Introduction: Donor fraction (DF) cell-free DNA (cfDNA) is an emerging tool for non-invasive rejection surveillance in heart transplantation (HTx). Little is known about the significance of DF and/or total cfDNA (TcfDNA) in the first month post HTx. We explored the relationship between early cfDNA results and later clinical events in adult and pediatric HTx recipients. Aims: 1. Explore the relationship between early cfDNA levels and late events after HTx 2. Describe the decline in DF during the first month post HTx Methods: Retrospective data from the multicenter prospective blinded DTRT study (DNA-based transplant rejection test) was used. DF and TcfDNA results from samples drawn post HTx day 1 (<24h), day 4 (+/- 12h), day 7 (+/- 12h), day 14 (+/- 24h) and day 28 (+/- 7 days) were compared in subjects with or without events >35d to 1 year post HTx. Captured events include cardiac arrest, mechanical circulatory support or death. Exclusions included absent TcfDNA, multiorgan Tx, any PTLD, or other cancer in previous 2 yrs. Cell-free DNA values were compared across event groups and time windows using GEE (generalized estimating equation) with Max Likelihood Estimation. Results: 190 subjects had 566 samples drawn < 35 days post HTx. Median age was 17.8 yrs with a range of 26 days - 73.4 yrs; 51.8% were pediatric. 16 subjects (8.4%) had events bridging or following day 35 post HTx; 23 with events prior to day 35 were included as non-event subjects. TcfDNA was significantly higher in event subjects on days 4, 7 and 28 post HTx. [Fig 1]. There was no difference in DF between event and non-event subjects. Median (IQR) DF at 14 days was 0.26% (0.20, 0.39) and 0.19% (0.13, 0.29) at 28 days. Conclusions: Elevated TcfDNA, even in the setting of normal DF, may be a useful early marker to identify patients at risk for later events in the first year post HTx. The majority of patients reach low DF levels by 28 days post HTx suggesting an opportunity to incorporate cfDNA earlier into rejection surveillance protocols.

Pulmonary vein stenosis (PVS) is a rare, serious, and progressive disease in the pediatric population. Evaluation is complex and involves multimodality imaging. Diagnosis is important as early treatment to prevent progressive pulmonary hypertension and right ventricular dysfunction is essential. Adult studies have shown good correlation between various imaging modalities; however, there are limited data in children. This is a single-center retrospective pilot study to determine the reliability of measurement of pulmonary vein stenosis and pulmonary hypertension across different imaging modalities—computed tomography angiography (CTA), echocardiography (echo), lung perfusion scan (LPS), and cardiac catheterization (cath). PVS was defined as > 2 mmHg by echo and cath and/or 50

