Simulated Reperfusion Research Articles

Role of sarcoplasmic reticulum in mitochondrial permeability transition and cardiomyocyte death during reperfusion

There is solid evidence that a sudden change in mitochondrial membrane permeability (mitochondrial permeability transition, MPT) plays a critical role in reperfusion-induced myocardial necrosis. We hypothesized that sarcoplasmic reticulum (SR) Ca(2+) cycling may induce partial MPT in microdomains of close anatomic proximity between mitochondria and SR, resulting in hypercontracture and cell death. MPT (mitochondrial calcein release), cell length, and sarcolemmal rupture (Trypan blue and lactate dehydrogenase release) were measured in adult rat cardiomyocytes submitted to simulated ischemia (NaCN/2-deoxyglucose, pH 6.4) and reperfusion. On simulated reperfusion, 83 +/- 2% of myocytes developed hypercontracture. In 22 +/- 6% of cases, hypercontracture was associated with sarcolemmal disruption [Trypan blue(+)]. During simulated reperfusion there was a 25% release of cyclosporin A-sensitive mitochondrial calcein (with respect to total mitochondrial calcein content). Simultaneous blockade of SR Ca(2+) uptake and release with thapsigargin and ryanodine, respectively, significantly reduced mitochondrial calcein release, hypercontracture, and cell death during simulated reperfusion. SR Ca(2+) blockers delayed mitochondrial Ca(2+) uptake in digitonin-permeabilized cardiomyocytes but did not have any effect on isolated mitochondria. Pretreatment with colchicine to disrupt microtubule network reduced the degree of fluorescent overlap between SR and mitochondria and abolished the protective effect of SR Ca(2+) blockers on MPT, hypercontracture, and cell death during reperfusion. We conclude that SR Ca(2+) cycling during reperfusion facilitates partial mitochondrial permeabilization due to the close anatomic proximity between both organelles, favoring hypercontracture and cell death.

Isoflurane induces a postconditioning effect on bovine pulmonary arterial endothelial cells exposed to oxygen–glucose deprivation

Application of volatile anesthetics during the onset of reperfusion reduced ischemia-induced cardiac and brain injury (anesthetic postconditioning). This study was designed to evaluate whether volatile anesthetics induced a postconditioning effect in endothelial cells. Bovine pulmonary arterial endothelial cell (BPAEC) cultures were exposed to oxygen–glucose deprivation, a condition to simulate ischemia in vitro, for 3 h. The volatile anesthetics isoflurane and desflurane were applied during the early phase of simulated reperfusion. Cell injury was quantified by lactate dehydrogenase (LDH) release and flow cytometrical measurement after annexin V and propidium iodide staining. Oxygen–glucose deprivation and the subsequent simulated reperfusion increased LDH release and annexin V-positive staining cells, a characteristic of cell apoptosis. Posttreatment with isoflurane, but not desflurane, reduced this cell injury. This protection was apparent even when 2% isoflurane was applied at 60 min after the onset of reperfusion. The isoflurane postconditioning effect was abolished by glybenclamide, a general ATP sensitive K + (K ATP) channel blocker, 5-hydroxydecanoate, a mitochondrial K ATP channel blocker, and chelerythrine, a protein kinase C inhibitor. Diazoxide, a mitochondrial K ATP channel activator, applied at the onset of reperfusion also decreased oxygen–glucose deprivation-induced endothelial cell injury. This diazoxide-induced protection was abolished by chelerythrine and 5-hydroxydecanoate. We conclude that isoflurane induced a postconditioning effect in BPAEC. The effective time window of isoflurane postconditioning was from 0 to 60 min after the onset of reperfusion. This isoflurane postconditioning effect may be mediated by mitochondrial K ATP channels and PKC. PKC may be downstream of mitochondrial K ATP channels for this isoflurane effect.

