Perfusion Apparatus Research Articles

Eprosartan improves cardiac function in swine working heart model of ischemia-reperfusion injury.

BackgroundEprosartan is an angiotensin II receptor antagonist used as an antihypertensive. We sought to evaluate the regional effect of Eprosartan on postinfarct ventricular remodeling and myocardial function in an isolated swine working heart model of ischemia-reperfusion injury.Material/Methods22 swine hearts were perfused with the Langendorff perfusion apparatus under standard experimental conditions. Myocardial ischemia was induced by a 10-min left anterior descending artery ligation. Hearts were reperfused with either saline (control group, n=11), or Eprosartan (treatment group, n=11). Left ventricular pressure (LVP) and regional heart parameters such as intramyocardial pressure (IMP), wall thickening rate (WTh), and pressure-length-loops (PLL) were measured at baseline and after 30 min of reperfusion.ResultsMeasured parameters were statistically similar between the 2 groups at baseline. The administration of Eprosartan led to a significantly better recovery of IMP and WTh: 44.4±2.5 mmHg vs. 51.2±3.3 mmHg, p<0.001 and 3.8±0.4 μm vs. 4.4±0.3 μm, p=0.001, respectively. PLL were also significantly higher in the treatment group following reperfusion (21694±3259 units vs. 31267±3429 units, p<0.01). There was no difference in the LVP response to Eprosartan versus controls (63.6±3.0 mmHg vs. 62.5±3.1 mmHg, p=0.400).ConclusionsPre-treatment with Eprosartan is associated with a significant improvement in regional cardiac function under ischemic conditions. Pharmacological treatment with eprosartan may exert a direct cardioprotective effect on ischemic myocardium.

Bipolar ablation for deep intra-myocardial circuits: human ex vivo development and in vivo experience

Current conventional ablation strategies for ventricular tachycardia (VT) aim to interrupt reentrant circuits by creating ablation lesions. However, the critical components of reentrant VT circuits may be located at deep intramural sites. We hypothesized that bipolar ablations would create deeper lesions than unipolar ablation in human hearts. Ablation was performed on nine explanted human hearts at the time of transplantation. Following explant, the hearts were perfused by using a Langendorff perfusion setup. For bipolar ablation, the endocardial catheter was connected to the generator as the active electrode and the epicardial catheter as the return electrode. Unipolar ablation was performed at 50 W with irrigation of 25 mL/min, with temperature limit of 50°C. Bipolar ablation was performed with the same settings. Subsequently, in a patient with an incessant septal VT, catheters were positioned on the septum from both the ventricles and radiofrequency was delivered with 40 W. In the explanted hearts, there were a total of nine unipolar ablations and four bipolar ablations. The lesion depth was greater with bipolar ablation, 14.8 vs. 6.1 mm (P < 0.01), but the width was not different (9.8 vs. 7.8 mm). All bipolar lesions achieved transmurality in contrast to the unipolar ablations. In the patient with a septal focus, bipolar ablation resulted in termination of VT with no inducible VTs. By using a bipolar ablation technique, we have demonstrated the creation of significantly deeper lesions without increasing the lesion width, compared with standard ablation. Further clinical trials are warranted to detail the risks of this technique.

Testicular Perfusion Apparatus

A reproductive biology laboratory requires to provide reliable instruments for regular use of research workers. To understand an organ and perform scientific study and reach on a conclusion good instruments and techniques are essential. Such a suitable one is not available in case of testicular study. The present study is aimed to clear this lacuna. A new testicular perfusion apparatus for rabbit and rat is designed and fabricated with glass. The glass blowing instrument available is used. The perfusion fluid preferred is Krebs Ringer bicarbonate buffer (pH 7.4). Quartz wool provided in stem tube filters emboli present in it. Perfusate temperature is maintained to that of testis. A 26-gauge needle is introduced into the spermatic artery to allow entry of perfusate into testis. Method for the collection of perfusate leaving testis is available. This permits to measure the level of an injected substance before its entry through perfusion fluid into the testis, which shall be compared in the fluid after completion of perfusion. Initially few animal testes were used and found the instrument to be functioning well. A new simple perfusion apparatus for testes of rabbit and rat is designed. The apparatus made out of glass could be prepared with the help of glass blowing facility and used regularly in any laboratory.

