Yury Ladilov

Soluble Adenylyl Cyclase Controls Mitochondria-dependent Apoptosis in Coronary Endothelial Cells

The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC- and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells.

Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes

Uncontrolled release of Ca2+ from the sarcoplasmic reticulum (SR) contributes to the reperfusion‐induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨm. Fura‐2 was used to monitor cytosolic [Ca2+]i or mitochondrial calcium [Ca2+]m, after quenching the cytosolic compartment with MnCl2. Mitochondrial ROS [ROS]m production was detected with MitoSOX Red and mag‐fura‐2 was used to monitor Mg2+ concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨm collapse and disturbance of ATP recovery. Simultaneously, Ca2+ oscillations occurred, [Ca2+]m and [ROS]m increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR‐driven Ca2+ cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca2+ uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca2+ cycling, hypercontracture and necrosis. ROS scavengers (2‐mercaptopropionyl glycine or N‐acetylcysteine) had no effect on these parameters, but reduced [ROS]m. In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca2+ oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca2+ cycling and MPTP promotes the reperfusion‐induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

Type 10 adenylyl cyclase mediates mitochondrial Bax translocation and apoptosis of adult rat cardiomyocytes under simulated ischaemia/reperfusion

AIMS Apoptosis of cardiomyocytes significantly contributes to the development of post-ischaemic cardiomyopathy. Although mitochondria have been suggested to play a crucial role in this process, the precise mechanisms controlling the mitochondria-dependent apoptosis in cardiomyocytes under ischaemia/reperfusion are still poorly understood. Here we aimed to analyse the role of the soluble adenylyl cyclase (sAC). METHODS AND RESULTS Adult rat cardiomyocytes were subjected to simulated in vitro ischaemia (SI) consisting of glucose-free anoxia at pH 6.4. Apoptosis was detected by DNA laddering, chromatin condensation, and caspases cleavage. SI led to the translocation of sAC to the mitochondria and mitochondrial depolarization followed by cytochrome c release, caspase-9/-3 cleavage and apoptosis during simulated reperfusion (SR). Pharmacological inhibition of sAC during SI, but not during SR, significantly reduced the SI/SR-induced mitochondrial injury and apoptosis. Similarly, sAC knock-down mediated by an adenovirus coding for shRNA targeting sAC prevented the activation of the mitochondrial pathway of apoptosis. Analysis of the link between sAC and apoptosis revealed a sAC and protein kinase A-dependent Bax phosphorylation at Thr(167) and its translocation to mitochondria during SI, which subsequently caused mitochondrial oxygen radical formation followed by cytochrome c release and caspase-9 cleavage during SR. CONCLUSION These results suggest a key role of sAC in SI-induced mitochondrial Bax translocation and activation of the mitochondrial pathway of apoptosis in adult cardiomyocytes.

Oxysterol-induced apoptosis of smooth muscle cells is under the control of a soluble adenylyl cyclase

AIMS Apoptosis of vascular smooth muscle cells (VSMC) in advanced atherosclerotic plaques is an important cause of plaque instability. Oxysterols have been suggested as important inducers of apoptosis in VSMC, but the precise mechanism is still poorly understood. Here we aimed to analyse the role of the soluble adenylyl cyclase (sAC). METHODS AND RESULTS VSMC derived from rat aorta were treated with either 25-hydroxycholesterol or 7-ketocholesterol for 24 h. Apoptosis was detected by TUNEL staining and caspases cleavage. Oxysterols treatment led to the activation of the mitochondrial pathway of apoptosis (cytochrome c release and caspase-9 cleavage) and mitochondrial ROS formation, which were suppressed by the pharmacological inhibition or knockdown of sAC. Scavenging ROS with N-acetyl-l-cysteine prevented oxysterol-induced apoptosis. Analyses of the downstream pathway suggest that protein kinase A (PKA)-dependent phosphorylation and the mitochondrial translocation of the pro-apoptotic protein Bax is a key link between sAC and oxysterol-induced ROS formation and apoptosis. To distinguish between intra-mitochondrial and extra-mitochondrial/cytosolic sAC pools, sAC was overexpressed in mitochondria or in the cytosol. sAC expression in the cytosol, but not in mitochondria, significantly promoted apoptosis and ROS formation during oxysterol treatment. CONCLUSION These results suggest that the sAC/PKA axis plays a key role in the oxysterol-induced apoptosis of VSMC by controlling mitochondrial Bax translocation and ROS formation and that cytosolic sAC, rather than the mitochondrial pool, is involved in the apoptotic mechanism.

