Hormone-responsive myofibrillar protease activity in cultured rat myoblasts

The phosphorylation of NADP-specific isocitrate dehydrogenase in an isocitrate lyase and in a malate synthase mutant of Escherichia coli has been investigated. The results clearly demonstrate that isocitrate dehydrogenase may undergo an acetate-induced phosphorylation in organisms which do not have a functional glyoxylate cycle. This observation, together with those reported in Salmonella typhimurium, suggest that the current notion concerning the interrelationship between the glyoxylate cycle and the reversible phosphorylation of NADP-isocitrate dehydrogenase in microbial physiology should be reevaluated, and that phosphoenolpyruvate may be a key factor in the regulation of the reversible covalent modification of this enzyme in vivo.     

Immunochemical characterisation of the myofibrillar proteinase from cultured rat myocytes as chymase

The proteinase extracted from the myofibrillar fraction of (a) primary rat myocytes and (b) the L-8 myogenic cell line, both maintained in culture, was identified by immunochemical analysis as chymase, the chymotrypsin-like serine proteinase of rat mast cells. Chymase would therefore appear to be an intrinsic protein in the rat myocyte also.     

Monoacylglycerol lipase activity in cardiac myocytes

Monoacylglycerol lipase activity in homogenates of isolated myocardial cells (myocytes) from rat hearts was recovered in both particulate and soluble subcellular fractions. The activity present in the microsomal (100,000 X g pellet) fraction was solubilized by treatment with Triton X-100 and combined with the 100,000 X g supernatant fraction; the properties of monoacylglycerol lipase were investigated with this soluble enzyme preparation. The Km for the hydrolysis of a 2-monoolein substrate was 16 microM. The rates of hydrolysis of 1-monoolein and 2-monoolein were identical, and 1-monoolein was a competitive inhibitor (Ki = 20 microM) of the hydrolysis of 2-monoolein. Monoacylglycerol lipase activity was regulated by product inhibition according to the following order of potency: fatty acyl CoA greater than free fatty acids greater than fatty acyl carnitine.     

Distribution of isomyosin in cultured cardiac myocytes as determined by monoclonal antibodies and adenosine triphosphatase activity

The distribution of isomyosin in cardiac muscle cells in culture has been investigated with monoclonal antibodies and Ca2+-activated myosin ATPase cytochemical staining. With immunofluorescent studies using monoclonal antibodies to isomyosins V1 and V3, the cardiac myocytes grown in a serum-free and thyroxine (T4)-free medium for 7 days contained a predominant population of cells which were strongly reactive to anti-V3 antibody. A small population of myocytes in this culture exhibited weak or no reaction to anti-V3 antibody. When cultures were exposed to anti-V1 antibody, the predominant cardiac myocyte population showed little or no reactivity to this antibody, whereas a small population of the myocytes were strongly reactive. The myosin ATPase staining reaction of the positive myocyte population was significantly less pronounced than that of the V3-negative population which showed a strong reaction. The staining pattern changed dramatically after exposure of cultured myocytes to thyroid hormone for 7 days. Most of the cells were found to react strongly with anti-V1 antibody, while some cells showed little reactivity and some were not stained at all. A small number of cardiac myocytes in this culture showed little or no reactivity to anti-V1 antibody but were strongly reactive to anti-V3 antibody. The predominant anti-V1-positive myocyte population exhibited strong myosin ATPase staining as compared to a smaller V3-positive myocyte population which showed very weak staining. The cytochemical results of ATPase staining in cardiac myocytes agreed well with ATPase activity as determined on pyrophosphate gels containing isomyosin derived from cultured cardiac myocytes with or without T4. This study has demonstrated that cultured myocytes contain a small population of muscle cells which is not responsive to thyroid hormone or to the lack of it.     

