Influence of Aortic Impedance on the Development of Pressure-Overload Left Ventricular Hypertrophy in Rats
Background Aortic input impedance, which represents LV afterload, is considered to be a major determinant for the development of pressure-overload left ventricular (LV) hypertrophy.
Methods and Results To test whether the sustained change in aortic input impedance might affect the mode of development of LV hypertrophy, coarctation of either the ascending aorta (G1, n=13) or suprarenal abdominal aorta (G2, n=12) was performed over 4 weeks in 6-week-old Wistar rats. Although peak LV pressure and total systemic resistance were increased similarly in G1 and G2, time to peak LV pressure was decreased by 24% (P<.01) in G1 compared with G2. The aortic input impedance spectra revealed that the early systolic loading in G1 was characterized by an increase in characteristic impedance, whereas the late systolic loading in G2 was by an augmented arterial wave reflection. G1 showed a smaller increase (P<.01) in either the ratio of LV weight (mg) to body weight (g) or LV wall thickness than G2 after aortic banding. Myocyte diameter was also smaller (P<.05) in G1 (14.3±0.7 mm) than in G2 (16.1±1.2 mm). The ex vivo passive pressure-volume relation had a rightward shift in G1 compared with G2, suggesting less concentric LV hypertrophy in G1.
Conclusions The sustained early systolic loading due to the increase in characteristic impedance was accompanied by less concentric, reduced hypertrophy, whereas the sustained late systolic loading due to the augmented arterial wave reflection was accompanied by concentric, adequate hypertrophy.
The LV is generally thought to adapt to sustained arterial hypertension by developing concentric hypertrophy.1 As a compensatory ventricular response to a chronic pressure overload, ventricular wall thickness increases to normalize the wall stress, and LV dilatation represents a late transition toward myocardial failure.
Although evidence has accumulated as to the roles of hemodynamic load and myocardial contractile state,2 3 little information is available about the influence of the properties of the arterial system on the development of LV hypertrophy and their possible pathophysiological mechanisms.
Aortic input impedance, which depends directly on the geometry and mechanical properties of the arterial network, represents LV afterload and hence is considered to be a major determinant for the development of LV hypertrophy.4 In this connection, several reports5 6 emphasized the important role of the arterial wall characteristics for the development of LV hypertrophy. However, the exact interrelationship between aortic impedance and the development of pressure-overload LV hypertrophy remains to be elucidated. In this study, by changing the coarctation site of the aorta, we produced a sustained increase in aortic impedance in two different ways. Our objective was to investigate the influence of the different patterns of aortic impedance and hence the different systolic loading sequences on the mode of development of LV hypertrophy.
In 6-week-old Wistar rats weighing 130 to 150 g, early systolic loading was produced by coarctation of the aortic arch (group 1) and late systolic loading by coarctation of the abdominal aorta (group 2). In age-matched control rats, sham operations were performed without the aortic constriction being induced for either group 1 or group 2. In addition, age-matched control rats even without sham operation were used to evaluate the basal condition in humoral or morphological studies.
In group 1, the left thorax was opened to expose the aortic arch under artificial ventilation with oxygen and ether. To produce a constriction, a wire 1.2 mm in diameter was placed alongside the aortic arch and was tightly fixed with surgical silk between the first and second branches of the aortic arch. The wire was removed, leaving the aortic arch constricted to an outer diameter equivalent to the diameter of the wire (Fig 1⇓).
In group 2, a wire 0.8 mm in diameter was placed around the abdominal aorta, and a similar constriction was performed as in group 1. Mortality of the rats was 10% with coarctation of the aortic arch, and 90% of these rats died within a day after the surgical procedure. In the case of coarctation of the abdominal aorta, the mortality rate was 0%.
After induction of anesthesia with pentobarbital 50 mg/kg IP, the trachea was cannulated and ventilated with a rodent respirator. A 2F ultraminiature catheter pressure transducer (PR 249, Millar Instruments) was positioned into the LV via the right carotid artery to measure LV pressure.
