Pegfilgrastim administered in an abbreviated schedule, significantly improved neutrophil recovery after high-dose radiation-induced myelosuppression in rhesus macaques.

Conventional daily administration of filgrastim is effective in reducing the duration of severe neutropenia and enhancing survival following lethal radiation, myelosuppressive cytotoxic therapy or myeloablation and stem cell transplantation. A sustained-duration form of filgrastim, pegfilgrastim has significantly simplified scheduling protocols after chemotherapy-induced neutropenia to a single injection while maintaining the therapeutic effectiveness of daily administration of filgrastim. We examined the ability of a single or double (weekly) administration of pegfilgrastim to significantly improve neutrophil recovery in a rhesus macaque model of severe radiation-induced myelosuppression. Animals were exposed to potentially lethal 6 Gy total-body X radiation. After irradiation all animals received supportive care and were administered either pegfilgrastim at 300 µg/kg on day 1 or day 1 and day 7 post exposure, or filgrastim at 10 µg/kg/day initiated on day 1 post exposure and continued daily through neutrophil recovery. Pharmacokinetic parameters and neutrophil-related values for duration of neutropenia, neutrophil nadir, time to recovery to an absolute neutrophil count ≥500/µL or ≥2000/µL, and days of antibiotic support were determined. Effective plasma concentrations of pegfilgrastim were maintained in neutropenic animals until after the onset of hematopoietic recovery, which is consistent with neutrophil-dependent properties of elimination. Administration of pegfilgrastim at day 1 and day 7 was most effective at improving neutrophil recovery compared to daily administration of filgrastim or a single injection of pegfilgrastim on day 1, after severe, radiation-induced myelosuppression in rhesus macaques.

Protecting the acutely ischemic myocardium beyond reperfusion therapies: are we any closer to realizing the dream of infarct size elimination?

Patients currently treated for acute myocardial infarction receive reperfusion therapy as their only anti-infarct intervention. Although pharmacologic agents have been evaluated in the past for their ability to salvage ischemic myocardium when administered at reperfusion, until very recently none has demonstrated clear efficacy in clinical trials. However, a new generation of interventions has emerged which protects the heart by activating the reperfusion-induced salvage kinase (RISK) pathway. Unlike the disappointing results documented with previously touted putative cardioprotective agents, the preclinical experience with these newer interventions is very consistent indicating that there is a high likelihood that they will be effective clinically. Ischemic postconditioning, which also acts by activating the RISK pathway, has shown marked reduction in infarct size in small-scale trials. Finally, if a strategy for rapidly cooling the heart can be devised so that the in-hospital normothermic ischemic time can be significantly reduced, then infarct size can be even further decreased. In our opinion it is well within our reach using existing technologies to see the day when infarction can be virtually eliminated in the patient with acute coronary occlusion.

Cardioprotection: spotlight on PKG.

Classical ischaemic preconditioning, delayed or second window preconditioning and postconditioning are forms of cardioprotection that are dependent on cell surface receptors, intracellular signalling molecules and kinases that ultimately block formation of the mitochondrial permeability transition. The latter is presumed to cause myocardial necrosis as well as apoptosis, so prevention of its formation upon resumption of perfusion after a prolonged coronary occlusion should be cardioprotective. In all of these forms of cardioprotection, formation of cGMP and activation of protein kinase G (PKG) are recognized to be key steps in the signal transduction pathway. Burley et al. highlight the roles of cGMP and PKG in their comprehensive review. They describe the basic biology of PKG and emphasize its compartmentalization, which may be responsible for the frustration induced by assays for PKG in whole cell lysates and for the spurious conclusions about the role of PKG in cardioprotection. This review will be useful to both the novice and the seasoned investigator.

Augmented taurine release is not the mechanism of ischemic preconditioning's cardioprotection.

