Degraded cardiomyocytes and dysfunctional energy metabolism; their relationship with canine cardiomyopathies

Tissue sections from hearts of dogs suffering from cardiomyopathies were studied histologically and ultrastructurally. Two types of mitochondrial changes were defined and quantitated. Mitochondrial hypertrophy occurred in cardiomyocytes of middle-aged and older dogs. Significant numbers of degraded cardiomyocytes related to hydropic degeneration of hypertrophied mitochondria associated with autolysis of contractile elements and loss of cellular physiochemical functions were seen in hearts of old dogs with cardiomyopathy. Dogs with cardiac disease that were treated to enhance dysfunctional energy production early in the course of disease had cellular changes similar to those seen in old dogs but they survived heart disease and ultimately died of other causes.     

Size of cardiomyocytes in different regions of the rat heart

In rats after alcaline dissociation of the heart at the state of systol and diastol, length and width of cardiomyocytes have been measured, their volume has been calculated. Statistical characteristics of distribution connected with the first four moments have been taken into consideration. Certain size differences of cardiomyocytes in five cardiac parts have been revealed, as well as peculiarites of their variability that is connected with an individual and internal changeability. In the atria predominates the first, in the ventricles--the second. If the internal changeability of the cardiomyocytes in all the cardiac parts is mainly the same, the individual one--is different (in the atrii it is 2 times as great, in the interventricular septum it is the same as in the atrii). The individual changeability of the cardiomyocytes volume is also greater than that of their size. This also proves the variability of their proportions. The degree of their contractility makes 11--20%, differences between the cardiac parts are not significant. The data obtained could be used as normatives for various experiments.     

Subcellular reorganization of cardiomyocytes during and after temporary experimental hypothermic circulatory arrest

Ultrastructure of cardiomyocytes of the left ventricle has been studied in dogs during the experiments, performed with a general external cooling, prolonged circulatory arrest, as well as during long term periods after the operation. In the experiment without cooling it is not possible to restore hemodynamics after 30 min of total ischemia. In cardiomyocytes severe, sometimes irreversible lytic lesions are registered. Opposite to this, by the end of one hour's cardiac arrest under total cooling up to 24-22 degrees C, changes in ultrastructure of cardiomyocytes are minimal. This is proved by a stabilizing action of hypothermia to membranous, fibrillar and even labile granular cellular components. More manifested changes occur in cells after restoration of the cardiac activity and worming, though even at this stage certain morphological signs of partial restoration of synthetic processes are noted. By the third day after the operation ultrastructure of cardiomyocytes is fully normalized at an essential hypertrophy and hyperplasia of protein synthesis organels and lysosomes. Thus, under conditions of aperfusional hypothermia (24-22 degrees C) and cardiac arrest, produced with pharmacological cooling, cardiomyocytes safely survive one hour's total ischemia, presenting their ability to intracellular regeneration in full.     

Spread of excitation through the heart upon electrical stimulation of the atrioventricular valves]

Experiments were conducted on the isolated hearts of rabbits and dogs. Synchronous recording of the electrical activity of the right auricle, the right ventricle, and tricuspid valve demonstrated the existence of definite functional associations between the auricular, ventricular and valvular depolarization. The spread of excitation in the heart in electrical stimulation of the atrio-ventricular valves was investigated. The impulses from the heterotopic excitation focus localized in these valves could be conducted to the other parts of the heart and thus lead to cardiac arrhythmias.     

Peroxynitrite and myocardial contractility: in vivo versus in vitro effects

Generation of peroxynitrite (ONOO-) as a result of altered redox balance has been shown to affect cardiac function; however, inconsistencies in the data exist, particularly for myocardial contractility. The hypothesis that the cardiac impact of ONOO- formation depends on its site of generation, intravascular or intramyocardial, was examined. Cardiac contractility was assessed by pressure-volume analysis to delineate vascular versus cardiac changes on direct infusion of ONOO- into the right atria of conscious dogs both with normal cardiac function and in heart failure. Additionally, ONOO- was administered to isolated murine cardiomyocytes to mimic in situ cardiac generation. When infused in vivo, ONOO- had little impact on inotropy but led to systemic arterial dilation, likely as a result of rapid decomposition to NO2- and NO3-. In contrast, infused ONOO- was long lived enough to abolish beta-adrenergic (dobutamine)-stimulated contractility/relaxation, most likely through catecholamine oxidation to aminochrome. When administered to isolated murine cardiomyocytes, ONOO- induced a rapid reduction in sarcomere shortening and whole cell calcium transients, although neither decomposed ONOO- or NaNO2 had any effect. Thus, systemic generation of ONOO- is unlikely to have primary cardiac effects, but may modulate cardiac contractile reserve, via blunted beta-adrenergic stimulation, and vascular tone, as a result of generation of NO2- and NO3-. However, myocyte generation of ONOO- may impair contractile function by directly altering Ca2+ handling. These data demonstrate that the site of generation within the cardiovascular system largely dictates the ability of ONOO- to directly or indirectly modulate cardiac pump function.     

