Prevention of disorders of cardiomyocyte ultrastructure in experimental myocardial infarction in immobilization-induced stress by administration of thyroid hormones

In the experiment, carried out on 48 non-inbred male rats ultrastructural changes in cardiomyocytes in non-ischemized parts of the heart at experimental infarction of myocardium under conditions of immobilization stress have been studied, as well as possibility to correct these changes by means of thyroid hormones. The stress intensifies dystrophic processes, developed outside the infarction zone, increases the mass of the necrotized tissue, essentially decreases the areas occupied by mitochondria and myofibrils, as well as their ratio in the section area. Small doses of thyroid hormones prevent the heart from the damaging effect of the stressor: decreasing area; occupied by mitochondria, myofibrils and their relation in the section, as well as they stimulate intracellular regenerative processes (accumulation of polymorphous mitochondria with clearly manifested cristae, membranes of the endoplasmic reticulum) and decrease the myocardial necrotized zone). Thus, structural lesions, resulted from the effect of ischemic necrosis and stress, can be prevented by small doses of thyroid hormones+.     

Ultrastructural stereological analysis of acute myocardial hypertrophy of hypertensive etiology

The myocardium of Wistar rats was studied by electron microscopy after ligation of the renal artery resulting in a stable elevation of blood pressure. The ultrastructural data on the development of heart hypertrophy were described within 5 to 35 days after operation using the morphometric and stereological methods and correlation analysis. The hypertrophied cardiomyocytes showed a reduction in the ratio of the total volume density of the mitochondria, sarcoplasmic reticulum and T system to the volume density of myofibrils. It was discovered that hypertrophy of cardiomyocytes is marked by an increase in the sarcoplasmic reticulum rather than in myofibrils and other cell compartments, whereas the relative volume of mitochondria decreases.     

Effect of acute exercise stress in cardiac hypertrophy: I. correlation of regional blood flow and qualitative ultrastructural changes.

Ultrastructural myocardial cell changes were determined in eight miniswine after the development of pressure-overload hypertrophy induced by supra-valvular aortic constriction. Four miniswine served as control animals. Regional myocardial blood flows were measured at rest and during exercise stress with radioactive microspheres after two days and one month of aortic constriction. Exercise stress, causing the heart rate to increase to 85 percent of its maximum, was imposed twice weekly for 7 minutes on four pressure-overloaded animals and the four control animals to elicit differences between the control and experimental groups that might not occur at rest. After one month of pressure overload the swine were killed and myocardial samples were processed for electron microscopy. Ultrastructural changes similar to those in hypertrophied hearts were present throughout the left ventricular walls of the pressure-overloaded animals. Other changes consistent with ischemic injury were present in the subendocardial regions of pressure-overloaded animals subjected to exercise stress. These changes included disorganization of myofibrils, disintegration and broadening of Z-bands, swelling and aggregation of mitochondria, electron-dense deposits in mitochondria, decreased cristal density and vacuolization of mitochondria, intracellular edema, margination and clumping of nuclear chromatin, and a decrease of glycogen granules. Regional ischemia in the subendocardium of these animals was confirmed by functional studies which showed decreased regional myocardial blood flow to the subendocardium during exercise and S-T segment elevation for the first 2-10 days after inducing pressure overload. The ischemia, as shown by flow studies, during exercise stress persisted in the compensatory stage of hypertrophy although S-T segments returned to normal. Thus, the combined effect of pressure overload and exercise stress can produce focal subendocardial ischemia in the compensated, hypertrophied heart.     

An ultrastructural stereological analysis of the myocardium in homoiothermal animals during cooling]

By methods of electron microscopy and stereology the ultrastructural cardiomyocyte reorganization of rats exposed to influence of low temperatures (exposure during 16 days at -7 degrees C) was studied. It was shown, that with this regime of cooling the disturbance of intracellular regeneration in cardiomyocytes occurred and the complex of morphological changes typical for the syndrome of regenerative-plastic deficiency was developed. The most essential changes were seen in myofibrils and mitochondria. According to the stereologic data a change in spatial reorganization of cardiomyocytes was connected largely with these organelles. With the increase in cooling duration an increase in the volume density of myofibrils and a decrease in this parameter for mitochondria were marked. As a result of these changes the mitochondria/myofibrils volume ratio was essentially decreased. As a whole, ultrastructural reorganization of cardiomyocytes under the influence of low temperatures on rats was characterized by a decrease in the ratio of the organelle volume to myofibrillar volume. The same quantitative reorganization was revealed in atrophied cardiomyocytes under the conditions of the decreased inflow of plastic substances to cells.     