Tolerance of Isolated Cardiac Myocytes From Male and Female Rats to Simulated Ischemia and Reperfusion

Tolerance of Isolated Cardiac Myocytes From Male and Female Rats to Simulated Ischemia and Reperfusion

Silencing PTEN, a negative regulator of the PI3K/AKT pathway, is not sufficient to confer protection against simulated ischaemia and reperfusion

Silencing PTEN, a negative regulator of the PI3K/AKT pathway, is not sufficient to confer protection against simulated ischaemia and reperfusion

Reperfusion and calculated RISKs: pharmacological postconditioning of human myocardium

The last five years have witnessed a remarkable resurgence of interest in myocardial reperfusion injury. Reperfusion is absolutely essential to salvage ischaemic myocardium but experimental and clinical studies show that reperfusion-associated injury may mask the full benefits of prompt reperfusion in acute myocardial infarction. In the current issue of the British Journal of Pharmacology, Mudalagiri et al demonstrate a protective effect against simulated reperfusion injury using exogenously applied erythropoietin in human isolated myocardium. Crucially, the benefits of erythropoietin were observed when it was administered specifically during re-oxygenation. The demonstration that the protective effects of the cytokine were dependent on PI3-kinase/Akt and ERK1/2 activation provides compelling evidence that reperfusion injury salvage kinases (RISKs) are key survival mechanisms in human myocardium, as they are in experimental animal species. Although erythropoietin may be only one of several potential pharmacological approaches in human patients, this study establishes the important proof-of-principle that activation of RISKs is protective in human myocardium and could be a promising therapeutic target in acute myocardial infarction.

Rho-kinase-dependent F-actin rearrangement is involved in the inhibition of PI3-kinase/Akt during ischemia–reperfusion-induced endothelial cell apoptosis

Activation of cytoskeleton regulator Rho-kinase during ischemia–reperfusion (I/R) plays a major role in I/R injury and apoptosis. Since Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, we hypothesized that inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase/Akt activity and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement. Human umbilical vein endothelial cells were subjected to 1 h of simulated ischemia and 1 or 24 h of simulated reperfusion after treatment with Rho-kinase inhibitor Y-27632, PI3-kinase inhibitor wortmannin, F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide. Intracellular ATP levels decreased following I/R. Y-27632 treatment reduced I/R-induced apoptosis by 31% (P < 0.01) and maintained Akt activity. Both effects were blocked by co-treatment with wortmannin. Y-27632 treatment prevented the formation of F-actin bundles during I/R. Similar results were observed with cytochalasinD treatment. In contrast, latrunculinA and jasplakinolide treatment did not prevent the formation of F-actin bundles during I/R and had no effect on I/R-induced apoptosis. Apoptosis and Akt activity were inversely correlated (R2 = 0.68, P < 0.05). In conclusion, prevention of F-actin rearrangement by Rho-kinase inhibition or by cytochalasinD treatment attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity.

Abstract 3435: GeneChip Analyses Identify Bone Marrow Cells as a Rich Source of Paracrine Factors: Insight From the BOOST Study Program

