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Goldstandard bei terminaler Herzinsuffizienz bleibt die Herztransplantation. Der massive Mangel an Spenderherzen zwingt Transplantationszentren, altere Spender und langere Ischamiezeiten zu akzeptieren. Die Behandlung mit mesenchymalen Stammzellen (MSC) oder deren konditioniertem Medium (CM) schutzt das Herz vor Ischamie-Reperfusion-Schaden („ischemia-reperfusion injury“, IRI). Zu prufen war, ob die Perfusion von Spenderherzen 15 Monate alter Ratten mit CM bei langerer Konservierung vor IRI schutzt. Es wurden Ratten-MSC isoliert und kultiviert. Das CM enthielt 28 Faktoren, die wahrend Apoptose, Inflammation und oxidativem Stress eine Rolle spielen. Herzen von 15 Monate alten Ratten wurden explantiert und kontinuierlich fur 5 h mit oxygenierter 4 °C kalter Kardioplegielosung perfundiert, die entweder mit normalem Zellkulturmedium (Kontrollgruppe) oder CM (CM-Gruppe) erganzt war. Die Herzen wurden heterotop transplantiert und 1,5 h nach der Transplantation wurde die linksventrikulare Transplantatfunktion in vivo und die myokardiale Expression von 120 Genen mithilfe von PCR-Arrays untersucht. Die systolischen Kontraktions- und Relaxationsparameter waren bei 15 Monate alten Ratten im Vergleich zu jungen Ratten signifikant verringert. Nach der Transplantation waren die systolische und die diastolische Funktion in der CM- im Vergleich zur Kontrollgruppe signifikant verbessert. Die myokardiale Expression von 66 Genen war verandert. Gene des proinflammatorischen Zytokins und Interleukin waren herunterreguliert, wahrend das Superoxiddismutase-2-Antioxidans-Gen in den CM-behandelten Transplantaten im Vergleich zur Kontrollgruppe hochreguliert wurde. Die Perfusion von 15 Monate alten Rattenspenderherzen mit CM hat einen vor myokardialen IRI schutzenden Effekt.

Hearts are usually procured from brain-dead (BD) donors. However, brain death may induce hemodynamic instability, which may contribute to posttransplant graft dysfunction. We hypothesized that BD-donor heart preservation with a conditioned medium (CM) from mesenchymal stem cells (MSCs) would improve graft function after transplantation. Additionally, we explored the PI3K pathway's potential role. Rat MSCs-derived CM was used for conservation purposes. Donor rats were either exposed to sham operation or brain death by inflation of a subdural balloon-catheter for 5.5 hours. Then, the hearts were explanted, stored in cardioplegic solution-supplemented with either a medium vehicle (BD and sham), CM (BD + CM), or LY294002, an inhibitor of PI3K (BD + CM + LY), and finally transplanted. Systolic performance and relaxation parameters were significantly reduced in BD-donors compared to sham. After transplantation, systolic and diastolic functions were significantly decreased, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells and endonuclease G positive cells were increased in the BD-group compared to sham. Preservation of BD-donor hearts with CM resulted in a recovery of systolic graft function (dP/dtmax : BD + CM: 3148 ± 178 vs BD: 2192 ± 94 mm Hg/s at 110 µL, P < .05) and reduced apoptosis. LY294002 partially lowered graft protection afforded by CM in the BD group. Our data suggest that PI3K/Akt pathway is not the primary mechanism of action of CM in improving posttransplant cardiac contractility and preventing caspase-independent apoptosis.

Demand for organs is increasing while the number of donors remains constant. Nevertheless, not all organs are utilized due to the limited time window for heart transplantation (HTX). Therefore, we aimed to evaluate whether an iron-chelator-supplemented Bretschneider solution could protect the graft in a clinically relevant canine model of HTX with prolonged ischemic storage. HTX was performed in foxhounds. The ischemic time was standardized to 4 h, 8 h, 12 h or 16 h, depending on the experimental group. Left ventricular (LV) and vascular function were measured. Additionally, the myocardial high energy phosphate and iron content and the in-vitro myocyte force were evaluated. Iron chelator supplementation proved superior at a routine preservation time of 4 h, as well as for prolonged times of 8 h and longer. The supplementation groups recovered quickly compared to their controls. The LV function was preserved and coronary blood flow increased. This was also confirmed by in vitro myocyte force and vasorelaxation experiments. Additionally, the biochemical results showed significantly higher adenosine triphosphate content in the supplementation groups. The iron chelator LK614 played an important role in this mechanism by reducing the chelatable iron content. This study shows that an iron-chelator-supplemented Bretschneider solution effectively prevents myocardial/endothelial damage during short- as well as long-term conservation.

OBJECTIVES HTK-Solution (Custodiol) is a well-established cardioplegic and organ preservation solution. We currently developed a novel HTK-based solution, Custodiol-N, which includes iron chelators to reduce oxidative injury, as well as l-arginine, to improve endothelial function. In this first-in-human study, Custodiol-N was compared to Custodiol in patients undergoing elective coronary artery bypass surgery. The aim of this comparison was to evaluate the safety and ability of Custodiol-N to protect cardiac tissue. METHODS The study was designed as a prospective randomized double-blind non-inferiority trial. Primary end point was area under the curve (AUC) of creatine kinase muscle-brain (CK-MB) within the first 24 h after surgery. Secondary end points included peak CK-MB and troponin-T and AUC of troponin-T release, cardiac index, cumulative catecholamine dose, intensive care unit stay and mortality. All values in the abstract are given as mean ± SD, P RESULTS Early termination of the trial was performed per protocol as the primary non-inferiority end-point was reached after inclusion of 101 patients. CK-MB AUC (878±549 vs 779±439 h U/l, non-inferiority P CONCLUSIONS This study shows that Custodiol-N is safe and provides similar cardiac protection as the established HTK-Custodiol solution. Significantly reduced peak CK-MB levels in the Custodiol-N group in the full analysis set may implicate a beneficial effect on ischaemia/reperfusion injury in the setting of coronary bypass surgery.

