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Biomolecular changes in the aging myocardium: the effect of enalapril

Chronic administration of enalapril in the aging mouse prevents myocardial fibrosis. To investigate the mechanisms involved, we studied 30 CF1 female mice that received enalapril (ENAL:20 mg/L) in their drinking water after weaning and 30 control
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  Biomolecular Changes in the AgingMyocardium The Effect of Enalapril Leon Ferder, Luis A. Romano, Liliana B. Ercole, Ine´s Stella, and Felipe Inserra Chronic administration of enalapril in the agingmouse prevents myocardial fibrosis. To investigatethe mechanisms involved, we studied 30 CF1female mice that received enalapril (ENAL:20 mg/ L) in their drinking water after weaning and 30control (CONT) mice. Ten animals from eachgroup were killed at 12, 18, and 24 months. Half ofthe samples were prepared for light microscopy(LM) and the other half for electron microscopy(EM). Cardiac histologic sections were studied byan image analyzer (Bioscan OPTIMAS 4.1). Weperformed the following measurements incardiomyocytes: mitochondrial number,mitochondrial superoxide dismutase (SOD) usingimmunohistochemical methods with EM, thepercentage of cell cyclin, and apoptosis. The resultsobtained for CONT and ENAL, respectively wereas follows. For cyclin (percentage of positive) ourresults were: 12 months 17.1  0.1% and 18.2  0.8%, 18 months 2.4  1.6% ( P  << .001), and 11.4  0.1% ( P  << .001), 24 months 1.2  1.3% ( P  << .001),and 8.2  1.2% ( P  << .001) with significantdifferences at 18 and 24 months. For the Feulgenmethod (cell/mm 2 ) we found: 12 months CONT89.7  1.2, ENAL 84.6  1.2; 18 months CONT 62.8  1.2, ENAL 98.7  1.3, and 24 months CONT 81.2  1.3, ENAL 112.3  1.4. Apoptosis (percentage ofpositive) was found to be 12 months 3.7  0.4%and 1.9  0.1%, 18 months 7.1  0.3% ( P  << .001),and 1.5  0.1% ( P  << .001), 24 months 10.9  0.5%( P  << .001) and 2.1  1.8% ( P  << .001), for CONTand ENAL, respectively; there were significantdifferences at 18 and 24 months. The number ofmitochondria per cardiomyocyte were: 12 months85.9  1.8 and 87.3  1.5, 18 months 69.2  1.5†and 82.2  1.8 ( P  << .001), 24 months 54.6  1.1( P  << .001) and 81.4  1.6 ( P  << .001) for CONT andENAL respectively, with significant differences at18 and 24 months. Mitochondrial SOD was foundto be: 12 months 13.6%  0.2% ( P  << .05) and 17.8%  1.3% ( P  << .05), 18 months 7.1%  1.0% ( P  << .001) and 16.7%  1.6% ( P  << .001), 24 months 4.1%  0.5% ( P  << .001), and 12.4%  0.9% ( P   .001) forCONT and ENAL respectively, with significantdifferences at 12 months and at 18 and 24 months(ANOVA and contrast Scheffe´’s test). We concludethat chronic administration of ENAL modifiesmitochondrial SOD at 12 months, whereas at 18and 24 months ENAL was associated with highermitochondrial SOD and a higher mitochondrialnumber with a greater cyclin expression, and alower percentage of apoptosis. Enalapril mayprevent myocardial fibrosis, possibly by causingchanges related to enzymatic-mitochondrial orcellular cycle modifications. Am J Hypertens 1998;11:1297–1304 © 1998 American Journal ofHypertension, Ltd. KEY WORDS : Aging, myocardiosclerosis, enalapril,angiotensin converting enzyme inhibitor,cardioprotection, myocardial apoptosis,mitochondria, myocardium.  AJH 1998;11:1297–1304© 1998 by the American Journal of Hypertension, Ltd. 0895-7061/98/$19.00Published by Elsevier Science, Inc. PII S0895-7061(98)00152-6  A ngiotensin converting enzyme inhibition(ACEi) prevents or reverses left ventricu-lar hypertrophy (LVH) and myocardialsclerosis in both animals and humans, 1–3 similar effects can be seen in a variety of conditions. 4–6 Furthermore, recent studies in experimental animalshave shown that ACEi reduces the incidence of ar-rhythmia and myocardial contractile dysfunctioncaused by ischemia reperfusion. 7,8 Similarly, ACEi,reduces mortality and postischemic myocardial dys-function 9 in acute myocardial infarction.Several studies suggest that Ang II acts as an intra-cellular growth and metabolism modulator. 10 Thus, astate similar to that of phase G1 of the cell cycle may be seen in Ang II-treated renal cells. 