Heterotopic Abdominal Heart Transplantation Research Articles

ERK5 promotes TLR2-dependent up-regulation of inflammatory mediator expression induces by global myocardial ischemia

Global myocardial ischemia and reperfusion [I/R] injury is predominantly associated with heart transplant and coronary artery bypass graft (CABG) surgery. Toll-like receptor-2 (TLR2) is a pivotal pathogen recognition receptor that has a key role in inflammation and injury. The proinflammatory role for ERK5 downstream of TLR2 in global myocardial I/R has not been reported previously. The aim of this study was to investigate to what extent inflammatory stimuli modify the expression by ERK5 of TLR2. Global myocardial I/R was induced in male TLR2+/+ and TLR2−/− mice using heterotopic abdominal heart transplantation model that allows to subject donor hearts to global hypothermic ischemia and blood reperfusion, which very closely stimulates I/R conditions associated with cardiac surgical operations. The results show that inhibition or siRNA knockdown of ERK5 reduces the TLR2-dependent activation of expression of cytokines, attenuated myocardial injury, less oxidative stress and decreased NF-κB activation and indicating that ERK5 promotes cytokine expression downstream of TLR2. We have identified novel aspects of TLR2 signaling in cold myocardial I/R, including that ERK5 activity is required for TLR2 activation.

Molecular Chimeric Recipient Precursor T Cells Promote Cardiac Allograft Survival in Mice

BackgroundMolecular chimerism has become a potential method to induce donor-specific transplant tolerance. We researched the prolongation of cardiac allograft survival by recipient mouse molecular chimeric precursor T cells (pre-T cells) or hematopoietic stem cells (HSCs) infusion in vivo. MethodsThe donor C57BL/6 mouse MHC-I gene (H-2Kb and H-2Db gene) were amplified by RT-PCR. The identified recipient BALB/c mouse pre-T cells and HSCs were transduced by the pMSCVneo retroviral vector of C57BL/6 mouse MHC-I gene (pMSCVneo-H-2Db/H-2Kb). Then the molecular chimeric cells were transfused back to the BALB/c mice. Allogeneic T-lymphocyte proliferation was assessed in mixed lymphocyte reactions (MLR). A mouse model of heterotopic abdominal heart transplantation was performed to evaluate survival times and histological grade of acute rejection at 7 days after transplantation. ResultsBALB/c mice molecular chimeric pre-T cells and HSCs were cultured successfully after pMSCV-H-2Db/H-2Kb transduction. After the molecular chimeric pre-T cell treatment, the result of MLR showed that the stimulating index of allogeneic T lymphocyte had a statistically significant decrease, which also exhibited a significant reduction after molecular chimeric HSC treatment. The survival time of cardiac allograft was prolonged after chimeric pre-T cell or HSC infusion; meanwhile, pathologic rejection grade decreased significantly. Nevertheless, molecular chimeric pre-T cells exhibited a longer median survival time. ConclusionThe molecular chimeric recipient mouse pre-T cell or HSC infusion reduced spleen T cells' response to allogeneic T cells in vitro and delayed cardiac allograft rejection in vivo. Pre-T cells have more advantages than HSCs on the prolongation of mouse cardiac allograft survival.

Varying Degrees of Ventricular Unloading in the Heterotopic Rat Heart Transplant Model Demonstrated by Magnetic Resonance Imaging

Objective: Left ventricular assist device placement is an increasingly common treatment for cardiac failure, resulting in cardiac unloading and potentially reversing the remodelling changes seen in heart failure. A popular animal model for human ventricular unloading is the rodent heterotopic non-working heart transplant; the volume loading status of this preparation is important to interpreting the resulting reverse remodelling yet has not been previously investigated. This study was designed to assess the variability of left ventricular volume loading in the rodent transplant model. Methods: Heterotopic abdominal heart transplant was performed on syngeneic rats; high resolution cine magnetic resonance imaging was subsequently performed on the heterotopic transplanted hearts in anesthetised rats, after variable post-transplant recovery times, in order to assess ventricular loading status. Results: Highly variable left ventricular volume loading status was demonstrated, with some hearts exhibiting considerable ventricular filling and ejection. Conclusions: These observations call into question the assumption that studies using this model are consistently examining fully unloaded ventricles, and indicate the desirability of in vivo imaging of such hearts to quantify the degree of ventricular loading.

