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Journal of Cardiovascular Pharmacology:
doi: 10.1097/01.fjc.0000211728.23304.ad
Original Articles

Comparative Study of the Effects of Lacidipine and Enalapril on the Left Ventricular Cardiomyocyte Remodeling in Spontaneously Hypertensive Rats

Cagalinec, Michal* †; Kyselovic, Jan PhD; Blaskova, Eva PhD; Bacharova, Ljuba MD*; Chorvat, Dusan Jr PhD*; Chorvatova, Alzbeta PhD§ ¶

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Author Information

*International Laser Centre, Bratislava, Slovakia

Faculty of Mathematics, Physics, and Informatics, Comenius University, Bratislava

Department of Pharmacology, Comenius University, Odbojarov 10, Bratislava

§Research Centre of Sainte-Justine Hospital, Montreal, Canada

Department of Pediatrics, University of Montreal, Montreal

This work was supported by Collaborative Linkage grant LST.CLG.979836 from NATO and partly by the grant 1/0507/03 from the Science Grant Agency VEGA, Slovakia. A.C. is a “Fonds de la Recherche en Santé du Québec” (No. 2948) fellow supported by the Canadian Institute for Health Research (MOP 74600).

Reprints: Dr. Chorvatova Alzbeta, Research Centre of Sainte-Justine Hospital, 3175 Côte Sainte Catherine, H3 T 1C5 Montreal, Canada (e-mail: alzbeta.chorvatova@recherche-ste-justine.qc.ca)

Received for publication October 24, 2005; accepted March 17, 2006

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Abstract

Antihypertensive medications are the most efficient drugs in achieving regression of myocardial hypertrophy in both clinical studies and animal models of hypertension. Nevertheless, there is a lack of clear and concise comparative study of their effects on the modulation of cardiomyocyte morphology and function. Here, we assessed the tissue-protective actions of 2 of these drugs, the calcium channel blocker lacidipine (3 mg/kg/day) and the angiotensin-converting enzyme-inhibitor enalapril (10 mg/kg/day) in vivo, after 8 weeks of treatment of 12-week-old spontaneously hypertensive rats, as well as in vitro, after short-term (4 min) application to isolated cardiomyocytes. Left ventricular hypertrophy (LVH) was compared at organ, tissue, and single-cell level. Our data showed that both drugs prevented the LVH of 20-week-old spontaneously hypertensive rats, but only lacidipine significantly decreased the cardiomyocyte size. Similarly, the single-cell contractility was significantly lowered in lacidipine-treated rats only. The effect of lacidipine was initiated shortly after exposure to the drug in a dose-dependent manner at 0.5 Hz, as well as at 2 Hz, with EC50 of 10−7 mol/L. These results can help in understanding the effects of these drugs on the prevention of LVH.

Antihypertensive drugs are probably the most efficient medications capable of achieving the regression of myocardial hypertrophy in both clinical studies and animal models of hypertension.1,2 Calcium channel blockers (CCBs) and angiotensin-converting enzyme (ACE) inhibitors are their well-known representatives with different therapeutic actions and distinct effects on left ventricular (LV) mass reduction. On one hand, the effect of CCBs is usually attributed to arterial vasodilatation and to the subsequent lowering of the blood pressure, leading to reduced cardiac load and the mechanical stress on the vessel wall.3 On the other hand, ACE inhibitors reduce blood pressure and cardiac mass by inhibition of the conversion of angiotensin I to angiotensin II.4 Despite the fact that these medications use diverse mechanisms of action, both CCBs and ACE inhibitors reduce the cardiac hypertrophy and fibrosis-related gene reprogramming and/or re-expression of fetal genes in their long-term treatment, as demonstrated in the animal model of spontaneously hypertensive rats (SHR) by decreased cardiac fibrosis5 and by lowering collagen deposits in the extracellular matrix.6 The heart regressed by CCBs and/or by ACE inhibition displays more patterns of normal physiology in comparison to hypertrophied myocardium, as a result of increased capillary density,7 improved diastolic and systolic performance,8,9 and energy production restoration.10

