vascular and myocardial protective effects of converting enzyme inhibition in experimental heart...

Vascular and Myocardial Preteetive Effects of Converting Enzyme Inhibition in

Experimental Heart Failure Paul Mulder, PhD, Bruno Devaux, MD, Lahcen El Fertak, PhD, Patricia Compagnon, Vincent Richard, PhD, Jean-Paul Henry, Elizabeth Scalbert, PhD, Pierre Desch6, MD,

Bertrand Ma&, MD, and Christian Thuillez, MD, PhD

Systemic vasoconstriction due to stimulation of the sympathetic and reni-ngiotensin-aldosterone systems is a hallmark of heart failure and this is accompanied by impaired endothelium-dependent relaxations at the level of large arteries. This study investigated, in a rat model of heart failure, whether such an endothelial dysfunction also exists at the level of the resistance artery, and whether this is associated with morphologic changes, as well as the effects of chronic treatment with the angiotensin- converting enzyme inhibitor perindopril(2 mg/kg/ day). After 12 months, arterial pressure, leftventricu- lar (LV) end diastolic pressure (LVEDP), and LV dP/& were measured in anesthetized rats. Re- sponses to acetylcholine and nitroprusside were determined in isolated and perfused mesenteric artery segments (diameter: 280 f 15 pm). After fixation, vessel diameter, media cross-sectional area, and media collagen and elastin densities were measured by image analysis. After 12 months, untreated rats showed signs of heart failure, i.e.,

reduced LV dP/dt, and increased LVEDP, heart weight/body weight, LV cavity circumference, and myocardial collagen density. In mesenteric vessels the endothelium-dependent vasodilator response to acetylcholine was impaired, whereas the response to the nitric oxide donor nitropntsside was unaffected. Heart failure did not affect vascular morphological parameters. Perindopril decreased blood pressure and LVEDP without any modification of LV dP/cft, and prevented cardiac remodeling. At the vascular level, perindopril improved the response to acetylcholine and reduced media cross- sectional area and collagen density without affect- ing internal vessel diameter or elastin density. Thus, heart failure decreases endothelium-dependent vasodilator response to acetylcholine without modification of vessel structure. The heart-failure induced endothelial dysfunction could be prevented by angiotensin-converting enzyme inhibition.

(Am J Cardioll995; 76:28E<3E)

C hronic heart failure is characterized hemo- dynamically by an increase in ventricular filling pressures and an increase in systemic

vascular resistance. The systemic vasoconstriction and the impaired vasodilation-both of which contribute to the abnormalities in vasomotor tone- have been well demonstrated in models of heart failure and in humans. These changes appear to play an important role in the development of many of the signs and symptoms of chronic heart failure by decreasing peripheral blood flow delivery at rest and during exercise.’

In addition to the well characterized contribu- tion of the sympathetic and renin-angiotensin systems to the increased peripheral resistance in heart failure, emerging evidence suggests that vascular endothelial dysfunction may also contribute to the increased vasomotor tone and thus to the classic vicious cycle seen in heart failure.2

However, in such a context, which combines

From the Department of Pharmacology, VACOMED-IFRMP, Rouen University Medico1 School, Rouen, France.

Address for reprints: Christian Thuillez, MD, Service de Pharmo- cologie, CHU de Rouen, 76031 Rouen Cedex, France.

functional alterations in vasomotricity and chronic increases in tissue catecholamines and angiotensin II that may act as growth factors, the potential vascular histomorphometric changes and the rela- tion between these morphologic changes and the functional alterations observed at the level of the endothelium have never been studied in resistance arteries. Therefore, using the experimental model of coronary artery ligation in rats to reproduce the hemodynamic conditions of chronic heart failure, the present experiments were designed to evaluate the effects of heart failure on endothelial function and on vascular morphology at the level of the mesenteric resistance artery and to investigate whether chronic treatment with the angiotensin- converting enzyme (ACE) inhibitor perindopril modifies these parameters.

