Toll-like receptor 9 protects non-immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2.

Toll-like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro-inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium-transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca(2+) handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non-immune cells--including cardiomyocytes--using the same ligand-receptor system.

TLR9 mediates cellular protection by modulating energy metabolism in cardiomyocytes and neurons.

Toll-like receptors (TLRs) are the central players in innate immunity. In particular, TLR9 initiates inflammatory response by recognizing DNA, imported by infection or released from tissue damage. Inflammation is, however, harmful to terminally differentiated organs, such as the heart and brain, with poor regenerative capacity, yet the role of TLR9 in such nonimmune cells, including cardiomyocytes and neurons, is undefined. Here we uncover an unexpected role of TLR9 in energy metabolism and cellular protection in cardiomyocytes and neurons. TLR9 stimulation reduced energy substrates and increased the AMP/ATP ratio, subsequently activating AMP-activated kinase (AMPK), leading to increased stress tolerance against hypoxia in cardiomyocytes without inducing the canonical inflammatory response. Analysis of the expression profiles between cardiomyocytes and macrophages identified that unc93 homolog B1 (C. elegans) was a pivotal switch for the distinct TLR9 responses by regulating subcellular localization of TLR9. Furthermore, this alternative TLR9 signaling was also found to operate in differentiated neuronal cells. These data propose an intriguing model that the same ligand-receptor can concomitantly increase the stress tolerance in cardiomyocytes and neurons, whereas immune cells induce inflammation upon tissue injury.

Epicardial placement of mesenchymal stromal cell-sheets for the treatment of ischemic cardiomyopathy; in vivo proof-of-concept study.

Transplantation of bone marrow mesenchymal stromal cells (MSCs) is an emerging treatment for heart failure. We have reported that epicardial placement of MSC-sheets generated using temperature-responsive dishes markedly increases donor MSC survival and augments therapeutic effects in an acute myocardial infarction (MI) model, compared to intramyocardial (IM) injection. This study aims to expand this knowledge for the treatment of ischemic cardiomyopathy, which is likely to be more difficult to treat due to mature fibrosis and chronically stressed myocardium. Four weeks after MI, rats underwent either epicardial MSC-sheet placement, IM MSC injection, or sham treatment. At day 28 after treatment, the cell-sheet group showed augmented cardiac function improvement, which was associated with over 11-fold increased donor cell survival at both days 3 and 28 compared to IM injection. Moreover, the cell-sheet group showed improved myocardial repair, in conjunction with amplified upregulation of a group of reparative factors. Furthermore, by comparing with our own previous data, this study highlighted similar dynamics and behavior of epicardially placed MSCs in acute and chronic stages after MI, while the acute-phase myocardium may be more responsive to the stimuli from donor MSCs. These proof-of-concept data encourage further development of the MSC-sheet therapy for ischemic cardiomyopathy toward clinical application.

The use of scaffold-free cell sheet technique to refine mesenchymal stromal cell-based therapy for heart failure.

Transplantation of bone marrow-derived mesenchymal stromal cells (MSCs) is an emerging treatment for heart failure based on their secretion-mediated "paracrine effects". Feasibility of the scaffoldless cell sheet technique to enhance the outcome of cell transplantation has been reported using other cell types, though the mechanism underpinning the enhancement remains uncertain. We here investigated the role of this innovative technique to amplify the effects of MSC transplantation with a focus on the underlying factors. After coronary artery ligation in rats, syngeneic MSCs were grafted by either epicardial placement of MSC sheets generated using temperature-responsive dishes or intramyocardial (IM) injection. Markedly increased initial retention boosted the presence of donor MSCs persistently after MSC sheet placement although the donor survival was not improved. Most of the MSCs grafted by the cell sheet technique remained resided on the epicardial surface, but the epicardium quickly regressed and new vessels sprouted into the sheets, assuring the permeation of paracrine mediators from MSCs into the host myocardium. In fact, there was augmented upregulation of various paracrine effect-related genes and signaling pathways in the early phase after MSC sheet therapy. Correspondingly, more extensive paracrine effects and resultant cardiac function recovery were achieved by MSC sheet therapy. Further development of this approach towards clinical application is encouraged.

