Postischemic cardiac recovery in heme oxygenase-1 transgenic ischemic/reperfused mouse myocardium.

Heme oxygenase-1 (HO-1) transgenic mice (Tg) were created using a rat HO-1 genomic transgene. Transgene expression was detected by RT-PCR and Western blots in the left ventricle (LV), right ventricle (RV) and septum (S) in mouse hearts, and its function was demonstrated by the elevated HO enzyme activity. Tg and non-transgenic (NTg) mouse hearts were isolated and subjected to ischemia/reperfusion. Significant post-ischemic recovery in coronary flow (CF), aortic flow (AF), aortic pressure (AOP) and first derivative of AOP (AOPdp/dt) were detected in the HO-1 Tg group compared to the NTg values. In HO-1 Tg hearts treated with 50 µmol/kg of tin protoporphyrin IX (SnPPIX), an HO enzyme inhibitor, abolished the post-ischemic cardiac recovery. HO-1 related carbon monoxide (CO) production was detected in NTg, HO-1 Tg and HO-1 Tg + SnPPIX treated groups, and a substantial increase in CO production was observed in the HO-1 Tg hearts subjected to ischemia/reperfusion. Moreover, in ischemia/reperfusion-induced tissue Na(+) and Ca(2+) gains were reduced in HO-1 Tg group in comparison with the NTg and HO-1 Tg + SnPPIX treated groups; furthermore K(+) loss was reduced in the HO-1 Tg group. The infarct size was markedly reduced from its NTg control value of 37 ± 4% to 20 ± 6% (P < 0.05) in the HO-1 Tg group, and was increased to 47 ± 5% (P < 0.05) in the HO-1 knockout (KO) hearts. Parallel to the infarct size reduction, the incidence of total and sustained ventricular fibrillation were also reduced from their NTg control values of 92% and 83% to 25% (P < 0.05) and 8% (P < 0.05) in the HO-1 Tg group, and were increased to 100% and 100% in HO-1 KO(-/-) hearts. Immunohistochemical staining of HO-1 was intensified in HO-1 Tg compared to the NTg myocardium. Thus, the HO-1 Tg mouse model suggests a valuable therapeutic approach in the treatment of ischemic myocardium.

Isolation and analysis of bioactive constituents of sour cherry (Prunus cerasus) seed kernel: an emerging functional food.

A plant-based diet reduces the risk for the development of several chronic diseases, such as ischemic heart disease or cancer due to natural compounds found in plants. Numerous cereals, berries, fruits, and vegetables, including sour cherry (Prunus cerasus), which is a favored fruit worldwide, contain biological active components. The antioxidant components of the sour cherry seed kernel have not been investigated until now. The aim of our study was to isolate and analyze the bioactive constituents of sour cherry seed kernel. We separated the oil fraction of the kernel; then the remaining solid fraction was dried, and the oil-free kernel extract was further analyzed. Our results show that sour cherry seed kernel oil contains vegetable oils including unsaturated fatty acids, oleic acids, alpha-tocopherol, tocotrienols, and tocopherol-like components. The components of the solid fraction include various bioactive structures such as polyphenols, flavonoids, vegetable acids, and pro- and anthocyanidins, which could have useful therapeutic effects in the prevention of various vascular diseases.

Protective mechanisms of resveratrol against ischemia-reperfusion-induced damage in hearts obtained from Zucker obese rats: the role of GLUT-4 and endothelin.

The resveratrol-induced cardiac protection was studied in Zucker obese rats. Rats were divided into five groups: group 1, lean control; group 2, obese control (OC); group 3, obese rats treated orally with 5 mg kg(-1) day(-1) of resveratrol (OR) for 2 wk; group 4, obese rats received 10% glucose solution ad libitum for 3 wk (OG); and group 5, obese rats received 10% glucose for 3 wk and resveratrol (OGR) during the 2nd and 3rd wk. Body weight, serum glucose, and insulin were measured, and then hearts were isolated and subjected to 30 min of ischemia followed by 120 min of reperfusion. Heart rate, coronary flow, aortic flow, developed pressure, the incidence of reperfusion-induced ventricular fibrillation, and infarct size were measured. Resveratrol reduced body weight and serum glucose in the OR compared with the OC values (414 +/- 10 g and 7.08 +/- 0.41 mmol/l, respectively, to 378 +/- 12 g and 6.11 +/- 0.44 mmol/l), but insulin levels were unchanged. The same results were obtained for the OG vs. OGR group. Resveratrol improved postischemic cardiac function in the presence or absence of glucose intake compared with the resveratrol-free group. The incidence of ventricular fibrillation and infarct size was reduced by 83 and 20% in the OR group, and 67 and 16% in the OGR group, compared with the OC and OG groups, respectively. Resveratrol increased GLUT-4 expression and reduced endothelin expression and cardiac apoptosis in ischemic-reperfused hearts in the presence or absence of glucose intake. Thus the protective effect of resveratrol could be related to its direct effects on the heart.