Size of cardiomyocytes of the left portion of the heart in adults

As demonstrates the investigation of cardiomyocytes of the left cardiac parts performed in 16 practically healthy mature persons died in accidents, the average area of the cells projections in the auricular atria makes 644 +/- 15 mcm2, and the portion of the multinuclear cells--2.27%. In the ventricular wall and in the papillar muscle these parameters are 831 +/- 16, 700 +/- 30 mcm2 and 11.25, 12.62%, respectively. Distribution of nuclei in the cardiomyocytes of all the cardiac zones studied is characterized by a predominance of tetra-, hypertetra- and octaploid classes, therefore the average amount of DNA in the nuclei reaches certain high values. Differences in size of the cardiomyocytes revealed in various parts of the heart are explained at their unequal loading under pressure.     

Changes in cardiac troponins with gestational age explain changes in cardiac muscle contractility in the sheep fetus

The development of the adult cardiac troponin complex in conjunction with changes in cardiac function and cardiomyocyte binucleation has not been systematically characterized during fetal life in a species where maturation of the cardiomyocytes occurs prenatally as it does in the human. The aim of this study was to correlate the expression of each of the major adult troponin isoforms (T, I, and C) during late gestation (term of 150 days) to changes in both Ca(2+) sensitivity and maximum Ca(2+)-activated force of the contractile apparatus and the maturation of cardiomyocytes. The percentage of mononucleated cardiomyocytes in the right ventricle decreased with gestational age to 46% by 137-142 days of gestation. The length of binucleated cardiomyocytes did not change with gestational age, but the length of binucleated cardiomyocytes relative to heart weight decreased with gestational age. There was no change in the expression of adult cardiac troponin T with increasing gestation. The contractile apparatus was significantly more sensitive to Ca(2+) at 90 days compared with either 132 or 139 days of gestation, consistent with an ～30% increase in the expression of adult cardiac troponin I between 90 and 110 days of gestation. Maximum Ca(2+)-activated force significantly increased from 90 days compared with 130 days consistent with an increase of ～40% in cardiac troponin C protein expression. These data show that increased adult cardiac troponin I and C protein expression across late gestation is consistent with reduced Ca(2+) sensitivity and increased maximum Ca(2+)-activated force. Furthermore, changes in cardiac troponin C, not I, protein expression track with the timing of cardiomyocyte binucleation.     

Analysis of the ultrastructure of atrial cardiomyocytes in rats after alimentary dehydration

By means of water deprivation primary- and secondary-compensated and decompensated degrees of dehydration have been caused in 24 rats. Electron microscopical investigation has demonstrated a more pronounced granularity of cardiomyocytes in the left than in the right cardiac auricles. During the alimentary dehydration granularity of the atrial cardiomyocytes in the right auricles increases at the primary- and secondary-compensated degrees of dehydration, in the stage of decompensated dehydration it decreases, the character of distribution of the secretory granules, however, shows certain processes of exhaustion. Granularity of the atrial cardiomyocytes in the left auricles during alimentary dehydration of the organism does not change. Changes of the endocrine apparatus in the atrial cardiomyocytes occur against the background of microcirculatory disturbances in both auricles of the heart and are combined with dystrophic changes in cardiomyocytes.     

Structural variability of cardiomyocytes of various parts of the heart in its hyperfunction

By means of morphometrical and histological methods hearts of 84 white rats have been studied in 3 months after right-sided pulmonectomy. Cardiac hyperfunction resulted is accompanied with an increased mass of its parts, where hypertrophy of the right ventricle and atrium predominate. Hypertrophy of myocardial parts takes place mainly at the expense of increasing length and width of cardiomyocytes; this causes disorganization and disordering of morphological systems and an essential decrease of compensatory possibilities of the hyperfunctioning heart parts.     

