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.

Alterations at the intercalated disk associated with the absence of muscle LIM protein.

In this study, we investigated cardiomyocyte cytoarchitecture in a mouse model for dilated cardiomyopathy (DCM), the muscle LIM protein (MLP) knockout mouse and substantiated several observations in a second DCM model, the tropomodulin-overexpressing transgenic (TOT) mouse. Freshly isolated cardiomyocytes from both strains are characterized by a more irregular shape compared with wild-type cells. Alterations are observed at the intercalated disks, the specialized areas of mechanical coupling between cardiomyocytes, whereas the subcellular organization of contractile proteins in the sarcomeres of MLP knockout mice appears unchanged. Distinct parts of the intercalated disks are affected differently. Components from the adherens junctions are upregulated, desmosomal proteins are unchanged, and gap junction proteins are downregulated. In addition, the expression of N-RAP, a LIM domain- containing protein located at the intercalated disks, is upregulated in MLP knockout as well as in TOT mice. Detailed analysis of intercalated disk composition during postnatal development reveals that an upregulation of N-RAP expression might serve as an early marker for the development of DCM. Altered expression levels of cytoskeletal proteins (either the lack of MLP or an increased expression of tropomodulin) apparently lead to impaired function of the myofibrillar apparatus and to physiological stress that ultimately results in DCM and is accompanied by an altered appearance and composition of the intercalated disks.

Mass production of embryoid bodies in microbeads.

Embryonic stem cells (ESC) are totipotent cells that can differentiate into a large number of different cell types. Stem cell-derived, differentiated cells are of increasing importance as a potential source for non-proliferating cells (e.g., cardiomyocytes or neurons) for future tissue engineering applications. Differentiation of ESC is initiated by the formation of embryoid bodies (EB). Current protocols for the generation of EB are either of limited productivity or deliver EB with a large variation in size and differentiation state. To establish an efficient and robust EB production process, we encapsulated mouse ESC into alginate microbeads using various microencapsulation technologies. Microencapsulation and culturing of ESC in 1.1% alginate microbeads gives rise to discoid colonies, which further differentiate within the beads to cystic EB and later to EB containing spontaneously beating areas. However, if ESC are encapsulated into 1.6% alginate microbeads, differentiation is inhibited at the morula-like stage, so that no cystic EB can be formed within the beads. ESC colonies, which are released from 1.6% alginate microbeads, can further differentiate to cystic EB with beating cardiomyocytes. Extended supplementation of the growth medium with retinoic acid promotes differentiation to smooth muscle cells.

Recombinant Sindbis virus allows expression and precise targeting of proteins of the contractile apparatus in cultured cardiomyocytes.

Expression of epitope-tagged sarcomeric proteins in cardiomyocytes is a powerful approach for the characterization of interacting domains. Here, we report a new strategy for the study of the targeting of contractile proteins in cardiomyocytes by Sindbis virus (SIN)-mediated gene transfer. Two recombinant SIN were generated, one encoding the myosin-light chain MLC3f-eGFP fusion protein (SINrep5/MLC3f-eGFP), and the other encoding the alpha-actinin-DsRed fusion protein (SINrep5/alpha-actinin-DsRed). After infection of long-term cultured neonatal and adult rat cardiomyocytes with SINrep5/MLC3f-eGFP, the exogenous MLC3f-eGFP fusion protein localized to the sarcomeres. Freshly isolated rod-shaped ventricular cardiomyocytes infected with SINrep5/alpha-actinin-DsRed exhibited a correct incorporation of the newly synthesized alpha-actinin-DsRed fusion protein at the Z-band of the sarcomere. This allows the assumption that the exogenous protein is assembled into myofibrils in living cardiomyocy-tes using the same molecular interactions equally to the endogenous counterpart. It has been thus demonstrated that the SIN expression system makes possible the straightforward analysis of the localization of sarcomeric proteins in cultured cardiomyocytes and may offer new possibilities for the characterization of mutant proteins involved in hypertrophic cardiomyopathies.

