The subcellular distribution of protein kinase Calpha, -epsilon, and -zeta isoforms during cardiac cell differentiation.

There is little information on the molecular events that control the subcellular distribution of protein kinase C during cardiac cell differentiation. We examined protein kinase C activity and the subcellular distribution of representatives of the "classical," "novel," and "atypical" protein kinase C's in P19 murine teratoma cells induced to undergo differentiation into cardiac myocytes by the addition of dimethylsulfoxide to the medium (Grepin et al., Development 124, 2387-2395, 1997). Differentiation was assessed by the presence of striated myosin, a morphological marker for cardiac cells. Addition of dimethyl sulfoxide to the medium resulted in the appearance of striated myosin by 10 days postincubation. Immunolocalization and Western blot studies revealed that a significant proportion of protein kinase Calpha, -epsilon, and -zeta were associated with the particulate fraction in P19 cells prior to differentiation. Differentiation into cardiac cells resulted in a translocation of protein kinase C activity from the particulate fraction to cytosol and localization of most of protein kinase Calpha, -epsilon, and -zeta to the cytoplasmic compartment. The total cellular protein kinase C activity was unaltered during differentiation. The translocation of protein kinase C activity during differentiation of P19 cells into cardiac myocytes was associated with a decrease in the levels of cellular 1, 2-diacyl-sn-glycerol. The cellular levels of phosphatidylserine and phosphatidylinositol did not change during differentiation. Addition of 1,2-dioctanoyl-sn-glycerol, a cell-permeant 1, 2-diacyl-sn-glycerol analog, reversed the differentiation-induced switch in the relative distribution of protein kinase C activity and dramatically increased the association of protein kinase Calpha with the particulate fraction. Addition of 1,2-dioctanoyl-sn-glycerol did not reverse the pattern of distribution for protein kinase Cepsilon or -zeta. The results indicate that protein kinase C activity and protein kinase Calpha, -epsilon and -zeta isoforms are redistributed from the particulate to the cytosolic fraction during differentiation of P19 cells into cardiomyocytes. The mechanism for the redistribution of protein kinase Calpha may be related to the reduction in the cellular 1,2-diacyl-sn-glycerol levels that accompany differentiation.     

Actin and myosin expression during development of cardiac muscle from cultured embryonal carcinoma cells

P19 embryonal carcinoma cells are multipotential stem cells that differentiate into striated muscle as well as some other cell types when aggregated and exposed to dimethyl sulfoxide (DMSO). Immunofluorescence experiments using monospecific antibodies indicated that the majority of muscle cells were mononucleate and contained four myosin isoforms normally found in cardiac muscle; atrial and ventricular myosin heavy chains, ventricular myosin light chain 1, and atrial myosin light chain 2. Northern blot analysis of RNA isolated from differentiating cultures indicated that cardiac actin and skeletal actin mRNAs were expressed at similar levels and with identical kinetics during the differentiation of P19-derived myocytes. These results demonstrate that most of the P19-derived myocytes are of the cardiac type and suggest that they closely resemble the cells of the early embryonic myocardium.     

Expression of metalloproteinases and inhibitors in the differentiation of P19CL6 cells into cardiac myocytes

P19CL6 are a clonal derivative of P19 embryonal carcinoma cells, a euploid, multipotent mouse cell line, that differentiate efficiently into cardiac myocytes, with spontaneous beating evident within 10 days, following DMSO treatment. Using real-time quantitative RT-PCR we have profiled the expression of the complete matrix metalloproteinase and tissue inhibitor of metalloproteinase gene families during P19CL6 differentiation to cardiac myocytes. The genes subdivide into eight groups based upon their expression profile. Their expression was both qualitatively and quantitatively highly homologous to that seen during mouse heart development.     

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes

Summary Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14–15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin andα-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycinc centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells my provide a useful experimental system for the study of human cardiac muscle cell biology.     

Etomoxir mediates differential metabolic channeling of fatty acid and glycerol precursors into cardiolipin in H9c2 cells

We examined the effect of etomoxir treatment on de novo cardiolipin (CL) biosynthesis in H9c2 cardiac myoblast cells. Etomoxir treatment did not affect the activities of the CL biosynthetic and remodeling enzymes but caused a reduction in [1-14C]palmitic acid or [1-14C]oleic acid incorporation into CL. The mechanism was a decrease in fatty acid flux through the de novo pathway of CL biosynthesis via a redirection of lipid synthesis toward 1,2-diacyl-sn-glycerol utilizing reactions mediated by a 35% increase (P < 0.05) in membrane phosphatidate phosphohydrolase activity. In contrast, etomoxir treatment increased [1,3-3H]glycerol incorporation into CL. The mechanism was a 33% increase (P < 0.05) in glycerol kinase activity, which produced an increased glycerol flux through the de novo pathway of CL biosynthesis. Etomoxir treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thereby channeling both glycerol and fatty acid away from 1,2,3-triacyl-sn-glycerol utilization toward phosphatidylcholine and phosphatidylethanolamine biosynthesis. In contrast, etomoxir inhibited myo-[3H]inositol incorporation into phosphatidylinositol and the mechanism was an inhibition in inositol uptake. Etomoxir did not affect [3H]serine uptake but resulted in an increased formation of phosphatidylethanolamine derived from phosphatidylserine. The results indicate that etomoxir treatment has diverse effects on de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir mediates a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL.     