Influence of hyperthyroidism on the effect of adenosine transport blockade assessed by a novel method in guinea pig atria.

The aim of the present study was to investigate the effect of hyperthyroidism on the trans-sarcolemmal adenosine (Ado) flux via equilibrative and nitrobenzylthioinosine (NBTI)-sensitive nucleoside transporters (ENT1) in guinea pig atria, by assessing the change in the Ado concentration of the interstitial fluid ([Ado]ISF) under nucleoside transport blockade with NBTI. For the assessment, we applied our novel method, which estimates the change in [Ado]ISF utilizing the altered inotropic response to N6-cyclopentyladenosine (CPA), a relative stable selective agonist of A1 Ado receptors, by providing a relative index, the equivalent concentration of CPA. Our results show an interstitial Ado accumulation upon ENT1 blockade, which was more extensive in the hyperthyroid samples (CPA concentrations equieffective with the surplus [Ado]ISF were two to three times higher in hyperthyroid atria than in euthyroid ones, with regard to the negative inotropic effect of CPA and Ado). This suggests an enhanced Ado influx via ENT1 in hyperthyroid atria. It is concluded that hyperthyroidism does not alter the prevailing direction of the Ado transport, moreover intensifies the Ado influx in the guinea pig atrium.

Beneficial effects of carbon monoxide-releasing molecules on post-ischemic myocardial recovery.

There is increasing evidence corroborating a protective role of carbon monoxide releasing molecules (CORMs) in injured tissues. Carbon monoxide (CO) carriers have been recently developed as a pharmacological tool to simulate the effect of heme oxygenase-1-derived CO. The effects of CORM-3, a water-soluble CO releaser, on the incidence of reperfusion-induced ventricular fibrillation (VF) and tachycardia (VT) were studied in isolated rat hearts. Hearts were treated with different doses of CORM-3 before the induction of 30 min global ischemia followed by 120 min reperfusion. We found that at concentrations of 25 microM and 50 microM of CORM-3 promoted a significant reduction in the incidence of VF and VT. Thus, the incidence of VF was reduced by 67% (p<0.05) and 92% (p<0.05) with 25 microM and 50 microM of CORM-3, respectively. The protective effect of CORM-3 on the incidence of VT followed the same pattern. The antiarrhythmic protection was associated with a marked attenuation in infarct size, significant decreases in cellular Na(+) and Ca(2+) gains and K(+) loss. Consequently, the recovery of post-ischemic function was significantly improved. In conclusion, CORM-3 exerts beneficial effects against ischemia/reperfusion-induced injury through its abilities to release CO which mediates a cardioprotective action by regulating tissue Na(+), K(+), and Ca(2+) levels.

Cardioprotective mechanisms of Prunus cerasus (sour cherry) seed extract against ischemia-reperfusion-induced damage in isolated rat hearts.

The effects of kernel extract obtained from sour cherry (Prunus cerasus) seed on the postischemic cardiac recovery were studied in isolated working rat hearts. Rats were treated with various daily doses of the extract for 14 days, and hearts were then isolated and subjected to 30 min of global ischemia followed by 120 min of reperfusion. The incidence of ventricular fibrillation (VF) and tachycardia (VT) fell from their control values of 92% and 100% to 50% (not significant) and 58% (not significant), 17% (P<0.05), and 25% (P<0.05) with the doses of 10 mg/kg and 30 mg/kg of the extract, respectively. Lower concentrations of the extract (1 and 5 mg/kg) failed to significantly reduce the incidence of VF and VT during reperfusion. Sour cherry seed kernel extract (10 and 30 mg/kg) significantly improved the postischemic recovery of cardiac function (coronary flow, aortic flow, and left ventricular developed pressure) during reperfusion. We have also demonstrated that the extract-induced protection in cardiac function significantly reflected in a reduction of infarct size. Immunohistochemistry indicates that a reduction in caspase-3 activity and apoptotic cells by the extract, beside other potential action mechanisms of proanthocyanidin, trans-resveratrol, and flavonoid components of the extract, could be responsible for the cardioprotection in ischemic-reperfused myocardium.

Heme oxygenase-1-related carbon monoxide production and ventricular fibrillation in isolated ischemic/reperfused mouse myocardium.