Donor cell-type specific paracrine effects of cell transplantation for post-infarction heart failure.

Cell transplantation is an emerging therapy for treating post-infarction heart failure. Although the paracrine effect has been proposed to be an important mechanism for the therapeutic benefits, details remain largely unknown. This study compared various aspects of the paracrine effect after transplantation of either bone marrow mononuclear cells (BMC) or skeletal myoblasts (SMB) into the post-infarction chronically failing heart. Three weeks after left coronary artery ligation, adult rats received intramyocardial injection of either BMC, SMB or PBS only. Echocardiography demonstrated that injection of either cell type improved cardiac function compared to PBS injection. Interestingly, BMC injection markedly improved neovascularization in the border areas surrounding infarcts, while SMB injection decreased fibrosis in both the border and remote areas. Injection of either cell type similarly reduced hypertrophy of cardiomyocytes as assessed by cell-size planimetry using isolated cardiomyocytes. Quantitative RT-PCR revealed that, among 15 candidate mediators of paracrine effects studied, Fgf2 and Hgf were upregulated only after BMC injection, while Mmp2 and Timp4 were modulated after SMB injection. Additional investigations of signalling pathways relevant to heart failure by western blotting showed that p38 and STAT3 were temporarily activated after BMC injection, in contrast, ERK1/2 and JNK were activated after SMB injection. There was no difference in activation of Akt, PKD or Smad3 among groups. These data suggest that paracrine effects observed after cell transplantation in post-infarction heart failure were noticeably different between cell types in terms of mediators, signal transductions and consequent effects.

A factor underlying late-phase arrhythmogenicity after cell therapy to the heart: global downregulation of connexin43 in the host myocardium after skeletal myoblast transplantation.

BACKGROUND: Arrhythmia occurrence is a variable but serious concern of cell therapy for treating heart failure. Using a rat postinfarction chronic heart failure model, we compared skeletal myoblast (SMB) with bone marrow cell (BMC) injection to highlight donor cell-specific, late-phase arrhythmogenesis and the underlying factors. METHODS AND RESULTS: SMBs or BMCs derived from male GFP-transgenic rats, or PBS were injected intramyocardially into female rat hearts 3 weeks after coronary artery occlusion. At 28 days after injection, echocardiography showed that the left ventricular ejection fraction was significantly improved in both the SMB and BMC groups, compared to PBS control despite poor graft survival as assessed by PCR for the male-specific gene. Radio-telemetry analysis revealed that the SMB group displayed a higher occurrence of ventricular premature contractions with an elongation of the QRS complex and the hearts were more susceptible to isopreterenol-induced ventricular tachycardia compared to the BMC and PBS groups. Western blot and immunoconfocal analysis showed that the gap junction protein, connexin43, was widely and persistently decreased in the SMB group compared to the other groups. IL-1beta was shown to be upregulated in hearts after SMB injection, and in vitro experiments demonstrated that exposure to IL-1beta caused a decrease in connexin43 and intercellular communication in cultured cardiomyocytes. CONCLUSIONS: Although cell therapy was capable of improving function of the postinfarction chronically failing heart, there was late-phase arrhythmogenicity specific to donor cell type. Global downregulation of connexin43 in the host myocardium was indicated to be an important factor underlying late-phase arrhythmogenicity after SMB transplantation.

Modulated inflammation by injection of high-mobility group box 1 recovers post-infarction chronically failing heart.