Hypoplastic left heart syndrome (HLHS) is a complex form of congenital heart disease (CHD) characterized by hypoplasia of the left ventricle and proximal aorta, as well as stenosis or atresia of the mitral and aortic valves. Our lab previously identified that rare, predicted-damaging variants in the gene encoding for α-myosin heavy chain ( MYH6, α-MHC), a key contractile protein in the heart, are enriched in HLHS. It has been shown that pathological variants in the MHC head domain directly alter force generation, but mechanisms by which tail domain variants cause contractile defects are less clear.In double-blind analyses, patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carrying the MYH6 E1584K tail domain variant (VAR) showed significant structural and functional deficits compared to wild-type (WT). Consistent with our previously reported findings in atrial tissue from a patient carrying MYH6 E1584K , VAR iPSC-CMs exhibited sarcomere disarray (p<0.001) and altered sarcomeric gene expression (p<0.05). VAR iPSC-CMs display a hypercontractile phenotype compared to WT, with increased amplitude (2.16μm VAR, 1.32μm WT; p<0.05) and duration (233 msec VAR, 173 msec WT; p<0.05) of contraction and elongated relaxation times (270 msec VAR, 190 msec WT; p<0.01). Hypercontractility was also observed in bulk culture, where VAR iPSC-CMs demonstrated increased amplitude (1413.4 a.u. VAR, 993.6 a.u. WT, p<0.05) and velocity of contraction (1493.7 a.u./msec VAR, 982.2 a.u./msec WT, p<0.05). Notably, this is different than our previous reports of iPSC-CMs carrying the head domain variant MYH6 R443P , which exhibited decreased contraction velocity.Our data demonstrate the mechanism of MYH6 pathogenicity is distinct between variants, emphasizing the importance of mechanistic studies to inform clinical decision-making. Furthermore, this work will direct future investigations into the genetic etiology and physiology of CHD and provide a framework upon which personalized treatment strategies can be developed. Future studies will examine the effect of myosin modulators omecamtiv mecarbil (OM) and mavacamten on MYH6 variant iPSC-CMs. Advancing a Healthier Wisconsin Endowment This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Introduction: Preoperative risk stratification in congenital cardiac surgery includes patient and procedure related factors, which may be used in clinical decision making as well program performance evaluation. Despite these tools, unidentified factors contribute to wide variation in outcomes both within and between centers. Identification of latent physiologic risk factors may strengthen predictive models. Hypothesis: Total cell-free DNA (TCF) functions as a biomarker for cellular injury as well as a pro-inflammatory cytokine. We hypothesized that elevated preoperative TCF would be associated with poor outcome following pediatric cardiac surgery requiring cardiopulmonary bypass (CPB). Methods: Prospective observational study of children age < 18 yr and wt > 3 kg undergoing planned CPB surgery. The Children’s Wisconsin Institutional Review Board approved the protocol . A serum TCF sample was obtained after induction of anesthesia prior to surgical incision. The primary outcome measure was a composite of postoperative cardiac arrest, ECMO, or death (CAED). Association of outcome to TCF was assessed by logistic regression with a cutpoint chosen by ROC curve exploration. Odds ratios with 95% CI were calculated. Results: Data were available in 117 patients, median age 0.9 years (range 0-17.4), median weight 7.8kg (range 3.2-98). The primary outcome (CAED) was met in 6/117 (5.1%). Table 1 summarizes characteristics of patients with and without CAED. Risk of CAED was 2% with TCF<20 ng/ml, and 27% with TCF>20 ng/ml (OR=18.2, CI 2.2- 212, p<0.01). Elevated TCF was associated to fewer hospital free days (GLM p<0.01). Data in table reported as median [IQR]. Conclusions: Preoperative TCF has an important association with postoperative cardiac arrest, ECMO, and death. Alternative or intensified treatment strategies could be considered in patients with elevated preoperative TCF.

Hypoplastic Left Heart Syndrome (HLHS) is a severe form of congenital heart disease (CHD), requiring prompt surgical intervention and eventual transplant. Despite significant advances in detection and treatment of HLHS, genetic risk factors have been identified in <5% of patients. Our prior studies of 190 HLHS patients identified 19 distinct, rare variants in the gene encoding for a‐myosin heavy chain (MYH6), with variant carriers exhibiting reduced transplant‐free survival (p < 1 × 10−3). In‐depth genetic analysis was performed on the family of one HLHS subject who exhibited a novel E1503V variant in the tail domain of MYH6. The variant was identified in six additional members of the family, all of whom were related to the proband via the maternal grandmother. Four of those individuals also exhibited adult heart disease, with varying ages of onset. Though MYH6 variants have been implicated in a number of heart diseases – including arrhythmias, familial cardiomyopathies, as well as various forms of CHD – to our knowledge this is the first association of a single variant with both adult heart disease and HLHS.α‐myosin heavy chain (α‐MHC) is a cardiac‐specific protein that functions as a component of the sarcomere, the major contractile unit of the heart muscle. We therefore sought to evaluate functional consequences of MYH6E1503V on a‐MHC in vitro. Using CRISPR‐Cas9 gene editing technology, we successfully introduced the variant into induced pluripotent stem cells (iPSCs) from an unrelated individual, which were then used to generate cardiomyocytes. Subsequent evaluation of these cells revealed key insights into the effect of MYH6E1503V on sarcomere organization and contractile properties in cardiomyocytes, as well as their proliferation and differentiation capacity.Support or Funding InformationChildren's Research Institute at the Children's Hospital of Wisconsin, Little Hearts for Life Foundation, M.A is a member of the Medical Scientist Training Program at MCW, which is partially supported by a training grant from NIGMS T32‐GM080202This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Difficulty weaning from cardiopulmonary bypass (CPB) or the need to return to CPB (collectively D-CPB) may occur after the Norwood procedure. We sought to evaluate the relationship between D-CBP and survival. This was a retrospective chart review of all patients undergoing a Norwood procedure at our institution during the interval 2005-2017. Primary outcome was survival for the Norwood procedure. Secondary outcomes included various measures of morbidity. Successful wean from CBP (S-CPB) was defined as no need to return to full-flow CPB during the initial definitive wean or after separation from CPB; otherwise, the classification was difficulty with wean (D-CBP). Successful rescue in the D-CPB group was defined as not requiring extracorporeal life support either in the operating room or within the first 3 postoperative days. Of the 196 patients in the cohort, 49 were D-CPB. Survival for S-CPB was 92.5% (136/147) vs 71.4% (35/49) for D-CPB (P = 0.001). Major morbidity occurred in 29.9% (44/147) in S-CPB vs 69.4% (34/49) in D-CPB (P < 0.001). With multivariable analysis, D-CPB was significantly associated with mortality (odds ratio = 8.09; confidence interval 2.72-24.05; P < 0.001). Successful rescue occurred in 30 of 49 patients in the D-CPB group and demonstrated survival similar to the S-CPB group. In the Norwood patient, D-CPB is an important intraoperative event and prognostic factor for mortality and morbidity. Successful rescue appears to ameliorate the impact of D-CPB on survival.

