Gene module regulation in dilated cardiomyopathy and the role of Na/K-ATPase.

Dilated cardiomyopathy (DCM) is a major cause of cardiac death and heart transplantation. It has been known that black people have a higher incidence of heart failure and related diseases compared to white people. To identify the relationship between gene expression and cardiac function in DCM patients, we performed pathway analysis and weighted gene co-expression network analysis (WGCNA) using RNA-sequencing data (GSE141910) from the NCBI Gene Expression Omnibus (GEO) database and identified several gene modules that were significantly associated with the left ventricle ejection fraction (LVEF) and DCM phenotype. Genes included in these modules are enriched in three major categories of signaling pathways: fibrosis-related, small molecule transporting-related, and immune response-related. Through consensus analysis, we found that gene modules associated with LVEF in African Americans are almost identical as in Caucasians, suggesting that the two groups may have more common rather than disparate genetic regulations in the etiology of DCM. In addition to the identified modules, we found that the gene expression level of Na/K-ATPase, an important membrane ion transporter, has a strong correlation with the LVEF. These clinical results are consistent with our previous findings and suggest the clinical significance of Na/K-ATPase regulation in DCM.

Evaluation of Cardiotonic Steroid Modulation of Cellular Cholesterol and Phospholipid.

We have previously shown that 21-benzylidene digoxin (21-BD) increases the total cholesterol and phospholipid content on the membrane of HeLa cells. Lipid modulation caused by cardiotonic steroids (CTS) is still unexplored. Therefore, the aim of the present study was to evaluate the cholesterol and phospholipid modulation of the cell membrane caused by ouabain and 21-BD and the possible involvement of the caveolae on this modulation. For this, one cell line containing caveolae (HeLa) and other not containing (Caco-2) were used. The modulation of the lipid profile was evaluated by total cholesterol and phospholipids measurements, and identification of membrane phospholipids by HPTLC. The cholesterol distribution was evaluated by filipin staining. The caveolin-1 expression was evaluated by Western Blotting. Ouabain had no effect on the total membrane lipid content in both cell lines. However, 21-BD increased total membrane phospholipid content and had no effect on the membrane cholesterol content in Caco-2 cells. CTS were not able to alter the specific phospholipids content. In the filipin experiments, 21-BD provoked a remarkable redistribution of cholesterol to the perinuclear region of HeLa cells. In Caco-2 cells, it was observed only a slight increase in cholesterol, especially as intracellular vesicles. The caveolin-1 expression was not altered by any of the compounds. Our data mainly show different effects of two cardiotonic steroids. Ouabain had no effect on the lipid profile of cells, whereas 21-BD causes important changes in cholesterol and phospholipid content. Therefore, the modulation of cholesterol content in the plasma membrane of HeLa cells is not correlated with the expression of caveolin-1.

Differences of lipid membrane modulation and oxidative stress by digoxin and 21-benzylidene digoxin.

Cardiotonic steroids (CTS) are compounds which bind to the Na,K-ATPase, leading to its inhibition and in some cases initiating signaling cascades. Long utilized as a treatment for congestive heart disease, CTS have more recently been observed to inhibit proliferation and cause apoptosis in several cancer cell lines. A synthetic derivative of the CTS digoxin, called 21-benzylidene digoxin (21-BD), activates the Na,K-ATPase rather than cause its inhibition, as its parent compound does. Here, the mechanism behind the unique effects of 21-BD are further explored. In HeLa cancer cells, low (5µM) and high (50µM) doses of 21-BD activated and inhibited the Na,K-ATPase, respectively, without altering the membrane expression of the Na,K-ATPase. While digoxin did not affect HeLa membrane cholesterol or phospholipid content, 50µM 21-BD increased both lipids via a mechanism reliant on an intact cell. Afterwards, the direct action of 21-BD was evaluated on erythrocyte membranes; however, no effect was observed. As CTS may generate reactive oxygen species (ROS) which can affect plasma membrane fluidity and therefore Na,K-ATPase activity, several markers involved in ROS generation were analyzed such as, lipid peroxidation (TBARS), reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD). GSH content and catalase activity were unaffected by digoxin or 21-BD. Surprisingly, TBARS and SOD activity was decreased with digoxin and with 50µM 21-BD. Thus, 21-BD and digoxin altered components involved in ROS generation and inhibition in a similar fashion. This study suggests alterations to the Na,K-ATPase and membrane lipids by 21-BD is not reliant on ROS generation.

The Influence of Fatty Acid Methyl Esters (FAMEs) in the Biochemistry and the Na(+)/K(+)-ATPase Activity of Culex quinquefasciatus Larvae.

Culex quinquefasciatus is the main vector of lymphatic filariasis and combating this insect is of great importance to public health. There are reports of insects that are resistant to the products currently used to control this vector, and therefore, the search for new products has increased. In the present study, we have evaluated the effects of fatty acid methyl esters (FAMEs) that showed larvicidal activity against C. quinquefasciatus, on glucose, total protein, and triacylglycerol contents and Na(+)/K(+)-ATPase activity in mosquito larvae. The exposure of the fourth instar larvae to the compounds caused a decrease in the total protein content and an increase in the activity of the Na(+)/K(+)-ATPase. Furthermore, the direct effect of FAMEs on cell membrane was assessed on purified pig kidney Na(+)/K(+)-ATPase membranes, erythrocyte ghost membranes, and larvae membrane preparation. No modifications on total phospholipids and cholesterol content were found after FAMEs 20 min treatment on larvae membrane preparation, but only 360 µg/mL FAME 2 was able to decrease total phospholipid of erythrocyte ghost membrane. Moreover, only 60 and 360 µg/mL FAME 3 caused an activation of purified Na(+)/K(+)-ATPase, that was an opposite effect of FAMEs treatment in larvae membrane preparation, and caused an inhibition of the pump activity. These data together suggest that maybe FAMEs can modulate the Na(+)/K(+)-ATPase on intact larvae for such mechanisms and not for a direct effect, one time that the direct effect of FAMEs in membrane preparation decreased the activity of Na(+)/K(+)-ATPase. The biochemical changes caused by the compounds were significant and may negatively influence the development and survival of C. quinquefasciatus larvae.

Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane.

Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1), iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 µM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5%) than in women and was associated with an increase (446%) in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS) and an increase (327%) in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 µM FeCl3 for 24 h showed an increase (132%) in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels.