Validation of a new model of selective antegrade cerebral perfusion with circulatory arrest in rats.

BACKGROUND: Selective antegrade cerebral perfusion (SACP) is adopted as an alternative to deep hypothermic circulatory arrest (DHCA) during aortic arch surgery. However, there is still no preclinical evidence to support the use of SACP associated with moderate hypothermia (28-30°C) instead of DHCA (18-20°C). The present study aims to develop a reliable and reproducible preclinical model of cardiopulmonary bypass (CPB) with SACP applicable for assessing the best temperature management. MATERIALS AND METHODS: A central cannulation through the right jugular vein and the left carotid artery was performed, and CPB was instituted.Animals were randomized into two groups: normothermic circulatory arrest without or with cerebral perfusion (NCA vs SACP). EEG monitoring was maintained during CPB. After 10 min of circulatory arrest, rats underwent 60 min of reperfusion. After that, animals were sacrificed, and brains were collected for histology and molecular biology analysis. RESULTS: Power spectral analysis of the EEG signal showed decreased activity in both cortical regions and lateral thalamus in all rats during the circulatory arrest. Only SACP determined complete recovery of brain activity and higher power spectral signal compared to NCA (p < 0.05). Histological damage scores and western blot analysis of inflammatory and apoptotic proteins like caspase-3 and Poly-ADP ribose polymerase (PARP) were significantly lower in SACP compared to NCA. Vascular endothelial growth factor (VEGF) and RNA binding protein 3 (RBM3) involved in cell-protection mechanisms were higher in SACP, showing better neuroprotection (p < 0.05). CONCLUSIONS: SACP by cannulation of the left carotid artery guarantees good perfusion of the whole brain in this rat model of CPB with circulatory arrest. The present model of SACP is reliable, repeatable, and not expensive, and it could be used in the future to achieve preclinical evidence for the best temperature management and to define the best cerebral protection strategy during circulatory arrest.

Efficacy of Pulsatile Flow Perfusion in Adult Cardiac Surgery: Hemodynamic Energy and Vascular Reactivity.

Background: The role of pulsatile (PP) versus non-pulsatile (NP) flow during a cardiopulmonary bypass (CPB) is still debated. This study's aim was to analyze hemodynamic effects, endothelial reactivity and erythrocytes response during a CPB with PP or NP. Methods: Fifty-two patients undergoing an aortic valve replacement were prospectively randomized for surgery with either PP or NP flow. Pulsatility was evaluated in terms of energy equivalent pressure (EEP) and surplus hemodynamic energy (SHE). Systemic (SVRi) and pulmonary (PVRi) vascular resistances, endothelial markers levels and erythrocyte nitric-oxide synthase (eNOS) activity were collected at different perioperative time-points. Results: In the PP group, the resultant EEP was 7.3% higher than the mean arterial pressure (MAP), which corresponded to 5150 ± 2291 ergs/cm3 of SHE. In the NP group, the EEP and MAP were equal; no SHE was produced. The PP group showed lower SVRi during clamp-time (p = 0.06) and lower PVRi after protamine administration and during first postoperative hours (p = 0.02). Lower SVRi required a higher dosage of norepinephrine in the PP group (p = 0.02). Erythrocyte eNOS activity results were higher in the PP patients (p = 0.04). Renal function was better preserved in the PP group (p = 0.001), whereas other perioperative variables were comparable between the groups. Conclusions: A PP flow during a CPB results in significantly lower SVRi, PVRi and increased eNOS production. The clinical impact of increased perioperative vasopressor requirements in the PP group deserves further evaluation.

Detection of Periodontal Pathogens in Oral Samples and Cardiac Specimens in Patients Undergoing Aortic Valve Replacement: A Pilot Study.

This observational study aimed to: (i) assess the presence of periodontal disease among patients requiring aortic valve replacement; (ii) investigate the presence of oral pathogens in aortic valve specimens and compare them with the microorganisms detected in the oral cavity. Twenty-six patients (15 men and 11 women) were scheduled to be visited the day before the cardiac surgery: periodontal conditions were accurately registered through clinical and radiographic examinations; dental plaque or salivary samples were collected. Valve specimens were collected during surgical aortic valve replacement and analyzed for pathogens detection through microbiological 16SrRna gene sequencing. Bacteria found in plaque samples and valve specimens were assessed according to oral and periodontal conditions. A qualitative comparison between oral and cardiac profiles of the microorganisms detected was performed. The overall number of patients examined for soft tissues conditions was 19, as 7 patients were edentulous. Twelve and three individuals, respectively, presented moderate and severe periodontitis. Nine valves were found to be positive for the presence of oral and periodontopathic bacterial DNA. The microbial species found in valve samples of patients with periodontitis suggest that the presence of these microorganisms in valvular tissue seems to be not coincidental.

