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Left ventricular hypertrophy in spontaneously hypertensive rat: Effects of ACE-inhibition on myocardiocyte ultrastructure
Pharmacological Research,
Vol. 31, No. 6,1995
315
LEFT VENTRICULAR HYPERTROPHY IN SPONTANEOUSLY HYPERTENSIVE RAT: EFFECTS OF ACE-INHIBITION ON MYOCARDIOCYTE ULTRASTRUCTURE V. VULPIS,
T. M. SECCIA,
B. NICO*,
S. RICCI,
L. RONCALI”
DIMO, Department of Biomedical Sciences and Human Oncology, “Chair of Histology and Embriology, University Accepted
and A. PIRRELLI
Chair of Internul Medicine,
qf Bari, Italy
I June 1995
The aim of this study is to investigate the effects of two ACE-inhibitors with different chemical formulae, cilazapril (CLZ) and captopril (CPT), on left ventricular myocardiocytes from spontaneously hypertensive rats (SHR), characterized by ultrastructural alterations associated with left ventricular hypertrophy, and from Wistar-Kyoto (WKY) rats, considered not remarkable changes are observed in WKY as controls. After CLZ-treatment, myocardiocytes, whereas SHR ones show a considerable reduction in their original alterations in ultrastructure. After CPT-treatment, both SHR and WKY myocardiocytes are altered in ultrastructure. The morphometric investigation confirms that CPT and CLZ produce different effects. Even if the drugs induce a similar decrease in blood pressure and left ventricular mass index, CLZ unlike CPT seems to improve the ultrastructural abnormalities associated with left ventricular hypertrophy. These changes could be related to the different chemical structure of CLZ and CPT, or to a different affinity of the two drugs for the local renin-angiotensin system. KEY WORDS:hypertension,
left ventricular
hypertrophy,
INTRODUCTION The development of left ventricular hypertrophy (LVH) caused by increased pressure load is associated with structural and functional adaptations of the myocardium cell components [l-6]. These changes, which gradually increase with the LVH progression, affect in particular the myocyte population and, at the subcellular level, myofibrils and mitochondria. Previous studies provide evidence that different antihypertensive treatments produce different effects on myocardial tissue, not always related to the changes of blood pressure values [7-121. The response of the ventricular myocardium would be the result of a direct effect of the drug or it would be the effect of the impairment of the systems controlling the tissue homeostatis. The renin-angiotensin system (RAS), known to be paracrine and autocrine as well as endocrine, has been assumed to play a possible role in the onset and progression of left ventricular hypertrophy [13, 141. Recent evidence suggests that the reduction of intramyocardial Ang II generated by an in situ RAS system is one mechanism by which angiotensin converting enzyme (ACE) inhibitors cause a reduction of left ventricular mass [ 151. Drugs within this class would have different effects, in
Correspondence to: Dr Vito Vulpis, DIMO, S&one di Medicina Interna, Universitb di Bari, Piazza G. Cesare, 11, 71024 Bari, Italy. 1043%6618/95/060375-07/$08.00/O
ultrastructure,
ACE-inhibition.
particular at the subcellular level where knowledge is limited [16-181. The aim of this study was to investigate the effects of cilazapril (CLZ) and captopril (CPT) on ultrastructural alterations caused by high blood pressure in left ventricular myocardiocytes from spontaneously hypertensive rats (SHR). CLZ and CPT have different chemical formulae, pharmacodynamics and tissue affinity; in addition, CLZ, one of the new inhibitors of ACE activity, has been shown to have a better interaction with ACE and a higher affinity for it [ 19,201.
MATERIALS
AND METHODS
Two strains of male rats (Charles River) were used: Wistar-Kyoto (WKY, n=30) and spontaneously hypertensive rats (SHR, n=30); the former considered as controls. The procedure of the study was programmed in accordance with institutional guidelines. The animals were housed individually from the fourth week of life and maintained under standard conditions (constant temperature 25°C and humidity, balanced diet including sodium intake, constant light/dark rhythm) until they were killed. From the twelfth week of life, six groups were considered: (a) 10 WKY and 10 SHR received vehicle (control animals); (b) 10 WKY and 10 SHR received cilazapril (CLZ, supplied by Roche, Basel, 01995
The Italian Pharmacological
Society
Pharmacological
316
Vol. 31, No. 6,1995
percentage area of myofibrils, mitochondria and matrix as well as myofibrillar diameter. A t-test or one-way analysis of variance and Bonferroni test was used for the statistical analysis, as appropriate. The level of significance considered was P=O.O5. All data were expressed as meankstandard deviation (SD).
