Hydrogen sulfide contributes to cardioprotection during ischemia-reperfusion injury by opening K ATP channels

The present study was undertaken to investigate the protective effect of H2S against myocardial ischemia-reperfusion (I/R) injury and its possible mechanism by using isolated heart perfusion and patch clamp recordings. Rat isolated hearts were Langendorff-perfused and subjected to a 30-minute ischemia insult followed by a 30-minute reperfusion. The heart function was assessed by measuring the LVDP, +/-dP/dt max, and the arrhythmia score. The results showed that the treatment of hearts with a H2S donor (40 micromol/L NaHS) during reperfusion resulted in significant improvement in heart function compared with the I/R group (LVDP recovered to 85.0% +/- 6.4% vs. 35.0% +/- 6.1%, +dP/dt max recovered to 80.9% +/- 4.2% vs. 43.0% +/- 6.4%, and -dP/dt max recovered to 87.4% +/- 7.3% vs. 53.8% +/- 4.9%; p < 0.01). The arrhythmia scores also improved in the NaHS group compared with the I/R group (1.5 +/- 0.2 vs. 4.0 +/- 0.4, respectively; p < 0.001). The cardioprotective effect of NaHS during reperfusion could be blocked by an ATP-sensitive potassium channel (K ATP) blocker (10 micromol/L glibenclamide). In single cardiac myocytes, NaHS increased the open probability of K ATP channels from 0.07 +/- 0.03 to 0.15 +/- 0.08 after application of 40 mumol/L NaHS and from 0.07 +/- 0.03 to 0.36 +/- 0.15 after application of 100 mumol/L NaHS. These findings provide the first evidence that H2S increases the open probability of K ATP in cardiac myocytes, which may be responsible for cardioprotection against I/R injury during reperfusion.     

Physiological insulin concentrations protect against ischemia-induced loss of cardiac function in rats

This study determined whether insulin at pre- (fasting) and post-prandial concentrations increases coronary blood flow and improves cardiac function after acute ischemia during a situation of myocardial stunning. The experiments were performed using an isolated, erythrocyte perfused, working rat heart model. To the perfusate we added erythrocytes and 1.5% bovine serum albumin to improve clinical relevance. The following protocol was used: 8 min baseline performance assessment, 10 min pre-ischemic treatment, 12 min global ischemia, 20 min post-ischemic treatment and 8 min recovery assessment. Vehicle, 10 mIU l(-1) and 100 mIU l(-1) human insulin were tested (all n=6). No significant vasodilator response to insulin was observed either pre- or post-ischemically. After the 12-min ischemic insult, cardiac function returned dose-dependently to pre-ischemic values (function loss with 100 mIU l(-1) insulin: -0.2+/-0.4% vs. vehicle: 10.7+/-0.8%). This study clearly shows that in our clinically relevant model of moderate ischemia (stunning), insulin is highly cardioprotective at physiological concentrations. This may be explained primarily by higher glucose uptake, improving the myocardial energetic state during ischemia. Therefore, insulin should be considered for use when the myocardium is at acute risk for ischemic incidents.     

Matrix metalloproteinase-2 contributes to tumor necrosis factor alpha induced apoptosis in cultured rat cardiac myocytes

Tumor necrosis factor alpha (TNFalpha) is associated with a higher risk of cardiovascular disease. Matrix metalloproteinase-2 (MMP-2) has been implicated in the pathophysiology of ischemic heart disease. However, the role of interactions between MMP-2 and TNFalpha, associated with cardiac apoptosis, is unknown. We hypothesized that MMP-2 will contribute to TNFalpha-induced myocardial apoptosis. After treatment with TNFalpha (1-20 ng/ml) for 24 h, or with TNFalpha (10 ng/ml) for 0, 6, 12, 24, or 48 h, MMP-2 activity, percent of TUNEL-positive myocytes, and DNA fragmentation dose, and time-dependently increased compared to control. However, TNFalpha blockade (neutralizing antibodies against human TNFalpha, 25 microg/ml) significantly reduced the activity of MMP-2 and markers of apoptosis induced by TNFalpha. Interestingly, MMP-2 antibody (30 microg/ml), or the MMP-2 inhibitors Doxycycline (Dox, 1-50 micromol/l) or GM6001 (GM, 10 micromol/l), prior to TNFalpha insult, decreased myocardial MMP-2 activity and reduced the percent of TUNEL-positive myocytes and DNA fragmentation. Moreover, MMP-2 inhibition reduced Bax expression and caspase3 activity, as well as increasing Bcl2 expression. MMP-2 inhibition was associated with decreased cardiac MMP-2 activity and decreased myocardial apoptosis induced by TNFalpha. These results suggest that MMP-2 contributes to TNFalpha-induced apoptosis in cultured rat cardiac myocytes.     