METHODS Experimental protocol and selection proce-

dure: Ten-week old male Wistar rats were sub- jected to left coronary artery ligation or sham surgery, using techniques similar to those described previously.3 Briefly, rats were anesthetized

28E THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 76 NOVEMBER 24, 1995

with ether and a left thoracotomy was performed. The heart was exteriorized and a ligature was placed around the proximal left coronary artery. This silk suture was tied securely to induce myocardial infarction (MI). In sham-operated animals the suture was passed but not tied. The heart was then rapidly replaced in the chest, which was closed. The rats were housed 5 per cage, fed ad libitum with standard diet, and had free access to tap water.

Seven days after surgery the surviving rats with electrocardiographic signs of MI were randomly divided into 2 groups, either untreated (placebo group) or treated with perindopril at the daily dose of 2 mg/kg given in drinking water.

Hemodynamic measurements: At 12 months after onset of treatment, sham and MI rats were anesthetized with pentobarbital(50 mg/kg intraper- itoneally). A micromanometer-tipped catheter (SPR 407; Millar Instruments, TX, USA) was placed in the right carotid artery for the measure- ment of arterial blood pressure. The catheter was then advanced into the left ventricle for the record- ing of left ventricular systolic and end-diastolic pressures, as well as the maximal rate of rise (@/d&J of left ventricular pressure.

All parameters were continuously recorded on a phy&ologic recorder (Windowgraph; Gould Instru- ment Co, Cleveland, Ohio) and heart rate was calculated from arterial pressure tracing.

Studies in isolated arteries: After completion of hemodynamic measurements, the mesenteric artery (third order, diameter 280 ? 15 km) was carefully isolated, and a 3-mm long segment was cannulated and placed at its physiologic length in a bath filled with physiological saline solution (in mmol/liter: NaCl 119; NaHC03 24; KC1 4.7; KH2P04 1.18; MgS04 0.7; CaCl2 1.6; glucose 5.5) kept at 37 “C, which was continuously gassed with a 95% 02/5% COZ mixture. The vessel was perfused at constant intraluminal pressure (30 mm Hg). The mesenteric artery was visualized with a video cam- era through an inverted microscope, and the internal diameter was continuously measured with an electronic analyzer (Living Systems Instrumenta- tion, Burlington, Vermont, USA). After an equili- bration period of 60 minutes, the artery was con- stricted with phenylephrine to approximately 50% of its initial diameter. Afterward, cumulative con- centrations of acetylcholine or the nitric oxide (NO) donor sodium nitroprusside was added and concentration-response curves were obtained.

Morphometric studies: The heart was arrested in diastole by an intravenous injection of KCl. The

heart was rinsed, blotted dry, and immediately weighed and immersed in a Bouin fixative solution; 24 hours later, the heart was cut into 3 slices perpendicular to the apex-to-base axis. From each slice, one 3-km thick section was obtained, mounted on glass slides, and stained with Sirius red.

Cardiac collagen density was evaluated in the subendocardial and subepicardial regions of the viable ventricular wall at a 500-fold enlargement by computerized analysis (Cyberview, Cervus Int. France).

After the in vitro study, mesenteric arterioles were fixed in Bouin fixative solution with an intraluminal pressure of 30 mm Hg; 3-urn thick slices were cut perpendicular to the longitudinal axis, mounted on glass, and stained with Sirius red or orcein to determine intraluminal diameter, media cross-sectional area (500-fold enlargement) and media collagen and elastin density (lOOO-fold enlargement), using the previously described image analyser.

Statistical analysis: All reported values are given as mean + standard error of the mean.

The relaxing responses to acetylcholine and nitroprusside are expressed as a percentage of the reversal of the contractile response. These responses and the hemodynamic and histomorphometric parameters were compared between the 3 groups of rats (untreated or treated MI, and sham) by means of a one-way analysis of variance (ANOVA). If the ANOVA revealed significant differences, it was followed by a Tukey test for multiple comparisons. Differences were consid- ered significant at a level of p < 0.05.

RESULTS Cardiac and hemodynamic parameters: As

summarized in Table I, the untreated heart failure rats had a slightly reduced body weight compared to the sham operated control rats, and perindopril did not modify this parameter. The heart weight/ body weight ratio was increased in rats with heart failure, thus indicating advanced cardiac hypertrophy. At 12 months of treatment, perindopril induced a significant decrease in this index of hypertrophy.

