Attenuation of Cardiac Failure, Dilatation, Damage, and Detrimental Interstitial Remodeling Without Regression of Hypertrophy in Hypertensive Rats
Whether left ventricular (LV) hypertrophy is important in the development of LV failure associated with advanced myocardial damage and detrimental chamber and interstitial remodeling in hypertension has not been established. We examined the effect of an antihypertensive agent without the ability to regress LV hypertrophy on the development of LV changes in spontaneously hypertensive rats (SHR). Hydralazine given to SHR from 5.2 to 26 months of age returned systolic blood pressure to Wistar Kyoto (WKY) control values but failed to prevent the increase in LV mass noted in SHR (at 26 months of age: WKY, 0.99±0.02 g; untreated SHR, 1.40±0.02 g; treated SHR, 1.36±0.02 g; P<0.001 in SHR versus WKY). In comparison to both 16-month-old SHR and age-matched WKY, 26-month-old untreated SHR developed signs consistent with heart failure, LV dilatation (an increased LV internal radius), an eccentric LV geometry, advanced myocyte necrosis, an increase in myocardial collagen solubility (an index of decreases in myocardial collagen cross-linking), and marked increases in myocardial total, type III, and non-cross-linked myocardial collagen concentrations. Despite the inability of hydralazine to regress LV hypertrophy, treated SHR did not develop signs of heart failure, myocyte necrosis, decreases in myocardial collagen cross-linking, or increases in myocardial total, type III, and non-cross-linked collagen at 26 months of age. Moreover, treatment attenuated the development of LV dilatation and an eccentric LV geometry. In conclusion, antihypertensive therapy that does not attenuate LV hypertrophy but achieves normal blood pressure in SHR, is able to hinder the development of heart failure associated with advanced myocardial damage and detrimental chamber and interstitial remodeling.
Patients with a history of hypertension have at least a 6-fold greater risk of developing heart failure than do individuals without such a history1. Heart failure in hypertension can occur as a consequence of either diastolic dysfunction or systolic abnormalities associated with detrimental chamber (left ventricle [LV] dilatation and the development of an eccentric cardiac geometry)2,3 and interstitial (reparative fibrosis with qualitative changes in collagen)4–6 remodeling. Although hypertensive patients with LV hypertrophy have a clear increased risk for cardiovascular events7–10 and the development of diastolic heart failure,11 clinical studies have provided only limited data regarding the importance of hypertensive LV hypertrophy as a risk factor for the development of heart failure associated with advanced cardiac damage and detrimental LV remodeling.7–10 The aim of the present study, therefore, was to determine whether the use of an antihypertensive agent with no significant effect on modifying LV hypertrophy is able to influence the development of detrimental LV remodeling, LV damage, and heart failure in hypertensive heart disease. Hydralazine, a direct-acting vasodilator, has been shown to be unable to influence LV hypertrophy in hypertension despite profound antihypertensive actions.12 We, therefore, evaluated the effect of hydralazine on the development of detrimental chamber and interstitial remodeling, advanced cardiac damage, and the appearance of clinical signs of heart failure in spontaneously hypertensive rats (SHR).
The present study was approved by the Animal Ethics Screening Committee (AESC) of the University of the Witwatersrand (AESC approval Nos. 93/19/2b and 97/44/5).
SHR (5.2 months old; n=51; OLAC, UK) and Wistar Kyoto controls (WKY) (n=36; Kleinterfarm Madorin Ltd, Germany) were assigned to a group of rats (12 SHR and 10 WKY) who received hydralazine in the drinking water (0.07 mmol · kg−1 · day−1) until they were 26 months of age; to a group of rats (21 SHR and 14 WKY) left untreated until they were 16 months of age (compensated hypertrophy); or to a group of rats (18 SHR and 12 WKY) left untreated until they were 26 months of age (decompensated hypertrophy). Of those rats that did not survive the study period and were not included in the sample numbers given, 8 untreated SHR died at between 23 and 26 months of age (2 of a respiratory pathogen and 6 with signs of heart failure), 2 WKY (1 untreated and 1 hydralazine treated) died of soft tissue tumors at variable time periods, and 2 untreated WKY died of a respiratory pathogen at variable time periods; no hydralazine-treated SHR died. Systolic blood pressure was measured as previously described13 at regular periods throughout the study.