Simple perfusion apparatus for the study of oocytes

Simple perfusion apparatus for the study of oocytes

Full-Length Human Chromogranin-A Cardioactivity: Myocardial, Coronary, and Stimulus-Induced Processing Evidence in Normotensive and Hypertensive Male Rat Hearts

Plasma chromogranin-A (CgA) concentrations correlate with severe cardiovascular diseases, whereas CgA-derived vasostatin-I and catestatin elicit cardiosuppression via an antiadrenergic/nitric oxide-cGMP mediated mechanism. Whether these phenomena are related is unknown. We here investigated whether and to what extent full-length CgA directly influences heart performance and may be subjected to stimulus-elicited intracardiac processing. Using normotensive and hypertensive rats, we evaluated the following: 1) direct myocardial and coronary effects of full-length CgA; 2) the signal-transduction pathway involved in its action mechanism; and 3) CgA intracardiac processing after β-adrenergic [isoproterenol (Iso)]- and endothelin-1(ET-1)-dependent stimulation. The study was performed by using a Langendorff perfusion apparatus, Western blotting, affinity chromatography, and ELISA. We found that CgA (1-4 nM) dilated coronaries and induced negative inotropism and lusitropism, which disappeared at higher concentrations (10-16 nM). In spontaneously hypertensive rats (SHRs), negative inotropism and lusitropism were more potent than in young normotensive rats. We found that perfusion itself, Iso-, and endothelin-1 stimulation induced intracardiac CgA processing in low-molecular-weight fragments in young, Wistar Kyoto, and SHR rats. In young normotensive and adult hypertensive rats, CgA increased endothelial nitric oxide synthase phosphorylation and cGMP levels. Analysis of the perfusate from both Wistar rats and SHRs of untreated and treated (Iso) hearts revealed CgA absence. In conclusion, in normotensive and hypertensive rats, we evidenced the following: 1) full-length CgA directly affects myocardial and coronary function by AkT/nitric oxide synthase/nitric oxide/cGMP/protein kinase G pathway; and 2) the heart generates intracardiac CgA fragments in response to hemodynamic and excitatory challenges. For the first time at the cardiovascular level, our data provide a conceptual link between systemic and intracardiac actions of full-length CgA and its fragments, expanding the knowledge on the sympathochromaffin/CgA axis under normal and physiopathological conditions.

The effect of reactive oxygen species on the myogenic tone of rat ophthalmic arteries with and without endothelium

Reactive oxygen species (ROS) play an important role in the pathogenesis of various ocular diseases. ROS can induce vasodilation or vasoconstriction depending on the species, the tested vessel bed, and the condition of the vessel. This study investigates the effect of different dosages of ROS on the tone of rat ophthalmic arteries. Freshly dissected rat ophthalmic arteries were pressurized in a perfusion setup in steps of 10 mmHg to 180 mmHg in three consecutive cycles. The first cycle was run under mostly physiological conditions, the second cycle was run after ROS treatment, and the third cycle as passive dilation after all Ca(2+) was removed from the solution. ROS-induced dilation or constriction was calculated in relation to the passive dilation. All experiments were performed with or without endothelium. For vessels with endothelium, dilation in control experiments was 20.0 ± 0.1%; after 5 s of ROS dilation was 74.4 ± 0.6%, and after 20 s 87.4 ± 0.3%. ANOVA revealed significant differences between these groups (P = 0.048). For vessels without endothelium, a slight dilation was seen in control experiments (14.5 ± 0.4%), which was also present after 5 s of ROS treatment (15.4 ± 0.4%). Treatment with ROS for 20 s led to a constriction of the vessel preparations (-16.6 ± 0.5%; P = 0.831). ROS led to a vasodilation in vessels with endothelium that was not seen in vessels without endothelium. Endothelial function seems to determine the effect of ROS on the vessel tone in isolated rat ophthalmic arteries.