It has been hypothesized that the improvement of left ventricular function observed after intracoronary bone marrow cell (BMC) transfer in patients with STEMI is related in part to paracrine effects. RNA was prepared from BMC that were obtained from patients with STEMI (n=3) included in the ongoing multicenter BOne marrOw transfer to enhance ST-elevation infarct regeneration (BOOST)-2 trial. Peripheral blood leukocyte (PBL) RNA was obtained from the same 3 patients. GeneChip analyses (Affymetrix HG-U133 Plus 2.0, ca. 47000 probe sets) were performed to characterize the transcriptome in BMC vs. PBL. Using GeneSpring software 7.2, we identified 56 secreted factors that were more strongly expressed (&gt;2-fold) in BMC as compared to PBL. In a second microarray (RNA from n=3 additional patients), 41 of these factors were confirmed. Twelve of these 41 factors were chosen for independent validation by real-time PCR using RNA from 6 additional patients; overexpression in BMC vs. PBL was confirmed for each of these 12 factors. Several factors overexpressed in BMC represented putative cardioprotective cytokines (e.g., BMP-2, CT-1, IGF-1, SDF-1α, VEGF-A). BMC from 7 additional patients were cultured for 24h in serum-free DMEM medium. Conditioned supernatants (SN) were filtered and pooled. BMC-SN (10μL/mL) reduced apoptotic cell death in neonatal rat ventricular cardiomyocytes exposed to simulated ischemia (3h) and reperfusion (1h), as shown by TUNEL (62 ± 7% vs. control, i.e. treatment with unconditioned DMEM medium; n=7; P&lt;0.05) and histone-ELISA (69 ± 12% vs. control; n=5; P&lt;0.05). Moreover, BMC-SN stimulated human umbilical vein endothelial cell (HUVEC) migration (transwell chambers; 293 ± 55% vs. control; n=5; P&lt;0.01) and in-matrigel tube formation (tubular length, 275 ± 35% vs. control; n=5; P&lt;0.05; tube/branch point ratio, 156 ± 10% vs. control; n=5; P&lt;0.05). Finally, BMC-SN promoted enhanced endothelial cell sprouting in the mouse aortic ring assay ex vivo. In conclusion, BMC obtained from patients with STEMI are a rich source of secreted factors. Conditioned BMC supernatants protect cardiomyocytes from I/R-induced apoptosis, and promote pro-angiogenic effects in vitro. Our data tend to support the paracrine hypothesis.

Oxidant Stress during Simulated Ischemia Primes Cardiomyocytes for Cell Death during Reperfusion

Ischemia-reperfusion injury induces oxidant stress, and the burst of reactive oxygen species (ROS) production after reperfusion of ischemic myocardium is sufficient to induce cell death. Mitochondrial oxidant production may begin during ischemia prior to reperfusion because reducing equivalents accumulate and promote superoxide production. We utilized a ratiometric redox-sensitive protein sensor (heat shock protein 33 fluorescence resonance energy transfer (HSP-FRET)) to assess oxidant stress in cardiomyocytes during simulated ischemia. HSP-FRET consists of the cyan and yellow fluorescent protein fluorophores linked by the cysteine-containing regulatory domain from bacterial HSP-33. During ischemia, ROS-mediated oxidation of HSP-FRET was observed, along with a decrease in cellular reduced glutathione levels. These findings were corroborated by measurements using redox-sensitive green fluorescent protein, another protein thiol ratiometric sensor, which became 93% oxidized by the end of simulated ischemia. However, cell death did not occur during ischemia, indicating that this oxidant stress is not sufficient to induce death before reperfusion. However, interventions that attenuate ischemic oxidant stress, including antioxidants or scavengers of residual O(2) that attenuate/prevent ROS generation during ischemia, abrogated cell death during simulated reperfusion. These findings reveal that, in isolated cardiomyocytes, sublethal H(2)O(2) generation during simulated ischemia regulates cell death during simulated reperfusion, which is mediated by the reperfusion oxidant burst.

Role of reverse mode Na +/Ca 2+ exchanger in the cardioprotection of metabolic inhibition preconditioning in rat ventricular myocytes