Impact of combined clenbuterol and metoprolol therapy on reverse remodelling during mechanical unloading.

BackgroundClenbuterol (Cl), a β2 agonist, is associated with enhanced myocardial recovery during left ventricular assist device (LVAD) support, and exerts beneficial remodelling effects during mechanical unloading (MU) in rodent heart failure (HF). However, the specific effects of combined Cl+β1 blockade during MU are unknown.Methods and ResultsWe studied the chronic effects (4 weeks) of β2-adrenoceptor (AR) stimulation via Cl (2 mg/kg/day) alone, and in combination with β1-AR blockade using metoprolol ((Met), 250 mg/kg/day), on whole heart/cell structure, function and excitation-contraction (EC) coupling in failing (induced by left coronary artery (LCA) ligation), and unloaded (induced by heterotopic abdominal heart transplantation (HATx)) failing rat hearts. Combined Cl+Met therapy displayed favourable effects in HF: Met enhanced Cl's improvement in ejection fraction (EF) whilst preventing Cl-induced hypertrophy and tachycardia. During MU combined therapy was less beneficial than either mono-therapy. Met, not Cl, prevented MU-induced myocardial atrophy, with increased atrophy occurring during combined therapy. MU-induced recovery of Ca2+ transient amplitude, speed of Ca2+ release and sarcoplasmic reticulum Ca2+ content was enhanced equally by Cl or Met mono-therapy, but these benefits, together with Cl's enhancement of sarcomeric contraction speed, and MU-induced recovery of Ca2+ spark frequency, disappeared during combined therapy.ConclusionsCombined Cl+Met therapy shows superior functional effects to mono-therapy in rodent HF, but appears inferior to either mono-therapy in enhancing MU-induced recovery of EC coupling. These results suggest that combined β2-AR simulation +β1-AR blockade therapy is likely to be a safe and beneficial therapeutic HF strategy, but is not as effective as mono-therapy in enhancing myocardial recovery during LVAD support.

Inhibiting cardiac allograft rejection with interleukin-35 therapy combined with decitabine treatment in mice

BackgroundRegulatory T (Treg) cells play a pivotal role in the maintenance of transplantation tolerance. It is of great interest to induce allograft tolerance mediated by regulatory CD4+CD25+ T cells. MethodsHere we investigated the effect of hydrodynamic IL-35-expressing plasmid injection in combination with a methyltransferase inhibitor (decitabine) on immune function and transplantation tolerance in mice. ResultsWe showed that IL-35 and decitabine stimulated the proliferation of CD4+CD25+ Tregs and suppressed CD8+ T cell proliferation in an allogenic mixed lymphocyte culture in vitro. IL-35 gene therapy and decitabine administration prolonged the survival of the transplanted heart in the heterotopic abdominal heart transplantation model in mice. ConclusionsThe possible mechanism through which IL-35 and decitabine treatment increased the survival of graft tissues is to enhance the proliferation of CD4+CD25+ Treg cells and suppress the generation and function of effector T cells. Thus, IL-35 gene therapy combined with decitabine provides a novel approach to induce transplantation tolerance.

