Functional integrity of honeybee (Apis mellifera L.) resistant to dieldrin γ-aminobutyric acid receptor channels conjugated with three fluorescent proteins.

To generate an efficient tool used in Xenopus oocyte expression for in situ investigation of channel receptor expression, distribution and function, the C-terminus of the honeybee (Apis mellifera L.) resistant to dieldrin (RDL) subunit was fused with *FP, including monomeric red, enhanced yellow or enhanced green fluorescent protein (referred to as mRFP, EYFP and EGFP, respectively). In the present study, all fused *FP-AmRDLs could be visualized using fluorescence and laser confocal microscopy in cRNA-injected oocytes. Fluorescence was distributed isotropically in the cellular membrane. The potencies of the agonist γ-aminobutyric acid (GABA), but not ß-alanine, and the test antagonists (fipronil, flufiprole, dieldrin, α-endosulfan, bifenazate and avermectin B1a) in the *FP-AmRDL receptor did not significantly differ from that of the untagged receptor with two-electrode voltage clamp detection. The half maximal effective concentrations (EC50 s) of GABA in AmRDL, EGFP-AmRDL, EYFP-AmRDL and mRFP-AmRDL receptors were 11.98, 12.61, 18.92 and 22.11 µM, respectively, and those of ß-alanine were 651.6, 629.6, 1643.0 and 2146.0 µM, respectively. Inhibition percentages of test antagonists against *FP-AmRDL and AmRDL were not significantly different from each other. Overall, the consistency in functional properties between *FP-AmRDL and AmRDL receptors makes pGH19-*FP a promising tool for further in situ investigation of GABA receptors.

Cardiovascular effects of a PEGylated apelin.

Several studies have documented cardiovascular effects of apelin, including enhanced inotropy and vasodilation. However, these cardiovascular effects are short lived due to the predicted short circulating half-life of the apelin peptide. To address this limitation of apelin, we pursued N-terminal PEGylation of apelin and examined the cardiovascular effects of the PEGylated apelin. A 40kDa PEG conjugated apelin-36 (PEG-apelin-36) was successfully produced with N-terminal conjugation, high purity (>98%) and minimum reduction of APJ receptor binding affinity. Using an adenylate cyclase inhibition assay, comparable in vitro bioactivity was observed between the PEG-apelin-36 and unmodified apelin-36. In vivo evaluation of the PEG-apelin-36 was performed in normal rats and rats with myocardial infarction (MI). Cardiac function was assessed via echocardiography before, during a 20 min IV infusion and up to 100 min post peptide infusion. Similar increases in cardiac ejection fraction (EF) were observed during the infusion of PEG-apelin-36 and apelin-36 in normal rats. However, animals that received PEG-apelin-36 maintained significantly increased EF over the 100 min post infusion monitoring period compared to the animals that received unmodified apelin-36. Interestingly, EF increases observed with PEG-apelin-36 and apelin-36 were greater in the MI rats. PEG-apelin-36 had a prolonged circulating life compared to apelin-36 in rats. There were no changes in aortic blood pressure when PEG-apelin-36 or apelin-36 was administered. To our knowledge this is the first report of apelin PEGylation and documentation of its cardiovascular effects.

Angiogenesis by endothelial cell transplantation.

PURPOSE: Myocardial angiogenesis may improve regional perfusion and perhaps function after cardiac injury. We evaluated the effect of endothelial cell transplantation into a myocardial scar on angiogenesis and ventricular function, as an alternative to angiogenic gene or protein therapy. METHODS AND RESULTS: A transmural myocardial scar was created in the left ventricular free wall of rat hearts by cryoinjury. Allogeneic aortic endothelial cells were injected into the scar 2 weeks after cryoinjury. A cluster of transplanted cells was identified at the site of injection 1 day and 1 week after transplantation, but not after 2 weeks. The size of this cluster of transplanted cells decreased as vascular density in the transplanted scar tissue increased with time. Six weeks after transplantation, vascular density was significantly greater in transplanted hearts than in control hearts. Regional blood flow, by microsphere analysis, was greater in the transplanted rats. Systolic and diastolic ventricular function was similar between groups. In a second series of experiments, syngeneic aortic endothelial cells labeled with bromodeoxyuridine were transplanted 2 weeks after cryoinjury. Vascular density in the transplanted scar was greater than in controls. Labeled transplanted endothelial cells were identified forming part of the newly developed blood vessels. No difference in vascular density was found between allogeneic and syngeneic cell transplantation. Vascular endothelial growth factor was not expressed at greater levels in the transplanted cells or the myocardial scar. CONCLUSION: Transplanted endothelial cells stimulated angiogenesis, were incorporated into the new vessels, and increased regional perfusion in myocardial scar tissue, but did not improve global function in this cryoinjury rat model.