Type 10 soluble adenylyl cyclase is overexpressed in prostate carcinoma and controls proliferation of prostate cancer cells

Background: Soluble adenylyl cyclase (sAC) may be an alternative intracellular localized source of cAMP controlling proliferation. Results: sAC is overexpressed in prostate carcinoma, and inhibition of sAC leads to cell cycle arrest. Conclusion: sAC controls proliferation of prostate carcinoma cells. Significance: sAC represents a novel pathway promoting proliferation in cancer cells and is a promising target for prostate cancer treatment. cAMP signaling plays an essential role in modulating the proliferation of different cell types, including cancer cells. Until now, the regulation of this pathway was restricted to the transmembrane class of adenylyl cyclases. In this study, significant overexpression of soluble adenylyl cyclase (sAC), an alternative source of cAMP, was found in human prostate carcinoma, and therefore, the contribution of this cyclase was investigated in the prostate carcinoma cell lines LNCaP and PC3. Suppression of sAC activity by treatment with the sAC-specific inhibitor KH7 or by sAC-specific knockdown mediated by siRNA or shRNA transfection prevented the proliferation of prostate carcinoma cells, led to lactate dehydrogenase release, and induced apoptosis. Cell cycle analysis revealed a significant rise in the G2 phase population 12 h after sAC inhibition, which was accompanied by the down-regulation of cyclin B1 and CDK1. sAC-dependent regulation of proliferation involves the EPAC/Rap1/B-Raf signaling pathway. In contrast, protein kinase A does not play a role. In conclusion, this study suggests a novel sAC-dependent signaling pathway that controls the proliferation of prostate carcinoma cells.

Mechanisms involved in postconditioning protection of cardiomyocytes against acute reperfusion injury

Experimental and clinical studies demonstrated that postconditioning confers protection against myocardial ischemia/reperfusion injury. However the underlying cellular mechanisms responsible for the beneficial effect of postconditioning are still poorly understood. The aim of the present study was to examine the role of cytosolic and mitochondrial Ca(2+)-handling. For this purpose adult rat cardiomyocytes were subjected to simulated in vitro ischemia (glucose-free hypoxia at pH6.4) followed by simulated reperfusion with a normoxic buffer (pH7.4; 2.5 mmol/L glucose). Postconditioning, i.e., 2 repetitive cycles of normoxic (5s) and hypoxic (2.5 min) superfusion, was applied during the first 5 min of reoxygenation. Mitochondrial membrane potential (ΔΨm), cytosolic and mitochondrial Ca(2+) concentrations, cytosolic pH and necrosis were analysed applying JC-1, fura-2, fura-2/manganese, BCECF and propidium iodide, respectively. Mitochondrial permeability transition pore (MPTP) opening was detected by calcein release. Hypoxic treatment led to a reduction of ΔΨm, an increase in cytosolic and mitochondrial Ca(2+) concentration, and acidification of cardiomyocytes. During the first minutes of reoxygenation, ΔΨm transiently recovered, but irreversibly collapsed after 7 min of reoxygenation, which was accompanied by MPTP opening. Simultaneously, mitochondrial Ca(2+) increased during reperfusion and cardiomyocytes developed spontaneous cytosolic Ca(2+) oscillations and severe contracture followed by necrosis after 25 min of reoxygenation. In postconditioned cells, the collapse in ΔΨm as well as the leak of calcein, the increase in mitochondrial Ca(2+), cytosolic Ca(2+) oscillations, contracture and necrosis were significantly reduced. Furthermore postconditioning delayed cardiomyocyte pH recovery. Postconditioning by hypoxia/reoxygenation was as protective as treatment with cyclosporine A. Combining cyclosporine A and postconditioning had no additive effect. The data of the present study demonstrate that postconditioning by hypoxia/reoxygenation prevents reperfusion injury by limiting mitochondrial Ca(2+) load and thus opening of the MPTP in isolated cardiomyocytes. These effects seem to be supported by postconditioning-induced delay in pH recovery and suppression of Ca(2+) oscillations.

SLC4A7 sodium bicarbonate co-transporter controls mitochondrial apoptosis in ischaemic coronary endothelial cells

AIMS Bicarbonate transport has been shown to participate in apoptosis under ischaemic stress. However, the precise transporting mechanisms involved in ischaemic apoptosis are unknown and were thus the aim of the present study. METHODS AND RESULTS Rat coronary endothelial cells (EC) were exposed to simulated in vitro ischaemia for 2 h, and apoptosis was subsequently determined by chromatin staining and caspase-3 activity analysis. By examining the expression of bicarbonate transporters (BT) in EC by reverse transcriptase polymerase chain reaction and western blotting, a marked expression of the electroneutral sodium bicarbonate co-transporter (SLC4A7) was defined. To analyse the potential role of this transporter during apoptosis, a selective inhibitor (S0859, Sanofi-Aventis) was applied. Treatment with S0859 significantly increased caspase-3 activity and elevated the number of apoptotic EC. These results were comparable with an unselective inhibition of all BT due to withdrawal of bicarbonate in the anoxic medium. Knockdown of SLC4A7 in EC by transfecting appropriate siRNA similarly increased apoptosis of EC under simulated ischaemia. The initial characterization of the participating mechanisms of SLC4A7-dependent apoptosis revealed an activation of the mitochondrial pathway of apoptosis, i.e. cleavage of caspase-9 and binding of Bax to mitochondria. In contrast, no activation of the endoplasmic reticulum-dependent pathway (caspase-12 cleavage) or the extrinsic apoptotic pathway (caspase-8 cleavage) was found. Finally, a mitochondrial localization of SLC4A7 was demonstrated. CONCLUSION The electroneutral sodium bicarbonate co-transporter SLC4A7 localizes in mitochondria and suppresses the ischaemia-induced activation of the mitochondrial pathway of apoptosis in coronary EC.