Enhanced Ca2+ influx through cardiac L-type Ca2+ channels maintains the systolic Ca2+ transient in early cardiac atrophy induced by mechanical unloading

Cardiac atrophy as a consequence of mechanical unloading develops following exposure to microgravity or prolonged bed rest. It also plays a central role in the reverse remodelling induced by left ventricular unloading in patients with heart failure. Surprisingly, the intracellular Ca2+ transients which are pivotal to electromechanical coupling and to cardiac plasticity were repeatedly found to remain unaffected in early cardiac atrophy. To elucidate the mechanisms underlying the preservation of the Ca2+ transients, we investigated Ca2+ cycling in cardiomyocytes from mechanically unloaded (heterotopic abdominal heart transplantation) and control (orthotopic) hearts in syngeneic Lewis rats. Following 2 weeks of unloading, sarcoplasmic reticulum (SR) Ca2+ content was reduced by ~55 %. Atrophic cardiac myocytes also showed a much lower frequency of spontaneous diastolic Ca2+ sparks and a diminished systolic Ca2+ release, even though the expression of ryanodine receptors was increased by ~30 %. In contrast, current clamp recordings revealed prolonged action potentials in endocardial as well as epicardial myocytes which were associated with a two to fourfold higher sarcolemmal Ca2+ influx under action potential clamp. In addition, Cav1.2 subunits which form the pore of L-type Ca2+ channels (LTCC) were upregulated in atrophic myocardium. These data suggest that in early cardiac atrophy induced by mechanical unloading, an augmented sarcolemmal Ca2+ influx through LTCC fully compensates for a reduced systolic SR Ca2+ release to preserve the Ca2+ transient. This interplay involves an electrophysiological remodelling as well as changes in the expression of cardiac ion channels.

Total Arterial Anastomosis Heterotopic Heart Transplantation Model

BackgroundMouse transplant models offer a valuable platform for studying the biology of a spectrum of diseases, particularly those of the immune system. We have developed a modified abdominal heterotopic heart transplantation (AHHT) model with a total arterial anastomosis and compared the results with the cervical heterotopic heart transplantation (CHHT) and the non-modified AHHT models. MethodsMice were randomly assigned to four groups: sham, AHHT, CHHT, and modified AHHT groups. Each group (except for the sham) included donor and recipient animals. Postoperative outcome, operative mortality, operative time, and tissue damage were assessed by measuring plasma levels of tumor necrosis factor α. ResultThe modified AHHT group had significantly lower values. However, hind limb paralysis was observed equally and only in AHHT and modified AHHT models. The modified AHHT group had the highest success rate of functioning hearts.

Induction of CD4+CD25+ T Cells and Control of Cardiac Allograft Rejection by CD40/CD40L Costimulatory Pathway Blockade in Mice

BackgroundRejection is a major problem in organ transplantation. We speculated that dendritic cells (DCs) with low expression of CD40 may inhibit T-cell responses providing protective effects on organ transplantions. MethodsRNA interference (RNAi) is an effective approach to degrade RNA levels of a target gene. We used RNAi to reduce CD40 levels in mouse bone marrow-derived DCs. Allogeneic T-lymphocyte proliferation stimulated by DCs was assessed in mixed lymphocyte reactions. A mouse model of heterotopic abdominal heart transplantation was performed to evaluate survival times and histologic grade of acute rejection at 7 days after transplantation. CD4+CD25+ regulatory T cells (Tregs) in peripheral blood were quantified by flow cytometry. ResultsMouse DCs with reduced CD40 expression lowered the proliferation of recipient T cells. More DCs with silenced CD40 delayed cardiac allograft rejection. ConclusionsReduction of CD40 in DCs induced peripheral Tregs and delayed cardiac allograft rejection.

Mechanical unloading reverses transverse tubule remodelling and normalises local calcium-induced calcium release in a rodent model of heart failure

