Effect of sarcolemmal rupture on myocardial electrical impedance during oxygen deprivation.

Plasma membrane disruption is a characteristic feature of cell death induced by hypoxia or ischemia. Here, we investigated whether analysis of tissue electrical impedance allows detection of ongoing cell membrane rupture and necrotic cell death in hypoxic or ischemic myocardium. Twenty-eight isolated rat hearts were submitted to 5 h of ischemia (n = 8) or hypoxia (n = 20). Myocardial electrical impedance and lactate dehydrogenase (LDH) release were monitored. The time course of hypoxia-induced cell death was modified by altering pH (pH 7.4 or 6.4, 5 h) or by adding 3 or 10 mM glycine. Ischemia and hypoxia induced an increase in electrical impedance, followed by a plateau, and later a reduction. During hypoxia, LDH release started after a prolonged lapse of time (80.00 +/- 8.37 min at pH 7.4 and 122.50 +/- 11.82 min at pH 6.4). The onset of LDH release was followed by the onset of the late reduction in electrical impedance, and both were delayed by acidic pH (P < 0.05) and by glycine (P < 0.05). The times of onset of LDH release and of late electrical changes were significantly correlated (r = 0.752, P < 0.001). In separate experiments, induction of sarcolemmal rupture with Triton X-100 (n = 6) mimicked the late effects of ischemia or hypoxia on tissue impedance. The protective effects of glycine and acidosis on membrane disruption were confirmed (propidium iodide) in energy-deprived HL-1 cardiomyocytes. These results describe for the first time a late fall in electrical impedance in myocardium submitted to prolonged oxygen deprivation and demonstrate that this fall allows detection of ongoing cell necrosis.

Ischemic preconditioning attenuates calpain-mediated degradation of structural proteins through a protein kinase A-dependent mechanism.

OBJECTIVES: It has been shown that sarcolemmal rupture can occur during reenergization in cardiomyocytes in which previous ischemia has induced sarcolemmal fragility by calpain-dependent hydrolysis of structural proteins. We tested the hypothesis that attenuated calpain activation contributes to the protection against reperfusion-induced cell death afforded by ischemic preconditioning (IPC), and investigated the involvement of protein kinase A (PKA) in this effect. METHODS: Calpain activity and degradation of different structural proteins were studied along with the extent of necrosis in isolated rat hearts submitted to 60 min of ischemia and 30 min of reperfusion with or without previous IPC (two cycles of 5 min ischemia-5 min reperfusion), and the ability of different treatments to mimic or blunt the effects of IPC were analyzed. RESULTS: IPC accelerated ATP depletion and rigor onset during ischemia but reduced LDH release during reperfusion by 69% (P<0.001). At the end off reperfusion, calpain activity was reduced by 66% (P<0.001) in IPC, and calpain-dependent degradation of sarcolemmal proteins was attenuated. Addition of the calpain inhibitor MDL-28170 mimicked the effects of IPC on protein degradation and reduced LDH release by 48% (P<0.001). The effects of IPC on calpain, alpha-fodrin, and LDH release were blunted by the application of the PKA inhibitor H89 or alprenolol during IPC, while transient stimulation of PKA with CPT-cAMP or isoproterenol before ischemia attenuated calpain activation, alpha-fodrin degradation, and markedly reduced LDH release (P<0.001). In hearts exposed to Na(+)-free perfusion, IPC attenuated calpain activation by 67% (P<0.001) and reduced by 56% (P<0.001) LDH release associated to massive edema occurring during Na(+) readmission without modifying its magnitude. CONCLUSION: These results are consistent with PKA-dependent attenuation of calpain-mediated degradation of structural proteins being an end-effector mechanism of the protection afforded by IPC.

Glycine protects cardiomyocytes against lethal reoxygenation injury by inhibiting mitochondrial permeability transition.