Heme oxygenase-1 (HO-1)-dependent carbon monoxide (CO) production related to reperfusion-induced ventricular fibrillation (VF) was studied in HO-1 wild-type (+/+), heterozygous (+/-), and homozygous (-/-) isolated ischemic/reperfused mouse heart. In HO-1 homozygous myocardium, under aerobic conditions, HO-1 enzyme activity, HO-1 mRNA, and protein expression were not detected in comparison with aerobically perfused wild-type and heterozygous myocardium. In wild-type, HO-1 hetero- and homozygous hearts subjected to 20 min ischemia followed by 2 h of reperfusion, the expression of HO-1 mRNA, protein, and HO-1 enzyme activity was detected in various degrees. A reduction in the expression of HO-1 mRNA, protein, and enzyme activity in fibrillated wild-type and heterozygous myocardium was observed. In reperfused/nonfibrillated wild-type and heterozygous hearts, a reduction in HO-1 mRNA, protein expression, and HO-1 enzyme activity was not observed, indicating that changes in HO-1 mRNA, protein, and enzyme activity could be related to the development of VF. These changes were reflected in the HO-1-related endogenous CO production measured by gas chromatography. In HO-1 knockout ischemic/reperfused myocardium, all hearts showed VF, and no detection in HO-1 mRNA, protein, and enzyme activity was observed. Thus, interventions that are able to increase endogenous CO may prevent the development of VF.

The role of heme oxygenase-related carbon monoxide and ventricular fibrillation in ischemic/reperfused hearts.

Reperfusion-induced ventricular fibrillation (VF) and heme oxygenase (HO)-related carbon monoxide (CO) production in isolated ischemic/reperfused rat hearts were studied by gas chromatography. Hearts were subjected to 30 min ischemia followed by 2 h reperfusion, and the expression of HO-1 mRNA (about 4-fold) was observed in ischemic/reperfused-nonfibrillated hearts. In fibrillated hearts, the reduction (about 75%) in HO-1 mRNA expression was detected. These changes in HO-1 mRNA expression were reflected in tissue CO production. Thus, in the absence of VF, CO production was increased about 3.5-fold, while in the presence of VF, CO production was under the detectable level in comparison with the control group. Our results suggest that the stimulation of HO-1 mRNA expression may lead to the prevention of reperfusion VF via an increase in endogenous CO production. To prove this, hearts were treated with 1 microM of N-tert-butyl-alpha-phenylnitrone (PBN) as an inducer of HO-1. PBN treatment resulted in about 20 times increase in HO-1 mRNA expression, and even a higher production rate in endogenous CO. HO protein level and enzyme activity followed the same pattern, as it was observed in HO-1 mRNA expression, in fibrillated and nonfibrillated myocardium. Five mM/l of zinc-protoporphyrin IX (ZnPPIX) significantly blocked HO enzyme activity and increased the incidence of VF, therefore the application of ZnPPIX led to a significant reduction in HO-1 mRNA and protein expression. Our data provide direct evidence of an inverse relationship between the development of reperfusion-induced VF and endogenous CO production. Thus, interventions that are able to increase tissue CO content may prevent the development of reperfusion-induced VF.

Mitochondrial gene expression and ventricular fibrillation in ischemic/reperfused nondiabetic and diabetic myocardium.

We investigated the mitochondrial gene expression related to cardiac function and ventricular fibrillation (VF) in ischemic/reperfused nondiabetic and diabetic myocardium. To identify potentially more specific gene responses we performed subtractive screening, Northern blotting, and reverse transcription-polymerase chain reaction (RT-PCR) of mitochondrial genes expressed after 30 min ischemia followed by 120 min reperfusion in isolated rat hearts that showed VF or did not show VF. Cytochrome oxidase B subunit III (COXBIII) and ATP synthase subunit 6, studied and selected out of 40 mitochondrial genes by subtractive screening, showed an expression after 30 min ischemia (no VF was recorded) in both nondiabetic and diabetic subjects. Upon reperfusion, the down-regulation of these genes was only observed in fibrillated hearts. Such a reduction in signal intensity was not seen in nonfibrillated myocardium. In additional studies, nondiabetic and diabetic hearts, without the ischemia/reperfusion protocol, were subjected to electrical fibrillation, and a significant reduction in COXBIII and ATPS6 mRNA signal intensity was observed indicating that VF contributes to the down-regulation of these genes. Cardiac function (heart rate, coronary flow, aortic flow, left ventricular developed pressure) showed no correlation between the up- and down-regulation of these mitochondrial genes in both nondiabetic and diabetic ischemic/reperfused myocardium. Our data suggest that COXBIII and ATPS6 may play a critical role in arrhythmogenesis, and the stimulation of COXBIII and ATPS6 mRNA expression may prevent the development of VF in both nondiabetic and diabetic ischemic/reperfused myocardium.