Calcium-induced calcium release (CICR) is crucial for contraction in cardiomyocytes. The transverse (t)-tubule system guarantees the proximity of the triggers for calcium release (L-type calcium channel, dihydropyridine receptors) and the sarcoplasmic reticulum calcium-release channels (ryanodine receptors). Transverse tubule disruption occurs early in heart failure. Clinical studies of left ventricular assist devices in heart failure indicate that mechanical unloading induces reverse remodelling. We hypothesise that unloading of failing hearts normalises t-tubule structure and improves CICR.Heart failure was induced in Lewis rats by left coronary artery ligation for 12 weeks; sham-operated animals were used as controls. Failing hearts were mechanically unloaded for 4 weeks by heterotopic abdominal heart transplantation (HF-UN). Heart failure reduced the t-tubule density as measured by di-8-ANEPPS staining in isolated left ventricular myocytes, and this was reversed by unloading. The deterioration in the regularity of the t-tubule system in heart failure was also reversed in HF-UN. Scanning ion conductance microscopy showed the reappearance of normal surface striations in HF-UN. Electron microscopy revealed recovery of normal t-tubule microarchitecture in HF-UN. L-type calcium current density, measured with whole-cell patch clamping, was reduced in heart failure but unaffected by unloading. The variance of the time-to-peak of the calcium transient, an index of CICR dyssynchrony, was increased in heart failure and normalised by unloading. The increased calcium spark frequency observed in heart failure was reduced in HF-UN. These results could be explained by the recoupling of orphaned ryanodine receptors in heart failure, as indicated by immunofluorescence.Our data show that mechanical unloading of the failing heart reverses the pathological remodelling of the t-tubule system and improves CICR. FundingBritish Heart Foundation.

Dynamic expression profiles of MMPs/TIMPs and collagen deposition in mechanically unloaded rat heart: implications for left ventricular assist device support-induced cardiac alterations

Left ventricular assist devices (LVADs) ameliorate heart failure by reducing preload and afterload. However, extracellular matrix (ECM) deposition after application of LVADs is not clearly defined. The purpose of the present study was to investigate ECM remodeling after mechanical unloading in a rat heart transplant model. Sixty male Lewis rats were subjected to abdominal heterotopic heart transplantation, and the transplanted hearts were pressure- and volume-unloaded. The age- and weight- matched male Lewis rats who had undergone open thoracic surgeries were used as the control. Left ventricle ECM accumulation and the expression/activity of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) were measured on the third, seventh, and fourteenth days after transplantation/sham surgery. Compared with the control group, myocardial ECM deposition significantly increased on the seventh and fourteenth days after heart transplantation (P < 0.05) and peaked on the 14th day. The gelatinase activity as well as mRNA expression of MMP-2 and MMP-9 significantly increased after transplantation (P < 0.05). Both mRNA and protein levels of TIMP-1 and TIMP-2 significantly increased compared with those of the control group. Mechanical unloading may lead to adverse remodeling of the ECM of the left ventricle. The underlying mechanism may due to the imbalance of the MMP/TIMP system, especially the remarkable upregulation of TIMPs in the pressure and volume unloaded heart.

Influence of ivabradine on reverse remodelling during mechanical unloading

Ivabradine (Iva) has shown beneficial structural and functional effects in clinical and experimental heart failure (HF), but its action in combination with mechanical unloading (MU), such as during treatment with left ventricular assist devices (LVAD), is unknown. The aim of this study was to investigate the effects of Iva during MU, in a rodent model of HF. We studied the chronic effects (4 weeks) of Iva (10 mg/kg/day) alone and in combination with MU [induced by heterotopic abdominal heart transplantation (HATx)] on whole-heart and cellular structure, function, and excitation-contraction (E-C) coupling in a rodent (rat) model of HF, 12 weeks post-left coronary artery (LCA) ligation. Effects of Iva were compared with those of β-blockade using metoprolol [(Met), 250 mg/kg/day]. Iva, but not Met, reversed myocardial fibrosis, alone and in combination with MU. MU-induced restoration of deranged E-C coupling was enhanced by Iva to a greater extent than Met: both Iva and Met enhanced the recovery of the Ca(2+) transient amplitude and the sarcoplasmic reticulum (SR) Ca(2+) content, but Iva alone maintained MU-induced normalization of L-type Ca(2+) current and t-tubule abnormalities. Met prevented MU-induced reduction in the myocardial size (myocardial atrophy); Iva had no effect on this parameter. Iva shows beneficial structural and E-C coupling effects during MU: Iva reverses myocardial fibrosis and enhances the restoration of deranged E-C coupling, displaying more beneficial effects than that of Met. These results suggest that Iva may prove effective in enhancing functional recovery in heart failure patients receiving LVAD therapy.

