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Abstract: Background: Apoptotic death of endothelial cells (EC) plays a crucial role for the development of ischemic injury. In the present study we investigated the impact of extracellular Adenosine-5′-triphosphate (ATP), either released from cells or exogenously added, on ischemia-induced apoptosis of human EC. Methods and results: To simulate ischemic conditions, cultured human umbilical vein endothelial cells (HUVEC) were exposed to 2h of hypoxia (Po2 <4mm Hg) in serum-free medium. Ischemia led to a 1.7-fold (+/−0.4; P <0.05) increase in EC apoptosis compared to normoxic controls as assessed by immunoblotting and immunocytochemistry of cleaved caspase-3. Ischemia-induced apoptosis was accompanied by a 2.3-fold (+/−0.5; P <0.05) increase of extracellular ATP detected by using a luciferin/luciferase assay. Addition of the soluble ecto-ATPase apyrase, enhancing ATP degradation, increased ischemia-induced caspase-3 cleavage. Correspondingly, inhibition of ATP breakdown by addition of the selective ecto-ATPase inhibitor ARL67156 significantly reduced ischemia-induced apoptosis. Extracellular ATP acts on membrane-bound P2Y- and P2X-receptors to induce intracellular signaling. Both, ATP and the P2Y-receptor agonist UTP significantly reduced ischemia-induced apoptosis in an equipotent manner, whereas the P2X-receptor agonist αβ-me-ATP did not alter caspase-3 cleavage. The anti-apoptotic effects of ARL67156 and UTP were abrogated when P2-receptors were blocked by Suramin or PPADS. Furthermore, extracellular ATP led to an activation of MEK/ERK- and PI3K/Akt-signaling pathways. Accordingly, inhibition of MEK/ERK-signaling by UO126 or inhibition of PI3K/Akt-signaling by LY294002 abolished the anti-apoptotic effects of ATP. Conclusion: The data of the present study indicate that extracellular ATP counteracts ischemia-induced apoptosis of human EC by activating a P2Y-receptor-mediated signaling reducing caspase-3 cleavage. [Copyright &y& Elsevier]

Abstract: Aims: Since adenine nucleotide translocase 1 (ANT1) overexpression improved cardiac function in rats with activated renin–angiotensin system (RAS) and angiotensin II is known to enhance transforming growth factor β (TGFβ) signaling in cardiomyocytes, we assumed that ANT1 might modulate the classical TGFβ/SMAD pathway. We therefore investigated whether the cardioprotective effect of ANT1 overexpression suppresses TGFβ1-induced apoptosis, whether mitochondrial permeability transition pore (MPTP) regulation is involved, and SMAD signaling pathway is affected. Methods and results: Ventricular cardiomyocytes isolated from wild-type (WT) and ANT1 transgenic rats were treated with the apoptosis-inducing agent TGFβ1 (1ng/ml). TGFβ1 treatment of WT cells enhanced the number of apoptotic cells by 31.8±11.7% (p<0.01 vs. WT) measured by chromatin condensation. Apoptosis was blocked by 1μM cyclosporine A and by ANT1 overexpression. The protecting effect of ANT1 overexpression on TGFβ1‐induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGFβ1 decreased mitochondrial membrane potential as measured by JC-1 staining by 18.0±3.7% in WT cardiomyocytes, but only by 7.2±2.8% (p<0.05 vs. WT) in ANT1 cardiomyocytes. Cyclosporine A also attenuated the decline in mitochondrial membrane potential under TGFβ1 in WT cardiomyocytes. Determination of MPTP opening by Calcein assay in isolated cardiomyocytes and calcium retention assay in isolated mitochondria revealed a reduced open probability of MPTP after ANT1 overexpression. In addition to the effects of ANT1 on MPTP opening we investigated if ANT1 may interfere with the classical TGFβ signaling pathway. Interestingly, ANT1-transgenic cardiomyocytes expressed less TGFβ receptor II than WT cells. However, SMAD2 phosphorylation was already enhanced without TGFβ1 stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGFβ1 treatment, SMAD signaling was still responsive to TGFβ1 indicated by an upregulation of SMAD7, a TGFβ1 target protein. Conclusion: Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGFβ1 by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGFβ signaling. [Copyright &y& Elsevier]

Abstract: Ischemic preconditioning has a powerful protective potential against a reperfusion-induced injury of the post-ischemic myocardium. Cardiomyocyte hypercontracture, i.e. excessive cell shortening, is an essential mechanism of the reperfusion-induced injury. Rigor contracture, i.e. Ca2+-independent contracture, has been shown to be an import component of the reperfusion-induced hypercontracture. Since rigor contracture is dependent on the rapidity of the metabolic recovery during reoxygenation, we hypothesized that preconditioning of the cardiomyocyte mitochondria may improve mitochondrial function to restore the energy balance during the initial phase of reoxygenation and may thus prevent rigor contracture. For this purpose adult rat cardiomyocytes were exposed to anoxia with subsequent reoxygenation. For preconditioning, cells were pre-treated with the mitochondrial ATP-sensitive K+ channel opener diazoxide. Pre-treatment with 100 μmol/l diazoxide significantly reduced the reoxygenation-induced hypercontracture of cardiomyocytes due to an attenuation of the Ca2+-independent rigor-type contracture, which was accompanied by an acceleration of the phosphocreatine resynthesis during the initial phase of reoxygenation. Treatment with the mitochondrial ATP-sensitive K+ channel antagonist 5-hydroxydecanoate (500 μmol/l) during preconditioning phase abolished these protective effects. Similarly, partial suppression of the mitochondrial function with 100 μmol/l NaCN during the reoxygenation phase abolished the diazoxide effects. Finally, in isolated rat hearts, preconditioning with diazoxide prior to global ischemia significantly improved left ventricular function and attenuated hypercontracture during reperfusion. This effect could be abolished by the treatment with 100 μmol/l NaCN during reperfusion. Taken together, pharmacological preconditioning of cardiomyocytes with diazoxide protects against the reoxygenation-induced rigor hypercontracture due to an improvement of the energy recovery at the onset of reoxygenation. [Copyright &y& Elsevier]