The Effects And Mechanism Of Tumor Necrosis Factor α On The Expression Of Calcium Handling Proteins And Concentration Of Intracellular Free Calcium In Cardiomyocytes

Recent data demonstrate that phospholamban (PLB) is a key regulator of cardiac contractility. PLB is a 52-amino acid protein that is localized in the sarcoplasmic reticulum (SR) membrane ,and regulates the kinetics of cardiac contractility through interferring with myocardial Ca²⁺ homeostasis by decreasing the affinity of SERCA2afor Ca²⁺ and reducing the rate of Ca²⁺ uptake into the SR. To alter the expression of PLB and/or SERCA2a will affect cardiac contractile function.Tumor necrosis factor (TNF)- a is a proinflammatory cytokine with pleiotropic biological effects. It mainly derived from activated macrophages and cardiomyocytes have the capacity to synthesize TNF a .It participate in a variety of cardiovascular diseases, including congestive heart failure, acute myocarditis and ischaemic cardiomyopathy. Many studies demonstrated that TNF a depress contractile function , and further investigation of the TNF a, revealed that TNF a depress contractile function through to interfere with myocardial calcium handling. However its molecular mechanism remains uncertain.Although PLB is a key regulator of cardiac contractility and TNF a has significantly negative inotropic effect on myocardium. It is remain unclear whether the diminished cardium function effected by TNF a is attributable to the expression changes at the level of PLB and/or SERCA2a. The present study was designed to determine whether and to what extent alterations in the expression of PLB and/or SERCA2a and the intracellular free calcium in cultured neonatal rat cardiac myocytes with TNF a .Synthetic oligonucleotides are utilized for a wide range of purposes in molecular biology. Rat neonatal cardiomyocytes in primary culture as a model used to study the effectes of TNF a on the expression of PLB and SERCA2a and alteration of intracellular free calcium in cardiomyocytes.To explore the molecular mechanism of negative inotropic of TNF a through to depress the expression of PLB with antisense oligonucleotides.Objectives1) To determine the effects of TNF a on the expression of PLB and SERCA2a, and whether there is concentration-dependent relationship.2) To investigate the effects of TNF a on intracellular free calcium in cultured neonatal rat cardiac myocytes.3) To explore whether PLB is the regulator of the effects of TNF a on intracellular free calcium in cardiomyocytes.Methods1) Neonatal rat Primary Cardiomyocyte Cultures l³-day-old Wistar rat neonates were euthanized by cervical dislocation. The hearts from the rat were removed aseptically and kept in Hanks' balanced salt solution without Ca²⁺ and Mg²⁺ on ice. The tissues were washed three to five times and the atrial tissue was removed, and the ventricles were cut into 1 mm³fragments and enzymatically digested through multiple rounds in 0.125% pancreatin and 0.05% collagenase in a balanced salt solution. The supernatant from the first incubation was discarded, 10 ml fresh enzyme solution was added, and the incubation procedure was repeated for 3 to 4 times. Subsequent supernatants were collected and centrifuged at 1000 rpm for 6 min and resuspended in DMEM media supplemented with 10% fetal bovine serum (FBS). Afterward, the cells were differentially plated for 1 h on uncoated cell culture dishes to remove contaminating nonmyocytes. Cells stained with trypan blue and counted. The enriched cardiomyocyte fractions were then plated at a density of l×l0⁶ cells/well on six-well tissue culture plates in DMEM media containing 10% FBS supplemented with 0.1 mmol/L bromodeoxyuridine for the first 72 h at 37° C in 5% CO₂. All cells were cultured in serum-free DMEM media supplemented with 5 μg/mL insulin, 1.5 μmol/L vitamin B 12, and 10 ug/mL transferrin for 24 hours before the initiation of experiments.2) Semi-quantitative RT-PCR Analysis Total RNA was isolated from neonatal cardiomyocytes by an UNIQ—10 Column Total RNA Isolation Kit (Shanghai Sangon), quantified by spectrophotometry and regulate the concentration of RNA to 0.1 μg/μl, followed the protocol of AMV Single S...