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Left ventricular function in physiologic and pathologic hypertrophy in Sprague–Dawley rats
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Science & Sports 23 (2008) 299–305
Original article
Left ventricular function in physiologic and pathologic hypertrophy in Sprague–Dawley rats Les différences des fonctions des ventricules gauches du cœur de l’hypertrophie fonctionnelle et de l’hypertrophie pathologique du Rattus Sprague-Dawley S.S. Zhu a , J.Z. Ma b , Y.H. Yong a , J. Niu b , J.N. Zhang a,∗ a
Institute of Cardiovascular Disease, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China b Department of Military Education and Training, the Basic College of the PLA University of Science and Technology, Nanjing, 211101, China Received 5 December 2007; accepted 14 April 2008 Available online 24 June 2008
Abstract Objective. – The aim of this study was to design a high intensity swimming training and two-kidney, one-clip (2K1C) hypertension protocol in Sprague–Dawley (SD) rats and to use current echocardiography techniques to examine the differential diagnosis between physiological and pathological left ventricular hypertrophy. Methods. – One group of SD rats performed swimming training at high intensive swimming training (HIGH) for eight weeks. In animals of the other arm of the study, a 2KIC hypertension was created and maintained for eight weeks. Results. – After eight weeks, all rats were studied by standard and tissue Doppler echocardiography. The heart weight/body weight ratio (HW/BW) of 2K1C and HIGH rats increased by 16% and 42%, respectively. Echocardiography showed increased septal and posterior wall thickness in both the 2K1C and HIGH rats. Left ventricular increased by 35 and 41% respectively. Left ventricular diameters, stroke volumes, cardiac output, and ejection fractions were unchanged in either group. Mitral inflow showed a decrease in late-wave velocity, thus increasing the E/A ratio in HIGH rats. However, mitral inflow showed an increase in late-wave velocity, thus decreasing the E/A ratio in 2K1C rats. There was a significant increase in Ea and early diastolic (Em)/late diastolic (Am) in HIGH rats in basal septum and lateral mitral valve annulus. And there was a significant increase in Am, which led to a significant decrease of Em/Am in 2K1C rats. No significant change occurred in pulmonary vein systolic velocity and diastolic velocity, in either of the three animal groups. However, there was significant increase in atrial reversal velocity in HIGH rats. Conclusions. – Doppler echocardiographic parameters of LV diastolic function can be of diagnostic importance for discrimination between pathologic and physiologic LV hypertrophy. © 2008 Elsevier Masson SAS. All rights reserved. Résumé Objectifs. – Chez des rats Spague-Dawley (SD), comparer les caractéristiques anatomiques et fonctionnelles de l’hypertrophie cardiaque gauche de l’entraînement en endurance avec celles de l’hypertension artérielle sévère. Méthodes. – Des rats ont été entraînés à nager avec une intensité de plus en plus grande (groupe HIGH). Un autre groupe a été rendu hypertendu en clampant une artère rénale (modèle 2K1C). Au bout de huit semaines, les modifications anatomiques et fonctionnelles cardiaques ont été quantifiées dans chaque groupe de rats par échographie-doppler. Résultats. – Le poids du cœur augmente de 16 % chez les animaux hypertendus et de 42 % chez les animaux entraînés. L’épaisseur de septum interventriculaire et celle de la paroi postérieure du ventricule gauche augmentent dans les deux groupes. Le poids du VG augmente de 35 % dans le groupe 2R1C et de 41 % dans le groupe HIGH. Le diamètre du VG, le volume d’éjection systolique, le débit cardiaque et la fraction d’éjection systolique ne sont modifiés dans aucun des deux groupes. Dans le groupe HIGH, la vitesse de fin de flux mitral est diminuée et le rapport E/A
∗
Corresponding author. E-mail address: [email protected] (J.N. Zhang).
0765-1597/$ – see front matter © 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.scispo.2008.04.004
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augmenté. L’inverse est observé dans le groupe 2K1C. Dans le groupe HIGH les index Ea et Em/Am d’échographie tissulaire sont augmentés pour la base du septum et le bord latéral de l’anneau mitral. Dans le groupe 2K1C, Am est augmenté et Em/Am diminué. La vélocité dans la veine pulmonaire n’est pas différente entre les groupes en systole ni en diastole. Mais le flux rétrograde est augmenté dans le groupe HIGH. Conclusions. – Les indices de fonction ventriculaire gauche obtenus en échographie-doppler différencient les hypertrophies ventriculaires physiologiques (induites par l’entraînement en endurance) d’une hypertrophie pathologique (HTA). © 2008 Elsevier Masson SAS. All rights reserved. Keywords: Physiological hypertrophy; Rat; Endurance exercise; Echocardiography; Exercise intensity Mots clés : Hypertrophie fonctionnelle ; Rattus ; Entraînement de l’endurance ; Échocardiogramme ; Intensité de l’exercice
1. Introduction
2. Materials and methods
Athlete’s heart is a cardiac adaptation to long-term intensive training, which includes changes such as increased ventricular cavity diameters, wall thickness and mass, produced with a degree consistent with sports activities and exercise programs [15,18]. Athletes involved in sports with a high dynamic component (e.g., running) develop predominantly increased left ventricular chamber size with a proportional increase in wall thickness caused by volume overload associated with the high cardiac output of endurance training. Athletes involved in mainly static or isometric exercise (e.g., weightlifting) develop predominantly increased left ventricular wall thickness with unchanged left ventricular chamber size, which is caused by pressure overload accompanying the high systemic arterial pressure found in this type of exercise [17]. The differentiation of traininginduced cardiac adaptations from pathological conditions is a key issue in sports cardiology. As morphological features do not allow for a clear delineation of early stages of relevant pathologies, the echocardiographic evaluation of left ventricular (LV) function is the technique of first choice in this regard. Tissue Doppler imaging (TDI) is a relatively recent method for the assessment of cardiac function that provides direct, local measurements of myocardial velocities throughout the cardiac cycle. Although it has shown a superior sensitivity in the detection of ventricular dysfunction in clinical and experimental studies, its application in sports medicine is still rare [6,7]. More recent arguments for hypertrophic hypertrophy in athletes are a high incidence of sudden cardiac death in high-level athletes, a higher incidence than normal of lethal arrhythmias among athletes, and the increased risk for sudden cardiac death associated with LV hypertrophy in the general population [15]. Because of the increased risk for adverse cardiac events, it is important to differentiate between physiologic and pathologic LV hypertrophy. Hypertension, the most common cause of LV hypertrophy in the general population, is associated with diastolic function abnormalities and an increased incidence of ventricular arrhythmias. Therefore, the aim of this study was to induce the development of endurance-induced physiologic and pathologic cardiac hypertrophy in Sprague–Dawley (SD) rats, through either a high intensity swimming training (HIGH), or a two-kidney one clip hypertension protocol (2K1C). Current echocardiographic techniques were used to assess the morphological and functional differences of heart between the two animal groups and so, further extend our understanding of physiological and pathologic cardiac hypertrophy.
