Heart Myoblasts Research Articles

Cytotoxicity and hemolytic activity of jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae) venom

The recent bloom of a giant jellyfish Nemopilema nomurai has caused a danger to sea bathers and fishery damages in the waters of China, Korea, and Japan. The present study investigated the cytotoxic and hemolytic activities of crude venom extract of N. nomurai using a number of in vitro assays. The jellyfish venom showed a much higher cytotoxic activity in H9C2 heart myoblast than in C2C12 skeletal myoblast (LC 50 = 2 µg/mL vs. 12 µg/mL, respectively), suggesting its possible in vivo selective toxicity on cardiac tissue. This result is consistent with our previous finding that cardiovascular function is a target of the venom. In order to determine the stability of N. nomurai venom, its cytotoxicity was examined under the various temperature and pH conditions. The activity was relatively well retained at low environmental temperature (≤ 20 °C) and dramatically lost at high temperature (≥ 60 °C). In pH stability test, the venom has abruptly lost its activity at low pH environment (pH ≤ 4). Interestingly enough, however, its activity was not significantly affected even at the highest pH environment tested (pH ≤ 12) in the present study. Additionally, hemolytic activity of the venom was examined using the erythrocytes of cat, dog, human, rabbit and rat. Venom concentration-dependent hemolysis could be observed from 10 µg/mL of protein equivalents or higher with variable potencies in different species, among which dog erythrocyte was the most susceptible to the venom (EC 50 = 151 µg/mL). SDS-PAGE analysis of N. nomurai venom showed the molecules of 20–40 kDa and 10–15 kDa appeared to be the major protein components of the venom.

Side Effects on the Heart and Skeleton of Growing Mice Attributed to Chronic Imatinib Exposure.

Objectives: Chronic myeloid leukemia (CML) is effectively treated by Imatinib (IM) via inhibition of the BCR-ABL tyrosine kinase. However, also related tyrosine kinases like abl, c-Kit, PDGF-R, and c-FMS are blocked by IM. As shown in adult humans and mice, abl-controlled protein folding as part of the endoplasmatic stress response in heart myoblasts as well as bone “remodeling” depending on PDGF-R and c-FMS is impaired under imatinib exposure (Dewar AL et al 2005, Kerkelä R et al 2006, Fitter S et al 2008). The influence of IM on the growing heart and skeleton of immature animals has not been studied so far. With respect to treatment of pediatric CML we report alterations in these organs of juvenile mice chronically exposed to IM during the growth period.Methods: From the age of 4–14 weeks (w) [development milestones of mice: weaning 3 w; puberty 7 w; epiphysial lines closure 18 w] C3H/Neu male and female wild-type mice were chronically exposed to IM via the drinking water at concentrations of 500 mg/l (group A), 750 mg/l (group B), and 1000mg/l (group C). Femur length and overall skeletal development was analysed by whole body X-ray analysis using a mammography device. Bone metabolic activity was assessed by total body Na18F PET and CT after 5w and 10w of exposure using dedicated small animal tomographs. Bone mineral density and microstructure of tibiae were analysed by pQCT and microCT (resolution 12.5μ m) while the number of osteoclasts and resorption lacunae in femora and vertebrae was assessed by histomorphometry. Plasma concentration of IM, osteocalcin, and activity of the tartrate resistant acid phosphatase (TRAP5b) was also determined. The heart was examined histologically and ultrastructurally by electron microscopy.Results: IM was tolerated well and mean uptake of 80 mg/kg/d110 mg/kg/dand 150 mg/kg/dresulted in serum levels of 60–674 ng/ml, 36–242 ng/ml and 51–534 ng/ml, respectively.Body weight gain was delayed in groups B and C until the age of 8 w while no change in overall growth, development and behaviour was observed at 14 w. At higher doses of IM and at younger age there was a non-significant trend to a reduction in femur length. Heart morphological examination exhibited an increased number (p<0.05) of hypertrophic cardiomyocytes (toxic damage) paralleled by ultrastructural alterations in mitochondria, myofibrils, and nucleus. In the skeleton, no significant differences compared to controls concerning 18F-kinetics and uptake in vertebrae and femura could be demonstrated. However, IM dose-dependently reduced the number of osteoclasts and resorption lacunae (p<0.05); these effects were less pronounced in female mice. Tibia cortical thickness was increased significantly in males by 6.1% (B) and 11.2% (C), respectively, and 7.5% in females (C). By microCT cancellous bone exhibited a significant increase in trabecular bone mass density and volume and number resulting in an increase in trabecular connectivity in males by 63% (B) and 64% (C), respectively, and in females by 22% (B) and 38% (C), respectively. Bone biomarkers indicated a significant reduction of TRAP5b activity while osteocalcin levels remained unchanged.Conclusion: In juvenile mice, a chronic exposure of IM resulted in toxic damage of the cardiomyocytes at higher dose rates. However, these alterations do not necessarily imply also a functional impairment which can only be studied in vivo. In the skeleton, IM reduced the number of osteoclasts and resorption lacunae in long bones but not in vertebrae. IM showed an antiresorptive effect in cancellous bone and increased cortical thickness and trabecular number by inhibiting the expansion of the marrow cavity. The effects were more pronounced in male mice and at younger age.

