Ryo Aeba

Induction of human cardiomyocyte-like cells from fibroblasts by defined factors

Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca2+ oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2′-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

Fibroblasts can be directly reprogrammed into cardiomyocyte‐like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR‐133a (miR‐133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial‐to‐mesenchymal transition. MiR‐133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR‐133 overexpression. In contrast, overexpression of Snai1 in GMT/miR‐133‐transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR‐133‐mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR‐133/Snai1, is a key molecular roadblock during cardiac reprogramming.

Detrimental Effects of Exogenous Glutamate on Spinal Cord Neurons During Brief Ischemia In Vivo

BACKGROUND Paraplegia remains a serious complication of thoracoabdominal aortic operations. However, despite growing in vitro evidence, it has been difficult to demonstrate glutamate neurotoxicity in vivo because of the reuptake activity that occurs. We hypothesized that glutamate can be toxic to the spinal cord under metabolic stress. METHODS Infrarenal aortic isolation was performed in New Zealand white rabbits. Group A animals (n = 7) then received a segmental infusion of glutamate (50 mmol/L) for 5 minutes. Group B animals (n = 7) received saline as a negative control. Group C animals (n = 6) were pretreated with a segmental infusion of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (4 mg/kg), a competitive alpha-amino-3-hydroxy-5-methylisoazole-4-propionic acid/kainate antagonist, followed by the segmental infusion of glutamate (30 mmol/L) for 4 minutes. Group D animals (n = 6) received the vehicle agents only, followed by the same glutamate infusion (30 mmol/L) as in group C as a control for group C. Neurologic status was assessed at 12, 24, and 48 hours after operation and scored using the Tarlov system. RESULTS Group A animals exhibited paraplegia or paraparesis with marked neuronal necrosis. Group B animals recovered fully. Group C animals had better neurologic function than group D animals (p = 0.0039). CONCLUSIONS Exogenous glutamate can have detrimental effects on spinal cord neurons during a brief period of ischemia. This model may be useful for the purpose of assaying a glutamate receptor antagonist in vivo.

Heterotopic transplantation of a decellularized and recellularized whole porcine heart

OBJECTIVES One of the final treatments for end-stage heart failure is heart transplantation. However, a shortage of donor hearts has created a long waiting list and limited benefits. Our ultimate goal is to create a whole beating heart fabricated on an organ scaffold for human heart transplantation. Here, we successfully performed the first transplantation using a decellularized whole porcine heart with mesenchymal stem cells. METHODS A porcine heart was harvested following cardiac arrest induced by a high-potassium solution and stored at -80°C for 24 h. The porcine heart was completely decellularized with 1% sodium dodecyl sulphate and 1% Triton X-100 under the control of perfusion pressure (100 mmHg) and maintained at 37°C. A decellularized whole-heart scaffold was sterilized with gamma irradiation. Cultured mesenchymal stem cells were collected and either infused into the ascending aorta or injected directly into the left ventricular wall. Finally, recellularized whole-heart scaffolds were transplanted into pigs under systemic anticoagulation treatment with heparin. Coronary artery angiography of the transplanted heart graft was performed. RESULTS In our decellularization method, all cellular components were removed, preserving the heart extracellular matrix. Heterotopic transplantations were successfully performed using a decellularized heart and a recellularized heart. The scaffolds were well perfused, without bleeding from the surface or anastomosis site. Coronary angiography revealed a patent coronary artery in both scaffolds. The transplanted decellularized heart was harvested on Day 3. Haematoxylin and eosin staining showed thrombosis in the coronary arteries and migrated inflammatory cells. Haematoxylin and eosin staining of the transplanted recellularized heart showed similar findings, with the exception of injected mesenchymal stem cells. CONCLUSIONS To the best of our knowledge, this is the first report of heterotopic transplantation of a decellularized whole porcine heart with mesenchymal stem cells. The scaffolds endured surgical procedures. We detected short-term coronary artery perfusion in the transplanted scaffolds by angiography. Future studies should analyse the histological features of transplanted decellularized scaffolds and optimize the system for recellularization to apply this unique technology clinically.

