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1Laboratory of Hemodynamics and Cardiovascular Technology, Ecole Polytechnique Fédérale de Lausanne, Lausanne; 2Department of Cardiovascular Surgery, Centre Hospitalière Universitaire Vardois, Lausanne; 4Department of Cardiovascular Surgery, Inselspital, Bern; 3Department of Cardiology, Centre Hospitalière Universitaire, Vardois, Lausanne, Switzerland; 6Pharmacy Faculty, Pharmacology Laboratory, Henri Poincaré University, Nancy, France; and 5Hydraulics Laboratory, Institute of Biomedical Technology, Ghent University, Gent, Belgium
Submitted 23 January 2006 ; accepted in final form 6 May 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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blood flow; calcium; hypertension; hemodynamics; ventricular function
During aging, alterations in aortic structure and function occur, leading to a decrease in aortic compliance. In particular, the arterial extracellular matrix undergoes many profound age-related changes responsible for wall stiffening. Age-dependent medial degeneration, including phenomena such as elastocalcinosis (calcification followed by degeneration and fragmentation of elastic fibers), is probably the most important element in increased arterial stiffness (2). More than six decades ago, Blumenthal et al. (5) have noted that the time course of the decrease of elasticity with age closely paralleled the curve of the intensity of medial calcification with age. Another interesting case is provided by patients suffering from end-stage renal disease, in which aortic PWV is related to aortic calcification (23).
To study the effects of arterial stiffening on arterial and cardiac function, many experimental animal models have been used, ranging from acute animal models where the aorta was either replaced by a stiff tube (18, 30) or wrapped with a noncompliant Dacron band (15) to chronic models where aortic stiffness is increased by inbreeding (14) as a result of hypertension-induced remodeling (16) through some chemical or biological treatment or old age (2, 7, 25). Of particular importance is a model of aortic elastocalcinosis induced by the administration of vitamin D3 and nicotine (VDN), developed originally by Hass et al. (11). The VDN model leads to arterial stiffening by decimation of the arterial wall elastic fiber network (29). Earlier studies on the effects of VDN on arterial hemodynamics showed that arterial stiffening caused by VDN decreased compliance and increased wave speed, aortic characteristic impedance (Zc), and wave reflections leading to isolated systolic hypertension (ISH) and the development of compensatory left ventricular (LV) hypertrophy (20, 21, 25).
To date, however, no data have been reported on the effects of VDN on cardiac dimensions using trans-thoracic echocardiography (TTE) and conductance catheter (CC) contractility and on the resulting changes in the interaction between the heart and the vessels, i.e., ventricular-arterial coupling. Therefore, the primary goal of this work is to assess the changes occurring at the arterial and cardiac levels after aortic stiffening with VDN and comprehensively quantify the resulting changes in arterial hemodynamics, cardiac function, and ventricular-arterial coupling.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Experimental protocol for the VDN treatment. The procedure for preparing the calcification model was described previously by Lartaud-Idjouadiene et al. (20). Briefly, on day 1, 15 rats were injected (300,000 IU/kg hindleg muscle) with vitamin D3 (Duphafral D3 1000; Duphar, Weesp, The Netherlands) at 9:00 A.M. and given 25 mg/kg nicotine (Nicotine hydrogen tartrate; Sigma Chemical). Nicotine was given orally by gavaging in 5 ml/kg sterile water two times at 9:00 A.M. and 5:00 P.M. (VDN group). The VDN is a 1-day only treatment. Another 10 rats received an injection with normal saline intramuscularly and underwent two gavages of distilled water (control placebo group, CTRL).
The VDN rats were 2 mo old with a body weight of 226 ± 8 (SD) g. Four rats died within the first 2 weeks and two died at week 5, thus leaving n = 9. The control group had the same age and similar body weight of 226 ± 7 g. No deaths occurred. The two groups were housed for an 8-wk period and were then killed, after the cardiac and vascular function were analyzed.
Assessment of arterial structure and function Aortic flow (Qao; Fig. 1A) was measured using a transit-time ultrasonic flowmeter (Transonic, Ithaca, NY) with the flow probe placed around the ascending aorta and filled with conducting gel. Aortic pressure (Pao; Fig. 1A) was measured by retracting the CC from the LV in the ascending aorta. As soon as the trace changed from an LV one to an aortic one, the CC was withdrawn 0.5 cm to ensure that the CC and flowmeter were positioned on the same spot. From the pressure signal, mean arterial pressure (MAP), SBP, and diastolic blood pressure (DBP), and PP were derived. Steady-state measurements containing 10 cycles were averaged to construct a representative steady-state beat of Pao and Qao. Discrete Fourier transform (Mathematica 5.2; Wolfram Research ), was applied to obtain pressure and flow in the frequency domain.