NT-proBNP as a biomarker for the assessment of a potential cardiovascular drug-induced liability in beagle dogs

The amino-terminal pro-brain natriuretic peptide (NT-proBNP) is released into the plasma predominantly from ventricular cardiomyocytes, particularly in patients with chronic cardiac diseases, although small amounts are detectable in the plasma of healthy subjects. While NT-proBNP has been widely exploited in human medicine, limited literature is available related to its characterization in veterinary medicine (e.g., correlation with damage and specificity) and, particularly, in the context of preclinical drug safety assessment. This paper describes the analytical performance characteristics and the biological variability of NT-proBNP in male beagle dogs by using a commercially available enzyme-linked immunosorbent assay. Male beagle dogs were treated with Casopitant, an NK1 receptor antagonist under development for depression and anxiety, which, when administered chronically to dogs, caused cardiac toxicity. Heart weight increase, myocardial necrosis, degeneration, and inflammation associated with high serum levels of cardiac troponin I characterized the end stage pathology observed in dogs treated orally at 40 mg/kg for 39 weeks. Based on these data, ad hoc studies were designed in order to evaluate the possible relationship between NT-proBNP serum levels and both standard toxicology endpoints, such as the organ weight and histology, as well as nonstandard endpoints such as macroscopic morphometry and echocardiography. Early changes of NT-proBNP serum levels were observed following 2 weeks of treatment onward, preceding most, if not all of the anatomical and functional changes. The results obtained demonstrate that NT-proBNP acts as an early biomarker of cardiac changes, representing a sensitive and predictive marker of drug-induced cardiac liability.     

Effects of positive end-expiratory pressure on the right ventricle

Transmural cardiac pressures, stroke volume, right ventricular volume, and lung water content were measured in normal dogs and in dogs with oleic acid-induced pulmonary edema (PE) maintained on positive-pressure ventilation. Measurements were performed prior to and following application of 20 cmH2O positive end-expiratory pressure (PEEP). Colloid fluid was given during PEEP for ventricular volume expansion before and after the oleic acid administration. PEEP significantly increased pleural pressure and pulmonary vascular resistance but decreased right ventricular volume, stroke volume, and mean arterial pressure in both normal and PE dogs. Although the fluid infusion during PEEP raised right ventricular diastolic volumes to the pre-PEEP level, the stroke volumes did not significantly increase in either normal dogs or the PE dogs. The fluid infusion, however, significantly increased the lung water content in the PE dogs. Following discontinuation of PEEP, mean arterial pressure, cardiac output, and stroke volume significantly increased, and heart rate did not change. The failure of the stroke volume to increase despite significant right ventricular volume augmentation during PEEP indicates that positive-pressure ventilation with 20 cmH2O PEEP decreases right ventricular function.     

Chymase Mediates Injury and Mitochondrial Damage in Cardiomyocytes during Acute Ischemia/Reperfusion in the Dog

Cardiac ischemia and reperfusion (I/R) injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI). 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1) and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore-unrecognized chymase entry into cardiomyocytes.     

Myoglobin concentration and the state of myocardial organelles following myocardial infarction]

A study was made of the quantitative changes in myoglobin and the subcellular organization of the myocardium at early periods of experimental infarction of the heart in dogs. There proved to be a correlative relationship between the myoglobin content and the state of subcellular organization of the cardiomyocytes in the ischemic area and the so-called intact portions of the right and the left ventricles.     

On the presence of serotonin in mammalian cardiomyocytes

Pleiotropic effects of serotonin (5-HT) in the cardiovascular system are well documented. However, it remains to be elucidated, whether 5-HT is present in adult mammalian cardiomyocytes. To address this issue, we investigated the levels of 5-HT in blood, plasma, platelets, cardiac tissue, and cardiomyocytes from adult mice and for comparison in human right atrial tissue. Immunohistochemically, 5-HT was hardly found in mouse cardiac tissue, but small amounts could be detected in renal preparations, whereas adrenal preparations revealed a strong positive immunoreaction for 5-HT. Using a sensitive HPLC detection system, 5-HT was also detectable in the mouse heart and human atrium. Furthermore, we could identify 5-HT in isolated cardiomyocytes from adult mice. These findings were supported by detection of the activity of 5-HT-forming enzymes-tryptophan hydroxylase and aromatic L-amino acid decarboxylase-in isolated cardiomyocytes from adult mice and by inhibition of these enzymes with p-chlorophenylalanine and 3-hydroxybenzyl hydrazine. Addition of the first intermediate of 5-HT generation, that is 5-hydroxytryptophan, enhanced the 5-HT level and inhibition of monoamine oxidase by tranylcypromine further increased the level of 5-HT. Our findings reveal the presence and synthesis of 5-HT in cardiomyocytes of the mammalian heart implying that 5-HT may play an autocrine and/or paracrine role in the heart.     

Apoptosis is required for the proper formation of the ventriculo-arterial connections.