The armadillo repeat region targets ARVCF to cadherin-based cellular junctions.

The cytoplasmic domain of the transmembrane protein M-cadherin is involved in anchoring cytoskeletal elements to the plasma membrane at cell-cell contact sites. Several members of the armadillo repeat protein family mediate this linkage. We show here that ARVCF, a member of the p120 (ctn) subfamily, is a ligand for the cytoplasmic domain of M-cadherin, and characterize the regions involved in this interaction in detail. Complex formation in an in vivo environment was demonstrated in (1) yeast two-hybrid screens, using a cDNA library from differentiating skeletal muscle and part of the cytoplasmic M-cadherin tail as a bait, and (2) mammalian cells, using a novel experimental system, the MOM recruitment assay. Immunoprecipitation and in vitro binding assays confirmed this interaction. Ectopically expressed EGFP-ARVCF-C11, an N-terminal truncated fragment, targets to junctional structures in epithelial MCF7 cells and cardiomyocytes, where it colocalizes with the respective cadherins, beta-catenin and p120 (ctn). Hence, the N terminus of ARVCF is not required for junctional localization. In contrast, deletion of the four N-terminal armadillo repeats abolishes this ability in cardiomyocytes. Detailed mutational analysis revealed the armadillo repeat region of ARVCF as sufficient and necessary for interaction with the 55 membrane-proximal amino acids of the M-cadherin tail.

Lack of nuclear apoptosis in cardiomyocytes and increased endothelin-1 levels in a rat heart model of myocardial stunning.

OBJECTIVE: Reperfusion injury may affect the cardiac NO and endothelin production. We investigated whether 20 min of total ischemia followed by 40 min of reperfusion can induce apoptosis in a Langendorff model of retrogradely perfused rat hearts (37 degrees C; paced at 300/'), and we attempted to correlate these findings with measured tissue NO and ET-1 levels. METHODS: An apoptosis detection system was utilized which catalytically incorporates fluorescein-12-dUTP at the 3'-OH DNA ends using the principle of the TUNEL assay, with direct visualization of the labeled DNA. ET-1 was measured by radioimmunoassay and NO3/NO2 by ion pairing HPLC on C18 reverse phase columns. RESULTS: None of the postischemic (n = 6) nor of the control perfused (90 min, n = 6) hearts showed signs of apoptosis, while those exposed to longer ischemia (40 min) and reperfusion (2 h) confirmed the presence of apoptotic cells. Myocardial ET-1 concentrations were 4.8 +/- 1.0 versus 8.3 +/- 2.5 pg/100 mg (control vs. ischemic hearts, respectively; mean +/- SD; p < 0.05). Myocardial NO contents showed no differences. CONCLUSION: These data suggest that the time window of apoptosis with detectable DNA fragmentation exceeds 20 min of global total ischemia and 40 min of reperfusion, a model frequently used for inducing myocardial stunning. While NO was not increased in postischemic hearts, increased ET-1 levels indirectly argue for a role of ET-1 as inducer of apoptosis, but only at a later stage of reperfusion.

Dynamics of early contact formation in cultured adult rat cardiomyocytes studied by N-cadherin fused to green fluorescent protein.

We investigated dynamic events during the formation of intercalated disc-like structures of adult rat cardiomyocytes (ARC) in long-term culture. Given the complexity of ARC cytoIarchitecture after de- and re-differentiation, and the non-uniform morphological development of individual cells, green fluorescent protein (GFP) technology was used to track N-cadherin in living cells. Sorting and functionality of the GFP fusion protein was tested in ARC. Isolated ARC were micro-injected with the expression construct at the onset of spreading in culture, and the fluorescence signals were tracked during contact formation and in fully redifferentiated living cells. The first contact sites were found to be established by cellular protrusions, which were marked by an ultrastructure similar to microspikes and probably have a role as exploratory units in the spreading phase. Subsequently, initial contact sites served as anchorage for the most prominent stress fibre-like structures. The fusion protein appeared before connexin-43 at newly established cell-cell contacts. Membrane invaginations at the sarcolemma facing the substratum of cultured ARC may be responsible for the appearance of a striped pattern of N-cadherin and other adherens junction proteins away from intercalated disc-like structures. The stripes were immobile in redifferentiated cells, while the distinct small fluorescent particles in the cell body were found to move directionally at speeds around 10 micro m/min. These results contribute to the understanding of the mechanisms of cell-cell contact formation of adult cardiomyocytes, which is a prerequisite for any future implantation technology.