Structural and functional heterogeneity of cardiomyocytes during hemodynamic loading of the rat heart

Ultrastructural studies of cardiomyocytes during experimental aorta coarctation enabled one to divide them into 6 types: with mitochondrial swelling and enlargement of the sarcoplasmic reticulum; with primary damage to myofibrils; with disintegration of ultrastructure because of edema; with hypertrophy and hyperplasia of ultrastructures; without essential changes in organelles; and with concomitant changes in mitochondria and myofibrils. Such different reactions of cardiomyocytes are regarded as an adaptation mechanism that ensures the maintenance of heart function under extreme conditions.     

Stress-induced changes in the area ratio or the quantitative ratio of myocardial mitochondria and myofibrils and their correction with thyroid hormones

An experimental study performed on 30 male white rats has demonstrated that immobilization stress was associated with a decrease in mitochondrial to myofibril area ratio (Smch/Smf) in the left ventricle and interventricular septum by 34.6% and 46.9%, respectively. Pretreatment with small doses of thyroid hormones which did not influence body weight, heart rhythm or serum thyroxin level prevented the decrease and caused Smch/Smf increase in the left ventricle and interventricular septum by 68.7% and 45.8%, respectively. The doses of thyroid hormones applied caused a more marked increase of mitochondrial to myofibril area ratio in the control rats.     

Ultrastructural stereological analysis of the absolute parameters of the cardiomyocytes in myocardial hypertrophy

Arterial hypertension was produced in male Wistar rats by abdominal aorta ligation. By the 35th day of experiment the animals had developed myocardial hypertrophy. The subcellular organization of cardiomyocytes was studied by electron microscopy and stereology (computation of relative and absolute parameters). In myocardial hypertrophy, the absolute volume of myofibrils, sarcoplasmic reticulum and T system in the left ventricle of the heart was increased, whereas the absolute volume of the mitochondria remained unchanged. The general surface area of all organelles was noticeably increased. The myofibrils and sarcoplasmic reticulum experienced greater changes, which may reflect the features of compensatory processes under the experimental conditions described.     

HYPERTROPHY OF THE HUMAN HEART AT THE LEVEL OF FINE STRUCTURE : An Analysis and Two Postulates

Muscle cells in the left ventricular walls of four markedly hypertrophied human hearts (above 600 gm) were compared with muscle cells in four non-hypertrophied hearts (up to 310 gm). Blocks of tissue obtained postmortem within 6 hours were processed for light and electron microscopy under conditions suitable for good preservation of myofibrils. A lattice parameter, q_h, was defined as the number of myosin filaments per square micron in either H zones or A bands. By the use of methods of electron microscopy, q_h was determined for perpendicular cross-sections of A bands in a large number of well preserved myofibrils of muscle cells in both groups of hearts. Statistical evaluation of the distributions of values of q_h revealed no significant difference between the two groups. Thus, the myofilament lattices in hypertrophied cells were geometrically within normal limits. Planimetric measurements of cross-sectional areas of muscle fibers were made, using photomicrographs obtained from one representative hypertrophied heart and from one control. The size-frequency distribution of the measurements showed a marked difference between the two hearts, and confirmed the presence of hypertrophy of muscle cells. Counts of the number of myofibrils per muscle cell were determined for samples from the same two hearts, evaluated statistically, and found to be significantly higher for the hypertrophied heart. It is proposed (a) that myofibrils in hypertrophied heart muscle cells have filament lattices with geometrical arrangement and macromolecular parameters that are the same as those found in myofibrils of normal heart muscle cells; and (b) that in hypertrophy the number of myofilaments increases through formation of new myofibrils, and possibly also by addition of filaments to preexisting myofibrils.     