Post-ischaemic reperfusion may precipitate cardiomyocyte death upon correction of intracellular acidosis due in part to mitochondrial permeability transition. We investigated whether glycine, an amino acid with poorly understood cytoprotective properties, may interfere with this mechanism. In cardiomyocyte cultures, addition of glycine during re-energization following 1 h of simulated ischaemia (NaCN/2-deoxyglucose, pH 6.4) completely prevented necrotic cell death associated with pH normalization. Glycine also protected against cell death associated with pH normalization in reoxygenated rat hearts. Glycine prevented cyclosporin-sensitive swelling and calcein release associated with re-energization in rat heart mitochondria submitted to simulated ischaemia or to Ca(2+) stress under normoxia. NMR spectroscopy revealed a marked glycine depletion in re-energized cardiomyocytes that was reversed by exposure to 3 mm glycine. These results suggest that intracellular glycine exerts a previously unrecognized inhibition on mitochondrial permeability transition in cardiac myocytes, and that intracellular glycine depletion during myocardial hypoxia/reoxygenation makes the cell more vulnerable to necrotic death.

Retinoic acid-induced differentiation into astrocytes and glutamatergic neurons is associated with expression of functional and activable phospholipase D.

Phospholipase D (PLD) activity in mammalian cells has been associated with cell proliferation and differentiation. Here, we investigated the expression of PLD during differentiation of pluripotent embryonal carcinoma cells (P19) into astrocytes and neurons. Retinoic acid (RA)-induced differentiation increased PLD1 and PLD2 mRNA levels and PLD activity that was responsive to phorbol myristate acetate. Various agonists of membrane receptors activated PLD in RA-differentiated cells. Glutamate was a potent activator of PLD in neurons but not in astrocytes, whereas noradrenaline and carbachol increased PLD activity only in astrocytes. P19 neurons but not astrocytes released glutamate in response to a depolarizing stimulus, confirming the glutamatergic phenotype of these neurons. These results indicate upregulation of PLD gene expression associated with RA-induced neural differentiation.

Cariporide preserves mitochondrial proton gradient and delays ATP depletion in cardiomyocytes during ischemic conditions.

The mechanism by which inhibition of Na+/H+ exchanger (NHE) reduces cell death in ischemic-reperfused myocardium remains controversial. This study investigated whether cariporide could inhibit mitochondrial NHE during ischemia, delaying H+ gradient dissipation and ATP exhaustion. Mouse cardiac myocytes (HL-1) were submitted to 1 h of simulated ischemia (SI) with NaCN/deoxyglucose (pH 6.4), with or without 7 microM cariporide, and mitochondrial concentration of Ca2+ (Rhod-2), 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and the charge difference across the mitochondrial membrane potential (Deltapsim, JC-1) were assessed. ATP content was measured by bioluminescence and mitochondrial swelling by spectrophotometry in isolated mitochondria. Cariporide significantly attenuated the acidification of the mitochondrial matrix induced by SI without modifying Deltapsim decay, and this effect was associated to a delayed ATP exhaustion and increased mitochondrial Ca2+ load. These effects were reproduced in sarcolemma-permeabilized cells exposed to SI. In these cells, cariporide markedly attenuated the fall in mitochondrial pH induced by removal of Na+ from the medium. In isolated mitochondria, cariporide significantly reduced the rate and magnitude of passive matrix swelling induced by Na+ acetate. In isolated rat hearts submitted to 40-min ischemia at different temperatures (35.5 degrees, 37 degrees, or 38.5 degrees C) pretreatment with cariporide limited ATP depletion during the first 10 min of ischemia and cell death (lactate dehydrogenase release) during reperfusion. These effects were mimicked when a similar ATP preservation was achieved by hypothermia and were abolished when the sparing effect of cariporide on ATP was suppressed by hyperthermia. We conclude that cariporide acts at the mitochondrial level, delaying mitochondrial matrix acidification and delaying ATP exhaustion during ischemia. These effects can contribute to reduce cell death secondary to ischemia-reperfusion.

