Working Heart Mode Research Articles

Twenty-four-hour Normothermic Ex Vivo Heart Perfusion With Low Flow Functional Assessment in an Adult Porcine Model.

Cold static storage and normothermic ex vivo heart perfusion are routinely limited to 6 h. This report describes intermittent left atrial (LA) perfusion that allows cardiac functional assessment in a working heart mode. Using our adult porcine model, general anesthesia was induced and a complete cardiectomy was performed following cardioplegic arrest. Back-table instrumentation was completed and normothermic ex vivo heart perfusion (NEHP) was initiated in a nonworking heart mode (Langendorff). After 1 h of resuscitation and recovery, LA perfusion was initiated and the heart was transitioned to a coronary flow-only working heart mode for 30 min. Baseline working heart parameters were documented and the heart was returned to nonworking mode. Working heart assessments were performed for 30 min every 6 h for 24 h. Twenty-four-hour NEHP on 9 consecutive hearts (280 ± 42.1 g) was successful and no significant differences were found between working heart parameters at baseline and after 24 h of perfusion. There was no difference between initial and final measurements of LA mean pressures (5.0 ± 3.1 versus 9.0 ± 6.5 mm Hg, P = 0.22), left ventricular systolic pressures (44.3 ± 7.2 versus 39.1 ± 9.0 mm Hg, P = 0.13), mean aortic pressures (30.9 ± 5.8 versus 28.1 ± 8.1 mm Hg, P = 0.37), and coronary resistance (0.174 ± 0.046 versus 0.173 ± 0.066 mL/min/g, P = 0.90). There were also no significant differences between lactate (2.4 ± 0.5 versus 2.6 ± 0.4 mmol/L, P = 0.17) and glucose (173 ± 75 versus 156 ± 70 mg/dL, P = 0.37). A novel model using intermittent LA perfusion to create a coronary flow-only working heart mode for assessment of ex vivo cardiac function has been successfully developed.

Calcium handling dysfunction and cardiac damage following acute ventricular preload challenge in the dystrophin-deficient mouse heart.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and is caused by mutations in the dystrophin gene. Dystrophin deficiency is associated with structural and functional changes of the muscle cell sarcolemma and/or stretch-induced ion channel activation. In this investigation, we use mice with transgenic cardiomyocyte-specific expression of the GCaMP6f Ca2+ indicator to test the hypothesis that dystrophin deficiency leads to cardiomyocyte Ca2+ handling abnormalities following preload challenge. α-MHC-MerCreMer-GCaMP6f transgenic mice were developed on both a wild-type (WT) or dystrophic (Dmdmdx-4Cv) background. Isolated hearts of 3-7-mo male mice were perfused in unloaded Langendorff mode (0 mmHg) and working heart mode (preload = 20 mmHg). Following a 30-min preload challenge, hearts were perfused in unloaded Langendorff mode with 40 μM blebbistatin, and GCaMP6f was imaged using confocal fluorescence microscopy. Incidence of premature ventricular complexes (PVCs) was monitored before and following preload elevation at 20 mmHg. Hearts of both wild-type and dystrophic mice exhibited similar left ventricular contractile function. Following preload challenge, dystrophic hearts exhibited a reduction in GCaMP6f-positive cardiomyocytes and an increase in number of cardiomyocytes exhibiting Ca2+ waves/overload. Incidence of cardiac arrhythmias was low in both wild-type and dystrophic hearts during unloaded Langendorff mode. However, after preload elevation to 20-mmHg hearts of dystrophic mice exhibited an increased incidence of PVCs compared with hearts of wild-type mice. In conclusion, these data indicate susceptibility to preload-induced Ca2+ overload, ventricular damage, and ventricular dysfunction in male Dmdmdx-4Cv hearts. Our data support the hypothesis that cardiomyocyte Ca2+ overload underlies cardiac dysfunction in muscular dystrophy.NEW & NOTEWORTHY The mechanisms of cardiac disease progression in muscular dystrophy are complex and poorly understood. Using a transgenic mouse model with cardiomyocyte-specific expression of the GCaMP6f Ca2+ indicator, the present study provides further support for the Ca2+-overload hypothesis of disease progression and ventricular arrhythmogenesis in muscular dystrophy.

CARD16: Prolonged Normothermic Ex Vivo Heart Perfusion with Hemofiltration in Resting and Intermittent Working-heart Models