The time constant (τ) of isovolumetric LV pressure decay from peak (−)dP/dt to 10 mm Hg above the minimal LV pressure was calculated by the Weiss semilogarithmic method.7
After left sternal thoracotomy, LV anterior wall thickness was measured by a single ultrasonic transducer (Wall Tracker Module, WT-20, Crystal Biotech Inc) attached to the middle portion of the epicardium of the LV anterior wall and triggered by peak (+)dP/dt of LV pressure8 (Fig 2⇓).
The ultraminiature catheter pressure transducer was pulled back and positioned in the ascending aorta to measure aortic pressure. The ultrasonic transit-time flow probe (T-106, Transonic Systems Inc) was placed for measurement of the phasic instantaneous aortic blood flow. The frequency response of the pressure recording channel was flat from 0 to 100 Hz. Pressure was low-pass filtered with a corner frequency at 100 Hz. Flow-velocity signal was recorded at a 100-Hz filter setting (frequency response, −6 dB at 100 Hz).
Ten percent of all the rats that underwent the catheterization, crystal attachment, and flowmeter implantation developed serious, persistent hypotension (<90 mm Hg) and/or bradycardia (<200 bpm); these rats were eliminated from the data analysis. Overall mortality during the hemodynamic measurement was 9%, and the mortality was simply due to technical problems. The analog signals were digitized at 1-ms intervals and stored on disk. Ten consecutive cardiac cycles were sampled at each stage and averaged to provide the hemodynamic values.
End diastole was defined by the beginning of (+)dP/dt of LV pressure, and end systole was defined by the time at 10 ms preceding the peak (−)dP/dt of LV pressure. Aortic input impedance was computed by Fourier analysis of the phasic aortic pressure and flow waves. Stroke volume was calculated by an integration of aortic flow signal during the systolic phase. Zc was estimated by averaging impedance moduli between 4 and 10 harmonics.9 10 The first harmonic of the impedance modulus (Z1) was used as an index of arterial wave reflection.11 Total systemic resistance was determined as input resistance (the modulus of the impedance at zero harmonic). The reproducibility for the calculation of Zc was assessed in all groups. After recording aortic pressure and flow at three times, we obtained Zc in triplicate. As a result, there was little variability in the triplicate for Zc.
Zc was also estimated from the aortic pressure-flow relationship during the early ejection period (4 to 14 ms after the onset of ejection), when arterial wave reflection can be neglected.12 The pressure-flow relationship during this period was fitted to a linear equation, and the P/F slope was used as Zc in the time domain.
Ex Vivo P-V Relations, Chamber Stiffness, and Myocardial Stiffness
After the heart was arrested by injection of potassium chloride (2 mEq/mL), it was rapidly removed, the right ventricle was incised, and the AV groove was isolated by a ligature. A double-lumen catheter attached to a pressure transducer (Statham 23 Id), and an infusion pump was passed into the LV. After gentle aspiration of the LV cavity to remove any residual blood, normal saline was infused at 0.70 mL/min into the suspended LV while pressure was recorded. Saline was infused until the pressure increased to 40 mm Hg. The procedure was performed a minimum of two and a maximum of three times within 10 minutes of cardiac arrest before the onset of rigor mortis.13 14
The overall chamber stiffness constant, Kc, was calculated from the P-V relation (P=P0eKcV, where P0 is a modeling constant).13
Ventricular cavity volume at a distending pressure of 10 mm Hg was determined from the passive P-V relation. LV Vw was determined from the mass of the LV as Vw=LV mass (g)/1.05 (the density of muscle).13 14
LV Myocardial Stiffness
LV myocardial stiffness constant (Km) was obtained from the incremental modulus-stress (Einc-σ) relation with a spherical model applied for the LV15 16 : Einc=(1/2)Δσ/(ΔR/R)=Km·s and σ=(3/2)P(V/Vw)(b/R)3, where b is the outer radius and R is the midwall radius.