BACKGROUND: Inflammation plays an important role in the progress of adverse ventricular remodeling after myocardial infarction. High-mobility group box 1 (HMGB1) is a nuclear protein, which has recently been uncovered to also act as a modifier of inflammation when released. We hypothesized that HMGB1 injection could preferentially modulate local myocardial inflammation, attenuate ventricular remodeling, and subsequently improve cardiac performance of postinfarction chronic heart failure. METHODS AND RESULTS: Three weeks after left coronary artery ligation, HMGB1 (2.5 mug) or PBS was intramyocardially injected into rat hearts. At 28 days after injection, left ventricular ejection fraction was significantly improved after HMGB1 injection compared to PBS (39.3+/-1.4 versus 33.3+/-1.8%; P<0.01). Accumulation of CD45(+) inflammatory cells, two thirds of which were OX62(+) dendritic cells, in the peri-infarct area was significantly attenuated by HMGB1 injection. Dramatic changes in the expression of major proinflammatory cytokines were not detected by microarray or RT-PCR. Adverse ventricular remodeling including cardiomyocyte hypertrophy (cardiomyocyte cross-sectional area; 439+/-7 versus 458+/-6 mum(2); P<0.05) and extracellular collagen deposition (collagen volume fraction; 11.9+/-0.4 versus 15.2+/-0.6%; P<0.01) was attenuated by HMGB1 injection. Analyses of signal transduction pathways revealed that HMGB1 injection activated ERK1/2, but not p38, Akt, and Smad3. Cardiac regeneration and neovascularization were not observed. CONCLUSIONS: HMGB1 injection modulated the local inflammation in the postinfarction chronically failing myocardium, particularly via reducing the accumulation of dendritic cells. This modulated inflammation resulted in attenuated fibrosis and cardiomyocyte hypertrophy, which thereby improved global cardiac function. These data suggest that HMGB1 may be valuable for the chronic heart failure treatment.

Direct intramyocardial but not intracoronary injection of bone marrow cells induces ventricular arrhythmias in a rat chronic ischemic heart failure model.

BACKGROUND: Therapeutic efficacy of bone marrow (BM) cell injection for treating ischemic chronic heart failure has not been established. In addition, experimental data are lacking on arrhythmia occurrence after BM cell injection. We hypothesized that therapeutic efficacy and arrhythmia occurrence induced by BM cell injection may be affected by the cell delivery route. METHODS AND RESULTS: Three weeks after left coronary artery ligation, wild-type female rats were injected with 1x10(7) mononuclear BM cells derived from green fluorescent protein-transgenic male rats through either a direct intramyocardial or a retrograde intracoronary route. Both intramyocardial and intracoronary injection of BM cells demonstrated similar improvement in left ventricular ejection fraction measured by echocardiography and a similar graft size analyzed by real-time polymerase chain reaction for the Y chromosome-specific Sry gene. Noticeably, intramyocardial injection of BM cells induced frequent ventricular premature contractions (108+/-73 per hour at 7 days after BM cell injection), including multiform, consecutive ventricular premature contractions and ventricular tachycardia for the initial 14 days; intracoronary injection of BM cells and intramyocardial injection of phosphate-buffered saline rarely induced arrhythmias. Immunohistochemistry demonstrated that intramyocardial BM cell injection formed distinct cell clusters containing donor-derived cells and accumulated host-derived inflammatory cells in the infarct border zone, whereas intracoronary BM cell injection provided more homogeneous donor cell dissemination with less inflammation and without disrupting the native myocardial structure. CONCLUSIONS: BM cell injection is able to improve cardiac function in ischemic chronic heart failure but has a risk of arrhythmia occurrence when the intramyocardial route is used. Such arrhythmias may be prevented by using the intracoronary route.

Heterogeneic nature of adult cardiac side population cells.

Side population cells have been found in various types of adult tissue including heart and are presumed to be tissue-specific stem/progenitor cells. In the present study, we confirmed the presence of cardiac side population (cSP) cells, which showed both the Hoechst 33342 efflux ability and ABCG2 expression, in adult murine heart. Flow cytometric analysis showed that more than half of cSP cells expressed the endothelial marker VE-cadherin or the smooth muscle markers, alpha-smooth muscle actin and desmin. In addition, immunohistochemical analysis demonstrated that ABCG2(+) cells were mainly localized within vascular walls. Quantitative RT-PCR analysis demonstrated that VE-cadherin(-) cSP cells progressively expressed Nkx2.5 and cardiac troponin T with time in culture. VE-cadherin(-) cSP cells also expressed mesodermal-mesenchymal-associated markers and differentiated into osteocytes and adipocytes. These results highlight the heterogeneic nature of cSP cells, consisting of vascular endothelial cells, smooth muscle cells, and mesenchymal stem/progenitor cells including potential cardiomyogenic cells.

