Lung allografts are susceptible to myriad injury types, including acute rejection (AR), infectious disease (ID), baseline lung allograft dysfunction (BLAD), and chronic lung allograft dysfunction (CLAD), that affect outcomes. Donor-derived cell-free DNA (dd-cfDNA) is validated for detecting AR after lung transplantation (LT). However, data are limited regarding the ability of dd-cfDNA or total cell-free DNA (TcfDNA) to detect or differentiate other clinical conditions. This study stratified patients into Stable, AR, CLAD, and ID cohorts. Stable double LT recipients were further stratified into BLAD and non-BLAD over different periods posttransplantation. dd-cfDNA and TcfDNA results were associated with the various cohorts. cfDNA was measured in 354 plasma samples from 66 LT recipients. Median dd-cfDNA was elevated in AR (2.08%; P = 0.014) and ID (1.19%; P = 0.065) versus Stable (0.60%) but not CLAD; TcfDNA was only elevated in the ID cohort (P = 0.0078). dd-cfDNA was analyzed before and after treatment of eight episodes of AR, during which the median dd-cfDNA fraction decreased from 2.41% to 0.80% (P = 0.004). No differences were observed during early time points for BLAD versus non-BLAD, whereas median TcfDNA, but not dd-cfDNA, was elevated for BLAD beyond 12 months (13,843 vs 7768 cp/mL; P = 0.005). Overall, this was the first study that explored dd-cfDNA and TcfDNA levels across AR, ID, BLAD, and CLAD cohorts. These data suggest that cfDNA-based biomarkers have value in assessing allograft dysfunction beyond AR.

Congenital heart disease (CHD) occurs in over half of individuals with 22q11.2 deletion syndrome (22q11.2DS), but lesion type varies widely. We analyzed 3,016 unrelated postnatal individuals with 22q11.2DS from specialized centers in the United States, Canada, Europe, South America, Israel, and Australia, including 1,868 with whole-genome sequencing. The typical 3 Mb LCR22 A-D deletion was present in 2,788 individuals (92.4%), with smaller A-B (n=172, 5.7%) and A-C (n=56, 1.9%) deletions comprising the remaining cohort. In multivariable mixed-effects logistic regression adjusting for sex, deletion group, genome-wide principal components (PCs), and recruitment site, four non-intercept associations, to test relationships, met study-wide FDR statistical significance. Compared with the A-B deletion, the A-D deletion was associated with lower odds of persistent truncus arteriosus (OR=0.37, 95% CI 0.18-0.75) and higher odds of isolated septal defects (OR=4.68, 95% CI 1.71-12.83), although precision was limited by the smaller A-B group. PC2 was associated with lower odds of pulmonary stenosis/atresia with additional lesions (OR=0.73, 95% CI 0.61-0.87), and PC4 with higher odds of abnormal origin of the subclavian arteries (OR=2.59, 95% CI 1.37-4.89). ADMIXTURE-derived continental ancestry proportions did not show independent associations with these two PC-associated outcomes. These lesion-specific findings suggest the hypothesis that deletion interval and broader genetic background may contribute to CHD variability in 22q11.2DS, pending future replication. KEY MESSAGES:What is already known on this topic: Chromosome 22q11.2DS is a rare genetic disorder associated with serious medical challenges. Most affected individuals have congenital heart disease but with variable phenotypic expression. One of the main questions still an open topic in the field is why individuals with 22q11.2DS show extensive phenotypic heterogeneity.What this study adds: Analysis of retrospective data on 3,016 individuals with 22q11.2DS and 1,868 with sequence data suggest that deletion type and genetic variation influence phenotypic expression of congenital heart disease.How this study might affect research, practice or policy: This work informs the clinician as to the importance of testing for deletion type and obtaining accurate cardiac phenotypes to diagnose 22q11.2DS that will improve the prognosis and disease progression. It also implicates the complexity of sex and ancestry as confounding factors that will help guide future research studies to identify genetic modifiers of congenital heart disease.

