Glucose and palmitic acid induce degeneration of myofibrils and modulate apoptosis in rat adult cardiomyocytes.

Several studies support the concept of a diabetic cardiomyopathy in the absence of discernible coronary artery disease, although its mechanism remains poorly understood. We investigated the role of glucose and palmitic acid on cardiomyocyte apoptosis and on the organization of the contractile apparatus. Exposure of adult rat cardiomyocytes for 18 h to palmitic acid (0.25 and 0.5 mmol/l) resulted in a significant increase of apoptotic cells, whereas increasing glucose concentration to 33.3 mmol/l for up to 8 days had no influence on the apoptosis rate. However, both palmitic acid and elevated glucose concentration alone or in combination had a dramatic destructive effect on the myofibrillar apparatus. The membrane-permeable C2-ceramide but not the metabolically inactive C2-dihydroceramide enhanced apoptosis of cardiomyocytes by 50%, accompanied by detrimental effects on the myofibrils. The palmitic acid-induced effects were impaired by fumonisin B1, an inhibitor of ceramide synthase. Sphingomyelinase, which activates the catabolic pathway of ceramide by metabolizing sphingomyeline to ceramide, did not adversely affect cardiomyocytes. Palmitic acid-induced apoptosis was accompanied by release of cytochrome c from the mitochondria. Aminoguanidine did not prevent glucose-induced myofibrillar degeneration, suggesting that formation of nitric oxide and/or advanced glycation end products play no major role. Taken together, these results suggest that in adult rat cardiac cells, palmitic acid induces apoptosis via de novo ceramide formation and activation of the apoptotic mitochondrial pathway. Conversely, glucose has no influence on adult cardiomyocyte apoptosis. However, both cell nutrients promote degeneration of myofibrils. Thus, gluco- and lipotoxicity may play a central role in the development of diabetic cardiomyopathy.

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.

IGF-I and bFGF differentially influence atrial natriuretic factor and alpha-smooth muscle actin expression in cultured atrial compared to ventricular adult rat cardiomyocytes.

In the present study, we compare expression, storage and secretion of the atrial natriuretic factor (ANF) in atrial and ventricular adult rat cardiomyocytes (aARC and vARC) in long-term culture. The influence of insulin-like growth factor-I (IGF-I) and of basic fibroblast growth factor (bFGF) on ANF production and secretion, as well as on the expression of a structural component, alpha-smooth muscle actin (alpha-sm actin), was studied in the two cell types. Antibodies against alpha-ANF were used for immunocytochemical localization of ANF. aARC contained more ANF-granules than vARC, and they were distributed throughout the cell bodies. Quantitative determination of ANF storage and secretion was done by radioimmunoassay (RIA; 125I), and it was demonstrated that aARC stored and secreted ANF 18- and 16-times more, respectively, when compared to vARC. Immuno-electron microscopy confirmed that ANF storing secretory granules were present in both types of cardiomyocytes. Expression of ANF and alpha-sm actin in aARC and vARC responded differently to treatment with either IGF-I or bFGF. In aARC, neither IGF-I nor bFGF had an influence on expression of ANF. In vARC, expression of ANF was downregulated by IGF-I and upregulated by bFGF with regard to both immunoreactivity and message. In contrast to vARC, expression of alpha-sm actin was not affected by IGF-I in aARC, whereas bFGF produced a strong upregulation similar to that found in vARC. Mitogen-activated protein kinases (MAPK) 42 and 44, though, were equally activated by bFGF and IGF-I in both aARC and vARC.

Influence of fibroblast growth factor (bFGF) and insulin-like growth factor (IGF-I) on cytoskeletal and contractile structures and on atrial natriuretic factor (ANF) expression in adult rat ventricular cardiomyocytes in culture.

The effects of basic fibroblast growth factor (bFGF) and of insulin-like growth factor-I (IGF-I) on structural (actin cytoskeleton and myofibrillar apparatus) remodeling and on the expression of atrial natriuretic factor (ANF) in adult rat ventricular cardiomyocytes have been followed during the hypertrophy reaction up to 3 weeks in culture. Cells attach to the substratum spread into polygonal shapes with pseudopodia and resume contractile function after 1 week. A well structured actin cytoskeleton with stress fiber-like structures fills the cell bodies and the extensions. In controls and with IGF-I cells grow to the double volume while bFGF induces a four-fold increase. The myofibrillar apparatus follows the actin stress fiber-like structures in growing out into the cell periphery. Immunoreactive ANF granules develop and are concentrated around the nuclear region. The fetally occurring alpha-smooth muscle actin (alpha-sm-actin) is re-expressed in stress fiber-like structures. IGF-I down-regulates alpha-sm-actin and ANF and promotes myofibrillar growth whereas bFGF has the opposite effect by up-regulating alpha-sm-actin (on average five to six times more than in controls as analysed by immunoblotting) and ANF. In addition, bFGF restricts myofibrillar growth with a sharp boundary in the perinuclear region. The most dense packing of alpha-sm-actin in the cytoskeleton is found just outside the area containing the myofibrils; so alpha-sm-actin seems to restrict myofibrillar assembly and growth. These cells are nevertheless beating like the controls. The relative increase of cytoskeletal structures with the concomitant lack of growth of myofibrils, is mostly due to an increase in alpha-sarcomeric actin (alpha-cardiac and alpha-skeletal muscle actin) and in alpha-sm-actin.

N-cadherin in adult rat cardiomyocytes in culture. I. Functional role of N-cadherin and impairment of cell-cell contact by a truncated N-cadherin mutant.

N-cadherin is a transmembrane Ca(2+)-dependent glycoprotein that is part of adherens junctions. It functions with the cell adhesion N-terminal extracellular domain as a site of homophilic cell-cell contacts. The intracellular C-terminal domain provides via a catenin complex the interaction with the cytoskeleton. Ectopic expression of chicken N-cadherin in adult rat cardiomyocytes (ARC) in culture was obtained after microinjection into non-dividing cardiomyocytes; it was demonstrated that the exogenous protein colocalized with the endogenous N-cadherin at the plasma membrane of the cell and formed contact sites. A dominant negative chicken N-cadherin mutant was constructed by a large deletion of the extracellular domain. This mutant was expressed and inhibited the function of the endogenous rat N-cadherin probably by competing for the catenin complex binding domain, which is essential for the formation of a stable cell-cell contact of ARC. The injected cells lost contact with neighbouring cells and retracted; the connexons of the gap junctions were pulled out as well. This could be avoided by another N-cadherin mutation, which, in addition to the N-terminal truncation, contained a deletion of the catenin binding domain. In the case of the truncated N-cadherin at the N terminus, the sarcomeric structure of the myofibrils of ARC was also affected. Myofibrils were the most vulnerable cytoskeletal structures affected by the overexpressed dominant negative N-cadherin mutation. Similar behaviour was shown when cardiomyocytes separated following Ca2+ depletion and when new cell-cell contacts were formed after Ca2+ replenishment. N-cadherin is thought to be the essential component for establishing new cell-cell contacts which eventually led to a new formation of intercalated disc-like structures in the cardiac cell culture.

N-cadherin in adult rat cardiomyocytes in culture. II. Spatio-temporal appearance of proteins involved in cell-cell contact and communication. Formation of two distinct N-cadherin/catenin complexes.

The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The 'redifferentiation model' of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanisms by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. Appearance of N-cadherin, the catenins and connexin43 within newly formed adherens and gap junctions was studied. Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and alpha-catenin which bind to either beta-catenin or plakoglobin in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized beta-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC. ARC formed a tissue-like pattern in culture whereby the new cell-cell contacts could be dissolved through Ca2+ depletion. Presence of cAMP and replenishment of Ca2+ content in the culture medium not only allowed reformation of cell-cell contacts but also affected the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized beta-catenin over plakoglobin at a particular stage of ARC differentiation. The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the following gap junction formation; a temporal sequence of the appearance of adherens junction proteins and of gap junctions forming connexin-43 is suggested.