Introduction: Cold static storage (5C) or normothermic (37C) ex vivo heart perfusion (NEVHP) in the beating but resting mode (Langendorff) successfully preserves hearts for transplantation for up to 6 hours. Previous attempts to prolong the preservation period increases morbidity and mortality following transplantation. In addition, the 6-hour limit imposes geographic restrictions for recipients, prevents optimal organ assessment, and reduces the number of hearts available for transplantation. We have successfully maintained 10 consecutive pediatric hearts (60±8.8 g) for 24 hours using NEVHP with hemofiltration in the beating, resting-heart mode. Although this prolonged NEVHP model was successful, cardiac function could not be comprehensively assessed in the beating, resting-heart mode. We sought to expand our prolonged NEVHP model to include intermittent left atrial (LA) perfusion that would allow cardiac assessment in the working-heart mode. Methods: We performed 24-hour NEVHP on 5 consecutive hearts (276 ±22.6 g). Following anesthetic induction, sternotomy, cardioplegia administration, explantation, and back-table instrumentation, NEVHP was initiated in beating, resting mode. After 1 hour the circuit was transitioned to LA perfusion working-heart mode for 30 minutes, baseline working-heart parameters were documented (Figure 1) and perfusion was returned to beating, resting-heart mode. Intermittent LA perfusion with working-heart assessment was performed every 4-6 hours. Final working-heart measurements were obtained at 24 hours. Results: All hearts survived 24 hours and all maintained working-heart parameters statistically identical to baseline. Mean LA pressure was 8.2±7.5 mmHg, peak left ventricle (LV) pressure was 33.9±8.7 mmHg, mean LV pressure was 12.0±13.7 mmHg, peak aortic pressure was 35.6±7.8 mmHg, mean aortic pressure was 22.8±7.7 mmHg, coronary resistance 0.15±0.05 mmHg/L/min, and lactate levels were 2.7±0.6 mmol/L. Conclusions: We have successfully developed and validated a porcine NEVHP model for assessment of cardiac function in the working-heart mode using intermittent LA perfusion. While intermittent LA perfusion is an important step toward comprehensive ex vivo assessment of donor heart function, proof of cardiac preservation following prolonged NEVHP still requires successful transplantation and complete assessment in the recipient. We plan to perform this as our next step toward prolonged ex vivo heart preservation. Techniques and cannula for intermittent LA perfusion in the pediatric model will also be developed.

Ischemia Minimization Reduces Cardiac Xenograft Injury

<h3>Purpose</h3> Previous work has shown the benefit of ischemia minimization (IM) to reduce initial xenograft dysfunction. We examined cardiac injury in an <i>ex vivo</i> xenoperfusion model comparing IM with cold storage (CS). <h3>Methods</h3> Hearts from pigs expressing TKO.GHRKO (QKO) were compared to wild-type (WT) using an <i>ex vivo</i> model. Three QKO and 1 WT hearts were treated with IM for 2.5 hours following procurement. IM consisted of continuous cold cardioplegia. Two QKO and 2 WT hearts were placed in iced saline for the same time period. Following IM or CS hearts were Langendorff perfused with warm human blood then converted to working heart mode (WHM) if able. Total perfusion time was 4 hours. Troponin levels and physiologic function were periodically observed. <h3>Results</h3> Release of troponin was significantly reduced in the WT IM group (IM 185, CS>1000; p=0.02). A similar yet non-significant observation was noted in the QKO IM hearts (IM 750, CS>1000; p=0.37) (Fig.1). Two WT hearts (1 CS and 1 IM) were physiologically unable to reach WHM. One WT CS heart achieved WHM though failed at 3 hours (Fig.2F). Physiologic function was maintained in 2 of 3 QKO IM hearts (Fig 2A, B), and 1 of 2 QKO CS hearts (Fig. 2E). All WT hearts (2 CS and 1 IM (Fig. 2F)) and 2 of 5 QKO hearts (1 CS (Fig. 2D) and 1 IM (Fig. 2C)) failed within 4 hours. The 4 hour survival rate was not significantly improved by IM. <h3>Conclusion</h3> IM was shown to reduce cardiac injury in WT hearts and promote an increase in functional outcomes. This was a limited pilot study, however, the results were consistent with prior reported observations showing that IM reduced heart xenograft injury and dysfunction and support further evaluation of IM in <i>ex vivo</i> and <i>in vivo</i> models.

Ventricular Dysfunction and Calcium Handling Derangements in Isolated Hearts From Duchenne Muscular Dystrophy (DMD mdx‐4CV ) Mice Following Sustained Ventricular Preload

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and is caused by mutations in the dystrophin gene. Subclinical signs of cardiac disease present early and usually progress to dilated cardiomyopathy in late stage DMD patients. Dystrophin deficiency associates with stretch-induced myocyte calcium entry, adverse calcium overload, and cardiomyocyte damage. In this investigation we test the hypothesis that dystrophin deficiency leads to ventricular dysfunction and calcium handling abnormalities following sustained ventricular preload challenge. Hearts of 3-10 month male Control and Dmdmdx-4CV dystrophic mice were isolated, cannulated via both the aorta and left atrium, and perfused with physiological saline solution in working heart mode. Following elevation in preload to 20 mmHg, hearts of Control and Dmdmdx-4CV mice exhibited similar systolic maximum pressure development (PMax: 105±13 mmHg Control versus 102±7 mmHg Dmdmdx-4CV). However, while hearts of Control mice maintained systolic performance following the 30 minute pre-load challenge (PMax, 30 minutes - PMax, peak response: -6±3 mmHg, N=5), hearts of Dmdmdx-4CV mice exhibited a marked decline in systolic performance (PMax,30 minutes - PMax, peak response: -29±6 mmHg, N=7, P<0.01 vs. Control). The Dmdmdx-4CV hearts also had visible signs of damage following preload challenge. Using Control and Dmdmdx-4CV mice with transgenic, cardiac-specific expression of the GCaMP6f calcium indicator (MerCreMer-GCaMP6f), we examined incidence of calcium overload behavior following preload challenge. Isolated working hearts were subjected to 20 mmHg preload in working heart mode for 30 minutes, switched to Langendorff mode with physiological saline containing 40 uM blebbistatin, and imaged using laser-scanning confocal fluorescence microscopy. Calcium overload behavior was observed in 21% of cardiomyocytes of Dmdmdx-4CV hearts (n=30/140 cells equally sampled from N=4 hearts) compared to 1% of cardiomyocytes of Control hearts (n=2/140 cells from N=4 hearts). Taken together, these data indicate susceptibility to stretch-induced calcium overload, ventricular damage, and ventricular dysfunction in male Dmdmdx-4CV hearts.