Acidic pre-conditioning suppresses apoptosis and increases expression of Bcl-xL in coronary endothelial cells under simulated ischaemia.

Ischaemic pre‐conditioning has a powerful protective potential against ischaemia‐induced cell death, and acidosis is an important featur of ischaemia and can lead to apoptosis. Here we tested whether pre‐conditioning with acidosis, that is, acidic pre‐conditioning (APC), may protect coronary endothelial cells (EC) against apoptosis induced by simulated ischaemia. For pre‐conditioning, EC were exposed fo 40 min. to acidosis (pH 6.4) followed by a 14‐hrs recovery period (pH 7.4) and finally treated for 2 hrs with simulated ischaemia (glucose‐free anoxia at pH 6.4). Cells undergoing apoptosis were visualized by chromatin staining or by determination of caspase‐3 activit Simulated ischaemia in untreated EC increased caspase‐3 activity and the number of apoptotic cell (31.3 ± 1.3%versus 3.9 ± 0.6% in control). APC significantly reduced the rate of apoptosis (14.2 ± 1.3%) and caspase‐3 activity. Western blot analysis exploring the under lying mechanism leading to this protection revealed suppression of the endoplasmic reticulum‐ (reduced cleavage of caspase‐12) and mitochondria‐mediated (reduced cytochrome C release) pathways of apoptosis. These effects were associated with an over‐expression of the anti‐apoptotic protein Bcl‐xL 14 hrs after APC, whereas no effect on the expression of Bcl‐2, Bax, Bak, procaspase‐12, reticulum‐localized chaperones (GRP78, calreticulin), HSP70, HSP32 and HSP27 could be detected. Knock‐down of Bcl‐xL by siRNA‐treatment prevented the protective effect of APC. In conclusion, short acidic pre‐treatment can protect EC against ischaemic apoptosis. The mechanism of this protection consists of suppression of the endoplasmic reticulum‐ and mitochondria‐mediated pathways. Over‐expression of the anti apoptotic protein Bcl‐xL is responsible for the increased resistance to apoptosis during ischaemic insult.

Preconditioning with diazoxide prevents reoxygenation-induced rigor-type hypercontracture.

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. Ca(2+)-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 micromol/l diazoxide significantly reduced the reoxygenation-induced hypercontracture of cardiomyocytes due to an attenuation of the Ca(2+)-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 micromol/l) during preconditioning phase abolished these protective effects. Similarly, partial suppression of the mitochondrial function with 100 micromol/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 micromol/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.

Importance of bicarbonate transport for ischaemia-induced apoptosis of coronary endothelial cells.

Bicarbonate transport (BT) has been previously shown to participate in apoptosis induced by various stress factors. However, the precise role of BT in ischaemia‐induced apoptosis is still unknown. To investigate this subject, rat coronary endothelial cells (EC) were exposed to simulated ischaemia (glucose free anoxia at Ph 6.4) for 2 hrs and cells undergoing apoptosis were visualized by nuclear staining or by determination of cas‐pase‐ 3 activity. To inhibit BT, EC were either treated with the inhibitor of BT 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS, 300 μmol/l) or exposed to ischaemia in bicarbonate free, 4‐(2‐hydroxyethyl)‐I‐piperazi‐neethanesulphonic acid (HEPES)‐buffered medium. Simulated ischaemia in bicarbonate‐buffered medium (Bic) increased caspase‐3 activity and the number of apoptotic cell (23.7 + 1.4%versus 5.1 + 1.2% in control). Omission of bicarbonate during ischaemia further significantly increased caspase‐3 activity and the number of apoptotic cells (36.7 1.7%). Similar proapoptotic effect was produced by DIDS treatment during ischaemia in Bic, whereas DIDS had no effect when applied in bicarbonate‐free, HEPES‐buffered medium (Hep). Inhibition of BT was without influence on cytosolic acidification during ischaemia and slightly reduced cytosolic Ca2+ accumulation. Initial characterization of the underlying mechanism leading to apoptosis induced by BT inhibition revealed activation of the mitochondrial pathway of apoptosis, i.e., increase of cytochrome C release, depolarization of mitochondria and translocation of Bax protein to mitochondria. In contrast, no activation of death receptor‐dependent pathway (caspase‐8 cleavage) and endoplasmic reticulum‐ dependent pathway (caspase‐12 cleavage) was detected. In conclusion, BT plays an important role in ischaemia‐induced apoptosis of coronary EC by suppression of mitochondria‐dependent apoptotic pathway.