Hypertrophic cardiomyopathy (HCM) is the most common monogenic inherited heart disease and is a major cause of sudden death in individuals under 35 years of age. HCM is associated with progressive tissue-level disarray and subcellular disorganization in individual cardiomyocytes. Mutations in β-cardiac myosin (MYH7), the second most common genetic cause of HCM, commonly result in changes in sarcomeric force production, but how this leads to altered cell- and tissue-level organization is unclear. Here, we use cryo-electron tomography (cryo-ET) to bridge the molecular and cellular scales by visualizing the nanoscale organization of individual myosin-containing thick filaments within sarcomeres of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Compared to isogenic wild-type controls, hiPSC-CMs expressing P710R MYH7 exhibit pronounced disruption of the hexagonal packing of thick filaments within individual sarcomeres, which would be difficult to visualize using conventional light microscopy or even room-temperature electron microscopy. We also observe ribosome infiltration into areas of sarcomeric disorder for both wild-type and P710R MYH7 hiPSC-CMs, suggesting that disordered regions may be sites of local proteostasis or remodeling. Together, these data illuminate how altered myosin activity can propagate to yield dramatic changes in sarcomeric organization in HCM.

ABSTRACT BACKGROUND AND PURPOSE β-adrenergic receptors (AR) regulate both cardiac function and remodeling. Many studies suggest that, in addition to their effects on heart rate and contractility, β 1 -ARs mediate cardiotoxic signaling, whereas β 2 -ARs are generally cardioprotective. However, there is conflicting data on the role of β 2 -ARs, differing dependent on the nature of the stress. Given the extremely common use of β-blockers and agonists clinically, we sought to understand the differential cardioprotective/cardiotoxic effects of β 2 -AR signaling dependent on timing (acute vs. chronic) and type of cardiotoxic stress. EXPERIMENTAL APPROACH Wild-type (WT) and β-AR knockout (β 1 -KO and β 2 -KO) mice were subjected to acute (15 mg·kg -1 x 1 dose) or chronic (2 mg·kg -1 ·wk -1 x 7 wks) oxidative stress using doxorubicin (DOX). Survival, cardiac function and histopathology were assessed and differential signaling activation determined by Western blot and gene expression by RNA-seq. KEY RESULTS We have shown that β 2 -KOs manifest extreme cardiotoxicity with acute DOX (100% mortality within 30 min), supporting a strong cardioprotective role of β 2 -signaling. In marked contrast, with chronic DOX, β 2 -KO had enhanced survival (t½ 54 d vs. 42 d in WT) and attenuated cardiac dysfunction. In β 2 -KO, acute DOX activated stress MAPKs (p38, ERK and JNK), whereas chronic DOX did not; furthermore, in the absence of β2-ARs, oxidative stress and lipid accumulation were reduced, genes regulating compensatory metabolic pathways (AMPK and insulin/PI3K) were upregulated, and genes regulating mitochondrial and contractile function were preserved, whereas they were downregulated in WT with chronic DOX. CONCLUSIONS β 2 -AR signaling switches from being cardioprotective during acute oxidative stress, to cardiotoxic during chronic stress. Inhibition of β 2 -AR signaling during chronic stress induces signaling and metabolic compensations that serve to reduce oxidative injury. This unexpected temporal switching has potential significant implications for all models of cardiovascular disease, as well as for the clinical use of subtype-specific β-blockers. CLINICAL PERSPECTIVE What is new? Our finding that β2-adrenergic receptor signaling can switch from being beneficial (cardioprotective) to detrimental (cardiotoxic) depending on the acuteness or chronicity of a cardiac stressor. Identification of the mechanisms by which this temporal switch is mediated could lead to new drug development. What are the clinical implications? Our findings provide potential guidance in choosing between a β1-specific vs. a β1/2-non-specific drug when treating specific cardiovascular diseases based on their temporal characteristics. The temporal protective/toxic switching that we describe could be a mechanism common to many other drugs, yet is rarely tested, suggesting the need for additional studies using temporal course as a factor.

The adrenergic receptor signaling system has been a prototype for understanding the structure, function, and pharmacokinetics of hormone–receptor interactions. In parallel with our advancing knowledge of cardiac β-receptor biology, β-blockers have become the standard of care for adults with heart failure, one of the best examples of translation of basic science knowledge to the clinical setting. Understanding the basic biology of β-receptor downregulation and cardiotoxic signaling has provided a mechanism for the clinical efficacy of β-blockers. As new questions have arisen, e.g., differences in response across ethnic/racial groups, they have been taken from the bedside back to the bench, leading to a new appreciation of β-receptor pharmacogenomics, and the potential for personalized medicine approaches. Similarly, developing a better basic understanding of β-arrestin-biased signaling, will allow the design of next generation β-blockers that will more directly target cardiotoxic pathways. However, as clinical research in adult heart failure moves forward at a rapid pace, how pediatric cardiologists can apply these data to children remains an ongoing challenge.