After the physiological studies, right and left ventricles (plus septum) were separated. Coronal sections of the LV were obtained and fixed in 10% formalin and embedded in paraffin. Sections 4 μm thick were cut and stained with hematoxylin-eosin. Diameters of myocardial cells were measured on a longitudinal section across the nucleus of the cells by a micrometer in the microscope. The mean value of cell diameter was calculated from 100 cells for each heart.17
Connective tissue volume fraction was also assessed by Azan staining.18 19 The section was divided into four quadrants from the center of the section, and four fields were randomly selected from the subendocardial (two fields) and the subepicardial (two fields) regions. Each field was then transferred to a digitizing pad connected to a cursor-computer assembly (LA-555, PIAS). Connective-tissue volume fraction was calculated as the sum of all connective tissue areas in the 16 fields divided by the sum of all connective tissue and muscle areas in all fields. Areas of connective tissue surrounding the intramyocardial coronary arteries were excluded from the calculation.20
For morphological analysis of the ascending aortic wall, 20 rats were perfusion-fixed with 4% paraformaldehyde in 0.1 mol/L PBS, then the aorta was removed. The ascending aortas were postfixed in paraformaldehyde for 24 hours and embedded in paraffin.21 22 Four complete 4-μm cross sections were stained with hematoxylin-eosin, then the medial thickness, medial area, and internal diameter were measured.
In each experimental group, circulating plasma angiotensin II and plasma aldosterone levels were determined 4 days, 10 days, and 4 weeks after surgery.23
Data were evaluated by the statistical power analysis24 to obtain the lowest number (N) of animals to be statistically compared. In our design of the experiments, sample size was determined under the condition that the level of significance was 5% and the power of the study was 80%. When the actual number of animals (n) was the same as or greater than N (n≥N), an unpaired t test was used for statistical comparison of the data. Results are expressed as mean±SD, and a value of P<.05 was accepted as statistically significant.
Both LV pressures and ratios of LV weight to body weight increased significantly in the pressure-overloaded groups as early as 4 days after the operation, and these increases were maintained over 4 weeks (Fig 3⇓). In group 1, the ratio of LV weight to body weight was significantly lower than in group 2 at any time studied (Fig 3⇓). There was no significant difference in the body weight, right ventricular weight, and atrium weight among all groups (not shown).
The parameters of hemodynamics and LV geometry are summarized in Table 1⇓. Although peak LV pressure was increased similarly in the two pressure-overloaded groups, time to peak LV pressure was shorter in group 1 than in group 2, indicating early systolic loading in group 1 and late systolic loading in group 2. The peak (+)dP/dt of LV pressure was increased in both pressure-overloaded groups. The time constant of LV pressure decay remained unchanged in group 1 and even decreased in group 2. There was no significant difference among all groups in heart rate and LV end-diastolic pressure. The LV end-diastolic wall thickness was increased in both group 1 and group 2 compared with the corresponding sham groups. However, end-diastolic wall thickness was significantly lower in group 1 than in group 2.
Aortic Input Impedance
The average aortic input impedance spectra for each group are shown in Fig 4⇓. In group 1, the moduli of input impedance increased significantly, from 0 to 26 Hz, and tended to increase even above 26 Hz. In group 2, the moduli of input impedance significantly increased at low frequencies between 0 and 10 Hz; however, they did not change above 10 Hz. In group 1, phase angle was shifted to the left compared with the sham-operated controls, whereas in group 2, it was shifted to the right.
Aortic impedance parameters and cardiac output are listed in Table 2⇓. Although total systemic resistance increased to similar extents in the two banding groups, cardiac output was comparable among all groups. When the data were compared between group 1 and group 2, Zc and P/F slope (expression of Zc in the time domain, see “Methods”) increased significantly in group 1, whereas the modulus of the first harmonic, Z1, increased in group 2.
P-V Relations, Chamber Stiffness Constant, and Myocardial Stiffness Constant
Fig 5⇓ shows the ex vivo passive P-V relations. Compared with group 2, group 1 had a rightward shift of the P-V relation and an increase in V/Vw (Table 3⇓). The chamber stiffness and myocardial stiffness constants were smaller in group 1 than in group 2 (Table 3⇓).
Table 4⇓ summarizes the histological data of the LV and the ascending aorta. The myocyte diameters of the LV in group 1 and group 2 increased significantly compared with the corresponding sham-operated controls. The myocyte diameter was smaller in group 1 than in group 2. The connective tissue volume fraction in the subendocardial region was lower in group 1 than in group 2, with no significant difference in the subepicardial connective tissue volume fraction.