Effect of in vitro ketoconazole on steroid production in rat testis

In an attempt to confirm where in the testosterone (T) biosynthetic pathway of the rat testis ketoconazole (KTZ) inhibits T production, rat testicular mince was incubated with either 10 micrograms/ml or 100 micrograms/ml KTZ in the presence and absence of hCG (1 IU), and intratesticular pregnenolone (delta 5P), progesterone (P), 17-alpha-hydroxyprogesterone (17 alpha-HP), androstenedione (A) and testosterone (T) were assayed. In the absence of hCG, 10 micrograms/ml KTZ was sufficient to reduce intratesticular T by 80%. At this concentration of KTZ, intratesticular 17 alpha-HP (ng/g testis, mean +/- SEM) increased from 0.3 +/- 0.1 to 1.3 +/- 0.2 (p less than 0.0025), whereas intratesticular A decreased from 84 +/- 7 to 17 +/- 1 (p less than 0.005). KTZ did not inhibit the conversion of P to 17 alpha-HP. From these data it was concluded that KTZ has its inhibitory effect on testosterone biosynthesis in the rat testis primarily at the step catalyzed by the 17,20 desmolase enzyme.     

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.     

Effect of myocardial ischemia on uridine incorporation and histone acetylation

The influence of transient myocardial ischemia on recovery uridine incorporation into RNA and histone acetylation was investigated in an isolated perfused rat heart. Hemodynamically, hearts recovered from 15 min of ischemic arrest and were stable for at least 60 min of perfusion. Uridine incorporation was reduced (P less than 0.05) in ischemic hearts by 24 and 26% after 30 and 60 min of recovery perfusion. The incorporation of uridine into RNA from purified myocytes was decreased by 50% in the ischemic muscle cells. The covalent acetylation of total nucleohistones was diminished by 37%. Histone fractionation by urea polyacrylamide gel electrophoresis clearly indicated that histones H3 and H4 preferentially incorporated less acetate during ischemic recovery. However, histone acetylation for proteins H2A + H2B was not effected. These data suggest that a brief period of ischemia disrupts nucleotide incorporation during the recovery phase, with marked decrease associated with the muscle cell. The similar change in histone acetylation indicates a possible link between nucleoproteins and chromatin function during ischemic insult to the heart.     

Progesterone is a mediator in the ovulatory process of the in vitro-perfused rat ovary

The role of steroids in the ovulatory process of the rat was explored in an in vitro perfusion system. Immature rat ovaries were primed with pregnant mare's serum gonadotropin (20 IU) and perfused in a recirculating perfusion system for up to 20 h. Unstimulated ovaries did not ovulate whereas the addition of luteinizing hormone (LH; 0.1 micrograms/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) resulted in 13.6 +/- 1.0 ovulations per treated ovary. Addition of an inhibitor of 3 beta-hydroxysteroid dehydrogenase (Compound A; 10 micrograms/ml) significantly (p less than 0.01) decreased the number of ovulations after LH plus IBMX stimulation (1.6 +/- 0.8 ovulations per treated ovary). This inhibition was reversed by the addition of progesterone, with 6.6 +/- 2.1 ovulations at approximately 100 ng/ml progesterone in the perfusion medium and 15.2 +/- 3.4 ovulations at approximately 3000 ng/ml progesterone. The addition of testosterone (10 micrograms/ml) did not reverse the inhibition of ovulations by Compound A. High levels of progesterone in the perfusion medium (greater than 3000 ng/ml) did not significantly (p greater than 0.05) increase the number of ovulations after stimulation with LH plus IBMX (20.2 +/- 4.8 ovulations), and progesterone (greater than 3000 ng/ml) was not by itself able to induce ovulations. Addition of LH plus IBMX resulted in a marked increase in the levels of progesterone, testosterone, and estradiol in the perfusion medium. The production of these steroids was almost completely inhibited by the addition of Compound A, and the levels of testosterone and estradiol were restored by the addition of high concentrations of progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo inhibition of thromboxane synthetase in infarcted canine myocardium