Titanium dioxide ceramics control the differentiated phenotype of cardiac muscle cells in culture.

A new approach, the cultivation of heart muscle cells on biocompatible scaffolds made from titanium dioxide ceramics was established to provide a mechanism for in vitro engineering of a vital heart tissue. Terminally differentiated ventricular myocytes isolated from hearts of adult rats were kept in primary culture for long periods of time and used as an experimental model. The microenvironmental properties of titanium dioxide ceramics helped to maintain the tissue-like structural organisation of the cardiac cells in vitro. Coating of the cell substrata with fine-grained titanium dioxide ceramics imitating cell surface topography favoured the formation of focal adhesion complexes in the ventral plasma membrane of cardiomyocytes. It also promoted the cellular expression of vinculin, a protein that connects the ECM integrin receptors to the network of cytoplasmic filaments, which define cell shape. This topographical reinforcement of cell-material interactions led to stabilisation of the molecular linkage between the extracellular contacts and the intracellular cytoskeleton and thus assisted the preservation and maintenance of the heart muscle cell differentiated phenotype in long-term primary culture. The results of this work demonstrate a promising pathway for the regulation of cellular organisation in vitro by local geometric control.

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.

A novel autoregulated proliferation-controlled production process using recombinant CHO cells.

Controlled proliferation bioprocesses have shown great enhancement of heterologous protein production. This novel technology has been implemented here using a multicistronic expression unit encoding the product gene and a cytostatic cell-cycle-arresting gene (p27) under control of a single tetracycline-repressible (tet(off)) promoter. The strict genetic linkage of both genes allows the dissection of the production process into a nonproductive growth phase (dicistronic expression unit repressed) followed by a proliferation-inhibited production phase (dicistronic expression unit induced) when the cells have reached an optimal cell density. Based on rapid degradation of the external repressible agents tetracycline (tet) and doxycycline (dox) in the cell culture medium, we developed a self-regulated process for transition from the growth phase to the production phase in a fashion that is dependent only on the starting cell population and the initial concentration of the tetracyclines. With this process, no change in medium is required to accomplish the transition from growth to production phase. The two-phase bioprocess achieved here by tet switch-controlled proliferation is reliable and allows a growth-arrested production phase of at least 7 days, during which cells remain in a well-defined, highly viable physiological state and show enhanced heterologous protein production. This Tet(SWITCH) process is readily adaptable to a variety of industrial processes designed for production of difficult-to-express protein pharmaceuticals.

In vitro reestablishment of cell-cell contacts in adult rat cardiomyocytes. Functional role of transmembrane components in the formation of new intercalated disk-like cell contacts.

Primary adult rat cardiomyocytes (ARC)in culture are shown to be a model system for cardiac cell hypertrophy in vitro. ARC undergo a process of morphological transformation and grow only by increase in cell size, however, without loss of the cardiac phenotype. The isolated cells spread and establish new cell-cell contacts, eventually forming a two-dimensional heart tissue-like synchronously beating cell sheet. The reformation of specific cell contacts (intercalated disks) is shown also between ventricular and atrial cardiomyocytes by using antibodies against the gap junction protein connexin-43 and after microinjection into ARC of N-cadherin cDNA fused to reporter green fluorescent protein (GFP) cDNA. The expressed fusion protein allowed the study of live cell cultures and of the dynamics of the adherens junction protein N-cadherin during the formation of new cell-cell contacts. The possible use of the formed ARC cell-sheet cells under microgravity conditions as a test system for the reformation of the cytoskeleton of heart muscle cells is proposed.