Ischemia/reperfusion injury (IRI) remains a challenge in coronary artery bypass grafting (CABG). Diabetic patients with coronary artery disease are more likely to require CABG and therefore run a high risk for cardiovascular complications. Conditioned medium (CM) from bone marrow-derived mesenchymal stem cells has been shown to have beneficial effects against IRI. We hypothesized that adding CM to physiological saline protects vascular grafts from IRI in diabetic rats. Bone-marrow derived cells were isolated from nondiabetic rat femurs/tibias, and CM was generated. As we previously reported, CM contains 23 factors involved in inflammation, oxidative stress, and apoptosis. DM was induced by streptozotocin administration. Eight weeks later, to measure vascular function, aortic rings were isolated and mounted in organ bath chambers (DM group) or stored in 4°C saline, supplemented either with a vehicle (DM-IR group) or CM (DM-IR+CM group). Although DM was associated with structural changes compared to controls, there were no functional alterations. However, compared to the DM group, in the DM-IR aortas, impaired maximum endothelium-dependent vasorelaxation in response to acetylcholine (DM 86.7 ± 0.1% vs. DM-IR 42.5 ± 2.5% vs. DM-IR+CM 61.9 ± 2.0%

An increasing organ demand is facing a constant number of donors. Nevertheless, not all organs are utilized due to a limited time window for heart transplantation (HTX). Therefore, we aimed to evaluate, if iron chelator supplemented Bretschneider solution can protect the graft in a clinically relevant canine model of HTX with prolonged ischemic storage.HTX was performed in foxhounds. The ischemic time was standardized to 4h, 8h, 12h or 16h depending on the experimental group. Left ventricular (LV) and vascular function were measured. Additionally, myocardial high energy phosphate and iron content and in-vitro myocyte force were evaluated.Iron chelator supplementation proved superior at a routine preservation time of 4h as well as for prolonged times of 8h and longer. The supplementation groups recovered quickly compared to their controls. The LV function was preserved and coronary blood flow increased. This was also confirmed by in vitro myocyte force and vasorelaxation experiments. Additionally, the biochemical results showed significantly higher adenosine-triphosphate content in supplementation groups. The iron chelator LK614 played an important role in this mechanism by reducing the chelatable iron content.This study shows that iron chelator supplemented Bretschneider solution effectively prevents myocardial/endothelial damage during short as well as long term conservation.

Vascular ischemia/reperfusion injury (IRI) contributes to graft failure and adverse clinical outcomes following coronary artery bypass grafting. Sodium-glucose-cotransporter (SGLT)-2-inhibitors have been shown to protect against myocardial IRI, irrespective of diabetes. We hypothesized that adding canagliflozin (CANA) (an SGLT-2-inhibitor) to saline protects vascular grafts from IRI. Aortic rings from non-diabetic rats were isolated and immediately mounted in organ bath chambers (control, n = 9–10 rats) or underwent cold ischemic preservation in saline, supplemented either with a DMSO vehicle (IR, n = 8–10 rats) or 50µM CANA (IR + CANA, n = 9–11 rats). Vascular function was measured, the expression of 88 genes using PCR-array was analyzed, and feature selection using machine learning was applied. Impaired maximal vasorelaxation to acetylcholine in the IR-group compared to controls was significantly ameliorated by CANA (IR 31.7 ± 3.2% vs. IR + CANA 51.9 ± 2.5%, p &lt, 0.05). IR altered the expression of 17 genes. Ccl2, Ccl3, Ccl4, CxCr4, Fos, Icam1, Il10, Il1a and Il1b have been found to have the highest interaction. Compared to controls, IR significantly upregulated the mRNA expressions of Il1a and Il6, which were reduced by 1.5- and 1.75-fold with CANA, respectively. CANA significantly prevented the upregulation of Cd40, downregulated NoxO1 gene expression, decreased ICAM-1 and nitrotyrosine, and increased PECAM-1 immunoreactivity. CANA alleviates endothelial dysfunction following IRI.

Background Brain death (BD) has been suggested to induce coronary endothelial dysfunction. Ischemia/reperfusion (IR) injury during heart transplantation may lead to further damage of the endothelium. Previous studies have shown protective effects of conditioned medium (CM) from bone marrow-derived mesenchymal stem cells (MSCs) against IR injury. We hypothesized that physiological saline-supplemented CM protects BD rats’ vascular grafts from IR injury. Methods The CM from rat MSCs, used for conservation purposes, indicates the presence of 23 factors involved in apoptosis, inflammation, and oxidative stress. BD was induced by an intracranial-balloon. Controls were subjected to a sham operation. After 5.5 h, arterial pressures were measured in vivo. Aortic rings from BD rats were harvested and immediately mounted in organ bath chambers (BD group, n = 7) or preserved for 24 h in 4 °C saline-supplemented either with a vehicle (BD-IR group, n = 8) or CM (BD-IR+CM group, n = 8), prior to mounting. Vascular function was measured in vitro. Furthermore, immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) have been performed. Results BD in donors was associated with significantly impaired hemodynamic parameters and higher immunoreactivity of aortic myeloperoxidase (MPO), nitrotyrosine, caspase-3, caspase-8, caspase-9, and caspase-12 compared to sham-operated rats. In organ bath experiments, impaired endothelium-dependent vasorelaxation to acetylcholine in the BD-IR group compared to BD rats was significantly improved by CM (maximum relaxation to acetylcholine: BD 81 ± 2% vs. BD-IR 50 ± 3% vs. BD-IR + CM 72 ± 2%, p Conclusions The preservation of BD rats’ vascular grafts with CM alleviates endothelial dysfunction following IR injury, in part, by reducing levels of inflammatory response and caspase-mediated apoptosis.

Objectives: Peripheral arterial disease affects mainly the aorta and lower extremities and commonly results in tissue ischemia. Medical interventions to restore blood flow can induce reperfusion injury. We investigated the effects of both hypertension and ageing on vascular graft responsiveness under normoxia and ischemia/reperfusion (IR) conditions. Methods: Hemodynamic changes and aortic morphometry were studied in 18-month-old spontaneously hypertensive stroke prone (SHRSP, n=8) and age-matched normotensive Wistar (control, n=8) rats. Whereas thoracic aortic rings of the control- and SHRSP groups were immediately mounted in organ bath chambers (normoxia-group), aortic segments in the control-IR and SHRSP-IR groups underwent cold ischemic preservation, prior to mounting. Results: Significantly increased intima-media thickness, intima-media cross-sectional area, and lumen normalized to body weight were observed in SHRSP displaying a systolic blood pressure of 205±16 mmHg compared to controls 124±8 mmHg, p < 0.05. Phenylephrine-induced vasoconstriction was greater in aorta of SHRSP compared to controls. IR injury increased contractile responses to phenylephrine in both control and SHRSP compared to the corresponding groups, which was further increased in SHRSP-IR compared to control-IR (as percentage of KCl-contraction: SHRSP-IR 167±26% vs. control-IR 101±5%, p < 0.05). Compared to controls, vasoconstrictive response to high K+-induced depolarization was significantly reduced in SHRSP, control-IR, and SHRSP-IR. Additionally, KCl-induced contraction was reduced in SHRSP-IR compared to control-IR (SHRSP-IR 0.7±0.1g vs. control-IR 2.5±0.1g, p < 0.05). IR reduced maximum endothelium-dependent relaxation to acetylcholine in the normoxia control group, whereas it had no effect in the aortic rings from SHRSP. Conclusions: IR increases the susceptibility of aortic rings to vascular smooth muscle contractile dysfunction in 18-month-old hypertensive rats.

OBJECTIVES Hypertension can impair structure and function of blood vessels. Experimental data describing the reverse remodeling process after a mechanical pressure unloading therapy in the vasculature is limited. We studied the influence of pressure unloading on both the structural and functional alterations of the aorta in a hypertensive rat model. METHODS Using isolated thoracic aortic rings in an in-vitro organ bath system, endothelium-dependent and endothelium-independent vasorelaxation were studied 6-weeks or 12-weeks after abdominal aortic banding (aortic banding-6-week or aortic banding-12-week), and 6-weeks after an aortic debanding procedure performed after the sixth experimental week of aortic banding (aortic banding + debanding-12-week). Age-matched rats were sham-operated (sham-6-week or sham-12-week). The aortic morphometry and histological fibrosis were studied, and the mRNA-expression of metalloproteinase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-2, and soluble guanylate cyclase subunits GUCY1a3 and GUCY1b3 were determined. RESULTS Aortic banding significantly increased systolic, diastolic, and pulse pressures. Structural changes (increased intima-media thickness and area normalized to body weight, aortic collagen content, higher MMP-2 and TIMP-2, and lower GUCY1a3 and GUCY1b3 mRNA-levels) and functional alterations (impaired endothelium-dependent and endothelium-independent vasorelaxation) have already taken place after 6 weeks of aortic banding. Pressure unloading, after established vascular changes, improved vascular function, resulted in reduced collagen content, and decreased both MMP-2 and TIMP-2 mRNA-expression. CONCLUSION Pressure-overload-induced vascular changes regressed due to mechanical unloading. Furthermore, debanding leads to a reductive tendency in fibrosis-associated gene expression and collagen accumulation. Collectively, the addition of drugs that target fibrosis to existing hypertensive treatment may present an attractive therapy against vascular remodeling.

Objectives Ischaemia reperfusion (IR) injury occurs during vascular graft harvesting and implantation during vascular/cardiac surgery. Elevated intracellular cyclic guanosine monophosphate (cGMP) levels contribute to an effective endothelial protection in different pathophysiological conditions. The hypothesis that the phosphodiesterase-5 inhibitor vardenafil would protect vascular grafts against IR injury by upregulating the nitric oxide–cGMP pathway in the vessel wall of the bypass graft was investigated. Methods Lewis rats (n = 6–7/group) were divided into Group 1, control; Group 2, donor rats received intravenous saline; Group 3, received intravenous vardenafil (30 μg/kg) 2 h before explantation. Whereas aortic arches of Group 1 were immediately mounted in an organ bath, aortic segments of Groups 2 and 3 were stored for 2 h in saline and transplanted into the abdominal aorta of the recipient. Two hours after transplantation, the implanted grafts were harvested. Endothelium dependent and independent vasorelaxations were investigated. TUNEL, CD-31, ICAM-1, VCAM-1, α-SMA, nitrotyrosine, dihydroethidium and cGMP immunochemistry were also performed. Results Compared with the control, the saline group showed significantly attenuated endothelium dependent maximal relaxation (Rmax) 2 h after reperfusion, which was significantly improved by vardenafil supplementation (Rmax control, 91 ± 2%; saline 22 ± 2% vs. vardenafil 39 ± 4%, p Conclusions The results support the view that impairment of intracellular cGMP signalling plays a role in the pathogenesis of the endothelial dysfunction of an arterial graft after bypass surgery, which can effectively be prevented by vardenafil. Its clinical use as preconditioning drug could be a novel approach in vascular/cardiac surgery.

Heart transplantation is the therapy of choice for end-stage heart failure. However, hemodynamic instability, which has been demonstrated in brain-dead donors (BDD), could also affect the posttransplant graft function. We tested the hypothesis that treatment of the BDD with the dopamine derivate n-octanoyl-dopamine (NOD) improves donor cardiac and graft function after transplantation. Donor rats were given a continuous intravenous infusion of either NOD (0.882 mg/kg/h, BDD+NOD, n = 6) or a physiological saline vehicle (BDD, n = 9) for 5 h after the induction of brain death by inflation of a subdural balloon catheter. Controls were sham-operated (n = 9). In BDD, decreased left-ventricular contractility (ejection fraction; maximum rate of rise of left-ventricular pressure; preload recruitable stroke work), relaxation (maximum rate of fall of left-ventricular pressure; Tau), and increased end-diastolic stiffness were significantly improved after the NOD treatment. Following the transplantation, the NOD-treatment of BDD improved impaired systolic function and ventricular relaxation. Additionally, after transplantation increased interleukin-6, tumor necrosis factor TNF-α, NF-kappaB-p65, and nuclear factor (NF)-kappaB-p105 gene expression, and increased caspase-3, TNF-α and NF-kappaB protein expression could be significantly downregulated by the NOD treatment compared to BDD. BDD postconditioning with NOD through downregulation of the pro-apoptotic factor caspase-3, pro-inflammatory cytokines, and NF-kappaB may protect the heart against the myocardial injuries associated with brain death and ischemia/reperfusion.

Purpose Transplantation programs are worldwide burdened by the limited availability of suitable donor hearts, despite extending the donation criteria. Organ donation after circulatory death (DCD) became a reliable alternative over the past few years. Nevertheless, the grafts fetched as DCDs are submitted to a further ischemic insult due to the progressive hypoxia and circulatory arrest occurring between withdrawal of care and declaration of death. Although diverse storage and perfusion strategies exist to preserve DCD hearts, no direct comparison has yet been performed. The aim of the present study is to examine the effects of a novel cold crystalloid preservation solution (Custodiol-N) and warm blood perfusion protocols compared to simple cold storage in a porcine model. Methods In 39 pigs, DCD was induced by asphyxia and the hearts underwent 30 min initial warm ischemia in the donor. The hearts were than explanted and divided in 4 groups: 1) control group (n=8) was placed directly in a Langendorff model. In group 2 (CSG, n=8) the hearts were flushed with 2L of 4°C Custodiol N solution and then stored in the same solution for 4h before evaluation. In group 3 (CCP, n=8), the hearts underwent a 4h continuous oxygenated 4°C cold machine perfusion cycle with Custodiol N. In group 4 (WBP, n=7) the hearts were placed on warm blood reperfusion for 4h. Left ventricular function was defined by dP/dt min & max at different ventricle filling volumes. Coronary blood flow (CBF) was measured at 100 mmHg perfusion pressure and Troponin T was collected up to 2 hours. Results Both dP/dt min & max were significantly higher in the CPP group compared to the WBP group [-1038±182 vs -344±38 mmHg/s; p=0.0356 and 1441±194 vs 498±53 mmHg/s; p=0.0123 respectively]. CBF was also significantly higher in the CCP group [1145±57vs 726±72ml/min; p=0.0032]. Moreover, Troponin T was significantly elevated in the WBP compared to the CCP group [27477±11777 vs 251034±90247 pg/ml, p=0.0239]. There were no significant differences among the WBP, CSG and control groups. Conclusion Continuous hypothermic oxygenated crystalloid perfusion with the novel preservation solution Custodiol N offers superior preservation and resuscitation of DCD hearts in terms of functional recovery and myocardial enzyme release.

The use of hearts with left-ventricular (LV) hypertrophy (LVH) could offer an opportunity to extend the donor pool for cardiac transplantation. We assessed the effects of LVH in 18-month-old spontaneously hypertensive stroke-prone (SHRSP) donor rats and following transplantation. In donors, cardiac function and structural alterations were assessed. Then, the hearts were transplanted into young normotensive-rats. We evaluated LV graft function 1 h after transplantation. The myocardial expression of 92 genes involved in apoptosis, inflammation, and oxidative-stress was profiled using PCR-array. Compared to controls, SHRSP-rats developed LVH, had increased LV systolic performance (slope of the end-diastolic pressure-volume (PV) relationship: 1.6±0.2 vs 0.8±0.1mmHg/μl,ppesratio after versus before transplantation: 32% vs 29%,p>0.05). Whereas nitrotyrosine immunoreactivity was higher, myeloperoxidase-positive cell infiltration was decreased in the SHRSP+transplanted compared to control+transplanted. Among the tested genes, LVH was associated with altered expression of 38 genes in donors, while transplantation of these hearts resulted in the change of four genes. Alterations in 18-month-old donor hearts, as a consequence of hypertension and LVH, were not associated with graft dysfunction in the early phase of reperfusion after transplantation.