Interestingly, ACE inhibitors and CCBs are capable of reducing the hypertrophy-induced hypertension even at dosages that are ineffective on blood pressure (BP),11–14 suggesting that these beneficial actions could be related to both short-term and long-term effects. The primary consequence of the short-term effects (seen in minutes to hours) is the decrease of blood pressure related to either selective interaction of CCBs with calcium channels or decreased production of angiotensin II by ACE inhibitors.15 The long-term effects (appearing in weeks to months) additionally controls the disease through the prevention of the end organ damage and the interaction of the drugs with hypertrophic pathways, thus leading to obstruction of the re-expression of embryonic genes and/or to the overexpression of cardiac-hypertrophy-dependent genes. However, there is a lack of comparative study of the 2 drugs in same experimental conditions at the single-cell level to understand their antihypertrophic effects during antihypertensive treatment.

In this study, our goal is therefore compare the actions of lacidipine and of enalapril on the morphology and function of single cardiac cells in their process of protection against the hypertension-induced hypertrophic remodeling of the LV, while investigating conditions in which the 2 drugs had similar effects on BP and on left ventricular hypertrophy (LVH). The model of SHR, in which both types of medications have been shown to reduce blood pressure and cardiac hypertrophy, was chosen. The long-lasting in vivo effects, as well as the short-term in vitro effects of the CCB lacidipine and the ACE inhibitor enalapril on the left ventricular structure/function modifications, were investigated.

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MATERIALS AND METHODS

Animals

The work was done on male normotensive Wistar-Kyoto rats (WKY) and SHR, either 12 or 20 weeks old (Dobra Voda, Slovakia). SHRs at the age of 12 weeks were randomly divided into 4 groups. One group (labeled SHR 12) was used immediately for experiments. Three remaining groups were kept for 8 more weeks. One (labeled SHR 20) served as control SHRs and received vehicle (methylcellulose, Slovakofarma, Slovakia). The remaining 2 SHR groups received lacidipine (3 mg/kg/day) or enalapril (10 mg/kg/day) for 8 weeks by oral gavage performed twice per day. All of the procedures were done in accordance with the Ethical Committee of the Pharmaceutical Faculty, Comenius University, Bratislava, Slovakia. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Systolic blood pressure (SBP) was measured by tail-cuff method in conscious animals prewarmed to 35°C in thermostatic cages.

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Histology

Standard histological methods were used to examine myocyte morphology and matrix abnormalities in LV sections. Briefly, LV tissue was obtained from at least 3 rats from each group and fixed in liquid nitrogen. LV sections (150 sections/animal in each group, thickness of 7 μm) were stained with hematoxylin & eosin and van Gieson for extracellular matrix and examined on a blinded basis for the level of extracellular matrix deposition in myocardium, using 40×1.3 oil objective. To determine cardiomyocyte diameter from histological sections, microscopic imaging was used (MeOpta microscope, equipped with objective 10×, NA 0.30). Diameter was measured from sections perpendicular to the longitudinal axis of the ventricular wall. Only sections showing a regular pattern without visible abnormal lesions were used for comparison. The detection system was based on CCD Minitron connected to Miro Video DC30 frame graber, interfaced to PC (Pentium II). All of the images were digitized with 24-bit resolution (768×576 pixels, 96 dpi). Captured image sequences were preprocessed with image-analysis software (Pentium IV) in Impor 4.0 professional (Kvant, SRO) and in Adobe Photoshop 5.0 LE. The size of objects was estimated by image processing pixel and data were expressed in microns.