This study determined the role of the reverse mode Na +/Ca 2+ exchanger (NCX) in cardioprotection of metabolic inhibition preconditioning in isolated ventricular myocyctes. Activity of the reverse mode NCX was assessed by changes of [Ca 2+] i upon withdrawal of extracellular Na +. [Ca 2+] i was measured by spectrofluorometry, using Fura-2 as Ca 2+ indicator. The amplitude of contraction and exclusion of trypan blue by myocytes served as indices of contractile function and viability, respectively. Firstly, NCX activity significantly decreased during simulated reperfusion after severe metabolic inhibition (index ischaemia) in myocytes subjected to metabolic inhibition preconditioning. This inhibitory effect on NCX activity correlated with the enhancing effect of metabolic inhibition preconditioning on cell viability following ischaemic insult. Treatment myocytes with E4031, an activator of reverse mode NCX, during index ischaemia and reperfusion attenuated the enhancing effects of metabolic inhibition preconditioning on cell contraction and viability. Secondly, NCX activity was significantly higher at the end of metabolic inhibition preconditioning. More importantly, E4031 pretreatment mimicked the beneficial effects of metabolic inhibition preconditioning in myocytes and ischaemic preconditioning in the isolated perfused heart, respectively, and these effects were abolished by KB-R7943, an inhibitor of reverse mode NCX. The results indicate that increased reverse mode NCX activity during preconditioning triggered cardioprotection, and reduced reverse mode NCX activity during reperfusion after index ischaemia conferred cardioprotection.

Mitochondrial Ca2+ uptake during simulated ischemia does not affect permeability transition pore opening upon simulated reperfusion☆

Reenergization of ischemic cardiomyocytes may be associated with acute necrotic cell death due in part to cytosolic Ca2+ overload and opening of a permeability transition pore (PTP) in mitochondria. It has been suggested that Ca2+ overload during ischemia primes mitochondria for PTP opening during reperfusion. We investigated the ability of mitochondria to uptake Ca2+ during simulated ischemia (SI) and whether this uptake determines PTP opening and cell death upon simulated reperfusion (SR). Rat heart mitochondria were submitted to either hypoxia (anoxic chamber) or to SI (respiratory inhibition, substrate depletion and acidosis) and subsequent SR. Mitochondrial Ca2+ uptake was monitored using Ca2+ microelectrodes after exposure to different [Ca2+] up to 25 microM during SI, and PTP opening was assessed by quantification of mitochondrial swelling (changes in absorbance rate at 540 nm) and calcein release. Mitochondrial Ca2+ uptake (Rhod-2 fluorescence) and cytosolic Ca2+ rise (Fura-2 ratio fluorescence) were further investigated in HL-1 cardiac myocytes submitted to SI/SR, and the effect of reducing mitochondrial Ca2+ load (with 25 microM ruthenium red) or blocking PTP opening (with 0.5 microM cyclosporin A) on the rate of cell death was investigated in adult cardiomyocytes exposed to SI/SR. SI induced a progressive dissipation of mitochondrial membrane potential (TMRE fluorescence); however, prior to the completion of depolarization, high levels of Ca2+ uptake were observed in mitochondria. SR induced PTP opening but this phenomenon was not influenced by the magnitude of mitochondrial Ca2+ uptake during previous SI. Blockade of the mitochondrial Ca2+ uniporter during SI in cardiomyocytes attenuated mitochondrial Ca2+ uptake but increased cytosolic Ca2+ overload and cell death upon subsequent SR. Mitochondrial Ca2+ uptake during SI buffers cytosolic Ca2+ overload but its magnitude appears not to be an important determinant of PTP opening upon subsequent SR.

Ischemia-induced increase of stiffness of αB-crystallin/HSPB2-deficient myocardium

The two small heat shock proteins (sHSPs), alphaB-crystallin and HSPB2, have been shown to translocate within a few minutes of cardiac ischemia from the cytosol to myofibrils; and it has been suggested that their chaperone-like properties might protect myofibrillar proteins from unfolding or aggregation during stress conditions. Further evidence of an important role for HSPs in muscle function is provided by the fact that mutations of the alphaB-crystallin gene cause myopathy and cardiomyopathy. In the present study, we subjected isolated papillary muscles of alphaB-crystallin/HSPB2-deficient mice to simulated ischemia and reperfusion. During ischemia in alphaB-crystallin/HSPB2-deficient muscles, the development of contracture started earlier and reached a higher value compared to the wildtype mice. The recovery of contracture of alphaB-crystallin/HSPB2-deficient muscles was also attenuated during the simulated reperfusion period. However, twitch force was not significantly altered at any time of the experiment. This suggests that during ischemic insults, alphaB-crystallin/HSPB2 may not be important for the contraction process itself, but rather serve to maintain muscular elasticity.

