Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest.
ABSTRACT: The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.
Project description:Simultaneous inhibition of the cardiac equilibrative-p-nitrobenzylthioinosine (NBMPR)-sensitive (es) type of the equilibrative nucleoside transport 1 (ENT1) nucleoside transporter, with NBMPR, and adenosine deaminase, with erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA), prevents release of myocardial purines and attenuates myocardial stunning and fibrillation in canine models of warm ischemia and reperfusion. It is not known whether prolonged administration of hypothermic cardioplegia influences purine release and EHNA/NBMPR-mediated cardioprotection in acutely ischemic hearts.Anesthetized dogs (n = 46), which underwent normothermic aortic crossclamping for 20 minutes on-pump, were divided to determine (1) purine release with induction of intermittent antegrade or continuous retrograde hypothermic cardioplegia and reperfusion, (2) the effects of postischemic treatment with 100 ?M EHNA and 25 ?M NBMPR on purine release and global functional recovery, and (3) whether a hot shot and reperfusion with EHNA/NBMPR inhibits purine release and attenuates ventricular dysfunction of ischemic hearts. Myocardial biopsies and coronary sinus effluents were obtained and analyzed using high-performance liquid chromatography.Warm ischemia depleted myocardial adenosine triphosphate and elevated purines (ie, inosine > adenosine) as markers of ischemia. Induction of intermittent antegrade or continuous retrograde hypothermic (4°C) cardioplegia releases purines until the heart becomes cold (<20°C). During reperfusion, the levels of hypoxanthine and xanthine (free radical substrates) were >90% of purines in coronary sinus effluent. Reperfusion with EHNA/NBMPR abolished ventricular dysfunction in acutely ischemic hearts with and without a hot shot and hypothermic cardioplegic arrest.Induction of hypothermic cardioplegia releases purines from ischemic hearts until they become cold, whereas reperfusion induces massive purine release and myocardial stunning. Inhibition of cardiac es-ENT1 nucleoside transporter abolishes postischemic reperfusion injury in warm and cold cardiac surgery.
Project description:The vascular endothelium of the coronary arteries has been identified as the important organ that locally regulates coronary perfusion and cardiac function by paracrine secretion of nitric oxide (NO) and vasoactive peptides. NO is constitutively produced in endothelial cells by endothelial nitric oxide synthase (eNOS). NO derived from this enzyme exerts important biological functions including vasodilatation, scavenging of superoxide and inhibition of platelet aggregation. Routine cardiac surgery or cardiologic interventions lead to a serious temporary or persistent disturbance in NO homeostasis. The clinical consequences are "endothelial dysfunction", leading to "myocardial dysfunction": no- or low-reflow phenomenon and temporary reduction of myocardial pump function. Uncoupling of eNOS (one electron transfer to molecular oxygen, the second substrate of eNOS) during ischemia-reperfusion due to diminished availability of L-arginine and/or tetrahydrobiopterin is even discussed as one major source of superoxide formation. Therefore maintenance of normal NO homeostasis seems to be an important factor protecting from ischemia/reperfusion (I/R) injury. Both, the clinical situations of cardioplegic arrest as well as hypothermic cardioplegic storage are followed by reperfusion. However, the presently used cardioplegic solutions to arrest and/or store the heart, thereby reducing myocardial oxygen consumption and metabolism, are designed to preserve myocytes mainly and not endothelial cells. This review will focus on possible drug additives to cardioplegia, which may help to maintain normal NO homeostasis after I/R.