2.1. Study design and animals A total of 20 male adult Sprague–Dawley rats (Shanghai SLAC Laboratory Animal Co. Ltd., Shanghai, China), age 60–70 days at start of training were randomized into three groups: high intensive swimming training (HIGH) group, 2K1C group, and sham-operated group (control). All procedures were in accordance with the guidelines of the Chinese Committee for Experiments on Animals. 2.2. 2K1C hypertensive and sham-operated model To create the model, rats were anesthetized with chloral hydrate 7.5%, and silver chips of 0.2 mm internal diameter were slipped around the left renal artery as close as possible to its exit from the aorta. Sham-operated rats were exposed to the same surgical manipulations, except the clipping. The wound was sutured, and the animals were allowed to recover. After return to their cage, rats were maintained on a regular diet for eight weeks. 2.3. High intensity swimming exercise program The swimming apparatus was 80 cm in length, 50 cm in width and 90 cm in depth. The water level was adjusted to 70 ± 5 cm and the water temperature was maintained at 35 ± 2 ◦ C. Rats exercised twice per day, six days per week for eight weeks. The training periods were 15 min, twice daily in Week 1; 30 min, twice daily in Week 2; 60 min, twice daily in Week 3; 90 min, twice daily in Week 4; 120 min, twice daily in Weeks 5, 6, 7 and 8. But in Week 4, a load of string ring weighting 0.5% of body weight was attached to proximal end of the tail. Then the load was adjusted to 1% of body weight in Week 5, 1.5% in Week 6, 2% in Weeks 6, 7, 8. Rats that appeared exhausted during swimming were taken out for a 5 min rest, after that, they were placed back into the water to finish the scheduled sessions time. 2.4. Echocardiography After eight weeks, rats were weighed and then anesthetized with chloral hydrate 7.5%. Rats were lightly secured in the supine position to a warming pad, and the precordium was shaved. Transthoracic echocardiography was performed using a commercially available echocardiographic system (GE Vivid
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7 with 7.5 MHz transducer). Transthoracic echocardiographic parameters or calculations were recorded according to American Society of Echocardiography guidelines [20]. A sonographer experienced in pediatric echocardiography was used. All measurements were made online with the optimal digital images selected by the sonographer from five cardiac cycles. Mmode and 2-dimensional (2D) echocardiography images were obtained in the parasternal long-axis views. The dimensions were determined at the tips of the papillary muscle. Left atrial (LAd) and aortic diameters (Aod) were measured in parasternal long-axis orientation. Transmitral Doppler flows (E and A velocities, and their ratio) were measured in an apical fourchamber orientation with the sample volume placed at the tips of the mitral leaflets. Pulmonary Doppler velocities were measured in parasternal short-axis orientation after color flow localization. For Doppler tissue imaging (DTI) the smallest sample volume was placed basal interventricular septum and lateral mitral valve annulus (MA) in an apical four-chamber orientation. Gains were adjusted to eliminate background noise and allow for clear tissue signal and five to 10 cycles were recorded. Measurements were performed included early diastolic (Em), late diastolic (Am) and systolic (Sm) myocardial peak velocities. LV mass was calculated using a standard cube formula: LV mass = 1.04[(IVSd + LVPWd + LVDd) 3 −LVDd3 ]; where LVDd is LV diameter at end-diastole, LVPWd is LV posterior wall thickness at end-diastole, IVSd is ventricular septum thickness at end-diastole, and 1.04 is the estimated specific gravity of the myocardium and the remaining constants are correction factors. One study has demonstrated a good correlation between LV mass calculated in this manner and postmortem LV mass in rats [13]. The derived LV mass was normalized for body weight. Fractional shortening (FS), a measure of LV systolic function, was calculated from the M-mode LV dimensions using the equation FS (%) = (LVDd−LVDs)/LVDd. Ejection fraction (EF) was calculated M-mode LV dimensions using the equation EF% = (EDV−ESV)/EDV, where EDV is LV diastolic volume, and ESV is LV systolic volume. EDV and ESV were measured using Simpson method. Stroke volume (SV) was calculated using equation SV = EDV−ESV. Cardiac output (CO) was calculated using equation CO = SV × HR, where HR is heart rate from Doppler measurements.
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Table 1 Body weights and heart weights (n = 20) Control (n = 6)
2K1C(n = 8)
HIGH (n = 6)
294.9 (16.58) 540.2 (45.20) 1.45 (0.13) 2.69 (0.20)
298.4 (6.28) 555.1 (37.23) 1.71 (0.09)* 3.11 (0.17)*
283.8 (9.19) 440.3 (56.41)*,# 1.69 (0.19)* 3.83 (0.13)*,#
BW0 (g) BW (g) HW (g) HW/BW
BW0 , body weight before the initiation of exercise training; BW, body weight eight weeks after exercise training; HW, heart weight; HW/BW, Heart weight (mg)/body weight (g), Control, sham-operated group; 2K1C, two-kidney, oneclip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. # P < 0.05 vs. respective 2K1C group.
2.5. Necropsy After imaging was complete, rats were immediately euthanized and their hearts removed, and then blotted of excess fluid and weighed. 2.6. Statistical methods Data are presented as mean ± S.D. One-way analysis of variance (ANOVA) was performed to assess differences in parameters between groups. Significant differences were then subjected to post hoc analysis using the Newman–Keuls method. Correlation coefficients were obtained using linear regression. P < 0.05 was considered statistically significant. 3. Results 3.1. Differences in BW and cardiac mass/adaptation in control, 2K1C rats and HIGH rats At the initial evaluation (8–9 weeks of age), no intergroup differences were found in total body weight. After eight weeks, the BW of the HIGH group was significantly lower than sedentary control or 2K1C group. Heart weight had increased by an average 18% in the 2K1C group, and by an average 17% in the HIGH group (Table 1, Fig. 1A). The heart weight/body weight ratio (HW/BW) had increased by an average 16% in the 2K1C
Fig. 1. Different on heart weight (A) and heart weight (mg)/body weight (g) (B) in HIGH and 2K1C and control rats. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group, # P < 0.05 vs. respective 2K1C group.
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Fig. 2. The correlation between echocardiogram-derived LV mass and actual heart wet weight in combined group of HIGH, 2K1C and controls rats. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training.
group, and by an average 42% in the HIGH group (Table 1, Fig. 1B). 3.2. Differences in cardiac structure in control, 2K1C rats and HIGH rats There was a close correlation between echocardiogramderived LV mass and actual heart wet weight in a combined group of exercise-trained rats, 2K1C rats and sedentary controls (r = 0.87, P < 0.001, n = 20) indicating the accuracy of echocardiographic measurements (Fig. 2). The morphological characteristics by echocardiography are shown in Table 2. After eight weeks, no significant differences were found in LVDd. The IVSd had increased by an average 33% in the 2K1C group, and by an average 19% in the HIGH group (Fig. 3 and Fig. 4A). LVPWd had increased by 33 and 25% in the 2K1C and HIGH groups respectively. The 2K1C and HIGH rats significantly increased LV mass by 35 and 41%, respectively. The LV mass/weight (mg/g) ratio was different between the three animal groups.
Fig. 3. Two-dimensional targeted M-mode echocardiograms from control (A), 2K1C rats (B) and high intensity swimming training (C) rats. Note the thickness of the interventricular septum and posterior wall increase in 2K1C and exercise rats. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training.
Table 2 Morphological characteristics by echocardiography (n = 20)
Table 3 Echocardiographic parameters determining systolic function (n = 20)
Aod (cm) LAd (cm) LVDd (cm) IVSd (cm) LVPWd (cm) LV mass (g) LV mass/BW (mg/g)
Sedentary (n = 6)
2K1C (n = 8)
HIGH (n = 6)
0.34 (0.05) 0.36 (0.04) 0.65 (0.02) 0.21 (0.02) 0.21 (0.01) 1.10 (0.09) 1.87 (0.13)
0.34 (0.04) 0.39 (0.04) 0.62 (0.04) 0.28 (0.02)* 0.28 (0.03)* 1.48 (0.18)* 2.67 (0.26)*
0.36 (0.04) 0.43 (0.08) 0.70 (0.06) 0.26 (0.01)* 0.27 (0.01)* 1.56 (0.16)* 3.35 (0.32)*,#
Aod, aortic diameter; Lad, left atrial diameter; IVSd, interventricular septal enddiastolic thickness; LVDd, left ventricular end-diastolic dimension; LVPWd, left ventricular posterior wall end-diastolic thickness; LV mass, left ventricular mass; LV mass/BW, left ventricular mass (mg)/body weight (g). Control, shamoperated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. # P < 0.05 vs. respective 2K1C group.