LV Geometry and Dimensions
LV dimensions and geometry were determined in anesthetized, ventilated, open-chest rats. The surgery, instrumentation, experimental techniques, and calculations used in our current study have previously been described and validated.12–14 LV end diastolic (LVED) radius was determined from the equation
where LV wall volume=0.943×LV wet weight.14 LVED geometry was determined from LVED wall thickness (h)-to-radius (r) ratios determined over a range of LVED pressure (LVEDP) values, where LVED h=[LVED external diameter−2r)/2].14
Myocardial Collagen and Histology
Samples of LV tissue were weighed and stored at −70°C for tissue analysis. Myocardial hydroxyproline concentrations ([HPRO]) were determined as previously described6,12 by the method of Stegemann and Stalder,15 following acid hydrolysis. Myocardial collagen was also extracted and digested with cyanogen bromide (CNBr) according to the procedure described by Mukherjee and Sen.16 Using a portion of the CNBr-digested sample, polyacrylamide gel electrophoresis was subsequently performed on vertical gels by stacking and separating gel concentrations of 3% and 12.5%, respectively, and the type I-to-type III collagen ratio was determined following gel scanning.12,16 The remaining portion of the CNBr-digested sample was subjected to acid hydrolysis and [HPRO] determination. The quantity of [HPRO] extracted after CNBr digestion was expressed as a percentage (% solubility) of [HPRO] extracted after acid hydrolysis alone. We used this as a measure of tissue collagen solubility and subsequently as an index of the degree of collagen cross-linking.6,12 Myocardial type I, type III, insoluble (cross-linked), and soluble (non-cross-linked) collagen concentrations were calculated as previously described.6
Before storing tissue for biochemical assessment, a longitudinal slice of the LV from the apex to the base through both the anterior and posterior LV wall was obtained from all rats for histology. LV tissue was stored, prepared, sectioned, and stained as previously described.6 A pathological grade, modified from Teerlink et al,17 was assigned to each slice as follows: 0, no damage; 1, patchy fibrosis in 1 to 5 areas (<20% of the field); 2, patchy fibrosis in >20% of the field; 3, diffuse, contiguous subendocardial fibrosis in <50% of the field; 4, diffuse, contiguous subendocardial fibrosis in >50% of the field; 5, transmural fibrosis in <50% of the field; and 6, transmural fibrosis in >50% of the field.
Differences in body and tissue weights, LV internal dimensions, LV geometry, myocardial collagen analysis, and pathological scores between the groups were assessed using a 1-factor ANOVA followed by a Tukey-Kramer multiple comparisons post hoc test. A repeated-measure ANOVA was used to assess the effect of hydralazine on blood pressure. All values in the text are represented as mean±SEM.
An expanded Methods section can be found in an online data supplement available at http://www.hypertensionaha.org.
Hydralazine given to SHR returned systolic blood pressure to WKY control values for the duration of the study (Figure 1). However, hydralazine given to WKY controls failed to influence blood pressure (data not shown).
Signs of Cardiac Decompensation
Eleven of 18 of the surviving untreated 26-month-old SHR had obvious atrial thrombi and pleuropericardial effusions. In the WKY groups and the hydralazine-treated SHR group, neither atrial thrombi nor pleuropericardial effusions were present. Consistent with hepatic congestion, 26-month-old SHR had increased wet (Table 1) but not dry(data not shown) liver weights compared with those of WKY controls. Hydralazine administration prevented the development of hepatic congestion (Table 1).
LV (including septum) weight was increased in all SHR groups compared with their age-matched WKY controls, with the LV hypertrophy being most marked in 26-month-old SHR (Table 1). Right ventricular (RV, free wall only) weight was increased in only the 26-month-old SHR group in comparison to WKY controls (Table 1). Despite producing antihypertensive effects, hydralazine given to SHR failed to modify either LV or RV weight (Table 1). However, hydralazine administered to WKY controls did not alter cardiac chamber weight (data not shown).
