Yaser Abdallah

Reperfusion injury as a therapeutic challenge in patients with acute myocardial infarction

Cardiomyocyte death secondary to transient ischemia occurs mainly during the first minutes of reperfusion, in the form of contraction band necrosis involving sarcolemmal rupture. Cardiomyocyte hypercontracture caused by re-energisation and pH recovery in the presence of impaired cytosolic Ca2+ control as well as calpain-mediated cytoskeletal fragility play prominent roles in this type of cell death. Hypercontracture can propagate to adjacent cells through gap junctions. More recently, opening of the mitochondrial permeability transition pore has been shown to participate in reperfusion-induced necrosis, although its precise relation with hypercontracture has not been established. Experimental studies have convincingly demonstrated that infarct size can be markedly reduced by therapeutic interventions applied at the time of reperfusion, including contractile blockers, inhibitors of Na+/Ca2+ exchange, gap junction blockers, or particulate guanylyl cyclase agonists. However, in most cases drugs for use in humans have not been developed and tested for these targets, while the effect of existing drugs with potential cardioprotective effect is not well established or understood. Research effort should be addressed to elucidate the unsolved issues of the molecular mechanisms of reperfusion-induced cell death, to identify and validate new targets and to develop appropriate drugs. The potential benefits of limiting infarct size in patients with acute myocardial infarction receiving reperfusion therapy are enormous.

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.

Parathyroid hormone improves contractile performance of adult rat ventricular cardiomyocytes at low concentrations in a non-acute way

AIMS In patients with congestive heart failure, plasma parathyroid hormone (PTH) levels are positively associated with cardiac function. PTH, used to mobilize stem cells from the bone marrow after myocardial infarction, causes an increased left ventricular ejection fraction. The aim of this study was to investigate whether low but plasma-relevant concentrations of PTH directly influence the contractile properties of cardiomyocytes. METHODS AND RESULTS Isolated adult rat ventricular cardiomyocytes were exposed to PTH(1-34) or full-length PTH at picomolar concentrations for 24 h. Cell shortening was measured at 2 Hz as a cellular correlate of inotropic responsiveness. Intracellular calcium was measured in Fura-AM-loaded cells. PTH(1-3) (20-200 pM) and full-length PTH (200 pM) increased cell shortening within 24 h. PTH had no effect on cell size, but resting and peak systolic calcium concentrations were elevated. The beneficial effect of PTH was mediated via its cAMP/protein kinase A-activating domain and attenuated by addition of a protein kinase A inhibitor. In contrast, PTH peptides representing a protein kinase C-activating domain but not a cAMP/protein kinase A-activating domain or peptides that represent none of these domains had no effect on cell shortening. The effect of PTH on cell shortening was strong at low concentrations of extracellular calcium but declined at higher calcium concentrations. PTH downregulated the expression of the calcium sensing receptor, a receptor known to antagonize the action of PTH on calcium transport. Furthermore, PTH antagonized the angiotensin II-induced loss of cell function. CONCLUSION Low concentrations of PTH improve cell shortening by increasing calcium load at rest. By this mechanism cardiomyocytes compensate reduced extracellular calcium levels as they occur in patients with heart failure.

cAMP/PKA antagonizes thrombin-induced inactivation of endothelial myosin light chain phosphatase: role of CPI-17

AIMS Activation of cAMP signalling abrogates thrombin-induced hyperpermeability. One of the mechanisms underlying this protective effect is the inactivation of endothelial contractile machinery, one of the major determinants of endothelial barrier function, mainly via the activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors, PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied. METHODS AND RESULTS Endothelial contractile machinery and barrier function were analysed in cultured human umbilical vein endothelial cells (HUVEC). The cAMP analogues 8-CPT-cAMP and 6-Bnz-cAMP were used to activate Epac and PKA, respectively, and forskolin (FSK) was used to activate adenylyl cyclase. The cells were challenged by thrombin to inhibit MLCP via the RhoA/Rock pathway. Activation of either PKA or Epac partially blocked thrombin-induced hyperpermeability. Simultaneous activation of PKA and Epac had additive effects that were comparable to that of FSK. Activation of PKA but not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, partly via inhibition of the RhoA/Rock pathway. FSK activated the MLCP catalytic subunit PP1 via dephosphorylation and dissociation of the PP1 inhibitory protein CPI-17. FSK blunted thrombin-induced CPI-17 phosphorylation, CPI-17/PP1 complex formation, and PP1 inactivation. Down-regulation of CPI-17 attenuated thrombin-induced hyperpermeability and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect. CONCLUSION cAMP/PKA regulates the endothelial barrier via inhibition of the contractile machinery, mainly by the activation of MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability-lowering effect of the cAMP/Epac pathway is independent of CPI-17.