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Isolation of Single Cardiomyocytes

Animals were anesthetized by thiopental 45 mg/kg in the presence of heparin 100 mg/kg. The heart was removed and LV myocytes were isolated following retrograde perfusion of the heart with proteolytic enzymes,16,17 using 0.9 mg/mL collagenase (type II; Gibco BRL) and 0.01 mg/mL protease (Sigma-Aldrich, Germany) in low CaCl2 (50 μmol/L) at 36°±1°C. The pellets of myocytes were resuspended in enzyme-free isolation solution containing 0.75 mmol/L CaCl2 (see Solutions for composition) and maintained in petri dishes at 4°C until use. Only cells with clearly defined sarcomere striations were used. All of the experiments were performed up to 10 h following dissociation.

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Single Cardiomyocyte Volume, Contractility, and Intracellular Calcium Measurements

For determination of single cardiomyocyte volume, cells were stained with Fluo-3 fluorescent probe (1 μmol/L for 10 min at room temperature). Fluorescence was collected at 505 to 525 nm using laser scanning confocal microscope LSM 510 Meta (Zeiss, Germany) with C-Apochromat 40×/NA=1.2 water immersion objective in response to excitation by Ar:ion laser line 488 nm (Lasos Lasertechnik). 3D images of the cardiomyocyte were obtained by successive scanning of 2D confocal optical planes with 2-μm steps in the Z axis using the pinhole opening of 1 Airy unit corresponding to optical slices of 0.9 μm. Volume was calculated using geometrical approximation of a cell as an elliptic cylinder18 (eg, cell volume=length×width×depth×π/4). For length and width data, some measurements obtained from 2D images were also added.

Cells were field stimulated to contract at frequency 0.5 or 2.0 Hz using Pt electrodes incorporated into a homemade bath, stimulated by pulse generator (DS-2A, Digitimer, Canada) operated via trigger generator (DG-2, Digitimer). Rapid line-scan confocal imaging protocol (5 ms) was used to record contractions together with calcium transients. Cell shortening was calculated as a percentage of Δ cell length=(length−maximum shortening)/length. Intracellular calcium changes were assessed in Fluo-3-loaded cells by analysis of calcium transients as (systolic F)/(diastolic F0). Only cells with 2 contracting edges were taken into consideration.

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RNA Extraction and Real-Time-Polymerase Chain Reaction

Rats were sacrificed by decapitation and left ventricles were rapidly dissected, weighed, snap-frozen in liquid nitrogen and stored at −80°C until RNA extraction. Gene expression was analyzed in 3 to 6 animals per group. Total RNA was isolated from LVs using the TriPure reagent (Gibco BRL) essentially according to manufacturer's instructions and dissolved in an RNAse-free buffer. Aliquots (4 μL) were used in hot-start polymerase chain reaction (PCR; PLATINUM). Quantitative real-time (RT)-PCR THERMOSCRIPT One-Step System (Gibco BRL) with 5 pmol of each sense and anti-sense gene-specific primers for transforming growth factor-β1 (TGF-β1)19 and α-subunit of dihydropyridine (DHP) receptor.20 As an internal standard, we have used glyceraldehyde-3-phosphate dehydrogenase (GADPH) cDNA, 1.3 kb PstI/PstI fragment.21 After an initial denaturation of 5 min at 95°C, the mixture was subjected to cycles of denaturation (30 s at 95°C), annealing (30 s at 60°C), and primer extension (68°C, 2 min) in a thermal cycler (Biometra Personal cycler 2). PCR products were separated on a polyacrylamide gel. PCR product bands in dried gels were detected and quantified by OptiQuant software. In preliminary experiments, the number of PCR cycles yielding exponential amplification was determined for each gene and tissue. When total RNA without reverse transcription was used in the PCR reaction, no signal was detected, thus showing that genomic DNA possibly contaminating the RNA solution did not present a source of error in our study. Gene expression was corrected for GADPH and expressed relative to control, which was arbitrarily set to one. GADPH mRNA levels did not differ significantly between groups (data not shown).