Nitric Oxide in Selective Cerebral Perfusion Could Enhance Neuroprotection During Aortic Arch Surgery.

BACKGROUND: Hypothermic circulatory arrest (HCA) in aortic arch surgery has a significant risk of neurological injury despite the newest protective techniques and strategies. Nitric oxide (NO) could exert a protective role, reduce infarct area and increase cerebral perfusion. This study aims to investigate the possible neuroprotective effects of NO administered in the oxygenator of selective antegrade cerebral perfusion (SCP) during HCA. METHODS: Thirty male SD adult rats (450-550 g) underwent cardiopulmonary bypass (CPB), cooling to 22°C body core temperature followed by 30 min of HCA. Rats were randomized to receive SCP or SCP added with NO (20 ppm) administered through the oxygenator (SCP-NO). All animals underwent CPB-assisted rewarming to a target temperature of 35°C in 60 min. At the end of the experiment, rats were sacrificed, and brain collected. Immunofluorescence analysis was performed in blind conditions. RESULTS: Neuroinflammation assessed by allograft inflammatory factor 1 or ionized calcium-binding adapter molecule 1 expression, a microglia activation marker was lower in SCP-NO compared to SCP (4.11 ± 0.59 vs. 6.02 ± 0.18%; p < 0.05). Oxidative stress measured by 8oxodG, was reduced in SCP-NO (0.37 ± 0.01 vs. 1.03 ± 0.16%; p < 0.05). Brain hypoxic area extent, analyzed by thiols oxidation was attenuated in SCP-NO (1.85 ± 0.10 vs. 2.74 ± 0.19%; p < 0.05). Furthermore, the apoptotic marker caspases 3 was significantly reduced in SCP-NO (10.64 ± 0.37 vs. 12.61 ± 0.88%; p < 0.05). CONCLUSIONS: Nitric oxide administration in the oxygenator during SCP and HCA improves neuroprotection by decreasing neuroinflammation, optimizing oxygen delivery by reducing oxidative stress and hypoxic areas, finally decreasing apoptosis.

Slow versus fast rewarming after hypothermic circulatory arrest: effects on neuroinflammation and cerebral oedema.

OBJECTIVES: Among the factors that could determine neurological outcome after hypothermic circulatory arrest (HCA) rewarming is rarely considered. The optimal rewarming rate is still unknown. The goal of this study was to investigate the effects of 2 different protocols for rewarming after HCA on neurological outcome in an experimental animal model. METHODS: Forty-four Sprague Dawley rats were cooled to 19 ± 1°C body core temperature by cardiopulmonary bypass (CPB). HCA was maintained for 60 min. Animals were randomized to receive slow (90 min) or fast (45 min) assisted rewarming with CPB to a target temperature of 35°C. After a total of 90 min of reperfusion in both groups, brain samples were collected and analysed immunohistochemically and with immunofluorescence. In 10 rats, magnetic resonance imaging was performed after 2 and after 24 h to investigate cerebral perfusion and cerebral oedema. RESULTS: Interleukin 6, chemokine (C-C motif) ligand 5, intercellular adhesion molecule 1 and tumour necrosis factor α in the hippocampus are significantly less expressed in the slow rewarming group, and microglia cells are significantly less activated in the slow rewarming group. Magnetic resonance imaging analysis demonstrated better cerebral perfusion and less water content in brains that underwent slow rewarming at 2 and 24 h. CONCLUSIONS: Slow rewarming after HCA might be superior to fast rewarming in neurological outcome. The present experimental study demonstrated reduction in the inflammatory response, reduction of inflammatory cell activation in the brain, enhancement of cerebral blood flow and reduction of cerebral oedema when slow rewarming was applied.

Cardioprotective Effects of Sphingosine-1-Phosphate Receptor Immunomodulator FTY720 in a Clinically Relevant Model of Cardioplegic Arrest and Cardiopulmonary Bypass.