Abstract 856: Mitochondrial Protein Kinase B (akt) Translocation Mediates Insulin-stimulated Cardiac Glucose Oxidation

Introduction: Insulin stimulates glucose oxidation, an effect which is associated with simulating pyruvate dehydrogenase (PDH). However, how the insulin signal is transduced from the cell membrane to the mitochondria to stimulate PDH is not known. Protein kinase B (Akt), protein kinase C-delta (PKCδ) and glycogen synthase kinase-3beta (GSK-3β) are main components of the cytosolic insulin signalling pathway and it has been suggested that they can be translocated to the mitochondria following insulin receptor activation in noncardiac tissue. Therefore, we investigated whether any of these kinases has a role mediated cardiac insulin-stimulated glucose oxidation in the heart. Methods and Results: Male and female C57BL/6 mice were anesthetized and hearts were collected and perfused in the isolated working heart mode. Hearts were perfused with [5- 3 H] glucose and [U- 14 C] glucose to simultaneously measure glycolysis and glucose oxidation rates, respectively, in the presence and absence of insulin. Insulin enhanced the phosphorylation and translocation of Akt Ser473 , PKCδ Tyr311 and GSK-3β Ser9 to the cardiac mitochondria along with enhancing PDH activity by decreasing its phosphorylation. Pharmacological inhibition of Akt using AktiVIII completely abolish the stimulatory effect of insulin on cardiac glucose oxidation rates (354 ± 145 vs 2307 ± 185 nmol. g dry wt -1 . min -1 in vehicle-treated hearts, p&lt;0.05). However, pharmacological inhibition of either PKCδ or GSK-3β using Bisindolylmaleimide I or 3F8, respectively, did not have a significant effect on insulin-stimulated glucose oxidation rates. None of the pharmacological inhibitors had any significant effect on cardiac glycolysis. Conclusion: Insulin mediates its stimulatory effect on cardiac glucose oxidation via a mechanism which involve the activation and translocation of Akt to the mitochondria.

Effect of an artificial ring on mitral valve function.

ABSTRACTDifferences of the effect of annuloplasty rings on the mitral annulus and leaflets, and differences between types of annuloplasty rings are not well known. We analyzed annular motion and leaflet movement with a rigid or flexible ring and without a ring using an isolated swine working heart model. Hearts of 10 swine (weight: 40–50 kg) were used for a rigid ring (n=5) and a flexible ring (n=5). Four ultrasound crystal tips were fixed around the annulus and an annuloplasty ring was implanted in the isolated heart. In the working heart mode, measurement of mitral annular dimension was acquired by sonomicrometry. Images of mitral valve motion were acquired by a high-speed video camera. The same analyses were performed after removing the artificial ring. The antero-posterior diameter of the diastole distance was significantly reduced in the flexible ring (21.59±0.71 mm) and rigid ring (15.93±1.88 mm) compared with no ring (23.51±2.01 mm). The flexible ring made the transverse diameter shrink significantly more than did the rigid ring. The contraction range of the transverse diameter was significantly smaller in the flexible ring compared with no ring. The duration of opening to closing of the mitral leaflet with the rigid (124.7±4.4 ms) and flexible rings (107.9±3.5 ms) was significantly shorter than that with no ring (168±36.5 ms). Annuloplasty rings allow simplicity of leaflet motion, regardless of the type of artificial ring. In a flexible ring, the mitral annulus shows a vertically long shape, suggesting preservation of posterior annular movement.

Multiple genetically modified GTKO/hCD46/HLA-E/hβ2-mg porcine hearts are protected from complement activation and natural killer cell infiltration during ex vivo perfusion with human blood.

In pig-to-human xenotransplantation, early cellular rejection reactions are mediated by natural killer cells (NK cells). Human NK cells are inhibited by HLA-E via CD94/NKG2A receptors. To protect porcine grafts against human NK cell responses, transgenic GTKO pigs expressing hCD46 and HLA-E have been generated. The aim of this study was to test the effect of this genetic modification on xenogeneic, and in particular human NK cell response, using an ex vivo perfusion model of pig hearts with human blood. Cardiopleged and explanted genetically modified (gm) pig hearts (GTKO/hCD46/HLA-E/hβ2-microglobulin) and wild-type (wt) controls (n=6 each) were reperfused and tested in an 8hours ex vivo perfusion system using freshly drawn human blood. Cardiac function was evaluated during a 165-minute period in working heart mode. Myocardial damage, antibody deposition, complement activation, and coagulation parameters were evaluated histologically at the end of perfusion. The number of NK cells in the perfusate was determined by flow cytometry at baseline and at 8hours; tissue infiltration by NK cells was quantified by immunofluorescence microscopy using NKp46 staining of frozen sections. Deposition of IgG (1.2±1×107 vs 8.8±2.9×106 ; P<.01), IgM (4.4±3.7×106 vs 1.7±1.2×106 ; P<.01), and the complement activation product C4b/c (3.5±1.3×106 vs 2.3×106 ±9.4×105 ; P>.01) was lower in gm than wt hearts. NK cell percentages of leukocytes in the perfusate decreased from 0.94±0.77% to 0.21±0.25% (P=.04) during xenoperfusion of wt hearts. In contrast, the ratio of NK cells did not decrease significantly in the gm hearts. In this group, NK cell myocardial infiltration after 480minutes of perfusion was lower than in wt organs (2.5±3.7×104 /mm3 vs 1.3±1.4×105 /mm3 ; P=.0001). The function of gm hearts was better preserved compared to wt organs, as demonstrated by higher cardiac index during the first 2hours of ex vivo perfusion. GTKO, hCD46, and HLA-E expression in porcine hearts reduced complement deposition, complement dependent injury, and myocardial NK cell infiltration during perfusion with human blood. This tested combination of genetic modifications may minimize damage from acute human-anti-pig rejection reactions and improve myocardial function after xenotransplantation.

Complement C3 inhibitor Cp40 attenuates xenoreactions in pighearts perfused with human blood.

The complement system plays a crucial role in acute xenogeneic reactions after cardiac transplantation. We used an ex vivo perfusion model to investigate the effect of Cp40, a compstatin analog and potent inhibitor of complement at the level of C3. Fifteen wild-type pig hearts were explanted, cardiopleged, and reperfused ex vivo after 150minutes of cold ischemia. Hearts were challenged in a biventricular working heart mode to evaluate cardiac perfusion and function. In the treatment group (n=5), the complement cascade was blocked at the level of C3 using Cp40, using diluted human blood. Untreated human and porcine blood was used for controls. Throughout the perfusion, C3 activation was inhibited when Cp40 was used (mean of all time points: 1.11±0.34% vs 3.12±0.48% control activation; P<.01). Compared to xenoperfused controls, the cardiac index improved significantly in the treated group (6.5±4.2 vs 3.5±4.8mL/min/g; P=.03, 180minutes perfusion), while the concentration of lactate dehydrogenase as a maker for cell degradation was reduced in the perfusate (583±187U/mL vs 2108±1145U/mL, P=.02). Histological examination revealed less hemorrhage and edema, and immunohistochemistry confirmed less complement fragment deposition than in untreated xenoperfused controls. Cp40 efficiently prevents C3 activation of the complement system, resulting in reduced cell damage and preserved function in wild-type porcine hearts xenoperfused ex vivo. We suggest that this compstatin analog, which blocks all main pathways of complement activation, could be a beneficial perioperative treatment in preclinical and in future clinical xenotransplantation.

Preload-Induced Changes in Systolic and Diastolic Performance in the Young and Aged Murine Heart

The aged heart exhibits modifications limiting cardiac performance. The purpose of this study was to test the hypothesis that aged hearts exhibit impaired systolic and diastolic function in response to elevated preload. To test this, hearts of Young (3-6 month) and Aged (22-27 month) C57Bl/6 mice were excised and cannulated via aorta and left atrium. In working heart mode (8 Hz stimulation), defined preloads were applied via atrial cannula while peak pressure (mmHg), rate of pressure development (+dP/dt), and rate of pressure decay (-dP/dt) were monitored via intraventricular pressure catheter. Hearts underwent progressive preload increases from 5-20 mmHg, followed by preload challenge (30 mmHg, 20 minutes).Young hearts (6/6) responded to greater preloads (5-20 mm Hg) with increased pressure (from 73±4 to 96±7 mmHg), rate of development (from 2542±247 to 3628±328 mmHg/s), and rate of decay (from −2194±290 to −3370±368 mmHg/s). In contrast, divergent responses were observed in Aged, one population (5/9) increasing in peak pressure (from 67±5 to 109±8 mmHg) and rates (+dP/dt 2384±184 to 4317±347 mmHg/s, -dP/dt −1476±244 mmHg/s to −3783±275 mmHg/s)(i.e., positive responsive) and a second population (4/9) decreasing in peak pressure (from 81±10 to 75±9 mmHg) and rates (+dP/dt 2653±295 to 2378±378 mmHg/s, -dP/dt −1972±462 to −1914±716 mmHg/s)(i.e., negative responsive).Interestingly, during sustained preload challenge, positive responsive Aged exhibited greater decline in rate of decay (−3402±510 initial to −2457±336 mmHg/s final) compared to Young (−3023±328 initial to −2758±362 mmHg/s final) and negative responsive Aged (−1450±532 initial to −1325±433 mmHg/s final).In summary, our data suggest the Aged heart manifests two forms of diastolic dysfunction; one characterized by an inability to respond to preload and a second by the capacity to respond to preload with augmented hemodynamics but inability to sustain diastolic function.