A novel approach to the Langendorff technique: preparation of isolated cardiomyocytes and myocardial samples from the same rat heart

Researchers in biomedical sciences must continually re-evaluate their investment in experiments using laboratory animals. Our group is interested in various signalling pathways that underlie physiological and pathophysiological functioning of the mammalian heart. Two important resources for this type of work are isolated cardiomyocytes and homogenized or preserved sections of whole myocardium. In order to improve our experimental approach ethically, we devised an adaptation of the Langendorff whole-heart retrograde perfusion technique that allows the isolation of adult rat ventricular cardiomyocytes and processing/storage of myocardium from the same heart. This could result in a 50% reduction in the number of animals required for certain experiments. We believe that a generalized adoption of this method would represent a meaningful reduction of animal use in our field of research and, furthermore, strengthen data sets by permitting correlation between myocyte function and various parameters of myocardial biochemistry/structure in individual hearts. This approach is of particular relevance for studies of cardiac pathology, given the cost and time involved in generating experimental disease models.

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb(-/-)). Rheb(-/-) mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb(-/-) was lower than that in the control (Rheb(+/+)) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb(+/+) mice but not in Rheb(-/-) mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb(-/-) hearts during the neonatal period. Rheb(-/-) hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb(-/-) hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb(-/-) mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption. A variety of specific antigens have already been identified and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca(2+)) contents. In the last step, the effect of patient IgG on the cell shortening and Ca(2+) transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

Abstract 19275: T-type Calcium Channel Overexpression in Mice Attenuates Recovery from Bupivacaine-Induced Cardiac Arrest

Background _ The cardiotoxicity from the local anesthetic bupivacaine is predominantly a result of sodium channel blockade, though calcium channel inhibition has been implicated as well. Mechanisms of reversal of this cardiotoxicity are unclear. We investigated the toxicity of bupivacaine on FVB isolated mouse hearts that were either wild type (WT) or overexpressors for T-type calcium channel (TTCC). We subsequently attempted reversal of cardiotoxicity with a combination of intralipid and epinephrine. We hypothesized that the increased calcium metabolism of the transgenic hearts would augment the adverse effects of bupivacaine. Methods and Results _ Hearts from anesthetized WT and transgenic FVB mice aged 4 months were excised and placed on a Langendorff perfusion apparatus. The left atrial auricle was excised, the mitral valve was perforated, and a fluid-filled balloon was placed in the left ventricle to capture left ventricular pressure and heart rate data. After 10 min of baseline perfusion at an EDP of 10mmHg, bupivacaine was added at 100μM, causing arrest of cardiac activity for both mouse lines. After 3 min of cardiac arrest, the heart was perfused with a solution of bupivacaine (30μM), intralipid (0.2%) and epinephrine (0.15μg/ml) with cardiac recovery following. At baseline, there were no differences in left ventricle function between WT and transgenic mice. After recovery, the left ventricle end diastolic pressure in the transgenic mice was significantly higher (29.18 ± 4.05 mm Hg) than in the WT mice (17.15 ± 0.83 mmHg), P=0.02). Furthermore, the left ventricle developed pressure was significantly reduced (P=0.03) in the transgenic mice (28.61 ± 5.966 mmHg) when compared to WT (57.17 ± 8.052 mmHg). When incorporating heart rate, the Rate Pressure Product (HR x LVESP) was significantly decreased (P=0.03) in the transgenic mice (6455 ± 748.3) versus the WT (11396 ± 1646). Electrophysiological analysis revealed significantly increased suppression of calcium current in the L Type Calcium Channel (P=0.03) Conclusions _ These data show that bupivacaine-induced cardiotoxicity is more difficult to reverse in hearts overexpressing TTCC, possibly reflecting a stronger inhibition of Calcium release from the Sarcoplasmic Reticulum in them.

