Chemical reprogramming presents an innovative approach for generating induced pluripotent stem cells (iPSCs), bypassing the genetic instability and safe concern associated with viral vector approach. We describe a novel, efficient chemical method for reprogramming human umbilical cord tissue-derived mesenchymal stem cells (MSCs) into induced pluripotent stem cells (iPSCs). Compared to previous sources like adipose tissue and skin, frozen umbilical cord tissue offers an abundant, non-invasive, long-term storable, and ethically sound cell source. Our findings not only showcase the feasibility and safety of utilizing chemical reprogramming on cells from frozen umbilical cords but also underscore its potential in regenerative medicine, especially for developing safer and more effective therapies for cardiovascular diseases.### Competing Interest StatementThe authors have declared no competing interest.

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease (CHD). We previously demonstrated that genetic variants in the alpha myosin heavy chain (MYH6) gene are significantly associated with HLHS as well as poor outcomes in patients. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carrying an MYH6-R443P head domain variant demonstrated an impaired CM phenotype including dysmorphic sarcomere structure, altered contractility, and upregulated MYH7 expression. Mesenchymal stem cells (MSCs) and their secretome are currently being explored as a potential therapeutic for cardiac injury. In this study, a possible treatment strategy for iPSC-CMs with a MYH6 tail domain variant was examined through investigation of co-culturing umbilical cord tissue derived MSCs from a healthy newborn. iPSC-CMs from an unaffected family member were included as a normal control to compare cellular RNA and protein changes observed in the MYH6-E1584K line. The MYH6-E1584K variant line demonstrated significant upregulation of sarcomere, calcium channel, and inflammation/immune related gene expression in both mRNA and protein levels. Co-culturing with MSCs rescued expression of several genes and was confirmed through label free proteomic analysis. Co-culturing iPSC-CMs with MSCs also significantly improved contraction (contraction maximum displacement and velocity) in MYH6-E1584K iPSC-CMs. Finally, measurements of microRNA, cytokines, and exosomes secreted into cultured media indicated significant changes. This study suggests that MSC secreted factors improve CM expression and function and may elucidate a new mechanistic target for patients with HLHS. AHW/HHI Innovation Pilot Award. This is the full abstract presented at the American Physiology Summit 2024 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.

Cardiovascular calcification can occur in vascular and valvular structures and is commonly associated with calcium deposition and tissue mineralization leading to stiffness and dysfunction. Patients with chronic kidney disease and associated hyperphosphatemia have an elevated risk for coronary artery calcification (CAC) and calcific aortic valve disease (CAVD). However, there is mounting evidence to suggest that the susceptibility and pathobiology of calcification in these two cardiovascular structures may be different, yet clinically they are similarly treated. To better understand diversity in molecular and cellular processes that underlie hyperphosphatemia-induced calcification in vascular and valvular structures, we exposed aortic vascular smooth muscle cells (AVSMCs) and aortic valve interstitial cells (AVICs) to high (2.5 mM) phosphate (Ph) conditions in vitro, and examined cell-specific responses. To further identify hyperphosphatemic-specific responses, parallel studies were performed using osteogenic media (OM) as an alternative calcific stimulus.Consistent with clinical observations made by others, we show that AVSMCs are more susceptible to calcification than AVICs. In addition, bulk RNA-sequencing reveals that AVSMCs and AVICs activate robust ossification-programs in response to high phosphate or OM treatments, however, the signaling pathways, cellular processes and osteogenic-associated markers involved are cell- and treatment-specific. For example, compared to VSMCs, VIC-mediated calcification involves biological processes related to osteo-chondro differentiation and down regulation of 'actin cytoskeleton'-related genes, that are not observed in VSMCs. Furthermore, hyperphosphatemic-induced calcification in AVICs and AVSMCs is independent of P13K signaling, which plays a role in OM-treated cells. Together, this study provides a wealth of information suggesting that the pathogenesis of cardiovascular calcifications is significantly more diverse than previously appreciated.