Flow cytometric detection of p53 protein after incubation of a pre-B cell line with antitumor agents.

BACKGROUND: The study of new substances capable of counteracting tumor development has focused, in recent years, on several of the steps in a cell's initiation of the process of apoptosis. One of the crucial events is the activation of p53, leading to a cell cycle G1/S block or to programmed cell death. METHODS: We report here a parallel flow cytometric method for semiquantitative detection of p53 protein and apoptosis (percent of apoptotic cells) in a pre-B leukemic cell line (NALM-6) exposed to various antitumor agents (2.35 microg/ml etoposide; 0.175 microg/ml FCE296; 0.4 microg/ml FCE624; and 1.5 microg/ml L-PAM). RESULTS: All of the substances proved to be capable of inducing an increase of p53 after 16 or 24 h of incubation. In all experiments with antitumor agents we also found an onset of apoptosis after 24 h of incubation with the substance, as determined by the annexin V flow cytometric assay and by DNA fragmentation. CONCLUSIONS: This technique, based on flow cytometric data of both p53 intracellular content and percentage of apoptotic cells, is suitable to determine the amount of antitumor agent needed to induce p53, and thus to dose the drug in relation to the sensitivity of a defined tumor as well as choose the more efficacious drug, depending on cell responsiveness. The study of antitumor substances that induce apoptosis, bypassing p53, could also be evaluated by this method, in view of the development of substances for the treatment of p53-mutated tumors.

Efficient gene delivery into adult cardiomyocytes by recombinant Sindbis virus.

Somatic gene therapy as a potential strategy for the treatment of myocardial diseases relies on an efficient gene transfer into cardiac muscle cells. The difficulty of delivering genes into adult cardiomyocytes exists not only in vivo but also in primary culture systems. Therefore, possibilities for ex vivo gene transfer and the in vitro study of physiological processes by reverse genetics are limited. We investigated the potential of an alphavirus-based vector system to transduce adult rat cardiomyocytes (ARC) in culture using a replication-deficient Sindbis virus (SIN) encoding beta-galactosidase (SIN-LacZ). Transduction efficiency depended on the virus concentration used, with expression of the reporter gene being detectable in up to 80% of cultured ARC as early as 24 h after infection. We observed a remarkably lower cytotoxicity of this viral vector in ARC than in other cells such as fibroblasts and neonatal cardiomyocytes. Additionally, no perceptible changes in the morphology of the nuclei or cytoskeleton were found in ARC 48 h after infection with SIN-LacZ. We conclude that SIN vectors are useful for gene delivery into adult cardiomyocytes and believe that improved versions of this viral system may be useful for cardiovascular gene therapy in the future.

Triiodothyronine restricts myofibrillar growth and enhances beating frequency in cultured adult rat cardiomyocytes.

Adult rat cardiomyocytes (ARC) isolated from ventricles follow a defined sequence of structural remodeling during culturing for 2-3 weeks. Rod-shaped cells round up, attach to the substratum, and start growing out in all directions until they form contacts with one another and resume rhythmic contractile activity. In general, myofibrils redevelop along the actin scaffold into the periphery. IGF-I enhances this process while bFGF restricts the outgrowing of myofibrils to the central cell area. Presence of T3 in the culture medium also restricts myofibrillar growth like bFGF. At the same time, T3 increases spontaneous beating frequency in a dose-dependent manner. With 10 nM T3 beating frequency is increased three-fold versus control. Addition of isoproterenol or of epinephrine further increases the frequency at all T3 concentrations tested. Propranolol inhibits the fully stimulated beating frequency to about the same extent at all T3 concentrations. Therefore, T3 seems to determine the beating frequency of ARC in culture directly and not by changing the composition of the adrenoceptor population nor by changing their responsiveness.

Differential protein localization in sarcomeric and nonsarcomeric contractile structures of cultured cardiomyocytes.