Substrate dependence of the postischemic cardiomyocyte recovery: Dissociation between functional, metabolic and injury markers

Defining the substrate that influences the most favourably the myocardial post-ischemic recovery is subject of debates, due to dissociation between functional and biochemical benefits. Hence, we studied the effects of either glucose or different fatty acids on the functional and metabolic recovery of post-ischemic cardiomyocytes in a substrate-free hypoxia model of simulated ischemia-reperfusion. Rat cardiomyocytes were submitted to a 2.5 h simulated ischemia followed by a 2 h reoxygenation without substrate (control), or with either glucose, octanoic acid, oleic acid, or elaidic acid. During simulated ischemia, electromechanical function gradually disappeared while the cellular viability and mitochondrial function declined. During control simulated reperfusion, cardiomyocytes recovered near normal function but a significant reduction in the action potential amplitude and rate persisted. The addition of glucose or oleic acid during simulated reperfusion promoted a faster, better and sustain functional recovery. Amongst the fatty acids, the functional recovery was slower with elaidic and octanoic acids as compared with oleic acid. The mitochondrial function was better improved during simulated reperfusion with glucose than with the tested fatty acids, among which elaidic acid was the less unfavourable. Paradoxically, the addition of whichever substrate during simulated reperfusion tended to worsen the cellular viability. Thus, cardiomyocytes recovery strongly relies on the characteristics of the substrate supplied at the onset of simulated reperfusion: glucidic or lipidic nature, chain-length, insaturation degree. Moreover, these data suggest that defining the appropriateness of a given substrate for the post-ischemic cardiomyocyte recovery is closely related to the functional and the biological endpoints in consideration.

Mitochondrial ATP-Sensitive K+ Channels Prevent Oxidative Stress, Permeability Transition and Cell Death

Ischemia followed by reperfusion results in impairment of cellular and mitochondrial functionality due to opening of mitochondrial permeability transition pores. On the other hand, activation of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)) protects the heart against ischemic damage. This study examined the effects of mitoK(ATP) and mitochondrial permeability transition on isolated rat heart mitochondria and cardiac cells submitted to simulated ischemia and reperfusion (cyanide/aglycemia). Both mitoK(ATP) opening, using diazoxide, and the prevention of mitochondrial permeability transition, using cyclosporin A, protected against cellular damage, without additive effects. MitoK(ATP) opening in isolated rat heart mitochondria slightly decreased Ca(2+) uptake and prevented mitochondrial reactive oxygen species production, most notably in the presence of added Ca(2+). In ischemic cells, diazoxide decreased ROS generation during cyanide/aglycemia while cyclosporin A prevented oxidative stress only during simulated reperfusion. Collectively, these studies indicate that opening mitoK(ATP) prevents cellular death under conditions of ischemia/reperfusion by decreasing mitochondrial reactive oxygen species release secondary to Ca(2+) uptake, inhibiting mitochondrial permeability transition.

Intermittent hypoxia protects the rat heart against ischemia/reperfusion injury by activating protein kinase C

