Abstract The effects of a 12-week endurance training programme (treadmill) upon the passive and the norad- renaline-activated properties of the aorta were ...
Eur J Appl Physiol (1995) 71:173-179
© Springer-Verlag 1995
G. Koutsis • F. Kadi • H. Vandewatle • P. Lechat P. Hadjiisky - H. M o n o d
Effects of an endurance training programme on the passive and noradrenaline-activated compliances of rat aorta
Accepted: 1 March 1995
Abstract The effects of a 12-week endurance training programme (treadmill) upon the passive and the noradrenaline-activated properties of the aorta were studied in 15 trained and 24 sedentary rats. Aortic compliance was studied by measuring the length-tension curves of rings of the descending aorta without (passive properties) and with noradrenaline (noradrenaline activated) in a bubbling Krebs bath kept at a temperature of 37 °. The training effect on aortic volume compliance was studied by transforming the tension-length curves into a cross-sectional area-pressure curve according to Laplace's law. The noradrenaline responsiveness was studied by the dose-effect curve. The mechanical data were correlated with the results of a histomorphometric study which measured the aortic wall thickness and the percentages and amounts of elastic, connective and muscle components. Passive aortic compliance and volume compliance were higher in endurance-trained rats whose tunica media presented a lower percentage of collagen and a larger amount of elastic tissue. The dose-effect curve showed that the maximal aortic response to noradrenaline was stronger in trained rats but that the half maximal effective dose was not different. As a consequence, the length-tension curves of the noradrenaline fully activated aorta were similar in trained and sedentary rats except at the highest tensions where collagen is the main factor determining aortic stiffness. The increased noradrenaline response
G. Koutsis • F. Kadi • H. Vandewalle (N~)- H. Monod Laboratoire de Physiologie, Facult6 de M6decine Piti~-salp6ti6re, 91 de l'HSpital, F-75013, Paris, France P. Lechat Laboratoire de Pharmacologie, Facult6 de M6decine Piti6-salp6ti6re, 91 bld de l'H6pital, F-75013, Paris, France P. Hadjiisky Centre de recherche sur les maladies cardiovasculaires, Facult~ de M~decine Piti~-salp6ti~re, 91 bld de l'H6pital, F-75013, Paris, France
in trained rats was probably the result of the hypertrophy of the smooth muscle cells as maximal active strain (Newtons per square metre) was similar in trained and sedentary rats. Key words Aorta • Artery • Cardiovascular • Exercise. Training
Introduction The compliance of the large arteries is a factor determining arterial impedance because of the intermittent blood flow at the heart level. In vivo, aortic compliance estimation has shown that athletes have a more compliant aorta than sedentary subjects (Eug6ne et al. 1986; Mohiaddin et al. 1989; Vaitkevicius et al. 1993). This better aortic compliance could be either genetically determined in endurance athletes or the result of training or both. There are a few studies on in vitro measurement of aortic compliance in trained and sedentary rats. In the study by Faris et al. (1971), aortic distensibility has been measured by means of its Young modulus and found to be decreased in trained rats. However, the results of more recent studies are in favour of an improved aortic distensibility after training. Trained rats have been found to have significantly greater aortic extensibility indices at aorta breaking load in the studies by Bridges and Westerfield (1984) and Matsuda et al. (1989). Incremental elastic moduli at low, intermediate and high degrees of distension have been found to be lower in trained rats than in sedentary rats in the study of Matsuda (1989). A lower Young's modulus (or incremental elastic modulus) of the aortic wall does not prove, however, that the aortic volume compliance (dV/dP) is increased after training. Because of the relationship between tension, pressure and radius (Laplace's law), an alteration in Young's modulus or incremental elastic modulus can be counterbalanced by
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anatomical changes in the radius of the aorta. For example, a lesser aortic wall distensibility in old subjects is partly compensated by an increase of the aortic diameter. Moreover, only the passive properties of the aorta were studied in the previous experiments although the importance of smooth muscles is not negligible. In our experiment, we have studied the effects of an endurance training programme upon the passive properties and the noradrenaline-activated properties of rat aortic rings. Moreover, we have estimated the training effect on aortic volume compliance by transforming the tension-length curve into a cross-sectional area-pressure curve according to Laplace's law. To explain the observed differences between the trained and sedentary rats, we give the results of an histomorphometric study (aortic wall thickness, percentages and amounts of elastic, connective and muscle components, maximal active strain) which have been carried out in these rats, in addition to the biomechanical study some results of which have previously been presented (Koutsis et al. 1992).