Objective: FTY720, an immunomodulator derived from sphingosine-1-phosphate, has recently demonstrated its immunomodulatory, anti-inflammatory, anti-oxidant, anti-apoptotic and anti-inflammatory properties. Furthermore, FTY720 might be a key pharmacological target for preconditioning. In this preclinical model, we have investigated the effects of FTY720 on myocardium during reperfusion in an experimental model of cardioplegic arrest (CPA) and cardiopulmonary bypass. Methods: 30 Sprague-Dawley rats (300-350 g) were randomized into two groups: Group-A, treated with FTY720 1 mg/kg via intravenous cannulation, and Group-B, as control. After 15 min of treatment, rats underwent CPA for 30 min followed by initiation of extracorporeal life support for 2 h. Support weaning was done, and blood and myocardial tissues were collected for analysis. Hemodynamic parameters, inflammatory mediators, nitro-oxidative stress, neutrophil infiltration, immunoblotting analysis, and immunohistochemical staining were analyzed and compared between groups. Results: FTY720 treatment activated the Akt/Erk1/2 signaling pathways, reduced the level of inflammatory mediators, activated antiapoptotic proteins, and inhibited proapoptotic proteins, leading to reduced nitro-oxidative stress and cardiomyocyte apoptosis. Moreover, significant preservation of high-energy phosphates were observed in the FTY720-treated group. This resulted in improved recovery of left ventricular systolic and diastolic functions. Conclusion: The cardioprotective mechanism in CPA is associated with activation of prosurvival cell signaling pathways that prevents myocardial damage. FTY720 preserves high-energy phosphates attenuates myocardial inflammation and oxidative stress, and improves cardiac function.

Peptidomimetic Targeting of Cavß2 Overcomes Dysregulation of the L-Type Calcium Channel Density and Recovers Cardiac Function.

BACKGROUND: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. METHODS: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavß2 chaperone regulates channel density at the plasma membrane. RESULTS: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavß2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavß2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavß2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavß2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavß2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. CONCLUSIONS: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavß2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.

Hemoglobin Concentration Affects Electroencephalogram During Cardiopulmonary Bypass: An Indication for Neuro-Protective Values.

Hemodilution during cardiopulmonary bypass (CPB) is widely used to decrease transfusion and improve microcirculation but has drawbacks, such as diminished hemoglobin levels. Among others, reduced brain oxygenation accounts for neurological adverse outcomes after CPB. The aim of the present study was to ascertain if and how continuous electroencephalogram (EEG) during CPB is affected by hematocrit level and what should be the minimum value to avoid significant frequency band shifts on the EEG. A comparative study design was used with 16 subjects undergoing elective mitral valve repair/replacement. EEG was continuously recorded during the surgical procedure (from anesthesia induction to 20 min after CPB end). Data were marked at relevant time points (T0: before CPB start; T1: after 30 min from CPB beginning; T2: at CPB end), and the following 2 min EEG analyzed with a fast Fourier transform to obtain relative power for delta, theta, alpha, and beta bands. A general linear model for repeated measure was used to study interactions of time (T0, T1, and T2, EEG frequency band, and topographical distribution. The relative powers for each electrode were calculated and represented using topographic maps. Power spectrum differences between time points (T2-T1; T2-T0; T1-T0) were calculated for each electrode, and differences >10%, considered indicative of neuronal sufferance, were included in further analysis. Cutoff hemoglobin values that maximize the proportion of correctly classified EEG band shifts were obtained by previous definition were obtained. At T2, diffuse EEG slowing in delta and theta bands was detected; a minor slowing over anterior regions was evident at T1 for the theta band. Decrements in EEG power greater than 10% were detected only for the delta band at T2. Hemoglobin concentration levels at which no slowing increase was evident were 9.4 mg/dL (Ht: 28.2%) at T1 and 9.2 mg/dL (Ht: 27.6%) at T2. EEG burst-suppression pattern related to a lesser degree of slowing at T2. In conclusion, we propose hemoglobin cutoff levels that prevent EEG slowing indicative of neuronal sufferance. In addition, burst-suppression EEG patterns offer higher central nervous system protection as measured on EEG.

Oxygenator Is the Main Responsible for Leukocyte Activation in Experimental Model of Extracorporeal Circulation: A Cautionary Tale.

