[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Clinical application of skeletal myoblast transplantation has been curtailed due to arrhythmogenicity and inconsistent therapeutic benefits observed in previous studies. However, these issues may be solved by the use of a new cell-delivery mode. It is now possible to generate "cell-sheets" using temperature-responsive dishes without artificial scaffolds. This study aimed to validate the safety and efficacy of epicardial placement of myoblast-sheets (myoblast-sheet therapy) in treating heart failure. METHODS AND RESULTS: After coronary artery ligation in rats, the same numbers of syngeneic myoblasts were transplanted by intramyocardial injection or cell-sheet placement. Continuous radio-telemetry monitoring detected increased ventricular arrhythmias, including ventricular tachycardia, after intramyocardial injection compared to the sham-control, while these were abolished in myoblast-sheet therapy. This effect was conjunct with avoidance of islet-like cell-cluster formation that disrupts electrical conduction, and with prevention of increased arrhythmogenic substrates due to exaggerated inflammation. Persistent ectopic donor cells were found in the lung only after intramyocardial injection, strengthening the improved safety of myoblast-sheet therapy. In addition, myoblast-sheet therapy enhanced cardiac function, corresponding to a 9.2-fold increase in donor cell survival, compared to intramyocardial injection. Both methods achieved reduced infarct size, decreased fibrosis, attenuated cardiomyocyte hypertrophy, and increased neovascular formation, in association with myocardial upregulation of a group of relevant molecules. The pattern of these beneficial changes was similar between two methods, but the degree was more substantial after myoblast-sheet therapy. CONCLUSION: The cell-sheet technique enhanced safety and therapeutic efficacy of myoblast-based therapy, compared to the current method, thereby paving the way for clinical application.
International journal of cardiology 10/2012; 168(1). DOI:10.1016/j.ijcard.2012.09.081 · 6.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent evidence suggests that cell therapy such as the injection of bone marrow-derived mononuclear cells (BMMNCs) can exert protective effects in various conditions associated with ischemia-reperfusion injury. Here, we investigate the effects of BMMNCs on the organ injury/dysfunction induced by hemorrhagic shock (HS). Thirty-seven anesthetized male Wistar rats were subjected to hemorrhage by reducing mean arterial pressure to 35 ± 5 mmHg for 90 min, followed by resuscitation with 20 mL/kg Ringer's lactate administered over 10 min and 50% of the shed blood over 50 min. Rats were killed 4 h after the onset of resuscitation. Bone marrow-derived mononuclear cells were freshly isolated from rat tibias and femurs using Percoll density gradient centrifugation, and BMMNCs (1 × 10 cells per rat in 1 mL/kg phosphate-buffered saline, i.v.) were administered on resuscitation. Hemorrhagic shock resulted in significant organ injury/dysfunction (renal, hepatic, neuromuscular) and inflammation (hepatic, lung). In rats subjected to HS, administration of BMMNCs significantly attenuated (i) organ injury/dysfunction (renal, hepatic, neuromuscular) and inflammation (hepatic, lung), (ii) increased the phosphorylation of Akt and glycogen synthase kinase-3β, (iii) attenuated the activation of nuclear factor-κB, (iv) attenuated the increase in extracellular signal-regulated kinase 1/2 phosphorylation, and (v) attenuated the increase in expression of intercellular adhesion molecule-1. Our findings suggest that administration of BMMNCs protects against the induction of early organ injury/dysfunction caused by severe HS by a mechanism that may involve activation of Akt and the inhibition of glycogen synthase kinase-3β and nuclear factor-κB.
[Show abstract][Hide abstract] ABSTRACT: Calcineurin is a calcium-regulated phosphatase that plays a major role in cardiac hypertrophy. We previously described that alternative splicing of the calcineurin Aβ (CnAβ) gene generates the CnAβ1 isoform, with a unique C-terminal region that is different from the autoinhibitory domain present in all other CnA isoforms. In skeletal muscle, CnAβ1 is necessary for myoblast proliferation and stimulates regeneration, reducing fibrosis and accelerating the resolution of inflammation. Its role in the heart is currently unknown.
We generated transgenic mice overexpressing CnAβ1 in postnatal cardiomyocytes under the control of the α-myosin heavy chain promoter. In contrast to previous studies using an artificially truncated calcineurin, CnAβ1 overexpression did not induce cardiac hypertrophy. Moreover, transgenic mice showed improved cardiac function and reduced scar formation after myocardial infarction, with reduced neutrophil and macrophage infiltration and decreased expression of proinflammatory cytokines. Immunoprecipitation and Western blot analysis showed interaction of CnAβ1 with the mTOR complex 2 and activation of the Akt/SGK cardioprotective pathway in a PI3K-independent manner. In addition, gene expression profiling revealed that CnAβ1 activated the transcription factor ATF4 downstream of the Akt/mTOR pathway to promote the amino acid biosynthesis program, to reduce protein catabolism, and to induce the antifibrotic and antiinflammatory factor growth differentiation factor 15, which protects the heart through Akt activation.
Calcineurin Aβ1 shows a unique mode of action that improves cardiac function after myocardial infarction, activating different cardioprotective pathways without inducing maladaptive hypertrophy. These features make CnAβ1 an attractive candidate for the development of future therapeutic approaches.
[Show abstract][Hide abstract] ABSTRACT: Adult bone marrow mononuclear cells (BMMNCs) can restore cardiac function following myocardial necrosis. Protocols used to date have administered cells relatively late after ischaemia/reperfusion injury, but there is the opportunity with elective procedures to infuse cells shortly after restoration of blood flow, for example after angioplasty. Our aim was therefore to try and quantify protection from myocardial injury by early infusion of BMMNCs in a rat ischaemia reperfusion (I/R) model.
Male Wistar rats underwent 25 min of ischaemia followed by 2 h reperfusion of the left anterior descending coronary artery. Ten million BMMNCs were injected i.v. at reperfusion. We found BMMNCs caused a significant reduction in infarct size at 2 h when assessed by staining the area at risk with p-nitro blue tetrazolium (42% reduction, P<0.01). Apoptosis and necrosis of isolated cardiomyocytes was significantly reduced in the area at risk. Functional assessment at 7 days using echocardiography and left ventricular catheterisation showed improved systolic and diastolic function in the BMMNC treatment group (LVEF: BMMNC 71 ± 3% vs. PBS 48 ± 4%, P<0.0001). In functional studies BMMNC injected animals showed increased activation of Akt, inhibition of GSK-3β, amelioration of p38 MAP kinase phosphorylation and NF-κB activity compared to control myocardium. Inhibition of PI3K with LY294002 abolished all beneficial effects of BMMNC treatment. Proteomic analysis also demonstrated that BMMNC treatment induced alterations in proteins within known cardioprotective pathways, e.g., heat shock proteins, stress-70 protein as well as the chaperone protein 14-3-3 epsilon.
Early BMMNC injection during reperfusion preserves the myocardium, with evidence of reduced apoptosis, necrosis, and activation of survival pathways.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Molecular and Cellular Cardiology 06/2009; 47(2):288-95. DOI:10.1016/j.yjmcc.2009.05.009 · 5.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.