Shriprasad Deshpande

Microscale Generation of Cardiospheres Promotes Robust Enrichment of Cardiomyocytes Derived from Human Pluripotent Stem Cells

Summary Cardiomyocytes derived from human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, disease modeling, and drug discovery, all of which require enriched cardiomyocytes, ideally ones with mature phenotypes. However, current methods are typically performed in 2D environments that produce immature cardiomyocytes within heterogeneous populations. Here, we generated 3D aggregates of cardiomyocytes (cardiospheres) from 2D differentiation cultures of hPSCs using microscale technology and rotary orbital suspension culture. Nearly 100% of the cardiospheres showed spontaneous contractility and synchronous intracellular calcium transients. Strikingly, from starting heterogeneous populations containing ∼10%–40% cardiomyocytes, the cell population within the generated cardiospheres featured ∼80%–100% cardiomyocytes, corresponding to an enrichment factor of up to 7-fold. Furthermore, cardiomyocytes from cardiospheres exhibited enhanced structural maturation in comparison with those from a parallel 2D culture. Thus, generation of cardiospheres represents a simple and robust method for enrichment of cardiomyocytes in microtissues that have the potential use in regenerative medicine as well as other applications.

Outcomes of heart transplantation in children with hypoplastic left heart syndrome previously palliated with the Norwood procedure

BACKGROUND Following the Norwood operation, unfavorable hemodynamic or anatomic factors might disqualify children from progressing through subsequent palliative surgeries necessitating listing for heart transplantation. Those patients often have immune, clinical, or anatomic risk factors that could preclude donor allocation, increase operative risk, and diminish late survival. We studied transplantation outcomes in those patients using the Pediatric Heart Transplant Study database. METHODS A total of 253 children who had prior Norwood were listed for transplantation (1993-2012). Competing risks analysis modeled events after listing (death, transplantation) and after transplantation (death, retransplantation) and examined factors affecting outcomes. RESULTS Patients were listed following Norwood (n = 89, 35%), Glenn (n = 96, 38%), and Fontan (n = 68, 27%). Competing risk analysis showed that 1 year after listing, 23% of patients had died, 70% had received transplantation, and 7% were alive without transplantation. Factors associated with death without transplantation included UNOS status I (HR 3.44 [1.58-7.49], P = .002) and mechanical circulatory support (HR 4.13 [2.04-8.34], P < .001). Overall, 188 patients received transplantation. Competing risk analysis showed that 10 years following transplantation, 40% had died, 7% had received retransplantation, and 53% were alive without retransplantation. Factors associated with death following transplantation were race other than white (HR 2.18 [1.19-3.99], P = .01), and donor mode of death other than anoxic brain injury (HR 2.23 [1.00-5.01], P = .05). CONCLUSIONS Heart transplantation can salvage children failing palliation following Norwood with outcomes comparable to those reported for other congenital cardiac anomalies. Efforts to stabilize patients and increase donor pool could improve survival by decreasing the high waiting list mortality. In patients who received transplantation, survival is not affected by last palliation stage or sensitization, reflecting current advances in perioperative management and immunosuppression management strategies.

The Effect of Noncardiac and Genetic Abnormalities on Outcomes Following Neonatal Congenital Heart Surgery