Mechanical unloading reverses transverse tubule remodelling and normalizes local Ca2+‐induced Ca2+release in a rodent model of heart failure

AimsCa2+-induced Ca2+ release (CICR) is critical for contraction in cardiomyocytes. The transverse (t)-tubule system guarantees the proximity of the triggers for Ca2+ release [L-type Ca2+ channel, dihydropyridine receptors (DHPRs)] and the sarcoplasmic reticulum Ca2+ release channels [ryanodine receptors (RyRs)]. Transverse tubule disruption occurs early in heart failure (HF). Clinical studies of left ventricular assist devices in HF indicate that mechanical unloading induces reverse remodelling. We hypothesize that unloading of failing hearts normalizes t-tubule structure and improves CICR.Methods and resultsHeart failure was induced in Lewis rats by left coronary artery ligation for 12 weeks; sham-operated animals were used as controls. Failing hearts were mechanically unloaded for 4 weeks by heterotopic abdominal heart transplantation (HF-UN). HF reduced the t-tubule density measured by di-8-ANEPPS staining in isolated left ventricular myocytes, and this was reversed by unloading. The deterioration in the regularity of the t-tubule system in HF was also reversed in HF-UN. Scanning ion conductance microscopy showed the reappearance of normal surface striations in HF-UN. Electron microscopy revealed recovery of normal t-tubule microarchitecture in HF-UN. L-type Ca2+ current density, measured using whole-cell patch clamping, was reduced in HF but unaffected by unloading. The variance of the time-to-peak of the Ca2+ transient, an index of CICR dyssynchrony, was increased in HF and normalized by unloading. The increased Ca2+ spark frequency observed in HF was reduced in HF-UN. These results could be explained by the recoupling of orphaned RyRs in HF, as indicated by immunofluorescence.ConclusionsOur data show that mechanical unloading of the failing heart reverses the pathological remodelling of the t-tubule system and improves CICR.

Heterotopic abdominal heart transplantation in rats for functional studies of ventricular unloading

IntroductionChronic changes in mechanical load regulate long-term cardiac function. Chronic overload of the ventricle results in myocardial failure. Clinical use of ventricular assist devices shows that chronic reduction in load has a number of different consequences on the myocardium, including beneficial reverse remodeling as well as undesired remodeling (e.g., myocardial atrophy and fibrosis, both of which could have negative functional implications). The complex response to mechanical unloading necessitates reproducible animal models of mechanical unloading for use in the laboratory. This article aims to describe the operative technique of two animal models of mechanical unloading in detail, to enable the reproducible use of these animal models. MethodsIn 1964, Abbott et al first described the heterotopic abdominal heart transplantation technique as a means to study the biology of transplanted cardiac grafts. This involves an aorto-aortic anastomosis and a pulmonary artery to inferior vena cava anastomosis. In this model, the left ventricle is virtually completely volume unloaded, receiving only thebesian venous return, and substantially but not entirely pressure unloaded. In this report we describe two refined techniques for mechanical unloading of healthy or failing hearts based on experience with over 500 operations. ResultsWe describe an operative technique, including cardioprotective strategies, that provides a model of mechanical unloading with no immunological rejection and allows measurements of parameters of myocardial structure and function for many months. We describe a refined technique that achieves a lesser degree of left ventricular volume unloading, involving transplantation of both heart and lungs via a single aorto-aortic anastomosis. ConclusionsThis article is the first to describe these two techniques in sufficient detail to enable novices to attempt and understand these operations and the differences between them. The technique we describe provides an effective and reproducible model of complete and partial mechanical unloading.