The fate of a tissue-engineered cardiac graft in the right ventricular outflow tract of the rat.

OBJECTIVE: The synthetic materials currently available for the repair of cardiac defects are nonviable, do not grow as the child develops, and do not contract synchronously with the heart. We developed a beating patch by seeding fetal cardiomyocytes in a biodegradable scaffold in vitro. The seeded patches survived in the right ventricular outflow tract of adult rats. METHODS: Cultured fetal or adult rat heart cells (1 x 10(6) cells) were seeded into a gelatin sponge (15 x 15 x 1 mm), and the cell number was expanded in culture for 1 or 3 weeks, respectively. The free wall of the right ventricular outflow tract in syngeneic adult rats was resected and repaired with either unseeded patches or patches seeded with either fetal or adult cardiomyocytes (n = 10 for each group). The patches were examined histologically over a 12-week period. RESULTS: A significant inflammatory reaction was noted in the patch at 4 weeks as the scaffold dissolved. At 12 weeks, the gelatin scaffold had completely dissolved. Both types of the seeded cells were detected in the patch with 5-bromo-2'-deoxyuridine staining, and they maintained their continuity. Unseeded patches had an ingrowth of fibrous tissue. The patches became thinner between the fourth and the twelfth weeks in unseeded (P =.003), fetal (P =.0001), and adult (P =.07) cardiomyocyte groups as the scaffold dissolved. The control patch, but not the cell-seeded patches, was thinner than the normal right ventricular outflow tract. The endocardial surface area of each patch was covered with endothelial cells identified by factor VIII staining. CONCLUSIONS: A gelatin patch was used to replace the right ventricular outflow tract in syngeneic rats. The seeded cells survived in the right ventricular outflow tract after the scaffold dissolved 12 weeks after implantation. In addition, the unseeded patches encouraged the ingrowth of fibrous tissue as the scaffold dissolved and the patches remained completely endothelialized.

Optimal time for cardiomyocyte transplantation to maximize myocardial function after left ventricular injury.

BACKGROUND: This study was designed to determine the optimal time for cell transplantation after myocardial injury. METHODS: The left ventricular free wall of adult rat hearts was cryoinjured and the animals were sacrificed at 0, 1, 2, 4, and 8 weeks for histologic studies. Fetal rat cardiomyocytes (transplant) or culture medium (control) were transplanted immediately (n = 8), 2 weeks (n = 8), and 4 weeks (n = 12) after cryoinjury. At 8 weeks, rat heart function, planimetry, and histologic studies were performed. RESULTS: Cryoinjury produced a transmural injury. The inflammatory reaction was greatest during the first week but subsided during the second week after cryoinjury. Scar size expanded (p < 0.01) at 4 and 8 weeks. Cardiomyocytes transplanted immediately after cryoinjury were not found 8 weeks after cryoinjury. Scar size and myocardial function were similar to the control hearts. Cardiomyocytes transplanted at 2 and 4 weeks formed cardiac tissue within the scar, limited (p < 0.01) scar expansion, and had better (p < 0.001) heart function than the control groups. Developed pressure was greater (p < 0.01) in the hearts with transplanted cells at 2 weeks than at 4 weeks. CONCLUSIONS: Cardiomyocyte transplantation was most successful after the inflammatory reaction resolved but before scar expansion.

Heart cell transplantation improves heart function in dilated cardiomyopathic hamsters.