In ischemic preconditioning (IPC) a brief ischemic period protects the heart from a subsequent ischemic insult by an unknown mechanism. Osmotic swelling has been proposed to be a major cause of cell death when ischemic tissue is reperfused. The present study tests whether the preconditioned heart during reperfusion might release more taurine, an important osmolyte in the cardiac myocytes, to decrease cellular osmolarity, oppose swelling, and preserve viability. We collected the coronary effluent from isolated rabbit hearts for 10 min before and 10 min after preconditioning with 5 min of global ischemia. The heart then experienced 15 min of global ischemia and effluent was collected during reperfusion for 40 min. A control group was studied similarly but without the preconditioning ischemia. Fifteen min of ischemia was chosen to avoid any taurine release caused by ischemic cell death. Taurine was measured with HPLC. In the IPC group there was a postischemic release over baseline of 5.09 +/- 1.51 micromol (approx 3.3% of the total taurine pool), whereas in the control group the release was not significantly different, 5.72 +/- 1.67 micromol. The percent of the taurine pool lost from each heart during reperfusion was calculated based on an assumption of a total content of 20 microM taurine/gm wet weight. Since the amount of taurine released by the isolated rabbit heart following ischemia was not different in preconditioned and non-preconditioned hearts, we conclude that reduced swelling through taurine release is not the mechanism of the cardioprotective effects of IPC.

Acute alcohol-induced protection against infarction in rabbit hearts: differences from and similarities to ischemic preconditioning.

Recent studies reveal that brief ethanol exposure induces cardioprotection against simulated ischemia in cardiomyocytes by the activation of protein kinase C- epsilon. The present study tests the ability of ethanol to induce protection in rabbit hearts in which infarct size was the end-point and explores the signal transduction pathways involved. In isolated rabbit hearts, 50 m m ethanol infused for 5 min with 10 min of washout prior to 30 min of regional ischemia reduced infarct size (triphenyltetrazolium chloride staining) by 49%. Neither adenosine receptor blockade with 8-(p -sulfophenyl) theophylline nor the free radical scavenger N-2-mercaptopropionyl glycine inhibited the protection triggered by ethanol. In contrast, protein kinase C inhibition with chelerythrine, protein tyrosine kinase inhibition with genistein, and blockade of ATP-sensitive potassium channels (K(ATP)) with either 5-hydroxydecanoate or glibenclamide did abolish protection. Thus, transient ethanol exposure followed by washout prior to ischemia elicits a preconditioning-like effect involving protein kinase C, at least one protein tyrosine kinase, and K(ATP)channels, but neither adenosine nor free radicals.

Attenuation of oxidant stress during reoxygenation by AMP 579 in cardiomyocytes.

AMP 579, an adenosine A(1)/A(2) receptor agonist, has a strong anti-infarct effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether AMP 579 can reduce the production of reactive oxidant species (ROS) during reoxygenation in cultured chick embryonic cardiomyocytes. The intracellular fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH) was used to detect ROS. The cells were subjected to 60 min of simulated ischemia, followed by either 15 min or 3 h of reoxygenation. AMP 579 (0.5 and 1 microM), when started 10 min before reoxygenation, significantly reduced ROS generation from 4.86 +/- 0.30 (arbitrary units) in untreated cells to 2.72 +/- 0.31 and 1.85 +/- 0.14, respectively (P < 0.05). Cell death that was assessed by propidium iodide uptake was markedly reduced by AMP 579 (49.6 +/- 4.7% of control cells vs. 25.4 +/- 2.4%, P < 0.05). In contrast, adenosine did not alter ROS generation or cell death. Attenuation of ROS production by AMP 579 was completely prevented by simultaneous exposure of cells to the selective adenosine A(2) antagonist 8-(13-chlorostyryl) caffeine. These results indicate that AMP 579 directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates intracellular oxidant stress. Furthermore, adenosine could not duplicate these effects.

Acetylcholine, bradykinin, opioids, and phenylephrine, but not adenosine, trigger preconditioning by generating free radicals and opening mitochondrial K(ATP) channels.