In order to assess mechanisms underlying inflammatory activation during extracorporeal circulation (ECC), several small animal models of ECC have been proposed recently. The majority of them are based on home-made, nonstandardized, and hardly reproducible oxygenators. The present study has generated fundamental information on the role of oxygenator of ECC in activating inflammatory signaling pathways on leukocytes, leading to systemic inflammatory response, and organ dysfunction. The present results suggest that experimental animal models of ECC used in translational research on inflammatory response should be based on standardized, reproducible oxygenators with clinical characteristics.

Pulsatile cardiopulmonary bypass and renal function in elderly patients undergoing aortic valve surgery†.

OBJECTIVES: To evaluate if pulsatile cardiopulmonary bypass (CPB) has any protective influence on renal function in elderly patients undergoing aortic valve replacement (AVR). METHODS: Forty-six patients (≥ 75 years old) with aortic valve stenosis underwent AVR with either pulsatile perfusion (PP) or non-pulsatile perfusion (NP) during CPB. Haemodynamic efficacy of the blood pump during either type of perfusion was described in terms of the energy equivalent pressure and the surplus haemodynamic energy. Urine samples were collected before surgery, at sternum closure, and at 2 and 18 h of intensive care unit stay to detect acute kidney injury markers. Perioperative urine levels of N-acetyl-ß-D-glucosaminidase (NAG), kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin (NGAL) were assessed together with plasma creatinine, creatinine clearance (CCr) and 24-h haemodynamic monitoring. Normally distributed continuous variables were described as mean ± standard deviation and non-normally distributed data were presented as the median [25th-75th percentiles]. RESULTS: PP was characterized by a significantly higher amount of surplus haemodynamic energy transferred to the patients (P < 0.001), with lower mean systemic vascular resistance during CPB (P = 0.020) and during 18 h postoperatively (group-P = 0.018). No difference was found between pre- and postoperative CCr in the PP group (71 ± 23 vs 60 ± 35 ml/min, P = 0.27), while its statistically significant perioperative decrement was observed in the NP group (67 ± 24 vs 45 ± 15 ml/min, P < 0.001). The PP group showed significantly lower urinary levels of NAG at 18 h postoperatively (P = 0.008), and NGAL at sternum closure (P = 0.010), 2 h (P < 0.001) and 18 h (P = 0.015) postoperatively. CONCLUSIONS: Short-term PP in elderly patients showed higher safety for renal physiology than NP, resulting in better maintenance of glomerular filtration and lower renal tissue injury.

G-CSF displays restricted ability to promote Sca-1(+) cardiac stem cell proliferation in vitro.

Granulocyte colony-stimulating factor (G-CSF) is a controversial chemical in cardiac cell therapy. Myocardial homing of mobilized bone marrow-derived cells is thought to play a critical role in observed G-CSF-induced cardiac repair; meanwhile, the activation of proliferative potential of cardiac stem cells (CSCs) residing in the heart is a significant challenge. The present study aims to investigate whether G-CSF receptor is expressed in adult resident Sca-1(+) CSCs and determine the effect of G-CSF treatment on the proliferation of CSCs. For cardiac cells isolation, 12-week-old male C57BL/6 mice were anesthetized in a chamber containing 2.5% isoflurane in oxygen, euthanized by CO2 inhalation and then sacrificed by cervical dislocation. Magnetic-activated cell sorting was employed to acquire highly purified Sca-1(+) CSCs. We found that G-CSF receptor was expressed in adult resident Sca-1(+) CSCs by immunofluorescence staining and Western blotting. Exposure of Sca-1(+) cells to G-CSF in the culture medium for 72 h induced time-dependent but self-limiting cell cycle acceleration with a restricted effect on the CSC proliferation. As a result, it has provided a new insight to focus on the association between cardiac G-CSF therapy and adult resident stem cell activation. It may suggest gaining a deeper insight into the mechanisms of the interaction between CSCs and G-CSF to develop a synergistic strategy based on resident stem cell and G-CSF therapy for heart disease.

Diabetic hearts have lower basal urocortin levels that fail to increase after cardioplegic arrest: association with increased apoptosis and postsurgical cardiac dysfunction.