Margatoxin Inhibits VEGF-Induced Hyperpolarization, Proliferation and Nitric Oxide Production of Human Endothelial Cells

Background: Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells (EC) in vitro and angiogenesis in vivo. Furthermore, a role of VEGF in K+ channel, nitric oxide (NO) and Ca2+ signaling was reported. We examined whether the K+ channel blocker margatoxin (MTX) influences VEGF-induced signaling in human EC. Methods: Fluorescence imaging was used to analyze changes in the membrane potential (DiBAC), intracellular Ca2+ (FURA-2) and NO (DAF) levels in cultured human EC derived from human umbilical vein EC (HUVEC). Proliferation of HUVEC was examined by cell counts (CC) and [3H]-thymidine incorporation (TI).Results: VEGF (5–50 ng/ml) caused a dose-dependent hyperpolarization of EC, with a maximum at 30 ng/ml (n = 30, p < 0.05). This effect was completely blocked by MTX (5 µmol/l). VEGF caused an increase in transmembrane Ca2+ influx (n = 30, p < 0.05) that was sensitive to MTX and the blocker of transmembrane Ca2+ entry 2-aminoethoxydiphenyl borate (APB, 100 µmol/l). VEGF-induced NO production was significantly reduced by MTX, APB and a reduction in extracellular Ca2+ (n = 30, p < 0.05). HUVEC proliferation, examined by CC and TI, was significantly increased by VEGF and inhibited by MTX (CC: –58%, TI –121%); APB (CC –99%, TI –187%); N-monomethyl-L-arginine (300 µmol/l: CC: –86%, TI –164%). Conclusions: VEGF caused an MTX-sensitive hyperpolarization which results in an increased transmembrane Ca2+ entry that is responsible for the effects on endothelial proliferation and NO production.

New insights into paracrine mechanisms of human cardiac progenitor cells.

Cardiac progenitor cells (CPCs) have been shown to promote cardiac regeneration in vivo. Understanding the function of CPCs is essential for further implementation of these cells in the treatment of cardiac diseases. The present study tested the hypothesis that adult CPC exert paracrine effects that lead to an improvement in the functional characteristics of cardiomyocytes. This study also investigated whether aging (we included patients aged between 4 months and 81 years) has any effect on the paracrine mechanisms of CPC.

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.

Stimulation of cGMP signalling protects coronary endothelium against reperfusion-induced intercellular gap formation

AIMS Ischaemia-reperfusion provokes barrier failure of the coronary microvasculature, impeding functional recovery of the heart during reperfusion. The aim of the present study was to investigate whether the stimulation of cGMP signalling by activation of soluble guanylyl cyclase (sGC) can reduce reperfusion-induced endothelial intercellular gap formation and to determine whether this is due to an influence on endothelial cytosolic Ca(2+) homeostasis during reperfusion. METHODS AND RESULTS Experiments were performed with cultured coronary endothelial monolayers and isolated saline-perfused rat hearts. HMR1766 (1 micromol/L) or DEAnonoate (0.5 micromol/L) were used to activate sGC. After exposure to simulated ischaemic conditions, reperfusion of endothelial cells led to a pronounced increase in cytosolic calcium levels and intercellular gaps. Stimulation of cGMP signalling during reperfusion increased Ca(2+) sequestration in the endoplasmic reticulum (ER) and attenuated the reperfusion-induced increase in cytosolic [Ca(2+)]. Phosphorylation of phospholamban was also increased, indicating a de-inhibition of the ER Ca(2+) pump (SERCA). Reperfusion-induced intercellular gap formation was reduced. Reduction of myosin light chain phosphorylation indicated inactivation of the endothelial contractile machinery. Effects on cytsolic Ca(2+) and gaps were abrogated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823. In reperfused hearts, stimulation of cGMP signalling led to decreased oedema development. CONCLUSION sGC/PKG activation during reperfusion reduces reperfusion-induced endothelial intercellular gap formation by attenuation of cytosolic calcium overload and reduction of contractile activation in endothelial cells. This mechanism protects the heart against reperfusion-induced oedema.

The K+-channel opener NS1619 increases endothelial NO-synthesis involving p42/p44 MAP-kinase

Ca(2+)-activated K(+) channels with large conductance (BK(Ca)) have been shown to play an important role in the regulation of vascular tone. We examined the role of the p42/p44 MAP-kinase (p42/p44(MAPK)) on nitric oxide (NO) production in human endothelial cells induced by the BK(Ca)-opener NS1619. Using DiBAC-fluorescence imaging a concentration-dependent (2.5-12.5 microM) hyperpolarization induced by NS1619 was observed. A significant increase of intracellular Ca(2+)-concentration by NS1619 was seen using Fura-2-fluorescence-imaging, which was blocked by 2-APB, or reduction of extracellular Ca(2+) (n=30; p<0.05). A cGMP-radioimmunoassay was used to examine NO synthesis. NS1619 significantly increased cGMP levels which was inhibited by LNMMA, iberiotoxin, BAPTA, 2-APB, reduction of extracellular Ca(2+), PD 98059, or U0126 (cGMP (pmol/mg protein): NS1619 3.25 +/- 0.85; NS1619 + L-NMMA 0.86 +/- 0.02; NS1619 + iberiotoxin 0.99 +/- 0.09; NS1619 + BAPTA 0.93 +/- 0.29; NS1619 + 2-APB 0.99 +/- 0.31; NS1619 + Ca(2+)-reduction 1.17 +/- 0.06; NS1619 + PD98059 1.06 +/- 0.49; NS1619 + U0126 1.10 +/- 0.24; n=10; p<0.05). The phosphorylation of eNOS and p42/p44(MAPK) was examined by immunocytochemistry. Phosphorylation of p42/p44(MAPK) was significantly increased after 10 minutes of NS1619 stimulation, whereas eNOS phosphorylation was not changed over a period of 1 to 30 minutes. NS1619-induced hyperpolarization was not affected by treatment with PD 98059 or U0126. Additionally, NS1619 inhibited endothelial proliferation involving a NO-dependent mechanism. Our data demonstrate that NS1619 causes a transmembrane Ca(2+)-influx leading to an increased NO production involving p42/p44(MAPK). This rise of NO formation is responsible for the NS1619 induced reduction of endothelial cell growth.