Background. Elevated total cell-free DNA (TCF) concentration has been associated with critical illness in adults and elevated donor fraction (DF), the ratio of donor specific cell-free DNA to total cell-free DNA present in the recipient's plasma, is associated with rejection following cardiac transplantation. This study investigates relationships between TCF and clinical outcomes after heart transplantation. Methods. A prospective, blinded, observational study of 87 heart transplantation recipients was performed. Samples were collected at transplantation, prior to endomyocardial biopsy, during treatment for rejection, and at hospital readmissions. Longitudinal clinical data were collected and entered into a RedCAP (Vanderbilt University) database. TCF and DF levels were correlated with endomyocardial biopsy and angiography results, as well as clinical outcomes. Logistic regression for modeling and repeated measures analysis using generalized linear modeling was used. The standard receiver operating characteristic curve, hazard ratios, and odds ratios were calculated. Results. There were 257 samples from 87 recipients analyzed. TCF greater than 50 ng/mL were associated with increased mortality (P = .01, area under the curve 0.93, sensitivity 0.44, specificity 0.97) and treatment for infection (P <.005, area under the curve 0.68, sensitivity 0.45, specificity 0.96). Increased DF was not correlated with treatment for infection. DF was associated with rejection and cardiac allograft vasculopathy (P < .001), but TCF was not. Conclusions. TCF elevation predicted death and treatment for infection. DF elevation predicted histopathologic acute rejection and cardiac allograft vasculopathy. Surveillance of TCF and DF levels may inform treatment after heart transplantation. (C) 2021 by The Society of Thoracic Surgeons

Abstract Background Ebstein's anomaly (EA) is a rare congenital heart disease of the tricuspid valve and right ventricle. Patients with EA often manifest with left ventricular noncompaction (LVNC), a cardiomyopathy. Despite implication of cardiac sarcomere genes in some cases, very little is understood regarding the genetic etiology of EA/LVNC. Our study describes a multigenerational family with at least 10 of 17 members affected by EA/LVNC. Methods We performed echocardiography on all family members and conducted exome sequencing of six individuals. After identifying candidate variants using two different bioinformatic strategies, we confirmed segregation with phenotype using Sanger sequencing. We investigated structural implications of candidate variants using protein prediction models. Results Exome sequencing analysis of four affected and two unaffected members identified a novel, rare, and damaging coding variant in the Kelch‐like family member 26 (KLHL26) gene located on chromosome 19 at position 237 of the protein (GRCh37). This variant region was confirmed by Sanger sequencing in the remaining family members. KLHL26 (c.709C > T p.R237C) segregates only with EA/LVNC‐affected individuals (FBAT p < .05). Investigating structural implications of the candidate variant using protein prediction models suggested that the KLHL26 variant disrupts electrostatic interactions when binding to part of the ubiquitin proteasome, specifically Cullin3 (CUL3), a component of E3 ubiquitin ligase. Conclusion In this familial case of EA/LVNC, we have identified a candidate gene variant, KLHL26 (p.R237C), which may have an important role in ubiquitin‐mediated protein degradation during cardiac development.

Lifelong noninvasive rejection monitoring in heart transplant patients is a critical clinical need historically poorly met in adults and unavailable for children and infants. Cell-free DNA (cfDNA) donor-specific fraction (DF), a direct marker of selective donor organ injury, is a promising analytical target. Methodological differences in sample processing and DF determination profoundly affect quality and sensitivity of cfDNA analyses, requiring specialized optimization for low cfDNA levels typical of transplant patients. Using next-generation sequencing, we previously correlated elevated DF with acute cellular and antibody-mediated rejection (ACR and AMR) in pediatric and adult heart transplant patients. However, next-generation sequencing is limited by cost, TAT, and sensitivity, leading us to clinically validate a rapid, highly sensitive, quantitative genotyping test, myTAIHEART®, addressing these limitations. To assure pre-analytical quality and consider interrelated cfDNA measures, plasma preparation was optimized and total cfDNA (TCF) concentration, DNA fragmentation, and DF quantification were validated in parallel for integration into myTAIHEART reporting. Analytical validations employed individual and reconstructed mixtures of human blood-derived genomic DNA (gDNA), cfDNA, and gDNA sheared to apoptotic length. Precision, linearity, and limits of blank/detection/quantification were established for TCF concentration, DNA fragmentation ratio, and DF determinations. For DF, multiplexed high-fidelity amplification followed by quantitative genotyping of 94 SNP targets was applied to 1168 samples to evaluate donor options in staged simulations, demonstrating DF call equivalency with/without donor genotype. Clinical validation studies using 158 matched endomyocardial biopsy-plasma pairs from 76 pediatric and adult heart transplant recipients selected a DF cutoff (0.32%) producing 100% NPV for ≥2R ACR. This supports the assay's conservative intended use of stratifying low versus increased probability of ≥2R ACR. myTAIHEART is clinically validated for heart transplant recipients ≥2 months old and ≥8 days post-transplant, expanding opportunity for noninvasive transplant rejection assessment to infants and children and to all recipients >1 week post-transplant.

Coarctation of the aorta (CoA) is a common congenital cardiovascular (CV) defect characterized by a stenosis of the descending thoracic aorta. Treatment exists, but many patients develop hypertension (HTN). Identifying the cause of HTN is challenging because of patient variability (e.g., age, follow-up duration, severity) and concurrent CV abnormalities. Our objective was to conduct RNA sequencing of aortic tissue from humans with CoA to identify a candidate gene for mechanistic studies of arterial dysfunction in a rabbit model of CoA devoid of the variability seen with humans. We present the first known evidence of natriuretic peptide receptor C ( NPR-C; aka NPR3) downregulation in human aortic sections subjected to high blood pressure (BP) from CoA versus normal BP regions (validated to PCR). These changes in NPR-C, a gene associated with BP and proliferation, were replicated in the rabbit model of CoA. Artery segments from this model were used with human aortic endothelial cells to reveal the functional relevance of altered NPR-C activity. Results showed decreased intracellular calcium ([Ca2+]i) activity to C-type natriuretic peptide (CNP). Normal relaxation induced by CNP and atrial natriuretic peptide was impaired for aortic segments exposed to elevated BP from CoA. Inhibition of NPR-C (M372049) also impaired aortic relaxation and [Ca2+]i activity. Genotyping of NPR-C variants predicted to be damaging revealed that rs146301345 was enriched in our CoA patients, but sample size limited association with HTN. These results may ultimately be used to tailor treatment for CoA based on mechanical stimuli, genotyping, and/or changes in arterial function.

There is a compelling clinical need for a noninvasive alternative to endomyocardial biopsy (EMB) for the surveillance of rejection in heart transplant recipients. We and others have reported on donor cell-free deoxyribonucleic acid (cfDNA), which is shed from the donor allograft and is elevated

There is a compelling clinical need for a noninvasive alternative to endomyocardial biopsy (EMB) for the surveillance of rejection in heart transplant recipients. We and others have reported on donor cell-free deoxyribonucleic acid (cfDNA), which is shed from the donor allograft and is elevated

Donor fraction cell free DNA (DF cfDNA) was investigated as a marker for cardiac injury in cardiac transplant recipients. We hypothesized that injury from routine biopsy (bx) would produce a detectable rise in circulating DF cfDNA.

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

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. The goal of this study was to characterize a risk gene in HLHS and its effect on HLHS etiology and outcome. We performed next-generation sequencing on a multigenerational family with a high prevalence of CHD/HLHS, identifying a rare variant in the α-myosin heavy chain ( MYH6) gene. A case-control study of 190 unrelated HLHS subjects was then performed and compared with the 1000 Genomes Project. Damaging MYH6 variants, including novel, missense, in-frame deletion, premature stop, de novo, and compound heterozygous variants, were significantly enriched in HLHS cases ( P < 1 × 10−5). Clinical outcomes analysis showed reduced transplant-free survival in HLHS subjects with damaging MYH6 variants ( P < 1 × 10−2). Transcriptome and protein expression analyses with cardiac tissue revealed differential expression of cardiac contractility genes, notably upregulation of the β-myosin heavy chain ( MYH7) gene in subjects with MYH6 variants ( P < 1 × 10−3). We subsequently used patient-specific induced pluripotent stem cells (iPSCs) to model HLHS in vitro. Early stages of in vitro cardiomyogenesis in iPSCs derived from two unrelated HLHS families mimicked the increased expression of MYH7 observed in vivo ( P < 1 × 10−2), while revealing defective cardiomyogenic differentiation. Rare, damaging variants in MYH6 are enriched in HLHS, affect molecular expression of contractility genes, and are predictive of poor outcome. These findings indicate that the etiology of MYH6-associated HLHS can be informed using iPSCs and suggest utility in future clinical applications.

