Mol Cell Biochem (2011) 358:215–220 DOI 10.1007/s11010-011-0937-8
Generation of hydrogen peroxide in the developing rat heart: the role of elastin metabolism Jirˇ´ı Wilhelm • Ivana Osˇt’a´dalova´ • Richard Vyta´sˇek Ludeˇk Vajner
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Received: 21 April 2011 / Accepted: 21 June 2011 / Published online: 19 July 2011 Ó Springer Science+Business Media, LLC. 2011
Abstract Reports describing production of reactive oxygen species in neonatal heart are missing. As lysyl oxidase is potentially important source of H2O2, we studied its role during ontogenic development of rat heart. H2O2 was detected in thin sections of developing rat heart by fluorescence microscopy with the use of fluorescence probe 20 -70 -dichlorofluorescin. The experimental design comprised foetuses 21 days after conception, and then the animals sampled on the 1st, 4th, 7th, 10th, 15th, 30th and 60th day after birth. We also used 7-month-old animals as an example of ageing effects. Since the day 4 on, H2O2 was produced only extracellularly up to the day 15, between days 30 and 60 intracellular production was detected as well, and in 7-month-old animals only extracellular production was observed. The specific inhibitors of lysyl oxidase almost completely quenched the H2O2-dependent fluorescence. Starting from day 7, blue autofluorescence specific to oxidized proteins developed in the vessel wall. Intracellular blue autofluorescence specific to autoxidation products developed after day 30. Chloroform extraction diminished the intracellular blue fluorescence, leaving the
J. Wilhelm (&) R. Vyta´sˇek Department of Medical Chemistry and Biochemistry, Centre of Cardiovascular Research, 2nd Medical School, Charles University, Plzenˇska´ 221, 150 00 Prague 5, Czech Republic e-mail:
[email protected] I. Osˇt’a´dalova´ Centre of Cardiovascular Research, Institute of Physiology, Academy of Sciences of the Czech Republic, Vı´denˇska´ 1083, 142 20 Prague 4, Czech Republic L. Vajner Department of Histology and Embryology, 2nd Medical School, Charles University, Plzenˇska´ 221, 150 00 Prague 5, Czech Republic
extracellular fluorescence intact. This confirmed the protein nature of the fluorophores. Lysyl oxidase is significant source of H2O2 in the heart vessel wall during development and H2O2 oxidatively modifies elastin producing protein blue autofluorescence. Keywords Rat heart Development Hydrogen peroxide Elastin Fluorescence
Introduction Cardiac development is very dramatic process, which represents series of different adaptations to increasing demands of growing organism. Perinatal stage is a period of extremely progressive development. All cardiomyocytes proliferate and number of myocytes increases [1]. The marked capillary growth and maturation of arteries proceeds in myocardium simultaneously [2]. The process of vasculogenesis occurs predominantly during embryonic development. These initial blood vessels consist purely of endothelial cells and are referred to as capillary plexus. The ultimate vessel structure is determined by the derivation of the endothelial cells and smooth muscle cells comprising the vessel wall [3]. After the proliferation, elongation and alignment of the endothelial cells follows the formation of capillary sprouts. The growing sprout eventually develops a lumen and consequently these tubular structures anastomose with neighbouring vessels. The resulting capillary loop then permits blood flow, and the formation of the basement membrane completes the maturation process [2, 4, 5]. Elastin is the dominant extracellular matrix protein deposited in the arterial wall and can contribute up to 50% of its dry weight. The protein product of the elastin gene is
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synthesized by fibroblasts and vascular smooth muscle cells and secreted as a tropoelastin monomer. In extracellular space, tropoelastin is crosslinked by the action of lysyl oxidase and organized into elastin polymers that form concentric rings of elastic lamellae around the medial layer of arteries. Elastin matrix is also a potent autocrine factor that regulates arterial morphogenesis by instructing vascular smooth muscle cells to localize around the elastic fibres and remain in a quiescent, contractile state [6]. Lysyl oxidase thus plays a crucial role in the maintenance of extracellular matrix stability and is involved in vessel wall remodelling. It is also chemotactic for vascular smooth muscle cells and myocytes. Besides that, it generates hydrogen peroxide during its action [7]. In most mammalian tissues, the bulk of elastogenesis occurs during perinatal period. As tissue development and growth are completed, elastin production is turned off [8]. However, this was observed in the lungs. There are no available studies of the situation in the heart. The end products of free radical attack, the so-called lipofuscin-like pigments (LFP), have been used as the marker of oxidative damage in several studies, e.g. [9]. They fluoresce in blue spectral region and are extractable to chloroform. Blue-fluorescing proteins also originate from free radical damage [10, 11]. The information concerning production of reactive oxygen species (ROS) and lysyl oxidase activity in the heart is scarce. Reports describing cardiac ROS production during ontogeny with a special consideration to perinatal stage are completely missing up to date. Therefore, we decided to study ROS generation related to lysyl oxidase activity and its sequelae during ontogenic development of rat heart.