LV Internal Dimensions
Suggestive of concentric LV remodeling, in 16-month-old SHR a reduced LVED r at comparable LVEDPs was noted compared with that of age-matched WKY control values (LVED r data at an LVEDP value of 0 mm Hg are provided in Figure 2). In contrast, indicative of LV dilatation, LVED r, as determined over a range of LVEDP values, was increased in the untreated 26-month-old SHR group compared with the age-matched WKY group and the 16-month-old SHR group (LVED r data at an LVEDP value of 0 mm Hg are provided in Figure 2). Hydralazine treatment returned LVED r values in 26-month-old SHR to values similar to those noted in WKY controls and to values not significantly different from data obtained in 16-month-old SHR (LVED r data at an LVEDP value of 0 mm Hg are provided in Figure 2).
As a consequence of LV hypertrophy, LVED h/r, as determined over a range of LVEDPs, was greater in the untreated and the hydralazine-treated SHR groups compared with age-matched WKY controls (Figure 2). However, indicative of LV dilatation, 26-month-old untreated SHR had a reduced LVED h/r compared to that of the 16-month-old SHR, despite having a greater LV weight (Figure 2). In contrast, hydralazine treatment to SHR resulted in an increase in LVED h/r, compared with that of the untreated 26-month-old SHR, to values not significantly different from 16-month-old SHR at LVEDPs of >2 mm Hg (Figure 2). Up to LVEDPs of 2 mm Hg, hydralazine-treated SHR had modest decreases in LVED h/r in comparison to 16-month-old untreated SHR (Figure 2, P<0.05).
Histological evidence of marked myocyte necrosis was noted in 26-month-old untreated SHR, but not in either the 16-month-old SHR group or the hydralazine-treated 26-month-old SHR group (Figure 3).
Quantity of Myocardial Collagen
Both 16- and 26-month-old untreated SHR had increased myocardial [HPRO], and the untreated 26-month-old group exhibited a proportionately greater increment in [HPRO] than the 16-month-old group (Figure 3). In contrast, hydralazine therapy to SHR reduced [HPRO] to values comparable with those of both the 26-month-old WKY group and the 16-month-old SHR group (Figure 3).
Quality of Myocardial Collagen
Although increases in myocardial types I and III were noted in 16-month-old SHR, no differences in phenotypic ratios occurred in untreated or hydralazine-treated 26-month-old SHR (Table 2). The overall effect of quantitative and phenotypic collagen changes was that type I, but not type III, collagen concentrations were increased in 16-month-old SHR, whereas both type I and III concentrations were increased in 26-month-old SHR (Table 2 and Figure 3). Moreover, hydralazine administration reduced both type I and III myocardial collagen concentrations in 26-month-old SHR to control values (Figure 3).
Myocardial collagen solubility was decreased in 16-month-old SHR (indicating an increase in collagen cross-linking), whereas collagen solubility was increased in untreated 26-month-old SHR (indicating a decrease in collagen cross-linking), an effect which was prevented by hydralazine treatment (Table 2). The overall effect of quantitative and cross-linked collagen changes was that cross-linked (insoluble) (Table 2) but not non-cross-linked (soluble) (Figure 3) collagen concentrations were increased in 16-month-old SHR, whereas both cross-linked (Table 2) and non-cross-linked (Figure 3) collagen concentrations were increased in 26-month-old SHR, an effect that was reversed by hydralazine therapy (Figure 3).
The main finding of the present study is that in SHR, antihypertensive therapy with the vasodilator hydralazine, despite being unable to influence the development of increases in LV weight, was able to attenuate the development of LV dilatation, chamber eccentricity, myocardial necrosis, detrimental interstitial remodeling, and signs of cardiac decompensation.