Automated surface area measurement of cultured cardiac myocytes

An automated method for rapidly measuring surface area of individual cardiac myocytes was used as an index of myocyte growth. Hearts from 2- to 4-day-old rats were digested by overnight incubation in cold trypsin solution. Enriched suspensions of myocytes were plated at 2×10⁵ cells/well in 12-well-culture plates. Cells were grown in M199 supplemented with 1%, 10% serum or 10% serum plus 10^–7 M norepinephrine. On days 1–4 after plating, cells were fixed in Bouin's Solution and stained with Weigert's Iron Hematoxylin and Biebrich Scarlet-Acid Fuchsin. An inverted microscope, video camera and monitor were coupled to a video image processor (Image Technology Corp.). The enhanced image of stained heart cells was digitized, and perimeter, length, width and area of each selected cell were calculated. One hundred randomly selected cells were measured in each of eight wells from each treatment-day group. Areas of individual myocytes varied widely in culture dishes and the distribition was skewed toward larger cells. The standard deviation increased in proportion to an increase in mean cell area. A logarithmic transformation of the data normalized the data and yielded a more homogeneous variance. The geometric mean area of heart cells supplemented with 1% serum increased only slightly, but significantly, during four days in culture. Geometric mean area of cells supplemented with 10% serum increased nearly four-fold. Supplementing cells with norepinephrine (10^–7 M) in addition to 10% serum did not induce a further increase in cell size. This technique has the potential to rapidly and objectively monitor heart cell growth following pharmacological or toxicological treatments.     

Cerium depresses protein synthesis in cultured cardiac myocytes and lung fibroblasts

The present study examined the effect of Cerium on protein synthesis in cultured cardiac myocytes and lung fibroblasts exposed to normal and markedly subnormal levels of Mg²⁺. Cerium was found to have a general inhibitory effect on protein synthesis in these cell types, including the synthesis of myofibrillar proteins in the cardiac myocytes. Further, the effect of the metal ion was more pronounced in cells exposed to the Mg²⁺-deficient medium. The possible implications of the observations are discussed.     

F-actin modulates swelling-activated chloride current in cultured chick cardiac myocytes

The integrity ofF-actin and its association with the activation of aCl current(I_Cl) incultured chick cardiac myocytes subjected to hyposmotic challenge weremonitored by whole cell patch clamp and fluorescence confocalmicroscopy. Disruption of F-actin by 25 µM cytochalasin B augmentedhyposmotic cell swelling by 51% (from a relative volume of 1.54 ± 0.10 in control to 2.33 ± 0.21), whereas stabilization of F-actinby 20 µM phalloidin attenuated swelling by 15% (relative volume of1.31 ± 0.05). Trace fluorochrome-labeled (fluoresceinisothiocyanate or tetramethylrhodamine isothiocyanate) phalloidinrevealed an intact F-actin conformation in control cells underhyposmotic conditions despite the considerable changes in cell volume.Sarcoplasmic F-actin was very disorganized and occurred only randomlybeneath the sarcolemma in cells treated with cytochalasin B, whereas nochanges in F-actin distribution occurred under either isosmotic orhyposmotic conditions in cells treated with phalloidin.Swelling-activatedI_Cl (68.0 ± 6.0 pA/pF at +60 mV) was suppressed by both cytochalasin B (22.7 ± 5.1 pA/pF) and phalloidin (22.5 ± 3.5 pA/pF). On the basis of theseresults, we suggest that swelling of cardiac myocytes initiates dynamic changes in the cytoarchitecture of F-actin, which may be involved inthe volume transduction processes associated with activation ofI_Cl.

     

Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.     

Contractile activity and smooth muscle alpha-actin organization in thrombin-induced human lung myofibroblasts

