Monika Eppenberger-Eberhardt

Reexpression of α-smooth muscle actin isoform in cultured adult rat cardiomyocytes☆

Abstract Expression of α-smooth muscle (sm) actin in regenerating adult cardiomyocytes in culture was investigated. No α-sm-actin could be detected in adult ventricular tissue or in newly dissociated rod-shaped cells, whereas a fraction of the polymorphic flattened out adult cardiac cells in culture did express the protein. Immunofluorescence studies revealed a characteristic staining pattern, suggesting the preferential presence of α-sm-actin in stress fiber-like structures, while newly formed myofibrils contained only little α-sm-actin isoprotein. Cell-cell contacts were resumed, but formation of new gap junctions, as revealed by microinjecting Lucifer yellow, was not dependent on α-sm-actin expression. The behavior corresponds to fetal cardiomyocytes either in tissue or as single cells in culture where expression of α-sm-actin can be observed. Such immunofluorescence staining patterns with corresponding immunoblot data can be expected when a return to a less differentiated, more fetal state of the adult cardiomyocyte in culture is assumed. The possible role of the α-sm-actin and α-sarcomeric actin isoforms during reformation of myofibrillar sarcomeres is discussed.

Remodelling of cardiomyocyte cytoarchitecture visualized by three-dimensional (3D) confocal microscopy

The break-down and reassembly of myofibrils in long-term cultures of adult rat cardiomyocytes was investigated by a novel combination of confocal laser scanning microscopy and three-dimensional image reconstruction, referred to as FTCS, to visualize the morphological changes these cells undergo in culture. FTCS is discussed as an alternative imaging mode to low-magnification scanning electron microscopy. The three-dimensional shape of the cells are correlated with the assembly state of myofibrils in different stages. Based on immunofluorescence and confocal laser scanning microscopy it was shown that myofibrils are degraded within a few days after plating and that newly assembled myofibrils are predominantly confined to the continuous area in the perinuclear region close to the membrane in contact with the substratum. The localization of myofibrils along the cells vertical axis has been investigated both by optical sectioning using confocal light microscopy and by physical sectioning following by transmission electron microscopy. Based on the distribution of myofibrillar proteins we propose a model of myofibrillar growth locating the putative assembly sites to a region concentric around the nuclei. We provide evidence that the cell shape is dominated by the myofibrillar apparatus.

N-Cadherin: structure, function and importance in the formation of new intercalated disc-like cell contacts in cardiomyocytes.

N-cadherin belongs to a superfamily of calcium-dependent transmembrane adhesion proteins. It mediates adhesion in the intercalated discs at the termini of cardiomyocytes thereby serving as anchor for myofibrils at cell-cell contacts. A large body of data on the molecular structure and function of N-cadherin exists, however, little is known concerning spatial and temporal interactions between the different junctional structures during formation of the intercalated disc and its maturation in postnatal development. The progression of compensated left ventricular hypertrophy to congestive left heart failure is accompanied by intercalated disc remodeling and has been demonstrated in animal models and in patients. The long-term culture of adult rat cardiomyocytes allows to investigate the development of de novo intercalated disc-like structures. In order to analyze the dynamics of the cytoskeletal redifferentiation in living cells, we used the expression of chimeric proteins tagged with the green fluorescent protein reporter. This technique is becoming a routine method in basic research and complements video time-lapse and confocal microscopy. Cultured cardiomyocytes have been used for a variety of studies in cell biology and pharmacology. Their ability to form an electrically coupled beating tissue-like network in culture possibly allows reimplantation of such cells into injured myocardium, where they eventually will form new contacts with the healthy muscle tissue. Several groups have already shown that cardiomyocytes can be grafted successfully into sites of myocardial infarcts or cryoinjuries. Autologous adult cardiomyocyte implantation, might indeed contribute to cardiac repair after infarction, thanks to advances in tissue engineering.

Adult rat cardiomyocytes in culture: A model system to study the plasticity of the differentiated cardiac phenotype at the molecular and cellular levels

Adult rat cardiomyocytes (ARCs) in long-term culture, which show a distinct adaptive flexibility, are presented as a system to study cardiac cell hypertrophy in vitro. In the first 1-2 weeks after isolation, ARCs undergo a process of de- and redifferentiation during which the cell morphology is remodeled and the myofibrillar apparatus is restructured, accompanied by a cell enlargement. The growing cells spread and eventually establish new cell-cell contacts, which display newly formed intercalated discs; synchronous cell beating is resumed in the resulting tissuelike sheet. During myofibrillogenesis, the early fetal program of gene expression is reactivated for several genes, as is observed during hemodynamic overload hypertrophy. The cells resume hormonal activity and express atrial natriuretic factor (ANF); the expression pattern of ANF is also reminiscent of that seen in hypertrophy. In cells grown in a medium conditioned by 12-day ARCs, though, myofibrillogenesis is accelerated and accompanied by a downregulation of ANF. In a creatine-deficient medium, on the other hand, the ARCs display giant mitochondria with paracrystalline inclusions imitating a situation found, for example, in mitochondrial myopathies.