It has been assumed that all G(i)-coupled receptors trigger the protective action of preconditioning by means of an identical intracellular signaling pathway. To test this assumption, rabbit hearts were isolated and perfused with Krebs buffer. All hearts were subjected to a 30-minute coronary artery occlusion followed by 120 minutes of reperfusion. Risk area was measured with fluorescent particles and infarct size with triphenyltetrazolium chloride staining. Control hearts showed 29.1+/-2.8% infarction of the risk zone. A 5-minute infusion of acetylcholine (0.55 mmol/L) beginning 15 minutes before the 30-minute occlusion resulted in significant protection (9.2+/-2.7% infarction). This protection could be blocked by administration of 300 micromol/L N-2-mercaptopropionyl glycine (MPG), a free radical scavenger, or by 200 micromol/L 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) antagonist, for 15 minutes beginning 5 minutes before the acetylcholine infusion (35.2+/-3.9% and 27.8+/-2.4% infarction, respectively). Similar protection was observed with other known triggers, ie, bradykinin (0.4 micromol/L), morphine (0.3 micromol/L), and phenylephrine (0.1 micromol/L), and in each case protection was completely abrogated by either MPG or 5-HD. In contrast, protection by adenosine or its analog N(6)-(2-phenylisopropyl) adenosine could not be blocked by either MPG or 5-HD. Therefore, whereas most of the tested agonists trigger protection by a pathway that requires opening of mitochondrial K(ATP) channels and production of free radicals, the protective action of adenosine is not dependent on either of these steps. Hence, it cannot be assumed that all G(i)-coupled receptors use the same signal transduction pathways to trigger preconditioning.

Amp 579 reduces contracture and limits infarction in rabbit heart by activating adenosine A2 receptors.

To determine the mechanism by which AMP 579, an adenosine A1/A2 agonist, administered at reperfusion protects ischemic myocardium, buffer-perfused rabbit hearts were subjected to 30 min of global ischemia and 2 h of reperfusion. AMP 579 (500 nM) was included in the reperfusate for the first 70 min. Average left ventricular diastolic pressure during reperfusion in hearts receiving AMP 579 was lower than that in control hearts (17.9 +/- 2.4 vs. 39.0 +/- 6.5 mm Hg, p < 0.05), indicating attenuation of contracture. Left ventricular developed pressure and coronary flow during reperfusion were also significantly improved with AMP 579 treatment. AMP 579's anti-contracture effect was blocked by the adenosine A2-receptor antagonist 8-(3-chlorostyryl)caffeine (CSC), but not by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). CSC, but not DPCPX, also blocked AMP 579's ability to preserve developed pressure and coronary flow in these hearts. AMP 579 significantly reduced infarction in isolated hearts subjected to regional ischemia. The anti-infarct effect again was abolished by CSC but not by DPCPX. Finally, we tested whether 5'-(N-ethylcarboxamido)adenosine (NECA), another A1/A2 agonist, also administered for the initial 70 min of reperfusion, could duplicate the anti-infarct effect of AMP 579. One-hundred-nanomolar NECA duplicated the protection, but neither 50 nM CGS21680, a selective A2 agonist, nor 100 microM adenosine was protective. Therefore, AMP 579 given at reperfusion reduces contracture and infarction. Anti-contracture and anti-infarct effects require the adenosine A2, but not the A1, receptor suggesting that prevention of contracture and tissue salvage are mechanistically related. Not all A2 agonists were able to duplicate the anti-infarct effect, suggesting something unique about AMP579.

Menadione mimics the infarct-limiting effect of preconditioning in isolated rat hearts.