3.3. Differences in cardiac systolic function in control, 2K1C rats and HIGH rats Data are shown in Table 3, no significant differences were found in SV, CO, EF and FS between the three animal groups. Sm velocity was higher in HIGH than control and 2K1C animals.
HRDoppler (beats/min) EDV (ml) ESV (ml) SV (ml) CO (ml/min) EF% FS% Sm (m/s)
Control (n = 6)
2K1C (n = 8)
HIGH (n = 6)
339.83 (55.37) 0.66 (0.06) 0.10 (0.01) 0.56 (0.05) 187.49 (23.16) 84.50 (2.38) 46.48 (3.52) 0.05 (0.01)
398.38 (65.62) 0.64 (0.11) 0.11 (0.03) 0.54 (0.09) 215.71 (49.67) 83.54 (3.82) 45.61 (3.01) 0.06 (0.01)
387.67 (56.32) 0.66 (0.09) 0.09 (0.03) 0.57 (0.08) 222.87 (48.26) 86.49 (3.67) 52.41 (5.04) 0.07 (0.01)*,#
HRDoppler , heart rate during Doppler measurements; EDV, end diastolic volume; ESV, end-systolic volume; SV, stroke volume; CO, cardiac output; EF, ejection fraction; FS, fractional shortening; Sm, systolic myocardial velocity. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. # P < 0.05 vs. respective 2K1C group.
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Fig. 4. Different on interventricular septal end-diastolic thickness (IVSd) (A), left ventricular posterior wall end-diastolic thickness (LVPWd) (B) in HIGH, 2K1C and rats. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. Table 4 Echocardiographic parameters determining diastolic function (n = 20) Control (n = 6)
2K1C (n = 8)
HIGH (n = 6)
Peak E (m/s) Peak A (m/s) E/A
0.74 (0.14) 0.59 (0.21) 1.30 (0.25)
0.68 (0.23) 0.93 (0.24)* 0.72 (0.34)*
0.84 (0.15) 0.41 (0.09)# 2.11 (0.38)*,#
MA septal TDI Em (m/s) Am (m/s) Em/Am
0.05 (0.02) 0.05 (0.02) 0.89 (0.21)
0.05 (0.01) 0.09 (0.01)* 0.50 (0.08)*
0.10 (0.01)*,# 0.05 (0.01)# 1.61 (0.29)*,#
MA lateral TDI Em (m/s) Am (m/s) Em/Am
0.05 (0.01) 0.07 (0.01) 0.71 (0.06)
0.05 (0.01) 0.11 (0.01)* 0.45 (0.13)
0.09 (0.01)*,# 0.05 (0.01)*,# 2.06 (0.77)*,#
A, Transmitral late filling velocity; Am, late diastolic maximal velocity; E, transmitral early filling velocity; Em, early diastolic maximal velocity; TDI, Doppler tissue imaging; MA, mitral valve annulus. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. # P < 0.05 vs. respective 2K1C group.
3.4. Differences in cardiac diastolic function in control, 2K1C rats and HIGH rats Data are shown in Tables 4 and 5. No significant differences were found in Peak E between the three groups. Peak A was lower in HIGH rats than 2K1C, and higher in this latter group than in control animals. Consequently, the E/A ratio was lower in 2K1C than controls and higher in HIGH than in the two other groups. Compared with the Ea in sedentary group MA lateral TDI, there was a significant increase in Ea Table 5 Echocardiographic parameters in pulmonary vein flow (n = 20)
S (m/s) D (m/s) Ar (m/s)
Control (n = 6)
2K1C (n = 8)
HIGH (n = 6)
0.33 (0.10) 0.29 (0.04) 0.13 (0.03)
0.41 (0.13) 0.36 (0.19) 0.18 (0.05)
0.31 (0.10) 0.47 (0.14) 0.27 (0.12)*,#
S, Pulmonary vein systolic velocity (m/s); D, Pulmonary vein diastolic velocity (m/s); Ar, Pulmonary vein atrial reversal velocity (m/s).Control, sham-operated group; 2K1C = two-kidney; one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. # P < 0.05 vs. respective 2K1C group.
Fig. 5. Pulsed Doppler tissue imaging in rats at baseline (A), 2K1C rats (B) and high intensity swimming training (C). Compared with baseline, early diastolic myocardial velocity (Em) in HIGH group in MA lateral TDI is increased, whereas late diastolic velocity (Am) in 2K1C is increased. Control, shamoperated group; K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training.
in HIGH rats (Figure 5, Figure 6A). And there was a significant increase in Am in 2K1C group, this led to significantly increased Em/Am in HIGH rats and decreased Em/Am in 2K1C group (Figure 5, Figure 6A). Simply, these changes were found in MA Septal TDI between HIGH rats and Em/Am in 2K1C group. No significant differences were found in pulmonary vein systolic velocity and pulmonary vein diastolic velocity between the three animal groups. However, there was significant increase in the pulmonary vein atrial reversal velocity in HIGH rats. 4. Discussion The sudden death in a young athlete during competition always drives the question as to what more could have been done to identify this person who, apparently, was at higher risk. LV hypertrophy (LVH) in athletes often mimics disease states (hypertension or hypertrophic cardiomyopathy), and the distinction may have important implications, particularly when adults practice regular physical activity. Recently, new echocar-
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Fig. 6. Different on early diastolic myocardial velocity (Em) (A) and early diastolic myocardial velocity/systolic velocity (Em/Am) (B) in HIGH, 2K1C and control rats. Control, sham-operated group; 2K1C,two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group, # P < 0.05 vs. respective 2K1C group.*
diographic techniques (as integrated backscattering and Doppler tissue imaging), have been proposed to study structural and functional changes of the myocardium in athlete and to distinguish physiological from pathological hypertrophy [2,4,9,12,14]. In our study, we demonstrated an increase of wall thickness and LV mass in swimming group as compared with control animals. The thickening of the septum and posterior wall has already been found in previous studies [3,21]; in contrast with others studies [3,5], we have found a LV concentric hypertrophy in HIGH group, which may be due to the training mode. Furthermore, in human subelite distance runners, further increasing the training volume results in concentric cardiac hypertrophy [22]. In addition, we found comparable values for the LV muscle mass and muscle mass index in the trained rats and hypertensive rats, which were by far higher compared with those of the control group. No detectable impairment in the LV filling accompanied the LV hypertrophy in the HIGH, and there was no trend for those rats with the greatest increase in LV mass to exhibit any filling abnormality. In contrast, 2K1C hypertensive rats developed a similar LV hypertrophy with an impaired LV diastolic function. This diastolic dysfunction is detectable even before the increase of left ventricular dimensions beyond clinical limits [19]. In the present study, Doppler study showed that ventricular diastolic function was not influenced by hypertrophy in the HIGH group. When we assessed pulmonary venous flow, we found faster retrograde atrial wave in HIGH group. These results suggest that diastolic filling may be more efficient in HIGH group, probably due to a lower intracavitary ventricular pressure during protodiastolic phase and to a stronger atrial contraction or other unknown factors compared with normal subjects. Similar results were previously observed by Palazzuoli, et al. [16]. Indeed, it is well known that many patients with hypertension and LV hypertrophy have abnormalities of transmitral and pulmonary venous flows [19]. The pulsed DMI (PW-DMI) permits characterization of the movement of each ventricular segment by placing the sample volume at the centre of the cardiac muscle. It normally determines the three main waves of the pulsed Doppler: one positive systolic wave, and two negative diastolic waves, respectively correlating to the myocardial systolic period (Sm) and the myocardial diastolic early phase (Em) and late phase (Am). In adjunction to ECG, this method also permits quantification of the temporary difference between the electric activation of myocardium and the mechanic systolic and diastolic response