Switzerland; 10 mg kg-’ per day); and (c) 10 WKY and 10 SHR received captopril (CPT, supplied by Squibb & Sons, Princeton, NJ, USA; 50 mg kg-’ per day). Drugs were administered orally dissolved in the water ration at the same time every day. Water requirement was previously determined in our laboratory in SHR as well as in WKY rats, at different ages of life. Water intake was daily monitored by residual water. The treatment was measuring continued for 10 weeks. Arterial blood pressure, heart rate and body weight were monitored regularly at weekly intervals. Arterial blood pressure was measured in conscious animals by using a tail-cuff plethismographic method (LE 5000 digital pressure meter, Letica). The values were taken at least three times and the mean value was reported. During the twenty-second week of life, control and drug-treated rats were anaesthetized with ether and, after thoracotomy, the still-beating heart was removed and the left ventricle quickly isolated. The weights of the heart and the left ventricle were measured; left ventricle mass index (LVMI) was calculated using the following formula: left ventricle weight/body weightx 103. Small fragments (4-6) were taken from the free wall of the left ventricle, then fixed by immersion in 0.1 mol phosphate buffer containing 3% glutaraldehyde and rinsed in the same buffer for 12 h. They were post-fixed in 1% osmium tetroxide, dehydrated in graded ethanol and embedded in Epon 812. Ultrathin sections were cut with a LKB V Ultramicrotome, stained with uranyl acetate and lead citrate and examined under a Zeiss 9A electron microscope. Only tissue blocks with myofibrils in the longitudinal orientation were employed (at least three for each group); 10 micrographs (6000x) randomly selected from each tissue block were printed at a calibrated magnification of 24 000x and then analysed morphometrically using Autocad computer software. The parameters recorded for each selected area were
Systolic
Research,
RESULTS
Systolic blood pressure (SBP), heart rate (HR) and left ventricular mass index (LVMI) Table I shows blood pressure and heart rate data for both groups of animals. SBP was progressively increased from the eighth week of life in SHR, compared to age-matched WKY (P
Ultrastructural analysis Morphologic analysis Twenty-two-week-old
WKY control
group (Fig.
la)
The myocardiocytes appear regularly shaped slightly indented nuclear with elongated nucleus, completely and sarcoplasm almost membrane occupied by myofibrils and mitochondria. The myofibrils are aligned with each other and uniformly sized; the mitochondria, arranged in longitudinal rows between myofibrils, contain numerous cristae. The intercalated discs show normal features: the
Table I blood pressure @BP) and heart rate (HR) values at different ages. Vehicle- or drug-(captopril, or cilazapril, CLZ) treatment was taken up from the twelfth week
Group
Fifth
week
Eighth
week
Twelfth
week
Twenty-second
CPT week
SBP
SBP
HR
SBP
HR
SBP
HR
CnzmHgJ
CmmHgJ
@pm)
CmmHg)
@pm)
CmmHgJ
@pm)
WKY (control) WKY (CPT) WKY (CLZ)
95klO 98f12 96+11
402+15 400*14 405f16
124+13 125f15 126+16
422+25 428+26 425f28
136+8 138+10 139&l 1
310+60 315+64 308f63
142flO 128+10 135110
370+45 363f14 35x*15
SHR (control) SHR (CPT) SHR (CLZ)
127+10 130fll 128rt12
41227 415flO 410+12
170+10t 173k12.f 175+13t
387s3 1 383s34 385+85
185,301 187k321_ 184+3Ot
372,50 380+47 378+5 1
214*401_ 139-c12** 134k15””
382flO 406+14 411+14
**P
control group.
Pharmacological Research, Vol. 31, No. 6,1995
377
Fig. 1. Left myocardium of 22-week-old WKY (a) and SHR (b, c) control rats. (a) Myofibrils are arranged in register. Mitochondria are regularly packed and contain numerous cristae in a quite electron transparent matrix. Intercalated discs (asterisks) show apparently normal features. x9750. (b) Somewhat swollen mitochondria and not aligned myofibrils are scattered in an abundant and very electron-lucent sarcoplasm. x9750. (c) Detachments of the junctional plasma membranes (asterisks) at intercalated disc level. x30 875.
neighbouring
cells
are
tightly
joined
with
regularly
Twenty-two-week-old
SHR control group (Fig. 1 h and
shaped projections from one cell interdigitating with those of the next. An electron-dense material is
cl The myocardiocytes appear somewhat altered. An electron-lucent sarcoplasm, poor in glycogen granules
where
contains mitochondria not regularly packed; in some places they appear enlarged, with disarranged cristae.
markedly
evident
myofilaments
at disc
transversal
region
are attached.
Table II Body weight, ventricle weight and left ventricle mass index (LVMI) measured at the twenty-second after a IO-week treatment with vehicle or drug [captopril (CPT) or cilazapril (CLZ)] Group
Body weight (g)
Ventricle weight(g)
LVMI
WKY (control) WKY (CPT) WKY (CLZ)
486f25 425+30 489+20
0.85+0.2 0.75fO. 1 0.76kO.2
1.7+0. I 1.7fO.l 1.6fO.l
SHR (control) SHR (CPT) SHR (CLZ)
321?24 313_+11 312+20
0.89+0.2 0.67fO. l* 0.65+0.1*
2.8,0.21_ 2.1kO.l” 2.lfO.l”
*P
control group.
week,
378
Pharmacological
cells are detached poorly distinguishable.