Hypothermic Machine Perfusion Preservation Is Efficient for Non-Heart-Beating Donor Kidney Transplantation in Japan

Background: In Japan, brain-dead donors have been increased by revised Act of Organ Transplantation, however, insufficiency of deceased donors is still serious problem. In kidney transplantation, it is important to use marginal donors such as non-heart-beating donors for solving this problem. Static cold storage (SCS) is the most widely used organ preservation method for deceased donor, and in Japan it is now the only technique for kidney preservation, however, hypothermic machine perfusion (HMP) preservation technique may improve better outcomes than SCS. Because, HMP technology has had a major impact in circumventing ischemic injury in kidney transplantation in western countries. In this presentation, we report efficiency of HMP method for preservation of long ischemic kidney grafts in beagle and a case report of clinical usage to kidney transplantation of non-heat-beating donor in Japan. Methods: Young beagles were used as autonomal HMP transplantation model. After general anesthesia of beagles, left renal artery and vein were made ligation, after 30 min warm ischemia time (WIT) in situ, the left kidney was harvested. Each harvested kidney was assigned to one of two preservation treatment groups; SCS group (storage in University of Wisconsin solution at 2-4°C for 24h, n=5); HMP group (storage on LifePort® (Organ Recovery Systems), a portable hypothermic machine perfusion apparatus, at 4-6°C on pulsatile pressure (30 mmHg) for 24h with KPS-1 solution, n=5). After the storage, wedge biopsies were taken for histological evaluation (HE staining and TUNEL staining). The preserved graft was transplanted to the same beagle in left iliac fossa and removed the other kidney. After the operation, recipient survival and graft qualities (urine output, serum creatinine, sodium and potassium) were checked as the clinical outcomes. Results: All recipients of HMP group survived, however, three recipients of SCS group were dead with uremia. Serum creatinine level of HMP group was under 4mg/dL in all post-operation period and reduced under 1.5mg/dL after 11days of post-operation. The other hand, that of SCS group went up over 5mg/dL and did not reduce under 3mg/dL. In histological findings, HE staining revealed that a little bit interstitial edema was exist with HMP groups, and remarkable interstitial edema and vaculolation were exist with SCS group. TUNEL staining showed that apoptosis cells were scattered in tubular epithelium of only SCS group. [Case report] First clinical case of LifePort® preservation in Japan is a 39-year-old woman who had accepted hemodialysis with unexplained renal failure. She underwent deceased donor renal transplantation with preserved kidney with LifePort®. She had immediate urine and discharged hospital without any complications. Withdrawal of hemodialysis was achieved, and the allograft renal function presents well and stable after one-year post-operation. Discussions and conclusions: In this experiment, we indicate that HMP would be better for long term WIT kidney model compared with SCS method in beagles. We experienced first clinical case of deceased donor (non-heart-beating donor) kidney transplantation with LifePort® preserved graft in Japan. It suggests that with marginal donor that is non-heart-beating and is with long agony, HMP should be paid more tribute to than SCS technique.

A New Approach to Contain Costs for the Establishment of a Clinical Islet Transplant (ITX) Program

Background: Despite improvements in pancreatic islet auto- and allotransplantation the diffusion of ITX programs has been limited. This is in part due to the high cost of building a specialized islet isolation facility and its operation. Facilities are built in areas requiring demolition and a complete refitting of large spaces to accommodate the isolation room, air filtration system, all ancillary services (pre-post clean area, scrubbing, changing, etc.) and new certifications that can cost $1-2 million. In addition these facilities require dedicated personnel for maintenance and operation (cleaning, monitoring, setting up etc.) 24/7. In our Institution, we developed a novel strategy for cost-containment of a clinical ITX program. Methods and Results: We designed the islet isolation facility in the operating room (OR) area at our Institution. The facility consists of ˜250 sq ft. that includes an ante-room (computer/monitor, storage, back-up refrigerator/freezer, CO2 tanks) and the fully equipped isolation facility. Floor, walls and ceiling were built of non porous surfaces for cleaning. Equipment included: 6 ft safety cabinets (2), COBE 2991 cell separator (2), mechanical shaker, organ perfusion apparatus, centrifuges (2), microscope with remotely connected camera and room screen, cabinets/storage, refrigerator/freezer, incubators, pass-through window, hands free intercom and door operation, room/equipment monitoring system (24/7) with alarm report for temperature, humidity, CO2, pressure. The cost of this conversion and equipping was approximately $200K, with an additional $100K to classify the room at 10.000 (ISO-7) particle level standards with HEPA filters. Air handler and power supply connected to emergency generator, sterilization equipment, changing and scrubbing area, storage, room monitoring system, ice maker, and certain certifications as well as personnel for cleaning, setting up and monitoring were already available in the OR reducing the cost of dedicated FTEs to 50%. Same location of cell preparation and recipient reduced risk of contamination and improved coordination between teams. We performed 8 human islet isolations and 3 clinical auto-ITX with no complication. Positive pressure was maintained at all times and microbiologic cultures were negative in all samples. Conclusions: Integration of an islet isolation facility in the OR area presents many functional advantages and a significant cost containment.

