Morphometric, quantitative, and threedimensional

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Nov 24, 2012 - Ultrastruct Pathol 26:177–183. Torrent-Guasp F, Kocica MJ, Corno AF, Komeda M, Carreras-Costa F,. Flotats A, Cosin-Aguillar J, Wen H. 2005.
MICROSCOPY RESEARCH AND TECHNIQUE 76:184–195 (2013)

Morphometric, Quantitative, and Three-Dimensional Analysis of the Heart Muscle Fibers of Old Rats: Transmission Electron Microscopy and High-Resolution Scanning Electron Microscopy Methods ´ DIAS,2 MARCIA CONSENTINO KRONKA SOSTHENES,3 DIEGO PULZATTO CURY,1 FERNANDO JOSE CARLOS ALEXANDRE DOS SANTOS HAEMMERLE,2 KOICHI OGAWA,4 MARCELO CAVENAGHI PEREIRA DA ˜ O PAULO MARDEGAN ISSA,6 MAMIE MIZUSAKI IYOMASA,6 AND II-SEI WATANABE2* SILVA,5 JOA 1

Department of Surgery, Faculty of Veterinary Medicine and Animal Science, University of Sa˜o Paulo, Brazil Department of Anatomy, Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil Institute of Biological Sciences, Lab. of Investigations in Neurodegeneration and Infection, University Hospital ‘‘Joa˜o de Barros Barreto,’’ Federal University of Para´, Bele´m, Brazil 4 Department of Anatomy, School of Medicine, Fukuoka University, Fukuoka, Japan 5 Department of Morphology and Genetics, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil 6 Department of Morphology, Stomatology, and Physiology, Faculty of Dentistry, University of Sa˜o Paulo, Ribeira˜o Preto, Brazil 2 3

KEY WORDS

heart; old rats; TEM; HRSEM; mitochondria

ABSTRACT This research investigated the morphological, morphometric, and ultrastructural cardiomyocyte characteristics of male Wistar rats at 18 months of age. The animals were euthanized using an overdose of anesthesia (ketamine and xylazine, 150/10 mg/kg) and perfused transcardially, after which samples were collected for light microscopy, transmission electron microscopy, and highresolution scanning electron microscopy. The results showed that cardiomyocyte arrangement was disposed parallel between the mitochondria and the A-, I-, and H-bands and their M- and Z-lines from the sarcomere. The sarcomere junction areas had intercalated disks, a specific structure of heart muscle. The ultrastructural analysis revealed several mitochondria of various sizes and shapes intermingled between the blood capillaries and their endothelial cells; some red cells inside vessels are noted. The muscle cell sarcolemma could be observed associated with the described structures. The cardiomyocytes of old rats presented an average sarcomere length of 2.071 6 0.09 lm, a mitochondrial volume density (Vv) of 0.3383, a mitochondrial average area of 0.537 6 0.278 lm2, a mitochondrial average length of 1.024 6 0.352 lm, an average mitochondrial cristae thickness of 0.038 6 0.09 lm and a ratio of mitochondrial greater length/lesser length of 1.929 6 0.965. Of the observed mitochondrial shapes, 23.4% were rounded, 45.3% were elongated, and 31.1% had irregular profiles. In this study, we analyzed the morphology and morphometry of cardiomyocytes in old rats, focusing on mitochondria. These data are important for researchers who focus the changes in cardiac tissue, especially changes owing to pathologies and drug administration that may or may not be correlated with aging. Microsc. Res. Tech. 76:184–195, 2013. V 2012 Wiley Periodicals, Inc. C

INTRODUCTION The heart is considered as the main muscle of the circulatory system, presenting a complex organization of cardiomyocytes that accounts for approximately 75– 80% of the total myocardial volume (Bell and Yellon, 2012). The nature of these fibers has been known for a long time and was first reported by William Harvey in 1628 in a manuscript entitled Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus (Leake, 1930). The architecture of cardiomyocytes revealed a reinforced organization of cardiac myofibers (LeGrice et al., 1995). The walls of the heart are characterized by gradual changes of their fiber angles, with the axis rotating approximately 1208 from epicardium to endocardium. The fibers are situated in a clockwise arrangement on the surface of the epicardium, whereas in the endocardium, they can be observed in a counterclockwise disposition (Hooks et al., 2007; Lombaert et al., 2012; Streeter et al., 1969). C V