Significant noncardiac and genetic abnormalities (NC and GA) are common in neonates with congenital heart defects. We sought to examine current-era effect of those abnormalities on early and late outcomes following cardiac surgery. The method from 2002-2012, 1538 neonates underwent repair (n = 860, 56%) or palliation (n = 678, 44%) of congenital heart defects. Regression models examined the effect of NC and GA on operative results, resource utilization, and late outcomes. Neonates with NC and GA (n = 312, 20%) had higher incidence of prematurity (21% vs 13%; P < 0.001) and weight ≤2.5kg (24% vs 12%; P < 0.001) than neonates without NC and GA (n = 1226, 80%). Although the incidence of single ventricle was comparable (34% vs 31%; P = 0.37), neonates with NC and GA underwent more palliation (52% vs 42%; P = 0.001) and subsequently had higher percentage of STAT mortality categories (Society of Thoracic Surgeons (STS) and the European Association for Cardio-thoracic Surgery (EACTS) Congenital Heart Surgery Mortality Categories) 4 and 5 procedures (78% vs 66%; P < 0.001). Adjusted logistic regression models that included disparate patient and operative variables showed that the presence of NC and GA was associated with increased unplanned reoperation (odds ratio = 1.7; 95% CI: 1.1-2.7; P = 0.03) and hospital mortality (odds ratio = 2.2; 95% CI: 1.3-3.6; P = 0.002). Adjusted linear regression models showed significant association between NC and GA and increased postoperative mechanical ventilation duration, intensive care unit, and hospital stays (P < 0.001 each). Adjusted hazard analysis showed that the presence of NC and GA was associated with diminished late survival (hazard ratio = 2.4; 95% CI: 1.9-3.1; P < 0.001) and that was evident in all subgroups of patients (P < 0.001 each). Conclusion is neonates with NC and GA commonly have associated risk factors for morbidity and mortality such as prematurity and low weight. After adjusting for those factors, the presence of NC and GA continues to have significant association with increased unplanned reoperation, hospital mortality, and resource utilization after palliative and corrective cardiac surgery. Importantly, the hazard of death in those patients continues beyond the perioperative period for at least 1 year. Our findings show that the presence of NC and GA should be emphasized during parent counseling and decision making; and underscore the need to explore strategies to improve outcomes for this high-risk population that must address perioperative care, outpatient surveillance, and management.

Results of heart transplantation following failed staged palliation of hypoplastic left heart syndrome and related single ventricle anomalies.

OBJECTIVES Multistage palliation is the mainstay management strategy of children with hypoplastic left heart syndrome (HLHS) and related single ventricle anomalies. If this palliation strategy fails, heart transplantation (HT) is required. The results of HT in children who had a prior Norwood operation are reportedly poor due to several immune, clinical and anatomical risk factors. We report our institutional outcomes following HT in children who had a prior Norwood operation. METHODS Between 1994 and 2013, 107 children with congenital heart disease underwent HT. We examined early and late outcomes in our study cohort of children who had a prior Norwood operation (n = 24), and analysed risk factors affecting survival. Survival was subsequently compared with a control group of 83 children with congenital heart disease without a prior Norwood operation who received HT. RESULTS Twenty-four children with a prior Norwood operation underwent HT. The majority (22/24, 92%) had HLHS. Children were listed following Norwood (n = 2, 8.3%), Glenn (n = 17, 70.8%) or Fontan (n = 5, 20.8%) operation. Ten (42%) patients had panel reactive antibodies (PRAs) >10%. Median age at listing was 2.7 (range 0.4-16.8) years and median age at the time of HT was 3.0 (range 0.6-16.8) years, with the median waiting list duration of 63.5 (range 1-554) days. Hospital mortality was 1/24 (4%). Overall parametric survival estimates at 1, 5 and 10 years were 85, 65 and 52%. Survival was not affected by listing status, last palliation stage, age or high PRA. The only significant factor affecting survival was the later era in our series with significantly superior 5-year survival (100 vs 42%, P = 0.0003). Overall survival was comparable with the control group of children with congenital heart disease and no prior Norwood operation (52 vs 53% at 10 years, P = 0.97). Overall, 3 of 24 patients required retransplantation with only one late survivor. CONCLUSIONS Children failing multistage palliation of HLHS may require HT, often following the Glenn operation. HT results in this group are comparable with those in other children with congenital heart disease. Improvements in pretransplant management, immune suppression and outpatient care in the later era might have specifically benefited this particularly risky group of patients.

Outcomes and risk factors for heart transplantation in children with congenital heart disease

