Computational and Experimental Analyses for Pathogenicity Prediction of ACVRL1 Missense Variants in Hereditary Hemorrhagic Telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is a vascular disease caused by the defects of ALK1/ACVRL1 receptor signaling. In this study, we evaluated 25 recently identified ACVRL1 missense variants using multiple computational pathogenicity classifiers and experimentally characterized their signal transduction capacity. Three extracellular residue variants showed no detectable cell surface expression and impairment of bone morphogenetic protein 9 (BMP9) responsiveness of SMAD-dependent transcription in luciferase assays. Four variants with amino acid replacement in the motifs essential for the intracellular kinase function lost SMAD-dependent signaling. Most of other variations in the kinase domain also caused marked downregulation of signaling; however, two variants behaved as the wild-type ACVRL1 did, while computational classifiers predicted their functional abnormalities. Three-dimensional structure prediction using the ColabFold program supported the significance of the L45 loop and NANDOR domain of ACVRL1 for its association with SMAD1 and BMPR2, respectively, and the variations in these motifs resulted in the reduction of SMAD signaling. On the other hand, two of the GS domain variants maintained high signal transduction capacity, which did not accord with their computational pathogenicity prediction. These results affirm the requirement of a combinatory approach using computational and experimental analyses to accurately predict the pathogenicity of ACVRL1 missense variants in the HHT patients.

Pulmonary Arteriovenous Fistulae After Fontan Operation: Incidence, Clinical Characteristics, and Impact on All-Cause Mortality.

Background: The Fontan operation is a surgical procedure used in children with univentricular hearts. Pulmonary arteriovenous fistulae (PAVF) is a major complication after a Fontan operation. However, the incidence and related clinical pathophysiology of PAVF remain unclear. Purpose: This study aimed to clarify the incidence of PAVF, its clinical characteristics, and its influence on all-cause mortality. Methods and Results: We serially assessed the presence of PAVF using pulmonary artery angiography and/or contrast echocardiography during catheterization in 391 consecutive patients who underwent the Fontan procedure and compared the results with the Fontan pathophysiology and all-cause mortality. PAVF developed in 36 patients (9.2%), including 30 diffuse- and six discrete-PAVF types. The PAVF-free rates at 1, 5, 10, 15, 20, and ≥25 years after Fontan operation were 97, 96, 93, 88, 87, and 83%, respectively. The mean arterial blood oxygen saturation (SaO2) in patients with diffuse PAVF at each corresponding postoperative stage were 90, 91, 91, 91, 89, and 88%, respectively, indicating lower SaO2 levels than those in patients without PAVF (all p < 0.01). However, there was no difference in the SaO2 levels between patients with discrete PAVF and those without PAVF. During a median follow-up period of 2.9 years after the last catheterization, 31 patients, including 12 patients with PAVF, died. Patients with PAVF, especially those with diffuse PAVF, had a higher mortality rate (p = 0.01) than those without PAVF (hazard ratio: 3.6, 95% confidence interval: 1.6-7.8, p = 0.0026). Conclusion: Patients who underwent Fontan surgery had an increased incidence of PAVF as they aged. Discrete PAVF did not influence SaO2 or mortality, whereas the presence of diffuse PAVF caused hypoxia and was associated with all-cause mortality.

Cerebral embolism accompanied by remarkable diffusion-weighted imaging reversal in a 10 month-old infant with congenital heart disease.

•The case of infant ischemic stroke with remarkable DWI reversal.•A mismatch between core volume and visually DWI lesion predicts DWI reversal.•Chronic hypoxia and early recanalization may contribute to large DWI reversal.•Mismatch between DWI-ASPECTS and core volume may occur in infant brain.

Effect of Stiffened and Dilated Ascending Aorta on Aerobic Exercise Capacity in Repaired Patients With Complex Congenital Heart Disease.

Several studies have reported aortic dilation and increased stiffness of the ascending aorta in patients after repair of congenital heart disease (CHD), which may be a predominant cardiovascular risk. However, the clinical significance has not been described in detail. In this retrospective study, 175 repaired patients with complex CHD achieving biventricular circulation and age-matched 39 control subjects were reviewed (median age: 14.9 and 15.7 years, respectively). We measured the diameters of the ascending aorta and descending aorta from catheterization angiograms to yield Z-scores and stiffness indexes (ß) using diameter fluctuations corresponding to pulsatile pressures. Clinical profile, peak oxygen uptake during the cardiopulmonary exercise test, and incidence of unscheduled hospitalization during follow-up was also reviewed. Compared with controls, patients with complex CHD, except for those with aortic coarctation, exhibited significant dilation and increased stiffness of the aortic root and ascending aorta, but not of the descending aorta. In this CHD population (n = 147, including 112 conotruncal anomalies), exercise capacities correlated independently with the diameter Z-score and stiffness index of the ascending aorta along with the history of repetitive thoracotomies, reduced forced vital capacity, and right ventricular hypertension. During a follow-up period (median 15.6 years), either dilation (Z-score >3.5) or increased stiffness (ß >6.0) of the ascending aorta stratified morbidity, but no synergistic impact was detected. In conclusion, in repaired patients with complex CHD, a stiffened and dilated ascending aorta was frequently found, exerting significant adverse impacts on diminished exercise capacity and morbidity.

Progressive stiffening and relatively slow growth of the dilated ascending aorta in long-term Fontan survivors-Serial assessment for 15 years.

BACKGROUND: A stiffened, dilated ascending aorta may represent an important predictor of cardiovascular mortality, and has been reported in patients with congenital heart disease, including single ventricle. However, the serial conformational changes and determinants of reduced distensibility in ascending aorta have not been clarified. METHODS: This retrospective study investigated 115 postoperative Fontan survivors (median age at Fontan: 3.7 years). All patients underwent cardiac catheterization before and 1, 5, 10, and 15 years after the Fontan operation. We measured Z-scores for diameters and stiffness indexes (ß) of the ascending aorta and descending aorta from angiograms. We also reviewed the clinical profiles, hemodynamic parameters, and exercise capacities of patients and compared them with results from 47 control subjects. RESULTS: Fontan survivors displayed significantly larger Z-score and ß of the ascending aorta from before to 15 years after surgery than controls, whereas values for the descending aorta were comparable. Z-score for the ascending aorta was decreased, but ß was elevated significantly according to the trend test. In multivariable analysis, ß of the ascending aorta at 15 years after Fontan operation and its increasing trend were associated with older age at Fontan operation and elevated ventricular end-diastolic pressure. Reduced exercise capacity also correlated with stiffening of the ascending aorta. CONCLUSIONS: Fontan survivors showed progressive stiffening and relatively slow growth of the dilated ascending aorta. Progressive stiffening of the ascending aorta may be coupled to diastolic dysfunction and reduced exercise capacity, suggesting the importance of lifelong management of subclinical Fontan pathophysiology.

Prognostic value of von Willebrand factor in adult patients with congenital heart disease.

OBJECTIVES: von Willebrand factor (vWF) has prognostic value in patients with heart failure (HF) and in those with liver disease. Liver congestion, due to right-sided HF (RHF), is one of the major clinical pathophysiologic manifestations in adults with congenital heart disease (ACHD). The present study's purpose was to clarify the prognostic value of plasma levels of vWF antigen (vWF:Ag) in ACHD. METHODS: We measured vWF:Ag (%) in 382 consecutive patients (20 unrepaired cyanotic ACHD, 172 Fontan patients and 190 ACHD after biventricular repair) and compared the results with the clinical profiles and prognosis. RESULTS: The plasma vWF:Ag level was 130±53 (normal range: 55%-190%), and 48 patients (13%) showed high levels of vWF:Ag (≥190%). Older age, Fontan circulation, higher central venous pressure, lower arterial oxygen saturation and lower plasma levels of albumin were independently associated with high log (vWF:Ag) (p<0.05-0.0001). During the follow-up of 2.4±1.4 years, 15 patients died. High log (vWF:Ag) predicted the all-cause mortality (HR 1.63 per 0.1, 95% CI 1.40 to 1.96, p<0.0001). Specifically, patients with high vWF:Ag (≥165%) had a substantially higher risk of all-cause mortality (HR 56.4, 95% CI 11.4 to 1020, p<0.0001), and this prognostic value was independent of plasma levels of brain-type natriuretic peptide. CONCLUSIONS: High vWF:Ag may reflect RHF severity and related liver dysfunction with a strong prognostic value of all-cause mortality in ACHD. Thus, vWF:Ag might be an excellent biomarker for monitoring ACHD with RHF.