Hypoxia and acidosis impair cGMP synthesis in microvascular coronary endothelial cells.

To characterize the effects of ischemia on cGMP synthesis in microvascular endothelium, cultured endothelial cells from adult rat hearts were exposed to hypoxia or normoxia at pH 6.4 or 7.4. Cellular cGMP and soluble (sGC) and membrane guanylyl cyclase (mGC) activities were measured after stimulation of sGC (S-nitroso-N-acetyl-penicillamine) or mGC (urodilatin) or after no stimulation. Cell death (lactate dehydrogenase release) was negligible in all experiments. Hypoxia at pH 6.4 induced a rapid approximately 90% decrease in cellular cGMP after sGC and mGC stimulation. This effect was reproduced by acidosis. Hypoxia at pH 7.4 elicited a less pronounced (approximately 50%) and slower reduction in cGMP synthesis. Reoxygenation after 2 h of hypoxia at either pH 6.4 or 7.4 normalized the response to mGC stimulation but further deteriorated the sGC response; normalization of pH rapidly reversed the effects of acidosis. At pH 7.4, the response to GC stimulation correlated well with cellular ATP. We conclude that simulated ischemia severely depresses cGMP synthesis in microvascular coronary endothelial cells through ATP depletion and acidosis without intrinsic protein alteration.

Effect of inhibition of Na(+)/Ca(2+) exchanger at the time of myocardial reperfusion on hypercontracture and cell death.

OBJECTIVE: There is recent evidence that Ca(2+) influx via reverse mode Na(+)/Ca(2+) exchange (NCX) at the time of reperfusion can contribute to cardiomyocyte hypercontracture. However, forward NCX is essential for normalization of [Ca(2+)](i) during reperfusion, and its inhibition may be detrimental. This study investigates the effect of NCX inhibition with KB-R7943 at the time of reperfusion on cell viability. METHODS: The effect of several concentrations of KB-R7943 added at reperfusion was studied in Fura-2 loaded quiescent cardiomyocytes submitted to 40 min of simulated ischemia (NaCN 2 mM, pH 6.4), and in rat hearts submitted to 60 min of ischemia. [Ca(2+)](i) and cell length were monitored in myocytes, and functional recovery and LDH release in isolated hearts. From these experiments an optimal concentration of KB-R7943 was identified and tested in pigs submitted to 48 min of coronary occlusion and 2 h of reperfusion. RESULTS: In myocytes, KB-R7943 at concentrations up to 15 microM reduced [Ca(2+)](i) rise and the probability of hypercontracture during re-energization (P<0.01). Nevertheless, in rat hearts, the effects of KB-R7943 applied during reperfusion after 60 min of ischemia depended on concentration and timing of administration. During the first 5 min of reperfusion, KB-R7943 (0.3-30 microM) induced a dose-dependent reduction in LDH release (half-response concentration 0.29 microM). Beyond 6 min of re-flow, KB-R7943 had no effect on LDH release, except at concentrations > or = 15 microM, which increased LDH. KB-R7943 at 5 microM given during the first 10 min of reflow reduced contractile dysfunction (P=0.011), LDH release (P=0.019) and contraction band necrosis (P=0.014) during reperfusion. Intracoronary administration of this concentration during the first 10 min of reperfusion reduced infarct size by 34% (P=0.033) in pigs submitted to 48 min of coronary occlusion. CONCLUSIONS: These results are consistent with the hypothesis that during initial reperfusion NCX activity results in net reverse mode operation contributing to Ca(2+) overload, hypercontracture and cell death, and that NCX inhibition during this phase is beneficial. Beyond this phase, NCX inhibition may impair forward mode-dependent Ca(2+) extrusion and be detrimental. These findings may help in the design of therapeutic strategies against lethal reperfusion injury, with NCX as the target.