Postnatal Changes in the Morphology of the Myocardium in Rat Ventricles

Background: Aging of the myocardium is a dynamic process which involves progressive loss of cardiomyocytes due to necrosis and apoptosis, interstitial fi brosis and reactive hypertrophy of the remaining vital cardiomyocytes. In our study, we investigated the postnatal changes in the myocardium of 15 adult male Wistar rats, distributed in the following age groups: 2 weeks, 1, 3, 6 and 12 months old. We used routine hematoxylin and eosin staining and Mallory’s trichrome stain. Results: Hypertrophy of the cardiomyocytes and accumulation of extracellular matrix proteins in the interstitial space in the aging myocardium were observed. The occurrence of these processes was more dynamic in the left ventricle as opposed to the right ventricle owing to the functional dissimilarities between the two ventricles and a more pronounced effect of afterload on the left ventricle. Conclusions: In conclusion, it can be stated that aging of the myocardium is a dynamic process, characterized by hypertrophy of the cardiomyocytes, reduced capillary density and increased deposition of collagen. Research Article Postnatal Changes in the Morphology of the Myocardium in Rat Ventricles Georgi Kotov1, Alexandar Iliev1*, Boycho Landzhov1, Lazar Jelev1, Iva N. Dimitrova2, Dimka Hinova-Palova1 1Department of Anatomy, Histology and Embryology, Medical University of Sofi a, Bulgaria 2Department of Cardiology, University Hospital “St. Ekaterina”, Medical University of Sofi a, Bulgaria Dates: Received: 17 February, 2017; Accepted: 06 March, 2017; Published: 10 March, 2017 *Corresponding author: Alexandar Iliev, Professor, Department of Anatomy, Histology, and Embryology, Medical University of Sofi a, Bulgaria, Tel: +35929172641; Email:


Introduction
Comprehensive studying of the myocardial architecture is of key importance for the understanding of the precise mechanisms of cardiac function and the pathogenetic processes which form the basis of cardiovascular disorders.
Multiple contemporary studies reveal the participation of the myocardium in virtually every disease -either as a primary site of the pathophysiological changes or as an affected organ secondary to the pathological process. It is true that the myocardium possesses a remarkable diagnostic and therapeutic potential. Studying of the arrangement and the specifi c characteristics of the cardiac muscle cells can provide a morphological basis for the diagnostics of various disorders of the heart. The present study refl ects our efforts to understand the morphology of cardiomyocytes and the myocardium as a whole in hearts from male rats from different periods of the postnatal development and to elucidate the importance of morphological differences between the left and the right ventricle. To do so, we used different staining techniques which allowed us to clearly observe sections of the cardiac muscle cells in different planes and changes in the myocardial structure.
Although our studies were conducted on experimental animals, the environment in which the cardiomyocytes existed was identical to the conditions in the human body and complied with the universal biological principles.
The cardiomyocyte, or the cardiac muscle cell, is a cylindrically shaped cell with approximate diameter 10 -15 μm and length of about 100 μm [1]. Their mean diameter is intermediate as compared to that of the small atrial cells and the wider Purkinje fi bers. On light microscopy the cardiomyocyte appears as a cross-striated cell and has one or two centrally positioned, slightly elongated nuclei with low density [1,2]. Muscle fi bers in the ventricles are located in very close proximity to one another, thus forming wide bundles of branching cells, which more often than not connect in longitudinal direction through specialized structures known as intercalate discs. Inside, the ventricular cardiomyocyte is fi lled with alternating chains of sarcomeres and mitochondria [3]. An often encountered pattern of alignment is in a Y-shape, with the participation of three Purkinje fi bers. There are also zones with mosaic alignment of the cells. This type of cellular organization, as well as the presence of a complex intercalate disc play a key role in the rapid conduction between the Purkinje fi bers [3].
The alignment of the cardiomyocytes leads to the formation of a network or a functional syncytium which represents the main building unit of the myocardium. Other cells are also present in the structure of the myocardium: fi broblasts, endothelial cells and smooth muscle cells [4,5].
The connective tissue surrounding the cardiomyocyte includes the following three layers, listed herein starting from the outermost layer: epimysium, perimysium and endomysium.
The normal extracellular matrix is produced by the myocardial fi broblasts as well as by the cardiomyocytes themselves. It is built of collagen types I, III and IV, laminin, fi bronectin and proteoglycans. It seems that it plays a key role in the formation of the myofi brils and the intercalate discs during the differentiation and maturation of the myocardial cells from cardiac stem cells [6,7].
The aim of the present study was to elucidate the specifi cs of the normal morphology of the individual cardiomyocytes and the myocardium as a whole during different periods of the postnatal development and to compare the nature of the changes in myocardial structure between the left and the right ventricle.