OBJECTIVE Heart transplantation in children with underlying congenital heart disease has traditionally been associated with inferior outcomes. We report our single institution experience. METHODS Between 1988 and 2013, 124 children with congenital heart disease underwent heart transplantation. Competing risks analysis modeled events after heart transplantation (retransplantation, death without retransplantation). Multivariable regression analysis examined risk factors affecting outcomes. RESULTS There were 76 male patients (61%); median age was 3.8 years (interquartile range, 0.6-11.5). Sixteen patients (13%) underwent primary heart transplantation, and 108 patients (87%) underwent prior surgical repair/palliation. Thirty-eight patients (31%) had 2 ventricles, and 86 patients (69%) had a single ventricle, including 33 (27%) who underwent a prior Fontan procedure. Competing risks analysis showed that at 10 years after heart transplantation, 13% of patients had undergone retransplantation, 43% of patients had died without retransplantation, and 44% of patients were alive without retransplantation. After retransplantation, 9 of 17 patients were alive and 3 of 17 patients had undergone a second retransplantation. Overall 15-year survival after initial heart transplantation was 41%. On multivariable analysis, risk factors for early-phase mortality were age less than 1 year (hazard ratio [HR], 7.2; 95% confidence interval [CI], 2.4-22.0; P < .001) and prolonged cardiopulmonary bypass (HR, 5.0; 95% CI, 2.1-11.8; P < .001). Risk factors for late-phase mortality were age more than 1 year (HR, 3.0; 95% CI, 1.1-7.7; P = .025) and donor-recipient race mismatch (HR, 2.2; 95% CI, 1.2-4.1; P = .016). Survival was not affected by era, underlying anomaly, prior Fontan procedure, sensitization, or pulmonary artery augmentation. CONCLUSIONS The outcomes of heart transplantation in children with congenital heart disease have not improved in the current era. Survival was not affected by the underlying anomaly, prior Fontan procedure, or sensitization. Strategies to improve the outcomes in patients with congenital heart disease may need to address selection criteria, transplantation timing, and pretransplant and post-transplant care. The effect of donor-recipient race mismatch warrants further investigation and might affect organ allocation algorithms or immunosuppression management.

Safe and effective use of a glycemic control protocol for neonates in a cardiac ICU.

Objective: To investigate the safety and efficacy of a hyperglycemia protocol in neonates with critical cardiac illness. Neonates are often regarded as high risk for hypoglycemia while receiving continuous insulin infusions and thus have been excluded from some clinical trials. Design: A retrospective review. Setting: A pediatric cardiac ICU in a tertiary academic center. Interventions: Neonates with critical cardiac illness who developed hyperglycemia were placed on an insulin-hyperglycemia protocol at the attending physician’s discretion. Insulin infusions were titrated based on frequent blood glucose monitoring. Measurements: Critical illness hyperglycemia was defined as a blood glucose less than 140 mg/dL. Hypoglycemia was defined as moderate (⩽ 60 mg/dL) or severe (⩽ 40 mg/dL). Initiating blood glucose, lowest blood glucose during insulin infusion, doses of insulin, duration of insulin, and time to blood glucose greater than 140 mg/dL were evaluated. Main Results: A total of 44 patients were placed on the protocol between January 2009 and October 2011. The majority of insulin infusions were initiated in the early postoperative period (33 of 44, 75%). Moderate hypoglycemia occurred in two patients (4.5%), with blood glucose levels of 49 and 53 mg/dL. No episodes of severe hypoglycemia occurred. A total of 345 discrete blood glucose levels were analyzed; two of these being greater than 60 mg/dL (0.58%). Mean blood glucose prior to starting insulin was 252 ± 45 mg/dL and time until euglycemia was 6.1 ± 3.9 hours. The mean duration of insulin infusion was 24.6 ± 38.7 hours, mean peak dose was 0.10 ± 0.05 units/kg/hour, and mean insulin dose was 0.06 ± 0.02 units/kg/hour. For postoperative patients, mean time after bypass until onset of hyperglycemia was 2.2 ± 2.6 hours. Conclusions: A glycemic control protocol can safely and effectively be applied to neonates with critical cardiac disease. Neonates with critical cardiac illness should be included in clinical trials evaluating the benefits of glycemic control.

Late Outcomes of Infants Supported by Extracorporeal Membrane Oxygenation Following the Norwood Operation

Background: Hospital survival for infants who require extracorporeal membrane oxygenation (ECMO) following the Norwood operation is 30% to 60%. However, little is known about late outcomes of hospital survivors and their ability to progress through subsequent palliative stages. Methods: Between 2002 and 2012, 38 (13.4%) of the 284 neonates with hypoplastic left heart syndrome or other single ventricle variants received ECMO support following Norwood. We examined factors affecting hospital death and compared postdischarge events between hospital survivors who received postoperative ECMO (n = 16 of 38) and a control of hospital survivors who did not receive ECMO (220 of 246). Results: Unplanned cardiac reoperation was the only predictor of postoperative ECMO requirement. Overall, 22 (58%) of the 38 patients were weaned from ECMO support and 16 (42%) of the 38 survived to hospital discharge. The ECMO duration was a significant factor for hospital mortality (odds ratio = 1.52 per 1-day increase [1.03-2.24], P = .035). Following discharge, 15 (94%) of the 16 underwent Glenn and 1 (6%) of the 16 had interstage mortality. In the control group, 194 (88%) of the 220 underwent Glenn and 26 (12%) of the 220 had interstage mortality or received transplantation (P = .499). Following Glenn, 3 (20%) of the 15 patients had interstage mortality or received transplantation and 12 (80%) of the 15 proceeded to Fontan or were alive awaiting Fontan. In the control group, 23 (12%) of the 194 had interstage mortality or received transplantation and 171 (88%) proceeded to Fontan or were alive awaiting Fontan (P = .357). Overall, 81% of hospital survivors were alive 5 years following discharge in both ECMO and non-ECMO groups. Conclusions: ECMO support following Norwood is associated with high probability of hospital death. Nonetheless, interstage mortality, progression to subsequent palliative stages, intermediate survival, and freedom from heart transplantation are comparable to those in patients who did not require postoperative ECMO support.