Abnormal glucose metabolism in patients with Fontan circulation: Unique characteristics and associations with Fontan pathophysiology.

BACKGROUND: Fontan patients exhibit a high prevalence of abnormal glucose metabolism (AGM). We aimed to characterize AGM and clarify its association with Fontan pathophysiology. METHODS: We prospectively evaluated AGM with plasma glucose dynamics [mg/dL; fasting glucose (FPG), and maximum glucose increase (PG-spike)] during oral glucose tolerance test and hemoglobin A1c (HbA1c) in 276 consecutive Fontan patients (aged 19 ±â€¯7 years). Of these, 176 patients had serial AGM assessments with a mean interval of 6.5 years. RESULTS: Initial analysis revealed a high prevalence of impaired glucose tolerance (38.4%) and diabetes mellitus (DM) (4.7%), and positive family history, high HbA1c, and high central venous pressure independently predicted presence of DM. HbA1c was independently determined by hypersplenism and presence of DM (P < .05). Serial assessments revealed an increased PG-spike and a decreased HbA1c (P < .001 for both). Prevalence of DM increased (6.3% to 10.3%), and positive family history, high liver enzymes, and AGM predicted new onset of DM (P < .05 for all). Twenty-one patients died during 7.1-year follow-up. FPG (P < .01) and PG-spike (P < .05) independently predicted all-cause mortality. Particularly, patients with FPG ≤ 74 and/or PG-spike ≥85 had a mortality rate 8.7 times higher than those without (P = .0129). CONCLUSIONS: AGM progressed even in young adult Fontan patients, and HbA1c showed limited predictive value for progression. Oral glucose tolerance test plays important roles in uncovering unique Fontan AGM as well as predicting all-cause mortality.

Positive pediatric exercise capacity trajectory predicts better adult Fontan physiology rationale for early establishment of exercise habits.

OBJECTIVE: Exercise training is recommended for its possible favorable effects on Fontan pathophysiology. This study aimed to elucidate the impact of pediatric exercise capacity trajectory, which may mimic the effect of exercise training, on late adult Fontan pathophysiology. METHODS: Since 1990, 97 Fontan patients had consecutively undergone two serial cardiopulmonary exercise tests (CPX1 and CPX2) during childhood (ages 8 ±â€¯2 and 14 ±â€¯2 years) and one during adulthood (CPX3; age 23 ±â€¯5 years). The changes in peak oxygen uptake (PVO2: % of normal value) from CPX1 to CPX2 (1-dPVO2) and from CPX2 to CPX3 (2-dPVO2) were calculated, and then the patients were divided into four subgroups according the 1-dPVO2 and 2-dPVO2. RESULTS: In their adulthood, when compared with groups with negative 1-dPVO2, the central venous pressure, plasma brain natriuretic peptide level, and renal resistive index were lower, whereas liver synthetic function, body fat-free percentage, and PVO2 were higher in those with positive 1-dPVO2 (p < 0.05-0.0001). However, these favorable associations of 2-d-PVO2 with adult Fontan pathophysiology were not observed, except for the PVO2. After CPX3, 13 unexpected events occurred, and the risk was 76% lower in the groups having positive 1-dPVO2 than in those with negative 1-dPVO2 (hazard ratio, 0.24; 95% confidence interval, 0.09-0.62; p = 0.0035). CONCLUSIONS: A positive exercise capacity trajectory during childhood predicts better adult Fontan pathophysiology, including better prognosis. Thus, prescription of exercise could be a promising strategy in the management of pediatric Fontan patients.