Insulin-like growth factor I stimulates myofibril development and decreases smooth muscle alpha-actin of adult cardiomyocytes.

Adult rat cardiomyocytes in long-term culture express type 1 insulin-like growth factor (IGF) receptors. In contrast to insulin receptors, type 1 IGF receptors are up-regulated during culturing. IGF-I added to the cells at plating increased granular density and pseudopodia number per cell after 7 days. After 16 days, IGF-I-treated cells showed, as compared with controls, a dramatic increase of the number of newly built sarcomeres and were packed with myofibrils. At the same time, IGF-I suppressed the accumulation of smooth muscle alpha-actin (sm-alpha-actin) in a dose-dependent manner. Under the conditions of this in vitro system, growth hormone had no effect on cell morphology or sm-alpha-actin. sm-alpha-Actin, a nonsarcomeric isoform of actin expressed in early fetal cardiac development, reappears both during long-term culture of adult rat cardiomyocytes and during heart hypertrophy. This study shows that type 1 IGF receptors are up-regulated in adult rat cardiomyocytes in long-term culture and that IGF-I enhances myofibril development and concomitantly down-regulates sm-alpha-actin. This protein forms stress-fiber-like structures and may temporarily serve as a scaffold for the formation of new sarcomeres until myofibrils have developed throughout the cell and the scaffold is no longer needed. Our findings thus allow us to propose another hypothesis for the mechanism leading to overload heart hypertrophy.

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.

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 cell's vertical axis has been investigated both by optical sectioning using confocal light microscopy and by physical sectioning followed 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.

New occurrence of atrial natriuretic factor and storage in secretorially active granules in adult rat ventricular cardiomyocytes in long-term culture.

Under normal physiological conditions atrial natriuretic factor (ANF) in rat is stored in secretory granules in fetal and adult atrial cardiomyocytes (Cantin et al., 1984; de Bold, 1985), whereas in fetal and neonatal ventricular cardiomyocytes secretory granules are practically absent and ANF is directly secreted via the constitutive pathway (Bloch et al., 1986; Cantin et al., 1987). In ventricular adult rat cardiomyocytes however, ANF is down-regulated and only expressed in cells of interventricular septum (Reinecke, 1989). These ANF-containing cells seem to constitute part of the Purkinje fibre system. As ventricular adult rat cardiomyocytes (ARC) in longterm culture in many ways re-express the fetal program (Eppenberger et al., 1988; Eppenberger-Eberhardt et al., 1990), it was of interest to investigate whether this was also the case for ANF. Data in the present study show that immunoreactive (IR) ANF is upregulated in cultured ventricular ARC and is stored just as in cultured atrial ARC in "atrial-type" granules and secreted into the medium. A possible analogy with overload heart hypertrophy in vivo, where a reactivation of an early gene program including re-expression of ANF in ventricular myocytes has been described (Izumo et al., 1987; Chien et al., 1991), is proposed.

Molecular analysis of protein sorting during biogenesis of muscle cytoarchitecture.