A novel strategy for myocardial protection by combined antibody therapy inhibiting both P-selectin and intercellular adhesion molecule-1 via retrograde intracoronary route.

BACKGROUND: Antibody therapy to inhibit either P-selectin or intercellular adhesion molecule-1 (ICAM-1) has been reported to provide myocardial protection against leukocyte-mediated reperfusion injury. Because these molecules play different roles in the leukocyte-endothelial interaction, co-inhibition of both may achieve further enhanced cardioprotection. In addition, the therapeutic efficacy of such antibody therapy may be affected by the delivery route used. Retrograde intracoronary infusion will offer an effective, direct access to the postcapillary venules, where the target event (leukocyte-endothelial interaction) takes place. We investigated the feasibility and efficiency of the combined antibody therapy targeting both P-selection and ICAM-1 via the retrograde intracoronary route to attenuate myocardial ischemia-reperfusion injury. METHODS AND RESULTS: Lewis rats underwent 30-minute left coronary artery occlusion. Just before reperfusion, anti-P-selectin monoclonal antibody (150 microg/kg), anti-ICAM-1 monoclonal antibody (200 microg/kg), both antibodies together, or control antibody were retrogradely infused into the left cardiac vein. At 24 hours after reperfusion, administration of either anti-P-selectin or anti-ICAM-1 antibody significantly (P<0.05) improved left ventricular ejection fraction and attenuated infarct size (40.6+/-3.2% and 34.8+/-3.5%, respectively) compared with the control (56.8+/-3.4%). This was associated with reduced leukocyte accumulation and improved regional blood flow in the ischemic area. Noticeably, co-administration of both antibodies achieved a much greater reduction in infarct size (19.1+/-3.6%), associated with greater attenuation in leukocyte infiltration, compared with administration of either single antibody. CONCLUSIONS: Combined antibody therapy inhibiting both P-selectin and ICAM-1 via the retrograde intracoronary route could be a promising new strategy for myocardial protection against ischemia-reperfusion injury.

Evaluation of frequency, type, and function of gap junctions between skeletal myoblasts overexpressing connexin43 and cardiomyocytes: relevance to cell transplantation.

Cell transplantation of skeletal myoblasts (SMs) is one possible treatment for repairing cardiac tissue after myocardial injury. However, inappropriate electrical coupling between grafted SMs and host cardiomyocytes may be responsible for the arrhythmias observed in clinical trials of SM transplantation. Whether functional gap junctions occur between the two cell types remains controversial. We have studied the ability of SMs to electrically couple with isolated adult rat cardiomyocytes (CMs) and assessed whether connexin43 (Cx43) overexpression enhanced gap junctional conductance (Gj). C2C12 myoblast lines overexpressing Cx43 were generated by gene transfection and clonal selection. CMs were cocultured with either SMs overexpressing Cx43 (CM-SM(Cx43)) or control SMs (CM-SM(WT)) in vitro. Gj between pairs of SMs and CMs was quantified with dual whole cell patch clamping. Formation of Gj occurred between 22% of CM-SM(WT) pairs (n=73) and 48% of CM-SM(Cx43) pairs (n=71, P<0.001). The Gj of CM-SM(Cx43) pairs (29.7+/-4.3 nS, n=21) was greater than that of CM-SM(WT) pairs (14.8+/-2.0 nS, n=12, P<0.05). The overexpression of Cx43 in SMs increased the formation of electrical communication and the steady-state conductance between SMs and CMs. Enhanced gap junctional conductance may be useful to promote the integration of transplanted SMs into the myocardium.

Enhanced efficiency of superoxide dismutase-induced cardioprotection by retrograde intracoronary administration.