The major source of heart transplantation comes from brain-dead (BD) donors. However, brain death and myocardial ischemia/reperfusion injury during transplantation may lead to cardiac dysfunction and hemodynamic instability. A previous work demonstrated that pre-treatment of BD donors with dopamine improved the graft survival of heart allograft in recipient after transplantation. However, low-dose dopamine treatment might result in tachycardia and hypertension. Our previous experimental study showed that pre-treatment of BD donor rats with the dopamine derivate N-octanoyl dopamine (NOD), devoid of any hemodynamic effects, improved graft function after transplantation. Herein, we hypothesized that NOD confers superior myocardial protection than dopamine, in terms of graft function. Male Lewis donor rats were either subjected to sham-operation or brain death via a subdurally placed balloon followed by 5.5 h monitoring. Then, the hearts were explanted and heterotopically transplanted into Lewis recipient rats. Shortly before the onset of reperfusion, continuous intravenous infusion of either NOD (14.7 μg/kg/min, BD + NOD group, n = 9), dopamine (10 μg/kg/min, BD + Dopamine group, n = 8) or physiological saline vehicle (sham, n = 9 and BD group, n = 9) were administered to the recipient rats. In vivo left-ventricular (LV) graft function was evaluated after 1.5 h reperfusion. Additionally, immunohistochemical detection of 4-hydroxy-2-nonenal (HNE, an indicator of oxidative stress) and nitrotyrosine (a nitro-oxidative stress marker), was performed. After heart transplantation, systolic and diastolic functions were significantly decreased in the BD group compared to sham. Treatment with NOD but not dopamine, resulted in better LV graft systolic functional recovery (LV systolic pressure BD + NOD 90 ± 8 vs BD + Dopamine 66 ± 5 vs BD 65 ± 4 mmHg; maximum rate of rise of LV pressure dP/dtmax BD + NOD 2686 ± 225 vs BD + Dopamine 2243 ± 70 vs BD 1999 ± 147 mmHg/s, at an intraventricular volume of 140 μl, p

OBJECTIVES Heart transplantation is the standard treatment in end-stage heart failure and at shortage of cardiac allografts is its major limiting factor. Striving to optimize the use of this limited resource, the aspect that long distance procurement may increase the available donor pool must be taken into consideration. As poly(ADP-ribose)polymerase (PARP)-activation has been identified as a key pathway of reperfusion injury, we assessed the hypothesis that its inhibition would allow an extension of cold preservation time and protect the graft against ischaemia/reperfusion injury. METHODS Hearts from donor rats were explanted, stored in a preservation solution (Custodiol) at 4 °C for 4 h or 8 h, and heterotopically transplanted. A vehicle or the PARP-inhibitor, INO-1001 (5 mg/kg), was administered during the reperfusion period. We evaluated post-transplant graft function with a Millar micromanometer at different left-ventricular volumes. Additionally, in organ bath experiments the effect of PARP-inhibition on endothelium-dependent and -independent vasorelaxation was evaluated after long-term cold ischaemic storage/warm reperfusion. RESULTS PARP-inhibition resulted in a better systolic functional recovery of grafts submitted to 4 h and 8 h ischaemia. Furthermore, INO-1001 decreased the left-ventricular end-diastolic pressure after 8 h of ischaemia. Coronary blood flow was significantly higher after PARP-inhibition in comparison to controls. Endothelium-dependent vasorelaxation was significantly better in the INO-1001-groups than in the vehicle-treated transplant groups. After 24-h hypothermic storage, treatment of aortic ring with INO-1001 during reoxygenation significantly improved endothelial dysfunction. CONCLUSIONS By inhibiting the PARP activation, INO-1001 can protect the graft and endothelium from the injury that is caused by prolonged cold myocardial ischaemia/reperfusion, thereby improving post-transplant graft function.

Background: Pressure unloading induces the regression of left ventricular myocardial hypertrophy (LVH). Recent findings indicate that pharmacological activation of the soluble guanylate cyclase (sGC) - cyclic guanosine monophosphate (cGMP) pathway may also exert reverse-remodeling properties in the myocardium. Therefore, we aimed to investigate the effects of the sGC activator cinaciguat in a rat model of LVH and compare it to the "gold standard" pressure unloading therapy. Methods: Abdominal aortic banding was performed for 6 or 12 weeks. Sham operated animals served as controls. Pressure unloading was induced by removing the aortic constriction after week 6. The animals were treated from week 7 to 12, with 10 mg/kg/day cinaciguat or with placebo p.o., respectively. Cardiac function and morphology were assessed by left ventricular pressure-volume analysis and echocardiography. Additionally, key markers of myocardial hypertrophy, fibrosis, nitro-oxidative stress, apoptosis and cGMP signaling were analyzed. Results: Pressure unloading effectively reversed LVH, decreased collagen accumulation and provided protection against oxidative stress and apoptosis. Regression of LVH was also associated with a full recovery of cardiac function. In contrast, chronic activation of the sGC enzyme by cinaciguat at sustained pressure overload only slightly influenced pre-established hypertrophy. However, it led to increased PKG activity and had a significant impact on interstitial fibrosis, nitro-oxidative stress and apoptosis. Amelioration of the pathological structural alterations prevented the deterioration of LV systolic function (contractility and ejection fraction) and improved myocardial stiffness. Conclusion: Our results indicate that both cinaciguat treatment and pressure unloading evoked anti-remodeling effects and improved LV function, however in a differing manners.