and
Research,
the
Vol. 31, No. 6,1995
junctional
complexes
Twenty-two-week-old WKY after CLZ treatment (Fig. 2a) The myocardiocytes do not show considerable changes. Their sarcoplasm is rich in glycogen and contains myofibrils and mitochondria regularly packed between them, with normal shape and structure. The intercalated discs are unimpaired; nexuses, fasciae adhaerentese and desmosomes are well recognized. Twenty-two-week-old SHR after CLZ treatment (Fig. 2b and c) No substantial differences in the myocardiocyte ultrastructure are discernible between SHR and WKY CLZ-treated specimens. Myofibrils and mitochondria, regularly sized and shaped, are scattered in an electron-lucent sarcoplasm. The intercalated discs show cell interconnections with well preserved junctional structures. Twenty-two-week-old WKY after CPT-treatmerzt (Fig. 3a) The myocardiocytes contain abundant and electron-lucent sarcoplasm with few organelles. The mitochondria show changes in sizes, structure and distribution. Some of them appear swollen, others are characterized by electron-dense matrix without cristae. They are randomly scattered between myofibrils disarranged and variously oriented. The plasma membranes are thickened and poorly interconnected at level of the intercellular junctions. Twenty-two-week-old SHR after CPT treatment (Fig. 3b) The myocardiocytes contain large amounts of electron-lucent sarcoplasm with mitochondria of different sizes, enlarged and somewhere characterized by fragmentation of the cristae. The myofibrils are small, loosely distributed and variously sized; some of them are very thin and obliquely oriented. Plasma membrane detachments are seen in the intercalated discs where few myofilaments end on the junctional systems. Breaks of the plasma membranes are also observed. Some cell nuclei are affected by kariolysis, or fragmented. Fig. 2.
Left ventricular myocardium of 22-week-old WKY (a) and SHR (b, c) rats after cilazapril treatment. (a) In two myocardiocytes, sealed by intercalated discs (asterisks), rows of mitochondria appear regularly distributed between packed and aligned myofibrils. x9750. (b) An electron-lucent cytoplasm with glycogen intervenes between myofibrils and mitochondria. x9750. (c) Fasciae adhaerentes (arrows) and desmosomes (arrowheads) are recognizable in an intercalated disc. x30 875.
The configuration in sarcomeres of the myofibrils is preserved, whereas their thickness is varied, smaller and disarranged myofibrils are intermingled with normally-sized ones. The intercalated discs show impaired feature, the plasma membranes of adjacent
Morphometric
analysis
(Table
3)
Twenty-two-week-old WKY and SHR control in groups The myofibrillar area SHR myocardiocytes is lesser than in WKY ones (SHR 45.7f7.6, WKY 65.5k9.6, A -3O%, P
379
Pharmacological Research, Vol. 31, No. 6,1995
Fig. 3. Left ventricular myocardium of 22-week-old WKY (a) and SHR (b) rats after captopril treatment. (a) Two myocardiocytes enveloped by a thickened plasma membranes (arrows) are filled with clear sarcoplasm. The myofibrils are disarranged and some mitochondria, devoid of cristae, contain an electron-dense amorphous matrix (arrowheads). x9750. (b) Thin myofibrils spaced out by mitochondria of different sizes in an electron transparent sarcoplasm with few glycogen granules. x9750.
Percentage
Table III area of myofibrils, mitochondria and matrix recorded in vehicle-treated drug-treated [captopril (CPT) or cilazapril (CLZ)] Myofibr-illar area (%)
Mitochondrial
area (%)
(control
group) and
Matrix area (%)
WKY (control) WKY (CPT) WKY (CLZ)
6.5.5f9.6 36.9f7.0*** 54.2+10.0**
20.0+8.4 28.6+9.6* 25.8+6.7*
14.5+3.1 34.5+9.0*** 20.0+5.0**
SHR (control) SHR (CPT) SHR (CLZ)
45.7f7.7.1 42.7k9.6 52.7k7.1”
22.8+7.8 29.3+5.2* 23.4f4.1
31.5+10.0t 28.Ozb12.0 23.9f7.2
*P
compared to SHR control groups (CLZ-SHR: 52.7f 7.1, control SHR: 45.7k7.6, A +15%, P
control group.