[3,4,19]. In our study, we have found a ‘supernormal’ pattern of PW-DMI in the hypertrophy of the HIGH rats, characterized by an elevated Em, and an increase in the Em/Am ratio of the basal septal wall as well as on the lateral walls, which greatly increases the contribution of early-diastolic phase (i.e. E wave) in baseline conditions. On the contrary, we have found that the diastolic dysfunction in 2K1C rats was characterized by a significant increase of Am and Em/Am ratio significantly decreased after eight weeks. Several studies in human have shown that a decrease of Em and Em/Am ratio of DTI are related to impaired diastolic function, which could help in the differential diagnosis of physiologic and pathologic LV hypertrophy [7,8]. In contrast, there was no impairment in systolic contraction in 2K1C group. Consequently, left ventricular TDI velocities are decreased during early diastolic filling but increased or unchanged during atrial contraction and ventricular systole. Once LV hypertrophy has developed, diastolic dysfunction can be reliably verified by conventional echocardiography. Moreover, study has shown that the long-axis systolic and early diastolic velocities are decreased in patients with pathologic hypertrophy, but preserved in athletes [23]. The best differentiation of pathologic from physiologic hypertrophy was provided by a mean systolic annular velocity less than 9 cm/s (sensitivity 87%, specificity 97%) [23]. Therefore, the differentiation of hypertensive LV hypertrophy from athlete’s heart is not a major problem. The concentric hypertrophy of the athlete’s heart has a different behavior from hypertensive patients where this type of remodeling influences negatively the left ventricular filling profile. The systemic renin-angiotensin system plays an important role in cardiac remodeling during the development of 2K1C hypertension [10]. Renovascular hypertension in the 2K1C model is characterized by elevated angiotensin II production/concentration triggered by the release of prorenin caused by ischemia in the clipped kidney and shear stress in the nonclipped kidney [10]. The process of cardiac remodeling in renin-dependent renovascular hypertension is characterized by an increased LV mass [10]. Our results also confirmed this change. Furthermore, with fibrotic remodeling processes, an increase of the stiffness both of the total ventricle and of the LV myocardium can lead to impairment of the diastolic function [1,24]. The changed tissue texture in the case of arterial hypertension, along with an increase of the collagen content as a sign of reparative fibrosis, causes a decrease of the myocardia flexibility of the left ventricle and, thereby, an increase of the myocardial stiffness [24]. On the
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contrary, cardiac hypertrophy induced by exercise was associated with less cardiac fibrosis and better systolic and diastolic function, suggesting that the adaptive mechanisms may exist in exercise-induced hypertrophy [11,17]. In summary, whereas pathologic LV hypertrophy results from maladaptation of the heart to overload, physiologic hypertrophy usually occurs in trained rats and can be considered as a normal adaptation to a chronic pressure (e.g., strength) or volume (e.g., running) overload. The differential diagnosis between exercise- and disease-induced pathologic LV hypertrophy with the potential of ventricular arrhythmias is an important clinical problem. It is not possible to distinguish physiologic and pathologic hypertrophy based on morphologic criteria alone. Our data support the usefulness of a Doppler echocardiographic distinction between physiologic and pathologic LV hypertrophy. With the extent of the hypertrophy being comparable, in our hypertensive rats a pathologic diastolic filling pattern was documented relating to pathologic hypertrophy induced by 2K1C, however, in HIGH rats, a normal or ‘supernormal’ LV diastolic filling pattern and myocardial velocities were found. The data from our study also suggest the usefulness of DMI in the assessment of the myocardial systolic and diastolic function to detect the early identification of specific pattern associated with hypertension. Also, TDI should be used as a diagnostic criteria for differentiating physiologic hypertrophy from the pathologic to detect or prevent functional cardiac abnormalities in athletes. 5. Conclusion In this regard, our results indicate the left ventricular concentric hypertrophy develops in response to both overload regimens, but is associated with clearly different diastolic ventricular filling patterns in “athletic versus severely hypertensive rats”. And the differences of left ventricular diastolic function between the three groups studied can be of diagnostic importance for discrimination between pathologic and physiologic LV hypertrophy. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 30570745). References [1] Brilla CG, Janicki JS, Weber KT. Impaired diastolic function and coronary reserve in genetic hypertension: role of interstitial fibrosis and medial thickening of intramyocardial coronary arteries. Circ Res 1991;69: 107–15. [2] Caso P, D’Andrea A, Caso I, Severino S, Calabro P, Allocca F, et al. The athlete’s heart and hypertrophic cardiomyopathy: two conditions which may be misdiagnosed and coexistent. Which parameters should be analysed to distinguish one disease from the other? J Cardiovasc Med (Hagerstown) 2006;7:257–66. [3] Colan SD, Sandres SP, Borow KM. Physiologic hypertrophy: effects on left ventricular systolic mechanism in athletes. J Am Coll Cardiol 1987;9:776–83.
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[4] D’Andrea A, D’Andrea L, Caso P, Scherillo M, Zeppilli P, Calabro R. The usefulness of Doppler myocardial imaging in the study of the athlete’s heart and in the differential diagnosis between physiological and pathological ventricular hypertrophy. Echocardiography 2006;23:149–57. [5] Fenning A, Harrison G, Dwyer D, Rose’Meyer R, Brown L. Cardiac adaptation to endurance exercise in rats. Mol Cell Biochem 2003;251:51–9. [6] Garcia-Fernandez MA, Azevedo J, Moreno M, Arroja I, Zamorano J, Caso P. Doppler tissue imaging. Rev Port Cardiol 2001;20:133–47. [7] Garcia-Fernandez MA, Azevedo J, Moreno M, Bermejo J, Moreno R. Regional left ventricular diastolic dysfunction evaluated by pulsed-tissue Doppler echocardiography. Echocardiography 1999;16:491–500. [8] Garcia-Fernandez MA, Azevedo J, Moreno M, Bermejo J, Perez-Castellano N, Puerta P, et al. Regional diastolic function in ischaemic heart disease using pulsed wave Doppler tissue imaging. Eur Heart J 1999;20:496–505. [9] Krieg A, Scharhag J, Kindermann W, Urhausen A. Cardiac tissue Doppler imaging in sports medicine. Sports Med 2007;37:15–30. [10] Lee TM, Lin MS, Tsai CH, Chang NC. Effect of pravastatin on left ventricular mass in the two-kidney, one-clip hypertensive rats. Am J Physiol Heart Circ Physiol 2006;291:2705–13. [11] Levy WC, Cerqueira MD, Abrass IB, Schwartz RS, Stratton JR. Endurance exercise training augments diastolic filling at rest and during exercise in healthy young and older men. Circulation 1993;88:116–26. [12] Limongelli G, Verrengia M, Pacileo G, Da Ponte A, Brancaccio P, Canonico R, et al. Left ventricular hypertrophy in Caucasian master athletes: Differences with hypertension and hypertrophic cardiomyopathy. Int J Cardiol 2006;11:113–9. [13] Litwin SE, Katz SE, Morgan JP, Douglas PS. Serial echocardiographic assessment of left ventricular function after large myocardial infarction in the rat. Circulation 1994;89:345–54. [14] Maron BJ. Distinguishing hypertrophic cardiomyopathy from athlete’s heart: a clinical problem of increasing magnitude and significance. Heart 2005;91:1380–2. [15] Maron BJ, Pelliccia A. The heart of trained athletes: cardiac remodeling and the risks of sports, including sudden death. Circulation 2006;114:1633–44. [16] Palazzuoli A, Puccetti L, Pastorelli M, Pasqui AL, Auteri A, Bruni F. Transmitral and pulmonary venous flow study in elite male runners and young adults. Int J Cardiol 2002;84:47–51. [17] Pluim BM, Zwinderman AH, van der Laarse A, van der Wall EE. The athlete’s heart. A meta-analysis of cardiac structure and function. Circulation 2000;101:336–44. [18] Rodrigues AC, de Melo Costa J, Alves GB, Ferreira da Silva D, Picard MH, Andrade JL, et al. Left ventricular function after exercise training in young men. Am J Cardiol 2006;97:1089–92. [19] Schannwell CM, Schneppenheim M, Plehn G, Marx R, Strauer BE. Left ventricular diastolic function in physiologic and pathologic hypertrophy. Am J Hypertens 2002;15:513–7. [20] Schiller NB, Shah PM, Crawford M, DeMaria A, Devereux R, Feigenbaum H, et al. Recommendations for quantitation of the left ventricle by twodimensional echocardiography: American Society of Echocardiography committee on standards, subcommittee on quantitation of two-dimensional echocardiograms. J Am Soc Echocardiogr 1989;2:358–67. [21] Spirito P, Pelliccia A, Proschan MA, Granata M, Spataro A, Bellone P, et al. Morphology of the “Athlete’s heart” assessed by echocardiography in 947 elite athletes representing 27 sports. Am J Cardiol 1994;74:802–6. [22] Venckunas T, Stasiulis A, Raugaliene R. Concentric myocardial hypertrophy after one year of increased training volume in experienced distance runners. Br J Sports Med 2006;40:706–9. [23] Vinereanu D, Florescu N, Sculthorpe N, Tweddel AC, Stephens MR, Fraser AG. Differentiation between pathologic and physiologic left ventricular hypertrophy by tissue Doppler assessment of long-axis function in patients with hypertrophic cardiomyopathy or systemic hypertension and in athletes. Am J Cardiol 2001;88:53–8. [24] Weber KT. Cardiac interstitium in health and disease: the fibrillar collagen network. J Am Coll Cardiol 1989;13:1637–52.