Exposure to diagnostic and therapeutic radiation introduces risks for development of diseases later in life by causing DNA damage in cells. Currently, there is no clinical method for determining exposure risk caused by radiation toxicity to DNA. Cell-free DNA (cfDNA), a marker of DNA damage, is currently used to assess risk for long-term effects following organ transplantation, surgery and inflammation. The goal of our proposed study is to develop cfDNA as an early biomarker for assessing risk for cardiovascular disease and cancer from radiation exposure so that strategies to mitigate the damaging effects of medical radiation can be assessed. Hearts from male and female WAG/RijCmcr rats (n = 6-10/group) were exposed to increasing doses of X-radiation (50 mGy and 3.5 Gy). Blood was collected prior to and after (15 minutes-96 hours) irradiation, and cell-free plasma was prepared. Primers and probes were designed for quantitative analysis of sequences of mitochondria (12S rRNA) and nuclear (Gapdh) cfDNA present in rat plasma using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Exposure of hearts to radiation increased nuclear and mitochondrial cfDNA in a dose-dependent manner. Three point five grays from X-radiation increase cfDNA for Gapdh in plasma after 1 hour with a 15.8-fold increase (P < 0.001) after 6 hours. The earliest time nuclear and mitochondrial cfDNA increases were detected in plasma was at 60 minutes following exposure to 3.5 Gy. cfDNA has potential to advance as a biomarker of exposure to medical doses of radiation in patients.

Background: MYH6 variants are the most well-known genetic risk factor (10%) for hypoplastic left heart syndrome (HLHS) and are associated with decreased cardiac transplant-free survival. MYH6 encodes for α-myosin heavy chain (α-MHC), a contractile protein expressed in the neonatal atria. We therefore assessed atrial function in HLHS patients with MYH6 variants. Methods: We performed a retrospective, blinded assessment of pre-stage I atrial function using 2D speckle-tracking echocardiography (2D-STE). Variant carriers were control-matched based on AV valve anatomy, sex, and birth year. Studies were obtained postnatally from awake patients prior to surgical intervention. Right atrial (RA) and right ventricular (RV) strain and strain rate (SR) were measured from the apical four-chamber view. Results: A total of 19 HLHS patients with MYH6 variants had echocardiograms available; 18 were matched to two controls each, and one had a single control. RA active strain (ASct) was decreased in variant carriers (−1.41%, IQR −2.13, −0.25) vs. controls (−3.53%, IQR −5.53, −1.28; p = 0.008). No significant differences were identified in RV strain between the groups. RA reservoir strain (ASr) and conduit strain (AScd) positively correlated with heart rate (HR) in MYH6 variant carriers only (ASr R = 0.499, p = 0.029; AScd R = 0.469, p = 0.043). RV global longitudinal strain (GLS) as well as RV systolic strain (VSs) and strain rate (VSRs) correlated with HR in controls only (GLS R = 0.325, p = 0.050; VSs R = 0.419, p = 0.010; VSRs R = 0.410, p = 0.012). Conclusions: We identified functional consequences associated with MYH6 variants, a known risk factor for poor outcomes in HLHS. MYH6 variant carriers exhibit impaired RA contractility despite there being no differences in RV function between variant carriers and controls. MYH6 variants are also associated with an ineffective RA reservoir and conduit function at high heart rates, despite preserved RV diastolic function. RA dysfunction and reduced atrial “kick” may therefore be a significant contributor to RV failure and worse clinical outcomes in HLHS patients with MYH6 variants.

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.