Materials and Methods
Fifteen adult male Wistar rats, distributed in the following age groups: 2 weeks, 1, 3, 6 and 12 months old, were used for this study.
The animals were anesthetized with ether and cervical dislocation was performed. The chest cavity was opened and the hearts were isolated, placed in 0.9% solution of sodium chloride (physiological saline) to wash away the blood in the heart chambers and a transverse incision was performed through the middle of the heart, slightly below the level of the Mallory's trichrome stain; all according to standard methods.

Results
On H&E stained slides in 2-week-old rats, it was diffi cult to observe clearly defi ned longitudinal and transverse layers of cardiomyocytes in the wall of the left as well as the right ventricle. Cardiomyocytes appeared to be slightly eosinophilic and pronounced cross striation was not observed. They usually had one centrally located basophilic nucleus; in some cells, one or more nucleoli were noted. The perinuclear zone was well seen and was stained less intensively. The muscle fi bers were enveloped by fairly thin connective tissue and a perimysium with abundant capillary network. It was fairly rare that fi broblastic nuclei were observed among the cardiac muscle cells (Figures 1,2).
Mallory's trichrome stain in 2-week-old animals revealed a scarce amount of fi ne collagen fi bers in the walls of the two ventricles. Very few thin and quite short connective tissue fi bers were noted in the interstitial space of the left ventricle.
More collagen fi bers were situated predominantly in the perivascular zone and in the walls of the blood vessels and were stained in an intensive blue color; we noted a slight tendency for collagen fi bers to grow from the perivascular zone towards the interstitial space (Figures 3,4).
In the age groups of young rats (1-and 3-month-old), the H&E stain revealed a well-developed morphological organization of the layers of the myocardium in both ventricles.
Bundles of longitudinally aligned cardiomyocytes were clearly observed, as well as subepicardial and subendocardial layers of transversely cut cardiac muscle cells. On a longitudinal section,         (Figures 13,14).
In the group of senescent twelve-month-old rats, agerelated changes observed in the wall of the left and the right ventricle on H&E staining were best expressed. Bundles of large longitudinally positioned hypertrophic cardiac muscle cells with large basophilic nuclei with reticular structure were seen.
We also noted bundles of hypertrophic cardiomyocytes from the transverse layers and further narrowing of the interstitial space. Capillaries in the connective tissue surrounding the muscle fi bers were found less often. A well-pronounced neutrophil infi ltration and the presence of pyknotic nuclei were observed. Subendocardial and interstitial fi brosis was more manifest and presented as clearly visible eosinophilic fascicles. A major fi nding was the focal myocytolysis: individual cardiomyocytes which appeared 'empty', i.e. no nuclei and cross striations were noted in them, while their cytoplasm stained more intensively eosinophilic (Figures 15,16). When comparing the two ventricles we noted that these changes in myocardial     According to literature data, life expectancy in rats is between 2 and 3.5 years [8]. After two years of age, the frequency of infectious and neoplastic diseases increases and a large portion of animals die. Therefore, the studied animals in the period of senescence in the present work were twelve months old. All rats selected for this study were in good overall condition and with no physically noticeable signs of disease.
In line with our results and literature data, aging of the myocardium is related to hypertrophy of the left ventricle and the development of fi brosis [9,10]. The gradual agerelated deposition of collagen in the walls of the vessels,        [9]. The increased development of fi brosis is a fundamental prerequisite for myocardial rigidity which, together with the impaired relaxation of the ventricles, leads to myocardial dysfunction [11]. Over the course of development of fi brotic changes, an increased tendency for deposition of collagen in the periand endomysium is noted, which leads to interstitial fi brosis, while the deposition of collagen in the adventitia of intramural coronary vessels leads to perivascular fi brosis [12]. These data are supported by the present study.