A LABORATORY MODEL OF ORGAN DONOR CIRCULATORY DEATH WITH DIRECT MEASUREMENT OF CARDIAC GRAFT FUNCTION

At present, the only clinical pathway for cardiac transplantation involves the use of an organ donor who meets criteria for neurologic death. Unfortunately, there is a consistent shortage of donor hearts despite a growing number of potential transplant recipients. This supply-demand mismatch is associated with significant waitlist mortality. An alternative to neurologic death, referred to as organ donation after circulatory death (DCD), is currently in use for lung, liver and kidney transplantation. The DCD clinical pathway, however, is associated with a variable and unpredictable duration of donor hypoxia and hypotension, culminating in 5 minutes of total circulatory standstill. As a result of the heart’s unique susceptibility to injury under these conditions, cardiac grafts from DCD donors are currently wasted. Sprague-Dawley rats are deeply anesthetized, tracheotomized and mechanically ventilated. Rats are systemically anticoagulated with unfractionated heparin. Control subjects receive a median sternotomy. The heart is harvested and flushed with ice-cold cardioplegic solution prior to functional assessment. DCD subjects receive invasive blood pressure monitoring via the left femoral artery. Subsequently, a median sternotomy is performed and both pleural spaces are opened. The DCD pathway is initiated by discontinuing mechanical ventilation. Vital sign parameters are monitored regularly until the graft is harvested. When asystole is achieved, 5 minutes of circulatory standstill is observed (to mimic the clinical requirement) prior to harvesting and flushing the cardiac graft. Cardiac grafts are reperfused on a Langendorff rig. After 10 minutes of reperfusion, the rig is switched into a working-heart mode. The left ventricle is catheterized with a calibrated pressure-volume conductance catheter to generate baseline and preload occlusion data. We have established a clinically relevant rodent model of donor circulatory death. Furthermore, we have coupled our DCD protocol with direct phyiosologic assessment of cardiac function, demonstrating a significant difference between control and DCD subjects. Vital sign parameters recorded during the DCD protocol have enabled us to crudely correlate the extent and duration of donor instability with cardiac graft function. Our model of DCD with direct measurement of cardiac graft function is important and clinically relevant. It will be valuable in determining the limits of cardiac viability in the DCD setting, and also in assessing the ability of pre- and post-DCD treatment strategies to salvage cardiac function.

Glucagon and a glucagon‐GLP‐1 dual‐agonist increases cardiac performance with different metabolic effects in insulin‐resistant hearts

The prevalence of heart disease continues to rise, particularly in subjects with insulin resistance (IR), and improved therapies for these patients is an important challenge. In this study we evaluated cardiac function and energy metabolism in IR JCR:LA-cp rat hearts before and after treatment with an inotropic compound (glucagon), a glucagon-like peptide-1 (GLP-1) receptor agonist (ZP131) or a glucagon-GLP-1 dual-agonist (ZP2495). Hearts from IR and lean JCR:LA rats were isolated and perfused in the working heart mode for measurement of cardiac function and metabolism before and after addition of vehicle, glucagon, ZP131 or ZP2495. Subsequently, cardiac levels of nucleotides and short-chain CoA esters were measured by HPLC. Hearts from IR rats showed decreased rates of glycolysis and glucose oxidation, plus increased palmitate oxidation rates, although cardiac function and energy state (measured by ATP/AMP ratios) was normal compared with control rats. Glucagon increased glucose oxidation and glycolytic rates in control and IR hearts, but the increase was not enough to avoid AMP and ADP accumulation in IR hearts. ZP131 had no significant metabolic or functional effects in either IR or control hearts. In contrast, ZP2495 increased glucose oxidation and glycolytic rates in IR hearts to a similar extent to that of glucagon but with no concomitant accumulation of AMP or ADP. Whereas glucagon compromised the energetic state of IR hearts, glucagon-GLP-1 dual-agonist ZP2495 appeared to preserve it. Therefore, a glucagon-GLP-1 dual-agonist may be beneficial compared with glucagon alone in the treatment of severe heart failure or cardiogenic shock in subjects with IR.