Long-term respiratory outcomes after primary total correction for tetralogy of Fallot and absent pulmonary valve in patient with respiratory symptoms.

OBJECTIVE: To review long-term respiratory outcomes for tetralogy of Fallot and absent pulmonary valve (TOF/APV) in respiratory symptomatic populations. METHODS: Of 25 consecutive patients undergoing primary total correction for TOF/APV between 1987 and 2016, Sixteen patients (64%) with a preoperative respiratory disturbance were enrolled. The median age at operation was 1.9 months old, including 4 neonates and 12 infants. Ten patients (62.5%) preoperatively necessitated mechanical ventilator support. During operation, dilated central pulmonary arteries (cPAs) were plicated and retracted anteriorly in all patients, except for the first patient of the study cohort. VSD was completely closed, and the right ventricular outflow tract was reconstructed with a handmade valved conduit before 1990, or a transannular patch with a handmade monocusp after 1991. RESULTS: The actuarial survival rate at 20 years was 86.7%. One patient without cPAs plication and another requiring mechanical ventilator support right after his delivery died. The median duration of postoperative mechanical ventilator support was 14 days (range, 1-183). Readmission for respiratory disturbance after discharge was frequently observed before five years of age, however, no patients were readmitted to the hospital after six years of age, during the median follow-up period of 14.7 years, with a maximum of 27.2 years. Although persistent dilatation of cPAs was common, respiratory symptoms never recurred. CONCLUSIONS: Prognostic and respiratory outcomes after primary total correction for TOF/APV in respiratory symptomatic populations were good. Successful relief of tracheal/main bronchial compression by primary total correction at the neonatal or infantile period provided delayed, but nonrecurrent improvement of respiratory disturbances.

Heart failure with preserved right ventricular ejection fraction in postoperative adults with congenital heart disease: A subtype of severe right ventricular pathophysiology.

BACKGROUND: Right ventricle (RV) may determine heart failure (HF) severity in adults with congenital heart disease (ACHD). However, the association of RV properties with clinical profiles remains unclear. PURPOSE: To clarify the associations of RV properties with biomarkers, exercise capacity, and unscheduled hospitalization (USH) in postoperative ACHD patients. METHODS AND RESULTS: We evaluated determinants of RV end-diastolic volume (EDVI, ml/m(2)) and pressure (EDP, mmHg) in 260 patients who were divided into 4 groups, i.e., those with RVEDVI<100 and EDP≥10 (A, n=49, 19%), those with RVEDVI≥100 with EDP≥10 (B, n=22), those with RVEDVI<100 and EDP<10 (C, n=134), and those with RVEDVI≥100 and EDP<10 (D, n=55). EDVI, EDP, and ejection fraction (EF, %) of the RV were independently associated with the corresponding value of the left ventricle (LV) (p<0.0001 for all). Group A had a RV-EF of 53±8 and was defined HF with preserved RV-EF (RV-HFpEF). Younger age, lower platelet count, and elevated plasma γ-glutamyltransferase independently predicted the RV-HFpEF. When the presence of pulmonary hypertension and LV-HFpEF was included in the analysis, these two factors independently predicted the presence of RV-HFpEF (p<0.05-0.001). On multivariate analysis, older age, number of surgeries, cardiac index, and RV systolic pressure independently determined peak oxygen uptake (PVO2, p<0.05) and RV outflow reconstruction independently predicted USH. The RV-HFpEF group showed the lowest PVO2 and highest incidence of USH among the 4 groups (p<0.05). CONCLUSIONS: Impaired RV characteristics, especially RV-HFpEF, with liver dysfunction and lower platelet count predicted lower exercise capacity with a poorer prognosis in postoperative ACHD patients.

Left ventricular reverse remodeling with infantile dilated cardiomyopathy and pitfalls of carvedilol therapy.