Rat Cardiomyocytes Express a Classical Epithelial Beta-Defensin

Beta-defensins (BDs) are classical epithelial antimicrobial peptides of immediate importance in innate host defense. Since recent studies have suggested that certain BDs are also expressed in non-traditional tissues, including whole heart homogenate, and because effector molecules of innate immunity and inflammation can influence the development of certain cardiovascular disease processes, we hypothesized that BDs are produced by cardiomyocytes as a local measure of cardioprotection against danger signals. Here we report that at least one rat beta- defensin, rBD1, is expressed constitutively in cardiomyocytes specifically isolated using position- ablation-laser-microdissection (P.A.L.M. Microlaser Technologies). RT-PCR analysis showed expression of a single 318 bp transcript in adult rat heart (laser-excised cardiomyocytes) and H9c2 cells (neonatal rat heart myoblasts). Moreover, the full length cDNA of rBD1 was established, and translated into a putative peptide with 69 amino acid residues. The predicted amino acid sequence of the adult rat cardiac BD-1 peptide displayed 99% identity with the previously reported renal rBD1, and 88%, 53%, 53% and 50% identity with mouse, human, gorilla, and rhesus monkey BD1 respectively. Furthermore, structural analysis of the cardiac rBD1 showed the classical six- cysteine conserved motif of the BD family with an alpha-helix and three beta-sheets. Additionally, rBD1 displayed a significantly greater number of amphoteric residues than any of the human analogs, indicating a strong pH functional dependence in the rat. We suggest that rBD1, which was initially believed to be a specific epithelium-derived peptide, may be also involved in local cardiac innate immune defense mechanisms.

Characterization of adenosine transport in H9c2 cardiomyoblasts

Adenosine plays a significant role in various physiological processes including cardioprotection. Nucleoside transporters modulate adenosine levels in the vicinity of adenosine receptors, which in turn modulate adenosine functional efficacy. In the current study, adenosine transport in the rat heart myoblast cell line H9c2 was characterized. Kinetic analysis of adenosine transport in H9c2 cells revealed a K m of 8.9 ± 0.001 μM and a V max of 32.1 ± 0.65 pmol/mg protein/min. Adenosine transport in H9c2 cells was Na +-independent. About 6% of the total adenosine uptake was sensitive to nitrobenzylmercaptopurine riboside (NBMPR); however, 94% was insensitive, suggesting that adenosine uptake by H9c2 cells was predominantly mediated by the equilibrative nucleoside transporter (ENT)-2 and only mildly by ENT-1. Results of RT–PCR demonstrated the presence of mRNA for ENT-1, ENT-2 and ENT-3. Upon culture in a cell differentiation medium containing fetal bovine serum (1%) and retinoic acid (10 nM), both the activity and mRNA expression of ENT-1 increased 3-fold, however, ENT-2 was unaffected. Pharmacological studies revealed that ENT-1 activity was stimulated by PKA and PKC-δ/ε, however, ENT-2 activity was unaffected. Taken together, the exceptionally high expression level of ENT-2 in H9c2 cells raises questions regarding the use of H9c2 cells as a model for physiological adenosine activity in the heart. Furthermore, this study may form the basis for further investigation into the effect of cell differentiation and protein kinases on the regulation of nucleoside transporters.

