Myofibrillogenesis and formation of cell contacts mediate the localization of N-RAP in cultured chick cardiomyocytes

The expression of N-RAP was investigated in immuofluorescently stained embryonic chick cardiomyocyte cultures. After 1 day in culture, the cardiomyocytes were spherical and N-RAP, titin, alpha-actinin, and vinculin were all diffusely distributed. As the cardiomyocytes spread and formed myofibrils and cell contacts, N-RAP became localized to distinct areas in the cells. During myofibrillogenesis, N-RAP was found concentrated in premyofibrils. As the premyofibrils transformed into bundles of mature myofibrils, N-RAP became concentrated at the longitundal ends of the cells, and was not found in the mature sarcomeres. At sites of cell-cell contacts, N-RAP was localized to the cell junction even in cells without any significant myofibril formation. As the cell-cell contacts became more extensive and formed structures resembling the intercalated disks found in hearts, N-RAP became even more specifically concentrated at these junctions. The results show that myofibrillogenesis and cell contact formation can each independently target N-RAP to the longitudinal ends of cardiomyocytes.     

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.     

Restriction of placental function alters heart development in the sheep fetus

Placental insufficiency, resulting in restriction of fetal substrate supply, is a major cause of intrauterine growth restriction (IUGR) and increased neonatal morbidity. Fetal adaptations to placental restriction maintain the growth of key organs, including the heart, but the impact of these adaptations on individual cardiomyocytes is unknown. Placental and hence fetal growth restriction was induced in fetal sheep by removing the majority of caruncles in the ewe before mating (placental restriction, PR). Vascular surgery was performed on 13 control and 11 PR fetuses at 110-125 days of gestation (term: 150 +/- 3 days). PR fetuses with a mean gestational Po(2) < 17 mmHg were defined as hypoxic. At postmortem (<135 or >135 days), fetal hearts were collected, and cardiomyocytes were isolated and fixed. Proliferating cardiomyocytes were counted by immunohistochemistry of Ki67 protein. Cardiomyocytes were stained with methylene blue to visualize the nuclei, and the proportion of mononucleated cells and length and width of cardiomyocytes were measured. PR resulted in chronic fetal hypoxia, IUGR, and elevated plasma cortisol concentrations. Although there was no difference in relative heart weights between control and PR fetuses, there was an increase in the proportion of mononucleated cardiomyocytes in PR fetuses. Whereas mononucleated and binucleated cardiomyocytes were smaller, the relative size of cardiomyocytes when expressed relative to heart weight was larger in PR compared with control fetuses. The increase in the relative proportion of mononucleated cardiomyocytes and the relative sparing of the growth of individual cardiomyocytes in the growth-restricted fetus are adaptations that may have long-term consequences for heart development in postnatal life.     

Correction of stress-induced disorders of the ultrastructure of hypertrophied myocardium with thyroid hormones

In the experiment performed on 25 non-inbred male rats ultrastructural changes in cardiomyocytes of the hypertrophied heart have been studied under conditions of stress caused by immobilization and possibility to correct these changes by means of thyroid hormones. The stress intensifies destructive lesions in a number of organelles++, which develop at a prolonged hypertrophy, decreases essentially the ratio mitochondria/myofibrils in section area. Small doses of the thyroid hormones protect the hypertrophied heart from the damaging effect of the stress: prevent the stress-induced+ decrease in the ratio mitochondria/myofibrils, as well as stimulate development of the regenerative-adaptive processes (increase in size and number of mitochondria and their crists, elements of the sarcoplasmic reticulum, glycogen granules, increase in section areas of their nuclei and chromatin in them). The thyroid hormones restrict essentially decrease in correlation of organelles++, resulted from hypertrophy. Thus, the stress-induced disturbances in ultrastructure of the hypertrophied heart can be prevent by means of the thyroid hormones, administered in small doses.     

Contractile activity and cell-cell contact regulate myofibrillar organization in cultured cardiac myocytes

Adult feline ventricular myocytes cultured on a laminin-coated substratum reestablish intercellular junctions, yet disassemble their myofibrils. Immunofluorescence microscopy reveals that these non- beating heart cells lack vinculin-positive focal adhesions; moreover, intercellular junctions are also devoid of vinculin. When these quiescent myocytes are stimulated to contract with the beta-adrenergic agonist, isoproterenol, extensive vinculin-positive focal adhesions and intercellular junctions emerge. If solitary myocytes are stimulated to beat, an elaborate series of vinculin-positive focal adhesions develop which appear to parallel the reassembly of myofibrils. In cultures where neighboring myocytes reestablish cell-cell contact, myofibrils appear to reassemble from the fascia adherens rather than focal contacts. Activation of beating is accompanied by a significant reduction in the rate of total and cytoskeletal protein synthesis; in fact, myofibrillar reassembly, redevelopment of focal adhesions and fascia adherens junctions require no protein synthesis for at least 24 h, implying the existence of an assembly competent pool of cytoskeletal proteins. Maturation of the fasciae adherens and the appearance of vinculin within Z-line/costameres, does require de novo synthesis of new cytoskeletal proteins. Changes in cytoskeletal protein turnover appear dependent on beta agonist-induced cAMP production, but myofibrillar reassembly is a cAMP-independent event. Such observations suggest that mechanical forces, in the guise of contractile activity, regulate vinculin distribution and myofibrillar order in cultured adult feline heart cells.     