Effect of acute exercise stress in cardiac hypertrophy. II. Quantitative ultrastructural changes in the myocardial cell

Stereological techniques were used to determine quantitatively the changes in subcellular organelles in the hypertrophied swine heart. Hypertrophy was induced by aortic stenosis. In addition, the animals were stressed by exercising twice weekly on the treadmill for 30 days. The controls were normal animals which were neither exercised nor had aortic stenosis. Tissue samples from the left ventricle and interventricular septum were processed for electron microscopy. Relative volumes of myofibrils, mitochondria, transverse tubular system (T-system) sarcoplasmic reticulum (SR), and clear intracellular space (ICS) were measured. Significant differences in the volumes of all the components except the T-system were found between experimental and control animals in the epicardial and endocardial regions of the left ventricle and interventricular septum. Mitochondria and myofibrils were significantly decreased in the exercise stressed hypertrophied hearts as reported in ischemia, while SR and ICS were significantly increased. These findings suggest that ischemic injury occurs in all regions of the hypertrophied heart wall subjected to acute exercise stress, not solely the endocardial region as previous qualitative studies suggested.     

Stereological analysis of the ultrastructural organization of the cardiomyocytes in the red-cheeked suslik in different seasons of the year

The myocardium of red-cheeked sousliks was studied at different seasons with the use of stereological analysis. The volume and surface densities of the myofibrils, mitochondria, T system, sarcoplasmic reticulum as well as the surface-volume ratios of the main organelles were measured. Pronounced seasonal changes in physiological activity of the heterothermal animals were accompanied by marked reorganization of the spatial ultrastructure of the cardiomyocytes. The seasonal regression of the heart weight and diminution of the cardiomyocyte diameter were recorded during hibernation. The authors believe that the increased volume ratio of the cytoplasmic organelles to the myofibrils forms the basis for a rapid adaptive reaction of the heart during hibernation and waking up.     

Ultrastructural-morphometric characterization of adaptive and compensatory myocardial changes using heart enlargement as an example

A morphometric analysis of the heart muscle cell shows significant differences in the reaction of different cellular structures in different experimental stress models of the myocardium (swimming exercise and ligature infarction) in the rat. For instance, the number of mitochondria is 44% higher only 2 days after infarction than after 45 h of swimming exercise over a period of 4 weeks. The mitochondria/myofibrils ratio is higher than in the case of training. But later on in the course of the experiment the number of mitochondria and their membrane surface density diminishes compared with training. The differences in the rate of formation and in the kind and intensity of the changes are probably attributable to different adaptive mechanisms by means of which the heart reacts to acute and chronic loads.     

Relative hypertrophy of the right ventricle in silver foxes selected for domestication behavior

Silver foxes selected for domestication behavior were found to have relative hypertrophy of the right heart ventricle, which was 21% as enlarged in males and 18% as enlarged in females as compared with non-domesticated animals. It was established by stereological methods that hypertrophy occurs mainly at the expense of an increase in the absolute and relative content of myofibrils in cardiomyocytes, with the absolute total volume of the mitochondria being equal both in domesticated and non-domesticated animals. It was shown by means of dissociated cell counts that in both animal groups, the absolute number of cardiomyocytes and their nuclei in the right ventricles is approximately similar. It is suggested that there is a relationship between right heart ventricle hypertrophy in domesticated foxes and variation in the balance of sympathetic and parasympathetic effects.     

3-dimensional ultrastructural characteristics of the cardiomyocytes of SHR strain rats with spontaneous genetic hypertension

The quantitative morphological analysis of hypertrophied cardiomyocytes was carried out in SHR rats (spontaneously hypertensive rats). The features of spatial ultrastructural organization of heart muscle cells were shown as manifested in the increased synthesis of contractile proteins and focal disorders of myofilament arrangement. Stereological analysis revealed the decrease of the ratio of the summary volume of the main cardiomyocyte organelles (mitochondria, smooth sarcoplasmic reticulum and T-system) to the myofibril volume.     

Myocardial damage during immobilization stress and the protective effect of thyroid hormones

In the experiments performed on 30 white inbred male rats, the stress produces an essential increase of dilated canals in the T-system and sarcoplasmic reticulum, lesions of sarcolemma and nucleolemma, fragmentation and homogenization of mitochondrial cristae; there are many pinocytic vesicles in the capillary walls. Preliminary injection of thyroid hormones in small doses, that do not result in any noticeable influence on the body mass, cardiac contraction rate and thyrotoxin concentration in blood serum, at the stress causes less pronounced changes in ultrastructure of cardiomyocytes, demonstrating as a poor swelling of the T-system elements, preservation of the sarcolemmic integrity at its larger length; besides, large mitochondria with compact arrangement of cristae appear, amount of the sarcoplasmic reticulum elements increases, chromatin margination becomes evident, i. e. a complex of adaptive processes develops. The data presented demonstrate a defensive role of physiological concentrations of the thyroid hormones under the stress.     