The role of apoptosis in cardiac morphogenesis has not been directly tested. Cardiomyocyte apoptosis is prevalent during the remodeling of the embryonic chicken cardiac outflow tract (OFT) in the transition from a single to a dual circulation. We tested the hypothesis that OFT cardiomyocyte apoptosis drives the shortening and rotation of the embryonic cardiac OFT and is required to achieve the mature ventriculo-arterial configuration. Chick embryos were treated with the peptide Caspase inhibitors zVAD-fmk or DEVD-cho at HH stages 15-20 (looped heart). Morphology of control and experimental embryos was assessed at HH stage 35, at which time the control hearts have developed a dual circulation. Infection of the hearts with a recombinant adenovirus expressing green fluorescent protein was used to follow the fate of the OFT cardiomyocytes. Affected embryos displayed abnormal persistence of a long infundibulum (OFT myocardial remnant) beneath the great vessels, indicating failure of OFT shortening. In some instances, the infundibulum connected both great vessels to the right ventricle in a side-by-side arrangement with transposition of the aorta, indicating a failure of rotation of the OFT, and modeling human congenital double outlet right ventricle. Defects were also observed at other sites in the heart where apoptosis is prevalent, such as in the formation of the cardiac valves and trabeculae. To more specifically target the apoptosis of the OFT cardiomyocytes, recombinant adenovirus was used to express the X-linked inhibitor of apoptosis protein in these cells. This resulted in an effect on outflow tract shortening and rotation similar to that of the peptide inhibitors, while the effects on the other cardiac structures were not observed. These results demonstrate that elimination of OFT cardiomyocytes by apoptosis is necessary for the proper formation of the ventriculo-arterial connections, and suggest apoptosis as a potential target of teratogens and genetic defects that are associated with congenital human conal heart defects.     

Haloperidol increases expression of the inositol 1,4,5-trisphosphate receptors in rat cardiac atria, but not in ventricles

Numerous ligands of sigma receptors are known to prolong the QT interval and therefore cause a variety of arrhythmias. High affinity binding sites for the prototypical sigma ligand haloperidol were found in membranes of cardiac myocytes from adult rats. Activation of sigma 1 receptor leads to a release of calcium from the endoplasmic reticulum that follows increased synthesis of inositol 1,4,5-trisphosphate (IP3). We studied the effect of long-term haloperidol treatment on the expression of sigma 1 receptors, IP3 receptors of type 1 and 2 in the individual parts of the rat heart, in isolated rat cardiomyocytes and in PC12 cells. We have found that prolonged treatment with haloperidol significantly increased mRNA levels of sigma 1 receptors in both atria and ventricles. Sigma 1 receptor's mRNA was increased also in isolated cardiomyocytes. Haloperidol treatment affects the expression of IP3 receptors of type 1 and 2 in cardiac atria, but not in cardiac ventricles. We observed increase in IP3 receptors in differentiated PC12 cells, but not in isolated cardiomyocytes. We propose that this increase might participate in triggering cardiac arrhythmias during haloperidol treatment, which has to be further verified.     

Succinate dehydrogenase activity in various parts of the rat heart in systole and diastole

After fixation of the rat heart at the state of systole and diastole, succinate dehydrogenase (SDH) activity has been revealed. In distributions of the muscle fibers according to their optic density, statistic characteristics connected with the first four moments have been taken into consideration. SDH activity is different in five cardiac parts. Individual changeability is more pronounced in the all cardiac parts at diastole. In comparison to the internal one at systole, it is lower in the all parts, and at diastole--in the atria only. The ratio of the internal and individual changeability is in such a state, that it should be taken into account in all statistical calculations, connected with SDH activity determination in cardiomyocytes.     

Right ventricular function in acute myocardial ischaemia

A haemodynamic examination of 10 dogs was carried out at rest, during volume loading and after ligation of the right coronary artery in the presence of a closed pericardium. Ligation of the right coronary artery led to haemodynamic signs of depression of right ventricular function--a drop in systolic pressure and an increase in end diastolic pressure, together with a shift of the functional curve to the right and downwards. Overall performance of the heart (cardiac output and the mean systemic pressure, also fell. Our results show that the depression of the systolic function of the myocardium in the presence of right ventricular infarction can be an important factor in the genesis of low cardiac output syndrome observed in clinical situations. Its pathophysiological mechanisms and some of the clinical consequences are discussed.     

Myocardial reaction of the right ventricle to lung resection

Right-sided pulmonectomy (resection of 63-65% of the lung parenchyma) in white noninbred rats resulted in development of chronic cor pulmonale, that develops according to the stages: I--from the time of the operation up to the 10th-15th days after the operation--the stage of acute disturbances and mobilization forces of the organism; II--from the 11th-15th up to the 90th day is the stage of a relative steady compensatory hypertrophy of the cardiac right ventricle; III--after the 90th day--the stage of decompensation. The hypertrophy of the right ventricle myocardium transfers into its dilatation. Amount of cardiomyocytes and their nuclei in 1 mg of the right ventricle tissue progressively decreases, quantity of multinuclear cardiomyocytes increases, ploidy of the nuclei changes: number of tetraploid nuclei decreases, octaploid nuclei appear. Lethality among the animals is 56%.