The role of mitochondrial free radicals in the cardioprotective effect of ischemic preconditioning was examined in isolated buffer-perfused rat hearts. Infarct size in control rat hearts subjected to 30 min of regional ischemia and 120 min of reperfusion was 32.6 +/- 3.4% of the risk zone. Ischemic preconditioning (3 cycles of 5-min global ischemia/5-min reperfusion) before the same regional ischemia and reperfusion protocol significantly reduced infarct size to 2.6 +/- 0.8% of the risk zone. Perfusion with menadione (3.0 microM), a generator of mitochondrial free radicals, in lieu of preconditioning ischemia significantly reduced infarction to 10.9 +/- 2.7%. N-2-mercaptopropionylglycine (1.0 mM), a free radical scavenger, blocked the protection of menadione, significantly increasing infarction to 23.5 +/- 1.1%. Myxothiazol (0.6 microM), a site III mitochondrial inhibitor, blocked the protection of menadione and significantly increased infarction to 25.2 +/- 3.8%. The infarct-limiting effect of menadione was attenuated to 19.7 +/- 1.5% of the risk zone by 10 microM SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Furthermore, menadione significantly increased p38 MAPK phosphorylation to a level 5.6-fold over basal. These results indicate that free radicals that originate within mitochondria can activate p38 MAPK and protect hearts against infarction.

Acute ethanol exposure fails to elicit preconditioning-like protection in in situ rabbit hearts because of its continued presence during ischemia.

OBJECTIVES: Is the timing of exposure critical for ethanol's ability to induce cardioprotection? BACKGROUND: Acute ethanol exposure has been reported to mimic ischemic preconditioning in vitro, but it failed to protect in situ. We hypothesized that these conflicting findings were related to ethanol's presence during ischemia in situ. METHODS: The effect on infarct size (triphenyltetrazolium chloride) of acute ethanol exposure (0.35, 0.7, and 1.4 g/kg IV) 10 min before ischemia was measured in open-chest rabbits after 30 min of regional ischemia and reperfusion and was compared to ethanol's ability to reduce infarct size in isolated hearts in which the timing of ethanol exposure could be varied. RESULTS: Ethanol exposure in situ shortly before ischemia did not reduce infarct size. Moreover, ethanol abolished protection from both ischemic preconditioning and mitochondrial KATP channel activation. In contrast, in buffer-perfused hearts exposed to 10 to 50 mmol/liter ethanol for 5 min followed by washout before ischemia, infarct size was significantly reduced. When ethanol exposure was prolonged until the end of ischemia in isolated hearts, protection was abolished. Conversely, protection was seen when ethanol was infused in situ followed by removal of the heart and perfusion with ethanol-free buffer prior to ischemia in a Langendorff preparation. When 50 min were allowed to metabolize the ethanol prior to ischemia, protection could also be shown in situ. CONCLUSIONS: Ethanol exposure followed by washout or sufficient time to metabolize the alcohol prior to ischemia induces preconditioning-like myocardial protection. However, if present throughout ischemia, ethanol actually blocks all preconditioning-related protection.

Effect of weight loss on the ECG of normotensive morbidly obese patients.

BACKGROUND: Morbid obesity produces a variety of ECG alterations, including leftward shifts of the P-wave, QRS, and T-wave axes; disproportionately high frequencies of low QRS voltage; left ventricular hypertrophy and left atrial abnormality; and a high frequency of T-wave flattening in the inferior and lateral leads. This study was designed to assess the effects of substantial weight loss on the ECG in morbid obesity. METHODS: We performed a resting 12-lead ECG on 60 normotensive patients (48 women and 12 men; mean +/- SD age, 37 +/- 7 years), whose body weight was twice their ideal body weight prior to and at the time of maximum weight loss after bariatric surgery. RESULTS: Mean weight decreased from 136 +/- 7 to 85 +/- 3 kg. Weight loss produced significant decreases in the frequencies of low QRS voltage; Romhilt-Estes point score > or = 5 points; SV(1) + RV(5) or V(6) > 35 mm; RV(5) or V(6) > 26 mm; RaVL > 11 mm; RaVL > or = 7.5 mm; SaVR > 14 mm; P-terminal force more negative than - 0.04 mm.s in lead V(1); and T-wave flattening in the inferior, lateral, and inferolateral leads. Weight loss significantly shifted the mean P-wave, QRS, T-wave axes rightward, and significantly reduced mean RaVL and mean SaVR voltage. CONCLUSION: Substantial weight loss is capable of reversing many of the ECG alterations associated with morbid obesity.