Isolated, rod-shaped adult rat cardiomyocytes (ARC) were kept in long-term cell cultures and the changes of the cardiomyocyte structure were investigated by confocal microscopy. The cells round up and make contact with the substrate by very flat, foot-like structures. After prolonged culture the amorphous cells regenerate a cardiomyocyte-like cytoarchitecture and myofibrils reemerge. In the perinuclear region myofibrils form continuously while in other cells discontinuous myofibrillogenesis was observed, where short sarcomeric segments occur all over the cytoplasmic space. During the regeneration of myofibrils certain proteins like a smooth muscle actin sort to non sarcomeric region, while myomesin or heart C-protein localize on myofibrils with high specificity. This culture system combined with method of epitope-tagging of contractile proteins are ideally suited to monitor the intracellular localization sites of exogenously introduced constructs to different cytoskeletal, since ARC exhibit at the same time stress fiber-like filaments (SFLF) and nascent myofibrils. The molecular properties of the different members of the myosin light chain isoprotein family were investigated by transfection experiments using epitope-tagged myosin light chain (MLC) cDNA. The sorting of the different types of MLC was shown to be isoprotein specific and with chimeric constructs it was shown that the isoprotein-specific incorporation into myofibrils was dependent on the presence of the middle domain of MLC-1f/3f. These MLC isoproteins can be arranged into a sequence of increasing affinity to myofibrils. A hierarchical order of myofibrillar assembly is postulated based on the association affinity. Similar experiments with constructs containing alpha-cardiac, alpha-smooth muscle and gamma-cytoplasmic actins have shown that expression of epitope-tagged actins in ARC result in different epitope staining patterns. While the alpha-cardiac actin showed a marked preference for sarcomeres, the alpha-smooth muscle isoproteins had an intermediate specificity and could either be preferentially incorporated into stress fiber-like filaments (SFLF) and in some cells to a lesser extent into myofibrils as well. Most striking results were obtained with gamma-cytoplasmic actin carrying a 5 or 11-mer epitope. This actin gave rise to large cells, induced the formation of filopodia filled with the transfected actin and depletion of the transfected actin from the perinuclear myofibrillar region.

Adult rat cardiomyocytes cultured in creatine-deficient medium display large mitochondria with paracrystalline inclusions, enriched for creatine kinase.

In adult regenerating cardiomyocytes in culture, in contrast to fetal cells, mitochondrial creatine kinase (Mi-CK) was expressed. In the same cell, two populations of mitochondria, differing in shape, in distribution within the cell and in content of Mi-CK, could be distinguished. Immunofluorescence studies using antibodies against Mi-CK revealed a characteristic staining pattern for the two types of mitochondria: giant, mostly cylindrically shaped, and, as shown by confocal laser light microscopy, randomly distributed mitochondria exhibited a strong signal for Mi-CK, whereas small, "normal" mitochondria, localized in rows between myofibrils, gave a much weaker signal. Transmission EM of the giant mitochondria demonstrated paracrystalline inclusions located between cristae membranes. Immunogold labeling with anti-Mi-CK antibodies revealed a specific decoration of these inclusions for Mi-CK. Addition of 20 mM creatine, the substrate of Mi-CK, to the essentially creatine-free culture medium caused the disappearance of the giant cylindrically shaped mitochondria as well as of the paracrystalline inclusions, accompanied by an increase of the intracellular level of total creatine. Replacement of creatine in the medium by the creatine analogue and competitor beta-guanidinopropionic acid caused the reappearance of the enlarged mitochondria. It is believed that the accumulation of Mi-CK within the paracrystalline inclusions, similar to those observed in certain myopathies, represents a compensatory effect of the cardiomyocytes to cope with a metabolic stress situation caused by low intracellular total creatine levels.

Reexpression of alpha-smooth muscle actin isoform in cultured adult rat cardiomyocytes.

Expression of alpha-smooth muscle (sm) actin in regenerating adult cardiomyocytes in culture was investigated. No alpha-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 alpha-sm-actin in stress fiber-like structures, while newly formed myofibrils contained only little alpha-sm-actin isoprotein. Cell-cell contacts were resumed, but formation of new gap junctions, as revealed by microinjecting Lucifer yellow, was not dependent on alpha-sm-actin expression. The behavior corresponds to fetal cardiomyocytes either in tissue or as single cells in culture where expression of alpha-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 alpha-sm-actin and alpha-sarcomeric actin isoforms during reformation of myofibrillar sarcomeres is discussed.