2012 WILEY PERIODICALS, INC.

Several organelles can be detected in cardiac cells, and the presence of mitochondria corroborated the hypothesis for the common origin of all eukaryotic cells (Martin and Muller, 1998). Two hypotheses for the origin of eukaryotic cells have been proposed. In the first one, the simultaneous creation of eukaryotic nuclei and mitochondria occurred by required hydrogen fusing. Methanogenic Archaebacteria, hosting a-Proteobacteria via symbiosis, produced the necessary hydrogen for metabolism and thus gave rise to a eukaryotic cell possessing a *Correspondence to: Ii-sei Watanabe, Department of Anatomy, Institute of Biomedical Sciences, Av Prof. Lineu Prestes, 2415 – Ed. Biome´dicas III, University of Sa˜o Paulo, Sa˜o Paulo 05508-900, Brazil. E-mail: [email protected] Received 11 October 2012; accepted in revised form 25 October 2012 Contract grant sponsors: FAPESP, The Faculty of Medicine, Fukuoka University DOI 10.1002/jemt.22151 Published online 24 November 2012 in Wiley Online Library (wileyonlinelibrary.com).

TEM, HRSEM AND MORPHOMETRIC STUDY IN OLD RATS

mitochondrion. The second hypothesis involves an initial eukaryotic cell that did not contain mitochondria, which is associated with a fusion of an Archaebacteria and a Proteobacteria, followed by acquisition of the organelle via endosymbiosis with an a-Proteobacteria (Gray et al., 1999). DNA mitochondrial analysis (mtDNA) demonstrated an important eubacterial correlation among mitochondria, strengthening the cited hypotheses (Gray et al., 1999; Oka et al., 2012). Each mitochondrion contains multiples copies of mtDNA (Loeb et al., 2005). The mitochondria are enclosed in two functionally distinct populations: subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) (Palmer et al., 1977). The SSM are located beneath the plasma membrane, and the IFM are located among the myofibrils of cardiomyocytes (Lemieux et al., 2010). These organelles are the main sources of cellular adenosine triphosphate, which is related to the reactions of hydrocarbons with oxygen, and they play a central role in a variety of cellular processes (Kujoth et al., 2005; Loeb et al., 2005). The aging process leads to a progressive decline in cellular functions and often manifests as a loss of muscle strength and cardiovascular activities (Dillon et al., 2012; Lemieux et al., 2010). According to this context, deprivation of mitochondrial function is a major factor in aging (Loeb et al., 2005). The aim of our study was to describe and analyze the morphology and morphometry of cardiac mitochondria of old rats using light microscopy and ultrastructural approach with transmission and scanning electron microscopes. These data may serve as a basis for further studies and help in understanding the changes in these organelles owing to aging process.

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slices 5 lm thick. Next, the hematoxylin–eosin and picrosirius red staining methods were applied for the examination under polarized light (Nikon Eclipse E1000, 1 Software Image-Pro Plus 4.5). Transmission Electron Microscopy The hearts of the aged rats were removed after perfusion with a modified Karnovsky solution containing 2.5% glutaraldehyde and 2% formalin in 0.1 M sodium phosphate buffer at pH 7.4 (Watanabe and Yamada, 1983). The tissue was rinsed in phosphate buffer solution for 15 min at 48C. Postfixation occurred in 1% osmium tetroxide in phosphate buffer solution (0.1 M) at 48C for 2 h. The tissues were dehydrated in a graded1 series of alcohol (70–100%) and embedded in Spurr resin. Thick sections were obtained using glass knives 1 in a Reichert Ultra Cut microtome and the ultrathin sections (thickness, 90 hm) were mounted on copper grids (200-mesh grid). The grids were counterstained with 4% uranyl acetate and 0.4% lead citrate solutions (Watanabe and Yamada, 1983) and examined in a Jeol JEM 1010 transmission electron microscope at 80 kV at the Institute of Biomedical Sciences, University of Sa˜o Paulo.