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Solutions

The physiological salt solution used for isolation and storage of myocytes contained (in mmol/L): NaCl, 130.0; KCl, 5.4; MgCl2·6H2O, 1.4; NaH2PO4, 0.4; creatine, 10.0; taurine, 10.0; glucose, 10.0; and 4-(2-hydroxyethyl)-1-piperazineethenesulfonic acid (HEPES), 10.0; CaCl2, 0.75; titrated to pH 7.3 with NaOH. This was supplemented with EGTA or Ca2+ at different stages of the isolation procedure, as described previously.16,17 Basic external solution perfused on single cardiomyocytes during recording contained (mmol/L): NaCl, 130.0; KCl, 5.4; CaCl2, 1.8; MgCl2, 1.4; NaH2PO4, 0.4, glucose, 10.0; HEPES, 10.0; adjusted to pH 7.3 with NaOH. KCl, CaCl2, and glucose were purchased from Merck (Germany), collagenase was from Gibco BRL, NaCl, MgCl2, and NaH2PO4 were from Lachema (Czech Republic). All other drugs were purchased from Sigma, Germany. Thiopental and heparin were obtained from Lachema (Czech Republic). Enalapril was obtained from Krka (Slovenia). Lacidipine was provided by Glaxo-Wellcome (Italy).

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Data Analysis

Data are shown as mean±SEM. Comparison between means was made by 1- or 2-way ANOVA followed by Tukey post-test, unless stated otherwise. Data were obtained from at least 5 different animals in each group.

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RESULTS

Biometric and Hemodynamic Changes in SHR

First, biometric and hemodynamic parameters were analyzed in normotensive WKY and SHR of 12 and/or 20 weeks of age (Table 1). SBP was measured together with heart rate and the data were collected in the same animals where LV weight (LVW) was compared against body weight (BW). As expected, we observed significant increase in LVW/BW, as well as in SBP and heart rate in both 12-week-old SHR (SHR 12) and 20-week-old SHR (SHR 20) when compared to WKY of the same age, confirming the presence of hypertension and LVH in these animals.

Table 1
Table 1
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Changes in Cardiomyocyte Morphology in SHR

Cardiomyocyte diameter was established from histological images of sections of LV myocardium. We recorded its significant increase in SHR (Fig. 1A), but no significant changes were noted during growth from 12 to 20 weeks in WKY or in SHR animals. To question possible fibrotic changes in the myocardium at this stage of the disease, expression of TGF-β1 was determined by RT-PCR (Fig. 1Ba). These experiments showed no significant differences in the TGF-β1 mRNA (Fig. 1Bb) among the studied groups, and this result, backed by histological analysis (Fig. 1C), suggests that at this stage, fibrosis did not contribute to increased LV mass of SHR.

Figure 1
Figure 1
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The results obtained from the present study suggest that, at investigated stage of disease, modifications in single LV cardiomyocytes alone are responsible for observed LVH. However, histological staining is not precise estimation of cardiomyocyte changes because it does not allow the determination of the length of single cells and/or the ability to distinguish between changes in the cell length versus cell width. For these reasons, we isolated single cardiac myocytes from the LV (Fig. 2A). Using conventional laser scanning microscopy, confocal images were then taken in 3 dimensions in freshly isolated cells after staining with intracellular calcium marker Fluo-3. As illustrated in Figure 2B, successive scanning of 2D confocal optical planes with 2-μm steps in the Z axes was applied to obtain the image. Cardiomyocyte length, width, and depth were determined from these images, whereas the cell volume was calculated using approximation for elliptic cylinder. Obtained data, summarized in Table 2, revealed significantly greater cell length in SHR 12, as well as in SHR 20, confirming that cellular hypertrophy underlies LVH observed in these rats. Despite no changes in LVW/BW during growth from 12 to 20 weeks, cardiomyocytes isolated from 20-week-old rats were significantly longer in comparison to 12-week-old rats in the WKY as well as in the SHR group. Data obtained in studied SHR confirmed that LVH results from hypertrophic remodeling of single cells.