Activated fibroblasts, or myofibroblasts, are crucial players in tissue remodeling, wound healing, and various fibrotic disorders, including interstitial lung fibrosis associated with scleroderma. Here we characterize the signaling pathways in normal lung fibroblasts exposed to thrombin as they acquire two of the main features of myofibroblasts: smooth muscle (SM) alpha-actin organization and collagen gel contraction. Our results show that the small G protein Rho is involved in lung myofibroblast differentiation. Thrombin induces Rho-35S-labeled guanosine 5'-O-(3-thiotriphosphate) binding in a dose-dependent manner. It potently stimulates Rho activity in vivo and initiates protein kinase C (PKC)-epsilon-Rho complex formation. Toxin B, which inactivates Rho by ADP ribosylation, inhibits thrombin-induced SM alpha-actin organization, collagen gel contraction, and PKC-epsilon-SM alpha-actin and PKC-epsilon-RhoA coimmunoprecipitation. However, it has no effect on PKC-epsilon activation or translocation of PKC-epsilon to the membrane. Overexpression of constitutively active PKC-epsilon and constitutively active RhoA induces collagen gel contraction or SM alpha-actin organization, whereas, individually, they do not perform these functions. We therefore conclude that the contractile activity of myofibroblasts induced by thrombin is mediated via PKC-epsilon- and RhoA-dependent pathways and that activation of both of these molecules is required. We postulate that PKC-epsilon-RhoA complex formation is an early event in thrombin activation of lung fibroblasts, followed by PKC-epsilon-SM alpha-actin coimmunoprecipitation, which leads to the PKC-epsilon-RhoA-SM alpha-actin ternary complex formation.     

Mechanism of peroxide-induced cellular injury in cultured adult cardiac myocytes

Reactive oxygen species contribute to the tissue injury seen after reperfusion of ischemic myocardium. We propose that toxicity originates from the effect that mitochondrial peroxide metabolism has on substrate entry into oxidative pathways. To support our contention, cultured adult rat cardiomyocytes were incubated with physiological concentrations of peroxide. The cellular extract and incubation medium were analyzed for adenine nucleotides and purines by reverse-phase high-pressure liquid chromatography. Cellular glutathione efflux was determined by enzymatic analysis of the incubation medium. Pyruvate dehydrogenase (PDH) activity was determined in the cultured myocytes as well as in freshly isolated cardiac mitochondria using [1-C14]pyruvate. Extracellular glutathione rose 3.3-fold in response to small doses of peroxide (approximately 108 nmol/mg protein). Likewise, small quantities of peroxide reduced total cellular adenine nucleotides to 50-60% of control values with only a modest (0.95-0.91) reduction in energy charge [ATP + 1/2 ADP)/(ATP + ADP + AMP]. Peroxide-treated myocytes selectively release inosine and adenosine, as only these two purine degradation products were detected in the incubation medium. The most dramatic response was a peroxide dose-dependent inhibition of PDH activity in cultured myocytes as well as freshly isolated mitochondria; just 65 and 30 nmol peroxide/mg protein induced a 50% reduction in cellular and mitochondrial PDH activity, respectively. In conclusion, physiological quantities of peroxide potently inhibit PDH in cultured cardiomyocytes and isolated cardiac mitochondria. PDH inhibition blocks the aerobic oxidation of glucose and inhibits the oxidative phosphorylation of ADP, which in turn leads to cellular adenine nucleotide degradation.     

Maintenance and characterization of spontaneous contraction rhythm in cultured cardiac myocytes fused with cardiac fibroblasts

Cardiomyocytes (CMs) fuse with various cells including endothelial cells, cardiac fibroblasts (CFs). In addition, recent studies have shown that stem cells fuse spontaneously with cells remaining in the damaged tissues, and restore tissue functions after myocardial infarction. In this study, we investigated whether cultured cardiomyocytes fused with proliferative cardiac fibroblasts maintained the phenotype of functional myocytes by analyzing the spontaneous contraction rhythm after fusion with CFs lacking a beating capability. CMs and CFs cultured for 4 days in vitro were used in this study. The fusion of cultured CMs and CFs was achieved with polyethylene glycol (PEG) and hemagglutinating virus of Japan (HVJ). Analyses of CMs fused with CFs by using either PEG or HVJ to imitate spontaneous fusion in vivo demonstrated that CMs and CFs actually fused together and fused cells expressed lineage marker proteins of both CMs and CFs. In addition, fused cells reentered the G2-M phase of the cell cycle. Furthermore, fused cells retained the spontaneous contraction activity. The present study demonstrated that CMs fused with proliferative CFs showed the phenotype of both CMs and CFs and spontaneous rhythmic contraction.     