Effect of U50,488H, a κ-opioid receptor agonist on myocardial α-and β-myosin heavy chain expression and oxidative stress associated with isoproterenol-induced cardiac hypertrophy in rat

Both oxidative stress and β-MHC expression are associated with pathological cardiac hypertrophy. β-adrenergic receptor stimulation plays an important role in cardiac hypertrophy. Recent studies have reported a negative interplay between opioid receptors and adrenoceptors in heart. This study investigated the effect of U50,488H (a selective κ-opioid receptor agonist) on myocardial oxidative stress and α- and β-MHC expression in isoproterenol-induced cardiac hypertrophy. Male Wistar rats were administered normal saline (control), isoproterenol (ISO) (5 mg/kg BW s.c. OD), and isoproterenol with U50,488H (0.4 and 0.6 mg/kg BW, i.p. OD) for 14 days. In a separate group, nor-binaltorphimine (nor-BNI) (0.5 mg/kg, BW, i.p.) (κ-receptor antagonist) was administered along with ISO and U50,488H. ISO administration caused significant increase in left ventricular (LV) wall thicknesses, LV mass in echocardiography, heart weight to body weight ratio, and myocyte size as compared to control. Both the doses of U50,488H offered significant protection against these changes. The higher dose of U50,488H significantly prevented ISO-induced increase in myocardial lipid peroxidation and depletion of myocardial antioxidants (glutathione, superoxide dismutase, and catalase), while a similar trend (although not significant) was observed with the lower dose also. ISO-induced myocardial fibrosis was also significantly attenuated by both the doses of U50,488H. Isoproterenol-induced β-MHC expression in the hypertrophied heart was not altered by either doses of U50,488H, however, the latter prevented the loss of myocardial α-MHC expression. All these effects of U50,488H were blocked by nor-BNI. This study provides the evidence that U50,488H reduced oxidative stress and preserved expression of α-MHC in isoproterenol-induced cardiac hypertrophy.     

Decreased protein kinase C-epsilon expression in hypertrophied cardiac ventricles induced by triiodothyronine treatment in the rat

To examine the effect of thyroid hormone-induced cardiac hypertrophy on PKC expression, changes in the expression of PKC isoforms were studied in hypertrophied cardiac ventricles induced by triiodothyronine (T3) injection in the rat. Injection with T3 for 8 days induced 49% increase in cardiac weight compared to controls. Immunoblot analysis of cardiac ventricular extracts showed the expression of PKC-delta, -epsilon, and -zeta in both control and T3-treated groups. The expression of PKC-epsilon decreased by 40% in hyperthyroid rat cardiac ventricles, while PKC-delta and -zeta expressions were barely affected. PKC-epsilon immunoreactivity decreased in both cytosol and membrane fractions. On the contrary, PKC-epsilon expression did not decrease in the extract of hypertrophied cardiac ventricles produced by aortic banding or aortocaval shunt. These results indicate that thyroid hormone down regulates PKC-epsilon expression in the hyperthyroid-mediated cardiac hypertrophy.     

Sucrose feeding prevents changes in myosin isoenzymes and sarcoplasmic reticulum Ca2+-pump ATPase in pressure-loaded rat heart

Pressure-overload due to banding of the abdominal aorta in rats for 10 weeks resulted in cardiac hypertrophy, redistribution of myosin isoenzymes and reduction in the sarcoplasmic reticulum (SR) Ca2+-stimulated ATPase activity. Administration of sucrose in the drinking water (0.8%, w/v) to rats prevented changes in myosin isoenzymes and SR Ca2+-stimulated ATPase in hypertrophied hearts. This beneficial effect of sucrose feeding with respect to remodeling of the subcellular organelles in the myocardium was not associated with any significant changes in plasma glucose or thyroid hormone levels. It is suggested that the prevention of subcellular changes in the hypertrophied hearts due to sucrose feeding may be due to a shift in fuel utilization by the myocardium.     