Infarct size was similar in the 2 heart failure groups and averaged 51% in the untreated group and 54% of the left ventricle in the perindopril- treated group.

Left ventricular circumference increased in the heart failure group (+96% vs sham), thus indicating a marked left ventricular dilation. This dilation

A SYMPOSIUM: ENDOTHELIAL DYSFUNCTION 29E

TABLE I Morphologic Cardiac Parameters in Sham-Operated 75 - cc Rats and in Rats with Heart Failure, Treated or Not with Perindopril

for 12 Months 60-

Heoli Heart Failure +

Sham Failure Perindopril

Body weight (g) 600 k 15 579 + 15 583 f 19

Heart weight/body weight

(w/g) 2.35 f 0.06 3.30 f 0.09’ 2.82 f O.l5*t

Myocardiol infarct size (%) - 51 +6 54 2 4

Left ventricular cavity

circumference (mm) 10.2 f 0.4 20.0 + 0.7’ 16.2 f 0.9’t

Valuesare meon 2 SEM. Sham = sham-operated rots. ‘p < 0.05 versus sham; tp < 0.05 versus heortfailure.

r=

I I I I I 8 7 6 5 4

Acetylcholine (-log M)

FIGURE 2. Concentratiotwesponse curves to acetylcholine in mesenteric resistance arteries taken from sham-opemted & (solid circles), or from heart failure mts, either untmated (open circles) or treated with perindopril (open triangles). Results are expressed as mean value f SEM. * p <0.05 vs sham. 150

2 120

i 90 60

30

0

$ 12.5

2 10.0

f 7.5

2 5.0

5 2.5

3 % 0.0

* * #Yl!lB

I

FIGURE 1. Mean arterial blood pressure (MAP), heart rate (HR), left ventricular end diastolic pressure (LVEDP), and left ventricular dP/dt,, in sham-opetuted mt~ (solid bars, n = 16) and in heart failure mts either untreated (open bars, n = 17) or treated with perindopril (striped bars, n = 18). Results are expressed as mean value f SEM. *p co.05 vs sham; t p co.05 vs untreated heart failure rats.

was significantly reduced by perindopril (- 19% vs untreated heart failure group).

Hemodynamic variables in the study groups are illustrated in Figure 1. There were no significant differences in heart rate between the 3 groups. As compared to sham-operated animals, heart failure induced a slight decrease in mean arterial pressure, a significant decrease in left ventricular dP/ dt, and a significant increase in left ventricular and diastolic pressure. Perindopril significantly decreased mean arterial pressure and normalized left ventricular end diastolic pressure, without affect-

15 -

25 -

O-

Nitroprusside (-log M)

FIGURE 3. Concentmtion+esponse curves to nitroprusside in mesenteric resistance arteries taken from sham-opemted rats (solid circles), or from heart failure rats, either untreoted (open circles) or tmated with perindopril (open triangles). Results are expressed as mean value + SEM.

I I I I I

8 7 6 5 4

ing left ventricular dPldt compared with untreated

Responses to acetylcholine and nitroprusside: heart failure rats.

Concentration-response curves to acetylcholine and nitroprusside were obtained in pressurized vessels preconstricted with phenylephrine to about 50% of their initial diameter (166 + 10; 160 f 12; 172 + 16 pm in the sham, untreated, and treated heart failure groups, respectively). The acetylcholine concentration-response curve (Figure 2) was markedly shifted to the right in the untreated heart failure group, whereas the maximal vasodilation decreased from 66 + 5% in the sham group to


7.5 1 2

$ 6.0-

$ a 4.5 - 8 2 2 3.0-

3 2 2 1.5 - & f2 * o.o-

7.5

3 z 6.0 % B 1

*

L FIGURE 4. Subepicardial and subendocardial myocardial collagen content of left ventricle (?Yo) in sham-opemted rats (solid bars, n = 16), or from heart failure rats, either untreated (open bars, n = 17) or treated with perindopril (striped bars, n = 18). Results are expressed as mean value -C SEM. *p eO.05 vs sham; tp ~0.05 vs untreated heart failure rats.