Aims:Hypertrophic cardiomyopathy (HCM) is the most prevalent genetic heart disorder, characterized by significant phenotypic variability even among individuals with identical MYH7 mutations. This study aims to elucidate factors contributing to this variability and identify drivers of phenotype penetrance. We compared the baseline phenotypes of a highly penetrant MYH7 H251N mutation and the variably penetrant MYH7 G256E mutation and investigated the impact of adding beta-adrenergic stimulation and homozygosity on disease phenotype penetrance using cardiomyocytes from an isogenic line of human induced pluripotent stem cells (hiPSC-CMs). Methods and Results:Isogenic hiPSCs with MYH7 H251N and MYH7 G256E mutations were generated using CRISPR/Cas9 technology and differentiated into cardiomyocytes (CMs). Single-cell RNA sequencing (scRNAseq) and functional analysis of contractile function revealed consistent HCM phenotype presentation in H251N CMs, whereas G256E CMs exhibited a subtle and more variable phenotype. Beta-adrenergic stimulation induced a distinct metabolic stress response in G256E CMs, characterized by impaired mitochondrial ATP upregulation. Increasing mutant gene dosage from hetero- to homozygosity led to consistent increase in hypertrophic and structural gene expression changes in G256E CMs at RNA and protein levels. These changes were distinct from the changes observed with stress response. Importantly, homozygous G256E CMs exhibited a hypercontractile functional and disorganized structural phenotype. Across multiple experimental conditions, we identified consistent increase in cardiomyocyte specific transcriptomic markers such as NPPB, APOE, PDLIM3 and ANKRD1. Conclusions:Our study highlights the use of a variably penetrant MYH7 mutation to investigate factors that influence HCM phenotype penetrance. Specifically, we found that mutant gene dosage and beta-adrenergic stimulation induce distinct HCM disease phenotypes, providing novel insights into mechanisms that may contribute to variable disease expression in HCM.

Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy a platform for studying cardiovascular disease by combining cryo-ET with human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). After developing a cryo-ET workflow for visualizing macromolecules in hiPSC-CMs, we reconstructed sub-nanometer resolution structures of the human thin filament, a central component of the contractile machinery. We also visualized a previously unobserved organization of a regulatory complex that connects muscle contraction to calcium signaling (the troponin complex), highlighting the value of our approach for interrogating the structures of cardiac proteins in their cellular context.

BACKGROUND:By age 40, roughly half of the individuals with Fontan circulation will have died or undergone heart transplantation. Poor exercise capacity and endothelial dysfunction accelerate disease progression. OBJECTIVE:This study aims to assess the systolic function of the single ventricle (SV) in pediatric Fontan patients entering an exercise intervention (RE-ENERGIZE FONTAN) and how it is associated with exercise capacity and endothelial function. METHODS:This cohort comes from an ongoing randomized trial in Fontan patients, using live video conferencing for supervised exercise. Participants (ages 8-19) cleared for exercise underwent 2D/3D echocardiograms, cardiopulmonary testing, and endothelial function (RHI). 2D longitudinal strain (LS, right ventricle), 2D global longitudinal strain (GLS, left ventricle), and circumferential strain were measured with TomTec, and 3D ejection fraction (3D-EF) and 3D-GLS were calculated. RESULTS:We have enrolled 114 Fontan patients. Median age was 12.7 years (IQR 10.2, 15.6). The median time from Fontan operation was 8.8 years (IQR 6.2, 12.0). Fifty-seven patients (50%) had a single right ventricle. SV systolic performance measures were: 2D-LS/GLS -15.6% ± 4.19%, circumferential strain was -18.7% ± 6.83%, 3D-EF 49.9% ± 7.26%, and 3D-GLS -16.8% ± 4.37%. Peak VO2 was 1,290 ± 502 mL/min, and percent predicted peak VO2 was 67.8% ± 15.6%. RHI was 1.44 ± 0.576. 2D-LS/GLS and 3D EF correlated with percent predicted peak VO2 (R = -0.28, P = .007 and R = 0.24, P = .019). 3D-EF correlated positively with RHI (R = 0.29, P = .0071). CONCLUSIONS:In this cohort of pediatric Fontan patients, SV systolic function was diminished at baseline, and there was a direct correlation between 2D strain and 3D-EF with percent predicted peak VO2, and additionally, 3D-EF with endothelial function. CLINICAL TRIAL NUMBER:NCT04195451.