The medial wall thickness of the aorta increased to a similar extent in both aortic banding groups compared with the corresponding sham groups. There was no significant difference in the internal diameters of the aortas among all groups.
Plasma Angiotensin II and Aldosterone
Fig 6⇓ shows the levels of plasma angiotensin II and aldosterone at 4 days, 10 days, and 4 weeks after the operation in all groups. In both banding groups, the transient comparable increases of plasma angiotensin II and aldosterone were seen at 4 days after the operation compared with the sham groups or controls. These increases had disappeared at 10 days and 4 weeks after the operation.
The major findings of this study were as follows. First, the sustained early systolic loading produced by ascending aortic constriction was characterized primarily by the increase in Zc, whereas the sustained late systolic loading by abdominal aortic constriction reflected the increase in arterial wave reflection. Second, the sustained early systolic loading was associated with the reduced, less concentric LV hypertrophy in contrast to the adequate, concentric hypertrophy with late systolic loading. Third, the vascular hypertrophy in the aorta was also produced by either early or late systolic loading. However, there was no significant difference in the extent of vascular hypertrophy between the two hypertensive groups, suggesting the specific hypertrophic response of the LV to the different systolic loading sequence.
Systolic Loading Sequence and Aortic Input Impedance
In group 1, the impedance modulus was larger at high frequencies (>10 Hz), indicating a stiffening of the proximal aorta, whereas in group 2, the increase in the impedance modulus was prominent at low frequencies (<10 Hz), reflecting an increase in arterial wave reflection.25 On the basis of the finding of aortic impedance spectra, we interpret the LV pressure profile with early or late systolic loading as follows. Since Zc plays a significant role as a load against LV during the early ejection phase,12 26 LV pressure produced a peak during the early ejection phase, with ascending aortic banding in which Zc increased. However, in the case of abdominal aortic banding, arterial wave reflection increased without a change in Zc. Therefore, LV pressure produced a peak during the late ejection phase, reflecting the increase in arterial wave reflection, probably from the constriction site.
LV Hypertrophy and Pressure Overload
In the rats with early systolic loading, the ratio of LV weight to body weight was already decreased compared with late systolic loading even at 4 days after aortic banding. Therefore, it is suggested that the difference in aortic input impedance may influence the triggering process of LV hypertrophy. From the viewpoint of the difference in pressure profile or the impedance spectra between groups 1 and 2, we speculate that late systolic loading or the increase in arterial wave reflection may be necessary for the adequate development of LV hypertrophy. The reduced hypertrophic responses, namely, the decrease in LV weight, less hypertrophied myocytes, and the smaller connective tissue volume fraction, are likely to be responsible for the less concentric hypertrophy and also for the smaller chamber and myocardial stiffness in the rats with early systolic loading.
Vascular Hypertrophy and Pressure Overload
Previous investigations9 27 showed that vascular hypertrophy as well as LV hypertrophy was produced in the animals with hypertension. In this study, both types of pressure overload (early and late systolic loading) indeed caused an increase in the medial thickness of the ascending aorta. However, since there was no significant difference in the degree of vascular hypertrophy, it is suggested that at least in the present rat models, the development of vascular hypertrophy might not be influenced by the difference in the systolic loading sequence, in contrast to the LV, which clearly showed the different hypertrophic responses.
Effects of the Renin-Angiotensin-Aldosterone System and/or Growth Factors on Pressure-Overload Hypertrophy
In the renovascular hypertensive9 or aortic banding rat model,28 29 it has been reported that ACE inhibitor induced a prevention or regression of pressure-overload hypertrophy unrelated to its blood pressure–lowering effect, suggesting the possible involvement of plasma or locally formed cardiac angiotensin II in the development of LV hypertrophy. Indeed, we observed a transient increase in plasma angiotensin II at the early stage of LV hypertrophy. This increased angiotensin II may trigger the LV hypertrophy. However, since there was no significant difference in the level of angiotensin II between the two aortic banding groups, the plasma angiotensin II might not be responsible for the difference in the degree of hypertrophy observed in the two types of pressure overload. Nonetheless, it is possible that the observed reduced hypertrophy with sustained early systolic loading is induced through the lesser stimulation of the local renin-angiotensin system in cardiac tissue.
