Rat Heart Myoblasts Research Articles

Ozone Attenuated H9c2 Cell Injury Induced by Doxorubicin

Doxorubicin (DOX) is a commonly used drug in the treatment of cancers, whereas its application in the clinical stage is restricted because of side effects such as cardiomyocyte injury. Increasing studies indicated that ozone may protect cardiomyocytes from injuries. This study aimed to explore the effects of ozone on cardiotoxicity induced by DOX treatment. Rat heart myoblasts (H9c2) were treated with increasing concentrations of DOX (0.5, 1, 1.5, and 2 μM) to induce cell injury. 3-(4,5)-dimethylthiahiazo(-2)-3,5-diphenytetrazoliumromide assay and flow cytometry analysis were used to measure the viability and apoptosis of H9c2 cells. The mRNA and protein levels of proinflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-(IL)1β, and IL-6, matrix metalloproteinases (MMP-2 and MMP-9), and the key factors on the TLR4/NF-kB signaling (TLR4, p-p65, and p65) were measured by reverse transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and western blot. The result showed that DOX promoted apoptosis and increased the expression of TNF-α (by 3.65-fold changes), IL-1β (by 4.98-fold changes), IL-6 (by 3.44-fold changes), MMP-2 (by 1.98-fold changes), and MMP-9 (by 1.98-fold changes) levels in H9c2 cells. Moreover, the introduction of ozone reversed these changes in gene expression and suppressed the activation of the TLR4/NF-kB signaling, which indicated that ozone may exert protective effects on H9c2 heart myoblasts by relieving the cardiotoxicity induced by DOX. Our study provides theoretical basis for the significance of ozone in managing doxorubicin-induced H9c2 heart myoblast injury.

Sodium Aescinate Protects Myocardial Injury Induced by Methylparathion Poisoning by Regulating The miR-301/Rock1 Axis

Methylparathion is a highly toxic insecticide known to cause lung, liver, and myocardial injuries, could lead to myocardial injury. These injuries are known to be ameliorated by sodium aescinate. In this study, we have examined the mechanism of diminution of sodium aescinate-mediated myocardial injury. To this end, we have examined the biochemical changes in the heart muscles isolated from rats, and in rat heart myoblast cell, H9c2. Both rats and H9c2 cells were treated with methylparathion followed by increasing dosage of sodium aescinate. Data from quantitative reverse transcription polymerase chain reaction or Western blot analyses show that miR-301 expression was decreased, while Rock1 was increased, in methylparathion-induced heart tissues or H9c2 cells. Treatment with sodium aescinate increased miR-301 expression and decreased Rock1 expression in a dosage-dependent manner. MicroRNAs bind to the 3′ - untranslated region of the target gene leading to the degradation of target mRNA. Methylparathion treatment reduced miR-301 expression in H9c2 cells, while increased Rock1 expression. However, sodium aescinate treatment enhanced miR-301 expression and repress Rock1 expression in a dosage-dependent manner. Methylparathion reduces cell viability of H9c2, while promotes the cell apoptosis. However, treatment with sodium aescinate rescues from methylparathion-induced cytotoxicity, while forced Rock1 expression aggravates methylparathion-induced cytotoxicity. In conclusion, sodium aescinate amelioration of methylparathion-induced myocardial injury by miR-301/Rock1 axis provides a new therapeutic strategy for the management of methylparathion poisoning.

EPAC IS A KEY MEDIATOR OF cAMP-INDUCED CARDIOPROTECTIVE SIGNALLING

It has been shown that cyclic AMP (cAMP)-induced protein kinase A (PKA) activation is very important in a variety of cardioprotective protocols including ischaemic and temperature preconditioning. However, it has been recently found that cAMP can also directly activate Epac. Our data show that a cell permeable cAMP analogue 8-Br-cAMP-AM (8-BR), which activates both PKA and Epac, can protect heart against ischaemia/reperfusion injury. The purpose of our study was to determine whether Epac activation is involved in the cAMP-induced cardioprotection. Experiments were carried out on Langendorff-perfused rat heart and on rat heart myoblasts H9C2. Along with 8-Br, the following cAMP analogues and inhibitors were used: an activator of PKA only, 6-Bnz-cAMP-AM (6-Bnz); activators of Epac only, 8-Br-2′-O-Me-cAMP-AM (O-Me) and 8-CPT-2′-O-Me-cAMP-AM (CPT); a PKA inhibitor H-89 and an Epac inhibitor ESI-09. Exposure of the H9C2 cells to these compounds revealed that 10 µM O-Me and 5 µM 8-Br activate Epac to a similar extent whilst ESI-09 completely blocks this effect of 8-Br. Perfusion of hearts with 10 µM 6-Bnz appeared to activate PKA to the same extent as 5 µM 8-Br. Further experiments demonstrated that neither 6-Bnz nor O-Me can produce cardioprotective effect similar to 8-Br. However, both H-89 and ESI-09 completely abolish 8-Br-induced cardioprotection whilst a mixture of 6-Bnz and CPT induced a strong protective effect. These data imply that activation of Epac alone is unable to protect the heart against ischaemia/reperfusion injury but it represents an essential link in cAMP-induced cardioprotection when activated together with PKA.