BACKGROUND: Little is known about the effect of heart cell transplantation into the dilated cardiomyopathic myocardium. This study was designed to evaluate the effect of heart cell transplantation into dilated cardiomyopathic hamsters. METHODS AND RESULTS: Ventricular heart cells were isolated from 4-week-old BIO 53. 58 hamsters and cultured for 2 weeks before transplantation. The cells were labeled with bromodeoxyuridine (BrdU) before transplantation for identification. Adult hamsters (17 weeks old) were used as recipients. Heart cells (4 x 10(6) cells) or culture medium was transplanted into the left ventricular free wall (transplantation and control groups, respectively, n=12 each). Sham-operated hamsters (n=12) underwent the surgery but not the transplantation. Cyclosporine A was administered subcutaneously to all hamsters daily after the operation. Four weeks after the transplantation, heart function was evaluated with the use of a Langendorff preparation. Histology showed severe focal myocardial necrosis in all groups. BrdU-stained tissue was found at the cell transplantation sites. The transplanted hearts had greater (P:<0. 001) developed pressures at all balloon volumes and improved dP/dt (transplantation 915+/-253 versus control 453+/-120 and sham 530+/-187 mm Hg/s, P:<0.001, balloon volume of 15 microL). No differences in ventricular function were found between control and sham-operated hamsters. CONCLUSIONS: The transplanted ventricular heart cells formed cardiac-like tissue in cardiomyopathic myocardium and improved its contractile function.

Construction of a bioengineered cardiac graft.

OBJECTIVES: Currently available graft materials for repair of congenital heart defects cause significant morbidity and mortality because of their lack of growth potential. An autologous cell-seeded graft may improve patient outcomes. We report our initial experience with the construction of a biodegradable graft seeded with cultured rat or human cells and identify their 3-dimensional growth characteristics. METHODS: Fetal rat ventricular cardiomyocytes, stomach smooth muscle cells, skin fibroblasts, and adult human atrial and ventricular cardiomyocytes were isolated and cultured in vitro. These cells were injected into or laid onto biodegradable gelatin meshes, and their rate of proliferation and spatial location within the mesh was evaluated by using a cell counter and histologic analysis. RESULTS: Rat cardiomyocytes, smooth muscle cells, and fibroblasts demonstrated steady proliferation over 3 to 4 weeks. The gelatin mesh was slowly degraded, but this process was most rapid after seeding with fibroblasts. Human atrial cardiomyocytes proliferated within the gelatin meshes but at a slower rate than that of fetal rat cardiomyocytes. Human ventricular cardiomyocytes survived within the gelatin mesh matrix but did not increase in number during the 2-week duration of evaluation. Grafts seeded with rat ventricular cells exhibited spontaneous rhythmic contractility. All cell types preferentially migrated to the uppermost surface of each graft and formed a 300- to 500-microm thick layer. CONCLUSIONS: Fetal rat ventricular cardiomyocytes, gastric smooth muscle cells, skin fibroblasts, and adult human atrial cardiomyocytes can grow in a 3-dimensional pattern within a biodegradable gelatin mesh. Similar autologous cell-seeded constructs may eventually be applied to repair congenital heart defects.

Autologous porcine heart cell transplantation improved heart function after a myocardial infarction.

OBJECTIVE: Fetal cardiomyocyte transplantation improved heart function after cardiac injury. However, cellular allografts were rejected despite cyclosporine (INN: ciclosporin) therapy. We therefore evaluated autologous heart cell transplantation in an adult swine model of a myocardial infarction. METHODS: In 16 adult swine a myocardial infarction was created by occlusion of the distal left anterior descending coronary artery by an intraluminal coil. Four weeks after infarction, technetium 99m-sestamibi single photon emission tomography showed minimal perfusion and viability in the infarcted region. Porcine heart cells were isolated and cultured from the interventricular septum at the time of infarction and grown in vitro for 4 weeks. Through a left thoracotomy, either cells (N = 8) or culture medium (N = 8) was injected into the infarct zone. RESULTS: Four weeks after cell transplantation, technetium 99m-sestamibi single photon emission tomography demonstrated greater wall motion scores in the pigs receiving transplantation than in control animals (P =.01). Pigs receiving transplantation were more likely to have an improvement in perfusion scores (P =.03). Preload recruitable stroke work (P =.009) and end-systolic elastance (P =. 02) were greater in the pigs receiving transplantation than in control animals. Scar areas were not different, but scar thickness was greater (P =.02) in pigs receiving transplantation. Cells labeled with bromodeoxyuridine in vitro could be identified in the infarct zone 4 weeks after transplantation. Swine receiving transplantation gained more weight than control animals (P =.02). CONCLUSION: Autologous porcine heart cell transplantation improved regional perfusion and global ventricular function after a myocardial infarction.