OBJECTIVES: The present study investigated the cardioprotective role of urocortin (Ucn) and its relationship with protein kinase C (PKC)ε and PKCδ in patients with (DMPs) and without (NDMPs) diabetes mellitus after on-pump cardiac surgery (OPCS). The molecular mechanisms responsible for the reported worse outcomes of DMP after OPCS remain unknown. METHODS: Two sequential biopsy specimens were obtained from the right atrium of 27 DMPs and 22 NDMPs before and after cardiopulmonary bypass. RESULTS: Postcardioplegic induction of Ucn in NDMPs (P<.01) was not observed in the DMPs, whose precardioplegic Ucn levels were 50% lower than those in the NDMPs (P<.05). In the NDMPs, cardioplegic arrest increased PKCε mRNA and protein (P<.05); overexpression of PKCδ was not seen. In contrast, DMPs showed increased PKCδ expression (P<.01), with no change in PKCε. Apoptosis was more than twofold greater in the postcardioplegic samples from the DMPs than in those from the NDMPs. The apoptotic myocytes were Ucn negative and exhibited nuclear relocation of PKCδ. Enhanced PKCε/mitochondrial co-localization was observed in viable, Ucn-positive, myocytes. The leakage of troponin I documented in the DMPs was greater than that in the NDMPs, although the difference was not statistically significant (P=.06). Furthermore, despite a similar incidence of perioperative acute myocardial infarction, the DMPs did not show postoperative improvement of systolic or diastolic function, although that was seen in the NDMPs (P<.05). CONCLUSIONS: Cardioplegic arrest failed to induce in DMPs myocyte overexpression of Ucn or PKCε but was associated with induction and mitochondrial relocation of PKCδ, resulting in apoptosis. Failure to overexpress Ucn in the DMPs was associated with apoptosis and cardiac dysfunction and, thus, might contribute to worse postoperative outcomes.

Role of calcium desensitization in the treatment of myocardial dysfunction after deep hypothermic circulatory arrest.

INTRODUCTION: Rewarming from deep hypothermic circulatory arrest (DHCA) produces calcium desensitization by troponin I (cTnI) phosphorylation which results in myocardial dysfunction. This study investigated the acute overall hemodynamic and metabolic effects of epinephrine and levosimendan, a calcium sensitizer, on myocardial function after rewarming from DHCA. METHODS: Forty male Wistar rats (400 to 500 g) underwent cardiopulmonary bypass (CPB) through central cannulation and were cooled to a core temperature of 13°C to 15°C within 30 minutes. After DHCA (20 minutes) and CPB-assisted rewarming (60 minutes) rats were randomly assigned to 60 minute intravenous infusion with levosimendan (0.2 µg/kg/min; n = 15), epinephrine (0.1 µg/kg/min; n = 15) or saline (control; n = 10). Systolic and diastolic functions were evaluated at different preloads with a conductance catheter. RESULTS: The slope of left ventricular end-systolic pressure volume relationship (Ees) and preload recruitable stroke work (PRSW) recovered significantly better with levosimendan compared to epinephrine (Ees: 85 ± 9% vs 51 ± 11%, P<0.003 and PRSW: 78 ± 5% vs 48 ± 8%, P<0.005; baseline: 100%). Levosimendan but not epinephrine reduced left ventricular stiffness shown by the end-diastolic pressure-volume relationship and improved ventricular relaxation (Tau). Levosimendan preserved ATP myocardial content as well as energy charge and reduced plasma lactate concentrations. In normothermia experiments epinephrine in contrast to Levosimendan increased cTnI phosphorylation 3.5-fold. After rewarming from DHCA, cTnI phosphorylation increased 4.5-fold in the saline and epinephrine group compared to normothermia but remained unchanged with levosimendan. CONCLUSIONS: Levosimendan due to prevention of calcium desensitization by cTnI phosphorylation is more effective than epinephrine for treatment of myocardial dysfunction after rewarming from DHCA.

Targeting urocortin signaling pathways to enhance cardioprotection: is it time to move from bench to bedside?