The use of human pluripotent cell progeny for cardiac disease modeling, drug testing and therapeutics requires the ability to efficiently induce pluripotent cells into the cardiomyo-genic lineage. Although direct activation of the Activin-A and/or Bmp pathways with growth factors yields context-dependent success, recent studies have shown that induction of Wnt signaling using low molecular weight molecules such as CHIR, which in turn induces the Activin-A and Bmp pathways, is widely effective. To further enhance the reproducibility of CHIR-induced cardiomyogenesis, and to ultimately promote myocyte maturation, we are using exogenous growth factors to optimize cardiomyogenic signaling downstream of CHIR induction. As indicated by RNA-seq, induction with CHIR during Day 1 (Days 0-1) was followed by immediate expression of Nodal ligands and receptors, followed later by Bmp ligands and receptors. Co-induction with CHIR and high levels of the Nodal mimetic ActivinA (50-100 ng/ml) during Day 0-1 efficiently induced definitive endoderm, whereas CHIR supplemented with Activin-A at low levels (10 ng/ml) consistently improved cardiomyogenic efficiency, even when CHIR alone was ineffective. Moreover, co-induction using CHIR and low levels of Activin-A apparently increased the rate of cardiomyogenesis, as indicated by the initial appearance of rhythmically beating cells by Day 6 instead of Day 8. By contrast, co-induction with CHIR plus low levels (3-10 ng/ml) of Bmp4 during Day 0-1 consistently and strongly inhibited cardiomyogenesis. These findings, which demonstrate that cardiomyogenic efficacy is improved by optimizing levels of CHIR-induced growth factors when applied in accord with their sequence of endogenous expression, are consistent with the idea that Nodal (Activin-A) levels toggle the entry of cells into the endodermal or mesodermal lineages, while Bmp levels regulate subsequent allocation into mesodermal cell types.

Postsurgical bleeding causes significant morbidity and mortality in children undergoing surgery for congenital heart defects (CHD). 22q11.2 deletion syndrome (DS) is the second most common genetic risk factor for CHD. The deleted segment of chromosome 22q11.2 encompasses the gene encoding glycoprotein (GP) Ibβ, which is required for expression of the GPIb-V-IX complex on the platelet surface, where it functions as the receptor for von Willebrand factor (VWF). Binding of GPIb-V-IX to VWF is important for platelets to initiate hemostasis. It is not known whether hemizygosity for the gene encoding GPIbβ increases the risk for bleeding following cardiac surgery for patients with 22q11.2 DS. We performed a case–control study of 91 pediatric patients who underwent cardiac surgery with cardiopulmonary bypass from 2004 to 2012 at Children’s Hospital of Wisconsin. Patients with 22q11.2 DS had larger platelets and lower platelet counts, bled more excessively, and received more transfusion support with packed red blood cells in the early postoperative period relative to control patients. Presurgical genetic testing for 22q11.2 DS may help to identify a subset of pediatric cardiac surgery patients who are at increased risk for excessive bleeding and who may require more transfusion support in the postoperative period.