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60 Hz; see Ref. 36). Because HR differed from animal to animal, impedance values were averaged per harmonic, with mean values and SD of modulus, phase, and frequency computed and plotted. Total arterial compliance (C) was estimated using the Pulse Pressure Method (40). PWV was estimated indirectly from the Zc and ascending aorta dimensions as PWV = Zc x A/
, where A = 
r2 is the luminal cross-sectional area of the ascending aorta measured with echocardiography and being the density of blood.
After the rat was killed, the aorta was excised, and a 5-mm sample of the descending aorta was dehydrated in graded ethanol solutions and embedded in paraffin. Three 20-µm-thick sections were stained with hematoxylin-eosin for measurements of internal diameter (Di) and medial thickness (h; Saisam; Microvision Instruments; see Ref. 20). Elastic modulus (EM; 106 dyne/cm2) was calculated according to the Moens-Korteweg equation: EM =(PWV2 x Di x )/h, where PWV is measured in cm/s and = 1.05 g/ml (blood density). Wall stress (WS; 106 dyne/cm2) was calculated from the Lamé equation: WS = (MAP x 1,333 x Di)/2h. The ratio h/Di was also calculated.
Assessment of cardiac function.
After the initial treatment (VDN or placebo; 8 wk), the rats were anesthetized and intubated. The right neck region was disinfected, and the skin was opened. The right jugular vein and right carotid artery were isolated. A 2-Fr CC (SPR 838 Aria; Millar Instruments) was inserted in the LV via the right carotid artery. Parallel conductance (Vp) was measured after injection of 10% saline in the jugular vein using a 1-ml syringe (20 µl Natrium chloratum; Sintetica, Mendrisio, Switzerland), in accordance to the method by Baan et al. (3). An occlusion analysis was performed by temporarily occluding the inferior vena cava through a small window made in the diaphragm via a mini laparotomy. For each animal, the CC calibration factor, 
, was assessed as the ratio of SV over CC-derived SV. SV was derived from Qao, which was measured simultaneously by means of an ultrasonic flowmeter (Transonic) placed around the ascending aorta.
From the simultaneous measurement of LV pressure and volume during steady-state conditions (Fig. 1C), the following parameters were derived: SV, end-diastolic volume (EDV), ejection fraction (EF = SV/EDV), peak positive (dP/dtmax) and peak negative (dP/dtmin) value of the time derivative of LV pressure, HR, left ventricular end-diastolic pressure (LVEDP), and end-systolic pressure (Pes). Cardiac output (CO) was calculated from HR multiplied by SV.
From the occlusion analysis, we calculated the slope (Ees) and volume axis intercept (V0) of the end-systolic pressure-volume relationship (ESPVR; Fig. 1D). We also derived the slope of the end-diastolic pressure-volume relationship (EDPVR), the preload-recruitable stroke work (PRSW), with stroke work assessed from the area enclosed by the pressure-volume loop, and the preload adjusted dP/dtmax expressed as the slope of the relation between dP/dtmax and EDV. Data acquisition was provided at a sampling rate of 1 kHz and analyzed using IOX (EMKA, Paris, France).
Assessment of cardiac structure. For histology, heart tissue samples were fixed in 4% formaldehyde and mounted in paraffin block, and slices were obtained with a microtome. After deparaffinization and hydration, the samples were treated with either periodic acid-Schiff staining (Sigma) protocol (to discriminate cell borders) or 0.1% picrosirius red (for collagen; Sigma). The mean of the cardiomyocyte cross-sectional area and diameter were calculated by photomicrographs of 100 cells/specimen with a computer-assisted image analysis system (Metamorph analysis).