Survival and function of bioengineered cardiac grafts.

INTRODUCTION: Patients with congenital heart disease frequently require graft material for repair of cardiac defects. However, currently available grafts lack growth potential and are noncontractile and thrombogenic. We have developed a viable cardiac graft that contracts spontaneously in tissue culture by seeding cells derived from fetal rat ventricular muscle into a biodegradable material. We report our investigations of the in vitro and in vivo survival and function of this bioengineered cardiac graft. METHODS AND RESULTS: A cardiomyocyte-enriched cell inoculum derived from fetal rat ventricular muscle was seeded into a piece of Gelfoam (Upjohn, Ontario, Canada), a biodegradable gelatin mesh, to form the graft. For in vitro studies, growth patterns of the cells within the graft were evaluated by constructing growth curves and by histologic examination; in in vivo studies, the graft was cultured for 7 days and then implanted either into the subcutaneous tissue of adult rat legs or onto myocardial scar tissue in a cryoinjured rat heart. Five weeks later, the graft was studied histologically. The inoculated cells attached to the gelatin mesh and grew in 3 dimensions in tissue culture, forming a beating cardiac graft. In both the subcutaneous tissue and the myocardial scar, blood vessels grew into the graft from the surrounding tissue. The graft implanted into the subcutaneous tissue contracted regularly and spontaneously. When implanted onto myocardial scar tissue, the cells within the graft survived and formed junctions with the recipient heart cells. CONCLUSIONS: Fetal rat ventricular cells can grow 3-dimensionally in a gelatin mesh. The cells in the graft formed cardiac tissue and survived and contracted spontaneously both in tissue culture and after subcutaneous implantation. Future versions of this bioengineered cardiac graft may eventually be used to repair cardiac defects.

Autologous transplantation of bone marrow cells improves damaged heart function.

BACKGROUND: Autologous bone marrow cells (BMCs) transplanted into ventricular scar tissue may differentiate into cardiomyocytes and restore myocardial function. This study evaluated cardiomyogenic differentiation of BMCs, their survival in myocardial scar tissue, and the effect of the implanted cells on heart function. IN VITRO STUDIES: BMCs from adult rats were cultured in cell culture medium (control) and medium with 5-azacytidine (5-aza, 10 micromol/L), TGFbeta1 (10 ng/mL), or insulin (1 nmol/L) (n=6, each group). Only BMCs cultured with 5-aza formed myotubules which stained positively for troponin I and myosin heavy chain. In vivo studies: a cryoinjury-derived scar was formed in the left ventricular free wall. At 3 weeks after injury, fresh BMCs (n=9), cultured BMCs (n=9), 5-aza-induced BMCs (n=12), and medium (control, n=12) were autologously transplanted into the scar. Heart function was measured at 8 weeks after myocardial injury. Cardiac-like muscle cells which stained positively for myosin heavy chain and troponin I were observed in the scar tissue of the 3 groups of BMC transplanted hearts. Only the 5-aza-treated BMC transplanted hearts had systolic and developed pressures which were higher (P<0.05) than that of the control hearts. All transplanted BMCs induced angiogenesis in the scar. CONCLUSIONS: Transplantation of BMCs induced angiogenesis. BMCs cultured with 5-aza differentiated into cardiac-like muscle cells in culture and in vivo in ventricular scar tissue and improved myocardial function.

Fetal cell transplantation: a comparison of three cell types.

