Ischemic preconditioning enhances scavenging activity of reactive oxygen species and diminishes transmural difference of infarct size

Abstract

Reactive oxygen species (ROS) enhance myocardial ischemia-reperfusion (I/R) injury. Ischemic preconditioning (PC) provides potent cardioprotective effects in I/R. However, it has not been elucidated whether PC diminishes ROS stress in I/R and whether PC protects the myocardium from ROS stress transmurally and homogeneously. Isolated rabbit hearts perfused with Krebs-Henseleit buffer underwent 30 min of ischemia and 60 min of reperfusion. Hemodynamic changes and myocardial damage extent were analyzed in four groups. The control group underwent I/R alone. The H2O2 group underwent I/R with H2O2 infusion (50 microM) in the first minute of reperfusion to enhance oxidative stress. The PC and H2O2+PC groups underwent 5 min of PC before control and H2O2 protocols, respectively. Extracted myocardial DNA was analyzed for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative DNA damage, with the use of the HPLC-electrochemical detection method. Glutathione peroxidase (GPX) activity and the reduced form of GSH were measured by spectrophotometric assays. The myocardial infarct size was significantly reduced in the PC group (19 +/- 2%) compared with the control group (37 +/- 4%; P < 0.05), particularly in the subendocardium. H2O2 transmurally increased the infarct size by 59 +/- 4% (P < 0.05), which was significantly diminished in the H2O2+PC group (31 +/- 4%; P < 0.01). The GSH levels, but not GPX activity, were well preserved transmurally in protocols with PC. The 8-OHdG levels were significantly decreased in PC and were significantly enhanced in H2O2 (P < 0.01). These changes in oxidative DNA damage were effectively diminished by PC. In conclusion, PC enhanced the scavenging activity of GSH against ROS transmurally, reduced myocardial damage, particularly in the subendocardium, and diminished the transmural difference in myocardial infarct size.

Oral Pretreatment with Ebselen Enhances Heat Shock Protein 72 Expression and Reduces Myocardial Infarct Size

“…The thorax was opened and the heart was quickly removed. Hearts were retrogradely perfused using a Langendorff preparation (14) with Krebs-Henseleit solution, gassed with 95% O2 and 5% CO2 and paced at 200 beats/min. Left ventricular (LV) pressure, LV systolic pressure (LVSP), LV end diastolic pressure (LVEDP) and LV developed pressure (LVDP: the difference of LVSP and LVEDP) were continuously monitored using an intraventricular latex balloon connected to a transducer (AP-601G; Nihon Kohden Co., Tokyo, Japan).…”

“…Rabbits were assigned to 6 groups: the control group (n= 11) underwent 30 min global ischemia and 60 min reperfusion (30-60 min I/R); the H2O2 group (n= 11) underwent 30-60 min I/R with H2O2 infusion (50 μmol/l, 15 ml/min) just after reperfusion for 1 min with a syringe pump (syringe infusion pump 22; Harvard Apparatus Inc., Millis, USA) in order to enhance oxidative stress during the initial phase of the reperfusion period. The dose of H2O2 was determined by preliminary experiments in which infusions of 10 to 100 μmol/l H2O2 were tested (14). We found that 50 μmol/l H2O2 infusion causes a modifiable enhancement of LV dysfunction, i.e., approximately 40% reduction in LVDP after 60 min of reperfusion.…”

“…The slices were incubated for 15 min at 37°C in freshly made and pre-warmed solution with 1% triphenyl tetrazolium chloride (TTC) in phosphate buffer (pH 7.4) solution (17). Viable tissue was stained brick red and the infarct tissue was left unstained, allowing the differentiation of infracted myocardium (14,18). Photos were taken and the infarct myocardial area was traced and measured by planimetry with NIH image software (http://rsb.info.nih.gov/nihimage).…”

“…Myocardial samples (200 mg), taken after the 30-60 I/R or H2O2 protocol and separated into epicardial and endocardial parts (14), were homogenized in 4 volumes of 5% metaphosphoric acid and centrifuged for 10 min at 3,500 rpm, and the supernatants were subjected to assay according to the manufacturer's protocol. The assay was performed in two steps, with the first step leading to the formation of substitution products (thioethers) with the sample mercaptans (GSH) and the second step transforming thioethers to thiones whose absorbance at 400 nm was measured.…”

“…To accomplish these goals, we used a global ischemic model of isolated rabbit hearts to eliminate some effects of neuro-humoral factors which potentially modify the cardioprotective effects (14). Rabbit myocardium is known to lack xanthine oxidase (XO), which is largely responsible for the production of ROS such as H2O2 (15,16).…”