The clinical significance of copy number variants (CNVs) in congenital heart disease (CHD) continues to be a challenge. Although CNVs including genes can confer disease risk, relationships between gene dosage and phenotype are still being defined. Our goal was to perform a quantitative analysis of CNVs involving 100 well-defined CHD risk genes identified through previously published human association studies in subjects with anatomically defined cardiac malformations. A novel analytical approach permitting CNV gene frequency "spectra" to be computed over prespecified regions to determine phenotype-gene dosage relationships was employed. CNVs in subjects with CHD (n = 945), subphenotyped into 40 groups and verified in accordance with the European Paediatric Cardiac Code, were compared with two control groups, a disease-free cohort (n = 2,026) and a population with coronary artery disease (n = 880). Gains (≥200 kb) and losses (≥100 kb) were determined over 100 CHD risk genes and compared using a Barnard exact test. Six subphenotypes showed significant enrichment (P ≤ 0.05), including aortic stenosis (valvar), atrioventricular canal (partial), atrioventricular septal defect with tetralogy of Fallot, subaortic stenosis, tetralogy of Fallot, and truncus arteriosus. Furthermore, CNV gene frequency spectra were enriched (P ≤ 0.05) for losses at: FKBP6, ELN, GTF2IRD1, GATA4, CRKL, TBX1, ATRX, GPC3, BCOR, ZIC3, FLNA and MID1; and gains at: PRKAB2, FMO5, CHD1L, BCL9, ACP6, GJA5, HRAS, GATA6 and RUNX1. Of CHD subjects, 14% had causal chromosomal abnormalities, and 4.3% had likely causal (significantly enriched), large, rare CNVs. CNV frequency spectra combined with precision phenotyping may lead to increased molecular understanding of etiologic pathways.

Aim: To visualize copy number associations and quantify relative risk of Copy Number Variants (CNVs) in 100 well-defined congenital heart disease (CHD) risk genes identified from literature sources. Method: CNVs in subjects with congenital heart malformations (n=741) and phenotyped in accordance with the European Paediatric Cardiac Code were compared to the frequencies of CNVs present in a control population of subjects with coronary artery disease (n=880). CNV gains (≥ 200 kb) and losses (≥ 100 kb) were determined using the Affymetrix 6.0 Gene Chip and confirmed with TaqMan copy number assays. In addition, each gene was tested using a two proportions z-test approach to determine if the gene frequency was significantly different in CHD vs control. To quantify the effect of observed CNVs in the CHD population, we collapsed the set of rare individual CNVs into a single group to test the collective frequency difference between cases and controls by adapting a previously described Cohort Allelic Sums Test (CAST). Results: The following phenotypes showed a significant enrichment of gains and losses in the CHD population: Aortic Stenosis, Coarctation of the Aorta, Ebstein's Anomaly, Tetralogy of Fallot, Truncus Arteriosus and Ventricular Septal Defect. The CNV percentage was increased in CHD subjects in the following genes (p<0.05 with a Bonferroni correction): Losses: FKBP6(-), ELN(s), GTF2IRD1(-), SOX7(-), GATA4(ns), NOTCH1(ns), MYH11(ns), SALL4(s), TBX1(s), CRKL(-), MAPK1(-); Gains: PRKAB2(-), FMO5(-), CHD1L(-), BCL9(-), ACP6(-), GJA5(ns), NPHP3(s), FOXL2(s), SEMA5A(-), NSD1(s), HOXA1(s), TBX20(ns), SOX7(-), GATA4(ns), HRAS(s), GATA6(ns), RUNX1(s), MAPK1(-), BCOR(s), ATRX(s), GPC3(s), ZIC3(ns), FLNA(s) where s is syndromic, ns is non-syndromic and (-) is not available on the CHD WIKI website ( http://www.chearted.eu ). The approach rapidly identified common, cytogenetically visible abnormalities (i.e. TBX1 in 22q11.2 deletion syndrome, n=30, and RUNX1 in Trisomy 21, n=66). Conclusion: This is the first study to establish a copy number spectrum in a CHD population. We conclude that the CAST approach shows promise for evaluating qualitative outcomes and statistical precision of prior studies which have focused on CNVs in CHD and other diseases.

