Release of Mitochondrial and Nuclear DNA During On-Pump Heart Surgery: Kinetics and Relation to Extracellular Vesicles.

During heart surgery with cardiopulmonary bypass (CPB), the release of mitochondrial (mtDNA) and nuclear DNA (nDNA) and their association to extracellular vesicles were investigated. In patients undergoing elective coronary artery bypass grafting (CABG, n = 12), blood was sampled before, during, and after surgery from peripheral artery, pulmonary artery, and the coronary sinus. Plasma was separated in three fractions: microvesicles, exosomes, and supernatant. mtDNA and nDNA were measured by qPCR. mtDNA and nDNA levels increased after start of surgery, but before CPB, and increased further during CPB. mtDNA copy number was about 1000-fold higher than nDNA. mtDNA was predominantly localized to the vesicular fractions in plasma, whereas nDNA was predominantly in the supernatant. The amount of free mtDNA increased after surgery. There was no net release or disappearance of DNAs across the pulmonary, systemic, or coronary circulation. Extracellular DNAs, in particular mtDNA, may be important contributors to the whole-body inflammation during CPB.

In vivo visualization and ex vivo quantification of experimental myocardial infarction by indocyanine green fluorescence imaging.

The fluorophore indocyanine green accumulates in areas of ischemia-reperfusion injury due to an increase in vascular permeability and extravasation of the dye. The aim of the study was to validate an indocyanine green-based technique of in vivo visualization of myocardial infarction. A further aim was to quantify infarct size ex vivo and compare this technique with the standard triphenyltetrazolium chloride staining. Wistar rats were subjected to regional myocardial ischemia (30 minutes) followed by reperfusion (n = 7). Indocyanine green (0.25 mg/mL in 1 mL of normal saline) was infused intravenously for 10 minutes starting from the 25th minute of ischemia. Video registration in the near-infrared fluorescence was performed. Epicardial fluorescence of indocyanine green corresponded to the injured area after 30 minutes of reperfusion. Infarct size was similar when determined ex vivo using traditional triphenyltetrazolium chloride assay and indocyanine green fluorescent labeling. Intravital visualization of irreversible injury can be done directly by fluorescence on the surface of the heart. This technique may also be an alternative for ex vivo measurements of infarct size.

Heparinase treatment of heparin-contaminated plasma from coronary artery bypass grafting patients enables reliable quantification of microRNAs.

BACKGROUND: microRNAs have recently been identified as powerful biomarkers of human disease. Reliable polymerase chain reaction (PCR)-based quantification of nucleic acids in clinical samples contaminated with polymerase inhibitor heparin requires deheparinization. However, the effects of deheparinization procedure on quantification of nucleic acids remain largely unknown. The aim of this study was to determine whether the deheparinization procedure completely eliminates the inhibition of amplification, while maintaining RNA integrity and technical variability of the measured microRNA levels. METHODS: Heparinized plasma from 9 patients undergoing coronary artery bypass grafting (CABG) and the heparin-free plasma from 58 rats were spiked with a synthetic RNA oligonucleotide and total RNA was extracted. The RNA solutions were then treated with heparinase I to remove contaminating heparin prior to reverse transcription. Levels of synthetic spike-in RNA oligonucleotide, as well as endogenous hsa-miR-1-3p and hsa-miR-208a-3p, were measured using quantitative reverse transcription PCR (RT-qPCR). The amplification efficiency and presence of inhibitors in individual samples were directly determined using calibration curves. RESULTS: In contrast to RNA samples from rat plasma, RNA samples derived from the CABG patient plasma contained inhibitors, which were completely eliminated by treatment with heparinase. The procedure caused a decrease in the amount of detected RNA; however, the technical variability of the measured targets did not change, allowing for the quantification of circulating endogenous hsa-miR-1-3p and hsa-miR-208a-3p in the plasma of CABG patients. CONCLUSIONS: The heparinase treatment procedure enables utilization of RT-qPCR for reliable microRNA quantification in heparinized plasma.

Remote vs. local ischaemic preconditioning in the rat heart: infarct limitation, suppression of ischaemic arrhythmia and the role of reactive oxygen species.

The unmet clinical need for myocardial salvage during ischaemia-reperfusion injury requires the development of new techniques for myocardial protection. In this study the protective effect of different local ischaemic preconditioning (LIPC) and remote ischaemic preconditioning (RIPC) protocols was compared in the rat model of myocardial ischaemia-reperfusion, using infarct size and ischaemic tachyarrhythmias as end-points. In addition, the hypothesis that there is involvement of reactive oxygen species (ROS) in the protective signalling by RIPC was tested, again in comparison with LIPC. The animals were subjected to 30-min coronary occlusion and 90-min reperfusion. RIPC protocol included either transient infrarenal aortic occlusion (for 5, 15 and 30 min followed by 15-min reperfusion) or 15-min mesenteric artery occlusion with 15-min reperfusion. Ventricular tachyarrhythmias during test ischaemia were quantified according to Lambeth Conventions. It was found that the infarct-limiting effect of RIPC critically depends on the duration of a single episode of remote ischaemia, which fails to protect the heart from infarction when it is too short or, instead, too prolonged. It was also shown that RIPC is ineffective in reducing the incidence and severity of ischaemia-induced ventricular tachyarrhythmias. According to our data, the infarct-limiting effect of LIPC could be partially eliminated by the administration of ROS scavenger N-2-mercaptopropionylglycine (90 mg/kg), whereas the same effect of RIPC seems to be independent of ROS signalling.

New technique of local ischemic preconditioning induction without repetitive aortic cross-clamping in cardiac surgery.