FIGURE 5. Arterial diameter, cross sectional area (CSA), media collagen and elastin densities in mesenteric resistance arteries from sham- operated rats (solid bars, n = 16), and in heart failure rats either untreated (open bars, n = 17) or treated with perindopril (striped bars, n = 18). Results are expressed as mean value f SEM. *p <0.05 vs sham; ~ tp co.05 vs untreated heati failure rats.

40 rt 6% in the untreated heart failure group (p <0.05). Perindopril almost normalized the response to acetylcholine, as illustrated by the concentration-response curve that became similar to that obtained in the sham group (maximal vasodilation 60 2 8%).

In contrast to acetylcholine, the response to nitroprusside obtained in untreated rats with heart failure was not different from that obtained in the sham rats, and the maximal dilation was similar in the 2 groups (64 -C 7 vs 66 +- 6%; Figure 3). Further- more, perindopril did not modify the response to nitroprusside.

Myocardial and vascular morphologic studies: The interstitial collagen volume fraction of the left ventricle (Figure 4) was significantly elevated in untreated heart failure rats compared to sham animals, showing established interstitial fibrosis (which affected both subepicardium and subendo- cardium), and this parameter was strongly decreased by perindopril.

In mesenteric resistance arteries (Figure 5), the internal vessel diameter and the cross-sectional area, as well as the collagen and elastin density of the media, were not modified in untreated heart failure rats compared to sham rats. This indicates that heart failure did not induce any remodeling or hypertrophy in resistance arteries. However, despite the lack of effect of heart failure cross- sectional area and collagen, content of the media (but not that of elastin) were reduced by perindopril.

DISCUSSION The present study, using the rat model of

coronary artery ligation shows that, in the presence of hemodynamic signs of heart failure, the endothelium-dependent vasodilation induced by acetylcholine is markedly impaired in resistance arteries and that this impairment is reversed after 12 months of treatment with the ACE inhibitor perindopril. Simultaneously, heart failure induces classical changes in myocardial tissue, but does not modify the structure of the arterial wall, whereas perindopril affects the structure of both myocardium and resistance arteries.

Coronary artery ligation in the rat has been extensively used to elucidate the pathophysiology of heart failure and to demonstrate the effective- ness of ACE inhibitors in prolonging surviva14~5 and in preventing remodeling of the ventricular myocardium.6 The presence of heart failure, indirectly demonstrated by the increased left ventricular end diastolic pressure and the decreased dPldt, was associated with an increase in ventricular mass, which corresponds to results of previous studies.7-*0

In these conditions, our study shows a marked decrease in the response to the endothelium- dependent vasodilator acetylcholine in mesenteric resistance arteries. In the same arteries, the endothelium-independent vasodilation induced by sodium nitroprusside is preserved. Since the acetylcholine-induced release of NO causes vasodilation through stimulation of soluble guanylate cyclase in the smooth muscle cell-but vasodilation can also be stimulated independently of the endothelium by organic nitrates or NO donors such as sodium


nitroprusside”-these findings suggest that the decreased response to acetylcholine is not due to reduced responsiveness of the vascular smooth muscle to NO. Thus, chronic heart failure inter- feres with the stimulated release of NO, as previously shown in experimental studies12J3 and in humans.i4 However, this is the first study to assess the effect of heart failure on endothelial function at the level of the resistance arteries.

Our study was not designed to elucidate the underlying mechanisms of endothelial dysfunction. However, it is conceivable that chronically reduced flow may have contributed to reduce endothelial function. This hypothesis is supported by the fact that endothelium-dependent relaxations can be modulated by chronic changes in blood flow either in the absencei or the presence16 of cardiac dysfunction. However, other mechanisms can be evoked, such as alterations in acetylcholine receptor number of affinity and/or dysfunction of the NO synthase pathway. Indeed, it has been shown that tumor necrosis factor-for which plasma levels are elevated in chronic heart failure17+an impair the receptor-mediated stimulated release of N0.18 Finally, it cannot be excluded that abnormal diffusion of NO or its destruction by local factors such as superoxide anions19j20 participate to the endothelial dysfunction. Whatever the mechanism, it should be stressed that the endothelial dysfunction could contribute (1) to the impaired metabolic vasodilatation when the oxygen demand increases, for example during exercise1,21 and (2) to the progressive hemodynamic deterioration observed in severe heart failure.