Restoration of sarcoplasmic reticulum protein level by thyroid hormone contributes to partial improvement of myocardial function, but not to glucose metabolism in an early failing heart.

In heart failure, sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) activity is decreased, resulting in abnormal Ca(2+) handling and contractile dysfunction. We have previously reported that impaired glucose transporter (GLUT4) activity was an early indicator of progression of heart failure in pressure overload hypertrophied heart. This study was aimed to examine the contribution of both SERCA2 and glucose metabolism in pressure overload hypertrophied heart. Thyroid hormone, which is known to restore GLUT4 and/or SERCA2 function, was also tested. Hypertrophied rat heart was created by abdominal aortic banding for 16 and 26 weeks. Then 20-40 microg/kg of 3,5,3'-triiodo-L-thyronine (T3) was administered subcutaneously daily for the last 4 weeks. Hypertrophied myocytes were created by the stimulation of H9c2(2-1) rat heart myoblasts with 2 micromol/L of isoproterenol for 3, 7 and 10 days. Left ventricle function of the hypertrophied rat hearts were measured in Langendorff perfusion. Myocardial protein levels of GLUT4 and SERCA2 in two models were analyzed by Western immunoblotting. Glucose and lactate concentration of cultured medium of myocytes were measured enzymatically to determine the efficacy of glycolysis. Diastolic function (tau) was significantly deteriorated in 16-week heart with significantly lower SERCA2 protein (89.3%) than control. In 26-week heart, both systolic and diastolic function (+dP/dt max, -dP/dt max and tau) was significantly deteriorated. This was associated with significant decrease in both GLUT4 and SERCA2 protein (84.8 and 91.6%, respectively). In cultured hypertrophied myocytes, glycolysis was shifted from aerobic to anaerobic during progression of hypertrophy. GLUT4 protein was significantly decreased at day 7 (45.6% of control). This led to a down-regulation of SERCA2 protein at day 10 (51.8% of control). Although there was no impact of T3 treatment on GLUT4, SERCA2 protein level was almost reversed with partial improvement of myocardial function. We conclude that impairment of both glucose metabolism and SERCA2 function were seen in an early heart failure. Thyroid hormone partially improved myocardial function with successful improvement of SERCA2 protein but no impact on GLUT4 protein expression in hypertrophied rat heart. Restoration of glucose metabolism is a critical step to avoid further progression of heart failure.

Calpain is activated in experimental uremia: Is calpain a mediator of uremia-induced myocardial injury?