Update on Lung Transplantation using Donation after Cardiac Death in NSW

P< 0.05) with hydraulic permeability and burst pressure equal to autologous conduits (HP: IMA 0ml/min/cm2, EC 0 P=ns; BP: IMA 2267 mmHg, EC 1922 P=ns). Fluorescence microscopy confirmed endothelial proliferation on electrospun fibres, and thrombogenicity studies demonstrated reducedplatelet attachment on rHE scaffolds. Discussion: Synthetic coronary conduits constructed using rHE/PCL fibres match the mechanical properties of the IMA. In addition, these scaffolds exhibit reduced thrombogenicity and the ability to support endothelialisation. The elastic conduits will be implanted as carotid artery interposition grafts in rabbits for assessment in vivo before ultimate study as coronary bypass graft candidates. These novel conduits show great potential as a synthetic internal mammary artery, the “Holy Grail” of coronary revascularisation surgery. doi:10.1016/j.hlc.2010.10.034 Update on Lung Transplantation using Donation after Cardiac Death in NSW E. Granger ∗, P. Spratt, M. Harkess, P. Jansz, K. Dhital, P. Macdonald, M. Malouf, A. Glanville The Lung TransplantUnit, St. Vincent’s Hospital, Darlinghurst, ever it will probably augment transplant numbers by only 15%. Current results are excellent despite “warm ischaemia”. Donors with longer warm ischaemic times and more “marginal” gas exchange may now be considered. doi:10.1016/j.hlc.2010.10.035 Beating Heart Preservation of Donor Hearts for Transplantation—A Working Heart Perfusion System for Pig Hearts to Extend Organ Preservation Time P. Pritzwald-Stegmann1,∗, J. Passage1,2, T. Muller 2, A. Muhle2, S. Krabbes1,2, S. Dhein2, F.-W. Mohr2 1Monash Medical Centre, Clayton, Australia Q4 2 Heart Centre Leipzig, Leipzig, Germany Introduction:Donor heart ischaemic time places significant geographic constraints on organ retrieval. Constant pressure perfusion systems can double current time limits in a pig model of heart transplantation from 4 to 8h, but the yield of successfully preserved hearts is low. The aim of this studywas to develop aworking heart perfusion system that would extend donor heart preservation time and allow the subsequent assessment of organ function post reperfusion. Methods: Beating hearts were harvested from 61± 3 kg pigs (n= 6) andperfused via a double lumen cannula using Sydney, NSW, Australia Introduction: Donation after cardiac death (DCD) from Maastrict category 3 donors consists of approximately 12% of lung transplant activity in New South Wales. We review the results of DCD lung transplants and discuss the future direction of the program at St. Vincent’s Hospital. Methodology: Since commencing in November 2007 there have been 25 referrals for DCD, leading to 11 attempted retrievals. Only 9 retrievals resulted in lung donation. Elective withdrawal of life-sustaining support and certification of death (limit of 60min fromwithdrawal) occurred in the Intensive Care Unit or Operating Theatre. Results: Average recipient age was 35 years (range 15–61) and aetiology of respiratory failure included cystic fibrosis (2), bronchiectasis (1), bronchiolitis obliterans (1), emphysema (3), interstitial lung disease (1) and pulmonary hypertension (1). The donor group average age was 37 years (range 14–56) and average PaO2/FiO2 ratio 412 (±51). Causes of death included subarachnoid haemorrhage (2), Traumatic head injury (2), hypoxic brain injury (3) and embolic CVA (2). Mean time to asystole after withdrawal of treatment was 27min (range 9–57), mean warm ischaemic time 19min (range 10–31) and mean cold ischaemic time (left lung) 279min (range 241–320). Early gas exchange was excellent: an average PaO2/FiO2 of 324 (141–503). Average intensive care stay was 5 days (3–10) and length of stay 23 days (12–36). All patients are still alive. Conclusion: Organ donation remains at a challenging low level in Australia. Donation after cardiac death may increase the “donor organ pool”, howaLangendorff-basedperfusioncircuit.Workingheart conditionswere created, at physiological perfusion pressures. After 7 hours, the hearts were arrested with cold HTK cardioplegia solution and underwent 1 hour of warm ischaemia. The hearts were subsequently reperfused in a non-working state at constant pressure for 1 hour and then weaned to again perform under the original working heart conditions. Pmax, max dp/dt, min dp/dt were monitored throughout. Tissue adenosine triphosphate (ATP) and malondialdehyde (MDA) were measured to assess myocardial energy content and oxidative stress respectively. Induction of apoptosis was assessed by staining for AIF. Results: All 6 hearts were successfully harvested, preserved and weaned following reperfusion. Pmax achieved during the working heart mode was higher than in the donor animal (142 vs 86mmHg, p= 0.002). There was no significant overall deterioration in systolic (max dp/dt 2030 vs 1812mmHg/s, p= 0.521) or diastolic (min dp/dt 1200 vs 1225mmHg/s, p= 0.902) function throughout preservation. ATP stores recovered to pre harvest levels (23.6 vs 22.1 pmol/ g, p= 0.57) and oxidative stress was well controlled (1.6 vs 1.8 mol/g, p= 0.926). Apoptosis levels were comparable to hearts that underwent conventional 4 h hypothermic arrest prior to transplantation (15.7 vs 12.7/hpf, p= 0.741). Discussion: A working heart perfusion system can be utilised to successfully increase donor heart preservation times. The system allows the real-time monitoring of donor organ function and canmimic the ischaemia and reperfusion events that occur during transplantation. It permits the specific modification of preservation parame-

PBSH: A New Improved Cardiac Preservation Solution in Comparison With Three Clinically Proven Solutions

Introduction A solution under development at our institute, based on phosphate-buffered sucrose, has shown good preservation for kidneys and livers. This work used a refined version of this solution (PBSH—phosphate-buffered sucrose for the heart) in heart preservation, comparing it to solutions already widely used (University of Wisconsin, St. Thomas's, and Celsior solutions). Methods Following an initial washout phase and control working mode on a Langendorff system, hearts were flushed with preservation solution and after 6 hours at 4°C were then reperfused for 15 minutes followed by working heart mode for a further 30 minutes. Hemodynamic parameters were measured and compared with their preischemic values and expressed as percentage recovery. Enzyme measurement came from the collection of the initial 1.5 mL of the coronary effluent after the storage period. This was to test for creatine phosphokinase (CPK) and lactate dehydrogenase (LDH). Hearts were then placed immediately into liquid nitrogen for adenosine triphosphate (ATP), lactate, and creatine phosphate (CP) testing. Spectrophotometric analysis was used to assess both the release of CPK and LDH into the coronary effluent, and the level of ATP, lactate, and CP in the frozen heart tissue. Results These results show that hearts that are preserved in PBSH are hemodynamically as well preserved as the hearts preserved in other solutions tested and their enzyme and lactate content is lower while having higher levels of energy compounds in these hearts. Conclusion Overall these results show that PBSH is at least as effective in cardiac preservation in the rat model of 6 hours of cold ischemia as these other widely used solutions tested.