Assessment of cardiac structure using echocardiographic measurements. TTE was performed as previously described (17): in brief, at baseline and every 2 weeks for an 8-wk period with a commercially available echocardiographic system (C256 Sequoia; Acuson, Mountain View, CA) with the animal in the left lateral decubitus position. Light anesthesia was used during the analysis with isofluorane ventilated inside a nose cone at 0.5 l/min with 100% oxygen (Forene; Abbott, Baar, Switzerland). Once asleep, the rat was shaved with an electrical razor (Surgical clipper 9661; 3M health care). Ultrasound transonic blue gel (Tyco healthcare/Kendall) was placed on the thorax to optimize visibility of the cardiac chambers. A 15-MHz linear array transducer (15L8) was used with a frame rate of 100 Hz using bidimensional and color Doppler imaging. The probe was placed to obtain short- and long-axis and four-chamber views. From the long-axis view, an M mode trace of the LV was obtained, and LV end-diastolic diameter (LVDed), LV systolic diameter, and posterior and septal wall diastolic wall thickness (PWth and SWth, respectively) were measured. LV mass was calculated as {[(LVDed + SWth + PWth)3 –LVDed3] x 1.04} x 0.8+0.14 (in g, with LV dimensions expressed in mm). Relative wall thickness was assessed as RWT = (PWth + SWth)/(LVDed).
Wave reflection analysis.
Pao was separated into its forward (Pf) and backward (Pb) running components using the linear wave separation method (45):
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) was computed as (45):
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is a frequency-dependent complex quantity. To facilitate comparison between the groups, we computed and compared the modulus of the reflection coefficient at the first harmonic, , which is assumed to be representative of the general wave reflection properties of the arterial tree. The time of the arrival of the foot of the reflected wave was defined as 
t (Fig. 1E). The shorter the t, the stronger the SBP augmentation resulting from wave reflections. Coupling parameters. Arterial elastance (Ea) was calculated as the ratio of Pes divided by SV and the coupling parameter Ea/Ees computed (42). Efficiency, defined as the ratio of stroke work over pressure-volume area (PVA; stroke work plus potential energy), was calculated. In addition to the "conventional" coupling parameter, we also calculated two recently proposed ventricular-arterial coupling parameters (41). The first one is the "compliance coupling index" (CCI) expressed as the product of characteristic chamber elastance (Ees x C), with Ees being the inverse of ventricular compliance at end systole and C arterial compliance. The second is the "temporal coupling index" (TCI), expressed as the ratio of characteristic times (RC/T), with T being the heart period and RC the characteristic DBP decay time that is a combination of the capacitive resistive properties of the arterial tree (40).
Tissue calcium content. A 10-mm sample of the descending thoracic aorta and the cardiac apex was removed, and tissue calcium content (µmol/g dry wt) was determined by atomic absorption spectrophotometry (AA10; Varian) after mineralization and acid digestion of the tissue (13).
Statistical analysis. Values are given as means ± SD. Differences were determined by the unpaired Student's t-test, and the null hypothesis was rejected at P < 0.05. A linear regression analysis was also performed between calcium and all other parameters. All analysis was done using SPSS (SPSS 11.5; SPSS, Chicago, IL).
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RESULTS |
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30 Hz for the CTRL and 43 Hz for the VDN. This shift of the zero phase crossover toward higher frequencies is also indicative of higher wave velocity in the VDN group (Fig. 3). Calcium in the aorta increased by a factor of 28 in the VDN group compared with controls (Table 2).
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40% in the VDN group (P < 0.05, Table 4), and this is concurrent with an increase in the modulus of reflection coefficient (P < 0.05, Table 4). The time of arrival of the reflected wave, t, was somewhat shorter in the VDN group (P = ns).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Arterial stiffness, hypertension, and cardiovascular risk. Arterial stiffening, either as a result of aging or of pathological changes in the arterial wall, leads to increases in SBP and PP (27). SBP and PP, in particular, have been demonstrated to be strong independent predictors of cardiovascular mortality (10, 33). The mechanisms linking arterial stiffness to ISH have been clearly identified and discussed in detail (45). It is thus suggested that one should not only monitor PP and SBP to assess cardiovascular risk but also the hemodynamic factors influencing SBP and PP, namely ventricular ejection, aortic stiffness, and wave reflections (9, 33, 35, 37). In a recent review, aortic stiffness (PWV) and an early return of reflected waves to the heart have been established as independent predictors of cardiovascular risk (35).
Models of stiff arterial trees. Various animal models of chronic arterial stiffness have been reported in the literature, such as young spontaneously hypertensive rats (12), Dahl salt-sensitive rats (16), deoxycorticosterone acetate-salt hypertension rats (34), VDN rats (2), and others. In some of the above models, however, arterial stiffening is not the primary effect.