BACKGROUND: The left ventricular reverse remodeling (LVRR) in idiopathic dilated cardiomyopathy (DCM) and the treatment with carvedilol in infants with severe heart failure remain poorly understood. METHODS: We reviewed the medical records of 5 infants around 12 months old referred to our hospital with severe heart failure due to DCM. Increased left ventricular fractional shortening (LVFS) by more than 10% and the percent of normal of left ventricular end-diastolic dimension (%LVDd) less than 120% were defined as LVRR in this study. RESULTS: DCM onset ranged from 8 to 16 months. Initial treatment of their acute heart failure was successful in all 5 but 4 patients relapsed despite the usual dose of carvedilol (induction 0.02-maintenance 0.4mg/kg/day), and developed worsening heart failure. Brain natriuretic peptide (BNP) levels which increased again after the acute treatment had fallen subsequent to discontinuing or decreasing carvedilol. Over 24 months, LVFS had increased from 11±2% (mean±SD) to 34±5% (p<0.05), and %LVDd decreased from 149±27% to 108±11% (p<0.05). CONCLUSIONS: LVRR was found at 2 years after the onset of DCM. Usual dose induction of carvedilol therapy can sometimes worsen heart failure after successful initial conventional treatment for the acute heart failure in DCM. Close control of carvedilol treatment may determine the prognosis of infantile DCM around 12 months old. It is prudent to increase low-dose carvedilol slowly corresponding with the BNP level.

Low fasting plasma glucose level predicts morbidity and mortality in symptomatic adults with congenital heart disease.

BACKGROUND: Adults with complex congenital heart disease (ACHD) have a high prevalence of abnormal glucose regulation (AGR: impaired glucose tolerance and diabetes mellitus). However, the impact of AGR on the prognosis remains unclear. PURPOSE: Our purpose was to clarify the prognostic value of AGR in ACHD. METHODS AND RESULTS: We performed a 75 g oral glucose tolerance test in 438 consecutive patients with ACHD (age 26 ± 8 years), including 38 unrepaired, 148 Fontan, 252 biventricular, and 27 healthy subjects and investigated associations between AGR and clinical events that required hospitalization or caused deaths from all-causes. When compared with the healthy group, fasting blood glucose level (FPG, mg/dl) was lower in the unrepaired and Fontan subjects (p<0.05-0.01) and the prevalence of low FPG (≤ 80 mg/dl) was also higher in the unrepaired (58%), Fontan (47%), and biventricular group (33%) than in the healthy control (11%) (p<0.0001). Postprandial hyperglycemia (area under the curve of glucose: PG-AUC) was higher in all ACHD groups (p<0.0001 for all). New York Heart Association class and lower FPG independently predicted the hospitalization (FPG ≤ 84 mg/dl) and mortality (FPG ≤ 80 mg/dl) (p<0.05-0.0001), while the PG-AUC was not an independent predictor. When compared with the asymptomatic ACHD, symptomatic ACHD with lower FPG had high hazard ratios of 2.2 (95% confidence interval [CI]: 1.3-3.8, p<0.002) and 3.3 (95% CI: 1.2-11.9, p<0.03) for the hospitalizations and all-cause mortality, respectively. CONCLUSIONS: Low FPG is not uncommon in ACHD and the low FPG predicts the morbidity and all-cause mortality in symptomatic ACHD.

Comparison of prognostic variables in children and adults with Fontan circulation.

BACKGROUND: Non-cardiac complications, such as hepato-renal and metabolic problems, are emerging late after the Fontan operation due to its unique hemodynamics. Consequently, associations between clinical variables and postoperative outcome may change during the prolonged postoperative course. METHODS AND RESULTS: To determine if child and adult Fontan patients differ in the impact of cardiac and non-cardiac variables on clinical outcome, we prospectively evaluated associations between hemodynamics, neurohumoral factors, exercise variables, hepato-renal function and metabolic variables and unscheduled hospitalization, including death in 167 consecutive child and 116 adult Fontan patients. When compared with child patients, the adult patients showed higher rates of medications, lower cardiac index, higher values of natriuretic peptides, greater renal dysfunction, more cholestatic livers, and more impaired responses to exercise (p<0.05-0.0001). During the follow-up of 3.7 ± 2.1 years, 64 clinical events (37 in adults), including 13 deaths, occurred. A high CVP and low arterial oxygen satutration strongly predicted the child events (p<0.001), whereas these prognostic parameters were marginal in the adults. Instead, renal dysfunction and metabolic abnormality predicted adult events (p<0.05). Neurohumoral activation, low albumin, hyponatremia, and impaired exercise variables equally predicted clinical events in child and adult Fontan patients. CONCLUSIONS: Distinctive differences in predictive value of clinical variables exist between child and adult Fontan patients. In addition to cardiac issues, we should consider non-cardiac determinents of clinical outcome to maximize our efforts to improve prognosis for adult Fontan survivors.