While exome and whole genome sequencing have transformed medicine by elucidating the genetic underpinnings of both rare and common complex disorders, its utility to predict clinical outcomes remains understudied. Here, we use artificial intelligence (AI) technologies to explore the predictive value of whole exome sequencing in forecasting clinical outcomes following surgery for congenital heart defects (CHD). We report results for a prospective observational cohort study of 2,253 CHD patients from the Pediatric Cardiac Genomics Consortium with a broad range of complex heart defects, pre- and post-operative clinical variables and exome sequencing. Damaging genotypes in chromatin-modifying and cilia-related genes are associated with an elevated risk of adverse post-operative outcomes, including mortality, cardiac arrest and prolonged mechanical ventilation. The impact of damaging genotypes is further amplified in the context of specific CHD phenotypes, surgical complexity and extra-cardiac anomalies. The absence of a damaging genotype in chromatin-modifying and cilia-related genes is also informative, reducing the risk for some adverse postoperative outcomes. Thus, genome sequencing enriches the ability to forecast outcomes following congenital cardiac surgery.

Congenital heart disease (CHD) is a leading cause of infant mortality. We analyzed de novo mutations (DNMs) and very rare transmitted/unphased damaging variants in 248 prespecified genes in 11,555 CHD probands. The results identified 60 genes with a significant burden of heterozygous damaging variants. Variants in these genes accounted for CHD in 10.1% of probands with similar contributions from de novo and transmitted variants in parent–offspring trios that showed incomplete penetrance. DNMs in these genes accounted for 58% of the signal from DNMs. Thirty-three genes were linked to a single CHD subtype while 12 genes were associated with 2 to 4 subtypes. Seven genes were only associated with isolated CHD, while 37 were associated with 1 or more extracardiac abnormalities. Genes selectively expressed in the cardiomyocyte lineage were associated with isolated CHD, while those widely expressed in the brain were also associated with neurodevelopmental delay (NDD). Missense variants introducing or removing cysteines in epidermal growth factor (EGF)-like domains of NOTCH1 were enriched in tetralogy of Fallot and conotruncal defects, unlike the broader CHD spectrum seen with loss of function variants. Transmitted damaging missense variants in MYH6 were enriched in multiple CHD phenotypes and account for ~1% of all probands. Probands with characteristic mutations causing syndromic CHD were frequently not diagnosed clinically, often due to missing cardinal phenotypes. CHD genes that were positively or negatively associated with development of NDD suggest clinical value of genetic testing. These findings expand the understanding of CHD genetics and support the use of molecular diagnostics in CHD.

Variants with large effect contribute to congenital heart disease (CHD). To date, recessive genotypes (RGs) have commonly been implicated through anecdotal ascertainment of consanguineous families and candidate gene-based analysis; the recessive contribution to the broad range of CHD phenotypes has been limited. We analyzed whole exome sequences of 5,424 CHD probands. Rare damaging RGs were estimated to contribute to at least 2.2% of CHD, with greater enrichment among laterality phenotypes (5.4%) versus other subsets (1.4%). Among 108 curated human recessive CHD genes, there were 66 RGs, with 54 in 11 genes with >1 RG, 12 genes with 1 RG, and 85 genes with zero. RGs were more prevalent among offspring of consanguineous union (4.7%, 32/675) than among nonconsanguineous probands (0.7%, 34/4749). Founder variants in GDF1 and PLD1 accounted for 74% of the contribution of RGs among 410 Ashkenazi Jewish probands. We identified genome-wide significant enrichment of RGs in C1orf127, encoding a likely secreted protein expressed in embryonic mouse notochord and associated with laterality defects. Single-cell transcriptomes from gastrulation-stage mouse embryos revealed enrichment of RGs in genes highly expressed in the cardiomyocyte lineage, including contractility-related genes MYH6, UNC45B, MYO18B, and MYBPC3 in probands with left-sided CHD, consistent with abnormal contractile function contributing to these malformations. Genes with significant RG burden account for 1.3% of probands, more than half the inferred total. These results reveal the recessive contribution to CHD, and indicate that many genes remain to be discovered, with each likely accounting for a very small fraction of the total.