Quantitative profiling of the rat heart myoblast secretome reveals differential responses to hypoxia and re-oxygenation stress

Secretion of bioactive mediators regulates cell interactions with the microenvironment in tissue homeostasis and wound healing processes. We assessed the cardiomyocyte secretory response to hypoxia with the aim of identifying key mediators of tissue pathology and repair after ischemic heart attack. We profiled the secretome of rat H9C2 cardiomyoblast cells subjected to 16h hypoxia followed by 24h re-oxygenation using iTRAQ and label-free quantitative proteomics. A total of 860 and 2007 proteins were identified in the iTRAQ and label-free experiments respectively. Among these proteins, 1363 were identified as being secreted proteins, including mediators of critical cellular functions that were modulated by hypoxia/re-oxygenation stress (SerpinH1, Ppia, Attractin, EMC1, Postn, Thbs1, Timp1, Stip1, Robo2, Fat1). Further analysis indicated that hypoxia is associated with angiogenesis, inflammation, and remodeling of the extracellular matrix (ECM), whereas subsequent re-oxygenation was instead associated with modified secretion of proteins involved in suppression of inflammation, ECM modification, and decreased output of anti-apoptosis proteins. These data indicate that hypoxia and subsequent re-oxygenation modify the cardiomyocyte secretome in order to mitigate cellular injury and promote healing. The identified changes in cardiomyocyte secretome advance our current understanding of cardiac biology in ischemia/reperfusion injury and may lead to the identification of novel prognostic biomarker. Cardiovascular diseases (CVDs) are the leading cause of death globally. Myocardial infarction (MI) resulting from ischemic heart disease represents a substantial component of CVD-associated mortality, and is associated with obstruction of blood flow to the myocardium. Restoration of blood flow through the occluded coronary artery is the current most effective therapy to limit infarct size and preserve cardiac function after acute myocardial infarction. However, this treatment does not prevent subsequent development of heart failure in some patients. Reperfusion following ischemia causes additional cell death and increase in infarct size, a phenomenon called myocardial ischemia/reperfusion (I/R) injury. In order to advance our current understanding of cardiac biology in ischemia/reperfusion injury, we assessed the cardiomyocyte secretory response to hypoxia with the aim of identifying key mediators of tissue pathology and repair after ischemic heart attack. We profiled the secretome of rat H9C2 cardiomyoblast cells subjected to 16h hypoxia followed by 24h re-oxygenation using LC-MS/MS-based iTRAQ and label-free quantitative proteomics approaches. We identified many secreted proteins as mediators of critical cellular functions that were modulated by hypoxia and re-oxygenation stress. Further analysis of these modulated secretory proteins indicated that hypoxia is associated with angiogenesis, inflammation, and remodeling of the extracellular matrix (ECM), whereas subsequent re-oxygenation/reperfusion was instead associated with modified secretion of proteins involved in suppression of inflammation, ECM modification, and decreased output of anti-apoptosis proteins. These data indicate that hypoxia and subsequent re-oxygenation modify the cardiomyocyte secretome in order to mitigate cellular injury and promote healing. The identified changes in cardiomyocyte secretome may lead to the identification of novel prognostic biomarkers secreted from injured heart tissues into the circulation of patients with cardiovascular disease.

The Effect of Polymer Molecular Weight and UV Radiation on Physical Properties and Bioactivities of PCL Films

Biodegradable polymers, such as polycaprolactone (PCL), have been well studied in the application of tissue engineering and drug delivery systems due to their favourable biocompatibility profiles. However, little research has discussed the influence of the molecular weight of these polymers on cell behaviours. Here, PCL films were synthesized using polymers with various molecular weight and the physical characteristics of these films and their effect on cell growth were evaluated. Results showed that the tensile strength and elongation rate of PCL films increased from 1.82 MPa and 95.3% to 2.01 MPa and 741.6% when the molecular weight (Mn) of polymers increased from 40,000 to 80,000. A significant difference in polymer crystallinity was also observed between these formulations. Moreover, we found that prolonged exposure to UV radiation of these films could lead to the breakdown of polymer chains and consequently increased the crystallinity of PCL films with a corresponding reduction in the tensile strength. Using Hs68 human foreskin fibroblasts, PCL films prepared using high molecular weight polymers (CAPA 6800) displayed a higher cell proliferation rate compared to ones prepared using low molecular weight polymers (CAPA 6400 and 6500). In contrast, a higher cell proliferation rate was observed for PCL films made of low molecular weight polymers, especially CAPA 6500, when H9c2 rat heart myoblasts were used. In conclusion, polymer molecular weight can alter the

Efficiency of DNA Transfection of Rat Heart Myoblast Cells H9c2(2-1) by Either Polyethyleneimine or Electroporation