The cysteine proteases calpain and caspase-3 are known mediators of cell death. The aim of this study was to assess their contribution to the tissue damage found in experimental uremia. Calpain and caspase-3 activities were measured in the hearts of rats that were sham-operated (control), sham-operated and spontaneously hypertensive (SHR), and those rendered uremic by 5/6 nephrectomy (uremic). In an in vitro study, heart myoblasts (Girardi) were incubated with human serum from healthy subjects (control serum conditioned media, CSCM) or uremic patients (uremic serum conditioned media, USCM), in the presence and absence of calpain and caspase-3 inhibitors. After 48 hours the activity of calpain and caspase-3 was measured, and cell injury determined by DNA fragmentation (ELISA) and lactate dehydrogenase (LDH) release. An in situ assay was designed to study how USCM affects calpain activity over time. In the in vivo study, mean calpain activities were almost identical in the control and SHR groups, but calpain and caspase-3 activities were much elevated in the uremic group (P < 0.01 and 0.001 respectively vs. control). The SHR group had significantly higher mean arterial blood pressure (P < 0.001 vs. control, 0.01 vs. uremic). In the in vitro study calpain activity and DNA fragmentation were markedly higher in USCM treated cells compared to CSCM (both P<0.05). Both were reduced in USCM cells containing calpain inhibitors (E64d, calpastatin, or PD 150606). LDH release was raised also in USCM treated cultures (P < 0.05), which only the E64d treatment could significantly reduce (P < 0.02). Caspase-3 activities were similar in USCM and CSCM groups. The in situ assay showed significant increases in calpain activity in USCM treated cells compared to CSCM after just 3.5 hours (P<0.01). In vivo results suggest that the increases in calpain and caspase-3 activity in uremic rat hearts were primarily due to uremia and not to hypertension. In vitro data demonstrate that uremia-induced cell injury can be attenuated by calpain inhibition. Therefore, it is likely that calpain is a mediator of uremia-induced myocardial injury.

Continuous Measurement of Glucose Utilization in Heart Myoblasts

Understanding quantitative aspects of cell energy metabolism and how it is influenced by environment is central to biology, medicine, and biotechnology. Most methods used for measuring metabolic fluxes associated with energy metabolism require considerable personnel effort or high maintenance instrumentation. The microphysiometer is a commercially available instrument that measures acid extrusion rates, which are commonly used for drug screening. With the addition of oxygen sensors, the instrument can also be used to measure cell oxygen consumption rates and thereby calculate glycolytic fluxes. In the work described here, oxygen consumption and acid extrusion rates were used to measure glucose utilization by the H9c2 rat heart myoblast cell line and these results are compared with fluxes measured with a radiometric assay. Both assays were used to investigate changes in H9c2 energy metabolism due to cell stimulation with carbachol and insulin. The results demonstrate the utility of the microphysiometer method for measuring both transient and sustained changes in partitioning of glucose utilization between glycolysis and oxidation in live cells.

Trypanosoma cruzi Uses a 45-kDa Mucin for Adhesion to Mammalian Cells

A set of monoclonal antibodies that recognizes a Trypanosoma cruzi 45-kDa protein was produced and used to characterize this molecule and study its role in trypanosome adhesion to heart myoblasts. We found that the 45-kDa protein is a surface mucin, is expressed only in invasive trypomastigotes, but not in noninvasive epimastigotes or amastigotes, and is released by the trypanosome in culture medium. One of the monoclonal antibodies (Mab B5) from this set inhibits the attachment of trypomastigotes to heart myoblasts preventing trypanosome entry, whereas the others (Mabs B4 and F1) do not. This inhibition was seen with the B5 hybridoma culture supernatant, with the purified Mab B5 IgG or with Mab B5 Fab fragments. These novel findings identify the 45-kDa mucin as a new T. cruzi ligand that is used by invasive forms of this organism to adhere to heart myoblasts.

Constitutive hsp70 Attenuates Hydrogen Peroxide-Induced Membrane Lipid Peroxidation

Thermal pretreatment improves cardiac recovery from subsequent ischemia/reperfusion. Induction of heat shock proteins (hsps) may contribute to this protection. We have demonstrated that augmentation of the constitutive hsp70 (hsc70) in H9c2 heart myoblasts promotes oxidative resistance. We employed a model oxidant to explore potential target(s) of protection by hsc70. Upon exposure to 54 μM of hydrogen peroxide (H2O2), hsc70-overexpressing cells exhibited a lower lipid peroxidation than the sham-transfected control. Constituitive hsc70 overexpression, however, did not protect against H2O2-induced depletion of ATP and glutathione (GSH). Lipid protection also occurred in cells preconditioned at 39°C (selectively induces hsc70) during H2O2 exposure. Interestingly, the protection conferred by hsc70 was comparable in magnitude to that provided by α-tocopherol, and was followed with a reduced release of lactate dehydrogenase and a unaltered calcium uptake during H2O2 challenge. Collectively, our observations suggest that hsc70 may preserve membrane function via attenuation of lipid peroxidation during oxidative insult.