MATERIALS AND METHODS Experimental Groups In this study, six male Wistar rats (Rattus norvegicus) at 24 months of age were divided into two groups: three animals were prepared for light microscopy and three were prepared for scanning and transmission electron microscopy. Each group was maintained in plastic standard cages at the Institute of Biomedical Sciences (University of Sa˜o Paulo). The subjects had free access to food and water and were raised under a controlled room temperature (23 6 18C) and a light– dark cycle of 12 h. The experiment adhered to the Ethical Principles of Animal Experimentation adopted by the Brazilian School of Animal Experimentation (COBEA) and was previously submitted to the Ethics Committee on Using Animals for Experimentation (EAEC) at the Faculty of Veterinary Medicine and Animal Science of University of Sa˜o Paulo (no. 2536/2012). The experimental procedures were approved in full.

High-Resolution Scanning Electron Microscopy Animals were anesthetized using the same conditions, perfused, and fixed with a 2% osmium tetroxide solution in a 1/15 M sodium phosphate buffer (pH 7.4) at 48C for 2 h. Small samples were cut and treated according to the method described by Watanabe et al. (1992). The samples were rinsed in distilled water overnight and immersed successively in 12.5, 25, and 50% dimethylsulfoxide solution for 130 min each (Pı´coLi 1 et al., 2011). Using an Eiko TF-2 apparatus (Hitachi , Japan), the blocks were freeze-fractured and frozen with liquid nitrogen. The subjects were placed in a 50% dimethylsulfoxide solution and rinsed in distilled water. The following sequence was performed with the samples: maceration was performed in 0.1% osmium tetroxide solution for 24–48 h at 228C (Watanabe et al., 1992), rinsed in distilled water, postfixed in 2% buffered osmium tetroxide solution for 2 h at 48C, rinsed again, and immersed in a 2% tannic acid solution for 1 h at room temperature. The hearts were dehydrated in an increasing series of ethanol and tert-butyl alcohol 1 1 and dried in an Eiko ID-2 apparatus (Hitachi , Japan). Carbon paste was used to mount the samples on the gold plate and the samples were coated with plati1 num using an anion beam sputter (Hitachi VA 10S, Japan) (Tanaka, 1981, 1989). The samples were examined with high-resolution scanning electron microscopy 1 (HRSEM, Hitachi S-900, Japan) at 5 or 10 kV at the Department of Anatomy, School of Medicine, Fukuoka University, Fukuoka, Japan.

Histological Procedures All the subjects were euthanized with an overdose of ketamine (150 mg/kg) and xylazine (10 mg/kg) (Dias et al., 2012). The subjects were then perfused transcardially with heparinized saline for 10 min, followed by an aldehyde fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2–7.4) for 30 min. Subsequently, the samples were dehydrated in a graded alcohol series (70–100%), embedded in paraffin, and cut in

Morphometric and Quantitative Analysis In this study, the measurements and quantification of old rat cardiac muscles were obtained by applying images obtained by scanning and transmission electron microscopy, focusing on the mitochondria in the prepared tissues. The images were analyzed with ImageJ (Colmanetti et al., 2005). For the SEM images, the mitochondrial shapes (rounded, elongated, and irregular) were counted and classified, beyond the measurement

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Fig. 1. Light microscopy. A: Bundles of muscle fibers with the cores of the myocardial cells (larger arrows) and capillaries (small arrows). Stain: hematoxylin and eosin. Bar: 50 lm. B: Arrangement of bundles of cardiac muscle fibers (*) tissue of the endomysium in blue (arrowheads) and capillaries (small arrows). Stain: Azo-Carmin. Bar: 100 lm. C: General appearance of the muscular layer, revealing the arrange-

ment of cardiac muscle fibers (*) capillaries (small arrows), and collagen fibers of the endomysium (arrowhead). Stain: Picro-Sirius. Bar: 200 lm. D: Appearance of the previous fiber taking by polarized light. Evidence reddish (type I collagen) and green (type III collagen). Satin: Picro-Sirius (polarized light). Bar: 200 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

of mitochondrial cristae. For the transmission electron microscopy, the recorded parameters were the length of the sarcomere, the major and minor diameters of the mitochondria, and the mitochondrial volume density and area, which corresponded to a quantification of mitochondria that could be detected in an area by testsystem. The results are presented as averages with standard deviations and boxplot graphs to show the dispersion of the data.

tinctions could be made between type I (shades of red) and type III (shades of green). The amount and distribution of both collagen types had normal characteristics; thus, type I collagen appeared in greater quantity and location compared with type III collagen (Figs. 1C and 1D).