sampling proceeded as in our previous study [12]. Briefly, the animals were euthanized by decapitation in ether narcosis, the chest was quickly opened and the heart was excised and frozen in liquid nitrogen. The heart was cut at -20°C using the cryomicrotome Minotome (Damon, Needham, MA) as 6-lm thin sections. The non-fixed sections were used for the experiments.
Methods
Fluorescence microscopy
This study was conducted in accordance with 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).
The slides with heart thin sections were assessed by the Nikon Eclipse E-400 microscope in the bright field and by epifluorescence (filter UV-2A—excitation 330–380 nm, barrier 420 nm; filter B-2A—excitation 450–490 nm, barrier 520 nm; filter G-2A—excitation 510–560 nm, barrier 590 nm). Microphotographs were taken by the Nikon DSU1 digital camera using the NIS Elements AR software (Laboratory Imaging, Prague, Czech Republic). The extraction of autofluorescent fluorophores was done by rinsing the slides three times with 2 ml of chloroform– methanol mixture (2:1, v/v) as in our previous study [13]. Then the slides were washed with PBS and mounted. The photographs illustrating specific effects in the figures were chosen as the best examples of given series of slices. The same development was observed in all of the slices of each particular age group.
Animals and sampling A total of eight pregnant female Wistar rats were used throughout the experiments. They had free access to water and standard laboratory diet. Four male offsprings were used per experimental group. Thin sections of hearts from male Wistar rats were used for the study. We took whole hearts from animals on the 21st day after conception, and then on the 1st, 4th, 7th, 10th, 15th, 30th and 60th day after birth. We also used 7-month-old animals as an example of ageing effects. The
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Staining with fluorescent probe For the detection of hydrogen peroxide, we used 20 -70 dichlorofluorescin (DCFH) under conditions specified in our previous work [13]. In brief, the stock solution of DCFH was made by dissolution of 5 mg of the dye in 1 ml of ethanol, for staining it was diluted to 10 lg/ml in phosphate-buffered saline, pH 7.4 (PBS). Slides with frozen heart sections were washed in PBS and incubated with DCFH for 10 min, or left without any treatment for autofluorescence detection. Then they were mounted into buffered glycerol under a coverslip and sealed with a glue. As we did not observe any difference in staining of left or right ventricle, we examined several serial sections of left ventricle. Inhibition of lysyl oxidase The inhibition of lysyl oxidase was studied using copper chelator 2,20 -bipyridyl [14]. Thin section of the tissue on the slide was incubated for 1 h with 1.2 M urea and 50 mM 2,20 -bipyridyl at 37°C. Then it was rinsed with PBS and stained with DCFH as above. Another studied inhibitor was b-aminopropionitrile (BAPN) [15]. In this case, 10 lM BAPN in PBS was applied on the top of the heart section and incubated for 1 h at 37°C, rinsed with PBS and stained with DCFH.