Methods Protocol The experiments were undertaken with the approval of the University Committee for Experimental Animal Research. The experiments were carried out on 39 male Wistar rats aged 45 days, whose mean body mass was 150 g at the beginning of the study. The aortic compliance of 24 untrained rats was compared with the compliance of 15 rats which were trained on a treadmill 1 h a day, 6 days h week, for 12 weeks. The rats were trained to run on a treadmill at 20 m. min-1 and 0% gradient for 4 weeks at the beginning of the programme. Thereafter, the exercise intensity was gradually increased during the following weeks and corresponded to 32 m" min-1 and a 6% gradient for the last week. The rats were fed ad libitum. Because of a storage problem, the histomorphometric study was carried out on 10 trained and 15 sedentary rats only.
Measurements of aortic compliance A ring of the descending thoracic aorta was cut by two razor blades separated by a 4-ram plexiglass sheet. The rings were mounted on two thin stainless steel rods moved by a micrometre and incubated in a bubbling (02 95%, CO2 5%) Krebs solution (Na ÷ 140 mmol" 1-1, K + 4 mmol. 1-1, CaC12 1.3 mmol- 1-1, NaHzPO4 1.i mmol. 1- x, MgC12 1.2 mmol' 1- i, NaHCO3 24 retool" 1-1, Glucose 10 m m o H - 1) in a Celaster 20-ml cell kept at a temperature of 37°C. Force was measured by a Celaster isometric force transducer. The force exerted by the 4-mm aortic rings never exceeded 0.30 N. Passive compliance was studied by means of the tension-length relationship after 15 strain-recoil cycles. Then the aortic ring was strained in such a way that passive tension was equal to 0.03 N and thereafter was submitted to increasing concentrations of noradrenaline (1.10-1°-1.10- 5 m o l . l - 1) to study the responsiveness to catecholamines by determining the dose which corresponded to the half-maximal effective dose (EDso).
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Finally, the compliance of the noradrenaline activated aorta (compliance of the passive + active components of the aortic wall) was studied by means of the tension-length curve at the higher noradrenaline concentrations. The rate of length variation was 4 m m . m i n -1 for each 0.1-mm step followed by 4.5-s pauses between these steps, i.e. an averaged rate equal to 1 mm. m i n - 1. This rate was identical for the passive and noradrenaline activated tension-length curves. However, the length variation rate of the 15 strain-recoil cycles was 4 ram. min- 1 without pause. The active tension-length curve was obtained by substraction of the passive strain curve from the noradrenaline activated strain curve (Fig. 1).
Histomorphometry A small cylinder of descending aorta just under the aortic ring used in the compliance study was stored in a 10% formol solution. The thickness of the media was measured from the thickness of the elastic layer whose lamellae were strained in dark blue by means of Verhoeff staining (Bancroft and Stevens 1977). As the aortic wall thickness varied on the same slide because of aortic curvature, the values corresponded to the average of four measurements separated by approximately 90 ° (Fig. 2): Thickness measurements were carried out at x 620 magnification. The percentage of elastic tissue, collagen and smooth muscle cells were estimated from the Heidenhain's Azan staining(Gabe 1968) of the aortic wall. Smooth muscle cells and collagen were stained in red and blue, respectively, while elastic lamellae appeared as unstained white strips and patches. A 10 x 10 grid was superimposed on the microscopic view of the so-stained aortic walls at a x 1,250 magnification (Fig. 2). The component behind each of the 100-grid crossings was noted according to its colour. The percentages of each component corresponded to the average of four measurements carried out on four different parts of the same slide, i.e. corresponded to the measurement carried out on 400 crossings.