Cardiotonic effects of &lt;i&gt;Terminalia arjuna&lt;/i&gt; extracts on guinea pig heart &lt;i&gt;in vitro&lt;/i&gt;

We investigated the effects of fruit and bark extracts of Terminalia arjuna on the rate and contraction of guinea pig heart in vitro. Immediately after killing, the heart was connected to a perfusion apparatus containing oxygenated double-dextrose McWins solution with fresh arjun fruit and bark experimental extracts. The apex of the heart was then connected to a recording drum through a metallic hook and the rate and height (force) of contractions were recorded on a smoked drum. The heights of contraction for fruit extract and bark extract were significantly higher than that for normal saline (p&lt;0.001), while they were significantly lower than that for adrenaline (p&lt;0.01). Addition of verapamil caused a significant blockade in the heights of contraction produced by extracts of T. arjuna (p&lt;0.01), while heart rate was not affected. The cardiotonic effect of T. arjuna was probably mediated through high concentration of Ca++ present in the plant.

Postconditioning with glucagon like peptide-2 reduces ischemia/reperfusion injury in isolated rat hearts: role of survival kinases and mitochondrial KATP channels

We recently reported that heart expresses functional receptors for the anorexigenic glucagon-like peptide (GLP)-2. Activation of these cardiac receptors affected basal heart performance through extracellular regulated kinase (ERK1/2) activation. Since ERK1/2 is considered one of the prosurvival kinases of postconditioning cardioprotective pathways, we hypothesized that GLP-2 directly protects the heart against ischemia/reperfusion (I/R) injury via prosurvival kinases. Wistar rat hearts were retrogradely perfused on a Langendorff perfusion apparatus. After 40-min stabilization, hearts underwent 30-min global ischemia and 120-min reperfusion (I/R group). In GLP-2 group, the hearts received 20-min GLP-2 (10(-7)M) infusion at the beginning of the 120-min reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated by nitroblue-tetrazolium staining. Compared with the I/R group, GLP-2-treated hearts showed a significant reduction of infarct size and of postischemic diastolic LVP (index of contracture), together with a sharp improvement of developed LVP recovery (index of contractility). The protective effects were abolished by co-infusion with phosphatidylinositol 3-kinase inhibitor, Wortmannin (WT), the ERK1/2 inhibitor, PD98059, or the mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate. GLP-2 effects were accompanied by increased phosphorylation of protein kinase B (PKB/Akt), ERK1/2 and glycogen synthase kinase (GSK3β). After 7-min reperfusion, WT blocked Akt and GSK3β phosphorylation. After 30-min reperfusion, WT inhibited phosphorylation of all kinases. In conclusion, the data suggest that GLP-2, given in early reperfusion, as postconditioning, protects against myocardial I/R injury, limiting infarct size, and improving post-ischemic mechanical recovery. It seems that the GLP-2-protection of rat heart involves multiple prosurvival kinases and mitochondrial K(ATP) channels.

The administration of oxytocin during early reperfusion, dose-dependently protects the isolated male rat heart against ischemia/reperfusion injury