The in vivo effectiveness of the thromboxane synthetase inhibitor OKY-1581 was tested in normal and infarcted canine myocardium. A rapid in vitro assay was developed which permits an accurate assessment of the status of the tissue thromboxane synthetase at the time of sacrifice. Reperfused infarcts were created by two hours of coronary artery occlusion followed by release of occlusion and three days of recovery. OKY-1581 was infused at 100 micrograms/kg/min for 15 minutes, a dose previously found to cause an 85% inhibition of canine platelet thromboxane synthetase in vivo. The heart was rapidly excised and transmural tissue plugs of infarcted and normal areas were obtained. These were incubated for 5 minutes with prostaglandin endoperoxide (PGH2) in phosphate buffer. Thromboxane production was inhibited from 16 +/- 1 ng TxB2 per tissue plug to 5 +/- 1 in normal myocardium and from 27 +/- 5 to 6 +/- 1 in infarcted areas of myocardium. Control incubations showed no further inhibition with the in vitro addition of 20 micrograms/ml OKY-1581, confirming the completeness of in vivo inhibition. Thus significant inhibition of thromboxane synthetase by intravenous OKY-1581 occurs even in a reperfused zone of infarction.     

Direct protective action of epidermal growth factor on isolated gastric mucosal surface epithelial cells.

Epidermal growth factor (EGF) protects gastric mucosa against acute injury produced by a variety of damaging agents, but the mechanism of its protective action is not clear. Since the surface epithelial cells (SEC) are important component of gastric mucosal defence, we studied whether EGF may directly protect isolated gastric SEC against ethanol injury in vitro, in condition independent of systemic factors and whether endogenous prostaglandins may play a role in EGF's protective action. The isolated SEC from rat gastric mucosa were preincubated in medium only, or medium containing 0.0001-10.0 micrograms/ml of h-rEGF for 15 minutes, and incubated with 8% ethanol for 1 hour. In another study the above experiment was repeated but cells were pretreated with 10(-4) or 10(-5) M indomethacin before EGF treatment. The cell viability was assessed by fast green exclusion test. Incubation of SEC with 8% ethanol significantly reduced SEC cell viability to 50 +/- 2%: EGF 0.1 or 1.0 microgram/ml significantly reduced ethanol induced damage (cell viability 59 +/- 3 and 62 +/- 3% respectively). Pretreatment with 10(-4) M indomethacin (the dose which does not affect SEC viability but inhibit PGE2 and PGI2 generation), significantly reduced protective action of EGF against 8% ethanol injury. EGF 1.0 and 10.0 micrograms/ml alone without ethanol increased PGE2 and 6 keto PGF1 alpha generation by SEC. These studies demonstrated: 1) EGF is able to protect gastric surface epithelial cells directly without mediation by systemic factors. 2) EGF induced protection of SEC may in part be mediated by prostaglandins.     