Concerning the cardiac and vascular histologic changes observed in our study, it should be stressed that the left ventricular cavity circumference, the myocardial hypertrophic process,7 and the increased collagen content6,9-which represent the classic histomorphometric changes observed in post-MI heart failure-are strongly decreased by perindopril. This clearly represents a beneficial effect of chronic ACE inhibition, since the accumu- lation of collagen and the myocardial hypertrophy are deleterious in terms of ventricular compliance, oxygen delivery to myocardial tissue, and arrhyth- mogenicity. The normalization in preload and the decrease in afterload, both of which improve left ventricular workload, are involved in these beneficial effects, although the direct effect of converting enzyme blockade on collagen synthesis may also participate in these structural effects.22 In contrast to the myocardial effects, which have been extensively studied, the histologic changes in small

resistance arteries were never studied in heart failure. The fact that heart failure did not modify the arterial diameter and the structure of the arteriolar wall was intriguing, since the pathophysi- ologic context of heart failure-i.e., the increase in arterial resistance, together with the influence of trophic factors, such as angiotensin II,23 norepi- nephrine,24 and endothelin25-could have induced structural modifications, such as a decrease in arterial diameter and/or an increase in media cross-sectional area. Thus, it appears that there is no arteriolar remodeling or hypertrophy due to heart failure. Moreover, there is also no modification in collagen content. Since the increase in arterial tone per se can induce such a remodeling (as is observed in hypertension) this result suggests that despite endothelial dysfunction, there is prob- ably no marked modification in intraluminal pressure, a parameter that was not measured in our study.

Despite the absence of histologic modifications induced by heart failure, it should be stressed that converting enzyme blockade induced a decrease in media cross-sectional area and in collagen content of the arteriolar wall. Since perindopril induced a significant decrease in mean arterial pressure, it can be suggested that this decrease results both from a decrease in arteriolar pressure and from the direct effects of converting enzyme blockade on arterial tissues (as in other tissues such as the myocardium) and that these 2 effects are more marked than those of the excessive local release of trophic factors. The consequence of these histologic modifications cannot be easily appreciated, but it could be assumed that they induce an increase in arteriolar compliance, which can be beneficial in blood flow delivery and regulation and thus may also indirectly contribute to the improve- ment of endothelial function,

In conclusion, this study demonstrates that heart failure induced a decrease in endothelium-dependent vasodilation assessed at the level of peripheral resistance arteries, but that, in contrast to the classic functional and morphologic myocardial modifications, this vascular alteration is not associated with histomorphometric changes. In this context, chronic converting enzyme inhibition not only exerts the classic beneficial effects on myocardium, but also decreases media cross-sectional area and collagen content in resistance arteries and this- could contribute to the overall beneficial effects of this treatment in heart failure.

Acknowledgment: The authors thank Mrs. Poz- zan for typing the manuscript and Institut de


Recherches Internationales Servier France for pro- viding perindopril.

1. Lejemtel TH, Maskin CS, Lucido D, Chadwick BJ. FaiIure to augment maximal limb blood flow in response to one-leg versus two-leg exercise in patients with severe heart failure. C&&ion 1986,74:245-251. 2. Zelis R, FIaim SF. Alterations in vasomotor tone in congestive heart failure. Prog Cardiavasc Dis 1982;%437-4.59. 3. Pfeffer MA, Pfeffer JM, Fishbem MC, Fletcher PJ, Sparado J, KIoner RA, BramnvaId E. MyocardiaI infarct size and ventricular funaion in rats. Cti Res 1979;44:503-512. 4. Pfeffer JM, Pfeffer MA, Braumvald E. Hemodynamic benefits and pro longed survival with long-term captopril therapy in rats with myocardial infarction and heart failure. Circulation 1987;75(suppl 1):149-155. 5. Sweet CS, Emmett SE, Stabilito II, Ribeio LGT. Increased survival in rats with congestive heart failure treated with enalapril. J Cardiovasc Phummcol 1987;10:636642. 6. Richer C, Mulder P, Fames P, Domergue V, Heudes D, GiudiceIli JF. Long-term treatment with trandolapril opposes cardiac remodeling and pro-

