Cytoprotective mechanisms in cultured cardiomyocytes.
Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine mainly secreted by macrophages exerts pleiotropic effects on different cell types. However, the intracellular mediators of its action are not yet well characterized. To get an insight into endogenous cytoprotective mechanisms, we developed an in vitro model based on cultured cardiomyocytes treated with TNF-alpha at which we examined gene expression of heat shock proteins (HSP-27, HSP-70 and ubiquitin). Cardiomyocytes were isolated from the hearts of 18 day old fetal mice by enzymatic dissociation and grown in minimum essential medium containing 10% fetal calf serum. Spontaneously contractile cells were serum deprived for 24 h and treated with TNF-alpha (25 ng/ml) for 1, 2, 4, 6, 8, 12, and 24 h After each incubation, cells were processed to extract total proteins for Western and total RNA for Northern blot analyses. TNF-alpha induced arrhythmias and cessation of spontaneous contractions in a concentration and time dependent manner. Steady state (ubiquitin) or undetectable mRNA levels (HSP-27, HSP-70) were drastically induced (> 4 fold for all three genes vs untreated control cells) by TNF-alpha, reaching maximal values between 6-8 h of stimulation. Thereafter, the expression of these stress genes declined but remained elevated as compared to control. By Western blot analysis, we found increased multiple bands of ubiquitin protein conjugates in TNF-alpha treated cells whereas no significant change in HSP-27 protein accumulation until 12 h was observed as compared to control. 24 h of TNF-alpha incubation resulted in partial cellular necrosis. Our results indicate that TNF-alpha induces in cardiomyocytes transiently gene expression for cytoprotective molecules like HSP-27, HSP-70 and ubiquitin, suggesting these stress proteins to participate in subsequent defense mechanisms, for example in postischemic myocardial recovery.
Cardiovascular and Molecular Biology Laboratory, Erasmus University, Rotterdam, The Netherlands.
H S
HS
Sharma
J
J
Stahl
D
D
Weisensee
I
I
Löw-Friedrich
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D009206:Myocardium / Q000166:cytology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014452:Ubiquitins / Q000378:metabolism; D000099190:MAP-Kinase-Activated Kinase 2
D018840:HSP70 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D014452:Ubiquitins; D000099190:MAP-Kinase-Activated Kinase 2