–1 cmHg–1) of a patient monitor EAGLE 1000 (Marquette Hellige GmbH, Germany). After the measurement of blood pressure, the rats were anaesthetized with an intraperitoneal injection of sodium thiopental (50 mg kg–1), intubated and mechanically ventilated. The heart was exposed via midsternal thoracotomy, and the pericardium was excised. The body temperature of the animal was in the range 38–38.5°C; the heart was prevented from cooling and drying by means of a warm (38–39°C) 0.85% NaCl solution.

Blood pressure, morphometric and electrocardiographic measurements

Epicardial mapping was performed under sinus rhythm. The heart rate was 269 ± 64 beats min–1 (range, 151–337 beats min–1) and 294 ± 38 beats min–1 (range, 216–333 beats min–1) in the open-chest normotensive and hypertensive rats, respectively. Sixty-four electrodes were regularly distributed on the ventricular epicardium (Fig. 1) by means of a flexible array: eight rows and eight columns, with a distance of 1–4 mm between the electrodes. Sixty-four unipolar epicardial electrograms as well as standard bipolar and augmented limb lead ECG were recorded simultaneously. ECGs were recorded with the application of subcutaneous needle electrodes. The signals were isolated, amplified, multiplexed and recorded by means of a mapping system with a bandwidth of 0.05–1000 Hz at a sampling rate of 4000 Hz and an accuracy of 16 bits. Figure 2 shows the original graphs of epicardial electrograms of rat ventricles.



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  		Figure 1.  Positions of electrodes over ventricular epicardium in rats
RV, right ventricle; LV, left ventricle. Dashed line indicates an interventricular groove.

 


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  		Figure 2.  Epicardial electrograms recorded in normotensive (left) and hypertensive (right) rats (individual case studies)
The asterisks show uncorrected signals.

 
At the end of epicardial mapping, the animals were killed with an overdose of sodium thiopental (100 mg kg–1) injected intravenously, and the heart was removed. The left ventricular myocardium (including the interventricular septum) was weighed. The ratio of the left ventricular weight to the body weight was calculated in each rat to measure LVH.

Isochronous maps of ventricular epicardial activation were drawn. A local activation time was defined as the timing of the maximum negative derivative of the QRS complex (Steinhaus, 1989) and was determined automatically. The computer-chosen activation time of each electrogram was reviewed and manually corrected by the experimenter if required. All activation times were determined unequivocally. Activation maps were constructed on a rectangular array by a computer. Then, the isochrones were manually drawn on a diagram of the heart according to the electrode positions and interelectrode spacing.

Statistical analysis

All data are expressed as means ± S.D. Statistical comparisons were carried out by paired and unpaired Student's t test. P < 0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Systolic blood pressure was equal to or more than 150 mmHg in rats with a clipped renal artery (Table 1). LVH developed in those hypertensive rats. The ratio of the left ventricular weight to the body weight in the hypertensive rats was 23% (P < 0.01) greater than that in the normotensive animals (Table 2).


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  		Table 1. Blood pressure, morphometric and electrocardiographic data in the 2K1C hypertensive rats
 

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  		Table 2. Blood pressure, morphometric and electrocardiographic data in the normotensive and 2K1C hypertensive rats
 
The duration of the QRS complex measured by the standard II limb lead ECG (QRSII) was increased by 19 ± 9% during the development of hypertension after clipping of the left renal artery (Tables 1 and 2). The duration of the QRS complex was 19% greater in hypertensive rats than that in the control rats (Table 2). The waveform of QRSII did not alter. It was of R, Rs, qRs or qR type both in the normotensive and hypertensive rats.

Depolarization sequence of ventricular epicardium in hypertensive rats

One (n = 3), two (n = 4) or three (n = 1) breakthroughs were revealed on the ventral surface of the right ventricle. These breakthroughs were found on the basal (n = 4), central (n = 6) or apical (n = 5) parts of the right ventricle. The initial breakthrough (there were two initial breakthroughs in three rats) was observed on the apical (n = 3), central (n = 5) or basal (n = 3) areas of the right ventricular surface (Fig. 3). The left ventricular breakthrough appeared on the apical part of the left ventricular surface at 0–2 ms from the start of depolarization of the right ventricular epicardium. Excitation waves spread from the right and left ventricular breakthroughs in all directions and collided at 3–5 ms from the start of epicardial depolarization on the ventral surface of the heart, close to the interventricular groove (Fig. 4). The apical ventricular epicardium, the major part of the ventral epicardium of the right ventricle, the apical part of the left ventricular epicardium (up to two-thirds) had been depolarized after 6–7 ms. Then, excitation spread in the apex-to-base direction of the ventricles. Areas of the basal ventricular epicardium were the last to be depolarized. The ventricular epicardial depolarization process in hypertensive rats lasted 10.5 ± 1.3 ms (Table 2).



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  		Figure 3.  Areas of breakthroughs on ventricular epicardium in hypertensive rats
LVb, the area in which the left ventricular breakthrough appeared in each animal; n, number of animals.

 


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  		Figure 4.  Activation pattern of ventricular epicardium in the hypertensive rat (one experiment)
The time is given in milliseconds beginning from the initial breakthrough(s).

 
Depolarization sequence of ventricular epicardium in normotensive rats

One (n = 4), two (n = 3) or three (n = 1) breakthroughs were observed on the right ventricular epicardium in the normotensive rats. They were found on the basal (n = 5), central (n = 6) or apical (n = 2) parts of the right ventricle. The first breakthrough (there were two initial breakthroughs in one rat) was observed on the central (n = 6) or basal (n = 3) areas of the right ventricular epicardium. The left ventricular breakthrough appeared in the apical part of the left ventricle at 0–3 ms from the start of depolarization of the right ventricular epicardium (Fig. 5). The collision of the excitation waves spreading from the right and left ventricular breakthroughs occurred at 4–5 ms from the start of epicardial depolarization on the ventral surface of the heart, close to the interventricular groove. The apical and almost all ventral epicardium of the right ventricle and up to the two-thirds of the left ventricular epicardium (the apical part) had been excited after 5–6 ms. Then, the depolarization spread in the apex-to-base direction of the ventricles. The basal parts of the ventricular epicardium were depolarized last. The depolarization process of the ventricular epicardium in the normotensive rats lasted 7.8 ± 0.9 ms (Table 2).



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  		Figure 5.  Activation pattern of ventricular epicardium in the normotensive rat (one experiment)
The time is given in milliseconds beginning from the initial breakthrough(s).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Investigation of myocardial activation in model animals with different cardiac pathology helps to explain the mechanisms of alterations of the body surface potential distribution in patients with the identical heart diseases. For studying the epicardial activation pattern we used the hypertensive rat model of LVH.

Spatial characteristics of the body surface potential distribution do not alter during the development of LVH in rats (Hodgkin et al. 1981; Bernadic & Zlatos, 1996). In hypertensive patients with LVH, spatial characteristics of the body surface potential distribution during the initial ventricular activity are similar to those in non-hypertensive humans; locations of the potential extrema are analogous (Igarashi et al. 1987; Yamaki et al. 1989; Kornreich et al. 1989; Moroshkin et al. 1995). The absence of changes in displacement of the areas and extrema of the body surface potential distribution may be explained by unaltered cardiac excitation sequence. However, there is no experimental confirmation of this supposition. The present study is the first investigation that concerns the effect of hypertrophy on epicardial excitation of the ventricles.

Our data on the activation sequence of ventricular epicardium in normotensive rats are in agreement with earlier investigations (Suzuki et al. 1992; Roshchevskaya et al. 1999). The depolarization sequence of the ventricular epicardium in the hypertensive rats was similar to that in the normotensive rats. Differences in the excitation pattern of the ventricular epicardium between the hypertensive and normotensive rats were within the variability of the depolarization pattern of the ventricular epicardium among the animals in each group.

The excitation of the ventricular epicardium in the 2K1C hypertensive rats lasted about 35% (P < 0.05) longer than that in the normotensive animals (Table 2). It was accompanied by an increase of the QRS complex duration (about 20%) measured by the standard II limb lead ECG during the development of LVH. The increase of the QRS complex duration was shown by other investigators on different rat models of LVH (Sambhi & White, 1960; Dunn et al. 1978; Schoemaker & Smits, 1990; Bernadic & Zlatos, 1996). Changes of temporal parameters of the body surface potential distribution in rats (Hodgkin et al. 1981; Roshchevsky et al. 1988; Bernadic & Zlatos, 1996) and humans (Moroshkin et al. 1995) are consistent with our data concerning the increase of the duration of the epicardial excitation process in rats with 2K1C hypertensive LVH. The increase of the duration of the depolarization process of the ventricular epicardium could be accounted for by a decline in longitudinal conduction velocity in hypertrophied myocardium (Carey et al. 2001). It was shown on isolated preparations of human hypertrophied left ventricular myocardium that conduction velocity decreases progressively as cell diameter increases (McIntyre & Fry, 1997).

In conclusion, the results of the present study are the first to show the excitation sequence of the ventricular epicardium during LVH caused by 2K1C hypertension in rats. The depolarization pattern of the ventricular epicardium in the 2K1C hypertensive rats with LVH was similar to that in normotensive rats.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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Carey PA, Turner M, Fry CH & Sheridan DJ (2001). Reduced anisotropy of action potential conduction in left ventricular hypertrophy. J Cardiovasc Electrophysiol 12, 830–835.[CrossRef][Medline]

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Igarashi H, Kubota I, Ikeda K, Yamaki M, Tsuiki K & Yasui S (1987). Body surface mapping for the assessment of left ventricular hypertrophy in patients with essential hypertension. Jpn Circ J 51, 284–292.[Medline]

Kharin SN & Krandycheva VV (2004). Method of experimental constriction of renal artery for modeling of renovascular hypertension in rats. Bull Exp Biol Med 138, 103–105.[CrossRef][Medline]

Kornreich F, Montague TJ, Rautaharju PM, Kavadias M, Horacek MB & Taccardi B (1989). Diagnostic body surface potential map patterns in left ventricular hypertrophy during PQRST. Am J Cardiol 63, 610–617.[CrossRef][Medline]

Kornreich F, Montague TJ, van Herpen G, Rautaharju PM, Smets P & Dramaix M (1990). Improved prediction of left ventricular mass by regression analysis of body surface potential maps. Am J Cardiol 66, 485–492.[CrossRef][Medline]

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Roshchevskaya IM, Roshchevsky MP, Shmakov DN, Arteeva NV & Antonova NA (1999). Experimental and model investigation of cardiac electric field formation in Wistar rats at the period of ventricular depolarization. In Electrocardiology '98, ed. Préda I, pp. 173–177. World Scientific Publishing, Singapore, New Jersey, London, Hong Kong.

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   Acknowledgements
 
This work was supported by grants from the Russian Foundation for Basic Research (No 03-04-48001) and from the Ural Branch of the Russian Academy of Sciences (RAS) (the programme of support for basic research performed in the Ural Branch of RAS in association with the Far Eastern Branch of RAS during 2005–2006).





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