Purpose Although dual-drug immunosuppression is standard for rejection prophylaxis in pediatric heart transplant (HT), single-drug immunosuppression (monotherapy) may be sufficient for certain patients. We describe the experience with monotherapy at a single institution. Methods Retrospective review of HT patients at a single pediatric transplant center who underwent HT between from 1/2001- 12/2020 and were treated with calcineurin-inhibitor monotherapy for > 1 month. Patients were transitioned to monotherapy per primary clinician due to concerns for over-immune suppression or post-transplant lymphoproliferative disorder (PTLD). Clinical characteristics and patient outcomes were analyzed. Results During the study period, 12/333 (3.6%) patients were placed on monotherapy at a median of 4.6 years post-HT [IQR 1.9-6.7 years]. Ten (83%) were transplanted as infants and 6 (50%) were transplanted for congenital heart disease. Nine (75%) were on tacrolimus monotherapy and 3 were on cyclosporine monotherapy. Indication for monotherapy was serious or chronic infection in 6 (50%), PTLD in 4 (33%), and neutropenia in 2 (16%). At a median follow-up of 1.5 years [IQR 1.1-2.2 years], 2 (16%) patients experienced grade 1B/1R cellular rejection, one of which required mechanical support and was transitioned back to dual therapy immunosuppression. All patients remain alive, with the remaining 11 (91%) still on monotherapy at time of last follow-up. Conclusion Monotherapy immunosuppression with calcineurin inhibitors can be safely used in a subset of pediatric HT patients with evidence of over-immune suppression without significant increase in rejection.

PURPOSE:Long-term outcomes after pediatric heart transplant (pHT) are rarely reported owing to young programs and loss of follow-up after transition to adult care. We leveraged our program age, center volume, and linked electronic medical record to analyze 50-year outcomes after pHT, including events after adult care transfer. METHODS:Retrospective review of all pHT over 50 years at Stanford (August 19, 1974 to August 19, 2024). Patient characteristics and posttransplant outcomes, including death, retransplant, and subsequent kidney transplantation, were ascertained from the pediatric and adult electronic medical records, public records, and personal communication with other centers. RESULTS:There were 567 pHTs in 540 patients. Status as of August 19, 2024 was confirmed for 526 (97%),11 (2%) had partial follow-up, and 3 were missing. Over 1,888,341 follow-up days, 50 (9%) received a second heart transplant (27 pediatric, 23 adult) after a median of 9 (5, 14) years; 3 (1%) received a third heart transplant; 6 (1%) received a subsequent kidney transplant, and 212 (39%) died (172 pediatric, 40 adult). Median survival was 16.7 years; survival improved in successive eras (p<0.001). CONCLUSION:This linked analysis of pediatric and adult programs shows excellent long-term pHT outcomes with median survival exceeding 15 years and improving. Nearly half of retransplants occur after transfer; subsequent kidney transplant occurs infrequently.

Background:Fontan circulation carries high morbidity and mortality, driven in part by impaired exercise capacity, muscle deficits, and endothelial dysfunction. In a pediatric cohort with Fontan circulation, we examined associations between exercise capacity, endothelial function, and muscle parameters before enrollment in a randomized exercise intervention. Methods:Patients aged 8-19 years who have Fontan circulation were included. Exclusion criteria included New York Heart Association class IV, active illness, protein-losing enteropathy, pacemaker, or cognitive delay. Assessments included cardiopulmonary exercise testing (peak VO2), endothelial function testing (reactive hyperemia index [RHI]), fasting lipid profile, dual-energy x-ray absorptiometry and dynamometry (muscle mass and strength), and functional movement screening (FMS). Results:Among 137 participants (median age 12.7 years; 53% single right ventricle), median time since Fontan was 8.6 years. Peak VO2 was 1402 ± 509 mL/min (75.7% ± 17.9% predicted; 31.1 ± 7.4 mL/kg/min indexed). RHI was 1.45 ± 0.6; NOx 22.2 ± 17.4 μmol/L. The low-density lipoprotein cholesterol level was 65.8 ± 18.9 mg/dL. Length-adjusted leg lean mass, handgrip strength, and leg extension strength z scores were -1.02 ± 1.0, -0.76 ± 0.9, and -0.70 ± 1.0, respectively. The FMS score averaged 11.9 ± 3.0. Peak VO2 correlated with RHI (r = 0.50), lnRHI (r = 0.51), FMS (r = 0.30) (all P < 0.001), and length-adjusted leg lean mass z score (r = 0.25, P = 0.003). Indexed and percent-predicted peak VO2 also correlated with FMS (r = 0.31 and 0.30, respectively) and high-density lipoprotein (r = 0.24 and r = 0.25, respectively). Conclusion:In youth with Fontan circulation, lower aerobic capacity was associated with lower endothelial function, leg lean mass, strength, and mobility, underscoring the interdependence of cardiovascular, vascular, and musculoskeletal health. Clinical Trial Registration:NCT04195451.