Science & Sports 23 (2008) 299–305
Original article
Left ventricular function in physiologic and pathologic hypertrophy in Sprague–Dawley rats Les différences des fonctions des ventricules gauches du cœur de l’hypertrophie fonctionnelle et de l’hypertrophie pathologique du Rattus Sprague-Dawley S.S. Zhu a , J.Z. Ma b , Y.H. Yong a , J. Niu b , J.N. Zhang a,∗ a
Institute of Cardiovascular Disease, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China b Department of Military Education and Training, the Basic College of the PLA University of Science and Technology, Nanjing, 211101, China Received 5 December 2007; accepted 14 April 2008 Available online 24 June 2008
Abstract Objective. – The aim of this study was to design a high intensity swimming training and two-kidney, one-clip (2K1C) hypertension protocol in Sprague–Dawley (SD) rats and to use current echocardiography techniques to examine the differential diagnosis between physiological and pathological left ventricular hypertrophy. Methods. – One group of SD rats performed swimming training at high intensive swimming training (HIGH) for eight weeks. In animals of the other arm of the study, a 2KIC hypertension was created and maintained for eight weeks. Results. – After eight weeks, all rats were studied by standard and tissue Doppler echocardiography. The heart weight/body weight ratio (HW/BW) of 2K1C and HIGH rats increased by 16% and 42%, respectively. Echocardiography showed increased septal and posterior wall thickness in both the 2K1C and HIGH rats. Left ventricular increased by 35 and 41% respectively. Left ventricular diameters, stroke volumes, cardiac output, and ejection fractions were unchanged in either group. Mitral inflow showed a decrease in late-wave velocity, thus increasing the E/A ratio in HIGH rats. However, mitral inflow showed an increase in late-wave velocity, thus decreasing the E/A ratio in 2K1C rats. There was a significant increase in Ea and early diastolic (Em)/late diastolic (Am) in HIGH rats in basal septum and lateral mitral valve annulus. And there was a significant increase in Am, which led to a significant decrease of Em/Am in 2K1C rats. No significant change occurred in pulmonary vein systolic velocity and diastolic velocity, in either of the three animal groups. However, there was significant increase in atrial reversal velocity in HIGH rats. Conclusions. – Doppler echocardiographic parameters of LV diastolic function can be of diagnostic importance for discrimination between pathologic and physiologic LV hypertrophy. © 2008 Elsevier Masson SAS. All rights reserved. Résumé Objectifs. – Chez des rats Spague-Dawley (SD), comparer les caractéristiques anatomiques et fonctionnelles de l’hypertrophie cardiaque gauche de l’entraînement en endurance avec celles de l’hypertension artérielle sévère. Méthodes. – Des rats ont été entraînés à nager avec une intensité de plus en plus grande (groupe HIGH). Un autre groupe a été rendu hypertendu en clampant une artère rénale (modèle 2K1C). Au bout de huit semaines, les modifications anatomiques et fonctionnelles cardiaques ont été quantifiées dans chaque groupe de rats par échographie-doppler. Résultats. – Le poids du cœur augmente de 16 % chez les animaux hypertendus et de 42 % chez les animaux entraînés. L’épaisseur de septum interventriculaire et celle de la paroi postérieure du ventricule gauche augmentent dans les deux groupes. Le poids du VG augmente de 35 % dans le groupe 2R1C et de 41 % dans le groupe HIGH. Le diamètre du VG, le volume d’éjection systolique, le débit cardiaque et la fraction d’éjection systolique ne sont modifiés dans aucun des deux groupes. Dans le groupe HIGH, la vitesse de fin de flux mitral est diminuée et le rapport E/A
∗
Corresponding author. E-mail address: [email protected] (J.N. Zhang).
0765-1597/$ – see front matter © 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.scispo.2008.04.004
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augmenté. L’inverse est observé dans le groupe 2K1C. Dans le groupe HIGH les index Ea et Em/Am d’échographie tissulaire sont augmentés pour la base du septum et le bord latéral de l’anneau mitral. Dans le groupe 2K1C, Am est augmenté et Em/Am diminué. La vélocité dans la veine pulmonaire n’est pas différente entre les groupes en systole ni en diastole. Mais le flux rétrograde est augmenté dans le groupe HIGH. Conclusions. – Les indices de fonction ventriculaire gauche obtenus en échographie-doppler différencient les hypertrophies ventriculaires physiologiques (induites par l’entraînement en endurance) d’une hypertrophie pathologique (HTA). © 2008 Elsevier Masson SAS. All rights reserved. Keywords: Physiological hypertrophy; Rat; Endurance exercise; Echocardiography; Exercise intensity Mots clés : Hypertrophie fonctionnelle ; Rattus ; Entraînement de l’endurance ; Échocardiogramme ; Intensité de l’exercice
1. Introduction
2. Materials and methods
Athlete’s heart is a cardiac adaptation to long-term intensive training, which includes changes such as increased ventricular cavity diameters, wall thickness and mass, produced with a degree consistent with sports activities and exercise programs [15,18]. Athletes involved in sports with a high dynamic component (e.g., running) develop predominantly increased left ventricular chamber size with a proportional increase in wall thickness caused by volume overload associated with the high cardiac output of endurance training. Athletes involved in mainly static or isometric exercise (e.g., weightlifting) develop predominantly increased left ventricular wall thickness with unchanged left ventricular chamber size, which is caused by pressure overload accompanying the high systemic arterial pressure found in this type of exercise [17]. The differentiation of traininginduced cardiac adaptations from pathological conditions is a key issue in sports cardiology. As morphological features do not allow for a clear delineation of early stages of relevant pathologies, the echocardiographic evaluation of left ventricular (LV) function is the technique of first choice in this regard. Tissue Doppler imaging (TDI) is a relatively recent method for the assessment of cardiac function that provides direct, local measurements of myocardial velocities throughout the cardiac cycle. Although it has shown a superior sensitivity in the detection of ventricular dysfunction in clinical and experimental studies, its application in sports medicine is still rare [6,7]. More recent arguments for hypertrophic hypertrophy in athletes are a high incidence of sudden cardiac death in high-level athletes, a higher incidence than normal of lethal arrhythmias among athletes, and the increased risk for sudden cardiac death associated with LV hypertrophy in the general population [15]. Because of the increased risk for adverse cardiac events, it is important to differentiate between physiologic and pathologic LV hypertrophy. Hypertension, the most common cause of LV hypertrophy in the general population, is associated with diastolic function abnormalities and an increased incidence of ventricular arrhythmias. Therefore, the aim of this study was to induce the development of endurance-induced physiologic and pathologic cardiac hypertrophy in Sprague–Dawley (SD) rats, through either a high intensity swimming training (HIGH), or a two-kidney one clip hypertension protocol (2K1C). Current echocardiographic techniques were used to assess the morphological and functional differences of heart between the two animal groups and so, further extend our understanding of physiological and pathologic cardiac hypertrophy.