OBJECTIVE: We hypothesized that modification of the infusion route may improve the efficiency of superoxide dismutase (SOD)-induced cardioprotection against reperfusion injury. The routes for SOD delivery previously examined were intravenous, via the left atrium, or by a combination of these, all of which can deliver SOD into the ischemic myocardium only after reperfusion. In contrast, retrograde intracoronary infusion may be able to deliver SOD before reperfusion. We investigated the feasibility and efficiency of the retrograde intracoronary infusion of SOD to attenuate reperfusion injury. METHODS AND RESULTS: Lewis rats underwent 30-min left coronary artery occlusion followed by reperfusion for 24 h. Just before reperfusion, CuZn-SOD was administered intravenously (15,000 U/kg, V-SOD group) or by retrograde intracoronary infusion (1500 U/kg, R-SOD group) through a catheter inserted into left cardiac vein via left superior vena cava as we have previously reported. This method has been shown to perfuse the whole left ventricular free walls. Controls for each group were injected with phosphate buffer saline only via the same routes (V-PBS and R-PBS group). The R-SOD group demonstrated significantly preserved left ventricular ejection fraction (LVEF; 71.3+/-1.7% vs. 60.8+/-2.3%, p=0.028), reduced infarct size (23.3+/-2.3% vs. 42.4+/-3.5%, p<0.001), and attenuated polymorphonuclear leukocyte (PMNL) infiltration (11.8+/-0.4 vs. 14.8+/-0.2 10(3)/mm(2), p<0.001) compared to the V-SOD group. The V-SOD group demonstrated significantly improved reflow (64.3+/-2.1% vs. 53.4+/-2.4%, p=0.017) and attenuated PMNL infiltration (14.8+/-0.2 vs. 16.8+/-0.7 10(3)/mm(2), p=0.018) compared to the V-PBS group. CONCLUSION: Retrograde intracoronary infusion is a promising, clinically applicable method to enhance the efficacy of SOD-induced myocardial protection against ischemia-reperfusion injury.

Cell Size Critically Determines Initial Retention of Bone Marrow Mononuclear Cells in the Heart after Intracoronary Injection: Evidence from a Rat Model.

Intracoronary injection of bone marrow mononuclear cells (BMMNC) is an emerging treatment for heart failure. Initial donor cell retention in the heart is the key to the success of this approach, but this process remains insufficiently characterized. Although it is assumed that cell size of injected cells may influence their initial retention, no scientific evidence has been reported. We developed a unique model utilizing an ex-vivo rat heart perfusion system, enabling quantitative assessment of retention of donor cells after intracoronary injection. The initial (5 minutes after intracoronary injection) retention rate of BMMNC was as low as approximately 20% irrespective of donor cell doses injected (1×106, 8×106, 4×107). Quantitative cell-size assessment revealed a positive relationship between the size of BMMNC and retention ratio; larger subpopulations of BMMNC were more preferentially retained compared to smaller ones. Furthermore, a larger cell type-bone marrow-derived mesenchymal stromal cells (median size = 11.5µm versus 7.0µm for BMMNC)-had a markedly increased retention rate (77.5±1.8%). A positive relationship between the cell size and retention ratio was also seen in mesenchymal stromal cells. Flow-cytometric studies showed expression of cell-surface proteins, including integrins and selectin-ligands, was unchanged between pre-injection BMMNC and those exited from the heart, suggesting that biochemical interaction between donor cells and host coronary endothelium is not critical for BMMNC retention. Histological analyses showed that retained BMMNC and mesenchymal stromal cells were entrapped in the coronary vasculature and did not extravasate by 60 minutes after transplantation. Whilst BMMNC did not change coronary flow after intracoronary injection, mesenchymal stromal cells reduced it, suggesting coronary embolism, which was supported by the histological finding of intravascular cell-clump formation. These data indicate that cell-size dependent, passive (mechanical), intravascular entrapment is responsible for the initial donor cell retention after intracoronary injection of BMMNC in the heart having normal vasculatures (at least).