RESULTS Histological Morphology of Cardiac Tissue The histological analysis of old rat hearts showed a parallel arrangement between the muscle fibers and the nuclei of muscle cells in the central area of the sarcoplasm. Neither transverse striations nor branches, which are common characteristics of cardiac muscles, were evident (Figs. 1A and 1B). In the endocardium and the pericardium, it was possible to observe connective tissue, collagen fibers, and a number of small and large caliber blood vessels. Using polarized light microscopy, collagen fibers in the endocardium and the pericardium were observed, and dis-

High-Resolution Scanning Electron Microscopy High-resolution scanning electron microscopy provided a three-dimensional approach to examine the parallel configuration of the myofilament fibers, their intersections, and the arrangement of mitochondria in columns or in groups (Figs. 2A, 2C, and 2D–2F). It was evident from the images that varied mitochondrial formats existed: round (in which the major diameter was equal or close to the minor diameter), elongated (in which the major diameter was larger than the other), and irregular (in which the mitochondria showed no definite shape) (Fig. 2B). Samples that were freeze-cracked in the transverse direction revealed bundles of myofilaments and mitochondrial cristae inside the mitochondria (Figs. 3A–3D). Microscopy Research and Technique

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Fig. 2. High-resolution scanning electron microscopy. A: Bundles of myofilaments (*), fractured mitochondria (arrows), not fractured mitochondria (arrowhead). Bar: 3 lm. B: Rounded mitochondria (thin arrow), elongated mitochondria (large arrow) and irregular mitochon-

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dria (arrowhead) Bar: 3 lm. C: Mitochondria (M). Bar: 2 lm. D: Myofilaments beams (*), the intersection of the beams myofilaments (arrow), mitochondria (M). Bar: 2 lm. E: Myofilaments beams (*), mitochondria (M). Bar: 1 lm. F: Mitochondria (M). Bar: 1 lm.

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Fig. 3. HRSEM using cryofracture technique. A: Bundles of myofilaments (*), mitochondria (M). Bar: 1 lm. B: Bundles of myofilaments. Bar: 0.5 lm. C: Mitochondria (M). Bar: 1 lm. D: Mitochondrial cristae (arrows). Bar: 0.5 lm.

Transmission Electron Microscopy Analysis performed using transmission electron microscopy exposed a parallel distribution of the cardiomyocytes, SSM, and IFM, in addition to a basal lamina continuous with the external surface of the capillary endothelial cell (Fig. 4A). The particular branching of cardiac muscle fibers was observed, and the transmission electron microscopy (TEM) method revealed sarcomeres with their A-, I-, and H-bands, M- and Z-lines, and amyelinic nerve fibers (Fig. 4B). It was noted that a large number of mitochondria, in varied sizes and shapes, were arranged between the cardiomyocytes and inside the sarcolemma (Fig. 4C). In a longitudinal approach, endothelial cells, sarcolemma and endothelial cell basement membranes, caveolae and vacuoles were visualized (Fig. 4D).