The use of cardiomyocyte cell culture models allows the identification of various cell mediators that bring about changes in subcellular structures and gene expression associated with hypertrophy. The effects of insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), and triiodothyronine (T3) on gene expression and on the structural organization of myofibrillar and cytoskeletal proteins were compared in adult atrial (aARC) and ventricular (vARC) as well as in neonatal ventricular rat cardiomyocytes (vNRC) in long-term culture. Structural changes were evaluated by confocal microscopy and correlated to biochemical alterations. In vARC, IGF-I enhanced myofibrillar growth, whereas bFGF or T3 restricted sarcomere assembly to the central cell area, forming a sharp boundary in more than 50% of the cells. However, myosin occurred both in the cross-striated myofibrillar structures and in patches running along the nonsarcomeric fibrillar structures (also called stress fiber-like structures) in the cell periphery. In cells treated with either bFGF or T3, the expression of alpha-smooth muscle actin (alpha-sm actin) was greatly increased. This actin isoform was incorporated mainly into the nonsarcomeric contractile structures outside the area where myofibrils ended abruptly. alpha-sm actin protein increased up to 14- to 17-fold while the mRNA showed a moderate increase of 2- to 4-fold. This suggests that alpha-sm actin is mainly regulated at the translational or posttranslational level. In contrast, the cytoskeletal proteins alpha-actinin and vinculin increased only moderately (less than 2-fold) but also showed a relocalization in cells with restricted myofibrils. In aARC and in vNRC, alpha-sm actin was only moderately upregulated by bFGF or T3 and no drastic morphological changes were observed. In conclusion, IGF-I, bFGF, and T3 induced characteristic structural phenotypes depending on the type of cardiomyocyte. Large amounts of alpha-sm actin as expressed in bFGF and T3 treated vARC seem to be incompatible with sarcomere assembly.

A novel flow cytometric method for the quantification of p53 gene expression.

We describe a rapid and simple flow cytometry technique for the detection and quantification of p53 in several human cell lines, including an adenocarcinoma cell line (SW 626) having a mutant (m) p53, and a pre-B leukemia cell line (NALM-6) having wild-type (wt) p53. By introducing a second antibody coupled to RPE-fluorescence, the discrimination between control and specific peaks was improved over that achieved with methods used previously. To quantify the content of p53 molecules in the cells, we used a series of beads with the capability to bind mouse monoclonal IgG antibodies. p53 cell content, expressed as antibody binding capacity (ABC), was directly quantified from logarithmic scattergrams; the results were reproducible in all cell lines tested. Flow cytometric results were compared with those of a standard immunocytochemistry method routinely used for the detection of p53 in cells, and found to be in correlation. Furthermore, cytometric data also reflect ELISA determinations. In summary, we showed for the first time that (i) p53 can be clearly detected by flow cytometry in various cell lines, (ii) p53 can be quantitated in terms of number of molecules per cell, and (iii) it can be easily monitored as a function of time after stimulation.

Investigation of protein patterns in mammalian cells and culture supernatants by matrix-assisted laser desorption/ionization mass spectrometry.

The direct protein profiling of mammalian cells and bacteria has a growing influence in biotechnology as a high information bearing method for characterization of cells and cell states. Monitoring of proteins excreted in culture media not only serves to produce data on product yield and quality but provides important information on cell viability and nutrient supply that forms the basis for future process and expression optimization. Fast and simple MALDI mass spectrometry approaches were developed to efficiently characterize such complex biological systems. Several mammalian cell lines including CHO DXB11, CHOSSF3, and hybridomas were investigated; the lysis process, the sample pretreatment, and the matrix preparation were optimized for MALDI conditions. Initial experiments to observe the success of protein translation in gene expression experiments were performed. Using MALDI-compatible detergents, it was possible to extend the mass range detectable by MALDI mass spectrometry from the current range of 16,000 to 75,000 Da. In this mass range, the data are complementary (offering a better mass accuracy) to those obtained by SDS-PAGE electrophoresis experiments. These new methods were used to monitor a large-scale cultivation of hybridoma cells expressing an antibody of the IgG type. The increase in whole antibody and antibody light-chain protein, 8650 Da, and the decrease of insulin were followed during the monitoring period. Quantitative measurements of the IgG level during the cultivation compared favorably with those obtained by affinity HPLC.

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.

Various hypertrophic stimuli induce distinct phenotypes in cardiomyocytes.

Cardiac hypertrophy is characterized by an increase in cell size in the absence of cell division and is accompanied by a number of qualitative and quantitative changes in gene expression. Most forms of hypertrophy in vivo are compensatory or adaptative responses to increased workload resulting from various physiological and/or pathological etiologies. Until severe pathological alterations become apparent, myocytes show no drastic morphological changes. On the level of gene expression, upregulation of the so-called fetal genes, i.e., beta-myosin heavy chain, alpha-skeletal and alpha-smooth muscle actin, and atrial natriuretic factor (ANF) may be observed concomitant with a downregulation of alpha-myosin heavy chain and the Ca pump of sarcoplasmic reticulum. The use of primary cell culture systems for cardiomyocytes as an in vitro model for the hypertrophic reaction has identified a number of different stimuli as mediators of cardiac myocyte hypertrophy. The molecular dissection of the different intracellular signaling pathways involved herein has uncovered a number of branching points to cytosolic and nuclear targets and has identified many interactions between these pathways. The individual administration of these hypertrophic stimuli, i.e., hormones, cytokines, growth factors, vasoactive peptides, and catecholamines, to cultured cardiomyocytes, reveals that each stimulus induces a distinct phenotype as characterized by gene expression pattern and cellular morphology. Surprisingly, triiodothyronine (T3) and basic fibroblast growth factor (bFGF) effect a similar cellular phenotype although they use completely different intracellular pathways. This phenotype is characterized by drastic inhibition of myofibrillar growth and by upregulation of alpha-smooth muscle actin. On the other hand, insulin-like growth factor (IGF) I, a factor promoting muscle cell differentiation, and bFGF, an inhibitor of differentiation, cause completely different cardiomyocyte phenotypes although both are known to signal via receptor tyrosine kinases and have been shown to activate the Ras-Raf-MEK-MAP kinase pathway. However, both IGF-I and bFGF depend on T3 to bring about their typical responses, i.e., T3 is permissive for the action of these two growth factors on the expression of alpha-smooth muscle actin and cell morphology. Most of the hypertrophic stimuli are balanced under normal circumstances in vivo. When this balance is disturbed, however, a pathological heart phenotype may become dominant. Thus the knowledge of signaling pathways and cellular responses triggered by hypertrophic stimuli may be essential for the implementation of therapeutic strategies in the treatment of cardiac hypertrophy.

Signaling pathways in cardiac myocyte hypertrophy.

When a heart responds to increased workload it does so by hypertrophy. This is characterized by an increase in cell size in the absence of cell division, and is accompanied by distinct qualitative and quantitative changes in gene expression. The use of cardiomyocytes in cell culture has identified, besides mechanical loading, a range of substances, such as cytokines, growth factors, catecholamines, vasoactive peptides and hormones, involved in mediating cardiac myocyte hypertrophy, and has enabled the molecular dissection of the pathways involved in signal transduction. Many different pathways are activated in response to different hypertrophic stimuli, and a growing number of crosslinks are being characterized between these pathways. Recent evidence suggests a central role for Ras in transmitting signals from G-protein coupled receptors, from growth factor receptors and from cytokine receptors not only down the Raf-MEK-ERK pathway to the nucleus, but also to various other cytosolic effectors. The evaluation of distinct morphological phenotypes, together with biochemical data on gene regulation, suggests that interactions between different signaling pathways take place. Each stimulus provokes a typical cellular phenotype and different stimuli may act alone or in concert in a synergistic, antagonistic or permissive manner. Consequently, hypertrophy of cultured cardiomyocytes cannot simply be characterized as the reversal to the fetal gene expression program. Thus, hypertrophic growth of the heart may similarly be the result of a complex combinatorial action of various stimuli, which may also lead to different morphological and biochemical phenotypes with distinct physiological properties.