Project description:Potassium-based depolarizing St Thomas' Hospital cardioplegic solution No 2 administered as intermittent, oxygenated blood is considered as a gold standard for myocardial protection during cardiac surgery. However, the alternative concept of polarizing arrest may have beneficial protective effects. We hypothesize that polarized arrest with esmolol/adenosine/magnesium (St Thomas' Hospital Polarizing cardioplegic solution) in cold, intermittent oxygenated blood offers comparable myocardial protection in a clinically relevant animal model.Twenty anaesthetized young pigs, 42 ± 2 (standard deviation) kg on standardized tepid cardiopulmonary bypass (CPB) were randomized (10 per group) to depolarizing or polarizing cardiac arrest for 60 min with cardioplegia administered in the aortic root every 20 min as freshly mixed cold, intermittent, oxygenated blood. Global and local baseline and postoperative cardiac function 60, 120 and 180 min after myocardial reperfusion was evaluated with pressure-conductance catheter and strain by Tissue Doppler Imaging. Regional tissue blood flow, cleaved caspase-3 activity, GRK2 phosphorylation and mitochondrial function and ultrastructure were evaluated in myocardial tissue samples.Left ventricular function and general haemodynamics did not differ between groups before CPB. Cardiac asystole was obtained and maintained during aortic cross-clamping. Compared with baseline, heart rate was increased and left ventricular end-systolic and end-diastolic pressures decreased in both groups after weaning. Cardiac index, systolic pressure and radial peak systolic strain did not differ between groups. Contractility, evaluated as dP/dtmax, gradually increased from 120 to 180 min after declamping in animals with polarizing cardioplegia and was significantly higher, 1871 ± 160 (standard error) mmHg/s, compared with standard potassium-based cardioplegic arrest, 1351 ± 70 mmHg/s, after 180 min of reperfusion (P = 0.008). Radial peak ejection strain rate increased and the load-independent variable preload recruitable stroke work was increased with polarizing cardioplegia after 180 min, 64 ± 3 vs 54 ± 2 mmHg (P = 0.018), indicating better preserved left ventricular contractility with polarizing cardioplegia. Phosphorylation of GRK2 in myocardial tissue did not differ between groups. Fractional cytoplasmic volume in myocytes was reduced in hearts arrested with polarizing cardioplegia, indicating reduction of cytoplasmic oedema.Polarizing oxygenated blood cardioplegia with esmolol/adenosine/magnesium offers comparable myocardial protection and improves contractility compared with the standard potassium-based depolarizing blood cardioplegia.
Project description:INTRODUCTION:Surgical repair of congenital heart defects often requires the use of cardiopulmonary bypass (CPB) and cardioplegic arrest. Cardioplegia is used during cardiac surgery requiring CPB to keep the heart still and to reduce myocardial damage as a result of ischaemia-reperfusion injury. Cold cardioplegia is the prevalent method of myocardial protection in paediatric patients; however, warm cardioplegia is used as part of usual care throughout the UK in adults. We aim to provide evidence to support the use of warm versus cold blood cardioplegia on clinical and biochemical outcomes during and after paediatric congenital heart surgery. METHODS AND ANALYSIS:We are conducting a single-centre randomised controlled trial in paediatric patients undergoing operations requiring CPB and cardioplegic arrest at the Bristol Royal Hospital for Children. We will randomise participants in a 1:1 ratio to receive either 'cold-blood cardioplegia' or 'warm-blood cardioplegia'. The primary outcome will be the difference between groups with respect to Troponin T levels over the first 48 postoperative hours. Secondary outcomes will include measures of cardiac function; renal function; cerebral function; arrythmias during and postoperative hours; postoperative blood loss in the first 12?hours; vasoactive-inotrope score in the first 48?hours; intubation time; chest and wound infections; time from return from theatre until fit for discharge; length of postoperative hospital stay; all-cause mortality to 3 months postoperative; myocardial injury at the molecular and cellular level. ETHICS AND DISSEMINATION:This trial has been approved by the London - Central Research Ethics Committee. Findings will be disseminated to the academic community through peer-reviewed publications and presentation at national and international meetings. Patients will be informed of the results through patient organisations and newsletters to participants. TRIAL REGISTRATION NUMBER:ISRCTN13467772; Pre-results.