Figure 2
Figure 2
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Table 2
Table 2
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Long-Lasting Effects of Lacidipine and Enalapril on Cardiomyocyte Morphology in SHR

Our next goal was to prevent the LVH in SHR, described in the previous section, using 2 different antihypertensive drugs and to evaluate their ability to affect LV remodeling. We tested long-lasting effects of calcium antagonist lacidipine and ACE-inhibitor enalapril on cardiac hypertrophy in SHR. Both drugs were applied twice per day to 12-week-old SHR at a concentration of 3 mg/kg/day for lacidipine and 10 mg/kg/day for enalapril. Each treatment lasted 8 weeks. Animals were then evaluated and compared to nontreated 20-week-old SHR. As summarized in Table 1, biometric and hemodynamic parameters revealed significant positive effects of both drugs on hypertension, as well as on LVH, as characterized by the decrease in SBP and LVW/BW.

Furthermore, histological analysis also confirmed that both drugs prevented the deterioration of the LV tissue (Figs. 3Aa–Ac). Interestingly, cardiomyocyte diameter was significantly decreased in lacidipine-treated rats only (Fig. 3B). This result was confirmed by morphological analysis of isolated myocytes (Figs. 3Ca–Cb). Indeed, cardiomyocyte width (Fig. 3Cb) but not length (Fig. 3Ca) were reduced following treatment with lacidipine only. Treatment with enalapril failed to induce significant changes of single-cell morphology.

Figure 3
Figure 3
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Long-Lasting Effects of Lacidipine and Enalapril on Cardiomyocyte Function in SHR

Experiments described in the previous section demonstrated that both lacidipine and enalapril prevented hypertension and hypertrophy of SHR after 8 weeks of treatment. Although structural remodeling of LV myocytes followed application of lacidipine to SHR, no significant morphological changes were observed in single cells after treatment with enalapril. To further understand the effects of the 2 drugs, we questioned whether functional differences accompany the morphological ones at the single-cell level. Contractility is the main function of LV cardiomyocytes and results from the excitation–contraction coupling process, repeated at cardiac frequency. This process is initiated by opening DHP-sensitive L-type Ca2+ channels (ICaL), which induces trans-sarcolemmal Ca2+ entry,22,23 leading to the opening of ryanodine receptors on the sarcoplasmic reticulum, thereby permitting amplified release of Ca2+ that binds to the contractile apparatus and prompts the cell to contract.24 SHR are known to have larger calcium transients and hence increased single-cell contractility.25 As illustrated in Figure 4A, we observed that in our experimental conditions, the increase in the expression of the α-subunit of DHP receptors is reversed by enalapril and, to even more important extent, by lacidipine (Fig. 4Aa); at the same time the TGF-β1 is significantly reduced by both drugs (Fig. 4Ab) in 20-week-old SHR.

Figure 4
Figure 4
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Lacidipine belongs to the class of the DHP-sensitive L-type Ca2+ channel blocker26 and is therefore expected to affect the contractility of single cells. To examine single cardiomyocyte contractility, cells freshly isolated from the LV were field-stimulated at 2 different stimulation frequencies–0.5 Hz (to stabilize sarcoplasmic reticulum loading) and 2 Hz (to study frequency-dependence of the drug action and to induce stimulations closer to heart rate). As illustrated in Figure 4B, recorded data showed significantly lower contractility in lacidpine-treated but not in enalapril-treated rats when compared to 20-week-old SHR at both stimulation frequencies, suggesting that functional changes of single cells are correlated with their morphological modifications. Observed in vivo long-lasting functional effects of lacidipine are likely to precede the morphological changes and to be initiated shortly after exposure to these drugs. Indeed, both lacidipine and enalapril were capable of inducing immediate actions, namely rapid transient decrease in SBP, as illustrated by Figure 4C. Three hours after the first exposure to enalapril, BP was significantly decreased in treated animals, and this effect, also reported by others,15 was even more pronounced in response to the first dose of lacidipine (Fig. 4C, black triangles). This action suggests that functional effects of the drugs can be initiated rapidly after their exposure to cells.