-17%, PcO.025). Not significant difference is evident after CLZ-treatment between WKY and SHR specimens (CLZ-SHR: 52.7f7.1, CLZ-WKY: 54.2f 10.0, NS). The mitochondrial area is substantially similar in
380
CLZ-treated and control SHR (CLZ-SHR: 23.4k4.1, control SHR: 22.8f7.8, A +2% NS) whereas it is significantly greater in the CLZ-treated WKY than in the control ones (CLZ-WKY: 25.8k6.7, control WKY: 20.0f8.4, A +29%, P
DISCUSSION This study shows that the 22-week-old SHR myocardiocytes are affected by ultrastructural and morphomeric changes in their contractile apparatus. The myofibrils of the SHR myocardiocytes are in fact smaller and irregularly arranged in a more abundant cell matrix than those of the 22-week WKY myocardiocytes. This observation seems to conflict with previous data showing that wall stress is responsible for increase in myofibrils during myocardial hypertrophy [ 1, 211. However, the present results are in partial agreement with the hypothesis of Hamilton [22], suggesting that decrement in myofibril size, together with increase in mitochondrial area, may represent a compensatory response to an increased energy demand. The mitochondrial density augmentation might facilitate energy production while the myofibril size decrement might enhance the movement of both high energy phosphates and calcium to their respective sites. it is noteworthy to consider that Moreover, hypertension rate and duration play an important role in the degree of the morphological and functional
Pharmacological
Research,
Vol. 31, No. 6,1995
changes of hypertrophied myocardium the [l, 5,6,21]. The decreased myofibrillar area as well as the modified mitochondria observed in SHR myocardiocytes could be related to the duration of the hypertensive state and expression of a reduced cell ability to produce energy for contraction [23]. Previous studies carried out on human subjects and experimental animal models suggest that ACEinhibitors produce a decrease in left ventricular mass and improve the myocardial pumping function, even if there is no demonstration that the change in left ventricular mass is direct consequence of reduced blood pressure [24]. Captopril and cilazapril cause similar decrease in blood pressure and LVMI in SH rats, but the ultrastructural and morphometric data recorded after CLZ- and CPT-treatment indicate that captopril, unlike cilazapril, does not improve the morphological changes associated with LVH. The myofibrillar area augmentation as well as the feature of myofibrils and mitochondria regularly sized and shaped, both recorded in SHR myocardiocytes after CLZ treatment, suggest that the two drugs might have a different effect on the contractile system. Further investigations are required to determine whether the morphological changes are induced by CLZ are due to sarcomere addition and/or reorganization of myofibrils. The different effect evoked by the two ACE-inhibitors on the SHR myocardiocytes could be related to a CPT reactive group, such as the sulphydryl one, or to a different affinity of this molecule for the local renin-angiotensin system, resulting in a different interaction with growth factors [19,20,25]. The remarkable, different changes observed in WKY myocardiocytes after CPT and CLZ treatment, substantiate this hypothesis: myofibrils and mitochondria are regularly sized and shaped after CLZ treatment, whereas relevant abnormalities are seen in CPT-treated myocardiocytes. The evidence of a local renin-angiotensin system involved in the regulation of the cell growth and proliferation, as well as in the cell hypertrophy, could explain why ACE-inhibition induces modifications also in the myocardiocytes of control rates [26-301. The results of this investigation suggest that the ACE inhibitors interfere with the LVH regression and that the ventricular mass reduction may be apparently beneficial, since it is not always associated with improvement of myocardiocyte ultrastructure. This conclusion could explain why ACE inhibitors do not always correct the functional modifications occurring during LVH.
REFERENCES 1. Anversa P, Ricci R, Olivetti G. Quantitative structural analysis of the myocardium during physiologic growth and induced cardiac hypertrophy: a review. .I Am Co11 Cardiol
1986; 7:
1140-9.
Pharmacological Research, Vol. 31, No. 6, 1995 2. Weber KT, Janicki JS, Pick R, Abrahams C, Schroff SG, Bashley RI, Che RM. Collagen in hypertrophied, pressure overloaded myocardium. Circulation 1987; 75 (suppl 1): 40-7. EH, Olivetti G, 3. Anversa P, Palackal T, Sonnenblick Capasso JM. Hypertensive Cardiomyopathy. Myocyte nuclei hyperplasia in the mammalian rat heart. J Clin Irzvest 1990; 85: 994-7. 4. Weber KT, Anversa P, Armstrong PW, Brilla CG, Burnett JC, Cruickshank JM, Devereux RB, Giles TD, Korsgaard N, Leier CV, Mendelsohn FAO, Motz WH, Mulvany MJ, Strauer BE. Remodelling and reparation of the cardiovascular system. J Am Co11 Cardiol 1992; 20: 3-16. EH, Olivetti G, 5. Anversa P, Palackal T, Sonnenblick Meggs LG, Capasso JM. Myocyte cell loss and myocyte cellular hyperplasia in the hypertrophied aging rat heart. Circ Res 1990; 67: 871-85. AB. Myofibrilogenesis and reversible 6. Borisov disassembly of myofibrils as adaptative reactions of cardiac muscle cells. Acta Physiol Stand 1991; S559: 71-80. 7. Frohlich ED, Tarazi RC. Is arterial pressure the sole cardiac hypertensive factor responsible for hypertrophy? Am J Cardiol 1979; 44: 9.59-63. Y, Tarazi RC, Salcedo EE. 8. Fouad FM, Nakashima Reversal of left ventricular hypertrophy in hypertensive patients treated with methyldopa. Lack of association with blood pressure control. Am J Cardiol 1982; 49: 795-801. 9. Sen S. Regression of cardiac hypertrophy. Experimental animal model. Am J Med 1983; 75 (suppl3A): 87-93. 10. Nakashima Y, Fouad FM, Tarazi RC. Regression of left ventricular hypertrophy from systemic hypertension by enalapril. Am J Cardiol 1984; 53: 1044-9. 11. Gohlke P, Stoll M, Lamberty V. Mattfeld T, Mall G, Van Even P, Martorana P, Unger T. Cardiac and vascular effects of chronic angiotensin converting enzyme inhibition at subantihypertensive doses. J Hypertens 1992; 10 (suppl6): 141-4. 12. Black MJ, Campbell JH, Campbell GR. Effect of perindopril on cardiovascular hypertrophy of the SHR: respective roles of reduced blood pressure and reduced angiotensin II levels. Am J Cardiol 1993; 71: 17E-21E. 13. Re RN. The cellular biology of angiotensin: paracrine, autocrine and intracrine actions in cardiovascular tissues. JMol Cell Cardiol 1989; 21: 63-9. 14. Schelling P, Fischer H, Ganten D. Angiotensin and cell growth: a link to cardiovascular hypertrophy? .I Hypertens 1991; 9: 3-15. 15. Re RN, Chen L. Growth factors and cardiovascular structure. Implications for calcium antagonist therapy. Am J Hypertens 1991; 4: 46OS-465s.’