Countermeasures against physical motion in schoolchildren requiring no sedation during rest and stress myocardial perfusion imaging with technetium-99m-tetrofosmin.

For children, cardiac nuclear medicine imaging has not been widely used because of problems of physical motion, even in schoolchildren who require no sedation. In this study, rest-stress myocardial perfusion imaging (MPI) with technetium-99m-tetrofosmin (Tc-99m TF) was performed with the use of a Vac-Loc cushion, a patient immobilizer commonly used for radiotherapy, for immobilizing school-age patients. The immobilizer attenuated the gamma radiation by 6%. By visual assessment, physical motion-related artifacts were markedly improved in images acquired with the immobilizer, compared to those without. In the assessment of image reproducibility with the immobilizer, taking the reproducibility as 2.5Σ + 0.7σ, there were deviations of 4.45, 5.28, and 3.28 mm along the X-, Y-, and Z-axes, respectively, demonstrating a high reproducibility and a negligible rest-stress position error. It is suggested that for radiotherapy, the immobilizer could expand the versatility of MPI while allowing only minimal physical motion in children.

Neonatal mouse testis-derived multipotent germline stem cells improve the cardiac function of acute ischemic heart mouse model.

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. We previously reported that undifferentiated mGS cells are phenotypically similar to embryonic stem cells and that fetal liver kinase 1 (Flk1)(+) mGS cells have a similar potential to differentiate into cardiomyocytes and endothelial cells compared with Flk1(+) embryonic stem cells. Here, we transplanted these Flk1(+) mGS cells into an ischemic heart failure mouse model to evaluate the improvement in cardiac function. Significant increase in left ventricular wall thickness of the infarct area, left ventricular ejection fraction and left ventricular maximum systolic velocity was observed 4weeks after when sorted Flk1(+) mGS cells were transplanted directly into the hearts of the acute ischemic model mice. Although the number of cardiomyocytes derived from Flk1(+) mGS cells were too small to account for the improvement in cardiac function but angiogenesis around ischemic area was enhanced in the Flk1(+) mGS cells transplanted group than the control group and senescence was also remarkably diminished in the early phase of ischemia according to ß-galactosidase staining assay. In conclusion, Flk1(+) mGS cell transplantation can improve the cardiac function of ischemic hearts by promoting angiogenesis and by delaying host cell death via senescence.

Generation of skeletal muscle stem/progenitor cells from murine induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells, which are a type of pluripotent stem cell generated from reprogrammed somatic cells, are expected to have potential for patient-oriented disease investigation, drug screening, toxicity tests, and transplantation therapies. Here, we demonstrated that murine iPS cells have the potential to develop in vitro into skeletal muscle stem/progenitor cells, which are almost equivalent to murine embryonic stem cells. Cells with strong in vitro myogenic potential effectively were enriched by fluorescence-activated cell sorting using the anti-satellite cell antibody SM/C-2.6. Furthermore, on transplantation into mdx mice, SM/C-2.6(+) cells exerted sustained myogenic lineage differentiation in injured muscles, while providing long-lived muscle stem cell support. Our data suggest that iPS cells have the potential to be used in clinical treatment of muscular dystrophies.

Generation of transplantable, functional satellite-like cells from mouse embryonic stem cells.