Multiple CArG boxes in the human cardiac actin gene promoter required for expression in embryonic cardiac muscle cells developing in vitro from embryonal carcinoma cells.

Chimeric genes composed of the human cardiac actin promoter driving the Escherichia coli lacZ reporter gene were constructed, transfected, and stably integrated into genomes of P19 embryonal carcinoma cells. The transfected constructs were expressed actively in cardiac myocytes formed following dimethyl sulfoxide (DMSO)-induced cell differentiation but poorly in undifferentiated cultures and in cultures treated with retinoic acid to develop into derivatives of the neuroectoderm. A number of deletions of the promoter were constructed and tested. Three regions required for efficient expression in P19-derived cardiac muscle were identified, each containing sequences referred to as CArG boxes (CC[AT-rich]6GG). This analysis indicated that regulatory sequences important for expression in cardiac muscle were present upstream of the core promoter identified previously by transient assays in skeletal myoblasts. Expression of the cardiac actin promoter was enhanced 10-fold in undifferentiated P19 cells in the presence of the myoD protein. The promoter regions important for expression in P19-derived cardiocytes were similar to those important for myoD-induced enhancement, a result we interpret to be consistent with the idea that cardiac muscle might contain a myoD-like activity.     

The role of aggregation in embryonal carcinoma cell differentiation

Cultures of the P19 line of embryonal carcinoma cells differentiate into various cell types including cardiac muscle when aggregated and exposed to medium containing 1% dimethylsulfoxide (DMSO). DMSO-treated aggregates became completely covered with an epithelial cell type 3 to 4 days following drug exposure. This epithelial cell was tentatively identified as primitive extraembryonic endoderm by its ultrastructural appearance and its possession of cytokeratin intermediate filaments. Muscle cells developed within the interior of DMSO-treated aggregates. They first became apparent 5 to 6 days after DMSO exposure and were characterized by the presence of striated muscle-specific myosin, immature myofibrils, and intercalated discs. We determined the proportion of cells developing into epithelium and muscle in aggregates of various sizes and showed that the proportion of epithelium was highest in small aggregates whereas muscle cells developed only in aggregates of relatively large size. The muscle was usually associated with necrotic areas which developed within the interior of large aggregates. Our results suggest that cardiac muscle differentiation in the aggregates requires both the DMSO-induced formation of an epithelial cell coat and one other condition which may be the proximity to necrotic areas.     

<Emphasis Type="Italic">Fabp3</Emphasis> Inhibits Proliferation and Promotes Apoptosis of Embryonic Myocardial Cells

Fatty acid binding protein 3 (FABP3) is a member of a family of binding proteins. The protein is mainly expressed in cardiac and skeletal muscle cells, and it has been linked to fatty acid metabolism, trafficking, and signaling. Using suppression subtractive hybridization, we previously found that FABP3 is highly regulated in ventricular septal defect (VSD) patients and may play a significant role in the development of human VSD. We therefore aimed to identify the biological characteristics of the FABP3 gene in embryonic myocardial cells. On the basis of RT-PCR and western blotting analyses, we demonstrated that the expression levels of FABP3 mRNA and protein were up-regulated initially and then gradually decreased with P19 cell differentiation. MTT assays and cell cycle analysis showed that FABP3 inhibits P19 cell proliferation, and data from annexin V-FITC assays revealed that FABP3 can promote apoptosis of P19 cells. Further data from quantitative real-time RT-PCR revealed lower expression levels of cardiac muscle-specific molecular markers (cTnT, alpha-MHC, GATA4, and MEF2c) in FABP3-overexpressing cell lines than in the control cells during differentiation. Our results demonstrate that FABP3 may be involved in the differentiation of cardiac myocytes.     

Myosin light chain kinase mediates sarcomere organization during cardiac hypertrophy in vitro

During the development of hypertrophy, cardiac myocytes increase organization of the sarcomere, a highly ordered contractile unit in striated muscle cells. Several hypertrophic agonists, such as angiotensin II, phenylephrine, and endothelin-1, have been shown to promote the sarcomere organization. However, the signaling pathway, which links extracellular stimuli to sarcomere organization, has not been clearly demonstrated. Here, we demonstrate that myosin light chain kinase specifically mediates agonist-induced sarcomere organization during early hypertrophic response. Acute administration of a hypertrophic agonist, phenylephrine, or angiotensin II, causes phosphorylation of myosin light chain 2v both in cultured cardiac myocytes and in the adult heart in vivo. We also show that both sarcomere organization and myosin light chain 2v phosphorylation are dependent on the activation of Ca2+/calmodulin pathway, a known activator of myosin light chain kinase. These results define a new and specific role of myosin light chain kinase in cardiac myocytes, which may provide a rapid adaptive mechanism in response to hypertrophic stimuli.     