381 16. Eggena P, Zhu JH; Clegg K, Barrett JD. Nuclear angiotensin receptors induce transcription of renin and angiotensinogen mRNA. Hypertension 1993; 22: 496-501. and 17. Rogers TB, Gaa ST, Allen IS. Identification characterization of functional angiotensin II receptors on cultured heart myocytes. J Pharmacol Exper Ther 1986; 236: 438-44. 18. Marban E, Koretsune Y. Cell calcium, oncogenes, and hypertrophy. Hypertension 1990; 15: 652-8. 19. Francis RJ, Brown AN, Kler L, Fasanella d’Amore T, Brunner HR. Waeber B, Nussberger J, Pharmacokinetics of the converting enzyme inhibitor cilazapril in normal volunteers and the relationship to enzyme inhibition: development of a mathematical model. J Cardiovnsc Pharmacol 1987; 9: 32-8. MR, Eichler DA, Kogler P, 20. Natoff IL, Attwood Kleinbloesem CH, Van Brummelen P. Cilazapril. Cardiovasc Drug Reviews 1990; 8: l-24. EH. 21. Anversa P, Capasso JM, Olivetti G. Sonnenblick Cellular basis of ventricular remodeling in hypertensive Am J Hypertens 1992; 5: 758-70. cardiomyopathy. 22. Hamilton N, Ashton ML, Ianuzzo CD. Cell size of mammalian myocardia is not related to physiological demand. Experientin 1991; 47: 1070-2. 23. Vulpis V, Roncali L, Bertossi M, Nice B, Seccia T, Ricci S, Pirrelli A. Left ventricular hypertrophy in the rat: effect of ACE spontaneously hypertensive inhibitors on ultrastructural morphology. Cardiology 1994; 84: 14-24. ED, Iwata T, Sasaki 0. Clinical and 24. Frohlich physiological significance of local tissue reninangiotensin system. Am J Med 1989; 87 (suppl 6B): 19-23. 25. Jaffe IA. The adverse effects profile of sulfhydryl compounds in man. Am JMed 1986; 80: 471-6. 26. Dzau VJ, Re RN. Evidence for the existence of renin in the heart. Circulation 1987; 75 (suppl I): 1134-6. 27. Dzau VJ, Burt DW, Pratt RE. Molecular biology of the renin angiotensin system. Am J Physiol 1988; 255: F563-73. system. Molecular 28. Dzau VJ. Cardiac renin-angiotensin and functional aspects. Am .I Med 1988; 84 (suppl 3A): 22-7. B, Lindpaintner K, Ganten D. 29. Linz W, Scholkens Intracardiac renin-angiotensin system. J Hypertens 1989; 2: 307-10. A, Urata H, Bumpus FM, Husain A. 30. Kinoshita Measurement of angiotensin I converting enzyme inhibition in the heart. Circ Res 1993; 73: 5 l-60.
Vol. 31, No. 6,1995
315
LEFT VENTRICULAR HYPERTROPHY IN SPONTANEOUSLY HYPERTENSIVE RAT: EFFECTS OF ACE-INHIBITION ON MYOCARDIOCYTE ULTRASTRUCTURE V. VULPIS,
T. M. SECCIA,
B. NICO*,
S. RICCI,
L. RONCALI”
DIMO, Department of Biomedical Sciences and Human Oncology, “Chair of Histology and Embriology, University Accepted
and A. PIRRELLI
Chair of Internul Medicine,
qf Bari, Italy
I June 1995
The aim of this study is to investigate the effects of two ACE-inhibitors with different chemical formulae, cilazapril (CLZ) and captopril (CPT), on left ventricular myocardiocytes from spontaneously hypertensive rats (SHR), characterized by ultrastructural alterations associated with left ventricular hypertrophy, and from Wistar-Kyoto (WKY) rats, considered not remarkable changes are observed in WKY as controls. After CLZ-treatment, myocardiocytes, whereas SHR ones show a considerable reduction in their original alterations in ultrastructure. After CPT-treatment, both SHR and WKY myocardiocytes are altered in ultrastructure. The morphometric investigation confirms that CPT and CLZ produce different effects. Even if the drugs induce a similar decrease in blood pressure and left ventricular mass index, CLZ unlike CPT seems to improve the ultrastructural abnormalities associated with left ventricular hypertrophy. These changes could be related to the different chemical structure of CLZ and CPT, or to a different affinity of the two drugs for the local renin-angiotensin system. KEY WORDS:hypertension,
left ventricular
hypertrophy,
INTRODUCTION The development of left ventricular hypertrophy (LVH) caused by increased pressure load is associated with structural and functional adaptations of the myocardium cell components [l-6]. These changes, which gradually increase with the LVH progression, affect in particular the myocyte population and, at the subcellular level, myofibrils and mitochondria. Previous studies provide evidence that different antihypertensive treatments produce different effects on myocardial tissue, not always related to the changes of blood pressure values [7-121. The response of the ventricular myocardium would be the result of a direct effect of the drug or it would be the effect of the impairment of the systems controlling the tissue homeostatis. The renin-angiotensin system (RAS), known to be paracrine and autocrine as well as endocrine, has been assumed to play a possible role in the onset and progression of left ventricular hypertrophy [13, 141. Recent evidence suggests that the reduction of intramyocardial Ang II generated by an in situ RAS system is one mechanism by which angiotensin converting enzyme (ACE) inhibitors cause a reduction of left ventricular mass [ 151. Drugs within this class would have different effects, in
Correspondence to: Dr Vito Vulpis, DIMO, S&one di Medicina Interna, Universitb di Bari, Piazza G. Cesare, 11, 71024 Bari, Italy. 1043%6618/95/060375-07/$08.00/O
ultrastructure,
ACE-inhibition.