BACKGROUND: Several studies have demonstrated that local ischemic preconditioning can reduce myocardial ischemia-reperfusion injury in cardiac surgery patients; however, preconditioning has not become a standard cardioprotective intervention, primarily because of the increased risk of atheroembolism during repetitive aortic cross-clamping. In the present study, we aimed to describe and validate a novel technique of preconditioning induction. METHODS: Patients undergoing coronary artery bypass grafting (12 women and 78 men; mean age, 56 ± 11 years) were randomized into 3 groups: (1) Controls (n = 30), (2) Perfusion (n = 30), and (3) Preconditioning (n = 30). All patients were operated under cardiopulmonary bypass using normothermic blood cardioplegia. Preconditioning was induced by subjecting the hemodynamically unloaded heart to 2 cycles of 3 min of ischemia and 3 min of reperfusion with normokalemic blood prior to cardioplegia. In the Perfusion group, the heart perfusion remained unaffected for 12 min. Troponin I (TnI) levels were analyzed before surgery, and 12, 24, 48 h, and 7 days after surgery. The secondary endpoints included the cardiac index, plasma natriuretic peptide level, and postoperative use of inotropes. RESULTS: Preconditioning resulted in a significant reduction in the TnI level on the 7th postoperative day only (0.10 ± 0.05 and 0.33 ± 0.88 ng/ml in Preconditioning and Perfusion groups, respectively, P < 0.05). In addition, cardiac index was significantly higher in the Preconditioning group than in the Control and Perfusion groups just after weaning from cardiopulmonary bypass. The number of patients requiring inotropic support with ≥ 2 agents after surgery was significantly lower in the Preconditioning and Perfusion group than in the Control group (P < 0.05). No complications of the procedure were recorded in the Preconditioning group. CONCLUSIONS: The preconditioning procedure described can be performed safely in cardiac surgery patients. The application of this technique of preconditioning was associated with certain benefits, including improved left ventricular function after weaning from cardiopulmonary bypass and a reduced need for inotropic support. However, the infarct-limiting effect of preconditioning in the early postoperative period was not evident. The procedure does not involve repetitive aortic cross-clamping, thus avoiding possible embolic complications.

Myocardial protection against global ischemia with Krebs-Henseleit buffer-based cardioplegic solution.

BACKGROUND: The Krebs-Henseleit buffer is the best perfusion solution for isolated mammalian hearts. We hypothesized that a Krebs-Henseleit buffer-based cardioplegic solution might provide better myocardial protection than well-known crystalloid cardioplegic solutions because of its optimal electrolyte and glucose levels, presence of buffer systems, and mild hyperosmolarity. METHODS: Isolated Langendorff-perfused rat hearts were subjected to either global ischemia without cardioplegia (controls) or cardioplegic arrest for either 60 or 180 min, followed by 120 min of reperfusion. The modified Krebs-Henseleit buffer-based cardioplegic solution (mKHB) and St. Thomas' Hospital solution No. 2 (STH2) were studied. During global ischemia, the temperatures of the heart and the cardioplegic solutions were maintained at either 37°C (60 min of ischemia) or 22°C (moderate hypothermia, 180 min of ischemia). Hemodynamic parameters were registered throughout the experiments. The infarct size was determined through histochemical examination. RESULTS: Cardioplegia with the mKHB solution at moderate hypothermia resulted in a minimal infarct size (5 ± 3%) compared to that in the controls and STH2 solution (35 ± 7% and 19 ± 9%, respectively; P < 0.001, for both groups vs. the mKHB group). In contrast to the control and STH2-treated hearts, no ischemic contracture was registered in the mKHB group during the 180-min global ischemia. At normothermia, the infarct sizes were 4 ± 3%, 72 ± 6%, and 70 ± 12% in the mKHB, controls, and STH2 groups, respectively (P < 0.0001). In addition, cardioplegia with mKHB at normothermia prevented ischemic contracture and improved the postischemic functional recovery of the left ventricle (P < 0.001, vs. STH2). CONCLUSIONS: The data suggest that the Krebs-Henseleit buffer-based cardioplegic might be superior to the standard crystalloid solution (STH2).

Ischemic postconditioning: brief ischemia during reperfusion converts persistent ventricular fibrillation into regular rhythm.

OBJECTIVES: Brief episodes of myocardial ischemia-reperfusion employed during reperfusion after a prolonged ischemic insult may attenuate the total ischemia-reperfusion injury. This phenomenon has been termed ischemic postconditioning. In the present study, we studied the possible effect of postconditioning on persistent reperfusion-induced ventricular fibrillation (VF) in the isolated rat heart model. METHODS: Isolated Langendorff-perfused rat hearts (n = 46) were subjected to 30 min of regional ischemia and reperfusion. The hearts with persistent VF (n = 11) present after 15 min of reperfusion were then randomly assigned into one of the two groups: (1) control hearts (n = 6) in which perfusion was continued without intervention; (2) postconditioned hearts (n = 5) subjected to 2 min of global ischemia followed by reperfusion. Left ventricular pressures, heart rate, coronary flow, and electrogram were monitored throughout the experiment. RESULTS: Conversion of VF into regular rhythm was observed in all hearts subjected to postconditioning. Regular beating was maintained by all postconditioned hearts during the subsequent reperfusion. None of the hearts in the control group had normal rhythm at the end of the experiment. At the end of reperfusion, the left ventricular developed pressure was lower in beating postconditioned hearts compared to the hearts that did not develop persistent VF. CONCLUSIONS: Ischemic postconditioning possesses strong antiarrhythmic effect against persistent reperfusion-induced tachyarrhythmias. Postconditioning may be an interesting, novel adjunct strategy to protect the heart.