Involvement of lipid peroxidation in regression of heart hypertrophy

The regression of hypertrophied heart presupposes disassembling of all cardiomyocyte components including membrane structures. The involvement of free radical oxidation of membrane phospholipids was studied in the cardiac regression. Altitude hypertrophy was developed in barocamera (7000 m, 6 hours daily). 3 weeks of periodical hypoxia leads to 1.5-fold increase of heart weight (right ventricle weight was 2-fold increased). In 7-10 days after adaptation the heart weight reduced to normal. Both, the development and regression of myocardial hypertrophy was accompanied by changes in Mb and Hb organ concentration proportional to weight changes. In lipid extraction of maximal hypertrophied heart, the 30% decrease of lipid peroxidation product (diene conjugates) regularly occurred. The rate of regression had negative correlation with peroxidation products accumulation. Intraperitoneal injection of free radical scavenger BHT attenuate the regression rate. The results suggest that unlike the common knowledge about the membrane injury effect, lipid peroxidation can play positive role in disassembling of superfluous cell membrane structures.     

Non-enzymatic lipid peroxidation of microsomes and mitochondria from liver, heart and brain of the bird Lonchura striata: relationship with fatty acid composition

The aim of this study was to examine the fatty acid composition and non-enzymatic lipid peroxidation (LP) of mitochondria and microsomes obtained from liver, heart and brain of Lonchura striata. The percentage of total unsaturated fatty acid was approximately 30-60% in the organelles from all tissues studied. Brain mitochondria and both organelles of liver exhibited the highest percentage of polyunsaturated fatty acid (PUFA) (30 and 18%, respectively). The arachidonic acid (AA) content was 7% in mitochondria of liver and brain and 3% in heart mitochondria. The percentage of docosahexanoic acid (DHA) was 8% in brain mitochondria and approximately 2-3% in heart and liver mitochondria. The peroxidizability index (PI) of brain mitochondria and both organelles from liver was higher than that of organelles from heart and brain microsomes. Liver organelles and brain mitochondria were affected by LP, as indicated by the increase in chemiluminescence and a decrease of AA and DHA. These changes were not observed during LP of brain microsomes and both organelles from heart. These results indicate: 1) PI positively correlates with PUFA percentage and LP; 2) The resistance to LP detected in heart organelles would contribute to the cardiac protection against oxidative damage.     

Nanocurcumin Prevents Hypoxia Induced Stress in Primary Human Ventricular Cardiomyocytes by Maintaining Mitochondrial Homeostasis

Hypoxia induced oxidative stress incurs pathophysiological changes in hypertrophied cardiomyocytes by promoting translocation of p53 to mitochondria. Here, we investigate the cardio-protective efficacy of nanocurcumin in protecting primary human ventricular cardiomyocytes (HVCM) from hypoxia induced damages. Hypoxia induced hypertrophy was confirmed by FITC-phenylalanine uptake assay, atrial natriuretic factor (ANF) levels and cell size measurements. Hypoxia induced translocation of p53 was investigated by using mitochondrial membrane permeability transition pore blocker cyclosporin A (blocks entry of p53 to mitochondria) and confirmed by western blot and immunofluorescence. Mitochondrial damage in hypertrophied HVCM cells was evaluated by analysing bio-energetic, anti-oxidant and metabolic function and substrate switching form lipids to glucose. Nanocurcumin prevented translocation of p53 to mitochondria by stabilizing mitochondrial membrane potential and de-stressed hypertrophied HVCM cells by significant restoration in lactate, acetyl-coenzyme A, pyruvate and glucose content along with lactate dehydrogenase (LDH) and 5'' adenosine monophosphate-activated protein kinase (AMPKα) activity. Significant restoration in glucose and modulation of GLUT-1 and GLUT-4 levels confirmed that nanocurcumin mediated prevention of substrate switching. Nanocurcumin prevented of mitochondrial stress as confirmed by c-fos/c-jun/p53 signalling. The data indicates decrease in p-300 histone acetyl transferase (HAT) mediated histone acetylation and GATA-4 activation as pharmacological targets of nanocurcumin in preventing hypoxia induced hypertrophy. The study provides an insight into propitious therapeutic effects of nanocurcumin in cardio-protection and usability in clinical applications.