11 Furthermore,ACEi have shown renal protection in different models,either with or without systemic hypertension. 12–14 We developed an experimental model in CF1 mice,in which the angiotensin converting enzyme waschronically inhibited with enalapril. By using thismodel, we found that a lower mortality and a decreasein glomerular and myocardial sclerosis could be ob-served in mice treated with enalapril for 24 months,without changes in blood pressure. A larger numberof mitochondria in both myocardial and hepatic cellswas also observed, in comparison with control ani-mals. 15 On the other hand, we noticed an increase intissue antioxidative activity due to the increase in bothsuperoxide dismutase (SOD) and glutathion peroxi-dase (GPx) in hepatocytes of animals treated withenalapril and captopril for 11 weeks. 16 Bearing these phenomena in mind, we decided tostudy the number of mitochondria, mitochondrialSOD content, cyclin labeling as a probable marker of cell cycle, and apoptosis in cardiomyocytes of CF1mice. Our interest was focused on the effects of chronic ACEi reducing oxidative stress, which proba- bly leads to myocyte cell death as observed by apo-ptosis. We study differences in changes of these pa-rameters at 12, 18, and 24 months of life, and theeffects that might result from enalapril administration. MATERIALS AND METHODS Thirty 15-day-old female CF1 mice received 20 mg/Lenalapril (ENAL) in drinking water, immediately afterweaning. They were compared with a control group(CONT) of 30 animals of the same litter, age, and sex.Ten animals from each group were killed at 12, 18, and24 months of life by intraperitoneal administration of sodium pentobarbital (4 mg/100 g body weight). Fiveof 10 animals of each group were studied by lightmicroscopy and immunohistochemical methods,whereas the other five were studied using immunola- beling and electron microscopy. Cardiac tissue wastreated according to the following methodology. Light Microscopy and Immunohistochemistry Hearts from 15 animals of each group (five 12-, 18-,and 24-month-old mice, respectively) were perfused by catheterization of the inferior vena cava, first withsaline and then with Bouin’s fluid. Hearts were thenremoved and sectioned longitudinally; sections werethen kept in Bouin’s fluid for 3 h, processed accordingto the usual technique, and were finally included inparaplast. Twenty histological sections of left ventricleof each heart were prepared.Histological sections were stained according to anavidin-biotin-peroxidase complex modified tech-nique. 17 Cell cycle was analyzed by using cyclin associatedproliferating cell nuclear antigen (PCNA) monoclonalantibody (PC10; Novocastra, Newcastle, England) at a1:100 dilution primary antibody. The secondary anti- body was a horse antimouse immunoglobulin G (IgG).Sections were then incubated in avidin-peroxidasecomplex and exposed to 0.01% diaminobenzidine   3% NiCl and 0.02% H 2 O 2 , and counterstained withlight green. Negative controls included the absence of primary or secondary antibodies, and replacement of secondary antibody by rat IgG. 18 The Feulgen methodwas performed according to standard techniques. 19 To study programmed cell death (apoptosis), thetissue was previously treated with a protein digestingenzyme or proteinase K (20   g/mL) for 15 min atroom temperature. Apoptosis was detected usingApoTag Kit (In situ Apoptosis Detection Kit—Peroxi-dase, Oncor, Gaithersburg, MD) using antidigoxi-genic-peroxidase. Finally, treated tissue was exposedto 0.01% diaminobenzidine containing 3% NiCl and0.02% H 2 O 2 . 20 Immunolabeling and Electron Microscopy  Heartsfrom 15 mice of each group (three groups of five miceaged 12, 18, and 24 months, respectively) were per-fused by catheterization of the inferior vena cava, firstwith phosphate buffered saline (PBS) pH 7.2, and thenwith 0.2% glutaraldehyde. Perfusion was performeduntil the cardiac parenchyma appeared extremelypale, and was then removed. Sections of approxi-mately 1 mm 2 were kept in 0.2% glutaraldehyde for2 h, then washed in PBS, and treated with increasingconcentrations of ethyl alcohol. Tissue fragments wereincluded in Lowicryl K4M (Chemische Werke Lowi, Received December 16, 1997. Accepted June 12, 1998.From the Institute of Biomedical Research and Centro de Inves-tigaciones Medicas Albert Einstein, Argentine Hebrew University,Bar Ilan Foundation, Buenos Aires, Argentina.The abstract of this paper was presented at the 49th Annual FallConference and Scientific Session of the Council for High BloodPressure Research, and Dr. Luis Romano was chosen as a recipientof the 1995 Merck Young Investigator Award.Address reprint requests and correspondence to Felipe Inserra,MD, Virrey Loreto 3150, Buenos Aires (1426), Argentina.  