Effects of long-term administration of low-dose FTY720 on survival of murine cardiac allograft

This study examined the effect of long-term administration of low-dose FTY720 on survival of murine cardiac allograft and the possible mechanism. Murine models of abdominal heterotopic heart transplantation were established. Low-dose FTY720 (0.3 mg/kg) was administrated to the animals 4 days before the transplantation of cardiac allografts until the occurrence of rejection or the observation terminals. The animals without FTY720 treatment and those with syngeneic cardiac grafts transplanted served as controls. The mean survival time (MST) of grafts, and T lymphocyte subsets in grafts, peripheral blood and lymphoid organs were measured by histopathological examination or flow cytometry, and compared among groups. The results showed that the MST of allografts in FTY720-treated mice was more than 40 days, significantly longer than that in the untreated group (MST=8 days, P<0.01). After the long-term administration of FTY720, the proportion of CD4(+) and CD8(+) lymphocytes in peripheral blood was diminished significantly, but the proportion of CD4(+) lymphocytes was increased in mesenteric lymph nodes (MLNs) and spleen. Immunofluorescence staining revealed that the infiltration of CD4(+) and CD8(+) lymphocytes in allografts was significantly inhibited after long-term administration of low-dose FTY720. It was concluded that low-dose long-term administration of FTY720 could promote T lymphocytes in lymphatic organs and decrease their infiltration in allografts, resulting in the inhibition of rejection and the long-term survival of allografts.

Postinfarction healing dynamics in the mechanically unloaded rat left ventricle.

The healing process is a key determinant for postinfarction left ventricular (LV) remodeling and the development of heart failure, which could be influenced by mechanical (pressure and/or volume) load. So far, limited information exists regarding an indepth characterization of the postinfarct healing process in the mechanically unloaded state. In the present work, we performed isogenic Lewis-to-Lewis rat abdominal heterotopic heart transplantation, which is characterized by hemodynamic unloading in the left ventricle, and simultaneously ligated the left anterior descending coronary artery (T-infarct group). Pathological evolution was dynamically compared with that of in situ infarcted Lewis hearts (I-infarct group) on days 3, 7, 14, and 35. There was a remarkable myocardial salvage in the unloaded heart, as shown by the improvement in infarct size (T-infarct group: 25.47% ± 4.31% vs. I-infarct group: 38.46% ± 4.82%, P < 0.01) and the smaller fraction of fibrosis in infarct segments (T-infarct group: 42.12% ± 8.40% vs. I-infarct group: 75.65% ± 10.51%, P < 0.01). In addition, there was a progressive disorganization of the two-dimensional collagen fiber alignment as well as retarded collagen fiber maturation in the T-infarct group. We also observed enhanced angiogenesis, lymphangiogenesis, and inflammatory cell retention in the infarct region during mechanical unloading. Moreover, capillary density and collagen deposition were significantly increased in the noninfarcted area of the unloaded heart compared with the same region in the in situ infarcted heart. In conclusion, ischemic insult in the mechanically unloaded heart elicits an altered inflammatory and healing response, which is characterized by myocardial salvage, delayed resolution of inflammation, and disorganization of the collagen orientation in the infarcted region. These findings could provide novel insights into the contribution of hemodynamic load in the postinfarction healing process. Further studies are warranted to elucidate its potential mechanism.

Ivabradine and Mechanical Unloading Increase Sarcoplasmic Reticulum Calcium Content in a Rodent Model of Heart Failure