Cardiolipin: biosynthesis, remodeling and trafficking in the heart and mammalian cells (Review).

Cardiolipin is the principal polyglycerophospholipid found in the heart and most mammalian tissues. This phospholipid is the only phospholipid localized exclusively to the mitochondria of mammalian cells. Cardiolipin appears to be involved, either directly or indirectly, in the modulation of a number of cellular processes including the activation of mitochondrial enzymes and hence production of energy by oxidative phosphorylation. The regulatory properties which govern cardiolipin biosynthesis, its remodeling and trafficking are beginning to emerge. Studies in the isolated perfused rat heart and H9c2 cardiac myoblast cells have indicated that the rate-limiting step of cardiolipin biosynthesis, via the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway, is the conversion of phosphatidic acid and cytidine-5'-triphosphate to cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol. The cellular level of cytidine-5'-triphosphate appears to control the production of cardiolipin in H9c2 cells. The activities of the other enzymes of the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway of cardiolipin biosynthesis in the heart may be modulated by thyroid hormone and unsaturated fatty acids. In addition, extra-mitochondrial cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol and phosphatidylglycerol may be utilized for cardiolipin biosynthesis in the heart and permeabilized cells. Cardiolipin may be readily hydrolyzed by phospholipases and may be remodeled by a deacylation-reacylation pathway. Studies with a Chinese hamster lung fibroblast cell line CCL16-B2 have indicated that the remodeling of cardiolipin is markedly altered in the mitochondria of these cells and that this alteration in remodeling may be one of the underlying mechanisms for the mutation in oxidative energy production in these cells. Host cell cardiolipin may be trafficked from the mitochondria to an intracellular bacterial parasite Chlamydia trachomatis. The purpose of this review is to briefly discuss some of the more recent findings in cardiolipin metabolism in the heart and mammalian cells and to provide insight into their possible implications in the regulation of some cellular functions in mammalian tissues and cells.

Cloning of the rat fibroblast growth factor-2 promoter region and its response to mitogenic stimuli in glioma C6 cells.

Basic fibroblast growth factor (FGF-2) is abundant in the developing and adult brain and has been linked with the origin and growth of neuronal and glial cells. Glial cells produce high levels of FGF-2, stimulating autocrine growth as well as the survival and function of neurons in a paracrine manner. Abnormal levels of FGF-2 have been linked with Alzheimer's, Huntington's, and Parkinson's diseases. Recent evidence has suggested that a component of the mitogenic response of glial cells is exerted on FGF-2 at the transcriptional level. To assess transcriptional regulation of this potent growth factor, we cloned a 1.4-kb genomic fragment containing the rat FGF-2 promoter region. DNA blotting results indicated that the rat FGF-2 gene exists as a single copy in the genome. The promoter region contains no TATA box but appears to rely instead on multiple GC-rich start sites (P0, P1, and P2) for transcription initiation in rat brain as well as C6 glioma cells. One of these sites (P0) was located within four nucleotides of the reported 5' end of the rat brain cDNA and constituted part of a consensus Egr-1 binding site (5'-GCGGGGGCG-3'). Transcription from this site could be stimulated in C6 glioma cells in response to phorbol ester treatment. The induction of a "new" site (P1) with phorbol ester also suggested a mechanism to explain the discrepancy between the reported "starts" for the ovarian versus brain cDNAs. A hybrid luciferase gene directed by rat FGF-2 5'-flanking DNA (-1,058/+54) was expressed in rat glioma C6, heart myoblast H9c2, and human astrocytoma U87-MG cells after gene transfer. The level of transfected FGF-2 promoter activity was higher in glial cells (C6 and U87-MG) compared with nonglial (H9c2) cells. Also, expression of this hybrid FGF-2/luciferase gene was increased in response to phorbol ester or serum treatment of C6 cells. Deletion analysis revealed the presence of both positive and negative regulatory regions that are involved in the transcriptional control of rat FGF-2 gene by mitogenic stimuli.