OBJECTIVE: We have previously reported that fetal cardiomyocyte transplantation into myocardial scar improves heart function. The mechanism by which this occurs, however, has not been elucidated. To investigate possible mechanisms by which cell transplantation may improve heart function, we compared cardiac function after transplantation of 3 different fetal cell types: cardiomyocytes, smooth muscle cells (nonstriated muscle cells), and fibroblasts (noncontractile cells). METHODS: A left ventricular scar was created by cryoinjury in adult rats. Four weeks after injury, cultured fetal ventricular cardiomyocytes (n = 13), enteric smooth muscle cells (n = 10), skin fibroblasts (n = 10), or culture medium (control, n = 15 total) were injected into the myocardial scar. All rats received cyclosporine A (INN: ciclosporin). Four weeks after transplantation, left ventricular function was evaluated in a Langendorff preparation. RESULTS: The implanted cells were identified histologically. All transplanted cell types formed tissue within the myocardial scar. At an end-diastolic volume of 0.2 mL, developed pressures in cardiomyocytes group were significantly greater than smooth muscle cells and skin fibroblasts groups (cardiomyocytes, 134% +/- 22% of control; smooth muscle cells, 108% +/- 14% of control; skin fibroblasts, 106% +/- 17% of control; P =.0001), as were +dP/dt(max) (cardiomyocytes, 119% +/- 37% of control; smooth muscle cells, 98% +/- 18% of control; skin fibroblasts, 92% +/- 11% of control; P =. 0001) and -dP/dt(max) (cardiomyocytes, 126% +/- 29% of control; smooth muscle cells, 108% +/- 19% of control; skin fibroblasts, 99% +/- 16% control; P =.0001). CONCLUSIONS: Fetal cardiomyocytes transplanted into myocardial scar provided greater contractility and relaxation than fetal smooth muscle cells or fetal fibroblasts. The contractile and elastic properties of transplanted cells determine the degree of improvement in ventricular function achievable with cell transplantation.

Smooth muscle cell transplantation into myocardial scar tissue improves heart function.

This study was designed to evaluate the effect of smooth muscle cell transplantation into myocardial ventricular scar formed by cryo-necrosis. The left ventricular free wall (LVFW) of adult rats was cryo-necrosed. At 4 weeks after cryo-injury cultured fetal rat stomach smooth muscle cells (transplanted group, n = 10) or culture medium (control, n = 10) were transplanted. Sham animals (n = 8) were similarly operated but without cryo-necrosis and transplantation. The animals were administered a daily maintenance dose of cyclosporin A (5 mg/kg). At 8 weeks after cryo-injury, heart function was evaluated using a Langendorff preparation. Myocardial scar and transplanted cells were assessed histologically. Transplanted smooth muscle cells survived and formed smooth muscle cell tissue, as assessed by immunostaining against smooth muscle cell actin, within the myocardial scar. In the control hearts no muscle tissue was found in the scar. Angiogenesis occurred (P < 0.001) in the transplanted scar compared to the control scar. The transplanted cells increased the scar thickness (P < 0.01) by hyperplasia and prevented (P < 0.001) the dilatation of the ventricular chamber size compared to the controlled hearts. For physiological left ventricular volumes of 0.04 to 0.28 ml, the systolic and developed pressures in the transplanted group were greater (P < 0.001) than the control group, but less (P < 0.001) than those of the sham group. Transplanted smooth muscle cells formed smooth muscle tissue in myocardial scar tissue and improved contractile function compared to control hearts.

Autologous heart cell transplantation improves cardiac function after myocardial injury.

BACKGROUND: Fetal ventricular cardiomyocyte transplantation into a cardiac scar improved ventricular function, but these cells were eventually eliminated by rejection. We therefore examined the feasibility of autologous adult heart cell transplantation. METHODS: A transmural scar was produced in the left ventricular free wall of adult rats by cryoinjury. The left atrial appendage was harvested, and the atrial heart cells were cultured and their number expanded ex vivo. Three weeks after cryoinjury, either a cell suspension (2 x 10(6) cells, n = 12 rats, transplant group) or culture medium (n = 10 rats, control group) was injected into the scar. Rats having a sham operation (n = 5) did not undergo cryoinjury or transplantation with cells or culture medium. RESULTS: Five weeks after injection, ventricular function was evaluated in a Langendorff preparation, measuring systolic, diastolic, and developed pressures over a range of intraventricular balloon volumes. Systolic and developed pressures were greater in the transplant group than in the control group (p = 0.0001). Rats with a sham operation had the greatest systolic, diastolic, and developed pressures (p = 0.0001). Histologic studies demonstrated survival of the transplanted heart cells within the scar. The area of the scar was smaller (p = 0.0003) and its thickness greater (p = 0.0003) in rats in the transplant group. Left ventricular chamber volume was smaller in the transplant group (p = 0.043). CONCLUSIONS: Transplantation of autologous cultured adult atrial heart cells limited scar thinning and dilatation and improved myocardial function compared with results in control hearts. This technique may lead to a novel therapy to prevent scar expansion after a myocardial infarction and prevent the development of congestive heart failure.