Cardiac telocytes — their junctions and functional implications

Telocytes (TCs) form a cardiac network of interstitial cells. Our previous studies have shown that TCs are involved in heterocellular contacts with cardiomyocytes and cardiac stem/progenitor cells. In addition, TCs frequently establish 'stromal synapses' with several types of immunoreactive cells in various organs ( www.telocytes.com ). Using electron microscopy (EM) and electron microscope tomography (ET), we further investigated the interstitial cell network of TCs and found that TCs form 'atypical' junctions with virtually all types of cells in the human heart. EM and ET showed different junction types connecting TCs in a network (puncta adhaerentia minima, processus adhaerentes and manubria adhaerentia). The connections between TCs and cardiomyocytes are 'dot' junctions with nanocontacts or asymmetric junctions. Junctions between stem cells and TCs are either 'stromal synapses' or adhaerens junctions. An unexpected finding was that TCs have direct cell-cell (nano)contacts with Schwann cells, endothelial cells and pericytes. Therefore, ultrastructural analysis proved that the cardiac TC network could integrate the overall 'information' from vascular system (endothelial cells and pericytes), nervous system (Schwann cells), immune system (macrophages, mast cells), interstitium (fibroblasts, extracellular matrix), stem cells/progenitors and working cardiomyocytes. Generally, heterocellular contacts occur by means of minute junctions (point contacts, nanocontacts and planar contacts) and the mean intermembrane distance is within the macromolecular interaction range (10-30 nm). In conclusion, TCs make a network in the myocardial interstitium, which is involved in the long-distance intercellular signaling coordination. This integrated interstitial system appears to be composed of large homotropic zones (TC-TC junctions) and limited (distinct) heterotropic zones (heterocellular junctions of TCs).     

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.     

Re-expression of proteins involved in cytokinesis during cardiac hypertrophy

Cardiomyocytes stop dividing after birth and postnatal heart growth is only achieved by increase in cell volume. In some species, cardiomyocytes undergo an additional incomplete mitosis in the first postnatal week, where karyokinesis takes place in the absence of cytokinesis, leading to binucleation. Proteins that regulate the formation of the actomyosin ring are known to be important for cytokinesis. Here we demonstrate for the first time that small GTPases like RhoA along with their downstream effectors like ROCK I, ROCK II and Citron Kinase show a developmental stage specific expression in heart, with high levels being expressed in cardiomyocytes only at stages when cytokinesis still occurs (i.e. embryonic and perinatal). This suggests that downregulation of many regulatory and cytoskeletal components involved in the formation of the actomyosin ring may be responsible for the uncoupling of cytokinesis from karyokinesis in rodent cardiomyocytes after birth. Interestingly, when the myocardium tries to adapt to the increased workload during pathological hypertrophy a re-expression of proteins involved in DNA synthesis and cytokinesis can be detected. Nevertheless, the adult cardiomyocytes do not appear to divide despite this upregulation of the cytokinetic machinery. The inability to undergo complete division could be due to the presence of stable, highly ordered and functional sarcomeres in the adult myocardium or could be because of the inefficiency of degradation pathways, which facilitate the division of differentiated embryonic cardiomyocytes by disintegrating myofibrils.     

Formation of transversely oriented myofibrils in myocardiocytes in the presence of heart enlargement]

Myocardial hypertrophy was induced in rats and mice by training in the pressure chamber (high altitude hypoxia) or ligation of the apex of the heart. Transversely oriented myofibrils developed in the subsarcolemma area of some cardiomyocytes of the ventricles in a fortnight; the Z bands of the newly-formed myofibrils were connected with the T tubules as in the normal myofibrils. It is supposed that this phenomenon was due to the appearance under the sarcolemma of transversely directed mechanical forrces as a result of enlargement of the cell volume.     