BACKGROUND Heart transplantation is the definitive treatment for end-stage heart failure. A shortage of donor hearts forced transplant programs to accept older donors and longer ischemic times. Previous studies have suggested that administration of mesenchymal stem cells (MSCs) or their conditioned medium (CM) protects the heart against ischemia/reperfusion injury (IRI). We hypothesized that the preservation of donor hearts with a CM would protect the graft from IRI after prolonged storage in 15-month-old rats and investigated mRNA changes attributable to CM. METHODS Rat MSCs were isolated and cultured. The CM was used and characterized by a 90-antibody array, revealing the presence of 28 factors involved in apoptosis, inflammation, and oxidative stress. Hearts from 15-month-old donor rats were explanted and continuously perfused for 5 hours with oxygenated, 4°C cardioplegic solution, and supplemented with either regular cell culture medium (control group) or CM. The hearts were then heterotopically transplanted. We evaluated in-vivo left ventricular graft function 1.5 hours after transplantation and the myocardial expression of 120 genes using polymerase chain reaction arrays. RESULTS Systolic contractility and relaxation parameters were significantly reduced in 15-month-old rats compared with the young rats. After transplantation, systolic function (dP/dtmax: 1,197 ± 94 vs 1,825 ± 279 mm Hg/s at 140 µl; p < 0.05) and diastolic function (dP/dtmin: 737 ± 168 vs 1,200 ± 166 mm Hg/s at 140 µl, p < 0.05) were significantly improved in the CM group compared with controls. Among the genes surveyed, the expressions of 66 were altered. Genes of pro-inflammatory cytokines and interleukins were down-regulated, whereas expression of the anti-oxidant gene superoxide dismutase-2 was up-regulated in the CM-treated grafts compared with the control group grafts. CONCLUSIONS Perfusion of donor hearts with CM protects against myocardial IRI in 15-month-old rats.

Purpose Currently, only the hearts of brain-dead (BD) donors are used for transplantation. However, hemodynamic instability and cardiac dysfunction occur in 1/4 of these donors. Myocardial ischemia/reperfusion injury encountered at the time of heart transplantation (HTX) lead to further impairment, contributing to adverse short- and long-term graft outcome in the recipients. Our previous study demonstrated that BD donors’ treatment with dopamine derivate n-octanoyl dopamine (NOD) contributes to the functional recovery of grafts after transplantation. Taken together, we hypothesized that recipient treatment with NOD would improve graft function after transplantation. Methods Donor rats were either subjected to BD via inflation of subdurally placed balloon or sham operation, followed by 5.5h monitoring. After cardiac arrest, the hearts were explanted and stored for 1h in cold Custodiol cardioplegic solution, finally heterotopically transplanted. Five minutes before the onset of reperfusion, continuous intravenous infusion of NOD (0.882 mg/kg/h, BD+HTX+NOD group, n=9) or placebo (BD+HTX group, n=9) were administered to the recipient rats. In vivo left-ventricular (LV) graft function was evaluated 1.5h after reperfusion via a Millar catheter system at different LV volumes. Results BD adversely affected hemodynamic parameters in heart donors compared with the sham group. After transplantation, a significantly improved graft systolic function (LV systolic pressure 90±8 vs 65±4 mmHg; maximum rate of rise of LV pressure dP/dtmax 2686±225 vs 1999±147 mmHg/s, at an intraventricular volume of 140 µl, p Conclusion Our data demonstrate that recipient treatment with the dopamine derivate NOD before the onset of reperfusion improves posttransplant function of hearts derived from BD donors in rats.

OBJECTIVES The use of 'marginal' hearts, such as from donors with diabetes mellitus (DM), could offer an opportunity to expand the donor pool in cardiac transplantation. Previous studies have shown that the phosphatidylinositol-3-kinase (PI3K)/Akt pathway is altered after ischaemia/reperfusion injury in the diabetic myocardium. We hypothesized that DM-induced cardiac dysfunction in donors is further impaired after heart transplantation and that PI3K/Akt-pathway alterations may be one of the underlying pathomechanisms. METHODS In the donor rats, DM was induced with a single dose of streptozotocin. Non-diabetic rats only received citrate buffer. After 8 weeks, the donor left ventricular (LV) cardiac function was measured. Then, the hearts were heterotopically transplanted into non-diabetic recipients. We evaluated LV graft function 1.5 h after transplantation via a Millar catheter system at different LV volumes. Histological analyses were performed, and the expression of 84 genes involved in PI3K/Akt signalling was profiled. RESULTS DM was associated with significantly decreased LV contractility and impaired relaxation. After transplantation, in the DM group, the grafts' systolic function (LV systolic pressure 112 ± 31 vs 155 ± 60 mmHg; dP/dtmax 2676 ± 896 vs 3584 ± 1779 mmHg/s, P

Objectives The in situ internal thoracic artery (ITA) is recognized as the best conduit for coronary artery bypass surgery. The ITA—if it is used as an in situ graft—has a much higher late patency rate than any other arterial graft, including a free ITA graft. We sought to determine if the use of the ITA as an in situ/free graft and its storage in preservation solutions, have an effect on endothelial function. Methods The ITA was harvested as either a free or in situ graft in a porcine model. Free grafts were stored in different preservation solutions (saline, Custodiol and Tiprotec [both Kohler Chemie GmbH, Bensheim, Germany]). The ITA was anastomosed off pump to the left anterior descending artery (as in situ/free graft). Freshly harvested ITA served as a control. After 2 hours of reperfusion, the implanted grafts were harvested. The assessment of endothelial function, histopathological analysis, and gene expression were performed. Results Endothelial function and integrity were severely impaired after reperfusion in the free ITA groups, however, it was partially preserved in the Tiprotec group. Reperfusion injury resulted in increased nitro-oxidative stress, DNA breakage, vascular cell adhesion protein 1, intercellular adhesion molecule-1, and caspase-3 scores, and a decreased endothelial nitric oxide synthase score in the free ITA groups. The in situ ITA graft showed no signs of injury. mRNA levels were significantly altered among the groups. Conclusions An early, severe endothelial dysfunction of the stored, free ITA as described, could be completely prevented by the use of an in situ ITA graft. Tiprotec might be a feasible option for storage of free arterial grafts during coronary artery bypass grafting.