AJH–NOVEMBER 1998–VOL. 11, NO. 11, PART 1 1298  FERDER ET AL  Waldkreiburg, West Germany) for 48 h at 4°C underultraviolet light. Sections were performed with a LKBultramicrotome (LKB Instrument Inc.). Grids contain-ing tissue were washed in PBS, and incubated with 2% bovine serum albumin in PBS (BSA, type V, SigmaChemicals, St. Louis, MO) for 10 min. Grids wereincubated with Anti-Superoxide-Dismutase (SigmaChemical, St. Louis, MO) 21 at a 1:200 dilution in PBSfor 45 min. They were later washed in PBS and incu- bated with a 20-nm protein A-gold complex for 30 to35 min (10  L/grid). Grids were washed several timesin PBS, stained with uranyl-citrate for 2 to 3 min, andwith lead acetate for 20 sec. Finally, they were exam-ined with a Zeiss (Oberkochen, Germany) EM-109electron microscope. 22 Quantitative Analysis  Quantitative analysis by elec-tron microscopy (number of mitochondria/cardiomy-ocyte, and number of colloidal gold granules repre-senting mitochondrial SOD) was performed accordingto Weibel’s 23 and Tashiro et al’s 24 methods, as de-scribed previously. 15 Quantitative analysis by lightmicroscopy—ie, the percentage of cell nuclei/mm 2 labeled with PCNA and ApoTag kit—was performed by means of an image analyzer (consisting of a videocamera, a digitizer frame grabber card, an IBM AT-compatible personal computer and Bioscan OPTIMAS4.1 software [Bioscan, Edmonds, WA]). 25 Statistical Analysis  Results were compared usingANOVA and Scheffe´’s contrast test. Results corre-spond to the mean  SD. RESULTS The first and third columns of Table 1 show the per-centage of cyclin labeled cardiomyocytes and apopto-sis percentages, respectively. The second columnshows the results of Feulgen techniques. The fourthcolumn indicates the number of mitochondria/cardi-omyocyte; and the fifth column includes the numberof colloidal gold granules/mitochondria, correspond-ing to mitochondrial SOD immunolabeling.As far as cyclin is concerned, a progressive fall can be noticed that becomes highly prominent at 18 and 24months of life. Animals receiving ACEi exhibited amuch slower fall, the difference becoming statisticallysignificant at the ages mentioned. These results arecoincident with the Feulgen method (Table 1 and Fig-ures 1, 2, and 3).With regard to apoptosis, control animals showed aprogressive increase of labeled cardiomyocytes in sec-tions corresponding to 12, 18, and 24 months, whereasanimals receiving enalapril showed no labeling in-crease indicative of programmed cardiomyocyte deathat the times mentioned. Differences with control ani-mals were significant at 18 and 24 months (Table 1 andFigures 1 and 2).The number of mitochondria per cardiomyocyte fellprogressively in control animals at 18 and 24 months. TABLE 1. RESULTS AT DIFFERENT AGES OF THE ANIMALSAge(months)Cyclin(% ofpositive)Feulgen(cell/mm 2 )Apoptosis(% ofpositive)No.Mitochondria/ myocardiumMitochondrialSOD perMitochondrion 12 CONT 17.1  0.1 89.7  1.2 3.7  0.4 85.9  1.8 13.6  0.2*12 ENAL 18.2  0.8 84.6  1.3 1.9  0.2 87.3  1.5 17.8  1.3*18 CONT 2.4  1.6† 62.8  1.2† 7.1  0.3† 69.2  1.5† 7.1  1.0†18 ENAL 11.4  0.1† 98.7  1.3† 1.5  0.1† 82.2  1.8† 16.7  1.6†24 CONT 1.2  1.3† 81.2  1.3† 10.9  0.5† 54.6  1.1† 4.1  0.5†24 ENAL 8.2  1.2† 112.3  1.4† 2.1  1.8† 81.4  1.6† 12.4  0.9† ENAL versus CONT:  * P  .05. † P  0.001.Table 1 shows cyclin immunolabeling and apoptosis as a percentage of positive with light microscopy, the number of Feulgen positive cells/mm 2 , numberof mitochondria/cardiomyocyte, and number of gold colloidal granules labeled with monoclonal antibody for SOD, with electron microscopy. Results areexpressed as mean  SD. FIGURE 1.  Cyclin and apoptosis variations in control animals(in % of positive).  