2.1. Study design and animals A total of 20 male adult Sprague–Dawley rats (Shanghai SLAC Laboratory Animal Co. Ltd., Shanghai, China), age 60–70 days at start of training were randomized into three groups: high intensive swimming training (HIGH) group, 2K1C group, and sham-operated group (control). All procedures were in accordance with the guidelines of the Chinese Committee for Experiments on Animals. 2.2. 2K1C hypertensive and sham-operated model To create the model, rats were anesthetized with chloral hydrate 7.5%, and silver chips of 0.2 mm internal diameter were slipped around the left renal artery as close as possible to its exit from the aorta. Sham-operated rats were exposed to the same surgical manipulations, except the clipping. The wound was sutured, and the animals were allowed to recover. After return to their cage, rats were maintained on a regular diet for eight weeks. 2.3. High intensity swimming exercise program The swimming apparatus was 80 cm in length, 50 cm in width and 90 cm in depth. The water level was adjusted to 70 ± 5 cm and the water temperature was maintained at 35 ± 2 ◦ C. Rats exercised twice per day, six days per week for eight weeks. The training periods were 15 min, twice daily in Week 1; 30 min, twice daily in Week 2; 60 min, twice daily in Week 3; 90 min, twice daily in Week 4; 120 min, twice daily in Weeks 5, 6, 7 and 8. But in Week 4, a load of string ring weighting 0.5% of body weight was attached to proximal end of the tail. Then the load was adjusted to 1% of body weight in Week 5, 1.5% in Week 6, 2% in Weeks 6, 7, 8. Rats that appeared exhausted during swimming were taken out for a 5 min rest, after that, they were placed back into the water to finish the scheduled sessions time. 2.4. Echocardiography After eight weeks, rats were weighed and then anesthetized with chloral hydrate 7.5%. Rats were lightly secured in the supine position to a warming pad, and the precordium was shaved. Transthoracic echocardiography was performed using a commercially available echocardiographic system (GE Vivid
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7 with 7.5 MHz transducer). Transthoracic echocardiographic parameters or calculations were recorded according to American Society of Echocardiography guidelines [20]. A sonographer experienced in pediatric echocardiography was used. All measurements were made online with the optimal digital images selected by the sonographer from five cardiac cycles. Mmode and 2-dimensional (2D) echocardiography images were obtained in the parasternal long-axis views. The dimensions were determined at the tips of the papillary muscle. Left atrial (LAd) and aortic diameters (Aod) were measured in parasternal long-axis orientation. Transmitral Doppler flows (E and A velocities, and their ratio) were measured in an apical fourchamber orientation with the sample volume placed at the tips of the mitral leaflets. Pulmonary Doppler velocities were measured in parasternal short-axis orientation after color flow localization. For Doppler tissue imaging (DTI) the smallest sample volume was placed basal interventricular septum and lateral mitral valve annulus (MA) in an apical four-chamber orientation. Gains were adjusted to eliminate background noise and allow for clear tissue signal and five to 10 cycles were recorded. Measurements were performed included early diastolic (Em), late diastolic (Am) and systolic (Sm) myocardial peak velocities. LV mass was calculated using a standard cube formula: LV mass = 1.04[(IVSd + LVPWd + LVDd) 3 −LVDd3 ]; where LVDd is LV diameter at end-diastole, LVPWd is LV posterior wall thickness at end-diastole, IVSd is ventricular septum thickness at end-diastole, and 1.04 is the estimated specific gravity of the myocardium and the remaining constants are correction factors. One study has demonstrated a good correlation between LV mass calculated in this manner and postmortem LV mass in rats [13]. The derived LV mass was normalized for body weight. Fractional shortening (FS), a measure of LV systolic function, was calculated from the M-mode LV dimensions using the equation FS (%) = (LVDd−LVDs)/LVDd. Ejection fraction (EF) was calculated M-mode LV dimensions using the equation EF% = (EDV−ESV)/EDV, where EDV is LV diastolic volume, and ESV is LV systolic volume. EDV and ESV were measured using Simpson method. Stroke volume (SV) was calculated using equation SV = EDV−ESV. Cardiac output (CO) was calculated using equation CO = SV × HR, where HR is heart rate from Doppler measurements.
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Table 1 Body weights and heart weights (n = 20) Control (n = 6)
2K1C(n = 8)
HIGH (n = 6)
294.9 (16.58) 540.2 (45.20) 1.45 (0.13) 2.69 (0.20)
298.4 (6.28) 555.1 (37.23) 1.71 (0.09)* 3.11 (0.17)*
283.8 (9.19) 440.3 (56.41)*,# 1.69 (0.19)* 3.83 (0.13)*,#
BW0 (g) BW (g) HW (g) HW/BW
BW0 , body weight before the initiation of exercise training; BW, body weight eight weeks after exercise training; HW, heart weight; HW/BW, Heart weight (mg)/body weight (g), Control, sham-operated group; 2K1C, two-kidney, oneclip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. # P < 0.05 vs. respective 2K1C group.
2.5. Necropsy After imaging was complete, rats were immediately euthanized and their hearts removed, and then blotted of excess fluid and weighed. 2.6. Statistical methods Data are presented as mean ± S.D. One-way analysis of variance (ANOVA) was performed to assess differences in parameters between groups. Significant differences were then subjected to post hoc analysis using the Newman–Keuls method. Correlation coefficients were obtained using linear regression. P < 0.05 was considered statistically significant. 3. Results 3.1. Differences in BW and cardiac mass/adaptation in control, 2K1C rats and HIGH rats At the initial evaluation (8–9 weeks of age), no intergroup differences were found in total body weight. After eight weeks, the BW of the HIGH group was significantly lower than sedentary control or 2K1C group. Heart weight had increased by an average 18% in the 2K1C group, and by an average 17% in the HIGH group (Table 1, Fig. 1A). The heart weight/body weight ratio (HW/BW) had increased by an average 16% in the 2K1C
Fig. 1. Different on heart weight (A) and heart weight (mg)/body weight (g) (B) in HIGH and 2K1C and control rats. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group, # P < 0.05 vs. respective 2K1C group.
