Adenosine and TNF-α exert similar inotropic effect on heart cultures, suggesting a cardioprotective mechanism against hypoxia

When cardiomyocytes were subjected to hypoxia, tumor necrosis factor-α (TNF-α; 3–50 ng/ml) or adenosine (1–100 μM), decreased hypoxic damage as was detected by lactate dehydrogenase (LDH) release, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) absorbance, ROS (reactive oxygen species) measurement or desmin immunostaining. This cardioprotection was not prevented in TNF-α-treated cultures by 5-hydroxydecanoic acid (5-HD). Our aim was to elucidate whether adenosine and TNF-α mediate a similar protective mechanism against hypoxia in primary heart cultures and in H9c2 cardiomyocytes. Adenosine and TNF-α are known for their negative inotropic effects on the heart. We have suggested that deoxyglucose uptake reflects heart contractility in cell cultures; therefore, we assayed its accumulation under various conditions. Treatment for 20 min with adenosine, R-PIA [(−)-N(6)-phenylisopropyladenosine] (10 μM), or TNF-α reduced 3H-deoxyglucose uptake in primary heart cultures and also in H9c2 cardiomyocytes by 30–50%. Isoproterenol accelerated 3H-deoxyglucose uptake by 50%. Adenosine, R-PIA, or TNF-α attenuated the stimulatory effect of isoproterenol on 3H-deoxyglucose uptake to control levels. Hypoxia reduced 3H-deoxyglucose uptake by 50%, as in the treatment of the hypoxic cultures with TNF-α or adenosine. Glibenclamide (2 μM), 5-HD (300 μM), or diazoxide (50 μM) increased 3H-deoxyglucose uptake by 50–80%. Adenosine (100 μM) and TNF-α (50 ng/ml) stimulated 86Rb efflux. Glibenclamide attenuated this effect. We demonstrate that TNF-α, like adenosine, accelerated Ca2+ uptake into the sarcoplasmic reticulum (SR) by 50–100% and therefore prevented cardiomyocyte Ca2+ overload. Our findings further suggest that TNF-α, as well as adenosine, may mediate an adaptive effect in the heart by preventing Ca2+ overload via activation of SR Ca-ATPase (SERCA2a).