Short-time glucose exposure of embryonic carcinoma cells impairs their function as terminally differentiated cardiomyocytes

The fetal and postnatal phenotype is influenced by developmental conditions experienced prenatally. Among prenatal development metabolic factors are of particular importance as they are supposed to predispose for pathophysiological alterations later in life and to pioneer functional impairment in senescence (metabolic programming). Till now the mechanisms of metabolic programming are not well understood. We have investigated various concentrations of glucose during differentiation of pluripotent P19 embryonic carcinoma cells (ECC) into cardiomyocytes. Undifferentiated P19 cells were exposed to 5mM (low), 25 mM (control), 40 mM or 100mM (high) glucose for 48 h during embryoid body (EB) formation, followed by plating and differentiation into cardiomyocytes in vitro with standard glucose supplementation (25 mM) for 10-15 days. The amount of cardiac clusters, the frequency of spontaneous beatings as well as the expression of metabolic and cardiac marker genes and their promoter methylation were measured. We observed a metabolic programming effect of glucose during cardiac differentiation. Whereas the number of beating clusters and the expression of the cardiac marker alpha myosin heavy chain (α-MHC) were comparable in all groups, the frequencies of beating clusters were significantly higher in the high glucose group compared to low glucose. However, neither the insulin receptor (IR) or insulin like growth factor 1 receptor (IGF1R) nor the metabolic gene glucose transporter 4 (GLUT4) were influenced in RNA expression or in promoter methylation. Our data indicate that a short time glucose stress during embryonic cell determination leads to lasting effects in terminally differentiated cell function.     

Cyclosporin A Induces Cardiac Differentiation but Inhibits Hemato-Endothelial Differentiation of P19 Cells

Little is known about the mechanisms underlying the effects of Cyclosporin A (CsA) on the fate of stem cells, including cardiomyogenic differentiation. Therefore, we investigated the effects and the molecular mechanisms behind the actions of CsA on cell lineage determination of P19 cells. CsA induced cardiomyocyte-specific differentiation of P19 cells, with the highest efficiency at a concentration of 0.32 μM during embryoid body (EB) formation via activation of the Wnt signaling pathway molecules, Wnt3a, Wnt5a, and Wnt8a, and the cardiac mesoderm markers, Mixl1, Mesp1, and Mesp2. Interestingly, cotreatment of P19 cells with CsA plus dimethyl sulfoxide (DMSO) during EB formation significantly increases cardiac differentiation. In contrast, mRNA expression levels of hematopoietic and endothelial lineage markers, including Flk1 and Er71, were severely reduced in CsA-treated P19 cells. Furthermore, expression of Flk1 protein and the percentage of Flk1+ cells were severely reduced in 0.32 μM CsA-treated P19 cells compared to control cells. CsA significantly modulated mRNA expression levels of the cell cycle molecules, p53 and Cyclins D1, D2, and E2 in P19 cells during EB formation. Moreover, CsA significantly increased cell death and reduced cell number in P19 cells during EB formation. These results demonstrate that CsA induces cardiac differentiation but inhibits hemato-endothelial differentiation via activation of the Wnt signaling pathway, followed by modulation of cell lineage-determining genes in P19 cells during EB formation.     

On the mechanism of the phospholipase C-mediated attenuation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells

The effect of phospholipase C treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with phosphatidylcholine-specific Clostridium welchii phospholipase C reduced the pool size of phosphatidylcholine compared with controls whereas the pool size of cardiolipin and phosphatidylglycerol were unaffected. Pulse labeling experiments with [1,3-³H]glycerol and pulse-chase labeling experiments with [1,3-³H]glycerol were performed in cells incubated or pre-incubated in the absence or presence of phospholipase C. In all experiments, radioactivity incorporated into cardiolipin and phosphatidylglycerol were reduced in phospholipase C-treated cells with time compared with controls indicating attenuated de novo biosynthesis of these phospholipids. Addition of 1,2-dioctanoyl-sn-glycerol, a cell permeable 1,2-diacyl-sn-glycerol analog, to cells mimicked the inhibitory effect of phospholipase C on cardiolipin and phosphatidylglycerol biosynthesis from [1,3-³H]glycerol indicating the involvement of 1,2-diacyl-sn-glycerol. The mechanism for the reduction in cardiolipin and phosphatidylglycerol biosynthesis in phospholipase C-treated cells appeared to be a decrease in the activities of phosphatidic acid:cytidine-5triphosphate cytidylyltransferase and phosphatidylglycerolphosphate synthase, mediated by elevated 1,2-diacyl-sn-glycerol levels. Upon removal of phospholipase C from the incubation medium, phosphatidylcholine biosynthesis from [methyl-³H]choline was markedly stimulated. These data suggest that de novo phosphatidylglycerol and cardiolipin biosynthesis may be regulated by 1,2-diacyl-sn-glycerol and support the notion that phosphatidylglycerol and cardiolipin biosynthesis may be coordinated with phosphatidylcholine biosynthesis in H9c2 cardiac myoblast cells.