Improvements in Ca2+ handling, and sarcoplasmic reticulum (SR) Ca2+ regulation in particular, are associated with myocardial recovery in patients with end stage heart failure (HF) treated with left ventricular assist devices (LVAD). Ivabradine (IV), a novel heart rate reducing agent has been shown to have positive Ca2+ regulatory effects in rodent HF, and is used in human HF therapy; however, the effects of IV during LVAD treatment have not been studied.We studied the chronic effects of IV on cellular Ca2+ handling during mechanical unloading (MU) of HF hearts. HF was induced by left coronary artery ligation and was characterized by reduced ejection fraction (EF), smaller Ca2+ transient amplitude (CaT) and reduced SR Ca2+ content compared to age matched sham operated animals (SH). MU (4 weeks) was achieved by heterotopic abdominal heart transplantation and IV (10mg/kg/day) was administered for 4 weeks. Isolated ventricular myocytes were loaded with Indo-1 AM and field stimulated at 1 Hz.CaT was significantly elevated in the MU+IV group (indo-1 ratio units): MU+IV: 0.25 ± 0.06 (n=45) p< 0.001 vs MU: 0.18 ± 0.05 (n=40), and p<0.001 vs SH: 0.17 ± 0.05 (n=45)). IV augmented SR Ca2+ content in combination with MU compared with MU alone and to a level even greater than SH: SR Ca2+ content (indo-1 ratio units): MU+IV: 0.27 ± 0.07 (n=45) p< 0.05 vs MU: 0.24 ± 0.05 (n=40), and p<0.001 vs SH: 0.22 ± 0.07 (n=45))Our results show that IV treatment combined with mechanical unloading induces a larger increase of Ca2+ transient amplitude and SR Ca2+ content compared with mechanical unloading alone. These effects may be exploited to enhance myocardial recovery in patients receiving LVAD therapy for HF.

All-trans Retinoic Acid Attenuates Cardiac Allograft Vasculopathy in Rats

Objective We sought to study the inhibitory effects of all-trans retinoic acid (ATRA) on cardiac allograft vasculopathy in rats. Methods Inbred Wistar and Sprague-Dawley rats were used as donors and recipients, respectively. After abdominal heterotopic heart transplantation, animals were randomized to a cyclosporine (CsA) group versus a CsA+ATRA group: 10 mg/kg/d CsA versus the same CsA dose plus 10 mg/kg/d ATRA. Transplanted hearts were analyzed at 60 days. Cardiac allograft sections were treated with Van Giesson stain to examine vascular luminal occlusion, with immunohistochemistry for CD68 and proliferating cell nuclear antigen (PCNA), and with reverse-transcription polymerase chain reaction (RT-PCR) for platelet-derived growth factor A (PDGF-A) mRNA. Results Luminal occlusion in the CsA+ATRA group was significantly less than that in the CsA group (40.10 ± 8.20% vs 62.86 ± 17.18%; P < .01). The CsA+ATRA group showed a marked reduction in PCNA- and CD68-positive cells: namely, 33.96 ± 8.65% versus 60.17 ± 17.74% ( P < .01) and 17.63 ± 4.24% versus 32.13 ± 9.26 ( P < .01), respectively. RT-PCR analysis showed that relative PDGF-A mRNA content in the CsA+ATRA group was significantly decreased compared with the CsA group (0.46 ± 0.08 vs 0.94 ± 0.11; P < .01). Conclusion ATRA may attenuate rat cardiac allograft vasculopathy by inhibiting macrophage infiltration and cell proliferation.

Prolonged mechanical unloading affects cardiomyocyte excitation‐contraction coupling, transverse‐tubule structure, and the cell surface