Project description:INTRODUCTION::This experimental study compares myocardial function after prolonged arrest by St. Thomas' Hospital polarizing cardioplegic solution (esmolol, adenosine, Mg2+) with depolarizing (hyperkalaemic) St. Thomas' Hospital No 2, both administered as cold oxygenated blood cardioplegia. METHODS::Twenty anaesthetized pigs on tepid (34°C) cardiopulmonary bypass (CPB) were randomised to cardioplegic arrest for 120 min with antegrade, repeated, cold, oxygenated, polarizing (STH-POL) or depolarizing (STH-2) blood cardioplegia every 20 min. Cardiac function was evaluated at Baseline and 60, 150 and 240 min after weaning from CPB, using a pressure-conductance catheter and epicardial echocardiography. Regional tissue blood flow, cleaved caspase-3 activity and levels of malondialdehyde were evaluated in myocardial tissue samples. RESULTS::Preload recruitable stroke work (PRSW) was increased after polarizing compared to depolarizing cardioplegia 150 min after declamping (73.0±3.2 vs. 64.3±2.4 mmHg, p=0.047). Myocardial tissue blood flow rate was high in both groups compared to the Baseline levels and decreased significantly in the STH-POL group only, from 60 min to 150 min after declamping (p<0.005). Blood flow was significantly reduced in the STH-POL compared to the STH-2 group 240 min after declamping (p<0.05). Left ventricular mechanical efficiency, the ratio between total pressure-volume area and blood flow rate, gradually decreased after STH-2 cardioplegia and was significantly reduced compared to STH-POL cardioplegia after 150 and 240 min (p<0.05 for both). CONCLUSION::Myocardial protection for two hours of polarizing cardioplegic arrest with STH-POL in oxygenated blood is non-inferior compared to STH-2 blood cardioplegia. STH-POL cardioplegia alleviates the mismatch between myocardial function and perfusion after weaning from CPB.
Project description:Diabetic heart dysfunctions during cardiac surgeries have revealed several clinical problems associated with ion imbalance. However, the mechanism of ion imbalance mediated by cardioplegia and a diabetic heart is largely unclear. We hypothesized that ion transporters might be regulated differently in the diabetic heart and that the differentially regulated ion transporters may involve in ion imbalance of the diabetic heart after cardioplegic arrest. In this study, we modified the Langendorff-free cardioplegia method and identified the involved ion transporters after cardioplegia-induced arrest between wild type and db/db heart. Enhanced expression of Na+-K+-2Cl- cotransporter 1 (NKCC1) was observed in the db/db heart compared to the wild type heart. Enhanced NKCC1 activity was observed in the left ventricle of db/db mice compared to that of wild type after cardioplegia-induced arrest. The expression and activity of Slc26a6, a dominant Cl-/HCO3 - exchanger in cardiac tissues, were enhanced in left ventricle strips of db/db mice compared to that of wild type. The Cl- transporting activity in left ventricle strips of db/db mice was dramatically increased as compared to that of wild type. Interestingly, expression of Slc26a6, as well as carbonic anhydrase IV as a supportive enzyme of Slc26a6, was increased in db/db cardiac strips compared to wild type cardiac strips. Thus, the enhanced Cl- transporting activity and expression by NKCC1 and Slc26a6 in db/db cardiac tissues after cardioplegia-induced arrest provide greater insight into enhanced acidosis and Cl- movement-mediated db/db heart dysfunction. Thus, we suggested that enhanced Cl- influx and HCO3 - efflux through NKCC1 and Slc26a6 offer more acidic circumstances in the diabetic heart after cardioplegic arrest. These transporters should be considered as potential therapeutic targets to develop the next generation of cardioplegia solution for protection against ischemia-reperfusion injury in diabetic hearts.