Peptide growth factors are also possible candidates that could induce or modulate the different types of LV pressure-overload hypertrophy observed, since peptide growth factors might play an autocrine/paracrine role in mediating myocardial hypertrophy.30
Although we calculated Zc as an average of the impedance modulus from the 4th to the 10th harmonics,9 10 Mitchell et al31 recommended that estimates of Zc include the first impedance minimum in the average to prevent overestimation of Zc when reflected waves are prominent. In this regard, we estimated Zc by measuring the P/F slope during the early ejection period (see “Methods”), in which arterial wave reflection can be neglected.12 The P/F slope so obtained was closely correlated with Zc in the frequency domain (r=.90, P<.001, n=34), indicating the reasonable estimation of Zc in our study.
During the time course of hypertension, aortic input impedance is influenced by the vascular smooth muscle activity under the control of the vascular sympathetic nervous system.32 33 Since we observed a tendency for heart rate to increase with the aortic banding, consistent with the previous investigation,34 the activity of the vascular sympathetic nervous system may be higher with pressure overload, which in turn may affect the aortic input impedance and the mode of development of LV hypertrophy. Also, these changes in heart rate might contribute to modification of the dynamic rigidity of the aorta per se.
We should consider the effect of anesthesia. Anesthesia may possibly influence the aortic wall characteristics and hence aortic impedance. With regard to these observations, clearly more work is needed.
Selected Abbreviations and Acronyms
|P/F slope||=||slope of pressure-flow relation during early ejection period|
We thank Dr Minoru Nakamoto at The Department of Hygiene, Yamaguchi University, for his valuable comments.
- Received March 13, 1996.
- Revision received July 10, 1996.
- Accepted July 31, 1996.
- Copyright © 1996 by American Heart Association
Grossman W, Jones D, McLaurin LP. Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest. 1975;56:56-64.
Ganau A, Devereux RB, Pickering TG, Roman MJ, Schnall PL, Santucci S, Spitzer MC, Laragh JH. Relation of left ventricular hemodynamic load and contractile performance to left ventricular mass in hypertension. Circulation. 1990;81:25-36.
Levy BI, Safar ME. Ventricular afterload and aortic impedance. In: Swynghedauw B, ed. Research in Cardiac Hypertrophy and Failure. London, UK: John Libbey & Co Ltd; 1990:521-529.
Safar ME, Toto-Moukouo JJ, Bouthier JA, Asmar RE, Levenson JA, Simon AC, London GM. Arterial dynamics, cardiac hypertrophy, and antihypertensive treatment. Circulation. 1987;75(suppl I):I-156-I-161.
Levy BI, Babalis D, Lacolley P, Poitevin P, Safar ME. Cardiac hypertrophy and characteristic impedance in spontaneously hypertensive rats. J Hypertens. 1988;6(suppl 4):110-111.
Weiss JL, Frederiksen JW, Weisfeldt ML. Hemodynamic determinants of the time-course of fall in canine left ventricular pressure. J Clin Invest. 1976;58:751-760.
Zhu WX, Myers ML, Hartley CJ, Roberts R, Bolli R. Validation of a single crystal for measurement of transmural and epicardial thickening. Am J Physiol. 1986;251:H1045-H1055.
Levy BI, Michel JB, Salzmann JL, Azizi M, Poitevin P, Safar M, Camilleri JP. Effects of chronic inhibition of converting enzyme on mechanical and structural properties of arteries in rat renovascular hypertension. Circ Res. 1988;63:227-239.
Huijberts MSP, Wolffenbuttel BHR, Boudier HAJS, Crijns FRL, Kruseman ACN, Poitevin P, Levy BI. Aminoguanidine treatment increases elasticity and decreases fluid filtration of large arteries from diabetic rats. J Clin Invest. 1993;92:1407-1411.
Laskey WK, Kussmaul WG, Martin JL, Kleaveland JP, Hirshfeld JW, Shroff S. Characteristics of vascular hydraulic load in patients with heart failure. Circulation. 1985;72:61-71.
Raya TE, Gay RG, Lancaster L, Aguirre M, Moffett C, Goldman S. Serial changes in left ventricular relaxation and chamber stiffness after large myocardial infarction in rats. Circulation. 1988;77:1424-1431.