In healthy Wistar rats, aging is related to changes in myocardial morphology. Age-related remodeling of the cardiac wall is related to cardiomyocytic hypertrophy and interstitial fi brosis. In physiological conditions, the bundles of cardiomyocytes and the individual cells are enveloped by thin layers known as peri-and endomysium. In the aging myocardium, transformation of the fi broblasts into myofi broblasts and accumulation of extracellular matrix proteins in the interstitial space are observed. Histological analyses of aging hearts from Wistar rats show progressive loss of cardiomyocytes due to necrosis and apoptosis [13]. Preserved cardiac muscle cells undergo a process of compensatory hypertrophy [14]. Collagen content increases as aging progresses, therefore collagen fi bers in the endo-and perimysium of older animals are thicker [15]. This was well noted in the present study, where a tendency for collagen fi bers to become thicker and to acquire a complex spiral shape was observed as aging progressed in both ventricles but was signifi cantly more pronounced in the myocardium of the left ventricle, which was attributed to the higher afterload exerted on the left ventricle and consequently, the higher degree of injury suffered by the ventricle with progression to senescence.
The loss of cardiomyocytes due to necrosis and apoptosis leads to compensatory or reactive hypertrophy of the remaining cardiomyocytes [16]. Cardiomyocytic hypertrophy itself is a physiological process taking place in response to increased demands (such as physical exercise) and therefore, agerelated hypertrophy can be considered as developing through a similar mechanism. Unlike physiological hypertrophy, agerelated hypertrophy is a result of loss of cardiomyocytes due to aging and the decrease in their stability, which increases workload on the remaining healthy cells [17]. This hypothesis was confi rmed by the results of our study. We observed initial hypetrophic changes in the group of adult Wistar rats, which progressed to well-developed hypertrophy in the group of senescent animals and combined with the presence of focal myocytolysis, which is the histological equivalent of cell necrosis. These morphological changes were better expressed and had a more dynamic course in the wall of the left ventricle.
This led us to the hypothesis that the relative preservation of the normal morphology of the right ventricle may be associated with a higher reserve capacity to respond to increased stress. It can be stated that the processes of accumulation of interstitial collagen and cardiomyocytic hypertrophy with the onset of senescence show that the morphology and function of the heart becomes impaired not only at the level of the organ but at the level of the individual cardiomyocyte as well [17].
Literature data make it evident that in senescent Wistar rats, the formation of fi brosis in the right ventricle increases with age [17]. One study found development of fi brosis and accumulation of adipose tissue in the cardiac conduction system [18]. signifi cant difference between the intensity of staining between histological slides from the left and the right ventricle, with the left ventricle staining more intensively blue with notable visualization of the collagen fi bers which corresponded to a more pronounced accumulation of collagen.
It is considered that cardiomyocytic hypertrophy results from the impairment of the balance between the signal pathways of hypertrophy and atrophy [20]. The low rate of cellular death in the normal heart, as well as the presence of cardiomyocytic hypertrophy and fi brosis as mechanisms intended to compensate cellular loss probably decrease the need for cardiomyocytic regeneration [14]. New data present the hypothesis of a probable existence of two sources of cardiomyocytic renewal: division of already existing cardiomyocytes and differentiation of precursor cells [21]. The loss of cardiomyocytes as aging progresses probably results from two separate mechanisms: loss of cells due to depletion of adaptive mechanisms (impaired division of cardiomyocytes and/or of the maturation of the cardiac progenitor cells) and active loss of cells due to activation of the process of apoptosis [22]. All of this leads to impairment of the myocardial function and is therefore a key process in the overall incidence of pathological conditions such as heart failure and cardiac rhythm and conduction disorders.

Conclusion
In conclusion, aging of the myocardium is a dynamic process characterized by cardiomyocytic hypertrophy and increased deposition of collagen in the interstitial space.