Regulatory Effect of the Level of Free Ca 2+ of the Host Cell on the Capacity of Trypanosoma cruzi to Invade and Multiply Intracellularly

We studied whether modification of the free intracellular Ca2+ level of a mammalian host cell would affect its susceptibility to infection by Trypanosoma cruzi or its capacity to support trypomastigote-->amastigote transformation and amastigote replication. Pretreatment of rat heart myoblasts (RHM) with BAPTA.AM or Quin-2.AM, intracellular Ca2+ chelators, decreased the susceptibility of these cells to infection by untreated trypomastigotes. This was evidenced by a significant drop in both the percentage of infected RHM and the average number of organisms per 100 host cells relative to control values. Similar RHM treatment with the Ca2+ ionophore ionomycin had the opposite effects. The rate of trypomastigote-->amastigote transformation measured in RHM that had been treated with BAPTA.AM, Quin-2.AM, or ionomycin before and after, but not during co-culture with trypomastigotes was not significantly altered. The rate of intracellular amastigote multiplication measured in RHM exposed to the intracellular Ca2+ chelators only after virtually all of the internalized trypomastigotes had transformed into amastigotes was significantly decreased by incubation with BAPTA.AM or Quin-2.AM but was increased by ionomycin. None of the drug treatments affected RHM viability to any significant extent. These results suggest that T. cruzi relies on host cell Ca(2+)-dependent events, utilizes host cell free Ca2+ during invasion, or both, and highlight a requirement for an adequate free Ca2+ level for effective intracellular T. cruzi multiplication but not for trypomastigote-->amastigote transformation.

Effect of "Enhancer" Sequences on Ventricular Myosin Light Chain-2 Promoter Activity in Heart Muscle and Nonmuscle Cells

Positive (HF-1 and HF-2) and negative (HF-3) elements responsible for cardiac-specificity of the rat ventricular myosin light chain-2 (MLC-2v) promoter are contained in a 250 base pair region. The effect of the simian virus 40 (SV40) enhancer or 3 copies of the HF-1, HF-2 and HF-3 elements on MLC-2v promoter activity and specificity was assessed in neonatal rat cardiac myocytes and cardiac nonmuscle cells as well as rat heart myoblast H9c2 and glioma (nonmuscle) C6 cell Lines. The SV40 enhancer increased promoter activity by at least 10-fold in both muscle and nonmuscle cell types; however, there was a decrease in cardiac ventricular myocyte-specificity. In contrast, the 3 copies of HF-1, HF-2 and HF-3 elements stimulated MLC-2v promoter activity ∼3-fold in neonatal ventricular cardiac myocytes alone and, effectively, displayed about a 5-fold increase in specificity over the wild type MLC-2v promoter.

Changes in Trypanosoma cruzi infectivity by treatments that affect calcium ion levels