Project description:This study investigated whether the novel St. Thomas' Hospital polarizing cardioplegic solution (STH-POL) with esmolol/adenosine/magnesium offers improved myocardial protection by reducing demands for high-energy phosphates during cardiac arrest compared to the depolarizing St. Thomas' Hospital cardioplegic solution No 2 (STH-2).Twenty anaesthetised pigs on tepid cardiopulmonary bypass were randomized to cardiac arrest for 60?min with antegrade freshly mixed, repeated, cold, oxygenated STH-POL or STH-2 blood cardioplegia every 20?min. Haemodynamic variables were continuously recorded. Left ventricular biopsies, snap-frozen in liquid nitrogen or fixed in glutaraldehyde, were obtained at Baseline, 58?min after cross-clamp and 20 and 180?min after weaning from bypass. Adenine nucleotides were evaluated by high-performance liquid chromatography, myocardial ultrastructure with morphometry.With STH-POL myocardial creatine phosphate was increased compared to STH-2 at 58?min of cross-clamp [59.9?±?6.4 (SEM) vs 44.5?±?7.4?nmol/mg protein; P? < ? 0.025], and at 20?min after reperfusion (61.0?±?6.7 vs 49.0?±?5.5?nmol/mg protein; P? < ? 0.05), ATP levels were increased at 20?min of reperfusion with STH-POL (35.4?±?1.1 vs 32.4?±?1.2?nmol/mg protein; P? < ? 0.05). Mitochondrial surface-to-volume ratio was decreased with polarizing compared to depolarizing cardioplegia 20?min after reperfusion (6.74?±?0.14 vs 7.46?±?0.13?µm 2 /µm 3 ; P? = ? 0.047). None of these differences were present at 180?min of reperfusion. From 150?min of reperfusion and onwards, cardiac index was increased with STH-POL; 4.8?±?0.2 compared to 4.0?±?0.2?l/min/m 2 ( P? = ? 0.011) for STH-2 at 180?min.Polarizing STH-POL cardioplegia improved energy status compared to standard STH-2 depolarizing blood cardioplegia during cardioplegic arrest and early after reperfusion.
Project description:Identification of microRNAs (miRNA) associated with cardiopulmonary bypass, cardiac arrest and subsequent myocardial ischemia/reperfusion may unravel novel therapeutic targets and biomarkers. The primary aim of the present study was to investigate the effects of cardiopulmonary bypass and temperature of cardioplegic arrest on myocardial miRNA profile in pigs' left ventricular tissue. We employed next-generation sequencing to analyse miRNA profiles in the following groups: (1) hearts were arrested with antegrade warm St Thomas Hospital No. 2 (STH2) cardioplegia (n = 5; STH2-warm, 37 °C) and (2) cold STH2 (n = 6; STH2-cold, 4 °C) cardioplegia. Sixty min of ischemia was followed by 60 min of on-pump reperfusion with an additional 90 min of off-pump reperfusion. In addition, two groups without cardiac arrest (off-pump and on-pump group; n = 3, respectively) served as additional controls. STH2-warm and STH2-cold cardioplegia revealed no hemodynamic differences. In contrast, coronary venous creatine kinase-myocardial band (CK-MB) levels were significantly lower in pigs receiving STH2-warm cardioplegia (p < 0.05). Principal component analysis revealed that cardiopulmonary bypass and cardioplegic arrest markedly affected miRNAs in left ventricular tissue. Accordingly, ssc-miR-122, ssc-miR-10a-5p, ssc-miR-193a-3p, ssc-miR-499-3p, ssc-miR-374a-5p, ssc-miR-345-5p, ssc-miR-142-3p, ssc-miR-424-5p, ssc-miR-545-3p, ssc-miR-30b-5p, ssc-miR-145-5p, ssc-miR-374b-5p and ssc-miR-139-3p were differently regulated by cardiopulmonary bypass (false discovery rate (FDR) < 0.05 versus off-pump group). However, only ssc-miR-451 was differently expressed between STH2-warm and STH2-cold (FDR < 0.05). These data demonstrate for the first time that cardiopulmonary bypass and temperature of cardioplegic solution affected the expression of miRNAs in left ventricular tissue. In conclusion, specific miRNAs are potential therapeutic targets for limiting ischemia-reperfusion injury in patients undergoing cardiac surgery.