Calculation Approximately 35 measurements were collected for each condition. The 28 lengths which corresponded to an integer value of force
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Fig. 2 Diagram of the histomorphometric methods used for determining aortic wall thickness and the percentages of collagen, elastic tissue and smooth muscle cells in aorta, tunica media between 1 and 28 N-10 .2 were calculated by linear interpolation from the experimental data in the different conditions (passive or noradrenaline activated forces). Force F (in Newtons) was transformed into tension (T) according to the following equation which took into account the fact that the forces produced by a ring are equal to twice the force which would have been produced by an equivalent aortic strip:
175 (13.3-21.3 kPa) in all the individual curves, the volume compliance has been estimated from the slope of this part of the pressure-area curve. The cumulated thickness of a given component (collagen, elastic tissue or smooth muscle cells) in the aorta was estimated by multiplying aortic wall thickness and the percentage of this component in the media. The maximal strain of the smooth muscle cells (maximal active strain in Newtons per square meter) was estimated from the division of Tmax by the cumulated thickness of the smooth muscle cells.
Statistics Statistical differences in mechanical properties and histomorphometric data between trained and sedentary rats were tested with a Student's t-test or a Mann-Witney rank-sum-test when the normality test failed. Statistical differences between trained and sedentary rats were tested for each of the lengths corresponding to the different tensions.
Results
After 12 weeks of endurance training, the mass of the heart was 7.8% higher in the trained rats [-1.494 (SD 0.167) g compared to 1.386 (SD 0.133) g]. On the other hand, body mass was 10.5% heavier in the sedentary rats [527 (SD 37) g compared to 477 (SD 40) g]. Consequently, the heart of the trained rats was 19% larger when related to body mass {I-3.13 (SD 0.22)]-10 -3 compared to [2.63 (SD 0.17)]. 10-3}.
T = F/2h
where h is the height of aortic ring (4.10 .3 m). The different individual tension-length relationships were fitted with 3rd order polynomial equations according to the least squares method. The length corresponding to zero tension of the passive aortic ring (lEQ) was calculated from the 3rd order polynomial equation of the passive aorta. Thereafter, the relationship between tension and extension rate (length divided by IEQ)was calculated for the different conditions (passive or noradrenaline fully activated). The area between the length-tension curve and the tension axis (area test)was calculated for each rat, for the different conditions (passive or noradrenaline activated). A 3rd order polynomial equation was calculated by the least squares method from the experimental data which concerned the active curve. The length lo corresponding to maximal active tension (Zmax) was calculated as equal to the length at the maximum of this 3rd order equation. Pressure-volume curves have been estimated according to a simplified model. The aorta was assumed to be a cylinder, the radius of which was given by the Laplace's law and the height of which was not modified by radius variations. Consequently, the volume of the aortic cylinder was calculated as proportional to the square of the estimated radius. Radius was assumed to be equal to the length of the stretched aortic ring, divided by 3.1416. Pressure (equal to tension divided by radius) was obtained from the 3rd order lengthtension curve (see above). The pressure-volume curves have been estimated in passive and noradrenaline activated conditions. For the noradrenaline activated aorta, the calculated variation of aortic cross-sectional area corresponding to a calculated pressure increase within a physiological range [from 50 to 150 mm Hg (6.7 20.0 kPa)] has been used as an index of volume compliance. For the passive aorta, given that the computed pressure-area curves were approximately linear for pressures between 100 and 1 6 0 m m H g