A reduction in LV concentricity in decompensated pressure overload LVH, as determined in the present study, is consistent with previous reports in humans3 and is attributed to LV dilatation. Detrimental cardiac chamber remodeling in the form of LV dilatation is known to occur in various other models of cardiac decompensation, including postinfarction cardiac remodeling,18 tachycardia-induced heart failure,19 ß-agonist-induced cardiotoxicity following myocyte necrosis,17 and ß-agonist-induced cardiac hypertrophy without myocyte necrosis.6 LV dilatation and an eccentric geometry in hypertension reflects inappropriate chamber remodeling, in which despite cardiac growth, wall thickness-to-internal radius ratios decrease and the purpose of hypertrophy (ie, to maintain normal wall stress values despite high chamber systolic pressures) is attenuated.
The relationship between myocardial fibrosis and hypertensive heart failure associated with LV dilatation is well established.4,6 It is likely that the increases in myocardial collagen concentrations noted in 16-month-old SHR in the present study reflect reactive interstitial changes, because no histological evidence of myocyte necrosis or increases in type III collagen (which is typically produced during tissue reparation20) were detected. Moreover, in untreated 26-month-old SHR, because myocyte necrosis and both types I and III collagen concentrations were increased, a combination of both reactive and reparative changes is likely. Interestingly, hydralazine therapy, which has previously been shown to be unable to modify reactive fibrosis,12,16 prevented the development of reparative fibrosis in 26-month-old SHR in the present study. This would suggest that although reactive myocardial fibrosis depends on effects that are to some extent unrelated to changes in blood pressure alone, reparative fibrosis is likely to be proportional to the extent of myocardial damage mediated by blood pressure effects.
The morphological changes that result in LV dilatation in pressure overload hypertrophy have been suggested to be an enhanced myocyte cell length,21,22 rather than myocyte slippage. However, in the present study, although hydralazine administration failed to modify LV weight in SHR with advanced LV hypertrophy, hydralazine attenuated the development of LV dilatation. The results of the present study therefore indicate that although there is a close correlation between cardiomyocyte length and LV cavity size in some models of pressure overload hypertrophy,21,22 cardiac growth, determined as cardiac weight, a traditional clinical measure of LV hypertrophy, is not closely related to LV dilatation in established hypertensive heart failure. This notion is consistent with data obtained by Brooks et al,4 who have shown that the administration of a pharmacological agent that reduces LV weight in rats with POH after LV dilatation is established, is unable to modify ventricular end diastolic volumes. Rather, myocyte slippage mediated through either myocyte necrosis with reparative fibrosis (this study), increases in myocardial collagenase activities,23 or decreases in myocardial collagen cross-linking (as shown in this study and in other forms of pressure overload hypertrophy6), is a more likely morphological change responsible for LV dilatation in hypertensive heart disease.
In the present study, we failed to evaluate whether myocyte side-to-side slippage could explain LV dilatation. However, as pointed out by Tamura et al,21 there are a number of technical limitations to the assessment of myocyte slippage, all of which restrict the interpretation of these data. In our hands, we have not been able to reproducibly assess myocyte slippage.
The mechanisms responsible for the lack of effect of hydralazine on cardiac weight in hypertension have not been elucidated. Potential mechanisms include conversion of pressure to volume-overload hypertrophy, reflex sympathetic over-activity following excessive vasodilatation, and stimulation of the renin-angiotensin system. However, in the present study, the lack of effect of hydralazine on cardiac weight cannot be attributed to a specific action of hydralazine alone, as hydralazine failed to stimulate cardiac growth in normotensive rats. Moreover, the ability of hydralazine to prevent LV failure without mediating an effect on RV weight suggests that RV hypertrophy is not only attributed to LV failure in SHR.
Although there are data to show that the vasodilator hydralazine is unable to prevent or regress LV hypertrophy or influence reactive interstitial changes in pressure overload hypertrophy despite profound antihypertensive actions,12,16 this study is the first to show that hydralazine is able to attenuate chamber dilatation, myocyte necrosis, and detrimental interstitial remodeling associated with reparative changes, irrespective of its lack of effect on cardiac weight in LV hypertrophy. These findings support the notion that antihypertensive therapy that achieves marked blood pressure-lowering effects is more important than using agents that specifically have an ability to regress the development of LV hypertrophy when attempting to prevent the development of cardiac failure associated with LV dilatation, an eccentric cardiac geometry, advanced myocardial damage, and detrimental interstitial remodeling in hypertension.