In our previous study, the administration of oxytocin (OT) could precondition the heart against ischemia/reperfusion injury. In this study, to investigate the cardiac postconditioning effect of oxytocin, isolated rat hearts were mounted on a Langendorff perfusion apparatus. In all groups, the hearts underwent 30min of regional ischemia followed by 120min of reperfusion. In the ischemia/reperfusion (IR) group, ischemia and reperfusion was induced. In the ischemic postconditioning (Ipost) group, hearts underwent 6cycles of 10s reperfusion and 10s ischemia at the beginning of reperfusion. In the other groups (III-IX), OT was perfused 5min before the onset of reperfusion and continued for 25min with following doses (Molar): 10−12, 5×10−12, 8×10−12, 10−11, 2×10−11, 5×10−11, and 10−10. The infarct size and coronary effluent levels of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and malondialdehyde (MDA) were calculated at the end of reperfusion. The infarct size decreased considerably in Ipost group compared to IR group (P<0.05). Also, the infusion of oxytocin by doses of 8×10−12M, 10−11M and 2×10−11M dose-dependently reduced infarct size (P<0.05) significantly compared to the IR group. LDH level in coronary effluent was markedly decreased in Ipost group and treatment with oxytocin by doses of 8×10−12M, 10−11M, 2×10−11M and 5×10−11M (P<0.05) compared to IR group. Ipost, OT 2×10−11and 10−11M significantly decreased CK-MB level (P<0.05). Ipost, OT 8×10−12, 10−11 and 2×10−11M significantly decreased MDA level as compared to IR group. Our study shows that oxytocin dose-dependently exerts cardiac postconditioning.

Hydrogen as additive of HTK solution fortifies myocardial preservation in grafts with prolonged cold ischemia

BackgroundRecent evidences indicated that hydrogen (H2) can attenuate organ transplantation induced cold ischemia/reperfusion (I/R) injury if administrated perioperatively. In this study we evaluated whether administrating H2 during the prolonged cold ischemia stage by adding it to Histidine–Tryptophan–Ketoglutarate (HTK) solution fortifies preservation for cardiac grafts. MethodsOne hundred and twenty-eight Sprague–Dawley (SD) rats were equally randomized to four groups: three H2-rich HTK-treated groups with H2 of different concentrations and traditional HTK-treated group as the control group. Isolated hearts were mounted on the Langendorff apparatus for aerobic perfusion. Following baseline hemodynamic measurements, grafts were arrested and stored in HTK with or without H2 for 6h at 4°C. After this prolonged cold storage, grafts were reperfused and concerned parameters were examined. ResultsCompared with the control group, preservation in H2-rich HTK significantly enhanced the percentage recovery of hemodynamic parameters, which was parallel to the diminished re-beating time and improved microscopic morphology of myocardium. Oxidative stress associated parameters including 8-hydroxy-2′-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) were decreased while myocardial superoxide dismutase (SOD) activity was preserved. Concentrations of inflammatory mediators including tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6), percentage of TUNEL-positive cells, expression of pro-apoptotic molecule Bax, and caspase-3 activity were reduced while Bcl-2 mRNA and protein levels were up-regulated in H2-rich HTK groups. The protective effects of H2 were concentration dependant. ConclusionsHydrogen as additive of HTK solution fortifies HTK's preservation efficacy for cardiac grafts subjected to prolonged cold ischemia by inhibiting cold ischemia-induced up-regulation of oxidative stress, inflammation mediators, and apoptosis.

Abstract 17318: Caspase Inhibition Prevents Hypertrophy and Improves Cardiac Function with Chronic Pressure Overload

It is widely held that a major mechanism underlying the transition from stable left ventricular (LV) function to failure is myocyte loss through apoptosis. To test this hypothesis, we examined the effects of chronic transverse aortic constriction (TAC) in C57BL/6 mice with or without administration of the caspase inhibitor (CI) (Z-Asp-2,6-DCBMk) delivered through an intraperitoneal osmotic minipump. In mice treated with CI, LV function (LV ejection fraction) was preserved (73±1%) compared with the vehicle group (52±7%) (p&lt;0.05) after 2 weeks of TAC. One possibility was that CI prevented apoptosis of myocytes and thereby improved LV function. This was not likely, since with CI, apoptosis was reduced, but relatively trivially in myocytes; using double staining with TUNEL and wheat germ agglutinin, only 7% of the reduced cellular apoptosis in the heart could be attributed to myocytes as compared with 93% of non-myocytes protected. Furthermore, if myocyte apoptosis protection was the mechanism, then LV mass would likely have been larger with CI. However, the reverse occurred, i.e., CI actually prevented LV hypertrophy; LV weight/tibial length did not increase significantly with CI after TAC (5.26±0.30) vs. sham (4.95±0.30) as opposed to vehicle after TAC (7.49±0.43). This was not because of differences in LV/aortic pressure gradient in CI (102±4mmHg) vs. vehicle (100±2mmHg), but likely due to protected LV wall stress, which was less (p&lt;0.05) in the CI group (70.2±9.9 Kdyn/cm 2 ) than vehicle (132.2±19.4 Kdyn/cm 2 ). Therefore, the mechanism for improved function with TAC and CI was likely related to an effect of CI on myocytes. Accordingly, we examined isolated myocytes using a Langendorff perfusion apparatus, and measured isolated myocyte contractility by an optical edge-tracking method. After TAC the contractility (% of cell length) under 1mM calcium was increased in CI (15.5±1.2%) compared to vehicle (10.2±1.4%) (p&lt;0.01). Thus, CI improved LV function after chronic pressure overload, not by protecting against myocyte apoptosis, but rather by improving myocyte contractile function resulting in reduced LV wall stress and hypertrophy.