A broad view of the myocardium revealed a large number of mitochondria and capillaries with their nuclei and endothelial cells surrounded by a basement membrane, and muscle cell sarcolemmas were also evident (Fig. 5A). At high magnification, we observed the details of the nuclei of capillary endothelial cells, highlighting their lining basement membranes (Fig. 5B). Some erythrocytes were noted inside the capillaries, as were myofibrils sprouting out (Fig. 5C). Figures 5D, 6A, and 6B show vesicles, endothelial centrioles, and intercalated disks, the last of which are located at the interface of adjacent cells, are related to cell junctions and are identified between the Z-lines of the I-band. The connective tissue in the muscle cell basement membrane and the mitochondrial architecture in the columns were visualized (Fig. 6B). Microscopy Research and Technique

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Fig. 4. TEM. A: SSM (arrowhead), interfibrilar mitochondria (greater arrow), continuous basal lamina on the exterior surface of the endothelial cell capillary (lower arrow). Bar: 2 lm. B: Cardiomyocytes branch (arrow), bands A (A), I (I), H (H), M line (arrowhead), Z line (thin arrow) of sarcomeres, cardiomyocytes (*), unmyelinated

nerve fibers (n). Bar: 2 lm. C: Myofilaments (MF), rounded mitochondria (R), elongated (E), irregular (I). Bar: 1 lm. D: Junction of endothelial cells (arrow largest), Caveolae endothelial basement membrane (thin arrow), Caveolae sarcolemma (arrowhead). Bar: 1 lm.

A cardiac muscle cell nucleus inside the sarcolemma was demonstrated (Fig. 7A), and it was possible to visualize the intercellular space with collagen fibers and endothelial cells emitting pinocytosis vesicles (Fig. 7B). Included in the description obtained by TEM analysis, we observed a capillary cytoplasmic protrusion of endothelial cell and cardiomyocyte intercellular junctions in blood; an erythrocyte occupying almost the entire vessel lumen is shown (Fig. 7C).

Quantitative and Morphometric Analysis Length of Sarcomeres, Mitochondrial Volume Density, Mitochondrial Area and Length, Thickness of the Mitochondrial Cristae, and Classification and Analysis of the Mitochondrial Profile. The measurements and quantifications were evaluated for the cardiac tissue of old rats; the following descriptions, values, and graphic representations are of quantitative data.

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Fig. 5. TEM. A: Capillaries (*), endothelial cell nucleus (N). Bar: 4 lm. B: SSM (large arrow), IFM, nucleus of endothelial cells of the capillary (N), basal lamina of the capillary coating (thin arrow), basal lamina of the muscle cell (arrowhead). Bar: 2 lm. C: Erythrocyte (H), capillaries (*), nuclei (N.) Bar: 4 lm. D: Vesicle (large arrow), centriole (arrowhead), intermediate disk (thin arrows), IFM. Bar: 200 nm.

The average length of the sarcomeres of old rat hearts was 2.071 6 0.09 lm with a median value of 2.064 lm. The sarcomere length varied from 1.84 to 2.31 lm, with half of the data values between 2.015 and 2.13 lm. The dispersion of data from this measurement is shown in graphic 1 (Fig. 8A). Related to the mitochondria of cardiac muscle measurements, the mitochondrial volume density (Vv) of the old rat cardiomyocytes was 0.338. The mitochondrial area was a mean of 0.537 6 0.278 lm2 and the

median area was 0.491 lm2. The dispersion of the related data of the area is shown in graphic 2 (Fig. 8B). We observed that mitochondrial areas ranged from 0.039 to 0.938 lm2, and half of the values were identified between 0.318 and 0.583 lm2. For mitochondrial area measurements, outlier samples were identified with values up to 1.462 lm2. The mean of the major mitochondrial diameter present in the aged rat cardiac muscle was 1.024 6 0.352 lm, and the median major diameter was 0.981 lm. A statistical dispersion of that Microscopy Research and Technique

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Fig. 6. TEM. A: IFM, intermediate disk (arrow). Bar: 500 nm. B: Connective tissue (CT), sarcolemma (small arrows), and intermediate disk (arrow higher). Bar: 1 lm.