BACKGROUND:The aims of the study were to assess the performance of a clinically available cell-free DNA (cfDNA) assay in a large cohort of pediatric and adult heart transplant recipients and to evaluate performance at specific cut points in detection of rejection. METHODS:Observational, non-interventional, prospective study enrolled pediatric and adult heart transplant recipients from seven centers. Biopsy-associated plasma samples were used for cfDNA measurements. Pre-determined cut points were tested for analytic performance. RESULTS:A total of 487 samples from 160 subjects were used for the analysis. There were significant differences for df-cfDNA values between rejection [0.21% (IQR 0.12-0.69)] and healthy samples [0.05% (IQR 0.01-0.14), p < .0001]. The pediatric rejection group had a median df-cfDNA value of 0.93% (IQR 0.28-2.84) compared to 0.09% (IQR 0.04-0.23) for healthy samples, p = .005. Overall negative predictive value was 0.94 while it was 0.99 for pediatric patients. Cut points of 0.13% and 0.15% were tested for various types of rejection profiles and were appropriate to rule out rejection. CONCLUSION:The study suggests that pediatric patients with rejection show higher levels of circulating df-cfDNA compared to adults and supports the specific cut points for clinical use in pediatric and adult patients with overall acceptable performance.

We demonstrate here that iPSCs derived from patients with Ebstein’s anomaly and left ventricular noncompaction, when differentiated into cardiomyocytes, display significant structural and functional changes that offer insight into disease pathogenesis, including altered ER/SR and mitochondrial morphology, contractility, and calcium signaling.

BACKGROUND:Preoperative risk stratification in cardiac surgery includes patient and procedure factors that are used in clinical decision-making. Despite these tools, unidentified factors contribute to variation in outcomes. Identification of latent physiologic risk factors may strengthen predictive models. Nuclear cell-free DNA (ncfDNA) increases with tissue injury and drops to baseline levels rapidly. The goal of this investigation is to measure and to observe ncfDNA kinetics in children undergoing heart operations with cardiopulmonary bypass (CPB), linking biomarkers, organ dysfunction, and outcomes. METHODS:This is a prospective observational study of 116 children <18 years and >3 kg undergoing operations with CPB. Plasma ncfDNA samples were collected and processed in a stepwise manner at predefined perioperative time points. The primary outcome measure was occurrence of postoperative cardiac arrest or extracorporeal membrane oxygenation. RESULTS:Data were available in 116 patients (median age, 0.9 years [range, 0-17.4 years]; median weight, 7.8 kg [range, 3.2-98 kg]). The primary outcome was met in 6 of 116 (5.2%). Risk of primary outcome was 2% with ncfDNA <20 ng/mL and 33% with ncfDNA >20 ng/mL (odds ratio, 25; CI, 3.96-158; P = .001). Elevated ncfDNA was associated with fewer hospital-free days (P < .01). CONCLUSIONS:This study analyzes ncfDNA kinetics in children undergoing operations with CPB for congenital heart disease. Elevated preoperative ncfDNA is strongly associated with postoperative arrest and extracorporeal membrane oxygenation. Further studies are needed to validate this technology as a tool to predict morbidity in children after cardiac surgical procedures.

Severe right ventricular outflow tract (RVOT) obstruction developed in a 4-year-old boy with Schuurs-Hoeijmakers syndrome and history of double-outlet right ventricle, tetralogy of Fallot type, status post repair with transannular patch augmentation of the RVOT. Echocardiography and computed tomography defined the presence of a 1 × 1-cm sinus of Valsalva aneurysm protruding into the RVOT, causing obstruction. Resection and repair of the aneurysm by a 2-patch technique as well as resection of RVOT muscle bundles and revision of the transannular patch were performed. The postoperative course was uneventful, with no evidence of persistent RVOT obstruction.