In conclusion, the results of the present preclinical study suggest that irrespective of effects of antihypertensive agents on LV weight, therapy that achieves controlled blood pressures is able to attenuate the development of LV dilatation and consequently heart failure in hypertension, probably through effects on both myocyte integrity and the cardiac interstitium. These data caste doubt on the importance of LV weight as a predictor of subsequent LV dilatation and damage in hypertensive heart disease.
This research was supported by the Medical Research Council of South Africa, the H.E. Griffin Charitable Trust, and the University Research Council of the University of the Witwatersrand
- Received January 26, 2001.
- Revision received February 16, 2001.
- Accepted April 8, 2001.
Kannel WB, Castelli WP, McNamara PM, McKee PA, Feinleib M. Role of blood pressure in the development of congestive heart failure: the Framingham Study. N Engl J Med. 1972; 287: 781–787.
Brooks WW, Bing OHL, Robinson KG, Slawsky MT, Chaletsky DM, Conrad CH. Effect of angiotensin-converting enzyme inhibition on myocardial fibrosis and function in hypertrophied and failing myocardium from the spontaneously hypertensive rat. Circulation. 1997; 96: 4002–4010.
Mukherjee D, Sen S. Collagen phenotypes during development and regression of myocardial hypertrophy in spontaneously hypertensive rats. Circ Res. 1990; 67: 1474–1480.
Woodiwiss AJ, Tsotetsi OJ, Sprott S, Lancaster EJ, Mela T, Chung ES, Meyer TE, Norton GR. Reduction in myocardial collagen cross-linking parallels left ventricular dilatation in rat models of systolic chamber dysfunction. Circulation. 2001; 103: 155–160.
Casale PN, Devereux RB, Milner M, Zullo G, Harshfield GA, Pickering TG, Laragh JH. Value of echocardiographic measurements of left ventricular mass in predicting cardiovascular morbid events in hypertensive men. Ann Intern Med. 1986; 105: 172–178.
Koren MJ, Devereux RB, Casale PN, Savage DD, Laragh JH. Relation of left ventricular mass and geometry to morbidity and mortality in men and women with uncomplicated essential hypertension. Ann Intern Med. 1991; 114: 345–352.
Gilbert JC, Glantz SA. Determinants of left ventricular filling and of the diastolic pressure-volume relation. Circ Res. 1989; 64: 827–851.
Norton GR, Tsotetsi J, Trifunovic B, Hartford C, Candy GP, Woodiwiss AJ. Myocardial stiffness is attributed to alterations in cross-linked rather than total collagen or phenotypes in spontaneously hypertensive rats. Circulation. 1997; 96: 1991–1998.
Norton GR, Tsotetsi OJ, Woodiwiss AJ. Hydrochlorothiazide improves ventricular compliance and thus performance without reducing hypertrophy in renal artery stenosis in rats. Hypertension. 1993; 21: 638–645.
Woodiwiss AJ, Norton GR. Exercise-induced cardiac hypertrophy is associated with an increased myocardial compliance. J Appl Physiol. 1995; 78: 1303–1311.
Teerlink JR, Pfeffer JM, Pfeffer MA. Progressive ventricular remodeling in response to diffuse isoproterenol-induced myocardial necrosis in rats. Circ Res. 1994; 75: 105–113.
Litwin SE, Katz SE, Morgan JP, Douglas PS. Serial echocardiographic assessment of left ventricular geometry and function after large myocardial infarction in the rat. Circulation. 1994; 89: 345–354.
Spinale FG, Zellner JL, Johnson WS, Eble DM, Munyer PD. Cellular and extracellular remodeling with the development and recovery from tachycardia-induced cardiomyopathy: changes in fibrillar collagen, myocyte adhesion capacity and proteoglycans. J Mol Cell Cardiol. 1996; 28: 1591–1608.
Clore JH, Cohen IK, Diegelmann RF. Quantification of collagen types I and III during wound healing in rat skin. Proc Soc Exp Biol Med. 1979; 161: 337–340.