Despite the exponential growth in medical knowledge, cardiovascular diseases (CVDs) contribute to more than one-third of worldwide morbidity and mortality. A range of therapies already exist for established CVDs, although there is significant interest in further understanding their pathogenesis. The urocortins (Ucns) are peptide members of the corticotrophin-releasing factor family, a group of evolutionary conserved peptides with homologues in fish, amphibians and mammals and considered to play a pivotal role in energy homeostasis and local tissue repair. A number of preclinical studies in vitro, in-vivo and ex-vivo have defined a multifaceted effect of Ucns on the cardiovascular system. Different G-protein coupled signaling and protein-kinase pathways have been shown to be activated by Ucns, together with different transcriptional and translational effects, all of which preferentially converge on the mitochondria, where the modulation of apoptosis is considered their principal action. It has been demonstrated in experimental models, and consequentially suggested in human diseases, that Ucn-mediated inhibition of apoptosis can be exploited for the improvement of both therapeutic and preventative strategies against CVDs. Specifically, some unavoidable iatrogenic ischemia/reperfusion (I/R) injuries, e.g. during cardiac surgery or percutaneous coronary angioplasty, may greatly benefit from the anti-apoptotic effect of Ucns. However, few studies on the topic have been employed in humans to date. Therefore, this review will focus on the different intra-cellular mechanisms of action of Urocortins, and detail the different Ucn-mediated pathways identified so far. It will also highlight the limited evidence already existing in human clinical and surgical settings, as well as emphasize the potential uses of Ucns in human cardiac pathology.

Gaseous micro-emboli activity during cardiopulmonary bypass in adults: pulsatile flow versus nonpulsatile flow.

Cardiopulmonary bypass (CPB) has a risk of cerebral injury, with an important role of gaseous micro-emboli (GME) coming from the CPB circuit. Pulsatile perfusion is supposed to perform specific conditions for supplementary GME activity. We aimed to determine whether pulsatile CPB augments production and delivery of GME and evaluate the role of different events in GME activity during either type of perfusion. Twenty-four patients who underwent on-pump coronary artery bypass grafting surgery at the University of Verona were divided equally into two groups-pulsatile perfusion (PP) group and nonpulsatile perfusion (NP) group. The circuit included a JostraHL-20 roller pump set in pulsatile or nonpulsatile mode, an open Sorin Synthesis membrane oxygenator with integrated screen-type arterial filter, and phosphorylcholine-coated tubes. Hemodynamic flow evaluation was performed in terms of energy equivalent pressure and surplus hemodynamic energy (SHE). GME were counted by means of a GAMPT BCC200 bubble counter (GAMPT, Zappendorf, Germany) with two probes placed at postpump and postarterial filter positions. Results were evaluated in terms of GME number, GME volume, number of over-ranged GME from both probes, and series of filtering indexes. In PP mode, the pump produced and delivered along the circuit significantly higher amounts of SHE than in NP mode. At the venous postpump site, GME number was significantly higher during PP but no difference was found in terms of GME volume or number of over-ranged bubbles. No significant difference in GME number, GME volume, or number of over-ranges was found at the postarterial filter site. Filtering indexes were similar between the two groups. Neither type of perfusion was shown to contribute to excessive GME production during the most important perfusionist manipulation. Pulsatility leads to GME increment by splitting and size diminishing of the existing bubbles but not by additional gas production. PP augmented GME number at the venous postpump site, while mean volume remained comparable with NP. Sorin Synthesis oxygenator showed high efficacy in GME removal during either type of perfusion. Supplementary GME production and delivery during typical perfusionist manipulations did not depend on perfusion type.

Cardioprotective effect of δ-opioid receptor agonist vs. mild therapeutic hypothermia in a rat model of cardiac arrest with extracorporeal life support.

BACKGROUND: To compare the effect of δ-opioid receptor agonist, d-Ala2-d-Leu5 enkephalin (DADLE) with normothermic control and therapeutic hypothermia on post resuscitation myocardial function in a model of extracorporeal life support (ECLS). METHODS: Ventricular fibrillation (VF) was induced in male Wistar rats. After 10 min of untreated VF, venoarterial ECLS was instituted for 60 min. At the beginning of ECLS animals were randomized to three groups of ten: normothermia, hypothermia (32 °C) and DADLE intravenous infusion (1 mg/kg/h). Cooling to 32 °C or normothermia or drug infusion lasted for the entire ECLS. Plasma samples and myocardial biopsies were obtained and left-ventricular (LV) function was assessed by a conductance catheter at baseline and after weaning from ECLS. RESULTS: DADLE administration resulted in a significantly enhanced recovery of LV systolic function expressed by slope of the LV end-systolic pressure volume relationship (Ees) and preload recruitable stroke work (PRSW) than hypothermia and normothermia. LV stiffness indicated by end-diastolic pressure volume relationship (EDPVR) was significantly lower after DADLE administration (P<0.01). LV relaxation described by Tau was preserved after DADLE treatment but not after normothermia or mild hypothermia (P<0.01). Plasma lactate concentrations were lower in DADLE group (P<0.05). DADLE and not conventional hypothermia significantly increased phosphorylation of the kinases ERK1 and 2 (3.9±0.3 and 3.1±0.5 vs. 0.4±0.1 and 0.3±0.1-fold of baseline levels) (P<0.001). Both DADLE and hypothermia but not normothermia increase phosphorylation of Akt. CONCLUSIONS: DADLE was more effective than mild therapeutic hypothermia in recovering myocardial function and activation of the pro-survival kinases Akt and ERK after ECLS.