particular at the subcellular level where knowledge is limited [16-181. The aim of this study was to investigate the effects of cilazapril (CLZ) and captopril (CPT) on ultrastructural alterations caused by high blood pressure in left ventricular myocardiocytes from spontaneously hypertensive rats (SHR). CLZ and CPT have different chemical formulae, pharmacodynamics and tissue affinity; in addition, CLZ, one of the new inhibitors of ACE activity, has been shown to have a better interaction with ACE and a higher affinity for it [ 19,201.
MATERIALS
AND METHODS
Two strains of male rats (Charles River) were used: Wistar-Kyoto (WKY, n=30) and spontaneously hypertensive rats (SHR, n=30); the former considered as controls. The procedure of the study was programmed in accordance with institutional guidelines. The animals were housed individually from the fourth week of life and maintained under standard conditions (constant temperature 25°C and humidity, balanced diet including sodium intake, constant light/dark rhythm) until they were killed. From the twelfth week of life, six groups were considered: (a) 10 WKY and 10 SHR received vehicle (control animals); (b) 10 WKY and 10 SHR received cilazapril (CLZ, supplied by Roche, Basel, 01995
The Italian Pharmacological
Society
Pharmacological
316
Vol. 31, No. 6,1995
percentage area of myofibrils, mitochondria and matrix as well as myofibrillar diameter. A t-test or one-way analysis of variance and Bonferroni test was used for the statistical analysis, as appropriate. The level of significance considered was P=O.O5. All data were expressed as meankstandard deviation (SD).
Switzerland; 10 mg kg-’ per day); and (c) 10 WKY and 10 SHR received captopril (CPT, supplied by Squibb & Sons, Princeton, NJ, USA; 50 mg kg-’ per day). Drugs were administered orally dissolved in the water ration at the same time every day. Water requirement was previously determined in our laboratory in SHR as well as in WKY rats, at different ages of life. Water intake was daily monitored by residual water. The treatment was measuring continued for 10 weeks. Arterial blood pressure, heart rate and body weight were monitored regularly at weekly intervals. Arterial blood pressure was measured in conscious animals by using a tail-cuff plethismographic method (LE 5000 digital pressure meter, Letica). The values were taken at least three times and the mean value was reported. During the twenty-second week of life, control and drug-treated rats were anaesthetized with ether and, after thoracotomy, the still-beating heart was removed and the left ventricle quickly isolated. The weights of the heart and the left ventricle were measured; left ventricle mass index (LVMI) was calculated using the following formula: left ventricle weight/body weightx 103. Small fragments (4-6) were taken from the free wall of the left ventricle, then fixed by immersion in 0.1 mol phosphate buffer containing 3% glutaraldehyde and rinsed in the same buffer for 12 h. They were post-fixed in 1% osmium tetroxide, dehydrated in graded ethanol and embedded in Epon 812. Ultrathin sections were cut with a LKB V Ultramicrotome, stained with uranyl acetate and lead citrate and examined under a Zeiss 9A electron microscope. Only tissue blocks with myofibrils in the longitudinal orientation were employed (at least three for each group); 10 micrographs (6000x) randomly selected from each tissue block were printed at a calibrated magnification of 24 000x and then analysed morphometrically using Autocad computer software. The parameters recorded for each selected area were
Systolic
Research,
RESULTS
Systolic blood pressure (SBP), heart rate (HR) and left ventricular mass index (LVMI) Table I shows blood pressure and heart rate data for both groups of animals. SBP was progressively increased from the eighth week of life in SHR, compared to age-matched WKY (P
Ultrastructural analysis Morphologic analysis Twenty-two-week-old
WKY control
group (Fig.
la)
The myocardiocytes appear regularly shaped slightly indented nuclear with elongated nucleus, completely and sarcoplasm almost membrane occupied by myofibrils and mitochondria. The myofibrils are aligned with each other and uniformly sized; the mitochondria, arranged in longitudinal rows between myofibrils, contain numerous cristae. The intercalated discs show normal features: the
Table I blood pressure @BP) and heart rate (HR) values at different ages. Vehicle- or drug-(captopril, or cilazapril, CLZ) treatment was taken up from the twelfth week
Group
Fifth
week
Eighth
week
Twelfth
week
Twenty-second
CPT week
SBP
SBP
HR
SBP
HR
SBP
HR
CnzmHgJ
CmmHgJ
@pm)
CmmHg)
@pm)
CmmHgJ
@pm)
WKY (control) WKY (CPT) WKY (CLZ)
95klO 98f12 96+11
402+15 400*14 405f16
124+13 125f15 126+16
422+25 428+26 425f28
136+8 138+10 139&l 1
310+60 315+64 308f63
142flO 128+10 135110
370+45 363f14 35x*15
SHR (control) SHR (CPT) SHR (CLZ)
127+10 130fll 128rt12
41227 415flO 410+12
170+10t 173k12.f 175+13t
387s3 1 383s34 385+85
185,301 187k321_ 184+3Ot
372,50 380+47 378+5 1
214*401_ 139-c12** 134k15””
382flO 406+14 411+14
**P
control group.