The possible role of the intracellular Ca 2+ level in the regulation of Trypanosoma cruzi infectivity was explored by measuring the capacity of trypomastigote forms of this organism to invade mammalian host cells after treatments which decrease or elevate cytoplasmic Ca 2+. Parasites loaded with either bis-( o-aminophenoxy)-ethane- N, N, N′, N′ tetraacetic acid (BAPTA) or 2-{[2-bis(carboxymethyl)-amino-5-methylphenoxy]methyl}-6-nethoxy-8-bis(carboxymethyl)aminoquinoline (Quin-2) to chelate Ca 2+ displayed significantly decreased infectivity. This effect was denoted by reductions in both the proportion of rat heart myoblasts invaded by the parasite in vitro and the number of trypanosomes penetrating these host cells, the extents of which were BAPTA or Quin-2 concentration dependent. Consistent with these observations, inhibitory effects were also recorded when the parasite was pretreated with the calmodulin-binding phenothiazines trifluoperazine and chlorpromazine or with felodipine, a chemically different type of calmodulin antagonist, for as little as 5 min. In contrast, pretreatment with the Ca 2+ ionophore ionomycin, which elevated Ca 2+ levels in T. cruzi, significantly enhanced the infective capacity of the parasite. These results point to the existence of a Ca 2+-dependent mechanism that regulates the invasive capacity of T. cruzi.

Levels of the Mitochondrial Endonuclease during Rat Cardiac Development Implicate a Role for the Enzyme in Repair of Oxidative Damage in Mitochondrial DNA

Mammalian heart mitochondria (MT) contain a potent Mg 2+-dependent DNA endonuclease that becomes soluble once isolated mitochondria are disrupted using detergent. The level of this endonuclease was previously found to be markedly elevated in adult rat heart compared to other adult rat tissues. Among tissues, the level of the MT endonuclease does not appear to be correlated with the rate of MT DNA replication but rather with the rate of oxidative metabolism [Houmiel, K. L., Gerschenson, M. and Low, R. L., 1991. Biochimica Biophysica Acta 1079: 197-202]. In the present study, the level of the endonuclease has been quantitated both during rat cardiac development, from gestational day 18 through adulthood, and in cultured rat heart myoblasts. Surprisingly, the specific activity of the MT endonuclease in fetal and newborn mitochondria is high. The values are greater than 50% of that seen in the adult even though the mitochondria at this period of heart development are few and structurally disorganized. Remarkably, there is a burst of endonuclease activity at day 2 which accompanies a similar, transient elevation of respiratory complex I and IV activities. At later times, the endonuclease activity gradually increases until adulthood and correlates with steady increases in MT DNA and DNA polymerase-γ. In cultured myoblasts, the level of the endonuclease increases about seven-fold as the growing cells reach confluency and differentiate into myotubes. These variations in the specific activity of the endonuclease, when considered along with other properties of the enzyme suggest that the endonuclease may serve a role in the removal of oxidative damage in MT DNA incurred from respiration.

Purification of a 74-kilodalton surface glycoprotein from heart myoblasts that inhibits binding and entry of Trypanosoma cruzi into heart cells

We have identified and purified a 74 kDa surface glycoprotein from heart myoblasts that specifically binds to Trypanosoma cruzi trypomastigotes, and inhibits the attachment and internalization of trypomastigotes into these cells. The native form of the 74 kDa glycoprotein was purified to apparent homogeneity by preparative scale isoelectrofocusing and anion exchange chromatography. Pre-incubation of trypomastigotes with soluble 74 kDa glycoprotein strongly inhibited the binding and internalization of trypomastigotes into heart myoblast monolayers in a concentration dependent-manner. Pre-incubation of heart myoblast monolayers with antibodies specific to the purified 74 kDa glycoprotein also strongly inhibited trypomastigote binding and internalization into heart cells in a concentration dependent manner. These results support the notion that the surface 74 kDa glycoprotein is a target molecule on heart myoblast cells to which T. cruzi binds in order to invade them.

Inhibition of host cell invasion and intracellular replication of Trypanosoma cruzi by N,N'-bis(benzyl)-substituted polyamine analogs.