longs survival after myocardial infarction in rats. J Cnnfiov~c Phamzz~ol1992;2R 147-156. 7. Anversa P, Beghi C, Kikkawa Y, Olivetti G. Myocardial infarction in rats. Infarct size, myocyte hypertrophy and capillary growth. Circ J7e.s 1986,58:2637. 8. Anversa P, Loud AV, Levi&y V, Guideri G. Left ventricular failure induced by myocardial infarction. I. Myocyte hypertrophy. Am J Physiol1985;248:H876- H882. 9. Michel JB,‘Lattion AL, Salzmann JL, Carol ML, Philippe M, Camilleri JP, Corvol P. Hormonal and cardiac effects of converting enzyme inhibition in rat with myocardial infarction. Cim Res 1988;62:641-650. 10. Capasso JM, Malhorta A, Li P, Zhang X, Scheuer J, Anversa P. Chronic nonocclusive coronary artery constriction impairs ventricular function, myocardial structure, and cardiac contractile protein enzyme activity in rats. Cim Res 1992;70:14%162. 11. Katsuki S, Arnold WP, MittaI CK, Murad F. Stimulation of guanylate

cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine. J Cjck Nuclwtide Res 1977;3:2~35. 12. Ontkean MT, Gay RG, Greenberg B. Diminished endotheliumderived

relaxing factor activity in an experimental model of chronic heart failure. Cim Rer 1991;69:1tX%?-1096. 13. Drexler H, Hablawetz E, Lu W, Riede II, Chris&s A. Effects of inhibition of nitric oxide formation on regional blood flow in experimental myocardial infarction. Cinxlation 1992;8625>262. 14. Katz SD, Biasucci L, Sabba C, Strom JA, Jondeau G, GaIvao M, Solomon S, Nikiolic SD, Forman R, Le Jemtel TH. Impaired endothelium-mediated vasodilation in the peripheral vasculature of patients with congestive heart failure. JAm Cdl Car&l 1992;19:918925. 15. Miller VM, Vanhoutte PM. Enhanced release of endotheliumderived factor(s) by chronic increases in blood flow. Am J P&-id 1988;255:H446H451. 16. AmaI JF, Schoot C, Stoclet JC, Michel JB. Vascuhu relaxation and cyclic guanosine monophosphate in a rat model of high output heart failure. Cum!& vex Res 1993;27~1651-1656. 17. Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med 1990;323:236-241. 18. Aoki N, Siegfried M, Lefer AM. Anti-EDRF effect of tumor necrosis factor in isolated perfused cat carotid arteries. Am J Physic4 1989;256:H15t% 1512. 19. Rubanyi GM, Vanhoutte PM. Superoside anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physid 1986,25CkH822-H827. 20. Stewart DJ, Pohl U, Bassenge E. Free radicals inhibit endothelium-

dependent dilation in the coronary resistance bed. Am J Physid 1988;255:H765 H769. 21. Wilson JR Martin JL, Schwartz D, Ferraro N. Exercise tolerance in patients with chronic heart failure: Role of impaired nutritive flow to skeletal muscle. Cirrulahbn 1984;69:1079-1087. 22. Lii W, Schaper J, Wiemer G, AIbus U, SchiiIkens BA. Ramipril prevents left ventricular hypertrophy with myocardial fibrosis without blood pressure reduction: a one year study in rats. Br JPhmaco11992;107:97t&975. 23. Berck BC, Wekshtein V, Godron HM, Tsuda T. Angiotensin II-stimulates protein synthesis in cultured vascular smooth muscle ceils. @perrem& 1989;13: 305-314. 24. Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstitium. Cix Res 1991;83:184%1865. 25. Simonson MS, Warm S, Men6 P, Dubyak GR, Keater M, Nakamto Y, Seder JR. Endotherm stimulates phospholipase C, Na+/H+ exchange, c-fos expression, and mitogenesis in rat mesangial cells. J C[in Invest 1989;83:708- 712.


vascular and myocardial protective effects of converting enzyme inhibition in experimental heart...

Documents