Synemin isoforms differentially organize cell junctions and desmin filaments in neonatal cardiomyocytes

Intermediate filaments (IFs) in cardiomyocytes consist primarily of desmin, surround myofibrils at Z disks, and transmit forces from the contracting myofilaments to the cell surface through costameres at the sarcolemma and desmosomes at intercalated disks. Synemin is a type IV IF protein that forms filaments with desmin and also binds α-actinin and vinculin. Here we examine the roles and expression of the α and β forms of synemin in developing rat cardiomyocytes. Quantitative PCR showed low levels of expression for both synemin mRNAs, which peaked at postnatal day 7. Synemin was concentrated at sites of cell-cell adhesion and at Z disks in neonatal cardiomyocytes. Overexpression of the individual isoforms showed that α-synemin preferentially localized to cell-cell junctions, whereas β-synemin was primarily at the level of Z disks. An siRNA targeted to both synemin isoforms reduced protein expression in cardiomyocytes by 70% and resulted in a failure of desmin to align with Z disks and disrupted cell-cell junctions, with no effect on sarcomeric organization. Solubility assays showed that β-synemin was soluble and interacted with sarcomeric α-actinin by coimmunoprecipitation, while α-synemin and desmin were insoluble. We conclude that β-synemin mediates the association of desmin IFs with Z disks, whereas α-synemin stabilizes junctional complexes between cardiomyocytes.     

Modelling of myocardial hypertrophy in vitro for solving problems of medicinal correction

The work has been done on primary heart culture from neonatal rat ventricle. Cardiomyocyte hypertrophy was modelled using noradrenaline (NA), angiotensin II (AII) and fetal serum, respectively. Cell hypertrophy of primary heart cultures was assessed by measuring the surface area, the scope of protein synthesis estimated by 3H-leucine autoradiography and the contents of nucleic acids in gallocyanin-chromalum stained cardiomyocytes. The structure of myofibrillar apparatus was studied by rhodamine-conjugated phalloidin and indirect immunofluorescence of muscle alpha-actinin. Treatment with 10(-6) M NA increased 3H-leucine incorporation in 9-day old heart culture by 42% without changing cell size. AII in a dose 1 microM stimulated protein synthesis activity by 1.3 fold and the surface area by 1.7 fold, both in 2- and 9-day old primary heart cultures. The maximum stimulation of cell hypertrophy was provided by the medium supplemented with fetal serum. RNA contents in the cytoplasm of cardiomyocytes increased by 7.8 fold and the myocardial cell size by 2.9 fold in serum-supplemented culture by 9 days of cultivation. In the medium with fetal serum, amounts of cardiomyocytes with tetraploid nuclei reached 33%, against 14% in control. Coculturing of myocardiocytes and fibroblasts rendered effects of fetal serum on the growth of myocardiocytes. Cultivation in the presence of 1 microM enalapril, an ACE inhibitor, suppressed the development of cardiac muscle cells hypertrophy. The effect of enalapril depended on the degree of cellular hypertrophy. Addition of 10 microM amiloride to the medium lowered the protein synthesis by 29% independently on the initial cellular hypertrophy.     

Coxsackievirus-Induced miR-21 Disrupts Cardiomyocyte Interactions via the Downregulation of Intercalated Disk Components

Intercalated disks (ICDs) are substantial connections maintaining cardiac structures and mediating signal communications among cardiomyocytes. Deficiency in ICD components such as desmosomes, fascia adherens and gap junctions leads to heart dysfunction. Coxsackievirus B3 (CVB3) infection induces cardiac failure but its pathogenic effect on ICDs is unclear. Here we show that CVB3-induced miR-21 expression affects ICD structure, i.e., upregulated miR-21 targets YOD1, a deubiquitinating enzyme, to enhance the K48-linked ubiquitination and degradation of desmin, resulting in disruption of desmosomes. Inhibition of miR-21 preserves desmin during CVB3 infection. Treatment with proteasome inhibitors blocks miR-21-mediated desmin degradation. Transfection of miR-21 or knockdown of YOD1 triggers co-localization of desmin with proteasomes. We also identified K108 and K406 as important sites for desmin ubiquintination and degradation. In addition, miR-21 directly targets vinculin, leading to disturbed fascia adherens evidenced by the suppression and disorientation of pan-cadherin and α-E-catenin proteins, two fascia adherens-components. Our findings suggest a new mechanism of miR-21 in modulating cell-cell interactions of cardiomyocytes during CVB3 infection.     

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.     

Cardiomyocytes structure, function and associated pathologies

The heart is the first formed organ in the developing fetus. During fetal and postnatal development cardiomyocytes become terminally differentiated muscular cells that are connected end to end by gap junctions, allowing concerted contractile activity. The contraction-relaxation cycle of cardiomyocytes is orchestrated by cyclic increases and decreases in intracellular Ca(2+) initiated by depolarization of the sarcolemma and sustained by Ca(2+) release and re-uptake by the sarcoplasmic reticulum. When stressed, cardiomyocytes undergo hypertrophic growth and apoptotic responses in vivo as well as in cell culture models. Such changes predispose to heart failure in the longer term.     