Cellular engineering of ventricular adult rat cardiomyocytes

OBJECTIVE Preparation of viable cultured adult cardiomyocytes (vARCs) is a prerequisite for cell-based transplantation and tissue engineering. Ectopic gene expression is important in this context. Here, we present an in vitro cell replating strategy using Accutase for cultured vARCs, allowing ectopic gene expression. METHODS Cultured vARCs from 6- to 8-week-old rats were used. Transfections with EGFP (enhanced green fluorescent protein) constructs, Mlc-3f-EGFP or alpha-actinin-EGFP were performed using adenovirus-enhanced transferrin-mediated infection (AVET). Accutase (PAA Laboratories, Linz, Austria) was used for the detachment of cultured cells. Immunohistochemical analysis, together with confocal laser microscopy was used for structural analysis of the cells. RESULTS Cultured vARCs could be detached with a high yield (40 to 60%) from primary cultures using Accutase. The cultivation period plays an important role in the yield of viable cells. Resultant replated vARCs (rep-vARCs) rapidly (1-2 h) acquired a rounded up shape without degradation of their contractile apparatus, which is in contrast to the rod-shaped freshly isolated vARCs (fi-vARCs). The detached cells survived passage through a narrow syringe needle. After seeding, detached cells rapidly attached to various substrates, increased their content of the contractile apparatus, and formed cell-cell contacts within 3 days after reseeding. The detached cells survived passage through a narrow syringe needle. The high recovery of cells after replating enabled the use of the AVET system for gene delivery. AVET is free of infectious particles and does not lead to expression of viral proteins. Transfection of vARCs prior to detachment had a small effect on cell recovery and ectopically synthesized proteins were properly localized after replating. CONCLUSIONS Detachment of cultured vARCs using Accutase is well compatible with ectopic gene expression and yields a viable transgenic population of vARCs that eventually may be suitable as transgenic cardiomyocyte grafts.

Cytoskeletal Rearrangements in Adult Rat Cardiomyocytes in Culture

Growth of the heart in maturing mammals is primarily the result of hypertrophy of a pre-existing, fixed number of cardiomyocytes rather than cellular proliferation. Shortly after birth in mammals, ventricular myocytes rapidly and permanently leave the cell cycle and only infrequently undergo additional rounds of DNA replication associated with the process of endomitosis leading to binucleation, e . g . , in rat heart cells. Subsequently there begins a process of cellular hypertrophy of ventricular heart muscle cells until the adult heart size is reached. Adaptive compensatory hypertrophy of the adult heart on the other hand usually follows the increase in work load that has been imposed on the heart. Such a greater workload is mainly due to an increased afterload in patients with hypertension or after myocardial infarction, when the remaining muscle has to assume the work of that part of the myocard that was lost through myocardial cell death. The question arises as to how the involved cells do react to compensate for the increased work they have to produce. What are the signals required to transduce increased workload into cell growth? Is there, besides neuronal and hormonal influence, an intracellular signal generating mechanism based on, e . g . , mechanical stimuli? Stretch appears to induce autocrine and paracrine mechanisms which may generate those increased rates of protein synthesis or may be responsible for those decreased rates of protein degradation which are necessary to cope with an increasing size of cardiomyocytes.’ The cellular junctions between single cardiomyocytes, the so-called intercalated discs, as well as the interconnecting cytoskeleton and the contractile apparatus are also strongly involved when the size of cardiomyocytes increases. Since the complexity of the events that accompany hypertrophy in vivo imposes many limitations on studying interactions between single cardiomyocytes, more and more, isolated cardiac muscle cells are used when elaborating hypertrophic events.2 Isolated adult rat cardiomyocytes (ARC) in culture represent an excellent experimental system to monitor changes in the molecular and cellular behavior of cardiac cells. In addition, they allow the study of a number of fundamental problems in cardiology as well as of the differentiation of cross-striated muscle in general. ARC can generally be cultured in two different ways: first, in the “rapid attachment model” where the cells are kept in serum-free medium and where they maintain characteristic morphological features of intact, rod-shaped cells for about