Purpose The endothelium has a pivotal role in the maintenance of cardiac function after heart transplantation, mainly by controlling the coronary circulation. Brain death (BD) as well as ischemia/reperfusion (IR) injury compromise vascular endothelium and thereby myocardial protection. Beneficial effects of conditioned medium (CM) from mesenchymal stem cells (MSCs) against IR injury have been demonstrated. We hypothesized that physiological saline supplemented CM could protect vascular grafts of BD rats from IR injury. Methods Rat MSCs derived CM was used for conservation purposes. We detected 28 factors involved in inflammation, oxidative stress and apoptosis. Donors were either subjected to sham operation or BD by inflation of a subdural balloon for 5.5h. Then, thoracic aortic rings from BD rats were isolated and immediately mounted after preparation in an organ bath chamber (BD group) or underwent 24h cold ischemic preservation in physiological saline supplemented either with a vehicle (BD-IR group) or CM (BD-IR+CM group). Immunohistochemical staining for myeloperoxidase (MPO), nitrotyrosine (NT), caspase-8, -9, and -12 was performed. Results Characterization of BD donors: Compared to the sham group, BD leads to significantly impaired hemodynamic parameters, higher aortic MPO, NT, caspase-8, -9, and -12 contents. Organ bath experiments: The preservation of BD-IR aortic rings with CM significantly improved IR-induced decreased maximum endothelium dependent vasorelaxation (Rmax) to acetylcholine compared to the vehicle treated BD-IR group (Rmax to acetylcholine: BD 81±2 vs BD-IR 50±3 vs BD-IR+CM 72±2%, p Conclusion Endothelial dysfunction during IR injury is improved by preservation of vascular grafts from BD rats with CM. This protective effect may partly arise from a reduction of inflammation and nitro oxidative stress responses, and inhibition of caspase-8 and -9 mediated apoptosis.

Pressure unloading represents the only effective therapy in increased afterload-induced left ventricular hypertrophy (LVH) as it leads to myocardial reverse remodeling (reduction of increased left ventricular mass, attenuated myocardial fibrosis) and preserved cardiac function. However, the effect of myocardial reverse remodeling on cardiac mechanoenergetics has not been elucidated. Therefore, we aimed to provide a detailed hemodynamic characterization in a rat model of LVH undergoing pressure unloading. Pressure overload was induced in Sprague-Dawley rats by abdominal aortic banding for 6 (AB 6th wk) or 12 wk (AB 12th wk). Sham-operated animals served as controls. Aortic debanding procedure was performed after the 6th experimental week (debanded 12th wk) to investigate the regression of LVH. Pressure unloading resulted in significant reduction of LVH (heart weight-to-tibial length ratio: 0.38 ± 0.01 vs. 0.58 ± 0.02 g/mm, cardiomyocyte diameter: 18.3 ± 0.1 vs. 24.1 ± 0.8 μm debanded 12th wk vs. AB 12th wk, P < 0.05), attenuated the extracellular matrix remodeling (Masson's score: 1.37 ± 0.13 vs. 1.73 ± 0.10, debanded 12th wk vs. AB 12th wk, P < 0.05), provided protection against the diastolic dysfunction, and reversed the maladaptive contractility augmentation (slope of end-systolic pressure-volume relationship: 1.39 ± 0.24 vs. 2.04 ± 0.09 mmHg/μl, P < 0.05 debanded 12th wk vs. AB 6th wk, P < 0.05). In addition, myocardial reverse remodeling was also associated with preserved ventriculoarterial coupling and increased mechanical efficiency (50.6 ± 2.8 vs. 38.9 ± 2.5%, debanded 12th wk vs. AB 12th wk, P < 0.05), indicating a complete functional and mechanoenergetic recovery. According to our best knowledge, this is the first study demonstrating that the regression of LVH is accompanied by maintained cardiac mechanoenergetics.

The Goto-Kakizaki (GK) rat, a non-obese model of type 2 diabetes mellitus (T2DM), was generated by the selective inbreeding of glucose-intolerant Wistar rats. This is a convenient model for studying diabetes-induced cardiomyopathy independently from the effects of the metabolic syndrome. We investigated the myocardial functional and structural changes and underlying molecular pathomechanisms of short-term and mild T2DM. The presence of DM was confirmed by an impaired oral glucose tolerance in the GK rats compared with the age-matched nondiabetic Wistar rats. Data from cardiac catheterization showed that in GK rats, although the systolic indexes were not altered, the diastolic stiffness was increased compared with nondiabetics (end-diastolic-pressure-volume-relationship: 0.12 ± 0.04 vs. 0.05 ± 0.01 mmHg/μl, P < 0.05). Additionally, DM was associated with left-ventricular hypertrophy and histological evidence of increased myocardial fibrosis. The plasma pro-B-type natriuretic peptide, the cardiac troponin-T, glucose, and the urinary glucose concentrations were significantly higher in GK rats. Among the 125 genes surveyed using PCR arrays, DM significantly altered the expression of five genes [upregulation of natriuretic peptide precursor-A and connective tissue growth factor, downregulation of c-reactive protein, interleukin-1β, and tumor necrosis factor (TNF)-α mRNA-level]. Of the altered genes, which were evaluated by Western blot, only TNF-α protein expression was significantly decreased. The ECG recordings revealed no significant differences. In conclusion, while systolic dysfunction, myocardial inflammation, and abnormal electrical conduction remain absent, short-term and mild T2DM induce the alteration of cardiac TNF-α at both the mRNA and protein levels. Further assessments are required to reveal if TNF-α plays a role in the early stage of diabetic cardiomyopathy development.