No confirmation for a causal role of volume-regulated chloride channels in ischemic preconditioning in rabbits.

Volume-regulated chloride channels have recently been proposed to be end-effectors in ischemic preconditioning. The present study attempted to confirm this hypothesis by looking both at cardioprotection and channel activity. In isolated rabbit cardiomyocytes, hypo-osmotic stress (167 mosm/l) induced a current with a magnitude of 2-5 pA/pF at 60 mV. That current could be blocked by the selective chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) or indanyloxyacetic acid 94 (IAA-94), but only at 100 microM and 1 m M respectively. Lower concentrations were not effective. Because the channel-blocking concentrations were toxic in isolated perfused rabbit hearts, as evidenced by cessation of cardiac contraction and massive infarction, neither agent could be tested against preconditioning's anti-infarct effect. NPPB and IAA-94 at 1 microM and 10 microM, respectively (the doses used in a previous report), did not affect coronary flow, heart rate and developed pressure, and also did not prevent the infarct size reduction of ischemic preconditioning with 5 min global ischemia/10 min reperfusion preceding 30 min of regional ischemia and 120 min of reperfusion [11. 4(+/-3.6) and (11.1(+/-3.7)% infarction of risk area, respectively]. The volume-regulated chloride and organic osmolyte channel blocker 4, 4;-diisothiocyanostilbene-2,2;-disulfonic acid (DIDS) at 100 microM blocked the hypo-osmotically induced current in myocytes, but again could not be used, since it induced total cessation of cardiac contraction and reduced infarct size in non-preconditioned hearts. Our data do not confirm a prior study on a causal role for volume-regulated chloride channels in the protection of ischemic preconditioning. This hypothesis remains to be adequately tested.

Limitation of infarct size in rabbit hearts by the novel adenosine receptor agonist AMP 579 administered at reperfusion.

The novel A(1)/A(2)adenosine receptor agonist AMP 579 has been reported to reduce myocardial infarct size in pig and dog. The present study tested the effect of AMP 579 in two rabbit models. In open-chest rabbits undergoing 30 min of regional ischemia and 3 h of reperfusion AMP 579 (3 microg/min/kg) reduced infarct size when treatment was started either 10 min before ischemia or 10 min prior to reperfusion from 36.4+/-3.1% of the risk zone in untreated hearts to 11.8+/-4.4 and 12.3+/-1.0%, respectively. To determine whether protection observed when the drug was administered shortly before reperfusion represented a long-lasting effect rather than merely a transient delay of necrosis, the chest wound was closed in layers and the rabbits permitted to recover. After 3 days the hearts were removed to evaluate infarct size. Continued limitation of infarct size after 3 days of reperfusion (8.2+/-2.8% of the risk zone) confirmed that sustained tissue salvage had been conferred by the drug. In isolated, buffer-perfused rabbit hearts undergoing 30 min of regional ischemia and 2 h of reperfusion, AMP 579 again limited infarct size (8.6+/-2.9% of the risk zone) when treatment started 10 min prior to reperfusion, arguing against an anti-leukocyte mechanism of protection. AMP 579's protective effect in this in vitro model was abrogated by 8-(p-sulfophenyl)theophylline, indicating that it was mediated through adenosine receptors. We conclude that AMP 579 given just prior to reperfusion may be an effective anti-infarct intervention.

Opening of mitochondrial K(ATP) channels triggers the preconditioned state by generating free radicals.

The critical time for opening mitochondrial (mito) K(ATP) channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29+/-3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 micromol/L diazoxide, a mito K(ATP) channel opener, reduced infarction to 3+/-1% and 8+/-1%, respectively. The mito K(ATP) channel closer 5-hydroxydecanoate (200 micromol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24+/-3 and 28+/-6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 micromol/L), another K(ATP) channel closer, blocked the protection by PC only when administered early. These data suggest that K(ATP) channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8+/-3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 micromol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 micromol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with N:-(2-mercaptopropionyl) glycine (300 micromol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 micromol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K(ATP) channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.