Inadvertent phosphorylation of survival kinases in isolated perfused hearts: a word of caution

The isolated perfused heart is an important model in cardiovascular research. We hypothesized that the perfusion procedure per se will phosphorylate some protein kinases important in pre- and postconditioning. Isolated hearts were Langendorff-perfused for 20 min with or without an intraventricular balloon (rats and mice), or in the working heart mode (mice) and compared to non-perfused controls with respect to protein phosphorylation. Rat hearts were also perfused for 20 and 50 min in the Langendorff mode to investigate the effect of perfusion time on phosphorylation. Western blot analysis showed that perfusion per se induced a massive phosphorylation of ERK 1/2, P38-MAPK, JNK, AMPK, but decreased phosphorylation of AKT in the isolated rat and mouse heart. However, during ongoing perfusion the phosphorylation of these kinases was reduced. Langendorff-perfusion without the intraventricular balloon caused less phosphorylation of ERK 1/2, P38-MAPK and JNK, but had no effect on AMPK. In working hearts phosphorylation of kinases was similar to that of Langendorff-perfused hearts without the balloon. Our findings indicate that excising, handling and perfusion induce a time dependent phosphorylation of stress kinases. The presence of the intraventricular balloon caused the strongest phosphorylation, thus Langendorff-perfused hearts might be partly protected by the perfusion procedure if stress kinases are protective in pre- and postconditioning. This might explain conflicting results obtained with different models of both pre- and postconditioning, and the isolated heart might in some situations be suboptimal for such studies.

Criteria for the multiple use of isolated perfused hearts in electrophysiological and metabolic experiments / Kriterien für die Mehrfachnutzung von isoliert perfundierten Schweineherzen für elektrophysiologische und metabolische Untersuchungen

Both ethical and economic restrictions limit the availability of porcine hearts for in vitro perfusion experiments. Therefore, we tested the feasibility of multiple use of in vitro perfused working hearts for electrophysiological and metabolic investigations. Pig hearts (n=7) rejected for originally planned haemodynamic measurements because of exclusion criteria were perfused in a four-chamber working heart mode. All hearts were kept in steady-state conditions on a low haemodynamic level over 2 h during 75-channel ECG recordings and NADH fluorescence measurements before and after norepinephrine (NE) was administered. QRS and QT interval durations were in a range comparable to in vitro studies and, like QRS and T amplitudes, were found to be sensitive markers of the changing condition of the isolated heart preparation, as myocardial oedema leads to prolonged QRS and QT intervals and declining ECG voltage amplitudes. A change in NADH fluorescence following NE administration was observed in the first 150 min of perfusion, but not later. Considering a time frame of 120 min, multiple use of isolated perfused porcine hearts with low-level haemodynamics may allow a broad spectrum of investigations and could therefore represent a possibility of overcoming the restricted availability of porcine hearts.

Soluble Galα(1,3)Gal conjugate combined with hDAF preserves morphology and improves function of cardiac xenografts

Cytotoxic anti-Galalpha(1,3)Gal antibodies play a key role in the rejection of pig organs transplanted into primates. Regimens reducing anti-Galalpha(1,3)Gal antibodies were associated with severe side effects unable to prevent antibody rebound until soluble synthetic oligosaccharides with terminal Galalpha(1,3)Gal inhibiting antigen binding became available. We displayed kinetics of anti-pig and anti-Galalpha(1,3)Gal IgM and IgG antibody levels using GAS914, a Galalpha(1,3)Gal trisaccharide conjugated to poly-l-lysine, and investigated corresponding changes of parameters of heart function. Using a working heart model, hDAF pig hearts were perfused with human blood containing GAS914 (group 1). As controls hDAF pig hearts (group 2) and landrace pig hearts (group 3) were perfused with human blood only. Levels of anti-Galalpha(1,3)Gal (IgM, IgG) and anti-pig antibodies were assessed to prove the effectiveness of GAS914. As parameters of heart function, cardiac output (CO), stroke work index (SWI), coronary blood flow (CBF) and coronary resistance were measured. Creatine phosphokinases, lactate dehydrogenase and aspartate aminotransferase were evaluated as markers of myocardial damage. Histological and immunohistochemical investigations were performed at the end of perfusion. In group 1 an immediate and extensive reduction in both IgM and IgG anti-Galalpha(1,3)Gal was found. Anti-pig antibodies were eliminated accordingly. Antibody binding to GAS914 was complete before the start of organ perfusion. Corresponding to rapid antibody elimination in group 1 GAS914 not only was able to significantly prolong the beating time of the heart in hDAF pigs, but also to clearly improve functional parameters. When switching to the working heart mode hDAF pig hearts perfused with human blood containing GAS914 (group 1) revealed a CO starting at a significantly higher level than hDAF (group 2) and non-transgenic pig hearts (group 3) perfused with human blood only. Similarly, in group 1 SWI was significantly increased at the beginning of perfusion compared to that of group 2 and group 3. The increase in CBF during perfusion and the corresponding fall of coronary resistance occurred without significant differences between the groups revealing the independence of hDAF and GAS914. Due to an immediate and profound reduction in Galalpha(1,3)Gal-specific antibodies, soluble Galalpha(1,3)Gal conjugates not only prolong survival, but also improve the hemodynamic performance of the heart in DAF pigs.