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Fig. 2. The correlation between echocardiogram-derived LV mass and actual heart wet weight in combined group of HIGH, 2K1C and controls rats. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training.
group, and by an average 42% in the HIGH group (Table 1, Fig. 1B). 3.2. Differences in cardiac structure in control, 2K1C rats and HIGH rats There was a close correlation between echocardiogramderived LV mass and actual heart wet weight in a combined group of exercise-trained rats, 2K1C rats and sedentary controls (r = 0.87, P < 0.001, n = 20) indicating the accuracy of echocardiographic measurements (Fig. 2). The morphological characteristics by echocardiography are shown in Table 2. After eight weeks, no significant differences were found in LVDd. The IVSd had increased by an average 33% in the 2K1C group, and by an average 19% in the HIGH group (Fig. 3 and Fig. 4A). LVPWd had increased by 33 and 25% in the 2K1C and HIGH groups respectively. The 2K1C and HIGH rats significantly increased LV mass by 35 and 41%, respectively. The LV mass/weight (mg/g) ratio was different between the three animal groups.
Fig. 3. Two-dimensional targeted M-mode echocardiograms from control (A), 2K1C rats (B) and high intensity swimming training (C) rats. Note the thickness of the interventricular septum and posterior wall increase in 2K1C and exercise rats. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training.
Table 2 Morphological characteristics by echocardiography (n = 20)
Table 3 Echocardiographic parameters determining systolic function (n = 20)
Aod (cm) LAd (cm) LVDd (cm) IVSd (cm) LVPWd (cm) LV mass (g) LV mass/BW (mg/g)
Sedentary (n = 6)
2K1C (n = 8)
HIGH (n = 6)
0.34 (0.05) 0.36 (0.04) 0.65 (0.02) 0.21 (0.02) 0.21 (0.01) 1.10 (0.09) 1.87 (0.13)
0.34 (0.04) 0.39 (0.04) 0.62 (0.04) 0.28 (0.02)* 0.28 (0.03)* 1.48 (0.18)* 2.67 (0.26)*
0.36 (0.04) 0.43 (0.08) 0.70 (0.06) 0.26 (0.01)* 0.27 (0.01)* 1.56 (0.16)* 3.35 (0.32)*,#
Aod, aortic diameter; Lad, left atrial diameter; IVSd, interventricular septal enddiastolic thickness; LVDd, left ventricular end-diastolic dimension; LVPWd, left ventricular posterior wall end-diastolic thickness; LV mass, left ventricular mass; LV mass/BW, left ventricular mass (mg)/body weight (g). Control, shamoperated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. # P < 0.05 vs. respective 2K1C group.
3.3. Differences in cardiac systolic function in control, 2K1C rats and HIGH rats Data are shown in Table 3, no significant differences were found in SV, CO, EF and FS between the three animal groups. Sm velocity was higher in HIGH than control and 2K1C animals.
HRDoppler (beats/min) EDV (ml) ESV (ml) SV (ml) CO (ml/min) EF% FS% Sm (m/s)
Control (n = 6)
2K1C (n = 8)
HIGH (n = 6)
339.83 (55.37) 0.66 (0.06) 0.10 (0.01) 0.56 (0.05) 187.49 (23.16) 84.50 (2.38) 46.48 (3.52) 0.05 (0.01)
398.38 (65.62) 0.64 (0.11) 0.11 (0.03) 0.54 (0.09) 215.71 (49.67) 83.54 (3.82) 45.61 (3.01) 0.06 (0.01)
387.67 (56.32) 0.66 (0.09) 0.09 (0.03) 0.57 (0.08) 222.87 (48.26) 86.49 (3.67) 52.41 (5.04) 0.07 (0.01)*,#
HRDoppler , heart rate during Doppler measurements; EDV, end diastolic volume; ESV, end-systolic volume; SV, stroke volume; CO, cardiac output; EF, ejection fraction; FS, fractional shortening; Sm, systolic myocardial velocity. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. # P < 0.05 vs. respective 2K1C group.
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Fig. 4. Different on interventricular septal end-diastolic thickness (IVSd) (A), left ventricular posterior wall end-diastolic thickness (LVPWd) (B) in HIGH, 2K1C and rats. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. Table 4 Echocardiographic parameters determining diastolic function (n = 20) Control (n = 6)
2K1C (n = 8)
HIGH (n = 6)
Peak E (m/s) Peak A (m/s) E/A
0.74 (0.14) 0.59 (0.21) 1.30 (0.25)
0.68 (0.23) 0.93 (0.24)* 0.72 (0.34)*
0.84 (0.15) 0.41 (0.09)# 2.11 (0.38)*,#
MA septal TDI Em (m/s) Am (m/s) Em/Am
0.05 (0.02) 0.05 (0.02) 0.89 (0.21)
0.05 (0.01) 0.09 (0.01)* 0.50 (0.08)*
0.10 (0.01)*,# 0.05 (0.01)# 1.61 (0.29)*,#
MA lateral TDI Em (m/s) Am (m/s) Em/Am
0.05 (0.01) 0.07 (0.01) 0.71 (0.06)
0.05 (0.01) 0.11 (0.01)* 0.45 (0.13)
0.09 (0.01)*,# 0.05 (0.01)*,# 2.06 (0.77)*,#
A, Transmitral late filling velocity; Am, late diastolic maximal velocity; E, transmitral early filling velocity; Em, early diastolic maximal velocity; TDI, Doppler tissue imaging; MA, mitral valve annulus. Control, sham-operated group; 2K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. # P < 0.05 vs. respective 2K1C group.
3.4. Differences in cardiac diastolic function in control, 2K1C rats and HIGH rats Data are shown in Tables 4 and 5. No significant differences were found in Peak E between the three groups. Peak A was lower in HIGH rats than 2K1C, and higher in this latter group than in control animals. Consequently, the E/A ratio was lower in 2K1C than controls and higher in HIGH than in the two other groups. Compared with the Ea in sedentary group MA lateral TDI, there was a significant increase in Ea Table 5 Echocardiographic parameters in pulmonary vein flow (n = 20)
S (m/s) D (m/s) Ar (m/s)
Control (n = 6)
2K1C (n = 8)
HIGH (n = 6)
0.33 (0.10) 0.29 (0.04) 0.13 (0.03)
0.41 (0.13) 0.36 (0.19) 0.18 (0.05)
0.31 (0.10) 0.47 (0.14) 0.27 (0.12)*,#
S, Pulmonary vein systolic velocity (m/s); D, Pulmonary vein diastolic velocity (m/s); Ar, Pulmonary vein atrial reversal velocity (m/s).Control, sham-operated group; 2K1C = two-kidney; one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group. # P < 0.05 vs. respective 2K1C group.