We studied the effects of two N,N'-bis(benzyl)-substituted polyamine analogs on the capacities of Trypanosoma cruzi to invade and multiply within a mammalian host cell. At concentrations as low as 1 microM, these compounds reduced significantly the infectivity of the parasite for rat heart myoblasts in a time-dependent manner. Pretreatment of virulent T. cruzi trypomastigotes, but not myoblast pretreatment, reduced the level of infectivity. The inhibitory effects started to subside 3 h after removal of the drugs and were no longer detectable after 4 h. A significant decrease in the rate of intracellular amastigote multiplication was also seen when the drugs were added to myoblast cultures which had been previously infected with untreated T. cruzi. These results show that N,N'-bis(benzyl)-substituted polyamine analogs meet the two most important criteria for potential chemotherapeutic agents against T. cruzi infection, namely, inhibition of both host cell invasion and intracellular replication by this parasite.

Inhibition of S-Adenosyl-L-Methionine (AdoMet) Decarboxylase by the Decarboxylated AdoMet Analog 5'-[(Z)-4-Amino-2-Butenyl]Methylamino-5'-Deoxyadenosine (MDL 73811) Decreases the Capacities of Trypanosoma cruzi to Infect and Multiply within a Mammalian Host Cell

A decarboxylated S-adenosyl-L-methionine (AdoMet) analog, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL 73811), that specifically and irreversibly inhibits AdoMet decarboxylase (DC) was used to investigate the role of AdoMetDC in the regulation of host cell invasion and intracellular replication by Trypanosoma cruzi. The presence of MDL 73811 in cocultures of T. cruzi and rat heart myoblasts (RHM) significantly inhibited host cell infection in a dose-dependent manner. This effect, evidenced by reductions in the proportion of infected RHM and the number of parasites per 100 RHM, was due to MDL 73811 action on T. cruzi as it was reproduced when the parasites, but not the RHM, were pretreated with the inhibitor. Significant inhibition of infectivity required a 10-min treatment with MDL 73811 although greater effects ensued with additional incubation time. We could not detect signs of recovered infectivity up to 6 hr after the removal of nonincorporated MDL 73811, suggesting that either AdoMetDC turnover in T. cruzi is a relatively slow process or the amounts of MDL 73811 taken up could not be blocked or eliminated during this time period. MDL 73811-mediated inhibition of infectivity was not bypassed by the addition of exogenous spermidine or spermine, suggesting that the mechanism of action does not involve reduced production of these polyamines due to AdoMetDC inhibition. Intracellular accumulation of AdoMet appeared to be a more plausible explanation in view of the fact that exogenous AdoMet significantly inhibited infectivity. When added to infected RHM cultures, MDL 73811 or AdoMet inhibited also intracellular T. cruzi growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Attachment of Trypanosomacruzi to host cells: A monoclonal antibody recognizes a trypomastigote stage-specific epitope on the gp 83 required for parasite attachment

A set of monoclonal antibodies against the purified surface gp 83 of T. cruzi trypomastigotes was produced and the ability of these monoclonals to inhibit the attachment of trypomastigotes to heart myoblasts was investigated. Western blots of solubilized trypomastigotes, epimastigotes or amastigotes probed with this set of monoclonal antibodies show that the gp 83 is present in invasive trypomastigotes, but not in non-invasive epimastigotes or amastigotes. One monoclonal antibody (Mab 4A4) from this set inhibits the attachment of trypomastigotes to heart myoblasts, whereas the others (MAbs 2H6, 4B9, 2D11) do not. These results show that the Mab 4A4 recognizes an epitope on the gp 83 of invasive trypomastigotes required for parasite binding to host cells.

Purification of [formula omitted] surface proteins involved in adhesion to host cells

We have identified four surface 83 kDa proteins of pI values 6.3, 6.4, 6.5 and 6.6 in T. cruzi trypomastigotes which specifically bind to rat heart myoblasts. These proteins were purified by isoelectric focusing and anion-exchange chromatography in an FPLC system. These 83 kDa proteins inhibit the attachment of trypomastigotes to myoblasts in a concentration-dependent manner, indicating that these trypomastigote proteins mediate the attachment of trypomastigotes to heart myoblasts.