Stimulation of HCO3- secretion by the prostaglandin E2 analog enprostil: studies in human stomach and rat duodenum

This study investigated the action of enprostil, a synthetic analog of PGE2, on gastric HCO3- secretion in humans and on duodenal HCO3- secretion in the anesthetized rat. A previously validated 2-component model was used to calculate gastric HCO3- and H+ secretion in 10 human subjects. Compared to placebo, a single 70 micrograms oral dose of enprostil increased basal gastric HCO3- secretion from 1810 +/- 340 to 3190 +/- 890 mumol/hr (P less than 0.05). In addition, enprostil reduced basal gastric H+ secretion from 5240 +/- 1140 to 1680 +/- 530 mumol/hr (P less than 0.02). Enprostil also increased HCO3- secretion and reduced H+ secretion during intravenous pentagastrin infusion. In the rat, duodenal HCO3- secretion was measured by direct titration in situ using perfused segments of duodenum just distal to the Brunner gland area and devoid of pancreatic and biliary secretions. Addition of enprostil (10 micrograms/ml) to the duodenal bathing solution increased duodenal HCO3- secretion from 6.3 +/- 1.3 to 15.1 +/- 2.0 mumol/cm X hr (P less than 0.01, n = 6). The stimulatory action of enprostil on duodenal HCO3- secretion at 10 micrograms/ml was comparable in magnitude and duration to that of 10 micrograms/ml natural PGE2. In summary, the PGE2 analog enprostil stimulated gastroduodenal HCO3- secretion, effects which may be beneficial in protection of the gastroduodenal mucosa against luminal acid.     

Activation of corticotropin releasing factor receptor type 2 in the heart by corticotropin releasing factor offers cytoprotection against ischemic injury via PKA and PKC dependent signaling

Corticotrophin-releasing factor receptor 2β (CRFR2β) is expressed in the myocardium. In the present study we explore whether acute treatment with the neuropeptide corticotrophin-releasing factor (CRF) could induce cytoprotection against a lethal ischemic insult in the heart (isolated murine neonatal cardiac myocytes and the isolated Langendorff perfused rat heart) by activating CRFR2. In vitro, CRF offered cytoprotection when added prior to lethal simulated ischemic stress by reducing apoptotic and necrotic cell death. Ex vivo, CRF significantly reduced infarct size from 52.1±3.1% in control hearts to 35.3±3.1% (P<0.001) when administered prior to a lethal ischemic insult. The CRF peptide did not confer cytoprotection when administered at the point of hypoxic reoxygenation or ischemic reperfusion. The acute effects of CRF treatment are mediated by CRF receptor type 2 (CRFR2) since the cardioprotection ex vivo was inhibited by the CRFR2 antagonist astressin-2B. Inhibition of the mitogen activated protein kinase-ERK1/2 by PD98059 failed to inhibit the effect of CRF. However, both protein kinase A and protein kinase C inhibitors abrogated CRF-mediated protection both ex vivo and in vitro. These data suggest that the CRF peptide reduces both apoptotic and necrotic cell death in cardiac myocytes subjected to lethal ischemic induced stress through activation of PKA and PKC dependent signaling pathways downstream of CRFR2.     

Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration

Peptide nucleic acids (PNAs) are DNA analogs that hybridize to complementary nucleic sequences with high affinity and stability. In our previous work, we showed that a PNA complementary to a 12-base pair (bp) sequence of the coding region of the rat neurotensin receptor (rNTR1) mRNA is effective in significantly blocking a rat's central responses to neurotensin (NT), even when the PNA is injected intraperitoneally (i.p.). Using a novel gel shift detection assay to detect PNA, we have now used this same PNA sequence to derive its pharmacokinetic variables and its tissue distribution in the rat. The PNA has a distribution half-life of 3 +/- 3 minutes and an elimination half-life of 17 +/- 3 minutes. The total plasma clearance and volume of distribution of this PNA were 3.4 +/- 0.9 ml/min x kg and 60 +/- 30 ml/kg. Two hours after dosing, the PNA was found at detectable but low levels in all organs examined-in order of decreasing concentration: kidney, liver, heart, brain, and spleen. Approximately 90% of the PNA dose was recovered as unchanged parent compound in the urine 24 hours after administration.     