Natural history of fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue.

BACKGROUND: Fetal rat cardiomyocytes transplanted into left ventricular scar tissue of the adult rat heart limit scar expansion and improve heart function. This study determined morphologic changes of transplanted fetal rat cardiomyocytes in myocardial scar tissue. METHODS AND RESULTS: The left ventricles of 500-g Sprague-Dawley rats were cryodamaged. At 4 weeks after myocardial injury, a transmural scar (54+/-11 mm2) (mean+/-1 SDak) formed at the apex (n=6). Cardiomyocytes freshly isolated from 18-day-gestation Sprague-Dawley rat hearts were transfected with plasmid containing the beta-galactosidase and then injected into the 4-week-old scar tissue. Cell culture medium was injected into the scar tissue of control animals. At 4 weeks posttransplantation, the cardiomyocytes had formed cardiac tissue (20.7+/-6.9 mm2, n=14), which stained positively for beta-galactosidase activity in the scar (90.4+/-25 mm2, n=14). The transplanted cardiomyocytes formed sarcomeres and were linked by junctions composed of desmosomes and fascia adherens. Lymphocyte infiltration occurred despite use of cyclosporin A. No myocardial tissue was found in the scar tissue of the control animals (n=14). More arterioles and venules were found (P<.01) in the cardiomyocyte grafts (1.2+/-0.6 vessel/0.8 mm2; n=14) than in the control scar tissue (0.1+/-0.1 vessels/0.8 mm2; n=14). At 20 weeks post-transplantation, the transplant tissue size (6+/-6 mm2; n=7) was smaller (P=.007) than 4-week old transplant, and the scar (162+/-46 mm2; n=7) was larger (P=.005) than 4-week-old scar. Lymphocyte infiltration was still present among the remaining transplanted cells. CONCLUSIONS: This study demonstrated that cardiac tissue formed by transplanted fetal cardiomyocytes in the myocardial scar tissue decreased in size with time probably secondary to rejection.

The effects of artery occlusion on temperature homogeneity during hyperthermia in rabbit kidneys in vivo.

To investigate the role of arterial occlusion on temperature homogeneity during hyperthermia for deep seated tissue, a renal hyperthermia animal model has been established using New Zealand white rabbits. The effects of ultrasound-induced renal hyperthermia, with or without continuous and intermittent renal artery occlusion, were compared and analysed. Both continuous and intermittent occlusion showed certain protection of surrounding tissue and demonstrated improved temperature homogeneity and heating efficiency. The benefits of continuous vs. intermittent occlusion are compared and discussed as well.

Cardiomyocyte transplantation improves heart function.

BACKGROUND: Transplantation of cultured cardiomyocytes into myocardial scar tissue may prevent heart failure. METHODS: Scar tissue was produced in the left ventricular free wall of 15 rats (weight, 450 g) by cryoinjury. Seven animals had operation only and survived for 8 weeks (sham group). Four weeks after cryoinjury, cultured fetal rat cardiomyocytes or culture medium was injected into the scar tissue of transplantation (n = 5) and control (n = 5) animals, respectively. Five other rats were sacrificed for scar assessment. Eight weeks after cryoinjury heart function in the transplantation, control, and sham groups was measured using a Langendorff preparation. Histologic studies were performed to quantify the extent of the scar and the transplanted cells. RESULTS: Four weeks after cryoinjury, 36% +/- 4% (mean +/- 1 standard error) of the left ventricular free wall surface area was scar tissue. At 8 weeks, the scar size had increased (p < 0.01) to 55% +/- 3% in the control group. Although the scar size (43% +/- 2%) in the transplantation group at 8 weeks was not significantly different from that at 4 weeks, it was less (p < 0.05) than that in the control group. Hearts in the sham group had no scar tissue. The transplanted cardiomyocytes had formed cardiac tissue within the myocardial scar. Systolic and developed pressures in the transplantation group hearts were greater (p = 0.0001) than in the control group hearts but less (p < 0.01) than those in the sham group hearts. CONCLUSIONS: The transplanted cardiomyocytes formed cardiac tissue in the myocardial scar, limited scar expansion, and improved heart function compared with findings in the control hearts.