Use of Hemoglobin Vesicles During Cardiopulmonary Bypass Priming Prevents Neurocognitive Decline in Rats

Background— Homologous blood use is considered to be the gold standard for cardiopulmonary bypass (CPB) priming in infants despite exposure of the patient to potential cellular and humoral antigens. However, the use of hemoglobin vesicles (HbVs), artificial oxygen carriers that encapsulate a concentrated hemoglobin solution within phospholipid bilayer membranes, for CPB priming may prevent neurocognitive decline in infants. The goal of this study was to determine whether HbV use offsets hemodilution caused by patient/priming volume-mismatched CPB and thereby prevents the development of postoperative neurocognitive deficits. Methods and Results— CPB was established in 28 male Sprague-Dawley rats (age, 14 to 16 weeks; weight, 450 grams) after cannulation of the tail artery and right atrium. The animals were randomly assigned to 1 of 3 groups: sham surgery (n=9), HbV (−) prime (n=10), or HbV (+) prime (n=9). CPB was conducted for 90 minutes at 200 mL/kg per minute. The hematocrit during CPB was 10.0±1.2% in the HbV (+) prime group and 9.9±1.3% in the HbV (−) prime group (P=not significant). Learning and memory function were evaluated using 2 different maze tests (Maze-1 and Maze-2, in which the arrival times to the target were measured on the first, third, fifth, and seventh postoperative days). Learning and memory function were significantly better in the HbV (+) prime group than in the HbV (−) prime group (Maze-1, P=0.012; Maze-2, P=0.042); there was no difference between the HbV (+) prime and the sham surgery group. Conclusions— The use of HbV for CPB priming may serve as a substitute for homologous blood to prevent the unacceptable hemodilution and contribute to maintenance of intact neurocognitive function.

Surgical management of isolated congenital tricuspid regurgitation

BACKGROUND Isolated congenital tricuspid regurgitation without downward displacement of the leaflet is a rare clinical entity. Degenerative cusps and lack of chordae may preclude valvuloplasty and require valve replacement. METHODS Three consecutive patients with isolated congenital tricuspid regurgitation underwent surgical repair between May 1995 and April 1997. Their ages were 23, 15, and 8 years old. Tricuspid valvuloplasty was feasible in all of them, with use of a gathering suture of the anterior leaflet, artificial chordae implantation, and ring annuloplasty. RESULTS All 3 patients survived and recovered well after the operation. The cardiothoracic ratios on their chest roentgenograms decreased from 0.64 to 0.52 in patient 1, from 0.58 to 0.48 in patient 2, and from 0.60 to 0.44 in patient 3. Postoperative echocardiograms showed competent tricuspid valves and the disappearance of regurgitation in all cases. CONCLUSIONS Although malformation of the valve is extensive in isolated congenital tricuspid regurgitation, application of artificial chordae with conventional valvuloplasty technique can avoid the use of prosthetic valves by establishing the competence of the tricuspid valve.

Multislice computed tomography is useful for evaluating partial anomalous pulmonary venous connection

Volume-rendered images, derived from multidetector-row computed tomography (MDCT), can facilitate assessment of the morphology of partial anomalous pulmonary venous connection and are thus useful in pre-operative planning to prevent surgical morbidity and assist post-operative evaluations.