Tissue and plasma levels of endothelin in free flaps

The goal of the study was to assess whether endothelin-1 levels are increased in tissue and plasma in free flaps. To assess this hypothesis, blood samples were taken from the general circulation before and after reperfusion and from the flap after reperfusion in 20 patients undergoing breast reconstruction with free transverse rectus abdominis musculocutaneous or deep inferior epigastric perforator flaps. Tissue samples were also taken from the flap before and after the period of ischemia. Peripheral blood samples of 10 ml each were taken before the vessels were clamped and at 10 minutes and 1 hour after the flap was recharged. The flap vein was catheterized with a smooth catheter to avoid endothelial trauma, and ischemic blood from the flap was obtained immediately after the artery was unclamped and 10 minutes later. Two skin samples of 2 cm each were taken: one after dissection of the flap before division of the vessels and one after reanastomosis of the veins (one or two veins). Statistical analyses were performed with the (nonparametric) Wilcoxon signed rank test. Flap ischemia time, from vessel division to the completion of the arterial anastomosis, ranged from 35 to 120 minutes (mean, 48 minutes). The plasma endothelin-1 level extracted from the flap was 4.34 +/- 0.85 pg/ml, significantly higher than baseline, 3.87 +/- 0.81 pg/ml (p < 0.0001). There was a small increase, 4.5 +/- 1.03 pg/ml (p = NS), 10 minutes after reperfusion. The peripheral level after venous anastomosis was 3.78 +/- 0.79 pg/ml, not significantly different from the peripheral plasma level, before the flap was raised. The peripheral plasma level 1 hour after reperfusion was 3.83 +/- 0.8 pg/ml, with no difference from baseline. The tissue level of endothelin-1 before clamping was 3.8 +/- 0.8 pg/mg and in postischemic tissue, 5.2 +/- 0.6 pg/mg, a statistically significant increase. The authors concluded that endothelin-1 levels are elevated in free flaps. This could be an explanation for vasospasm and may lead to therapy directed against the no-reflow phenomenon.     

Protection of the reperfused heart by L-propionylcarnitine.

The effects of L-propionylcarnitine on mechanical function, creatine phosphate and ATP content, and lactate dehydrogenase leakage were studied in isolated perfused rat hearts exposed to global no-flow ischemia for 30 min followed by reperfusion for 20 min. Five and 10 mM L-propionylcarnitine resulted in a 100% recovery of left ventricular-developed pressure, whereas the recovery was only 40% in the hearts perfused without this agent. Ischemia-reperfusion caused a 85% loss of creatine phosphate and a 77% loss of ATP, which was prevented by 10 mM L-propionylcarnitine. Five millimolar L-propionylcarnitine protected the heart from the loss of creatine phosphate but not from the loss of ATP. Ten millimolar L-propionylcarnitine failed to improve the postischemic left ventricular-developed pressure, when it was added to the perfusate only after ischemia. L-propionylcarnitine alleviated the decrease of coronary flow in the reperfused hearts. Lactate dehydrogenase leakage was aggravated in the beginning of the reperfusion period by 10 mM L-propionylcarnitine. This adverse effect was, however, transient. L-Propionylcarnitine provides protection for the postischemic reperfused heart in a dose-dependent manner. The optimal time for administration is before the ischemic insult. High doses of this compound may perturb cell membrane integrity. Moreover, the present data point to an intracellular, metabolic, and perhaps anaplerotic mechanism of action of L-propionylcarnitine in cardiac ischemia-reperfusion injury.     

An inhibitor of p38 mitogen-activated protein kinase protects neonatal cardiac myocytes from ischemia

Cellular ischemia results in activation of a number of kinases, including p38 mitogen-activated protein kinase (MAPK); however, it is not yet clear whether p38 MAPK activation plays a role in cellular damage or is part of a protective response against ischemia. We have developed a model to study ischemia in cultured neonatal rat cardiac myocytes. In this model, two distinct phases of p38 MAPK activation were observed during ischemia. The first phase began within 10 min and lasted less than 1 h, and the second began after 2 h and lasted throughout the ischemic period. Similar to previous studies using in vivo models, the nonspecific activator of p38 MAPK and c-Jun NH2-terminal kinase, anisomycin, protected cardiac myocytes from ischemic injury, decreasing the release of cytosolic lactate dehydrogenase by approximately 25%. We demonstrated, however, that a selective inhibitor of p38 MAPK, SB 203580, also protected cardiac myocytes against extended ischemia in a dose-dependent manner. The protective effect was seen even when the inhibitor was present during only the second, sustained phase of p38 MAPK activation. We found that ischemia induced apoptosis in neonatal rat cardiac myocytes and that SB 203580 reduced activation of caspase-3, a key event in apoptosis. These results suggest that p38 MAPK induces apoptosis during ischemia in cardiac myocytes and that selective inhibition of p38 MAPK could be developed as a potential therapy for ischemic heart disease.     