The aim of this study was to investigate whether and how protein kinase C (PKC) was involved in the protection afforded by intermittent hypoxia (IH) and the subcellular distribution of different PKC isozymes in rat left ventricle. Post-ischemic recovery of left ventricular developed pressure and ±dP/dtmax in IH hearts were higher than those of normoxic hearts. Chelerythrine (CHE, 5 μM), a PKC antagonist, significantly inhibited the protective effects of IH, but had no influence on normoxic hearts. CHE significantly reduced the effect of IH on the time to maximal contracture (Tmc), but had no significant effect on the amplitude of maximal contracture (Amc) in IH group. In isolated normoxic cardiomyocytes, [Ca 2+] i, measured as arbitrary units of fluorescence ratio (340nm/380nm) of fura-2, gradually increased during 20 min simulated ischemia and kept at high level during 30 min reperfusion. However, [Ca 2+] i kept at normal level during simulated ischemia and reperfusion in isolated IH cardiomyocytes. In normoxic myocytes, [Na +] i, indicated as actual concentration undergone calibration, gradually increased during 20 min simulated ischemia and quickly declined to almost the same level as that of pre-ischemia during 30 min simulated reperfusion. However, in IH myocytes, [Na +] i increased to a level lower than the corresponding of normoxic myocytes during simulated ischemia and gradually reduced to the similar level as that of normoxic myocytes after simulated reperfusion. 5 μM CHE greatly increased the levels of [Ca 2+] i and [Na +] i during ischemia and reperfusion in normoxic and IH myocytes. In addition, we demonstrated that IH up-regulated the baseline protein expression of particulate fraction of PKC-α, ɛ, δ isozymes. There is no significant difference of protein expression of PKC-α, ɛ, δ isozymes in cytosolic fraction between IH and normoxic group. The above results suggested that PKC contributed to the cardioprotection afforded by IH against ischemia/reperfusion (I/R) injury; the basal up-regulation of the particulate fraction of PKC-α, ɛ, δ isozymes in IH rat hearts and the contribution of PKC to the elimination of calcium and sodium overload might underlie the mechanisms of cardioprotection by IH.

Selective inhibition of inward rectifier K+ channels (Kir2.1 or Kir2.2) abolishes protection by ischemic preconditioning in rabbit ventricular cardiomyocytes.

Volume regulatory Cl- channels are key regulators of ischemic preconditioning (IPC). Because Cl- efflux must be balanced by an efflux of cations to maintain cell membrane electroneutrality during volume regulation, we hypothesize that I(K1) channels may play a role in IPC. We subjected cultured cardiomyocytes to 60-minute simulated ischemia (SI) followed by 60-minute of simulated reperfusion (SR) and assessed percent cell death using trypan blue staining. Ischemic preconditioning (10-minute SI/10-minute SR) significantly (P<0.0001) reduced the percent cell death in nontransfected cardiomyocytes [IPC(CM) 18.0+/-2.1% versus control (C(CM)) 48.3+/-1.0%]. IPC protection was not altered by overexpression of the reporter gene (enhanced green fluorescent protein, EGFP). However, overexpression of dominant-negative Kir2.1 or Kir2.2 genes using adenoviruses (AdEGFPKir2.1DN or AdEGFPKir2.2DN) encoding the reporter gene EGFP prevented IPC protection [both IPC(CM)+AdEGFPKir2.1DN 45.8+/-2.3% (mean+/-SEM) and IPC(CM)+AdEGFPKir2.2DN 47.9+/-1.4% versus IPC(CM); P<0.0001] in cultured cardiomyocytes (n=8 hearts). Transfection of cardiomyocytes with AdEGFPKir2.1DN or AdEGFPKir2.2DN did not affect cell death in control (nonpreconditioned) cardiomyocytes (both C(CM)+ AdEGFPKir2.1DN 45.8+/-0.7% and C(CM)+AdEGFPKir2.2DN 46.2+/-1.3% versus C(CM); not statistically significant). Similar effects were observed in both cultured (n=5 hearts) and freshly isolated (n=4 hearts) ventricular cardiomyocytes after I(K1) blockade with 20 micromol/L BaCl2 plus 1 micromol/L nifedipine (to prevent Ba2+ uptake). Nifedipine alone neither protected against ischemic injury nor blocked IPC protection. Our findings establish that I(K1) channels play an important role in IPC protection.

Physiological and metabolic actions of mycophenolate mofetil on cultured newborn rat cardiomyocytes in normoxia and in simulated ischemia.