Fig. 7. TEM. A: Muscle cell nucleus (N), erythrocyte (H), and endothelial cells (arrows). Bar: 1 lm. B: Collagen fibers (C), endothelial cell nucleus (N), pinocytosis vesicles (arrows), and SSM. Bar: 400 nm. C: Cytoplasmic protrusion of the endothelial cell (arrow), intercellular junction (arrowhead), and erythrocyte (H). Bar: 1 lm.

parameter is shown in graphic 3 (Fig. 8C). The variable oscillated from 0.324 to 1.759 lm, with half of the values between 0.774 and 1.203 lm. The outliers were found with corresponding values of 1.873 and 2.124 lm. The mitochondrial cristae had a mean thickness of 0.038 6 0.09 lm and a median thickness of 0.038 lm. The data dispersion related to the thickness of the mitochondrial cristae is shown in graphic 4 (Fig. 8D). The thickness of the mitochondrial cristae ranged from 0.014 to 0.062 lm, and half of the values were between 0.032 and 0.044 lm. Microscopy Research and Technique

The ratio of the mitochondrial major diameter/minor diameter revealed a mean of 1.929 6 0.965 and the median of this ratio was 1.701 (dispersion in graphic 5, Fig. 8E). The ratio had values that ranged from 1.006 and 3.278, and half of the values were close to the median (1.333–2.145). Moreover, outliers (3.8–6.99) were detected. This information allowed for the classification of the mitochondrial shape: the lowest value of this ratio (1) represents round mitochondria, and the highest values represent mitochondria with elongated and irregular profiles. For further analysis of the mitochondrial profile, including the measurements of the major

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Fig. 8. Graphs of quantitative morphometric analysis of cardiac muscle fibers of aged rats. A: Boxplot graph of sarcomere length of cardiac muscle fibers data dispersion. B: Boxplot graph of mitochondrial area of heart muscle fibers data dispersion. C: Boxplot graph of

mitochondrion length of cardiac fibers data dispersion. D: Boxplot graph of the thickness of the mitochondrial cristae of heart muscle fibers data dispersion. E: Boxplot graph of the ratio major/minor diameter of the mitochondria of cardiac fibers data dispersion.

and minor diameters, mitochondria were classified (rounded, elongated, and irregular) and quantified. It was observed that 23.4% of the mitochondria had a round shape, 45.3% were elongated, and 31.1% had irregular profiles.

of cardiomyocytes. In another related study (Bennett, 2012), this union is intermediated by intercalated disks, which allows both structural and functional coordination. These results suggest that a remarkable feature of the transition from myofibril to intercalated disk is that sarcomere structure is maintained in an orderly manner all the way to the intercalated disk edge. In this study, we demonstrated that the IFM mitochondrial organization was situated among myofibrils in longitudinal columns with varying shapes and sizes. According to Birkedal and others (2006), IFM mitochondria are very well organized and sorted as strands in parallel; this mitochondrial arrangement, especially in rat cardiomyocytes, is linked to the disposal of global cellular and myofibrillar structure. The SSM mitochondria were observed just beneath the sarcolemma in a lesser amount than the IFM organelles. Riva et al. (2005) confirmed our findings, indicating that this type of mitochondrion relies directly on the sarcolemma. Adhihetty et al. (2005) explain that SSM organelles are more unstable than IFM organelles, which demonstrated the largest adaptive changes during chronic conditions. Regarding myofibrils and mitochondria, our study revealed a large amount in the myocardium compared to other findings (Forbes et al., 1990). The three major

DISCUSSION Our present data were demonstrated, applying electron microscopy techniques, the cardiomyocyte arrangement, observing the disposition in parallel between the mitochondria. Bursac et al. (2002) explained that cardiac cells show anisotropy in microscopic evaluations, which is a result of one spatial alignment and elongation and/or of preferential localization of intercellular junctions. The macroscopic anisotropic aspect is a consequence of the composition of cardiac muscle bundles and fibers that rotate and change from one form to another inside the heart wall. Added to the microscopic appearance, researchers (Lombaert et al., 2012) have affirmed that cardiac fibers are present in a laminar organization. In fact, a model suggested by Torrent-Guasp et al. (2005) hypothesizes the existence of a single muscle band folding on itself to form the whole wall heart. Through TEM analysis, we observed intercalated disks located among the Z-lines of the I-band, the specific structure of cardiac muscle related to the joining