Hypoplastic Left Heart Syndrome (HLHS) is a clinically and anatomically severe form of Congenital Heart Disease (CHD). Although prior studies suggest that HLHS has a complex genetic inheritance, its etiology remains largely unknown. We previously demonstrated that rare variants in the α‐myosin heavy chain (MYH6) gene are significantly enriched in HLHS (p<1×10−5, observed in 10.5% or 20/190 HLHS subjects). Analysis of clinical outcomes showed that survival without cardiac transplant was reduced in HLHS subjects with MYH6 variants (p=6×10−3) as compared to those without MYH6 variants. Transcriptome and protein expression analyses from cardiac tissue revealed differential expression of cardiac contractility genes, notably upregulation of the β‐myosin heavy chain (MYH7) gene in subjects with MYH6 variants. Visualizing α actinin in atria showed intrinsic disrupted sarcomere structure in HLHS patients with MYH6 variants.We observed that patient‐specific induced pluripotent stem cells (iPSCs) were defective in cardiomyogenic differentiation in vitro. Additionally, sarcomere structure in cardiomyocytes derived from iPSCs produced from a family trio (unaffected mother‐affected father‐patient) carrying a variant in MYH6 (MYH6‐R443P) was dysmorphic.We introduced the MYH6‐R443P variant into iPSCs derived from the unaffected parent using CRISPR/cas9 technology and observed that the MYH6‐R443P phenotype in HLHS‐iPSCs was recapitulated; specifically, gene‐edited MYH6‐R443P+/− and MYH6‐R443P−/− iPSCs displayed defective cardiomyogenic differentiation, and sarcomere structure in these cardiomyocytes was dysmorphic. We conclude that the etiology of MYH6‐associated HLHS may be informed utilizing iPSCs.Support or Funding InformationThe Advancing a Healthier Wisconsin Endowment (AHW)Wolfe Family FoundationGreat Milwaukee FoundationChildren's Research InstituteLittle Heart for LifeThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

•Kawasaki disease can present with incomplete clinical signs particularly in infants•Coronary complications are more common in infants with incomplete Kawasaki disease•Coronary anatomy must be carefully assessed in infants with cardiovascular collapse

Donor fraction (DF) of cell-free DNA (cf-DNA) has been proposed as a noninvasive surveillance method in heart transplantation. We present our pilot experience with a novel assay to quantify DF (%) with a clinically practical throughput and turnaround time. A blinded study was performed in which