Project description:We previously demonstrated that myocardial p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27) are phosphorylated following cardioplegic arrest in patients undergoing cardiac surgery and correlate with reduced cardiac function. The following studies were performed to determine whether inhibition of p38 MAPK and/or overexpression of nonphosphorylatable HSP27 improves cardiac function following cardioplegic arrest. Langendorff-perfused isolated rat hearts were subjected to 2 h of intermittent cold cardioplegia followed by 30 min of reperfusion. Hearts were treated with (CP+SB) or without (CP) the p38 MAPK inhibitor SB-203580 (5 ?M) supplied in the cardioplegia. Sham-treated hearts served as controls. In separate experiments, isolated rat ventricular myocytes infected with either green fluorescent protein (GFP) or a nonphosphorylatable HSP27 mutant (3A-HSP27) were subjected to 3 h of cold hypoxic cardioplegia and simulated reperfusion (CP) followed by video microscopy and length change measurements. Baseline parameters of cardiac function were similar between groups [left ventricular developed pressure (LVDP), 119 ± 4.9 mmHg; positive and negative first derivatives of LV pressure (± dP/dt), 3,139 ± 245 and 2, 314 ± 110 mmHg/s]. CP resulted in reduced cardiac function (LVDP, 72.2 ± 5.8 mmHg; ± dP/dt, 2,076 ± 231 and -1,317 ± 156 mmHg/s) compared with baseline. Treatment with 5 ?M SB-203580 significantly improved CP-induced cardiac function (LVDP, 101.9 ± 0 mmHg; ± dP/dt, 2,836 ± 163 and -2,108 ± 120 mmHg/s; P = 0.03, 0.01, and 0.04, CP+SB vs. CP). Inhibition of p38 MAPK significantly lowered CP-induced p38 MAPK, HSP27, and ?B-crystallin (cryAB) phosphorylation. In vitro CP decreased myocyte length changes from 10.3 ± 1.5% (GFP) to 5.7 ± 0.8% (GFP+CP). Infection with 3A-HSP27 completely rescued CP-induced decreased myocyte contraction (11.1 ± 1.0%). However, infection with 3A-HSP27 did not block the endogenous HSP27 response. We conclude that inhibition of p38 MAPK and subsequent HSP27 and cryAB phosphorylation and/or overexpression of nonphosphorylatable HSP27 significantly improves cardiac performance following cardioplegic arrest. Modulation of HSP27 phosphorylation may improve myocardial stunning following cardiac surgery.
Project description:This study investigates the effects of high glucose content on patients undergoing cold crystalloid versus cold blood cardioplegia in terms of early clinical results, functional myocardial recovery and ischaemia-reperfusion injury in patients undergoing repair of acyanotic cardiac lesions.Patients were randomly assigned to receive either crystalloid (n = 31) or blood cardioplegia (n = 31). Early clinical results were assessed. Changes in left ventricular fractional shortening, arterial blood lactate levels, central venous saturation, cardiac Troponin I release and blood glucose concentration were measured during the first 24 h after ischaemia.There was no significant difference in clinical outcomes and postoperative complication rates between groups. The postoperative changes in left ventricular function, lactate levels, central venous saturation and Troponin I were not significantly different between groups. The use of crystalloid cardioplegia was associated with significant increases in serum glucose compared with blood cardioplegia.A high glucose content blood cardioplegia does not show any advantage compared with crystalloid cardioplegia in terms of clinical outcomes, functional recovery and the degree of ischaemic injury in infants and children undergoing repair of acyanotic heart lesions. High glucose concentration of the cardioplegic solution might potentiate ischaemia-reperfusion injury and diminish the beneficial effects of blood cardioplegia.