Prolonged mechanical unloading (UN) of the heart is associated with detrimental changes to the structure and function of cardiomyocytes. The mechanisms underlying these changes are unknown. In this study, we report the influence of UN on excitation-contraction coupling, Ca2+-induced Ca2+ release (CICR) in particular, and transverse (t)-tubule structure. UN was induced in male Lewis rat hearts by heterotopic abdominal heart transplantation. Left ventricular cardiomyocytes were isolated from the transplanted hearts after 4 wk and studied using whole-cell patch clamping, confocal microscopy, and scanning ion conductance microscopy (SICM). Recipient hearts were used as control (C). UN reduced the volume of cardiomyocytes by 56.5% compared with C (UN, n=90; C, n=59; P<0.001). The variance of time-to-peak of the Ca2+ transients was significantly increased in unloaded cardiomyocytes (UN 227.4±24.9 ms2, n=42 vs. C 157.8±18.0 ms2, n=40; P<0.05). UN did not alter the action potential morphology or whole-cell L-type Ca2+ current compared with C, but caused a significantly higher Ca2+ spark frequency (UN 3.718±0.85 events/100 μm/s, n=47 vs. C 0.908±0.186 events/100 μm/s, n=45; P<0.05). Confocal studies showed irregular distribution of the t tubules (power of the normal t-tubule frequency: UN 8.13±1.12×105, n=57 vs. C 20.60± 3.174×105, n=56; P<0.001) and SICM studies revealed a profound disruption to the openings of the t tubules and the cell surface in unloaded cardiomyocytes. We show that UN leads to a functional uncoupling of the CICR process and identify disruption of the t-tubule-sarcoplasmic reticulum interaction as a possible mechanism.—Ibrahim, M., Al Masri, A., Navaratnarajah, M., Siedlecka, U., Soppa, G. K., Moshkov, A., Abou Al-Saud, S., Gorelik, J., Yacoub, M. H., Terracciano, C. M. N. Prolonged mechanical unloading affects cardiomyocyte excitation-contraction coupling, transverse-tubule structure, and the cell surface.

Preliminary study on cardiac allograft rejection in mice by Extractum trametes robiniophila murr

To study the effect of Extractum trametes robiniophila murr on cardiac allograft rejection in mice. All abdominal heterotopic heart transplantation models were divided into three groups as follows: (A) Extractum trametes robiniophila murr group. (B) Rejection group. (C) Isograft group. In each group, mean survival times (MST) of transplanted hearts and their pathologic histological changes at postoperative fifth day were observed. With fluoroimmunoassay, granzyme B and CD8(+) expressions were examined. The MST of heart allografts in group A were (6.38 +/- 0.69) d, significantly shorter than that of group B [(8.31 +/- 0.59) d] (P < 0.01). In group A, acute rejection was present in advance; transplanted hearts were seriously damaged into acute rejection pathological grade 3, and CD8(+) T lymphocytes infiltrated diffusely and the expression of granzyme B increased significantly as compared with other groups. Exclusive application of Extractum trametes robiniophila murr can promote the acute rejection of graft in early phase of postoperation, and the mechanism may be the promoted proliferation and infiltration of CD8(+) T lymphocytes and the increased expression of granzyme B.

Evaluation of a Peritoneal-Generated Cardiac Patch in a Rat Model of Heterotopic Heart Transplantation

Tissue engineering holds the promise of providing new solutions for heart transplant shortages and pediatric heart transplantation. The aim of this study was to evaluate the ability of a peritoneal-generated, tissue-engineered cardiac patch to replace damaged myocardium in a heterotopic heart transplant model. Fetal cardiac cells (1 x 10(6)/scaffold) from syngeneic Lewis rats were seeded into highly porous alginate scaffolds. The cell constructs were cultured in vitro for 4 days and then they were implanted into the rat peritoneal cavity for 1 week. During this time the peritoneal-implanted patches were vascularized and populated with myofibroblasts. They were harvested and their performance in an infrarenal heterotopic abdominal heart transplantation model was examined (n = 15). After transplantation and before reperfusion of the donor heart, a 5-mm left (n = 6) or right (n = 9) ventriculotomy was performed and the patch was sutured onto the donor heart to repair the defect. Echocardiographical studies carried out 1-2 weeks after transplantation showed normal LV function in seven of the eight hearts studied. After 1 month, visual examination of the grafted patch revealed no aneurysmal dilatation. Microscopic examination revealed, in most of the cardiac patches, a complete disappearance of the scaffold and its replacement by a consistent tissue composed of myofibroblasts embedded in collagen bundles. The cardiac patch was enriched with a relatively large number of infiltrating blood vessels. In conclusion, cardiac patches generated in the peritoneum were developed into consistent tissue patches with properties to seal and correct myocardial defects. Our study also offers a viable rat model for screening and evaluating new concepts in cardiac reconstruction and engineering.