Allogeneic Mesenchymal Stromal Cells Transplanted Onto the Heart Surface Achieve Therapeutic Myocardial Repair Despite Immunologic Responses in Rats.

BACKGROUND: Transplantation of allogeneic mesenchymal stromal cells (MSCs) is a promising treatment for heart failure. We have shown that epicardial placement of cell sheets markedly increases donor cell survival and augments therapeutic effects compared with the current methods. Although immune rejection of intramyocardially injected allogeneic MSCs have been suggested, allogeneic MSCs transplanted on the heart surface (virtual space) may undergo different courses. This study aimed to elucidate immunologic response against epicardially placed allogeneic MSCs, rejection or acceptance of these cells, and their therapeutic effects for heart failure. METHODS AND RESULTS: At 4 weeks after coronary artery ligation, Lewis rats underwent epicardial placement of MSC sheets from syngeneic Lewis or allogeneic Fischer 344 rats or sham treatment. At days 3 and 10 after treatment, similar ratios (≈50% and 30%, respectively) of grafted MSCs survived on the heart surface in both MSC sheet groups. By day 28, survival of syngeneic MSCs was substantially reduced (8.9%); survival of allogeneic MSCs was more extensively reduced (0.2%), suggesting allorejection. Correspondingly, allogeneic MSCs were found to have evoked an immunologic response, albeit low level, as characterized by accumulation of CD4(+) T cells and upregulation of interleukin 6. Despite this alloimmune response, the allogeneic MSC sheet achieved myocardial upregulation of reparative factors, enhanced repair of the failing myocardium, and improved cardiac function to the equivalent degree observed for the syngeneic MSC sheet. CONCLUSIONS: Allogeneic MSCs placed on the heart surface evoked an immunologic response; however, this allowed sufficient early phase donor cell survival to induce equivalent therapeutic benefits to syngeneic MSCs. Further development of this approach toward clinical application is warranted.

High-resolution en-face visualization of the cardiomyocyte plasma membrane reveals distinctive distributions of spectrin and dystrophin.

The actin-binding proteins, spectrin and dystrophin, are key components of the plasma membrane-associated cytoskeleton of the cardiac muscle cell. From confocal immunofluorescence studies, the distribution of spectrin appears to overlap with that of dystrophin, but the precise functional differentiation, molecular distributions and spatial relationship of these two cytoskeletal systems remain unclear. Freeze-fracture replica immuno-electron microscopy, in parallel with immunofluorescence/confocal microscopy, were applied to examine at high resolution the spatial relationships between the spectrin and dystrophin membrane-associated cytoskeleton systems in cardiac muscle. Application of freeze-fracture replica cytochemistry, with single and double immunogold labeling, permitted simultaneous examination of the organization of spectrin and dystrophin in en-face views of the plasma membrane at high resolution. In contrast to the close spatial relationship previously demonstrated for dystrophin and beta-dystroglycan, no association between the gold label marking dystrophin and that marking spectrin was observed. Our freeze-fracture cytochemical results suggest that the two membrane skeletal networks formed by dystrophin and spectrin in cardiac muscle are independently organized, implying that whatever overlap of function (e.g., in structural support to the plasma membrane) may exist between them, the two systems may each have additional distinctive roles.

Remodelling of gap junctions and connexin expression in heart disease.

Different combinations and relative quantities of three connexins-connexin43, connexin40 and connexin45-are expressed in different subsets of cardiomyocyte. In the healthy heart, gap junctions assembled from these different connexin combinations form the cell-to-cell pathways for the precisely orchestrated patterns of current flow that govern the normal heart rhythm. Remodelling of gap junction organization and connexin expression is a conspicuous feature of human heart disease in which there is an arrhythmic tendency. This remodelling may take the form of structural remodelling, involving disturbances in the distribution of gap junctions (i.e., disruption of the normal ordered pathways for cell-to-cell conduction), and remodelling of connexin expression, involving alteration in the amount or type of connexin(s) present. Most notable among quantitative alterations in connexin expression is a reduction in ventricular connexin43 levels in human congestive heart failure. By correlating data from studies in experimental animal models, gap junction and connexin remodelling emerges as a factor to be considered in understanding the pro-arrhythmic substrate characteristic of many forms of heart disease. However, our knowledge of the functional correlates of the specific patterns of multiple connexin expression found in different regions of the heart in health and disease remains rudimentary, and the development of new experimental cell models heralds advances in this area over the next few years.