RATIONALE: DiGeorge Syndrome (DGS1), chromosome 22q11.2 deletion syndrome, affects 1:4000 live births. Early diagnosis and medical intervention can prevent and treat many of the co-morbidities and mortalities associated with DGS1. The gold standard for detection is fluorescence in situ hybridization (FISH) with a probe at the TUPLE1 gene. However, FISH has a high false negative rate (15%), is costly, and requires several days to perform. As a result, FISH is underutilized and diagnosis is delayed (6 to 7 years of age). The goal of this study was to determine if multiplexed, quantitative, real-time PCR (MQPCR), a rapid and inexpensive assay, could be used to detect DGS1.METHODS: A blinded study was performed on 684 punches using DNA extracted from anonymous newborn screening cards (Guthrie cards) from 628 individuals. 59 punches came from three DGS1 subjects who had a FISH test indicating deletion. The MQPCR approach enabled the simultaneous use of a reference probe (chromosome 19) to compare with a probe of interest near the TBX1 gene on chromosome 22. An average relative gene copy number (rGCN) was calculated per plate with a threshold of 1.4 SDs to determine normal vs DGS1.RESULTS: 600 punches were identified as normal and 84 punches were identified as DGS1. The sensitivity and specificity of the assay for DGS1 was 100% and 96.04%, respectively.CONCLUSIONS: In summary, the MQPCR assay is a rapid (<4 hrs), inexpensive (< $6 per patient), and sensitive assay to diagnose DGS1. The assay has potential for population-based newborn screening. RATIONALE: DiGeorge Syndrome (DGS1), chromosome 22q11.2 deletion syndrome, affects 1:4000 live births. Early diagnosis and medical intervention can prevent and treat many of the co-morbidities and mortalities associated with DGS1. The gold standard for detection is fluorescence in situ hybridization (FISH) with a probe at the TUPLE1 gene. However, FISH has a high false negative rate (15%), is costly, and requires several days to perform. As a result, FISH is underutilized and diagnosis is delayed (6 to 7 years of age). The goal of this study was to determine if multiplexed, quantitative, real-time PCR (MQPCR), a rapid and inexpensive assay, could be used to detect DGS1. METHODS: A blinded study was performed on 684 punches using DNA extracted from anonymous newborn screening cards (Guthrie cards) from 628 individuals. 59 punches came from three DGS1 subjects who had a FISH test indicating deletion. The MQPCR approach enabled the simultaneous use of a reference probe (chromosome 19) to compare with a probe of interest near the TBX1 gene on chromosome 22. An average relative gene copy number (rGCN) was calculated per plate with a threshold of 1.4 SDs to determine normal vs DGS1. RESULTS: 600 punches were identified as normal and 84 punches were identified as DGS1. The sensitivity and specificity of the assay for DGS1 was 100% and 96.04%, respectively. CONCLUSIONS: In summary, the MQPCR assay is a rapid (<4 hrs), inexpensive (< $6 per patient), and sensitive assay to diagnose DGS1. The assay has potential for population-based newborn screening.

22q11.2 Deletion syndrome (22q11.2 DS) [DiGeorge syndrome type 1 (DGS1)] occurs in ∼1:3,000 live births; 75% of children with DGS1 have severe congenital heart disease requiring early intervention. The gold standard for detection of DGS1 is fluorescence in situ hybridization (FISH) with a probe at the TUPLE1 gene. However, FISH is costly and is typically ordered in conjunction with a karyotype analysis that takes several days. Therefore, FISH is underutilized and the diagnosis of 22q11.2 DS is frequently delayed, often resulting in profound clinical consequences. Our goal was to determine whether multiplexed, quantitative real-time PCR (MQPCR) could be used to detect the haploinsufficiency characteristic of 22q11.2 DS. A retrospective blinded study was performed on 382 subjects who had undergone congenital heart surgery. MQPCR was performed with a probe localized to the TBX1 gene on human chromosome 22, a gene typically deleted in 22q11.2 DS. Cycle threshold (C(t)) was used to calculate the relative gene copy number (rGCN). Confirmation analysis was performed with the Affymetrix 6.0 Genome-Wide SNP Array. With MQPCR, 361 subjects were identified as nondeleted with an rGCN near 1.0 and 21 subjects were identified as deleted with an rGCN near 0.5, indicative of a hemizygous deletion. The sensitivity (21/21) and specificity (361/361) of MQPCR to detect 22q11.2 deletions was 100% at an rGCN value drawn at 0.7. One of 21 subjects with a prior clinical (not genetically confirmed) DGS1 diagnosis was found not to carry the deletion, while another subject, not previously identified as DGS1, was detected as deleted and subsequently confirmed via microarray. The MQPCR assay is a rapid, inexpensive, sensitive, and specific assay that can be used to screen for 22q11.2 deletion syndrome. The assay is readily adaptable to high throughput.