Hypoplastic Left Heart Syndrome (HLHS) is a rare and complex congenital heart defect characterized by hypoplasia of the left ventricle, proximal aorta, as well as stenosis or atresia of the mitral and/or aortic valves. There is strong evidence for a genetic contribution to HLHS, including our lab’s previous discovery of 19 rare, predicted-damaging MYH6 variants that were significantly enriched in HLHS patients. The MYH6 gene encodes for the α-myosin heavy chain (α-MHC), a key contractile protein in cardiac sarcomere. Variants affecting cardiac MHC isoforms are thought to impact the strength, speed, and patterns of myocardial contractions. Reduced myocardial contractility due to MYH6 variants may impair cardiac blood flow and cause underdevelopment of the left heart. Mavacamten is a novel pharmacological agent that has shown promise in phase III clinical trials for treatment of hypertrophic obstructive cardiomyopathy. Mavacamten selectively acts on cardiac MHC to reduce cardiac muscle contractility. Given this specificity, Mavacamten may offer a promising way to treat diastolic dysfunction in (i.e. hypercontractility) in patients with HLHS, including those with MYH6 variants. We tested the in vitro efficacy of Mavacamten in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) exhibiting hypercontractility and investigated video-based analysis as a high-throughput method for evaluating iPSC-CM contractility, which would allow for repeated assessment of cells at different timepoints following drug treatment. Many packages for video-based contractility assessment lack precision and can be subject to disturbances such as cellular fragments, movement of cell media, and small differences in media color. They are also complex and require advanced programming knowledge. MYOCYTER is an open-source macro for ImageJ that improves upon previous plugins available by allowing for users to optimize parameters for contractility evaluation, while minimizing background noise. The aim of our study was to determine if MYOCYTER can be reliably used to detect small changes in iPSC-CM contractility following treatment with Mavacamten, and if the workflow is user-friendly enough such that reproducible analysis can be done by a high school science team.Our analysis revealed that MYOCYTER can detect baseline hypercontractility of iPSC-CMs carrying MYH6 variants compared to wild-type iPSC-CMs (p = 0.033). These differences in amplitude were eliminated following five, ten, and fifteen minutes of treatment with 500nM Mavacamten in a single experiment. Our results suggest that this method can be used to further evaluate the effect of myosin modulators, such as Mavacamten, in patient-specific iPSC-CMs. Children's Research Institute 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:Congenital heart disease is the leading cause of death related to birth defects and affects 1 out of every 100 live births. Induced pluripotent stem cell technology has allowed for patient-derived cardiomyocytes to be studied in vitro. An approach to bioengineer these cells into a physiologically accurate cardiac tissue model is needed in order to study the disease and evaluate potential treatment strategies.Methods:To accomplish this, we have developed a protocol to 3D-bioprint cardiac tissue constructs comprised of patient-derived cardiomyocytes within a hydrogel bioink based on laminin-521.Results:Cardiomyocytes remained viable and demonstrated appropriate phenotype and function including spontaneous contraction. Contraction remained consistent during 30 days of culture based on displacement measurements. Furthermore, tissue constructs demonstrated progressive maturation based on sarcomere structure and gene expression analysis. Gene expression analysis also revealed enhanced maturation in 3D constructs compared to 2D cell culture.Discussion:This combination of patient-derived cardiomyocytes and 3D-bioprinting represents a promising platform for studying congenital heart disease and evaluating individualized treatment strategies.

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.

Objectives: Donor-specific cell-free DNA shows promise as a noninvasive marker for allograft rejection, but as yet has not been validated in both adult and pediatric recipients. The study objective was to validate donor fraction cell- free DNA as a noninvasive test to assess for risk of acute cellular rejection and antibodymediated rejection after heart transplantation in pediatric and adult recipients. Methods: Pediatric and adult heart transplant recipients were enrolled from 7 participating sites and followed for 12 months or more with plasma samples collected immediately before all endomyocardial biopsies. Donor fraction cellfree DNA was extracted, and quantitative genotyping was performed. Blinded donor fraction cell-free DNA and clinical data were analyzed and compared with a previously determined threshold of 0.14%. Sensitivity, specificity, negative predictive value, positive predictive value, and receiver operating characteristic curves were calculated. Results: A total of 987 samples from 144 subjects were collected. After applying predefined clinical and technical exclusions, 745 samples from 130 subjects produced 54 rejection samples associated with the composite outcome of acute cellular rejection grade 2R or greater and pathologic antibody- mediated rejection 2 or greater and 323 healthy samples. For all participants, donor fraction cell-free DNA at a threshold of 0.14% had a sensitivity of 67%, a specificity of 79%, a positive predictive value of 34%, and a negative predictive value of 94% with an area under the curve of 0.78 for detecting rejection. When analyzed independently, these results held true for both pediatric and adult cohorts at the same threshold of 0.14% (negative predictive value 92% and 95%, respectively). Conclusions: Donor fraction cell-free DNA at a threshold of 0.14% can be used to assess for risk of rejection after heart transplantation in both pediatric and adult patients with excellent negative predictive value. (J Thorac Cardiovasc Surg 2023;165:460-8)

