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Objective: Nitric oxide (NO) has been shown to induce apoptosis in cardiomyocytes under normoxic conditions. The ability of NO to induce apoptosis after ischemia–reperfusion, a situation of increased NO release in vivo, has not been investigated. The present study was undertaken to characterize the pathway of induction of apoptosis by NO and the influence of ischemia on this pathway in cardiomyocytes. Methods: The study was performed on isolated adult cardiomyocytes of the rat. Ischemia was simulated by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected (1) by annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide and (2) by laddering of genomic DNA. Results: Incubation of cardiomyocytes with the NO-donor (±)-S-nitroso-N-acetylpenicillamine (SNAP, 100 μM) induced apoptosis in 14.1±1.9% of the cells and necrosis in 24.4±4.6%. The induction of apoptosis but not necrosis could be blocked by inhibition of soluble guanylyl cyclase or of protein kinase G. Apoptosis induction was mimicked by incubation of cardiomyocytes with 8-pCPT-cGMP (100 μM, 9.6±0.6% apoptotic cells) or YC-1 (75 μM, 14.6±2.8% apoptotic cells), a direct activator of soluble guanylyl cyclase. After 3 h of anoxia, cardiomyocytes were transiently protected against apoptosis induced by NO, but not by 8-pCPT-cGMP or YC-1 (8.9±0.7% or 13.4±2.4% apoptotic cells). A correlation of the apoptotic response to SNAP or YC-1 with an increased activity of soluble guanylyl cyclase, determined by measurements of intracellular cGMP contents, was found. Conclusions: NO induces apoptosis in a cGMP dependent manner in isolated adult cardiomyocytes whereas induction of necrosis seems cGMP-independent. After simulated in vitro ischemia the activation of soluble guanylyl cyclase by NO is transiently inhibited resulting in a transient anti-apoptotic protection. [ABSTRACT FROM PUBLISHER]

Objectives: Apoptosis is one feature of myocardial damage after ischemia–reperfusion, but the causes for its induction are unclear. The present study was undertaken to investigate whether apoptosis in cardiomyocytes is directly initiated by their sub-lethal injury that results from ischemia–reperfusion. Methods: Ischemia was simulated on isolated ventricular cardiomyocytes of adult rats by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected by (1) DNA laddering of genomic DNA, (2) TUNEL positive cells (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelling) and (3) annexinV–fluorescein isothiocyanate (annexinV–FITC) binding to cells under exclusion of propidium iodide. Necrotic cells were identified by (1) staining with both annexinV–FITC and propidium iodide, (2) unspecific DNA degradation and (3) enzyme release. Results: Simulated ischemia caused a >75% loss of high-energy phosphates within 2 h, which was reversible upon reoxygenation at pH 7.4. Even after 18 h of simulated ischemia, creatine phosphate contents recovered to 55.2±7.3% of control within 1 h. Apoptosis could be induced by UV irradiation (80 J/m2), H2O2 and the NO-donor N2-acetyl-S-nitroso-d,l-penicillinaminamide. In contrast to this, simulated ischemia and reoxygenation could not induce apoptosis in the cells, but with prolonged ischemia more cells became necrotic. After 18 hours of simulated ischemia and 4 h of reoxygenation 41.2±10.2% myocytes were necrotic (vs. 6.3±4.4% of control) and only 1.7±0.5% (vs. 8.7±4.6% of control) were apoptotic. The percentage of necrotic cells correlated with an increase in lactate dehydrogenase release from 9.9±0.6% (of total activity) of normoxic controls to 37.9±5.1% after 18 h of simulated ischemia and 12 h of reoxygenation. Conclusions: Simulated ischemia-reoxygenation causes necrosis of isolated cardiomyocytes but is not sufficient for induction of apoptosis. [ABSTRACT FROM PUBLISHER]

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]