Cardioprotection initiated by reactive oxygen species is dependent on activation of PKCepsilon

To examine whether cardioprotection initiated by reactive oxygen species (ROS) is dependent on protein kinase Cepsilon (PKCepsilon), isolated buffer-perfused mouse hearts were randomized to four groups: 1) antimycin A (AA) (0.1 microg/ml) for 3 min followed by 10 min washout and then 30 min global ischemia (I) and 2 h reperfusion (R); 2) controls of I/R alone; 3) AA bracketed with 13 min of N-2-mercaptopropionyl- glycine (MPG) followed by I/R; and 4) MPG (200 microM) alone, followed by I/R. Isolated adult rat ventricular myocytes (ARVM) were exposed to AA (0.1 microg/ml), and lucigenin was used to measure ROS production. Murine hearts and ARVM were exposed to AA (0.1 microg/ml) with or without MPG, and PKCepsilon translocation was measured by cell fractionation and subsequent Western blot analysis. Finally, the dependence of AA protection on PKCepsilon was determined by the use of knockout mice (-/-) lacking PKCepsilon. AA exposure caused ROS production, which was abolished by the mitochondrial uncoupler mesoxalonitrile 4-trifluoromethoxyphenylhydrazone. In addition, AA significantly reduced the percent infarction-left ventricular volume compared with control I/R (26 +/- 4 vs. 43 +/- 2%; P < 0.05). Bracketing AA with MPG caused a loss of protection (52 +/- 7 vs. 26 +/- 4%; P < 0.05). AA caused PKCepsilon translocation only in the absence of MPG, and protection was lost on the pkcepsilon(-/-) background (38 +/- 3 vs. 15 +/- 4%; P < 0.001). AA causes ROS production, on which protection and PKCepsilon translocation depend. In addition, protection is absent in PKCepsilon null hearts. Our results imply that, in common with ischemic preconditioning, PKCepsilon is crucial to ROS-mediated protection.     

Early inhibition of the Na+/K+-ATPase ion pump during acetaminophen-induced hepatotoxicity in rat

The status of Na+ regulation was examined during early stages of alkylation insult to rat liver. Na+/K+-ATPase activity in plasma membranes declined by 52% within 3 hr of treatment with 850 mg/kg acetaminophen. This loss preceded the release of alanine aminotransferase (2880 +/- 1550 U/ml) and necrosis (2+) seen at 24 hr. Activities of 5'-nucleotidase and Mg2+-ATPase and recovery of plasma membranes were comparatively unchanged at 3 hr. Because damage to Na+/K+-ATPase appeared early in the pathogenesis of acetaminophen hepatotoxicity, loss of hepatocellular Na+ regulation could represent one of the critical molecular consequences of lethal alkylation by acetaminophen.     

Inhibition of ischemia/reperfusion-induced damage by dexamethasone in isolated working rat hearts: the role of cytochrome c release

We investigated the contribution of dexamethasone treatment on the recovery of postischemic cardiac function and the development of reperfusion-induced arrhythmias in ischemic/reperfused isolated rat hearts. Rats were treated with 2 mg/kg of intraperitoneal injection of dexamethasone, and 24 hours later, hearts were isolated according to the 'working' mode, perfused, and subjected to 30 min global ischemia followed by 120 min reperfusion. Cardiac function including heart rate, coronary flow, aortic flow, and left ventricular developed pressure were recorded. After 60 min and 120 min reperfusion, 2 mg/kg of dexamethasone significantly improved the postischemic recovery of aortic flow and left ventricular developed pressure from their control values of 10.7 +/- 0.3 ml/min and 10.5 +/- 0.3 kPa to 22.2 +/- 0.3 ml/min (p < 0.05) and 14.3 +/- 0.5 kPa (p < 0.05), 19.3 +/- 0.3 ml/min (p < 0.05) and 12.3 +/- 0.5 kPa (p < 0.05), respectively. Heart rate and coronary flow did not show a significant change in postischemic recovery after 60 or 120 min reperfusion. In rats treated with 0.5 mg/kg of actinomycin D injected i.v., one hour before the dexamethasone injection, suppressed the dexamethasone-induced cardiac protection. Electrocardiograms were monitored to determine the incidence of reperfusion-induced ventricular fibrillation. Dexamethasone pretreatment significantly reduces the occurrence of ventricular fibrillation. Cytochrome c release was also observed in the cytoplasm. The results suggest that the inhibition of cytochrome c release is involved in the dexamethasone-induced cardiac protection.     