Litwin SE, Raya TE, Anderson PG, Litwin CM, Bressler R, Goldman S. Induction of myocardial hypertrophy after coronary ligation in rats decreases ventricular dilatation and improves systolic function. Circulation. 1991;84:1819-1827.
Mirsky I, Pfeffer JM, Pfeffer MA, Braunwald E. The contractile state as the major determinant in the evolution of left ventricular dysfunction in the spontaneously hypertensive rat. Circ Res. 1983;53:767-778.
Mirsky I. Assessment of diastolic function: suggested methods and future considerations. Circulation. 1983;69:836-841.
Sekiguchi M, Hiroe M, Morimoto S. On the standardization of histopathological diagnosis and semiquantitative assessment of the endomyocardium obtained by endomyocardial biopsy. Bull Heart Inst Jpn. 1979;21:55-85.
Heidenhaim M. U¨ber die Mallory'sche Binde-gewebsfa¨rbung mit Karmin und Azokarmin als Vorfarben. Z Wiss Mikrosk. 1915;32:361-372.
Kojima M, Shiojima I, Yamazaki T, Komuro I, Yunzeng Z, Ying W, Mizuno T, Ueki K, Tobe K, Kadowaki T, Nagai R, Yazaki Y. Angiotensin II receptor antagonist TCV-116 induces regression of hypertensive left ventricular hypertrophy in vivo and inhibits the intracellular signaling pathway of stretch-mediated cardiomyocyte hypertrophy in vitro. Circulation. 1994;89:2204-2211.
Weber KT, Janicki JS, Shroff SG, Pick R, Chen RM, Bashey RI. Collagen remodeling of the pressure-overloaded, hypertrophied nonhuman primate myocardium. Circ Res. 1988;62:757-765.
Todd ME, Gowen B. Arterial wall and smooth muscle cell development in young Wistar rats and the effects of surgical denervation. Circ Res. 1991;69:438-446.
Brilla CG, Pick R, Tan LB, Janicki JS, Weber KT. Remodeling of the rat right and left ventricles in experimental hypertension. Circ Res. 1990;67:1355-1364.
Cohen J. Statistical Power Analysis for the Behavioral Sciences. Revised ed. London, UK: Academic Press; 1977.
Nichols WW, O'Rourke MF. Mcdonald's Blood Flow in Arteries. 3rd ed. Baltimore, Md: Edward Arnold Publishers Ltd; 1990:251-269.
O'Rourke MF, Kelly RP, Avolio AP. The Arterial Pulse. London, UK: Lea & Febiger; 1992:146-176.
Levy BI, Duriez M, Phillipe M, Poitevin P, Michel JB. Effect of chronic dihydropyridine (isradipine) on the large arterial walls of spontaneously hypertensive rats. Circulation. 1994;90:3024-3033.
Linz W, Schaper J, Wiemer G, Albus U, Scho¨lkens BA. Ramipril prevents left ventricular hypertrophy with myocardial fibrosis without blood pressure reduction: a one year study in rats. Br J Pharmacol. 1992;1077:970-975.
Schunkert H, Jackson B, Tang SS, Schoen FJ, Smits JFM, Apstein CS, Lorell BH. Distribution and functional significance of cardiac angiotensin converting enzyme in hypertrophied rat hearts. Circulation. 1993;87:1328-1339.
Calderone A, Takahashi N, Izzo NJ Jr, Thaik CM, Colucci WS. Pressure- and volume-induced left ventricular hypertrophies are associated with distinct myocyte phenotypes and differential induction of peptide growth factor mRNAs. Circulation. 1995;92:2385-2390.
Mitchell GF, Pfeffer MA, Westerhof N, Pfeffer JM. Measurement of aortic input impedance in rats. Am J Physiol. 1994;267:H1907-H1915.
Cox RH. Effects of norepinephrine on mechanics of arteries in vitro. Am J Physiol. 1976;231:H420-H425.
Stone DN, Dujardin JPL. Changes in smooth muscle tone influence characteristic impedance of the aorta. Am J Physiol. 1984;246:H1-H7.
Beznak M. Cardiac output in rats during the development of cardiac hypertrophy. Circ Res. 1958;6:207-212.