Targeted cell delivery into infarcted rat hearts by retrograde intracoronary infusion: distribution, dynamics, and influence on cardiac function.

BACKGROUND: Intracoronary infusion for cell transplantation has potential advantages in disseminating cells globally into the myocardium with less injury over direct intramuscular injection. Arterial route, however, has a risk of coronary embolism and a limitation in cell delivery into ischemic or infarcted areas. We assessed the efficiency of retrograde intracoronary cell implantation into infarcted hearts using a novel rat model. METHODS AND RESULTS: After left coronary artery ligation in rat, a catheter was inserted into the left cardiac vein, which drains the left ventricular free wall. Through this, 1x10(6) skeletal muscle precursor cells expressing nuclear beta-galactosidase were infused retrogradely into the vein. In situ staining demonstrated that beta-galactosidase-expressing donor cells had disseminated throughout the left ventricular free wall, including both infarcted and surrounding border areas, at 10 minutes after infusion. At 28 days, in contrast, positively stained multinuclear myotubes were found in border zones, whereas no positive cells were seen in infarcted areas. Measurement of beta-galactosidase enzyme activity estimated that 29.8+/-6.9% of total infused cells were retained within the myocardium at 10 minutes and that this number decreased to 23.7+/-8.1% at 3 days but rapidly increased thereafter, reaching a plateau at 90.2+/-17.1% by 14 days. Echocardiography and Langendorff perfusion demonstrated that cell implantation improved cardiac function and dimensions by 28 days, compared with both sham-treated and phosphate-buffered saline-infused infarcted hearts, and this was associated with decreased collagen deposition. CONCLUSIONS: Retrograde intracoronary cell transplantation could provide an effective cell delivery into infarcted hearts and could be a useful strategy for treating myocardial infarction.

Role of interleukin-1beta in acute inflammation and graft death after cell transplantation to the heart.

BACKGROUND: Poor survival of grafted cells is a major factor hindering the therapeutic effect of cell transplantation; however, the causes of cell death remain unclear. We hypothesized that interleukin-1beta (IL-1beta) might play a role in the acute inflammatory response and graft death after cell transplantation and that inhibition of IL-1beta might improve graft survival. METHODS AND RESULTS: 14C-labeled male skeletal muscle precursor cells were implanted into female mouse hearts by direct intramuscular injection. The amount of 14C-label provides an estimate of the surviving cell number, whereas the amount of male-specific Smcy gene measured by polymerase chain reaction indicates the total (surviving+proliferated) number of donor-derived cells. At 10 minutes after implantation, 44.8+/-2.4% of the grafted cells survived and this steadily decreased to 14.6+/-1.1% by 24 hours, and to 7.9+/-0.6% by 72 hours (n=6 in each point). Proliferation of the surviving cells, which began after 24 hours, resulted in an increase in the total cell number from 15.5+/-0.8% at 24 hours to 24.4+/-1.6% at 72 hours. Acute inflammation was prominent at 24 hours and was reduced by 72 hours, in parallel with IL-1beta expression. Administration of anti-IL-1beta antibody improved graft survival at both 24 (25.6+/-1.6%) and 72 hours (14.8+/-1.1%) and resulted in a 2-fold increase in the total cell number at 72 hours (45.8+/-2.4%). The effects of IL-1beta inhibition corresponded with a reduced inflammatory response. CONCLUSIONS: IL-1beta is involved in acute inflammation and graft death after direct intramyocardial cell transplantation. Targeted inhibition of IL-1beta may be a useful strategy to improve graft survival.