An intrinsic timer that controls cell-cycle withdrawal in cultured cardiac myocytes

Developing cardiac myocytes divide a limited number of times before they stop and terminally differentiate, but the mechanism that stops their division is unknown. To help study the stopping mechanism, we defined conditions under which embryonic rat cardiac myocytes cultured in serum-free medium proliferate and exit the cell cycle on a schedule that closely resembles that seen in vivo. The culture medium contains FGF-1 and FGF-2, which stimulate cell proliferation, and thyroid hormone, which seems to be necessary for stable cell-cycle exit. Time-lapse video recording shows that the cells within a clone tend to divide a similar number of times before they stop, whereas cells in different clones divide a variable number of times before they stop. Cells cultured at 33 degrees C divide more slowly but stop dividing at around the same time as cells cultured at 37 degrees C, having undergone fewer divisions. Together, these findings suggest that an intrinsic timer helps control when cardiac myocytes withdraw from the cell cycle and that the timer does not operate by simply counting cell divisions. We provide evidence that the cyclin-dependent kinase inhibitors p18 and p27 may be part of the timer and that thyroid hormone may help developing cardiac myocytes stably withdraw from the cell cycle.     

Effect of ethanol on lipofuscin accumulation in cultured rat cardiac myocytes

The objective of this study was to determine the effect of ethanol on in vitro life span, rate of contraction and lipofuscin content of neonatal rat cardiac myocytes. Lipofuscin was quantified by microspectrofluorometry. The effects of 0, 3.1, 6.5, and 12.5 mM ethanol on myocytes, kept under an ambient oxygen concentration of 20% and 40%, were studied. Exposure to low concentrations of ethanol resulted in a decrease in the amount of lipofuscin whereas exposure to high concentration of ethanol caused an increase in the level of lipofuscin. The length of cell survival in controls and 3.1 mM ethanol exposed myocytes was similar under 20% oxygen, but was longer in the latter group under 40% oxygen, as compared to controls. The total number of contractions in 3.1 mM ethanol-exposed myocytes were, respectively, 4% and 8% higher under 20% and 40% oxygen atmosphere than in control cells.     

The vinculin/sarcomeric-alpha-actinin/alpha-actin nexus in cultured cardiac myocytes

Experiments are described supporting the proposition that the assembly of stress fibers in non-muscle cells and the assembly of myofibrils in cardiac cells share conserved mechanisms. Double staining with a battery of labeled antibodies against membrane-associated proteins, myofibrillar proteins, and stress fiber proteins reveals the following: (a) dissociated, cultured cardiac myocytes reconstitute intercalated discs consisting of adherens junctions (AJs) and desmosomes at sites of cell-cell contact and sub-sarcolemmal adhesion plaques (SAPs) at sites of cell-substrate contact; (b) each AJ or SAP associates proximally with a striated myofibril, and conversely every striated myofibril is capped at either end by an AJ or a SAP; (C) the invariant association between a given myofibril and its SAP is especially prominent at the earliest stages of myofibrillogenesis; nascent myofibrils are capped by oppositely oriented SAPs; (d) the insertion of nascent myofibrils into AJs or into SAPs invariably involves vinculin, alpha-actin, and sarcomeric alpha-actinin (s-alpha-actinin); (e) AJs are positive for A-CAM but negative for talin and integrin; SAPs lack A-CAM but are positive for talin and integrin; (f) in cardiac cells all alpha-actinin-containing structures invariably are positive for the sarcomeric isoform, alpha-actin and related sarcomeric proteins; they lack non-s-alpha-actinin, gamma-actin, and caldesmon; (g) in fibroblasts all alpha-actinin-containing structures are positive for the non-sarcomeric isoform, gamma-actin, and related non-sarcomeric proteins, including caldesmon; and (h) myocytes differ from all other types of adherent cultured cells in that they do not assemble authentic stress fibers; instead they assemble stress fiber-like structures of linearly aligned I-Z-I-like complexes consisting exclusively of sarcomeric proteins.