Cardioprotection Of Tumor Necrosis Factor-alpha And Its Signal Transduction

The heart is a tumor necrosis factor-alpha (TNF-a)-producing organ. Accumulating evidence indicates that myocardial TNF-a is an autocrine contributor that has been implicated in the pathogenesis of cardiovascular diseases, including myocardial dysfunction. Previous studies have suggested that TNF-a induces negative inotropic effects by disruption of calcium handling. But several studies showed that TNF-a is sufficient to protect cardiac myocytes against either hypoxic or ischemic injury by elevate mRNA and protein levels of manganous superoxide dismutase (Mn-SOD), which is regarded as the primary defensive enzyme against oxidative stress within mitochondria. However, the cell signaling mechanism involved TNF-a induced cardioprotection is not clear.Objectives:(1) To investigate the mechanism of the negative inotropic effect of TNF-a in cardiomyocytes;(2) To define the role of TNF-a in isolated rat heart and cultured neonatal ventricular myocytes during ischemia/reperfusion and hypoxia/reoxygenation;-5-(3) Testify whether the reactive oxygen species, nitric oxide or mitochondrial ATP-sensitive potassium (mitoKATP) channels are involved in the cardioprotection induced by TNF-o.Methords:(1) Papillary muscle preparation and measurement of contractionThe heart of male Sprague-Dawley rat was rapidly removed and transferred in ice-cold Tyrode solution. Papillary muscle was excised from the right ventricle and fixed in a tissue bath by two pieces of stainless steel pin. It was perfused with warmed Tyrode solution aerated with 95% O2 and 5% CO2, at a rate of 3 ml/min. The tendon was ligated with a silk lined to a tension transducer that was connected to the computer. Papillary muscle was stimulated with bipolar electrodes at twice threshold voltage with a pulse width of 1 ms. The contractile force was recorded and analyzed with MedLab system.(2) Preparation of isolated ventricular myocytesThe hearts were perfused with Ca2+-free Tyrode's solution with Langendorff apparatus. Then the perfusion solution was switched to a low Ca2+ (50 jiM) Tyrode's solution containing 0.03% collagenase and 1% bovine serum albumin (BSA) for 10 min. Then the ventricles were cut, minced, and gently triturated with a pipette in the low Ca2+Tyrode's solution containing BSA at 37癈 for 10 min. The cells were resuspended in the Tyrode's solution in which the Ca2+ concentration was gradually increased to 1.25 mM over 40 min.(3) Intracellular calcium recordingMonitoring of intracellular calcium was carried out using the fluorescent dye fura-2/AM. Fluorescence was measured on an Olympus inverted microscope equipped with a fluorometer system (T.I.L.L., Germany). The Ca2+-dependent signal of fura-2 was obtained by-6-illuminating at 340 and 380 nm and recording the emitted light at 510 nm. The [Ca2+], transient was induced by supra-threshold stimuli at 0.5 Hz delivered through two platinum field-stimulation electrodes in the bathing fluid.(4) Preparation of cardiac sarcoplasmic reticulum (SR) fractions(5) Preparation of cardiac plasma membrane(6) Determination of Ca2+-ATPase and Na+/K+-ATPase activityThe activities of rat myocardial SR Ca2+-ATPase, Ca2+-ATPase and Na+/K+-ATPase of plasma membrane were measured with colorimetrical methods by measuring the Pi liberated from ATP hydrolysis.(7) Isolated perfused rat heart preparationMale SD rat heart was retrogradely perfused with a 95%O2+5%CO2 equilibrated Krebs-Henseleit (K-H) solution via the aorta at a constant pressure of 74 mmHg. The Left ventricular developed pressure (LVDP), left ventricular end diastolic pressure (LVEDP), the maximal rates of left ventricular pressure elevation (+dp/dtmax) and depression (-dp/dtmax) were recorded by MedLab data acquisition system.(8) Neonatal rat cardiomyocyte cultureHearts were quickly removed from 1-day-old SD rats. The excised ventricles were cut into 1 to 2-mm cubes, then placed in 10ml D-Hanks containing 0.1% trypsin to dissociate. The dissociated cells were...