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Short-Term Effects of Lacidipine and Enalapril on Cardiomyocyte Function in SHR

Our next goal was to investigate in vitro, short-term, dose-dependent actions of lacidipine and enalapril on single-cell contractility and calcium transients. We questioned these effects in 12-week-old SHR, at the time point when the treatment with lacidipine and enalapril was initiated. Taking into consideration the importance of intracellular calcium cycling in these actions, calcium transients were determined in the same cells using fluorescent dye Fluo-3 and expressed as (systolic F)/(diastolic F0). All of the results were normalized in each cardiomyocyte before comparison of the means. As illustrated in Figure 5, the calcium channel blocker lacidipine induced a significant concentration-dependent inhibition of the single-cell contractility (filled circles) after 4 min of exposure at both stimulation frequencies 0.5 Hz (Fig. 5Aa) and 2 Hz (Fig. 5Ab). This effect accompanied the dose-dependent lacidipine-induced reduction of the calcium transient (open circles). Boltzmann fit was used to determine EC50 for the lacipine effects. Its logarithm reached 6.7±0.3 (n=10) at 0.5 Hz for contractility and 7.4±0.3 (n=9) for calcium (not significantly different). These results were comparable to those obtained at 2 Hz, namely 7.6±0.3 (n=8) for contractility and 7.3±0.3 (n=12) for calcium. Comparable (not significantly different) values were also obtained in 20-week-old WKY rats (data not illustrated). These results showed that the effect of lacidipine on contractility is initiated rapidly and dose dependently after application. They also confirmed that this effect is mediated via a calcium-dependent mechanism.

Figure 5
Figure 5
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Enalapril induced less spectacular effects on contractility. Although the short-term (4 min) exposure to the drug also led to some decrease in the single-cell contractility (Fig. 5B), the extent of this action was much less when compared to lacidipine. Interestingly, this small effect was observed in SHR only; no direct action was provoked in 12-week-old WKY rats (data not shown). However, the dose–response curve, illustrated in Figure 5B in 12-week-old SHR, indicated that this effect was not concentration dependent but was rather nonspecific at both studied frequencies of 0.5 Hz (Fig. 5Ba) and 2 Hz (Fig. 5Bb).

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DISCUSSION

Presented data confirmed that the calcium antagonist lacidipine, as well as the ACE inhibitor enalapril prevented the LVH, but only lacidipine significantly decreased the cardiomyocyte size in 20-week-old SHR. Similarly, the single-cell contractility was significantly lowered in lacidipine-treated rats only. After the direct application, the decrease in the contractility was initiated shortly after exposure to the lacidipine in a dose-dependent manner at 0.5 Hz, as well as at 2 Hz in response to lowering of intracellular calcium.

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LVH in SHR

The SHR model is a robust, commonly used genetic approach to study hypertension and cardiac hypertrophy. We questioned 2 different age groups of SHR: the first at 12 weeks of age, at the stage of the disease at which compensated hypertrophic remodeling is already in place, and the second at 20 weeks of age, closer to the stage at which the majority of studies in SHR are performed,27,28 while keeping in mind that SHR often develop heart failure at 18 to 24 months. Several authors reported significant increase in the heart:BW ratios in SHR in comparison to control WKY rats.25,29 As expected, we confirmed cardiac hypertrophy in our experimental conditions by the significant increase in LVW/BW in SHR versus WKY. In SHR 20, the LVH was accompanied by an increase in the cell diameter in tissue as well as in single cardiomyocyte length and width, whereas in SHR 12, only cell length was significantly prolonged. These changes are in agreement with LV remodeling in SHR that occurs to maintain the stroke volume and the cardiac output.30 However, myocardial properties are determined not only by changes in the muscular components, but may also be related to modifications of the fibrous connective tissue network composed of collagen and vasculature. Cardiac hypertrophy in SHR may thereby result, at least in part, from structural remodeling of the cardiac interstitium, a process widely known to take part in older SHR, where it implicates the renin-angiotensin system.31 Nevertheless, no changes in TGF-β, the cytokine principally responsible for fibrosis,32 or modifications in the myocardium histology were observed in SHR, suggesting that no changes in myocardial fibrosis are put in place in SHR at these early stages of the disease. These results indicate that structural modifications of cardiomyocytes underlie solely the LV hypertrophic enlargement in 20-week-old SHR, in accordance with others.25 In the same way, yet to a lesser extent, changes in single cardiomyocytes underlie hypertrophic LV modifications in the compensated stage in 12-week-old SHR.

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Long-Lasting In Vivo Effects of Lacidipine and Enalapril on Cardiomyocyte Morphology

We assessed the tissue-protective in vivo effects of the CCB lacidipine and the ACE inhibitor enalapril in SHR on LVH, as well as on underlying changes at tissue and cellular levels in concentrations at which the drugs induced significant reduction in the BP. As indicated in Table 1, applied doses were sufficient to prevent, after 8 weeks of treatment, both BP and LVH. We have chosen these doses to allow the complete normalization of LV mass in treated animals, in accordance with others.29 Our data revealed that the prevention of the LV remodeling differed following the 2 treatments. In comparison to enalapril, lacidipine had more pronounced long-lasting in vivo effects on cardiomyocyte morphology when examined in tissue, as well as in freshly isolated cells. The structural effects of lacidipine on cardiomyocytes were in line with observed reduction of morphological alterations seen in SHR in small-resistance arteries.29 However, treatment with enalapril induced no significant changes in single cardiomyocyte morphology under our experimental conditions. This result contrasts with others,33 and the difference may be attributable to age differences of the studied animals.

Despite the fact that high BP and LVH has been identified as a powerful risk factor for cardiovascular morbidity and mortality, the mechanistic link between the 2 is still not completely elucidated. Various pharmacological drugs have distinct hypertensive and hypertrophic effects and hypertrophy can be a pressure-independent cardiovascular risk factor.34 The dissociation between the effects of these drugs on the reduction of BP and/or LVH is proposed to involve their rapid direct actions. Lacidipine is shown to prevent cardiac remodeling and organ damage and inhibit salt-induced cardiac hypertrophy even at doses that do not prevent the development of hypertension.35,36 At concentrations (0.3 mg/kg/day) that affect only minimally the high SBP, the drug lowers gene expression of several gene products, including cardiac α-actin, β-myosin heavy chain, type I collagen, and DHP-sensitive L-type calcium channel, all induced by salt loading in stroke-prone SHR.11 These observations support the concept that the positive effects of lacidipine are not simply related to its antihypertensive efficacy but may also be linked to its direct rapid actions.37,38 Similarly, chronic ACE blockage is suggested to exert inhibitory modulation on the peripheral adrenergic transmission, which is not related to the BP reduction. However, in the case of ACE, this modulation does not appear to be a determinant in preventing the development of cardiac hypertrophy, although it is proposed to play a role in the regression of vascular structural alterations in SHR.39 These findings indicate that despite the rapid decrease in the SBP induced by both drugs in the hours following the first dose, BP reduction may not be the sole mechanism involved in the prevention of cardiac remodeling by CCBs and ACE inhibitors.12 Various classes of different antihypertensive drugs may prevent the LVH by different cellular actions initiated shortly after drug application. Data presented in this article strengthen the view that 2 different cellular actions are implicated in responses to calcium antagonists and/or ACE inhibitors.