Levosimendan is superior to epinephrine in improving myocardial function after cardiopulmonary bypass with deep hypothermic circulatory arrest in rats.

OBJECTIVE: To investigate effects of epinephrine and levosimendan on cardiac function after rewarming from deep hypothermia. METHODS: Forty-five male Wistar rats (400-500 g) underwent cardiopulmonary bypass and were cooled to a core temperature of 13°C to 15°C within 30 minutes. After 15 minutes of deep hypothermic circulatory arrest, they were randomly assigned to treatment with levosimendan (12 µg/kg; infusion of 0.2 µg · kg(-1) · min(-1)) (n = 15) or epinephrine (0.1 µg/kg; infusion of 0.1 µg · kg(-1) · min(-1)) (n = 15) or saline as control (n = 10). The rewarming lasted 60 minutes. Systolic and diastolic function was evaluated at different preloads with a conductance catheter, including the slope of the end-systolic pressure-volume relation (ESPVR) and end-diastolic pressure-volume relationship (EDPVR), preload recruitable stroke work, first derivative of left ventricular pressure (+dP/dt), and its relation to end-diastolic volume, as well as the time constant of left ventricular relaxation (Tau) and maximal slope of the diastolic pressure decrement (-dP/dt). Plasma lactate levels were collected. RESULTS: Stroke volume, ejection fraction and +dP/dt were significantly higher in the levosimendan-treated group than in the epinephrine group. The slope values of preload recruitable stroke work, ESPVR, and the relation of +dP/dt to end-diastolic volume were significantly higher, indicating a better contractility and increased systolic function. -dP/dt was significantly higher in the levosimendan group (3468 ± 320 vs 1103 ± 101 mm Hg/s; P < .01). Left ventricular stiffness expressed by EDPVR and relaxation (Tau) were significantly improved in levosimendan-treated group. Plasma lactated concentrations were lower in levosimendan group (2.03 ± 1.27 vs 4.64 ± 1.02; P < .05). CONCLUSIONS: Levosimendan has better inotropic and lusitropic effects than epinephrine during rewarming from deep hypothermic circulatory arrest with cardiopulmonary bypass.

Increased expression of adenosine triphosphate-sensitive K+ channels in mitral dysfunction: mechanically stimulated transcription and hypoxia-induced protein stability?

OBJECTIVES: The aim of this study was to test whether adenosine triphosphate-sensitive K(+) (KATP) channel expression relates to mechanical and hypoxic stress within the left human heart. BACKGROUND: The KATP channels play a vital role in preserving the metabolic integrity of the stressed heart. However, the mechanisms that govern the expression of their subunits (e.g., potassium inward rectifier [Kir] 6.2) in adult pathologies are mostly unknown. METHODS: We collected biopsies from the 4 cardiac chambers and 50 clinical parameters from 30 surgical patients with severe mitral dysfunction. Proteins and messenger ribonucleic acids (mRNAs) of KATP pore subunits and mRNAs of their known transcriptional regulators (forkhead box [FOX] F2, FOXO1, FOXO3, and hypoxia inducible factor [HIF]-1α) were measured respectively by Western blotting, immunohistochemistry, and quantitative real-time polymerase chain reaction, and submitted to statistical analysis. RESULTS: In all heart chambers, Kir6.2 mRNA correlated with HIF-1α mRNA. Neither Kir6.1 nor Kir6.2 proteins positively correlated with their respective mRNAs. The HIF-1α mRNA related in the left ventricle to aortic pressure, in the left atrium to left atrial pressure, and in all heart chambers to a decreased Kir6.2 protein/mRNA ratio. Interestingly, in the left heart, Kir6.2 protein and its immunohistochemical detection in myocytes were maximal at low venous PO(2). In the left ventricle, the Kir6.2 protein/mRNA ratio was also significantly higher at low venous PO(2), suggesting that tissue hypoxia might stabilize the Kir6.2 protein. CONCLUSIONS: Results suggest that post-transcriptional events determine Kir6.2 protein expression in the left ventricle of patients with severe mitral dysfunction and low venous PO(2). Mechanical stress mainly affects transcription of HIF-1α and Kir6.2. This study implies that new therapies could aim at the proteasome for stabilizing the left ventricular Kir6.2 protein.