Effects of ethinyl estradiol on isoproterenol-induced death in ventricular fibrillation in DOCA-salt pretreated male and female rats

To assess the effects of ethinyl estradiol on the incidence of death in ventricular fibrillation induced by isoproterenol in DOCA-salt pretreated rats we implanted male and female rats simultaneously with a 20 mg DOCA pellet and pellets containing either ethinyl estradiol or vehicle (wax). Rats drank saline after implantation. After 6 days rats were challenged with a single, sc dose of 150 micrograms of isoproterenol. The average daily dose of estradiol per rat was estimated on the basis of the quantity of pellet lost during 6 days. In male rats the average daily dose of 61.2 +/- 20.2 micrograms/rat of ethinyl estradiol decreased the incidence of mortality by 80%, from 73.3% (11/15) in vehicle treated to 13.3% (2/15) in estradiol treated rats. Death occurred within 19.2 +/- 8.0 minutes from the injection of isoproterenol and was due to ventricular fibrillation. Serum levels of magnesium and potassium were comparable in the two groups both before and after isoproterenol. Isoproterenol induced death in 9 of 11 DOCA-salt pretreated, ovariectomized rats within 22.3 +/- 9.8 minutes. Only 3 of 11 DOCA-salt ovariectomized rats receiving the average daily dose of 28.4 +/- 12.1 micrograms/rat of ethinyl estradiol died. None of 10 ovariectomized untreated rats died from isoproterenol challenge. Serum levels of magnesium and potassium were comparable in the estradiol and vehicle treated groups. The average daily dose of 2.8 +/- 0.42 micrograms/rat of ethinyl estradiol elicited uterine growth but did not influence the incidence of mortality, since 9 out of 16 and 10 out of 16 rats died following isoproterenol in vehicle and estradiol treated DOCA-salt ovariectomized rats. We conclude that only pharmacological doses of estradiol exert protective effects against DOCA-salt induced myocardial sensitization to isoproterenol and that this protection is not associated with relevant changes in serum potassium or magnesium.     

Metformin improves cardiac functional recovery after ischemia in rats.

The biguanide, metformin, is widely used for the treatment of type 2 diabetes mellitus. In the recently published United Kingdom Prospective Diabetes Study (UKPDS), it was shown that the use of metformin was associated with a reduction of macrovascular complications compared to other blood glucose-lowering strategies. The present study was aimed at determining whether metformin has direct beneficial effects on the heart. We tested the effects of metformin on cardiac functional recovery after a mild ischemic incident (stunning) in our isolated, erythrocyte perfused, rat working-heart model. Three groups were tested: vehicle, 50 and 500 micromol/l metformin (total n = 6). In diabetic rats, a concentration of 50 microM has been shown to reduce the blood glucose concentration. Slight metformin-induced increases in coronary blood flow during normoxia (pre-ischemically) and during reperfusion (post-ischemically) were observed and compared to vehicle (p < 0.05). Both metformin concentrations significantly reduced cardiac functional loss induced by the 12-min global ischemic incident compared with vehicle (3.4 +/- 1.0 % and 3.5 +/- 0.6 % loss during metformin versus 10.7 +/- 0.8 % during vehicle, p < 0.001). This study clearly shows that metformin acutely improves cardiac function after a mild ischemic incident (stunning) in rats.