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TEM, HRSEM AND MORPHOMETRIC STUDY IN OLD RATS

structures observed in cardiac tissue are myofibrils, mitochondria, and sarcoplasmic reticulum. Numerous capillaries were observed throughout the cardiac tissue, and it was also observed that endothelial cells adhered to its continuous basal lamina. These observations are corroborated by recent research (von Gise and Pu, 2012), which demonstrated that epithelial cells normally lie in the basement lamina and that the cell–cell and cell–basement lamina interactions coordinate and stabilize the epithelial architecture. We performed a morphometric and quantitative analysis of old rat cardiac tissue, with a focus on cardiomyocytes that are present in the right atrium and of mitochondria situated in the area. The average length of the sarcomeres was measured from cardiomyocytes. The volume density (Vv), average area, thickness of mitochondrial cristae, greater length, and ratio of mitochondrial greater length/lesser length were measured from mitochondria. These data, with the quantification of the mitochondrial types according to profile (rounded, elongated, and irregular), served to characterize the distribution of the organelle format in elderly rat heart tissue. Except for the volume density and the quantification of mitochondrial form types, almost all the quantitative morphometric data are presented as a standard deviation associated with a graphic type boxplot, which represents the dispersion of the measurement values. Typically, these dispersions of values are not represented in a boxplot graphical form (Barth et al., 1992; Bergman et al., 2003; Birkedal et al., 2006; Colmanetti et al., 2005; Corsetti et al., 2008; Kimpara et al., 1997; Knaapen et al., 1997; Li et al., 2011; Maharaj et al., 1993), but they are typically presented as a deviation value (Colmanetti et al., 2005; Corsetti et al., 2008; Kimpara et al., 1997; Knaapen et al., 1997) or a sample standard error (Barth et al., 1992). Using a boxplot chart to display the dispersion indicates very discrepant values (outliers) that, while often present in the samples, do not interfere with the median. However, the interpretation of these outliers is more complex, depending on the type of analysis. The rat cardiomyocytes are approximately 20 lm in diameter, and electron microscopy images show several lines of myofibrils. These myofibrils are exchanged with the lines of mitochondria and demonstrate that this mitochondrial arrangement is related to muscle cell structure and the disposition of cardiomyocyte myofibrils (Birkedal et al., 2006). The average length of sarcomeres that was previously reported (Quintao et al., 2012) in young rat cardiomyocytes was 1.7 6 0.1 lm. However, in this study, aged rat hearts had a mean sarcomere length of 2.071 6 0.09 lm and a median value of 2.064 lm. In this case, the mean and median values were very similar and demonstrated an increase in sarcomere length when comparing aged mice with young animals. The active force production in cardiac muscle is more dependent on the sarcomere length than in skeletal muscle. The elongation of the sarcomere length causes a decrease in space in the interfilament network connections that promote crossbridges (Kobirumaki-Shimozawa et al., 2012). The aged rat cardiomyocytes are characterized by large structural changes in the mitochondria and a Microscopy Research and Technique