Three-Year Follow-up of Sirolimus-Eluting Stents vs. Bare Metal Stents for Acute Myocardial Infarction

The long-term safety and efficacy of drug-eluting stents for patients with acute myocardial infarction (AMI) remain controversial. A total of 143 consecutive patients who presented between August 2004 and July 2006 with AMI and who underwent primary percutaneous coronary intervention (PCI) using sirolimus-eluting stents (SES), were compared with a historical control cohort of 129 consecutive patients who presented between August 2002 and July 2004 and who underwent primary PCI using bare metal stents (BMS). The rate of major adverse cardiovascular events at 3 years was significantly lower in the SES group than in the BMS group (20.3% vs. 33.1%, respectively; P=0.01). This reduction was mainly driven by a decrease in the rate of target vessel revascularization (12.3% vs. 22.4%, respectively; P=0.02). There was no significant difference in the rate of cardiovascular death (4.5% vs. 5.7%, respectively; P=0.67), non-fatal myocardial infarction (4.5% vs. 9.2%, respectively; P=0.16), coronary artery bypass grafting (2.3% vs. 2.5%, respectively; P=0.93), stroke (2.4% vs. 0.8%, respectively; P=0.35), and stent thrombosis (2.9% vs. 2.3%, respectively; P=0.80) between the 2 groups. SES can be used safely and effectively in patients with AMI.

Rooibos (Aspalathus linearis) offers cardiac protection against ischaemia/reperfusion in the isolated perfused rat heart

Rooibos, a unique South African herbal tea, is known to be an important source of unique polyphenolic compounds. In the present study we have quantified the main polyphenolic compounds in both fermented/traditional and unfermented/“green” rooibos ( Aspalathus linearis) and evaluated its cardioprotective effects against ischaemia/reperfusion injury. Male Wistar rats consumed aqueous rooibos and green tea ( Camellia sinensis) extracts (2%, w/v) for 7 weeks before their hearts were rapidly excised and perfused in a working heart perfusion apparatus. The results showed that the rooibos supplemented hearts significantly improved aortic output recovery after reperfusion when compared to the green tea supplemented hearts. Additionally, we showed that the rooibos extracts, containing the highest amount of flavonols, significantly decreased the level of cleaved caspase-3 and PARP, both pro-apoptotic proteins, during reperfusion when compared to green tea. Green tea supplementation increased phosphorylation of total PKB/Akt, Akt (threonine 308) and Akt (serine 473). The rooibos extracts did not cause significant change in the levels of the pro-survival PKB/Akt (threonine 308 and serinet 473). The GSH/GSSG ratio in the hearts of the green tea supplemented group was significantly ( p < 0.05) lower when compared to RF (37.78 ± 28.63), RU (33.20 ± 4.13) and C (45.50 ± 14.96). The results clearly demonstrate the cardio-protective properties of aqueous rooibos extracts via the inhibition of apoptosis which can possibly be related to the flavonol content of this unique South African herbal tea.

Allogeneic bone marrow mesenchymal stem cells over-expressing gap junction protein connexin 43 reduce ventricular arrhythmias following myocardial infarction in rats

BackgroundMesenchymal stem cell transplantation is a promising new therapy to improve cardiac function after myocardial infarction (MI). The electrophysiological consequences of MSC implantation have not been systematically studied.ObjectiveThis study strived...