Objectives: Mortality rates following pediatric cardiac surgery with cardiopulmonary bypass have declined over decades, but have plateaued in recent years. This is in part attributable to persistent issues with postoperative global inflammation and myocardial dysfunction, commonly manifested by systemic inflammatory response syndrome and low cardiac output syndrome, respectively. Quantified cell-free DNA (cfDNA), of nuclear or mitochondrial origin, has emerged as a biomarker for both inflammation and myocardial injury. Recent data suggest that nuclear cfDNA (ncfDNA) may quantify inflammation, whereas mitochondrial cfDNA (mcfDNA) may correlate with the degree of myocardial injury. We hypothesize that threshold levels of ncfDNA and mcfDNA can be established that are sensitive and specific for postoperative mortality mediated through independent pathways, and that association will be enhanced with combined analysis. Methods: Prospective observational study of infants younger than age 1 year undergoing planned surgery with cardiopulmonary bypass. The study received institutional review board approval. Samples were drawn before skin incision, immediately after completion of cardiopulmonary bypass, and subsequently at predetermined intervals postoperatively. Association of early postoperative ncfDNA and mcfDNA levels with mortality were assessed by logistic regression with cut-points chosen by receiving operating characteristic curve exploration. Results: Data were available in 59 patients. Median age and weight were 122 days (interquartile range, 63-154 days) and 4.9 kg (interquartile range, 3.9-6.2 kg). Median STAT category was 3 (interquartile range, 1-4). The primary outcome of death was met in 3 out of 59 (5%). Combined analysis of ncfDNA and mcfDNA levels at 12 hours after the initiation of cardiopulmonary bypass with death at a threshold of 50 ng/mL ncfDNA and 17 copies/mu L mcfDNA yielded 100% sensitivity and negative predictive value. The specificity (91%) and positive predictive value (38%) increased through combined analysis compared with univariate analysis. Combined analysis exhibited high specificity (93%) and negative predictive value (78%) for prolonged (>30 postoperative days) hospitalization. Conclusions: Combined analysis of early postoperative ncfDNA and mcfDNA can stratify risk of mortality and prolonged hospitalization following infant cardiac surgery. Evaluation of both ncfDNA and mcfDNA to identify states of generalized inflammation and myocardial injury may allow for targeted interventions and improved outcomes.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with complex genetic inheritance. HLHS segregates with other left ventricular outflow tract (LVOT) malformations in families, and can present as either an isolated phenotype or as a feature of a larger genetic disorder. The multifactorial etiology of HLHS makes it difficult to interpret the clinical significance of genetic variants. Specific genes have been implicated in HLHS, including rare, predicted damaging MYH6 variants that are present in >10% of HLHS patients, and which have been shown to be associated with decreased transplant-free survival in our previous studies. MYH6 (α-myosin heavy chain, α-MHC) variants have been reported in HLHS and numerous other CHDs, including LVOT malformations, and may provide a genetic link to these disorders. In this paper, we outline the MYH6 variants that have been identified, discuss how bioinformatic and functional studies can inform clinical decision making, and highlight the importance of genetic testing in HLHS.

A cDNA clone encoding grapevine (Vitis vinifera L. cv Sultanina) NAD(H)-glutamate dehydrogenase (GDH) was isolated from a cDNA expression library by immunoscreening with a polyclonal antibody raised against grapevine GDH. Nucleotide sequence analysis revealed an open reading frame (ORF) encoding a precursor protein of 411 amino acids (aa) with a calculated molecular mass of 44.517 kDa. The deduced aa sequence showed relatively higher homology to GDH from archaebacteria species, than to those from eukaryotes and eubacteria. This resemblance indicated a functional and/or evolutionary relationship in this class of enzymes which might be relevant to the stress-related function of plant GDH. We have shown that the bacterially produced plant GDH was thermostable.