AJH–NOVEMBER 1998–VOL. 11, NO. 11, PART 1  ENALAPRIL AND CHANGES IN AGING MYOCARDIUM  1299  The decrease was not as prominent in enalapril-treated animals as controls. The difference between both groups was significant at 18 and 24 months (Ta- ble 1 and Figure 3).With regard to mitochondrial (mit) SOD, there wasa progressive fall in control animals. This fall wasmuch slower in enalapril-treated animals. Significantdifferences were already noticeable at 12 months be-tween treated and control animals, which becamemore marked at 18 and 24 months (Table 1 and Fig-ure 4). DISCUSSION Aging is a natural process occurring in all species 26 :progressive morphological and functional modifica-tions of various organs take place during this pro-cess. 27,28 By and large, there is correlation between structuraland functional changes. The cardiovascular system isone of the most affected by the aging process. Struc-tural vascular changes and sclerosis of cardiac musclecause a decrease in cardiac output. 29–31 Angiotensin II may alter vascular structure (vascu-lar muscle hypertrophy and arteriosclerosis) either bya hemodynamic effect or by acting as a growth factoron the vascular smooth muscle. 32–37 It is known thatrenin-angiotensin system inhibition prevents vascularhypertrophy in a variety of experimental models. 36,37 As stated in our previous report, we have shownearlier that normotensive aged mice with chronic re-nin-angiotensin system inhibition exhibited a de-creased thickness of aorta middle layer, and musclelayers of middle arteries of heart, brain, and kidney.Moreover, we found similar changes in middle pul-monary arteries, which are not exposed to systemicpressure. Therefore, changes seem to be independentof arterial blood pressure. 6 On the other hand, it has been reported that ACEiprevents left ventricular hypertrophy and myocardialsclerosis both in animals and humans. 1,38 By using the above-mentioned model, we foundthat treated mice have a significantly lower heartweight and a significant decrease of myocardial fibro-sis in comparison with control animals. 6 Similar find-ings have been reported using other experimentalmodels. 4,5 Likewise, cardiomyocyte vital cyclechanges have been found in this model.Myocardiogenic differentiation has been consideredas an irreversible process associated with adult cardi-omyocyte possibility of initiating a new mitotic cycle.That is why DNA synthesis and cytokinetic activity inadult cardiac cell could not be proved. 39 Some facts FIGURE 2.  Cyclin and apoptosis variations in enalapril treatedanimals (in % of positive). FIGURE 3.  Changes in number of mitochondria per cardiomy-ocyte at 12, 18, and 24 months, comparing control and enalapril groups. (Enalapril versus Control  P  .001). FIGURE 4.  Mitochondrial SOD per mitochondria variation at12, 18, and 24 months in control and enalapril groups. (Enalversus Cont *  P  .05;  P  .001).  AJH–NOVEMBER 1998–VOL. 11, NO. 11, PART 1 1300  FERDER ET AL  seem to indicate that, under certain conditions, thecardiomyocyte cell is capable of synthesizing DNAand initiating a mitotic cycle. For this reason, a markedmyocardial cell proliferation is induced in amphibianswith experimental ischemic injuries or ventricle par-tial amputation. 40 A similar process occurs in reptilesand birds. 41 Although mammalian myogenic capacity is limited,myocardial cells can undergo DNA replication insome situations. 42 It has also been shown that somegrowth factors stimulate DNA synthesis in adult ratventricular myocardium: epithelial growth factor(EGF), IGF-1, and 12- O -tetradecanoyl phorbol-13-ace-tate (TPA). 43 Likewise, Kardami found that DNA syn-thesis in chicken cardiomyocyte is increased by basicfibroblastic growth factor (bFGF) and that there areinhibiting mechanisms like TGF-  . 44 There are alsodata (although scarce) of myocardial hypertrophy andhyperplasia in humans, with Feulgen staining andDNA content quantification using a biopsy tissue im-age analyzer. These data shows a cell proliferationincrement in cardiomyocytes. 