Pharmacological Research, Vol. 31, No. 6,1995
377
Fig. 1. Left myocardium of 22-week-old WKY (a) and SHR (b, c) control rats. (a) Myofibrils are arranged in register. Mitochondria are regularly packed and contain numerous cristae in a quite electron transparent matrix. Intercalated discs (asterisks) show apparently normal features. x9750. (b) Somewhat swollen mitochondria and not aligned myofibrils are scattered in an abundant and very electron-lucent sarcoplasm. x9750. (c) Detachments of the junctional plasma membranes (asterisks) at intercalated disc level. x30 875.
neighbouring
cells
are
tightly
joined
with
regularly
Twenty-two-week-old
SHR control group (Fig. 1 h and
shaped projections from one cell interdigitating with those of the next. An electron-dense material is
cl The myocardiocytes appear somewhat altered. An electron-lucent sarcoplasm, poor in glycogen granules
where
contains mitochondria not regularly packed; in some places they appear enlarged, with disarranged cristae.
markedly
evident
myofilaments
at disc
transversal
region
are attached.
Table II Body weight, ventricle weight and left ventricle mass index (LVMI) measured at the twenty-second after a IO-week treatment with vehicle or drug [captopril (CPT) or cilazapril (CLZ)] Group
Body weight (g)
Ventricle weight(g)
LVMI
WKY (control) WKY (CPT) WKY (CLZ)
486f25 425+30 489+20
0.85+0.2 0.75fO. 1 0.76kO.2
1.7+0. I 1.7fO.l 1.6fO.l
SHR (control) SHR (CPT) SHR (CLZ)
321?24 313_+11 312+20
0.89+0.2 0.67fO. l* 0.65+0.1*
2.8,0.21_ 2.1kO.l” 2.lfO.l”
*P
control group.
week,
378
Pharmacological
cells are detached poorly distinguishable.
and
Research,
the
Vol. 31, No. 6,1995
junctional
complexes
Twenty-two-week-old WKY after CLZ treatment (Fig. 2a) The myocardiocytes do not show considerable changes. Their sarcoplasm is rich in glycogen and contains myofibrils and mitochondria regularly packed between them, with normal shape and structure. The intercalated discs are unimpaired; nexuses, fasciae adhaerentese and desmosomes are well recognized. Twenty-two-week-old SHR after CLZ treatment (Fig. 2b and c) No substantial differences in the myocardiocyte ultrastructure are discernible between SHR and WKY CLZ-treated specimens. Myofibrils and mitochondria, regularly sized and shaped, are scattered in an electron-lucent sarcoplasm. The intercalated discs show cell interconnections with well preserved junctional structures. Twenty-two-week-old WKY after CPT-treatmerzt (Fig. 3a) The myocardiocytes contain abundant and electron-lucent sarcoplasm with few organelles. The mitochondria show changes in sizes, structure and distribution. Some of them appear swollen, others are characterized by electron-dense matrix without cristae. They are randomly scattered between myofibrils disarranged and variously oriented. The plasma membranes are thickened and poorly interconnected at level of the intercellular junctions. Twenty-two-week-old SHR after CPT treatment (Fig. 3b) The myocardiocytes contain large amounts of electron-lucent sarcoplasm with mitochondria of different sizes, enlarged and somewhere characterized by fragmentation of the cristae. The myofibrils are small, loosely distributed and variously sized; some of them are very thin and obliquely oriented. Plasma membrane detachments are seen in the intercalated discs where few myofilaments end on the junctional systems. Breaks of the plasma membranes are also observed. Some cell nuclei are affected by kariolysis, or fragmented. Fig. 2.
Left ventricular myocardium of 22-week-old WKY (a) and SHR (b, c) rats after cilazapril treatment. (a) In two myocardiocytes, sealed by intercalated discs (asterisks), rows of mitochondria appear regularly distributed between packed and aligned myofibrils. x9750. (b) An electron-lucent cytoplasm with glycogen intervenes between myofibrils and mitochondria. x9750. (c) Fasciae adhaerentes (arrows) and desmosomes (arrowheads) are recognizable in an intercalated disc. x30 875.