In microvessels of patients with coronary artery disease (CAD), flow-mediated dilation (FMD) is largely dependent upon the endothelium-derived hyperpolarizing factor H2O2. The goal of this study is to examine the influence of age and presence or absence of disease on the mechanism of FMD. Human coronary or adipose arterioles (~150 µm diameter) were prepared for videomicroscopy. The effect of inhibiting COX [indomethacin (Indo) or NOS (L-NAME), eliminating H2O2 (polyethylene glycol-catalase (PEG-CAT)] or targeting a reduction in mitochondrial ROS with scavengers/inhibitors [Vitamin E (mtVitamin E); phenylboronic acid (mtPBA)] was determined in children aged 0–18 years; young adults 19–55 years; older adults >55 years without CAD, and similarly aged adults with CAD. Indo eliminated FMD in children and reduced FMD in younger adults. This response was mediated mainly by PGI2, as the prostacyclin-synthase-inhibitor trans-2-phenyl cyclopropylamine reduced FMD in children and young adults. L-NAME attenuated dilation in children and younger adults and eliminated FMD in older adults without CAD, but had no effect on vessels from those with CAD, where mitochondria-derived H2O2 was the primary mediator. The magnitude of dilation was reduced in older compared to younger adults independent of CAD. Exogenous treatment with a sub-dilator dose of NO blocked FMD in vessels from subjects with CAD, while prolonged inhibition of NOS in young adults resulted in a phenotype similar to that observed in disease. The mediator of coronary arteriolar FMD evolves throughout life from prostacyclin in youth, to NO in adulthood. With the onset of CAD, NO-inhibitable release of H2O2 emerges as the exclusive mediator of FMD. These findings have implications for use of pharmacological agents, such as nonsteroidal anti-inflammatory agents in children and the role of microvascular endothelium in cardiovascular health.

Background: There remains limited options for end stage pulmonary arterial hypertension that is refractory to medical therapy. The reprisal of the Potts shunt (pulmonary artery to descending aorta anastomosis) has been used to decrease right ventricle (RV) afterload by creating a right to left shunt in effort to promote lung transplant free survival. Methods: Retrospective review of three patients ages 16, 19, and 27 years old who underwent the creation of a "reverse" Potts shunt at our institution in 2016. Results: The patients were WHO functional class III or IV and on three classes of pulmonary hypertension specific drug therapy including IV prostacyclin. All patients had RV dysfunction and suprasystemic RV pressures. Two patients had a catheterization for dilation and stenting of a tiny patent ductus arteriosus (PDA) and one had emergent surgical placement of a pulmonary artery to descending aorta conduit. The catheterization procedures were well tolerated without complications. The surgical procedure was complicated with heavy bleeding and respiratory failure. All patients recovered from their procedure and experienced improved functional class with decrease in RV pressures to systemic levels but no improvement in RV function. One catheterization patient required restenting due to stent fracture at 7 months. Our surgical patient died from massive hemoptysis 13 months after the procedure. Conclusions: Reverse Potts shunt physiology may be an option for end-stage PAH patients with suprasystemic RV pressures. Optimal timing of this procedure remains unclear, but if feasible, PDA stenting, even in adult patients may be accomplished with low morbidity.

Background: Congenitally corrected transposition of the great arteries (CCTGA) is a rare cardiac malformation resulting from abnormal looping of the embryonic cardiac tube that results in atrioventricular and ventriculoarterial discordance. Surgical options include a physiologic repair (PR) or

Purpose: To test the hypothesis that patients with hypoplastic left heart syndrome (HLHS) and developmental delay will have a higher average summative C-score in ciliopathy genes than patients with HLHS without developmental delay. Methods: Ciliopathy gene variant burden was determined utilizing a summative C-score for 14 ciliopathy genes in children with HLHS ( n = 24). Mean summative C-scores were compared between children with and without developmental delay. Genome-wide randomizing gene sets were evaluated as a scoring control. Results: Children with developmental delay had a mean summative C-score of 4.05 in ciliopathy genes as compared to a mean summative C-score of 2.02 for children without developmental delay. This difference in means was higher than 99.1% (empirical P value <0.01) of 2 million random lists of 14 genes. Conclusion: Genetically complex disorders such as ciliopathies can be assessed to determine phenotypic risk with summative C-score in appropriately chosen gene sets. If these results are replicated in subsequent cohorts, a diagnostic gene panel could identify risk for developmental delay and other ciliopathy-related comorbidities in infants with congenital heart disease. Genet Med advance online publication 27 October 2016