45 Our data match thosefindings. As shown in Table 1, Feulgen staining, aDNA content indicator, showed greater positivity inchronically enalapril-treated animals.According to these elements, we can assume that,under certain conditions, adult myocardium canshow, in addition to hypertrophy, hyperplasia withincreasing DNA, and mitogenic activity.Cyclin evaluation is a suitable method to analyzethe cell cycle. This polypeptide builds up during in-terphase and falls during the mitotic division; thus, itis directly related to DNA synthesis. It has beenshown 46 that nuclear levels of cyclin fluctuatethroughout the cell cycle; and their quantification be-comes useful as a cell mitotic activity indicator. 47 In the absence of pathologic processes, cellular cy-clin activity decreases with aging. 48 Similar results areseen in our model at 18 and 24 months (Table 1 andFigures 1 and 2).Our cell cycle analysis showed that enalapril-treatedanimals exhibited higher cyclin labeling than thoseobserved in control groups at 18 and 24 months,whereas cardiomyocyte regeneration was better pre-served (Table 1 and Figures 1 and 2). As shown inFigure 1, control animals show a decrease of cyclinactivity as they grow older; this phenomenon seems to be attenuated by chronic inhibition of the renin-angio-tensin system.In contrast to a decrease in PCNA with age (Table 1and Figures 1 and 2) the percentage of cells that arepositively labeled for programmed cell death (apopto-sis) increases as control animals grow older. Similarresults were found by Kajstura et al. 49 Conversely,cells that were positive for apoptosis decreased signif-icantly in animals receiving enalapril, especially insections obtained at 18 and 24 months. Even thoughapoptosis is a natural cellular process, by means of which other processes such as normal tissue turnover,immune system maturation, and embryo develop-ment may occur, it is likely that cardiomyocyte lifespan depends on a set of stimuli that programs deathwhenever that set is modified. As shown in Table 1and Figures 1 to 4, cardiomyocyte apoptosis increases,whereas the number of mitochondria and mitochon-drial SOD decrease with animal aging. It seems pos-sible that ACEi could delay or change this biologicalevent.A set of stimuli leading to apoptosis can be trig-gered, among other things, by oxidative stress 50 andangiotensin II by means of angiotensin II receptorstimulation. 51 For this reason, ACEi can achieve car-diomyocyte apoptosis reduction via any of thesemechanisms.Morphological, biochemical and molecular evi-dences indicate that oxidative phosphorylation 52 andreplicative activity 53 of mitochondria decline with ageand that such deterioration could be associated withincreased oxidative damage of mitochondrial DNA(mtDNA), leading to the concept that instability of themitochondrial genome 54 is the main cause of aging.In a previous study, we had found changes in thenumber of mitochondria/cardiomyocyte in micetreated with enalapril for 24 months. 15 This differencein the number of mitochondria/cardiomyocyte be-tween treated and control animals was also found inthis study, the difference becoming significant from 18months of life onward (Table 1 and Figure 3). Aginggenerates a decrease in mitochondrial mass in ani-mals. 55 As shown in Figure 3, results obtained withour experimental model are in accordance with thosepreviously obtained. It might be assumed that the fallin the number of mitochondria in treated animalscould probably arise from a better mitochondrial turn-over preservation in treated aging animals.Mitochondrial alteration is possibly related, amongother things, to the chronic impact produced by oxy-gen reactive species on it. In our laboratory, we havealso found that CF1 mice receiving enalapril or capto-pril for 11 weeks had enhanced tissue enzymatic an-tioxidant defenses in several organs, including theheart. 56 In this study, we found a mitochondrial SODimmunolabeling decrease with animal age in the con-trol group, whereas the enalapril treated group had agreater immunolabeling (Table 1 and Figure 4). Al-though Mn-SOD is known as inducible by diversestimuli, as we observed using electron microscopy, theincrease we previously found after 11 weeks was inCu-Zn-SOD activity, described as constitutive, as wellas in glutation peroxidase. 56  AJH–NOVEMBER 1998–VOL. 11, NO. 11, PART 1  ENALAPRIL AND CHANGES IN AGING MYOCARDIUM  1301
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