Expression of exogenous DNA in vitro is significantly affected by the particular transfection method utilized. In this study, we evaluated the efficiency of two transfection methods, chemically mediated polyethyleneimine (PEI) treatment and physically mediated electroporation, on a rat heart myoblast cell line, H9c2(2-1). After PEI transfection of pPgk-1/EGFP into H9c2(2-1) cells, EGFP expression could be easily detected by fluorospectrometer after 48 h (210 ± 12 RFU) and continued to increase after 72 h (243 ± 14 RFU). However, when H9c2(2-1) cells were transfected by electroporation (200 V, 500 μF, and one pulse), low level EGFP expression was observed after 48 h (49 ± 4 RFU) or 72 h (21 ± 14 RFU). In contrast, the easily transfectable control CHO-K1 cell line displayed a stronger EGFP expression than the H9c2(2-1) cells either by PEI or electroporation transfection. When transfection efficiencies were assayed by flow cytometry after 72 h, 13.6 ± 2.2% of PEI and 10.1 ± 1.5% of electroporation (250 V, 500 μF, and two pulses) transfected cells of H9c2(2-1) expressed EGFP, and PEI-transfected cells appeared to be less damaged (viability 93.6%) as compared to electroporation-transfected cells (39.5%). However, both PEI and electroporation (580 V, 50 Ω, and 50 μF) were effective for transfection of CHO-K1 with a higher efficiency, cell viability, and EGFP expression than H9c2(2-1). Our results indicate that the transfection efficiency of different methods varies among cell lines and that PEI is more efficient than electropolation for transfection of H9c2(2-1) whereas both PEI and electroporation are suitable for CHO-K1 transfection.

Tannic acid ameliorates doxorubicin-induced cardiotoxicity and potentiates its anti-cancer activity: Potential role of tannins in cancer chemotherapy

Doxorubicin, an anthracycline antibiotic, is widely used in the treatment of various solid tumors including breast cancer. However, its use is limited due to a variety of toxicities including cardiotoxicity. The present study aimed to evaluate the effect of tannic acid, a PARG/PARP inhibitor and an antioxidant, on doxorubicin-induced cardiotoxicity in H9c2 embryonic rat heart myoblasts and its anti-cancer activity in MDA-MB-231 human breast cancer cells as well as in DMBA-induced mammary tumor animals. Doxorubicin-induced cardiotoxicity was assessed by measurement of heart weight, plasma LDH level and histopathology. Bcl-2, Bax, PARP-1 and p53 expression were examined by western blotting. Our results show that tannic acid prevents activation of PARP-1, reduces Bax and increases Bcl-2 expression in H9c2 cells, thus, preventing doxorubicin-induced cell death. Further, it reduces the cell viability of MDA-MB-231 breast cancer cells, increases p53 expression in mammary tumors and shows maximum tumor volume reduction, suggesting that tannic acid potentiates the anti-cancer activity of doxorubicin. To the best of our knowledge, this is the first report which shows that tannic acid ameliorates doxorubicin-induced cardiotoxicity and potentiates its anti-cancer activity both in vitro (H9c2 and MDA-MB-231 cells) as well as in in vivo model of DMBA-induced mammary tumor animals.

Effect of Hyperglycemia on the Changes of Intracellular [Ca(superscript 2+)](subscript i) in Heart Myoblast

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in heart muscle. In the present study, we investigated changes of intracellular Ca2+ increased by potassium chloride (KCl) and phenylephrine (PE) under hyperglycemia in rat heart myoblast H9c2 cells (BCRC 60096), respectively. We employed the fluorescent Ca2+-indicator, fura-2, and digital imaging microscopy to measure [Ca2+]i in H9c2 cells. Cells were cultured in hyperglycemic (30 mM glucose) Dulbecco's Modified Eagle's Medium. The variation of [Ca2+]i induced by KCI and PE in hyperglycemia was examined, respectively. Moreover, tiron, one of the antioxidants, was pretreated in hyperglycemia-treated H9c2 cells to measure the role of free radicals in the changes of intracellular [Ca2+]i. An influx in intracellular Ca2+ induced by KCl or PE was observed in a dose-dependent manner and reached the highest concentration of 434 +/- 42.3 nM and 443 +/- 42.8 nM (n = 24 cells), respectively. Moreover, this increase of intracellular [Ca2+]i induced by KCl or PE was markedly reduced in cells exposed to hyperglycemia (434 +/- 42.3 vs. 1.26 +/- 0.21 nM and 443 +/- 42.8 vs. 2.54 +/- 0.25 nM, n = 24 cells, P < 0.001, respectively). Similar changes were not observed in cells received mannitol showing same osmolarity. However, the reduction of intracellular [Ca2+]i induced by hyperglycemia was abolished significantly in the presence of tiron. Our results suggest that an increase of intracellular Ca2+ by KCl or PE in heart cell was markedly reduced by hyperglycemic treatment; mediation of free radicals in this action can be considered because it was reversed in the presence of tiron.

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.

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.

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.

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.

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)

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.