Effect of VDN on baseline hemodynamics and arterial function. In our study, treatment with VDN leads to increases in SBP and PP (Table 1). This is in agreement with earlier studies in VDN-treated rats where SBP and PP were found to increase, whereas MAP remained constant and DBP decreased (20).
In previous studies, increase in aortic stiffness was represented by increases in aortic Zc, PWV, and elasticity modulus and occurred in the absence of any changes in mean blood pressure, geometry, or WS (20, 29). Our study shows similar findings except that we found a slight rise in Di in the VDN group that has, however, also been reported in a setting with 90 days VDN treatment (43). However, this rise in Di had no effect on WS, and its influence on the elasticity modulus is minimal. Furthermore, the thickness-to-diameter ratio, h/Di, is the same in the two groups, which leads to similar mean stresses in the arterial wall in control and VDN.
Lartaud-Idjouadienne et al. (20) found, however, that VDN affected only the elastic and not the resistive properties of the arterial system, with aortic Zc being increased by 56%, whereas total peripheral resistance remained unchanged. We know from earlier studies in the human (24) and other mammals (30, 31, 36) that, when CO and peripheral resistance are maintained while compliance is decreased, this results in an increased SBP and a decrease in DBP, which is consistent with the findings of Lartaud-Idjouadiene et al. (20, 21). This is in agreement with our findings (Fig. 3) showing that the modulus of the Zin of VDN rats for the first five harmonics was typically 35% higher than controls, and this corresponds well with the increase in PP (+36%). Zc, estimated as the average impedance modulus, rose but not significantly, with the significance being hindered by the large SDs in the impedance moduli of the VDN group (Table 2). The change in mean value of the Zc is, however, consistent with the decrease in compliance in the VDN rats (Table 2). Zc is proportional to the inverse square root of aortic compliance, and aortic compliance is the main determinant of total systemic compliance. Indeed, the ratio of Zc,CTRL/Zc,VDN = 0.060/0.082 = 0.73 is very close to the ratio 
= 
= 0.75, demonstrating that changes in compliance and aortic Zc are consistent.
In our study, aortic calcium compared well with previous reports on the effect of VDN on aortic wall composition and proved that elastocalcinosis is the major determinant of the induction of aortic stiffness in the VDN model (20, 21, 25, 29, 43). Also, when a subgroup analysis was performed separating low from high calcium levels in the aorta, the level of calcium was found to be directly proportional to the level of augmented SBP, being 148 ± 13 mmHg (low) and 171 ± 14 mmHg (high). More so, a direct correlation could be established between aortic calcium and SBP (r = 0.91, P = 0.005). Furthermore, in the present study, as in previous ones, calcinosis was stronger in the aortic wall than in the myocardium in VDN rats, since calcium deposits occur preferentially inside tissues containing elastic fibers, with a gradient of calcium content as follows: thoracic aorta > abdominal aorta 
carotid artery > kidney > caudal artery > myocardium > mesenteric bed (13).
Effect of VDN on cardiac structure and function. Lartaud-Idjouadiene et al. (4, 20, 32) and others studied the consequences of VDN-induced arterial stiffening on certain cardiac structure, composition, and performance parameters. Lartaud-Idjouadiene et al. found that, after VDN treatment, HR, SV, CO, and stroke work were maintained, whereas LV weight/body weight increased, suggesting hypertrophy. In our study, contrary to the above, we found that SV was compromised and CO maintained probably because of the fact that HR (+8%) was elevated after VDN treatment. More so, heart weight and its ratio with body weight rose after VDN treatment as did our nonbiochemical indicators of hypertrophy. This is also the first time cardiac volumes have been measured post-VDN treatment using CC techniques and validation with TTE.
Lartaud-Idjouadiene et al. (20) gave considerable information on LV tissue composition, but they did not quantify diastolic and systolic cardiac function in detail. We have found that diastolic filling pressure and EDPVR remained unchanged, meaning that the diastolic function of the heart is preserved in the VDN group (Tables 1 and 2). Cardiac contractility, however, is significantly enhanced in the VDN group; this is reflected by a 100% increase in both Ees and the slope of dP/dtmax-EDV relationship (Table 2). This is as expected from the previously published data from Lartaud-Idjouadienne et al. (20) because of the LV hypertrophy and rise in 
-myosin heavy chain isoform. The -myosin heavy chain isoform develops a slower, more efficient form of contraction. Another reason could be changes in LV stiffness resulting from the moderate but significant increase in myocardial collagen content; thus, interstitial fibrosis could be involved in the mechanism as reported in earlier studies (20).