Apoptosis in the developing human heart resembles apoptosis in epithelial tissues

It is widely accepted that apoptosis plays an important role in the development of the heart as well as in different heart diseases. Despite extensive research efforts, many issues regarding apoptosis in the heart remain unsolved, including the detection of apoptotic cardiomyocytes, their morphological features, the mechanisms of their removal and the clinical significance of apoptosis in the heart. It has been suggested that fetal cardiomyocytes resemble epithelial tissues. To test this hypothesis, we analyzed the expression of an epithelial marker cytokeratin 18 (CK18) and caspase-cleaved-CK18, recognized by antibody M30, as well as the expression of cleaved caspase-3 and desmosomal and classical cadherins, major components of desmosomes and adherens junctions in fetal hearts in comparison to infant and adult human hearts. We found that, in fetal hearts, cardiomyocytes expressed CK18 and that apoptotic cardiomyocytes expressed caspase-cleaved CK18, being recognized by antibody M30. Furthermore, desmosomal and classical cadherins exhibited a membraneous reaction similar to epithelial tissues. In adults and children after the age of 6 months, cadherins were localized in the intercalated disks, cardiomyocytes lost CK18 expression and apoptotic cardiomyocytes were no longer recognized by M30. We conclude that apoptosis in the developing human heart resembles apoptosis in epithelial tissues, exhibiting different characteristics than in the adult human heart.     

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.     

Relationship between cardiac protein tyrosine phosphorylation and myofibrillogenesis during axolotl heart development

The axolotl, Ambystoma mexicanum, is a useful system for studying embryogenesis and cardiogenesis. To understand the role of protein tyrosine phosphorylation during heart development in normal and cardiac mutant axolotl embryonic hearts, we have investigated the state of protein tyrosine residues (phosphotyrosine, P-Tyr) and the relationship between P-Tyr and the development of organized sarcomeric myofibrils by using confocal microscopy, two-dimensional isoelectric focusing (IEF)/SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting analyses. Western blot analyses of normal embryonic hearts indicate that several proteins were significantly tyrosine phosphorylated after the initial heartbeat stage (stage 35). Mutant hearts at stages 40-41 showed less tyrosine phosphorylated staining as compared to the normal group. Two-dimensional gel electrophoresis revealed that most of the proteins from mutant hearts had a lower content of phosphorylated amino acids. Confocal microscopy of stage 35 normal hearts using phosphotyrosine monoclonal antibodies demonstrated that P-Tyr staining gradually increased being localized primarily at cell-cell boundaries and cell-extracellular matrix boundaries. In contrast, mutant embryonic hearts showed a marked decrease in the level of P-Tyr staining, especially at sites of cell-cell and cell-matrix junctions. We also delivered an anti-phosphotyrosine antibody (PY 20) into normal hearts by using a liposome-mediated delivery method, which resulted in a disruption of the existing cardiac myofibrils and reduced heartbeat rates. Our results suggest that protein tyrosine phosphorylation is critical during myofibrillogenesis and embryonic heart development in axolotls.     

Proliferation of cardiomyocytes and interstitial cells in the cardiac muscle of the mouse during pre- and postnatal development

Proliferation of cardiomyocytes and interstitial cells in the cardiac ventricle of the mouse during pre- and postnatal development was studied. Furthermore, the number of cardiomyocyte and interstitial cell nuclei per unit area was determined on histological sections. The labelling index of cardiomyocytes decreases from 23% on day 14 of gestation to about zero at 3 weeks after birth. The number of cardiomyocyte nuclei per unit area increases up to day 16 of gestation and then continuously declines. This coincides with the concept that the increase in size of the heart during early fetal life is mainly due to hyperplasia, while during late fetal life and after birth it is mainly, and during adult life exclusively, due to hypertrophy of cardiomyocytes. Proliferation of interstitial cells continues up to 5 days after birth and then decreases. The ratio of cardiomyocytes to interstitial cells decreases by a factor of about 10 between day 14 of gestation and 3 weeks after birth.     

Proliferation of Cardiomyocytes and Interstitial Cells In the Cardiac Muscle of the Mouse During Pre- and Postnatal Development

Proliferation of cardiomyocytes and interstitial cells in the cardiac ventricle of the mouse during pre- and postnatal development was studied. Furthermore, the number of cardiomyocyte and interstitial cell nuclei per unit area was determined on histological sections. The labelling index of cardiomyocytes decreases from 23% on day 14 of gestation to about zero at 3 weeks after birth. the number of cardiomyocyte nuclei per unit area increases up to day 16 of gestation and then continuously declines. This coincides with the concept that the increase in size of the heart during early fetal life is mainly due to hyperplasia, while during late fetal life and after birth it is mainly, and during adult life exclusively, due to hypertrophy of cardiomyocytes. Proliferation of interstitial cells continues up to 5 days after birth and then decreases. the ratio of cardiomyocytes to interstitial cells decreases by a factor of about 10 between day 14 of gestation and 3 weeks after birth.