Satellite cells are myogenic stem cells responsible for the postnatal regeneration of skeletal muscle. Here we report the successful in vitro induction of Pax7-positive satellite-like cells from mouse embryonic stem (mES) cells. Embryoid bodies were generated from mES cells and cultured on Matrigel-coated dishes with Dulbecco's modified Eagle medium containing fetal bovine serum and horse serum. Pax7-positive satellite-like cells were enriched by fluorescence-activated cell sorting using a novel anti-satellite cell antibody, SM/C-2.6. SM/C-2.6-positive cells efficiently differentiate into skeletal muscle fibers both in vitro and in vivo. Furthermore, the cells demonstrate satellite cell characteristics such as extensive self-renewal capacity in subsequent muscle injury model, long-term engraftment up to 24 wk, and the ability to be secondarily transplanted with remarkably high engraftment efficiency compared to myoblast transplantation. This is the first report of transplantable, functional satellite-like cells derived from mES cells and will provide a foundation for new therapies for degenerative muscle disorders.

Flk1(+) cardiac stem/progenitor cells derived from embryonic stem cells improve cardiac function in a dilated cardiomyopathy mouse model.

OBJECTIVES: Flk1(+) cells derived from embryonic stem (ES) cells are known to differentiate into mesodermal lineages such as hematopoietic and endothelial cells. Here we demonstrate that they can develop into cardiomyocytes that support functional recovery in a dilated cardiomyopathy (DCM) C57/BL6 mouse model. METHODS: Flk1(+) and Flk1(-) cells were sorted at day 4 of differentiation, and cardiomyogenesis was assessed in vitro. Next, we transplanted these cells into the hearts of cardiomyopathy mice to assess improvement in cardiac function. RESULTS: Flk1(+) cells, but not Flk1(-) cells, isolated on day 4 after differentiation were efficiently converted into contractile cardiomyocytes. RT-PCR analysis and immunohistological assays demonstrated that contractile cells derived from Flk1(+) cells in vitro expressed mature cardiac markers on day 10 after differentiation. Transplantation of sorted Flk1(+) cells into DCM model mouse hearts improved cardiac function, as determined by echocardiography and cardiac catheterization. The in vivo differentiated Flk1(+) cells expressed cardiac markers and had gap junctions, as demonstrated by immunohistochemistry. Furthermore, these cells generated ventricular type action potentials similar to those of adult ventricle. CONCLUSION: These results indicate that Flk1 is a good marker for sorting cardiac stem/progenitor cells which can differentiate into mature cardiomyocytes both in vitro and in vivo.

Generation of cardiac and endothelial cells from neonatal mouse testis-derived multipotent germline stem cells.

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. Here, we compared mGS, embryonic stem (ES), and embryonic germ (EG) cells with regard to their ability to differentiate into mesodermal cells, namely, cardiomyocytes and endothelial cells. The in situ morphological appearances of undifferentiated mGS, ES, and EG cells were similar, and 4 days after being induced to differentiate, approximately 30%-40% of each cell type differentiated into Flk1(+) cells. The sorted Flk1(+) cells differentiated efficiently into cardiomyocytes and endothelial cells. By day 10 after differentiation induction, the three cell types generated equal number of endothelial colonies. However, by day 13 after differentiation induction, the Flk1(+) mGS cells generated more contractile colonies than did the Flk1(+) ES cells, whereas the Flk1(+) EG cells generated equivalent numbers as the Flk1(+) mGS cells. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of differentiation markers such as Rex1, FGF-5, GATA-4, Brachyury, and Flk1 revealed that mGS cells expressed these markers more slowly during days 0-4 after differentiation induction than did ES cells, but that this mGS cell pattern was similar to that of the EG cells. RT-PCR analysis also revealed that the three differentiation cell types expressed various cardiac markers. Moreover, immunohistochemical analysis revealed that the contractile colonies derived from Flk1(+) mGS cells express mature cardiac cell-specific markers. In conclusion, mGS cells are phenotypically similar to ES and EG cells and have a similar potential to differentiate into cardiomyocytes and endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.