Mycophenolate mofetil (MMF) is a new immunosuppressive drug used to reduce acute rejection after heart transplantation. As with other immunosuppressive drugs, MMF therapy is associated with several adverse effects. However, the direct effects of MMF on myocardial tissue has not been yet evaluated. The aim of the work was thus to evaluate the effects of MMF on isolated cardiomyocytes (CM) in normal conditions and in an in vitro model of simulated ischemia (SI; substrate-free hypoxia) and reperfusion (R; reoxygenation). Myocyte-enriched cultures were prepared from newborn rat heart ventricles. The transmembrane potentials were recorded using conventional microelectrodes and the cell contractions were monitored with a photoelectric device. In basal conditions, MMF (10(-6) and 10(-5) M) exerted no significant effects on the survival and on the electrical and contractile activities of CM in culture, even during long-term exposure (up to 48 h). SI per se led to a gradual decrease and then an abortion of the spontaneous automaticity and electromechanical activity of CM. Pretreating CM with either 10(-6) or 10(-5) M MMF was able to reduce the SI-induced cell dysfunctions. The presence of MMF at these concentrations did not hamper the post-SI functional recovery of CM during reoxygenation. At 10(-5) M, MMF applied during reoxygenation only permitted a better recovery of CM. However, the mitochondrial function after reoxygenation, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) test, was not significantly influenced by the addition of MMF before as well as after ischemia. Conversely, MMF was able to reduce in this model the postischemic rise in xanthine and hypoxanthine. These data from CM-enriched model show that MMF: (i) had no cytotoxic effect, (ii) displayed a cytoprotective effect during SI, and (iii) exerted its beneficial effect at least partly through the decrease in the xanthine oxidase-dependent free radical production.

Enhanced cell volume regulation: a key protective mechanism of ischemic preconditioning in rabbit ventricular myocytes

Accumulation of osmotically active metabolites, which create an osmotic gradient estimated at ~60 mOsM, and cell swelling are prominent features of ischemic myocardial cell death. This study tests the hypothesis that reduction of ischemic swelling by enhanced cell volume regulation is a key mechanism in the delay of ischemic myocardial cell death by ischemic preconditioning (IPC). Experimental protocols address whether: (i) IPC triggers a cell volume regulation mechanism that reduces cardiomyocyte swelling during subsequent index ischemia; (ii) this reduction in ischemic cell swelling is sufficient in magnitude to account for the IPC protection; (iii) the molecular mechanism that mediates IPC also mediates cell volume regulation. Two experimental models with rabbit ventricular myocytes were studied: freshly isolated pelleted myocytes and 48-h cultured myocytes. Myocytes were preconditioned either by distinct short simulated ischemia (SI)/simulated reperfusion protocols (IPC), or by subjecting myocytes to a pharmacological preconditioning (PPC) protocol (1 μM calyculin A, or 1 μM N 6-2-(4-aminophenyl)ethyladenosine (APNEA), prior to subjecting them to either different durations of long SI or 30 min hypo-osmotic stress. Cell death (percent blue square myocytes) was monitored by trypan blue staining. Cell swelling was determined by either the bromododecane cell flotation assay (qualitative) or video/confocal microscopy (quantitative). Simulated ischemia induced myocyte swelling in both the models. In pelleted myocytes, IPC or PPC with either calyculin A or APNEA produced a marked reduction of ischemic cell swelling as determined by the cell floatation assay. In cultured myocytes, IPC substantially reduced ischemic cell swelling (P < 0.001). This IPC effect on ischemic cell swelling was related to an IPC and PPC (with APNEA) mediated triggering of cell volume regulatory decrease (RVD). IPC and APNEA also significantly (P < 0.001) reduced hypo-osmotic cell swelling. This IPC and APNEA effect was blocked by either adenosine receptor, PKC or Cl – channel inhibition. The osmolar equivalent for IPC protection approximated 50–60 mOsM, an osmotic gradient similar to the estimated ischemic osmotic load for preconditioned and non-preconditioned myocytes. The results suggest that cell volume regulation is a key mechanism that accounts for most of the IPC protection in cardiomyocytes.