The configuration in sarcomeres of the myofibrils is preserved, whereas their thickness is varied, smaller and disarranged myofibrils are intermingled with normally-sized ones. The intercalated discs show impaired feature, the plasma membranes of adjacent
Morphometric
analysis
(Table
3)
Twenty-two-week-old WKY and SHR control in groups The myofibrillar area SHR myocardiocytes is lesser than in WKY ones (SHR 45.7f7.6, WKY 65.5k9.6, A -3O%, P
379
Pharmacological Research, Vol. 31, No. 6,1995
Fig. 3. Left ventricular myocardium of 22-week-old WKY (a) and SHR (b) rats after captopril treatment. (a) Two myocardiocytes enveloped by a thickened plasma membranes (arrows) are filled with clear sarcoplasm. The myofibrils are disarranged and some mitochondria, devoid of cristae, contain an electron-dense amorphous matrix (arrowheads). x9750. (b) Thin myofibrils spaced out by mitochondria of different sizes in an electron transparent sarcoplasm with few glycogen granules. x9750.
Percentage
Table III area of myofibrils, mitochondria and matrix recorded in vehicle-treated drug-treated [captopril (CPT) or cilazapril (CLZ)] Myofibr-illar area (%)
Mitochondrial
area (%)
(control
group) and
Matrix area (%)
WKY (control) WKY (CPT) WKY (CLZ)
6.5.5f9.6 36.9f7.0*** 54.2+10.0**
20.0+8.4 28.6+9.6* 25.8+6.7*
14.5+3.1 34.5+9.0*** 20.0+5.0**
SHR (control) SHR (CPT) SHR (CLZ)
45.7f7.7.1 42.7k9.6 52.7k7.1”
22.8+7.8 29.3+5.2* 23.4f4.1
31.5+10.0t 28.Ozb12.0 23.9f7.2
*P
compared to SHR control groups (CLZ-SHR: 52.7f 7.1, control SHR: 45.7k7.6, A +15%, P
control group.
-17%, PcO.025). Not significant difference is evident after CLZ-treatment between WKY and SHR specimens (CLZ-SHR: 52.7f7.1, CLZ-WKY: 54.2f 10.0, NS). The mitochondrial area is substantially similar in
380
CLZ-treated and control SHR (CLZ-SHR: 23.4k4.1, control SHR: 22.8f7.8, A +2% NS) whereas it is significantly greater in the CLZ-treated WKY than in the control ones (CLZ-WKY: 25.8k6.7, control WKY: 20.0f8.4, A +29%, P
DISCUSSION This study shows that the 22-week-old SHR myocardiocytes are affected by ultrastructural and morphomeric changes in their contractile apparatus. The myofibrils of the SHR myocardiocytes are in fact smaller and irregularly arranged in a more abundant cell matrix than those of the 22-week WKY myocardiocytes. This observation seems to conflict with previous data showing that wall stress is responsible for increase in myofibrils during myocardial hypertrophy [ 1, 211. However, the present results are in partial agreement with the hypothesis of Hamilton [22], suggesting that decrement in myofibril size, together with increase in mitochondrial area, may represent a compensatory response to an increased energy demand. The mitochondrial density augmentation might facilitate energy production while the myofibril size decrement might enhance the movement of both high energy phosphates and calcium to their respective sites. it is noteworthy to consider that Moreover, hypertension rate and duration play an important role in the degree of the morphological and functional
Pharmacological
Research,
Vol. 31, No. 6,1995
changes of hypertrophied myocardium the [l, 5,6,21]. The decreased myofibrillar area as well as the modified mitochondria observed in SHR myocardiocytes could be related to the duration of the hypertensive state and expression of a reduced cell ability to produce energy for contraction [23]. Previous studies carried out on human subjects and experimental animal models suggest that ACEinhibitors produce a decrease in left ventricular mass and improve the myocardial pumping function, even if there is no demonstration that the change in left ventricular mass is direct consequence of reduced blood pressure [24]. Captopril and cilazapril cause similar decrease in blood pressure and LVMI in SH rats, but the ultrastructural and morphometric data recorded after CLZ- and CPT-treatment indicate that captopril, unlike cilazapril, does not improve the morphological changes associated with LVH. The myofibrillar area augmentation as well as the feature of myofibrils and mitochondria regularly sized and shaped, both recorded in SHR myocardiocytes after CLZ treatment, suggest that the two drugs might have a different effect on the contractile system. Further investigations are required to determine whether the morphological changes are induced by CLZ are due to sarcomere addition and/or reorganization of myofibrils. The different effect evoked by the two ACE-inhibitors on the SHR myocardiocytes could be related to a CPT reactive group, such as the sulphydryl one, or to a different affinity of this molecule for the local renin-angiotensin system, resulting in a different interaction with growth factors [19,20,25]. The remarkable, different changes observed in WKY myocardiocytes after CPT and CLZ treatment, substantiate this hypothesis: myofibrils and mitochondria are regularly sized and shaped after CLZ treatment, whereas relevant abnormalities are seen in CPT-treated myocardiocytes. The evidence of a local renin-angiotensin system involved in the regulation of the cell growth and proliferation, as well as in the cell hypertrophy, could explain why ACE-inhibition induces modifications also in the myocardiocytes of control rates [26-301. The results of this investigation suggest that the ACE inhibitors interfere with the LVH regression and that the ventricular mass reduction may be apparently beneficial, since it is not always associated with improvement of myocardiocyte ultrastructure. This conclusion could explain why ACE inhibitors do not always correct the functional modifications occurring during LVH.
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