Effect of VDN on wave reflections. Arterial stiffening, such as in aging and hypertension, increases the amplitude of the arterial pulse wave and PWV, causing early return of reflected waves to the aorta. This, in turn, results in an associated increase in SBP and PP (35, 44). There is a strong correlation between the early return of wave reflections in the aorta and LV hypertrophy (22). Our results are in agreement with the above, and this is the first time comprehensive wave analysis was analyzed in the VDN model. Indeed, the decrease in compliance caused by VDN is associated with an increase in PWV (Table 2), an increase in the amplitude of the reflected wave, and a decrease in time of arrival of the reflected wave (Table 4), which indicates stronger wave reflections arriving earlier in systole. The larger amplitude of the reflected wave is further corroborated by the increased reflection coefficient. More so, a direct correlation could be established between aortic calcium and the amplitude of the reflected wave Pb (r = 0.84, P = 0.02).
Effect of VDN on ventricular-arterial coupling. Ventricular-arterial coupling is often judged upon optimal cardiac energetics, i.e., the ability of the heart to deliver optimal power or operate under maximal efficiency (46), characterized by the ratio of Ea/Ees. It has been suggested that the heart delivers maximum SW when Ea/Ees = 1, whereas optimal efficiency is obtained when Ea/Ees = 0.5 (6). As the ratio augments above 1.3 and below 0.3, the SW and efficiency are both compromised (16) as observed in patients with severe cardiac dysfunction (1). Changes in ventricular-arterial coupling resulting from arterial stiffening with VDN have not been reported so far. We found that, after treatment with VDN, both Ea and Ees increased significantly as seen in aging (Ref. 18 and Tables 2 and 3); thus, the ratio Ea/Ees was relatively preserved (Table 3). Because of the fact that Ea/Ees approaches 0.5 in the VDN group, optimal efficiency is attained and therefore rises significantly because of a reduction of PVA (Table 3). Optimal energetic coupling of the heart and the arterial system has been reported to be preserved in hypertension and aging (8, 18, 38). Although widely used, Ea/Ees has been criticized for not being a true coupling index (38). This is because Ea is approximately equal to R/T; therefore, the effective Ea is primarily a measure of peripheral resistance and does not reflect the elastic properties of large conduit arteries. Furthermore, Ea includes the effects of heart period, which is a cardiac parameter; therefore, Ea is not a pure arterial parameter. In that respect, we have computed the following two other nondimensional coupling parameters: the CCI and the TCI (41). We found that the CCI (Ees x C) was maintained in the VDN group (0.32 ± 0.08 in VDN vs. 0.34 ± 0.14 in CTRL; Table 4). This means that the stiffening of the arterial tree after VDN was followed by a proportional increase in systolic LV chamber stiffness. Although CCI has been identified as an important coupling parameter and independent determinant of SV and Pao (41), there are no reports on how this index behaves in arterial or cardiac disease. To the best of our knowledge, this is the first report showing that CCI is maintained during development of ISH after arterial stiffening. Contrary to CCI, the TCI (RC/T) was compromised after VDN treatment. This is because the decrease in compliance in the VDN group was not fully compensated by the increase in peripheral resistance and the slight increase in HR.
Clinical implications, novelty, and validity. The present work offers a comprehensive study of ventricular-arterial coupling in the presence of VDN-induced systolic hypertension. The effects of VDN treatment were analyzed at both the arterial level and the cardiac level, as well as in the framework of ventricular-arterial interaction. Most previous work focused on the effects of VDN treatment on heart and vessels, but there was neither a report on cardiac mechanics and energetics nor on the effect on ventricular-arterial coupling (2, 20). The comprehensive character of the study, which offers a consistent set of arterial, cardiac, and ventricular-arterial data on the same animal model, may be valuable for the global understanding of the pathophysiological phenomena involved in the development of systolic hypertension in the presence of a stiffened arterial system. Further research at a molecular level is encouraged to elucidate the effects of VDN on cardiac function (4).
In conclusion, we have studied hemodynamics, arterial function, cardiac function, and ventricular-arterial coupling in a rat model of reduced arterial compliance. The results show that arterial stiffening after VDN treatment provokes important changes in vascular impedance and wave reflections because of ISH and LV hypertrophy, whereas ventricular-arterial coupling was also altered. The effects are quantitatively similar to those of arterial stiffening with age.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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, Control group; 
, VDN group.