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considerable lytic area in the myofibril bundles (Nepomnyashchikh et al., 2011). The content of mitochondria (mitochondrial volume) in mammal hearts differs in several species, ranging from 22 to 37%. These values are very specific and constant for all species. Previous studies have reported that the mitochondrial volume of rat cardiac tissue was 32.03 6 1.83% (Barth et al., 1992) or 33.96 6 4.07% (Kimpara et al., 1997). In this study, the mitochondrial volume density in the aged rat heart was 33.83%, which is very similar to the previously reported values (Barth et al., 1992; Kimpara et al., 1997). Therefore, there is no significant loss of the mitochondrial volume density with increasing animal age. The mitochondrial volume density is considered to be an indicator of the cardiac muscle oxidative capacity, and the ultrastructural composition of normal cardiac muscle fibers of these species should be a useful baseline in heart pathophysiological studies in various animals (Barth et al., 1992). Owing to a high heart energy demand, a decline in mitochondrial function may result in a worsening of cardiac performance (Li et al., 2011). A great variability in the values was noticed for the measurements of the mitochondrial area in aged rat cardiomyocytes: mean, 0.537 6 0.278 lm2; median, 0.491 lm2; and values ranging from 0.039 to 1.462 lm2. Some studies have evaluated the mitochondrial volume (Corsetti et al., 2008; Knaapen et al., 1997) with a specific focus on the cardiomyocyte mitochondrial area. It was reported that the average area of healthy hamster mitochondria was 2.364 lm2 (Colmanetti et al., 2005), which is much larger than the values found in this study. However, comparison of these distinct values is restricted because of the different species and ages of the subjects. The mitochondrial volume in aged rat cardiac muscle (0.82 6 0.18 lm3) is moderately higher than in young rats (0.73 6 0.15 lm3), which can be compensated for by a significant decrease (34%) in the number of mitochondria (Corsetti et al., 2008). In this study, we did not perform the measurements of mitochondrial volume to allow for comparisons to the values that were previously disclosed; however, the volume and area in this case are more consistent because they were measured from the same species. The observed mitochondrial cristae thickness had a mean value of 0.038 6 0.09 lm and a median value of 0.038 lm. The measured values of the mitochondrial cristae thickness were not dispersed over a wide range (0.032–0.044). No other studies have evaluated mitochondrial cristae thickness, but it is interesting to note that in cases of megamitochondria (length, 3–14 lm) that are found in hearts of cardiomyopathic patients, only an increase in mitochondrial cristae was observed, whereas the related thickness remained unchanged (Tandler et al., 2002). The thickness of the mitochondrial cristae does not have a large variation, as observed in this study, suggesting that it is offset by the amount, such as in exceptional cases where these organelles change their size. Our results of the mitochondrial major diameter of the aged rat cardiomyocytes reveal that the values of central tendency (mean [1.024 lm] and median [0.981 lm]) have similar values with no extreme average discrepancies. The detected values ranged from 0.324 to

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2.124 lm, and although this variation was observed, half of the obtained values were between 0.774 and 1.203 lm, indicating that the mitochondrial average length did not have wide variation. The mitochondria present in normal skeletal muscle have a measured length ranging between 0.6 and 1.2 lm with an average of 0.9 lm (Bergman et al., 2003). These data corroborate our observations because the values are similar despite being from another muscle tissue type in adult rats. In the heart tissue of cardiomyopathic patients, mitochondria measuring up to 14 lm have been observed, the so-called megamitochondria (Tandler et al., 2002). In another study, researchers reported mitochondria with lengths up to 9.2 lm present in cardiomyocytes of the left ventricle from mice subjected to administration of hexane (Maharaj et al., 1993). These studies report mitochondria that have a length much greater than those observed in our results, which suggests that aging does not lead to the appearance of these mitochondria that are larger than those that are normally observed in muscle tissues, in general. In this study, mitochondrial shape was quantified and categorized into three profiles: rounded, elongated, and irregular. The shape was determined by the ratio between the largest and the smallest mitochondrial length. The smallest possible value was 1, representing a round shape. Values >1 represent elongated mitochondria; the higher the value the more elongated organelle will be. Our results indicate that in the aged rat myocardium (right atrium) only 23.4% of the mitochondria were round, most were elongated 45.3%, and 31.1% were classified as irregular. These data are confirmed by an analysis of the ratio between the greater and the lesser mitochondrial lengths, in which the values ranged from 1.006 to 6.99, and 50% of the values were between 1.33 and 2.145. The mean ratio was 1.929, and the median was 1.701. In both of the measures of central tendency, there was a predominance of spindle-shaped mitochondria. Previous reports revealed numerous mitochondria with round or oval shape and intact crests in skeletal muscle fibers (Bergman et al., 2003). In the left ventricular myocardium of healthy young mice, the observed mitochondria had spheroidal to elongated profiles with cristae surrounded by a dense mitochondriosol (Maharaj et al., 1993). These data corroborate our results to some extent, but no care was taken with regard to categorization, quantification, and understanding of the dispersion of characteristics in these studies. CONCLUSIONS In this study, we evaluated the histological and ultrastructural morphologies of aged rat cardiac muscle fibers and performed a morphometric assessment of cardiac muscle parameters. The data were presented to describe and contribute to the understanding of tissue and organelle characteristics, particularly mitochondria under physiological aging conditions. The results are important for researchers who are focusing on the changes in cardiac tissue, especially changes owing to pathologies and drug administration that may or may not be correlated with aging.

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