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Discernible endothelial layer and elastin deposits in the internal elastic lamina stained by orcein were observed on the 4th day after birth (Fig. 1a). In the prenatal animals and on the first day after birth, no elastin was detected by this method. The appearance of elastin is accompanied by strong fluorescence of oxidized DHCF which indicates generation of hydrogen peroxide (Fig. 1b). There was no observable production of hydrogen peroxide outside the vessel wall. The prenatal rat heart, just before birth, is characterized by developed endothelial tubes that contain deposits of collagen I that can be visualized by van Gieson staining
(not shown, because not discernible in a greyscale). When the sensitivity of the camera was increased by factor of 10, faint diffuse fluorescence of oxidized DHCF colocalized with collagen staining (Fig. 1c). It suggests lower concentration of hydrogen peroxide generated during collagen processing. In the next step of the study, the surprisingly high production of hydrogen peroxide by vessel wall was investigated during further development. Measurements on the 4th, 7th, 10th and 15th day of age showed that hydrogen peroxide was produced only in the vessel wall. On the 30th day, hydrogen peroxide production was observed also in myocytes and endothelial cells (Fig. 2a). On the 60th day, the production of hydrogen peroxide outside vessel wall decreased (Fig. 2b), and in 7-monthold animals only vessel wall was fluorescing, again (Fig. 2c).
Fig. 1 Elastin stained with orcein in 4-day-old animals (a), DCF fluorescence in 4-day-old animals (b), and DCF fluorescence in prenatal animals (c). The sensitivity of the camera in panel c was tenfold higher than in panel b
Fig. 2 DCF fluorescence in 30-day-old animals (a), 60-day-old animals (b), and 7-month-old animals (c)
Results Detection of elastin and hydrogen peroxide in heart vessel wall
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Inhibition of lysyl oxidase The possible source of hydrogen peroxide is lysyl oxidase that crosslinks elastin in the extracellular space. We therefore tested the effects of known inhibitors of lysyl oxidase on DCF fluorescence. As lysyl oxidase contains copper, copper chelator 2,20 -bipyridyl inhibits its activity. Figure 3a shows damped fluorescence of sample from 7-month-old rat treated with 2,20 -bipyridyl. Specific inhibitor of lysyl oxidase, b-aminopropionitrile, caused almost complete inhibition of DCF fluorescence (Fig. 3b). Compare the uninhibited light intensity in Fig. 2c.
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associated only with vessel wall. From that time on autofluorescence is observable also in extravascular cells as the result of ageing process. Extraction of blue fluorophores to chloroform In 7-month-old rats, chloroform extraction leaves the autofluorescence of large vessels intact, while the extravascular autofluorescence is washed away (Fig. 5a). In the case of a smaller vessel surrounded by autofluorescent cells (Fig. 5b), chloroform extraction decreases fluorescence even in the vessel wall (Fig. 5c).
Detection of blue autofluorescence Discussion Hydrogen peroxide is a strong oxidant and its presence could lead to protein oxidation. From this point of view, it is interesting that cell wall elastin develops blue autofluorescence during heart development. First faint signs of autofluorescence are observable in 7-day-old rats. This fluorescence is at the edge of visibility, therefore no picture is shown. On day 15, it is clearly discernible (Fig. 4a), on day 60, the intensity of autofluorescence reaches its maximum (Fig. 4b) and in 7-month-old rats it is still highly pronounced (Fig. 4c). Up to 60th day of age, the autofluorescence is
Fig. 3 Inhibition of lysyl oxidase in 7-month-old animals with 2,20 bipyridyl (a) and with BAPN (b)
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The oxidation of 20 ,70 -dichlorofluorescin (DCFH) to fluorescent dichlorofluorescein (DCF) was originally used for the detection of hydrogen peroxide [16]. In the recent
Fig. 4 Blue autofluorescence of heart sections. a 15-day-old animals, b 60-day-old animals, and c 7-month-old animals
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Fig. 5 Autofluorescence of 7-months old animals. a After extraction with chloroform (compare to Fig. 4c), b autofluorescence of a section with a small vessel, c the same area as in b, after extraction with chloroform
study, we demonstrated its applicability to heart thin sections [13]. In the present study, we have observed strong fluorescence due to oxidation of DHCF that was associated with development of internal elastic lamina of the vessel wall in the heart of 4-day-old rats. During investigation of prenatal heart (21st day after conception) and 1st day after parturition, no elastin was detected by orcein staining, and only minute amounts of hydrogen peroxide were produced. DCF fluorescence was associated with staining for elastin throughout the studied period of development, up to 7 months of age. It indicates that lysyl oxidase is active all the time, while other reports stated that the bulk of elastogenesis occurs during late foetal and early neonatal periods and except in response to injury or disease, no new elastin is made over the lifetime [8]. However, these studies were undertaken in the lungs and our result suggests an organ specificity of elastogenesis.