Fig. 5. Pulsed Doppler tissue imaging in rats at baseline (A), 2K1C rats (B) and high intensity swimming training (C). Compared with baseline, early diastolic myocardial velocity (Em) in HIGH group in MA lateral TDI is increased, whereas late diastolic velocity (Am) in 2K1C is increased. Control, shamoperated group; K1C, two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training.
in HIGH rats (Figure 5, Figure 6A). And there was a significant increase in Am in 2K1C group, this led to significantly increased Em/Am in HIGH rats and decreased Em/Am in 2K1C group (Figure 5, Figure 6A). Simply, these changes were found in MA Septal TDI between HIGH rats and Em/Am in 2K1C group. No significant differences were found in pulmonary vein systolic velocity and pulmonary vein diastolic velocity between the three animal groups. However, there was significant increase in the pulmonary vein atrial reversal velocity in HIGH rats. 4. Discussion The sudden death in a young athlete during competition always drives the question as to what more could have been done to identify this person who, apparently, was at higher risk. LV hypertrophy (LVH) in athletes often mimics disease states (hypertension or hypertrophic cardiomyopathy), and the distinction may have important implications, particularly when adults practice regular physical activity. Recently, new echocar-
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Fig. 6. Different on early diastolic myocardial velocity (Em) (A) and early diastolic myocardial velocity/systolic velocity (Em/Am) (B) in HIGH, 2K1C and control rats. Control, sham-operated group; 2K1C,two-kidney, one-clip (2K1C) hypertension; HIGH, high intensive swimming training. * P < 0.05 vs. respective control group, # P < 0.05 vs. respective 2K1C group.*
diographic techniques (as integrated backscattering and Doppler tissue imaging), have been proposed to study structural and functional changes of the myocardium in athlete and to distinguish physiological from pathological hypertrophy [2,4,9,12,14]. In our study, we demonstrated an increase of wall thickness and LV mass in swimming group as compared with control animals. The thickening of the septum and posterior wall has already been found in previous studies [3,21]; in contrast with others studies [3,5], we have found a LV concentric hypertrophy in HIGH group, which may be due to the training mode. Furthermore, in human subelite distance runners, further increasing the training volume results in concentric cardiac hypertrophy [22]. In addition, we found comparable values for the LV muscle mass and muscle mass index in the trained rats and hypertensive rats, which were by far higher compared with those of the control group. No detectable impairment in the LV filling accompanied the LV hypertrophy in the HIGH, and there was no trend for those rats with the greatest increase in LV mass to exhibit any filling abnormality. In contrast, 2K1C hypertensive rats developed a similar LV hypertrophy with an impaired LV diastolic function. This diastolic dysfunction is detectable even before the increase of left ventricular dimensions beyond clinical limits [19]. In the present study, Doppler study showed that ventricular diastolic function was not influenced by hypertrophy in the HIGH group. When we assessed pulmonary venous flow, we found faster retrograde atrial wave in HIGH group. These results suggest that diastolic filling may be more efficient in HIGH group, probably due to a lower intracavitary ventricular pressure during protodiastolic phase and to a stronger atrial contraction or other unknown factors compared with normal subjects. Similar results were previously observed by Palazzuoli, et al. [16]. Indeed, it is well known that many patients with hypertension and LV hypertrophy have abnormalities of transmitral and pulmonary venous flows [19]. The pulsed DMI (PW-DMI) permits characterization of the movement of each ventricular segment by placing the sample volume at the centre of the cardiac muscle. It normally determines the three main waves of the pulsed Doppler: one positive systolic wave, and two negative diastolic waves, respectively correlating to the myocardial systolic period (Sm) and the myocardial diastolic early phase (Em) and late phase (Am). In adjunction to ECG, this method also permits quantification of the temporary difference between the electric activation of myocardium and the mechanic systolic and diastolic response
[3,4,19]. In our study, we have found a ‘supernormal’ pattern of PW-DMI in the hypertrophy of the HIGH rats, characterized by an elevated Em, and an increase in the Em/Am ratio of the basal septal wall as well as on the lateral walls, which greatly increases the contribution of early-diastolic phase (i.e. E wave) in baseline conditions. On the contrary, we have found that the diastolic dysfunction in 2K1C rats was characterized by a significant increase of Am and Em/Am ratio significantly decreased after eight weeks. Several studies in human have shown that a decrease of Em and Em/Am ratio of DTI are related to impaired diastolic function, which could help in the differential diagnosis of physiologic and pathologic LV hypertrophy [7,8]. In contrast, there was no impairment in systolic contraction in 2K1C group. Consequently, left ventricular TDI velocities are decreased during early diastolic filling but increased or unchanged during atrial contraction and ventricular systole. Once LV hypertrophy has developed, diastolic dysfunction can be reliably verified by conventional echocardiography. Moreover, study has shown that the long-axis systolic and early diastolic velocities are decreased in patients with pathologic hypertrophy, but preserved in athletes [23]. The best differentiation of pathologic from physiologic hypertrophy was provided by a mean systolic annular velocity less than 9 cm/s (sensitivity 87%, specificity 97%) [23]. Therefore, the differentiation of hypertensive LV hypertrophy from athlete’s heart is not a major problem. The concentric hypertrophy of the athlete’s heart has a different behavior from hypertensive patients where this type of remodeling influences negatively the left ventricular filling profile. The systemic renin-angiotensin system plays an important role in cardiac remodeling during the development of 2K1C hypertension [10]. Renovascular hypertension in the 2K1C model is characterized by elevated angiotensin II production/concentration triggered by the release of prorenin caused by ischemia in the clipped kidney and shear stress in the nonclipped kidney [10]. The process of cardiac remodeling in renin-dependent renovascular hypertension is characterized by an increased LV mass [10]. Our results also confirmed this change. Furthermore, with fibrotic remodeling processes, an increase of the stiffness both of the total ventricle and of the LV myocardium can lead to impairment of the diastolic function [1,24]. The changed tissue texture in the case of arterial hypertension, along with an increase of the collagen content as a sign of reparative fibrosis, causes a decrease of the myocardia flexibility of the left ventricle and, thereby, an increase of the myocardial stiffness [24]. On the
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contrary, cardiac hypertrophy induced by exercise was associated with less cardiac fibrosis and better systolic and diastolic function, suggesting that the adaptive mechanisms may exist in exercise-induced hypertrophy [11,17]. In summary, whereas pathologic LV hypertrophy results from maladaptation of the heart to overload, physiologic hypertrophy usually occurs in trained rats and can be considered as a normal adaptation to a chronic pressure (e.g., strength) or volume (e.g., running) overload. The differential diagnosis between exercise- and disease-induced pathologic LV hypertrophy with the potential of ventricular arrhythmias is an important clinical problem. It is not possible to distinguish physiologic and pathologic hypertrophy based on morphologic criteria alone. Our data support the usefulness of a Doppler echocardiographic distinction between physiologic and pathologic LV hypertrophy. With the extent of the hypertrophy being comparable, in our hypertensive rats a pathologic diastolic filling pattern was documented relating to pathologic hypertrophy induced by 2K1C, however, in HIGH rats, a normal or ‘supernormal’ LV diastolic filling pattern and myocardial velocities were found. The data from our study also suggest the usefulness of DMI in the assessment of the myocardial systolic and diastolic function to detect the early identification of specific pattern associated with hypertension. Also, TDI should be used as a diagnostic criteria for differentiating physiologic hypertrophy from the pathologic to detect or prevent functional cardiac abnormalities in athletes. 5. Conclusion In this regard, our results indicate the left ventricular concentric hypertrophy develops in response to both overload regimens, but is associated with clearly different diastolic ventricular filling patterns in “athletic versus severely hypertensive rats”. And the differences of left ventricular diastolic function between the three groups studied can be of diagnostic importance for discrimination between pathologic and physiologic LV hypertrophy. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 30570745). References [1] Brilla CG, Janicki JS, Weber KT. Impaired diastolic function and coronary reserve in genetic hypertension: role of interstitial fibrosis and medial thickening of intramyocardial coronary arteries. Circ Res 1991;69: 107–15. [2] Caso P, D’Andrea A, Caso I, Severino S, Calabro P, Allocca F, et al. The athlete’s heart and hypertrophic cardiomyopathy: two conditions which may be misdiagnosed and coexistent. Which parameters should be analysed to distinguish one disease from the other? J Cardiovasc Med (Hagerstown) 2006;7:257–66. [3] Colan SD, Sandres SP, Borow KM. Physiologic hypertrophy: effects on left ventricular systolic mechanism in athletes. J Am Coll Cardiol 1987;9:776–83.
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