Exacerbation of acidosis during ischemia and reperfusion arrhythmia in hearts from type 2 Diabetic Otsuka Long-Evans Tokushima Fatty rats

BackgroundSensitivity to ischemia and its underlying mechanisms in type 2 diabetic hearts are still largely unknown. Especially, correlation between reperfusion induced ventricular arrhythmia and changes in intracellular pH has not been elucidated.Methods and resultsMale Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 16 and 32 weeks of age were used along with age-matched nondiabetic Long-Evans Tokushima Otsuka (LETO) rats. Hearts from rats in these 4 groups were perfused in the working heart mode, thus inducing whole heart ischemia. At 16 weeks of age, no differences in blood glucose levels or incidence and duration of reperfusion arrhythmia were found between the strains. At 32 weeks of age, both impaired glucose tolerance and obesity were observed in the OLETF rats. Further, the duration of reperfusion-induced ventricular fibrillation (VF) was significantly longer in the OLETF rats, while the pH level was significantly lower and proton contents were significantly higher in coronary effluent during ischemia in those rats. Following treatment with troglitazone, improvements in pH and proton level in coronary effluent during ischemia were observed, as was the duration of reperfusion-induced VF in OLETF rats at 32 weeks of age.ConclusionThe hearts of spontaneously diabetic OLETF rats were found to be more susceptible to ischemic insult. Troglitazone treatment improved ischemic tolerance by improving glucose metabolism in the myocardium of those rats.

Efficacy of ischaemic preconditioning in the eNOS overexpressed working mouse heart model

We recently demonstrated that exogenous nitric oxide (NO) acts as a trigger for preconditioning in the isolated rat heart model. There is however little data concerning the effects of elevated cardiac endothelial nitric oxide synthase (eNOS) expression on myocardial tolerance to ischaemia. Similarly, the effects of gender and eNOS overexpression on ischaemic preconditioning is unknown. We hypothesized that: 1) eNOS overexpression increases myocardial tolerance to ischaemia, and, 2) eNOS overexpressed hearts cannot be preconditioned, since the hearts are already maximally protected. Male and female wild-type and transgenic mice that overexpress eNOS exclusively in cardiac myocytes were perfused in the working heart mode with a modified Krebs–Henseleit buffer at a pre-load of 12.5 mm Hg and afterload of 50 mm Hg. Cardiac output, coronary flow, peak aortic systolic pressure and total work were determined before hearts were preconditioned by 4 × 5 min cycles of ischaemia/reperfusion, and then subjected to 20 min total global ischaemia, followed by reperfusion. Reperfusion function and myocardial infarct size were used as endpoints. Pre-ischaemic mechanical function (rate pressure product and cardiac output) was similar for wild-type and transgenic mice of both sexes. The eNOS overexpressed hearts had smaller infarcts than the hearts from their wild-type littermates (26.9 ± 1.4% vs. 37.0 ± 2.1% for controls, P < 0.05). Preconditioning the eNOS overexpressed hearts resulted in infarct sizes comparable with control non-preconditioned hearts (27.5 ± 2.0% vs. 26.9 ± 1.4% for controls). Myocardial cGMP levels were elevated during sustained ischaemia in the transgenic hearts when compared with wild-type hearts (22.43 ± 1.63 pmol/g ww vs 16.54 ± 1.48 pmol/g ww, P < 0.05). Preconditioning also elevated myocardial cGMP levels during sustained ischaemia in the wild-type hearts (26.77 ± 2.81 pmol/g ww, P < 0.05). We conclude that: 1) basal mechanical function is similar for both wild-type and transgenic mice of both sexes, 2) reperfusion function and infarct size was also similar for both sexes under both control conditions and after preconditioning, 3) the transgenic mice are more tolerant of ischaemia as reflected by their smaller myocardial infarcts, and, 4) the eNOS overexpressed mouse heart cannot be preconditioned regardless of whether mechanical function or infarct size is used as an end-point. These hearts may be maximally protected against ischaemia/reperfusion injury by their elevated endogenous NO levels.

Pressure–volume relationship in isolated working heart with crystalloid perfusate in swine and imaging the valve motion

It is widely accepted that both valve and cardiac functions are closely correlated. In order to investigate the relationship between the valve and cardiac functions, we developed a model of an isolated working heart with crystalloid perfusate in swine. We investigated the feasibility of this model to evaluate the precise left ventricular function using the pressure-volume relationship and metabolic measurement. Another objective was as a trial for the imaging and analysis of valvular interventions with a high-speed digital camera on this model. Six isolated working hearts were subjected in the pressure-volume study, and additional three hearts were used in the valve imaging study. Measurement of the pressure-volume relationship was undertaken in situ before the heart was removed, and on the working heart mode during the initial 30 min as the baseline, and at every 60 min. Lactate levels were measured at every stage in the working heart mode. Mitral valve interventions were performed in three hearts, and valve motions were observed by a high-speed digital camera via the left ventricle. The end-systolic elastance maintained a baseline level (5.17+/-2.25) until 180 min and decreased at 240 min (3.97+/-1.97, NS) and 300 min (2.85+/-1.29, P<0.01) of the working heart mode as compared with baseline. The Tau maintained a constant level until 180 min and increased at 240 min (62.2+/-13.3, NS) and 300 min (85.5+/-43.4, P<0.05) as compared with baseline (50.3+/-13.6). The slope of the end-diastolic pressure-volume relationship gradually increased with no significance until 180 min and increased significantly (0.147+/-0.066, P<0.01 vs 0.067+/-0.041 at baseline). Lactate increased accumulatively. The total heart energy was reduced from the initial phase of the working heart mode. The valves were well captured by the high-speed digital camera. The systolic and diastolic functions of an isolated heart were preserved at an acceptable level for 180 min. The practical reliability of the swine working heart model was demonstrated. This model will be used reliably for the investigation of the interaction of valve and cardiac functions.