Introduction: Type 2 diabetic patients are at an increased risk of cardiomyopathy and heart failure is a major cause of death amongst these patients. Growing evidence indicates that proinflammatory cytokines can cause sustained development of insulin resistance and anti-inflammatory medications may reverse this process. Hypothesis: We investigated the effects of an oral administration of zinc and acetylsalicylic acid in the form of bis(aspirinato)zinc(II) complex Zn(ASA)2 on different aspects of cardiac damage in the Zucker diabetic rat (ZDF), an experimental model of type 2 diabetic cardiomyopathy. Methods: The nondiabetic control and the diabetic ZDF rats were pretreated orally with vehicle or Zn(ASA)2 for 24 days. At the age of 30-32 weeks, both the electrical activity and the left ventricular (LV) structural/functional parameters were assessed via electrocardiogram and pressure volume (PV) conductance catheter system. Results: The Zn(ASA)2 treatment significantly decreased the blood glucose concentration (39.6±3.1 vs 50.4±2.6 mM), normalized the impaired LV contractility index (Emax 3.7±0.4 vs 1.9±0.6 mmHg/μl), the passive LV stiffness (end diastolic PV relationship: 0.064±0.008 vs 0.084±0.014 mmHg/μl), and the diastolic dysfunction (LV end diastolic pressure: 6.5±0.6 vs 7.9±0.7 mmHg). Furthermore, the ECG revealed a restoration of the prolonged corrected QT intervals (63±3 vs 83±4 ms, p Conclusions: The oral administration of Zn(ASA)2 may have therapeutic potential with the aim of preventing cardiac complications in type 2 diabetic patients.

Background: Type-2 diabetics have an increased risk of cardiomyopathy, and heart failure is a major cause of death among these patients. Growing evidence indicates that proinflammatory cytokines may induce the development of insulin resistance, and that anti-inflammatory medications may reverse this process. We investigated the effects of the oral administration of zinc and acetylsalicylic acid, in the form of bis(aspirinato)zinc(II)-complex Zn(ASA)2, on different aspects of cardiac damage in Zucker diabetic fatty (ZDF) rats, an experimental model of type-2 diabetic cardiomyopathy. Methods: Nondiabetic control (ZL) and ZDF rats were treated orally with vehicle or Zn(ASA)2 for 24 days. At the age of 29–30 weeks, the electrical activities, left-ventricular functional parameters and left-ventricular wall thicknesses were assessed. Nitrotyrosine immunohistochemistry, TUNEL-assay, and hematoxylin-eosin staining were performed. The protein expression of the insulin-receptor and PI3K/AKT pathway were quantified by Western blot. Results: Zn(ASA)2-treatment significantly decreased plasma glucose concentration in ZDF rats (39.0 ± 3.6 vs 49.4 ± 2.8 mM, P

We recently demonstrated that the pre-treatment of rats with zinc and acetylsalicylic acid complex in the form of bis(aspirinato)zinc(II) [Zn(ASA)2] is superior to acetylsalicylic acid in protecting the heart from acute myocardial ischemia. Herein, we hypothesized that Zn(ASA)2 treatment after the onset of an acute myocardial injury could protect the heart. The rats were treated with a vehicle or Zn(ASA)2 after an isoproterenol injection. Isoproterenol-induced cardiac damage [inflammatory infiltration into myocardial tissue, DNA-strand breakage evidenced by TUNEL-assay, increased 11-dehydro thromboxane (TX)B2-levels, elevated ST-segment, widened QRS complex and prolonged QT-interval] was prevented by the Zn(ASA)2 treatment. In isoproterenol-treated rats, load-independent left ventricular contractility parameters were significantly improved after Zn(ASA)2. Furthermore, Zn(ASA)2 significantly increased the myocardial mRNA-expression of superoxide dismutase-1, glutathione peroxidase-4 and decreased the level of Na(+)/K(+)/ATPase. Postconditioning with Zn(ASA)2 protects the heart from acute myocardial ischemia. Its mechanisms of action might involve inhibition of pro-inflammatory prostanoids and upregulation of antioxidant enzymes.

Aims Myocardial infarction (MI) is a common cause of mortality in patients with diabetes mellitus (DM) and vascular dysfunction is a major component of diabetic cardiomyopathy. We investigated the systemic influence of acute MI on the diabetes-induced pathogenic changes in the rat aorta. Methods Nondiabetic Wistar (W) and type-2 diabetic Goto–Kakizaki (GK) rats underwent 45 min of left anterior descending coronary artery occlusion followed by 24 h of reperfusion. Isometric force was measured using organ bath. Results Plasma glucose-levels were significantly higher in diabetic rats (GK + sham: 13 ± 2 mM; GK + MI: 19 ± 2 mM) compared to nondiabetic rats (W + sham: 8 ± 0 mM; W + MI: 8 ± 1 mM). Acetylcholine-induced relaxation was significantly weaker in rings from W + MI and GK + MI rats compared to corresponding sham-operated animals. Myocardial reperfusion injury was smaller in GK + MI than W + MI rats, and the concentration-response curves to acetylcholine were significantly enhanced in rings from GK + MI than W + MI rats. Nevertheless, the relaxation response to acetylcholine was similar in W + sham and GK + sham. Densitometric analysis of bands for endothelial nitric oxide synthase showed a significant decrease in W + MI rats compared to W + sham and GK + sham animals. Aortas from both GK + sham and GK + MI rats showed impaired contractile responses to phenylephrine in comparison with the nondiabetics. Conclusions For the first time we showed that short-term and mild type-2 DM improved remote endothelial dysfunction after reperfused acute MI.