Background: Following the successful aortic root replacement (ARR) in patients with familial forms of aortic aneurysms (FFAA), few studies have defined the long-term outcomes of the re-implanted coronary arteries. The goal of the study was to describe late coronary complications following ARR in patients with FFAA. Methods: 40 patients with a genetically confirmed form of FFAA S/P ARR whom had undergone at least 1 CT scan or cardiac MRI study following ARR were identified. All studies were reviewed retrospectively and the coronary ostium were measured offline by two independent observers. Results: The majority of patients had Marfan Syndrome (80%), 15% had Loeys-Dietz Syndrome (TGFBR1 or TGFBR2), and 2 patients a MYLK or ACTA2 mutation. Mean age at time of ARR was 30.7 years, range 10-65. Mean length of follow-up from ARR 6.2 years, range 0.5-19. At last follow-up, 55% had developed at least 1 coronary complication. Nineteen (48%) had developed a coronary ostium aneurysm and 3 patients coronary ostium stenosis. All patients were alive at last follow-up. No patient had developed a coronary button rupture or dissection. Patients with aneurysms were more likely to have a mutation other than Marfan Syndrome (p < .05) and were more likely to have undergone emergent ARR following dissection (p = .008). Progressive coronary ostium dilation was rare. Two patients however did develop severe dilation and required button revision, both with TGFBR2 mutation. Conclusion: Coronary ostium dilation is common following ARR in patients with FFAA. Progressive dilation and risk for coronary button rupture appears to be low. Patients with non-fibrillin 1 mutations, are at higher risk for severe dilation and some may require intervention. Long term surveillance in particular in patients with non-fibrillinl forms of familial aortic aneurysm and dissection syndrome should be considered.

Acute kidney injury (AKI) is common following cardiopulmonary bypass (CPB), and may result in increased morbidity and mortality. Definitions of AKI that rely solely on serum creatinine are inaccurate markers of early renal injury. We investigated the role of urine biomarkers and indices of renal

Our aim is to develop and validate an accurate method for estimating TCV using standard echocardiographic imaging that can be easily employed to aid in donor–recipient size matching in pediatric heart transplantation. Thirty patients who underwent Echo and cardiac magnetic resonance imaging (cMRI) were identified. TCV was measured on cMRI. TCV was determined echocardiographically by two methods: a volume measurement using the modified Simpson's method on a four‐chamber view of the heart; and a calculated volume measurement which assumed a true‐elliptical shape of the heart. These two methods where compared with the value obtained by cMRI using the concordance correlation coefficient (CCC). TCV by method 1 correlated well with cMRI (CCC = 0.98%, CI = 0.97, 0.99). TCV by method 2 had a CCC = 0.90 (CI = 0.9464, 0.9716) when compared to cMRI. Left ventricular end‐diastolic volume (LVEDV) also correlated as a predictor of TCV in patients with structurally normal hearts and could be described by the equation: TCV = 6.6 (LVEDV) + 12 (R2 = 0.97). Echocardiographic assessment of TCV for recipients and their potential donors is a simple process and can be prospectively applied as part of donor evaluation.

OBJECTIVE:To determine the effect of shipping blood in Streck blood collection tubes (BCT) prior to processing on cell-free DNA (cf-DNA) levels.METHODS:Blood was collected in ethylenediaminetetraacetic acid (EDTA) and BCT tubes from 10 pregnant women carrying male fetuses. One set of each tube for each subject was processed to plasma immediately (standard cf-DNA protocol), whereas the other set was shipped by air courier and then processed. DNA was extracted and total and fetal DNA concentrations were measured by TaqMan multiplexed quantitative real-time PCR.RESULTS:No significant difference was observed in total cf-DNA in plasma between immediately processed EDTA (control) and immediately processed BCT samples. Moreover, no significant change in total cf-DNA was detected in plasma of BCT samples shipped at room temperature. Significant differences in total cf-DNA leading to a significantly decreased fetal fraction were found in shipped EDTA samples and BCT samples shipped at 4°C.DISCUSSION:BCT tubes are suitable for shipping whole blood prior to processing with respect to cf-DNA levels. However, care should be taken to ensure that samples are not exposed to extreme temperatures during shipment. This finding will benefit the development and clinical application of noninvasive methods of fetal diagnosis that utilize cf-DNA.

ABSTRACTObjectiveTo develop a novel prenatal assay based on selective analysis of cell‐free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18).MethodsTwo hundred ninety‐eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR™). Cell‐free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. Products from 96 separate patients were pooled and sequenced together. A standard Z‐test of chromosomal proportions was used to distinguish aneuploid samples from average‐risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths.ResultsAt the lowest sequencing depth, corresponding to 204 000 sequencing counts per sample, average‐risk cases where distinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410 000 counts per sample substantially improved separation of aneuploid and average‐risk cases. A further increase to 620 000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgun sequencing approaches.ConclusionDigital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment. © 2012 John Wiley & Sons, Ltd.