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Effects of Lacidipine and Enalapril on Cardiomyocyte Contractile Function

To assess the actions of the 2 studied drugs on contractility, the principal function of cardiomyocytes, we compared both their long-term in vivo and short-term in vitro effects. Our in vivo data revealed that regardless of the fact that both drugs had the ability to prevent the LVH associated with increased BP, lacidipine had more important effects on the LV cardiomyocyte contractility. Although enalapril, following 8-week treatment, failed to induce significant effect on shortening of cardiomyocytes, lacidipine significantly decreased single-cell contractility. Previously, decreased contractility of Purkinje fibers was observed after lacidipine application in sheep.40 The drug is known to act through reduction of the L-type calcium channel amplitude.26 In SHR, lacidipine is therefore likely to affect contractility, increased because of enhanced calcium cycling,25 by its effect on intracellular calcium. Our data support such a concept. Lack of enalapril action is in agreement with previously performed studies using shorter treatments, revealing that daily treatment with enalapril for 3 weeks does not change basal cardiac performance or inotropic response to isoprenaline in the Langendorff preparation.41 Interestingly, our data also suggest that the structural changes in single cells correlated with functional modifications, more precisely lowering of single-cell contractility. Enalapril seemed more likely to prevent increase in LV mass by different means, which could not have been clearly elucidated from our results.

Study of short-term in vitro effects showed a significant decrease in single-cell contractility in response to lacidipine. The drug inhibited cardiomyocyte contractility in a dose-dependent manner, with EC50 of 10−7 mol/L, which is comparable to EC60 for inhibition of the L-type calcium current amplitude, which was found to be ≈0.1 μmol/L.40 Lacidipine is known to have a high affinity for vascular muscle,3 but it also affects the cardiac muscle.26 As expected, this concentration is much higher than that active on vasculature,3 and the decrease in the contractility resulted from reduction of intracellular calcium transients that were also inhibited in a concentration-dependent manner with the same EC50. Despite the fact that regression of hypertrophy induced by enalapril was shown to be accompanied by a change in myosin expression,42 we observed small changes in cardiomyocyte contractility after short-term in vitro application of the ACE inhibitor.

Our data indicate that long-term preventive effects on structure/function remodeling in SHR were initiated shortly after application of lacidipine but not of enalapril. This result suggests that mechanisms underlying early contractility changes in individual myocytes are also responsible for the long-term effects on cardiac remodeling. To better understand this action, regional differences, described in SHR versus WKY rats,25 need to be taken into consideration in the future. Inhibition of calcium channels, capable of triggering signaling pathways leading to prevention of the cellular hypertrophy seems to play an important role in the regulation of the single-cell morphology. Nevertheless, obtained results support the hypothesis that specific molecular mechanism of blockade of Ca2+ channels in myocardium by lacidipine may play a decisive role in protecting against LVH and thereby against excessive morphological changes induced by high BP during progress and/or development of LVH.

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CONCLUSIONS

Presented data confirmed that both lacidipine and enalapril were able to protect against hypertension-induced LVH at initial stages of the hypertension in SHR. Lacidipine induced functional effects, initiated shortly after application via rapid decrease in single-cell contractility and attenuation of calcium transients, lead to the prevention of cardiac hypertrophy in SHR by affecting single cells. Enalapril, despite having the same effects on SBP and LVW/BW, had a much lesser effect on single-cell structure/function remodeling. These results suggest that by modifying single-cell contractility, lacidipine also affected single-cell morphology and thus LVH, whereas smaller effects of enalapril on contractility resulted in lesser action of the drug on single-cell hypertrophy. A comparison of actions of both drugs under the same experimental conditions is crucial for their appropriate therapeutic application at different stages of cardiac hypertrophy. Further investigation is necessary to determine precise combinatory effects of these 2 antihypertensive drugs.

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Keywords:

cardiac myocytes; contractility; morphology; calcium channel blockers; angiotensin-converting enzyme inhibitors

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