Aorto–Right Atrial Tunnel: Fetal Heart Failure, Diagnosis, and Treatment

Aorto–right atrial tunnel (ARAT) is a rare clinical entity. Although it is a benign finding in most cases; it can present with significant right ventricular volume overload and congestive heart failure. This report describes the first fetal diagnosis of congenital aorto–right atrial tunnel and successful management of the heart failure by surgical intervention.

Sources of Circuit Thrombosis in Pediatric Extracorporeal Membrane Oxygenation.

Extracorporeal membrane oxygenation (ECMO) for cardiopulmonary support of critically ill patients is used frequently in the pediatric population. ECMO is burdened by complications, including thrombosis and hemorrhage. Here we demonstrate the focused location of clots, their histologic composition, and the relationship of in situ thrombus to local hemodynamics in ECMO circuits. Pediatric ECMO circuits from Children’s Healthcare of Atlanta, Emory University (Atlanta, GA) were obtained after removal from extracorporeal support over a 2.5 year period (n = 50). All clots and material deposited within the circuit were recorded. Location of clot was compared with local hemodynamics. Most clots were adherent to the junctions made by the tubing and connectors, as opposed to being randomly disturbed throughout the circuit tubing (p << 0.05). Loose, nonadherent clots were also found at the entry side of oxygenators. The clots colocated directly with zones of low shear rate. Histology revealed a fibrinous composition, consistent with coagulation potentiated by low shear. Centrifugal pump circuits (n = 16) had more clots than roller pump (n = 34) circuits (p << 0.05). In addition, all centrifugal pumps had clots that formed at the top of the pump shaft. The ECMO circuits from our single-center study demonstrate the concentrated location of fibrin clots at low shear zones created by tubing-connector junctions. Type of pump also influences the frequency of clot formation. Since the mechanism of the majority of ECMO circuit thrombosis is low shear and fibrin driven, optimization of hemodynamics and anticoagulation regimen may reduce clot formation and bleeding.

Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia (ARVC/D): Review of 16 Pediatric Cases and a Proposal of Modified Pediatric Criteria

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a heritable cardiomyopathy characterized by fibro-fatty replacement of right ventricular myocardium. Diagnostic criteria, established in 1994 and modified in 2010, are based on predominately adult manifestations of ARVC/D. The goal of this paper is to review a single-center experience with pediatric ARVC/D and propose modifications of current diagnostic criteria to appropriately include pediatric ARVC/D. We identified 16 pediatric cases of ARVC/D from our tertiary care center. Patient demographics, presentation, course, genetic testing, and family history were reviewed. Sixteen patients were diagnosed with ARVC/D through the modified diagnostic criteria, genetic testing, and pathology. Five patients had positive family histories. Five patients presented with cardiac arrest, and six were found to have ventricular tachycardia. Two patients presented with heart failure. Six autopsies, six explanted hearts, and three biopsies found massive fibro-fatty infiltration of the right ventricular wall. Six patients underwent heart transplantation, and two have received automatic implantable cardioverter defibrillator. Two patients had identifiable genetic mutations previously noted in the literature. One patient had a novel mutation of a known ARVC/D gene. Many pediatric patients do not meet the current ARVC/D diagnostic criteria, resulting in delays in diagnosis and treatment. The current criteria need further revision to encompass pediatric manifestations of ARVC/D. In our opinion, pathological and clinical findings alone are sufficient for accurate diagnosis of pediatric ARVC/D. Creating modified pediatric criteria would facilitate prompt diagnosis and management of ARVC/D and facilitate structured research with the goal of improving outcomes.