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Lysyl oxidase is involved also in crosslinking of collagen, so its activity in ageing animals could be explained in this way. In early perinatal period when collagen I deposition was detected by van Gieson staining and no elastin was present, DCF fluorescence was very weak. It appears that elastin fluorescence overshines that of collagen due to high concentration of elastin in the arterial vessel wall. Besides that, collagen is more dispersed throughout the extracellular space. Thus, the camera adjusted for the high intensity of elastin fluorescence does not pick up the light produced by collagen crosslinking. No signs of intracellular generation of hydrogen peroxide were detected up to postnatal day 30. Between day 30 and day 60, fluorescence originating from endothelial cells and cardiomyocytes was observed as well, however, it was much fainter on day 60, and 7-month-old hearts generated fluorescence only from vessel wall again. In this regard, it is important to note that rats are around the day 30 at the end of weaning period and that time is characterized by very intensive metabolism [17]. On day 60, development is complete and animals are sexually matured. Elastin matures in the extracellular space where lysyl oxidase crosslinks tropoelastin and during this process hydrogen peroxide is generated [7]. It was found that 50 mM 2,20 -bipyridyl (copper chelator) in the presence of 1.2 M urea produced about 80% inhibition of isolated lysyl oxidase [14] and upon incubation of isolated lysyl oxidase with 5 lM b-aminopropionitrile about 95% inhibition was achieved [15]. Application of these inhibitors in the thin sections of 7-month-old rat heart almost completely quenched the fluorescence produced by DCF which strongly supports the idea that hydrogen peroxide is produced predominantly by lysyl oxidase. It seems that lysyl oxidase plays a new role not only in cardiovascular diseases [7], but also in normal physiology. Exposure of proteins to free radical producing systems induces appearance of blue autofluorescence [10, 11]. In a previous study, we have observed blue fluorescence in isolated collagen I exposed to UV irradiation in vitro [18]. By use of confocal microscopy, elastin autofluorescence was found in mesenteric arteries of 10-day-old rats. The intensity of fluorescence was much higher in 1-month-old and 6-month-old animals [19]. We have observed first signs of autofluorescence of elastic lamina in 7-day-old heart, albeit very faint. On day 15, the autofluorescence was bright enough to be photographed, and the fluorescence intensity reached maximum on day 60. In 7-month-old animals, it was still highly pronounced. From day 60 on there is also observable blue autofluorescence originating from the cells. The intensity of this cellular autofluorescence increases with age and is higher
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in 7-month-old animals. The cellular autofluorescence is produced by LFP, the end products of lipid peroxidation [9]. These compounds are extractable to chloroform and after extensive washing of tissue thin sections with chloroform–methanol they cease to fluoresce. On the contrary, chloroform extraction does not influence protein autofluorescence in large vessels where evidently elastin was the fluorescing species. It is apparent that blue autofluorescence in small vessels is generated predominantly by oxidized phospholipids as it is sensitive to chloroform extraction. As the blue fluorescence of internal lamina gradually increases during heart development concomitantly with generation of hydrogen peroxide, we suggest that it originates from oxidative modification of elastin produced by hydrogen peroxide generated by lysyl oxidase. The very development of the vessel wall thus produces oxidative changes to its major component. Acknowledgments The study was supported by Grant Agency of Czech Republic, grant no. P303/11/0298.
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