H2O2-mediated oxidative stress versus cold ischemia-reperfusion: mitochondrial respiratory defects in cultured human endothelial cells.

Oxidative stress to vascular endothelium plays an important role in cold ischemia-reperfusion (CIR) injury. We compared mitochondrial and plasma membrane integrity in human endothelial cells after 20-min exposure to 500 microM H2O or 8-hr cold ischemia and simulated reperfusion. In both groups, plasma membrane integrity was maintained but respiration was significantly decreased, as measured by high-resolution respirometry. Uncoupling was more pronounced after H2O exposure compared with CIR. After H2O exposure, complex I respiration was significantly reduced, whereas CIR resulted additionally in a significant inhibition of complex II and IV respiration. Our results point to a partial overlap of the patterns of mitochondrial defects after H2O-mediated and CIR injury. In this respect, H2O exposure proved to be a useful model to study the mechanisms of CIR injury to human endothelial cells, whereas the full pattern of CIR injury could not be induced by a pulse of hydrogen peroxide exposure.

A Novel nonpeptidic caspase-3/7 inhibitor, ( S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin reduces myocardial ischemic injury

The efficacy of a novel, nonpeptidic, caspase 3/7-selective inhibitor, ( S)-(+)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (MMPSI) for reducing ischemic injury in isolated rabbit hearts or cardiomyocytes was evaluated. MMPSI (0.1–10 μM) evoked a concentration-dependent reduction in infarct size (up to 56% vs. control; IC 50=0.2 μM). Furthermore, apoptosis (DNA laddering, soluble nucleosomes) was reduced in the ischemic area-at-risk. MMPSI inhibited recombinant human caspase-3 with an IC 50=1.7 μM. Apoptosis in H9c2 cells after 16-h simulated ischemia and 2-h simulated reperfusion was significantly reduced by MMPSI in a concentration-dependent manner (IC 50=0.5 μM); similar effects were observed in isolated adult rabbit cardiomyocytes (IC 50=1.5 μM). These data support an important role for caspase-3/7 in mediating myocardial ischemic injury. Furthermore, these data indicate that cardioprotection via caspase-3/7 inhibition is attainable via a small molecule (nonpeptidic) inhibitor, a necessary step in making this approach therapeutically viable.

Annexin V staining during reperfusion detects cardiomyocytes with unique properties.

With the use of markers of sarcolemmal membrane permeability, cardiomyocyte models of ischemic injury have primarily addressed necrotic death during ischemia. In the present study, we used annexin V-propidium iodide staining to examine apoptosis and necrosis after simulated ischemia and simulated reperfusion in rat ventricular myocytes. Annexin V binds phosphatidylserine, a phosphoaminolipid thought to be externalized during apoptosis or programmed cell death. Propidium iodide is a marker of cell necrosis. Under baseline conditions, <1% of cardiomyocytes stained positive for annexin V. After 20 or 60 min of simulated ischemia, there was no increase in annexin V staining, although 60-min simulated ischemia resulted in significant propidium iodide staining. Twenty minutes of simulated ischemia, followed by 20 or 60 min of simulated reperfusion, resulted in 8-10% of myocytes staining positive for annexin V. Annexin V-positive cells retained both rod-shaped morphology and contractile function but exhibited the decreased cell width indicative of cell shrinkage. Baseline mitochondrial free Ca2+ (111 +/- 14 nM) was elevated in reperfused annexin V-negative cells (214 +/- 22 nM), and further elevated in annexin V-positive myocytes (382 +/- 9 nM). After 60 min of simulated reperfusion, caspase-3-like activity was observed in approximately 3% of myocytes, which had a rounded appearance and membrane blebs. These results suggest that the use of annexin V after simulated ischemia-reperfusion uncovers a population of cardiomyocytes whose characteristics appear to be consistent with cells undergoing apoptosis.