Passive length-tension curves The average value of lEQ was similar in trained and untrained rats. The average lengths corresponding to the different passive tension levels (1-35 N" m-~) were larger in the trained rats but these differences were significant at the five highest tensions only (Fig. 3). The relationships between tension and extension rate (length divided by IEQ)were significantly different in the trained and sedentary rats for the passive curve: all the average extension rates corresponding to the different tension levels were significantly larger in the trained rats than in the sedentary rats. The calculated areas were significantly different for the passive extension rate-tension curves (P < 0.01). Noradrenaline activated aorta The response to noradrenaline (Fig. 4) was significantly higher in the trained rats but the concentration corresponding to EDso was similar in trained and untrained rats. The lEQ were not significantly different in sedentary and trained rats. The length-tension relationships of trained and sedentary rats intersected and the average lengths corresponding to the different tensions were not
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Fig. 3 Length-tension curves of the aorta rings in the absence of noradrenaline in the trained, (black dots) and the sedentary rats (unfilled circles), mean and SEM 14
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Fig. 5 Extension rate-tension curves in the noradrenaline activated aorta rings in trained (black dots) and sedentary rats (unfilled circles), mean and SEM
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1 Comparison of some mechanical properties of aortic rings of the trained and the sedentary rats. Passive aorta and noradrenaline activated correspond to mechanical data obtained without noradrenaline and with noradrenaline in Krebs bath, respectively. IEQ Length corresponding to zero tension of the passive aortic ring, lo length, Ira, Xmaximal active tension, EDso half maximal effective dose, NS not significant Table
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(unfilled circles) significantly different. The relationship between extension-rates (length divided by /EQ) and tension were significantly different between trained and sedentary rats (P < 0.05) at the five highest tensions only, which generally corresponded to the descending part of the active curve (Fig. 5). The calculated areas were not significantly different for these noradrenaline activated curves. The lengths corresponding to half the maximal value in the active curve were similar in the trained and untrained rats (1.97 compared to 1.99 mm, NS). Likewise, the lengths lo corresponding to Tmaxwere similar. The Tmax was 11.3% higher in trained rats but, in contrast with the noradrenaline responsiveness test, this difference was not significant (P = 0.09). Volume compliance The estimated cross-sectional area corresponding to a given computed pressure was larger in trained rats
Passive aorta Mean IzQ(mm) 2.20 Volume compliance index 2.7 (10- 2 mm 2 "(mm H g - 1) Norepinephrine activated IEQ (ram) Volume compliance index ( 1 0 - - 2 mm 2 . mm H g - 1) EDso (10- s tool" 1- t) lo(mm) Tmax (N" m - 1)
Sedentary (n = 24) SD 0.12 0.4
Mean 2.17 2.35
SD 0.10 0.28
NS P = 0.01
1.57 0.96
0.13 0.15
1.64 0.98
0.10 0.23
NS NS
1.8 2.90 12.8
1.9 0.21 1.6
1.6 2.95 11.5
0.96 0.24 2.5
NS NS NS
but these differences were significant (P < 0.05) for pressures higher than 115 mm Hg (15.3 kPa) only. The index of passive volume compliance was significantly higher in trained than in sedentary rats (Table 1). In contrast, the volume compliance index was independent of the training status for the noradrenaline activated aorta (Table 1). Histomorphometric study The average aortic wall thickness was 7% greater in the trained than in the sedentary rats but this difference was not significant (P = 0.24). The percentage of collagen was 4.8% higher in sedentary rats (P < 0.05). However, the cumulated thickness of collagen in the media was as large in the trained as in the sedentary
177 Table 2 Comparison of the histomorphological data observed in the aorta of the trained and sedentary rats. IE0, Length corresponding to zero tension of the passive aortic ring
Trained (n = 15)
Sedentary (n = 24)
Wall thickness (ram) Thickness/lEo
Mean SD 0.112 0.016 0.052 0.006
Mean SD 0.105 0.016 0.048 0.007
NS NS
Collagen (%) Collagen (mm)
31.5 2.2 0.035 0.005
33.0 3.1 0.034 0.006
P < 0.05 NS
25.1 3.1 0.028 0.003 0.0131 0.001
24.7 3.2 0.26 0.004 0.0118 0.002
NS P < 0.05 P