Liberal use of tricuspid valve detachment for transatrial ventricular septal defect closure

BACKGROUND Although temporary tricuspid valve detachment is useful for improved visualization of ventricular septal defect through right atriotomy, liberal use of this adjunct is not widely supported, mainly because of concerns about iatrogenic complications such as heart blocks and tricuspid valve dysfunction. The objective of this study was to determine whether liberal use of this adjunct can improve operative outcome. METHODS Between January 1997 and March 2002, trans-atrial closure of isolated ventricular septal defect (conoventricular or canal type) was performed in 87 consecutive patients. Tricuspid valve detachment was used in 4 out of 44 patients (prudent-use group) and 19 out of 43 patients (liberal-use group) in the first and second half of this period, respectively (p = 0.0002). Patient demographics and use of other surgical and cardiopulmonary bypass techniques remained virtually unchanged during this period. RESULTS In the prudent-use group, there was one operative death with prolonged bypass time and one residual defect that required reoperation; neither of these patients underwent tricuspid valve detachment. All other patients (both groups) were free from mortality and clinically significant complications, including heart block, tricuspid regurgitation, and residual defect. The liberal-use group had shorter cardiopulmonary bypass time than the prudent-use group (59 +/- 14 vs 67 +/- 22 minutes, p = 0.037). CONCLUSIONS Tricuspid valve detachment should be used liberally for moderate- or even low-difficulty exposure of ventricular septal defect, regardless of patient background, because it is a safe and effective adjunct that can improve speed, programmability, reproducibility, and reliability.

Prognosis of Marfan and non-Marfan Patients With Cystic Medial Necrosis of the Aorta

The characteristics and prognosis of patients with cystic medial necrosis of the aorta were reviewed. Subjects were 46 patients who underwent aortic and/or aortic valve surgery between August 1965 and October 1994. All had histologically documented cystic medial necrosis including 22 Marfan patients. The patients with Marfan syndrome were substantially younger (median age, 32 vs 50 years; p < 0.05), and experienced annulo-aortic ectasia more frequently {81% (17/22) vs 46% (11/24); p < 0.05} than those without the syndrome. Sixty-eight percent (15/22) of the Marfan patients and 63% (15/24) of the non-Marfan patients experienced complications with aortic dissection, although not to a significant degree. The hospital mortality rate was 14% (3/22) in the Marfan group and 21% (5/24) in the non-Marfan group, which was also not significant. Of the 38 survivors, developments in the health of 37 were completely followed-up until October 1997. The cardiovascular event-free rate for Marfan patients at 10 years (28%) was lower than that for non-Marfan patients (68%, p = 0.057), whereas the actuarial survival rates at 10 years were nearly equal (72% for the Marfan patients and 74% for the non-Marfan patients). Reoperation was the first cardiovascular event in 77% (10/13) of the Marfan patients and in 14% (1/7) of the non-Marfan patients (p < 0.05). Cardiovascular event was the main cause of late death both for Marfan patients (80%; 4/5) and for non-Marfan patients (86%; 6/7). In conclusion, independent of the presence of Marfan syndrome, careful follow-up is necessary for patients with cystic medial necrosis of the aorta to eliminate serious late complications.

Left Atrial Appendage Insertion for Right Ventricular Outflow Tract Reconstruction

BACKGROUND The left atrial appendage (LAA) may serve as an alternative to the pulmonary arterial wall for right ventricular outflow tract (RVOT) reconstruction without an extracardiac conduit. METHODS Five consecutive patients with pulmonary atresia or severe stenosis underwent corrective (n = 4) or palliative (n = 1) RVOT reconstruction using an LAA insertion. Surgery was performed to treat tetralogy of Fallot, double-outlet right ventricle, or transposition of the great arteries. By inserting the LAA into the obstructed portion, the width of the posterior wall of the RVOT was 20 mm or more. The anterior half of the RVOT was then augmented with pericardial patch. RESULTS There were no early or late postoperative deaths, and no major complications (arrhythmias, thrombo-embolic episodes, infective endocarditis, need for reoperation). The postrepair systolic right ventricular-to-systemic arterial pressure ratio was 0.61 +/- 0.26. Color Doppler flow mapping revealed that the reconstructed RVOT was nonobstructive and had nonturbulent flow. No thrombus or pseudoneointimal formation was observed in the RVOT. CONCLUSIONS LAA insertion in the RVOT is an effective alternative to, or adjunct of, direct anastomosis. It offers several advantages, including fewer early and midterm complications and avoiding the use of an extracardiac conduit.