Preservation of endothelium nitric oxide release by pulsatile flow cardiopulmonary bypass when compared with continuous flow.

The aim of this work is to analyze endothelium nitric oxide (NO) release in patients undergoing continuous or pulsatile flow cardiopulmonary bypass (CPB). Nine patients operated under continuous flow CPB, and nine patients on pulsatile flow CPB were enrolled. Plasma samples were withdrawn for the chemiluminescence detection of nitrite and nitrate. Moreover the cellular component was withdrawn for the detection of nitric oxide synthase (NOS) activity in the erythrocytes, and an estimation of systemic inflammatory response was carried out. Significant reduction in the intraoperative concentration with respect to the preoperative was observed only under continuous flow CPB for both nitrite and NO(x) (nitrite + nitrate) concentration (P = 0.010 and P = 0.016, respectively). Significant difference in intraoperative nitrite concentration was also observed between the groups (P = 0.012). Finally, erythrocytes showed a certain endothelial NOS activity, which did not differ between the groups, and no differences in the inflammatory response were pointed out. The significant reduction of NO(2)(-) concentration under continuous perfusion revealed the strong connection among perfusion modality, endothelial NO release, and plasmatic nitrite concentration. The similar erythrocyte eNOS activity between the groups revealed that the differences in blood NO metabolites are mainly ascribable to the endothelium release.

Warm-blood cardioplegic arrest induces selective mitochondrial translocation of protein kinase Cepsilon followed by interaction with 6.1 inwardly rectifying potassium channel subunit in viable myocytes overexpressing urocortin.

OBJECTIVE: This study investigates the cardioprotective role and mechanism of action of urocortin in patients undergoing cardiac surgery, with respect to protein kinase Cepsilon expression, activation, and relocation. BACKGROUND: Cardioplegic arrest and subsequent reperfusion inevitably expose the heart to iatrogenic ischemia/reperfusion injury. We previously reported that iatrogenic ischemia/reperfusion injury caused myocyte induction of urocortin, an endogenous cardioprotective peptide. METHODS: Two sequential biopsies were obtained from the right atrium of 25 patients undergoing coronary artery bypass grafting at the start of grafting (internal control) and 10 minutes after release of the aortic clamp. RESULTS: In hearts exposed to iatrogenic ischemia/reperfusion injury, induction of urocortin was documented at both the mRNA (255% of basic levels; P < .05) and the protein (4-fold increase; P < .01) levels. Iatrogenic ischemia/reperfusion injury also induced a selective increase of protein kinase Cepsilon mRNA (225% of internal control; P < .05) and a 2-fold overexpression of total protein kinase Cepsilon (P < .05), which paralleled a 2.9-fold increase in protein kinase Cepsilon phosphorylation (P < .01). Mitochondrial translocation of activated protein kinase Cepsilon was observed only in postcardioplegic samples, using both subcellular fractionation (P < .05) and immunostaining techniques (P < .05). Enhanced protein kinase Cepsilon/mitochondria colocalization was selectively observed in viable myocytes, showing concurrently positive staining for urocortin (P < .05). Finally, co immunoprecipitation experiments documented an iatrogenic ischemia/reperfusion injury-enhanced physical interaction of phosphorylated protein kinase Cepsilon with the 6.1 inwardly rectifying potassium channel subunit of the K(ATP) channels (P < .05). CONCLUSION: After iatrogenic ischemia/reperfusion injury, urocortin expression in viable cells selectively colocalized with enhanced phosphorylation and mitochondrial relocation of protein kinase Cepsilon, suggesting a cardioprotective role for endogenous urocortin. The physical interaction of activated protein kinase Cepsilon with 6.1 inwardly rectifying potassium channel, enhanced by cardioplegic arrest, may represent a conjectural mechanism of urocortin-mediated cardioprotection.