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Conquering Atherosclerotic Cardiovascular Disease Substantial reductions in cardiovascular mortality in the United States reflect the power of a system that catalyzes the integration of basic scien...

Supplemental Digital Content is available in the text., Background: To assess the genetic architecture of hypertrophic cardiomyopathy (HCM) in patients of predominantly Chinese ancestry. Methods: We sequenced HCM disease genes in Singaporean patients (n=224) and Singaporean controls (n=3634), compared findings with additional populations and White HCM cohorts (n=6179), and performed in vitro functional studies. Results: Singaporean HCM patients had significantly fewer confidently interpreted HCM disease variants (pathogenic/likely pathogenic: 18%, P

Objective To explore the transcriptomic differences between patients with hypertrophic cardiomyopathy (HCM) and controls. Patients and Methods RNA was extracted from cardiac tissue flash frozen at therapeutic surgical septal myectomy for 106 patients with HCM and 39 healthy donor hearts. Expression profiling of 37,846 genes was performed using the Illumina Human HT-12v3 Expression BeadChip. All patients with HCM were genotyped for pathogenic variants causing HCM. Technical validation was performed using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. This study was started on January 1, 1999, and final analysis was completed on April 20, 2020. Results Overall, 22% of the transcriptome (8443 of 37,846 genes) was expressed differentially between HCM and control tissues. Analysis by genotype revealed that gene expression changes were similar among genotypic subgroups of HCM, with only 4% (1502 of 37,846) to 6% (2336 of 37,846) of the transcriptome exhibiting differential expression between genotypic subgroups. The qRT-PCR confirmed differential expression in 92% (11 of 12 genes) of tested transcripts. Notably, in the context of coronavirus disease 2019 (COVID-19), the transcript for angiotensin I converting enzyme 2 (ACE2), a negative regulator of the angiotensin system, was the single most up-regulated gene in HCM (fold-change, 3.53; q-value =1.30×10−23), which was confirmed by qRT-PCR in triplicate (fold change, 3.78; P=5.22×10−4), and Western blot confirmed greater than 5-fold overexpression of ACE2 protein (fold change, 5.34; P=1.66×10−6). Conclusion More than 20% of the transcriptome is expressed differentially between HCM and control tissues. Importantly, ACE2 was the most up-regulated gene in HCM, indicating perhaps the heart's compensatory effort to mount an antihypertrophic, antifibrotic response. However, given that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses ACE2 for viral entry, this 5-fold increase in ACE2 protein may confer increased risk for COVID-19 manifestations and outcomes in patients with increased ACE2 transcript expression and protein levels in the heart.

Visual Abstract, Highlights • Combining CHD phenotype–driven gene set enrichment and CRISPR knockdown screening in zebrafish is an effective approach to identifying novel CHD genes. • Mutations affecting genes coding for the WAVE2 protein complex and small GTPase-mediated signaling are associated with LVOTO lesions. • WAVE2 complex genes brk1, nckap1, and wasf2 and regulators of small GTPase signaling cul3a and racgap1 are critical to zebrafish heart development., Summary Genetic variants are the primary driver of congenital heart disease (CHD) pathogenesis. However, our ability to identify causative variants is limited. To identify causal CHD genes that are associated with specific molecular functions, the study used prior knowledge to filter de novo variants from 2,881 probands with sporadic severe CHD. This approach enabled the authors to identify an association between left ventricular outflow tract obstruction lesions and genes associated with the WAVE2 complex and regulation of small GTPase-mediated signal transduction. Using CRISPR zebrafish knockdowns, the study confirmed that WAVE2 complex proteins brk1, nckap1, and wasf2 and the regulators of small GTPase signaling cul3a and racgap1 are critical to cardiac development.

Background: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. Methods: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. Results: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. Conclusions: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.

Introduction: Biallelic damaging variants in ALPK3, encoding alpha-protein kinase 3, cause pediatric-onset cardiomyopathy with manifestations that are incompletely defined. Methods and Results: We analyzed clinical manifestations of damaging biallelic ALPK3 variants in 19 pediatric patients, including nine previously published cases. Among these, 11 loss-of-function (LoF) variants, seven compound LoF and deleterious missense variants, and one homozygous deleterious missense variant were identified. Among 18 live-born patients, 8 exhibited neonatal dilated cardiomyopathy (44.4%; 95% CI: 21.5%-69.2%) that subsequently transitioned into ventricular hypertrophy. The majority of patients had extracardiac phenotypes, including contractures, scoliosis, cleft palate, and facial dysmorphisms. We observed no association between variant type or location, disease severity, and/or extracardiac manifestations. Myocardial histopathology showed focal cardiomyocyte hypertrophy, subendocardial fibroelastosis in patients under 4 years of age, and myofibrillar disarray in adults. Rare heterozygous ALPK3 variants were also assessed in adult-onset cardiomyopathy patients. Among 1548 Dutch patients referred for initial genetic analyses, we identified 39 individuals with rare heterozygous ALPK3 variants (2.5%; 95% CI: 1.8%-3.4%), including 26 missense and 10 LoF variants. Among 149 U.S. patients without pathogenic variants in 83 cardiomyopathy-related genes, we identified six missense and nine LoF ALPK3 variants (10.1%; 95% CI: 5.7%-16.1%). LoF ALPK3 variants were increased in comparison to matched controls (Dutch cohort, P = 1.6×10−5; U.S. cohort, P = 2.2×10−13). Conclusion: Biallelic damaging ALPK3 variants cause pediatric cardiomyopathy manifested by DCM transitioning to hypertrophy, often with poor contractile function. Additional extracardiac features occur in most patients, including musculoskeletal abnormalities and cleft palate. Heterozygous LoF ALPK3 variants are enriched in adults with cardiomyopathy and may contribute to their cardiomyopathy. Adults with ALPK3 LoF variants therefore warrant evaluations for cardiomyopathy.

Precision medicine promises to dramatically improve patient outcomes and reduce health care costs through a shift in focus from disease treatment to prevention and individualized therapies. For families with inherited cardiomyopathies, efforts to date have been directed toward discovery and functional characterization of single disease-causing variants. With advances in sequencing, the cataloging of personal genetic variation has been expedited, providing improved insights into the key importance of the genes in which variants occur. These advances have propelled seminal opportunities for successful variant-targeted disease-reversing therapy. New challenges have also emerged-particularly interpretation of the rapidly rising numbers of "variants of unknown significance." For treatments based on patient genotype to be feasible on a wider scale, these obstacles need to be overcome. Here the authors focus on genetics of dilated cardiomyopathy and provide a roadmap for implementing genomic information into future patient management.

Background Human mutations in the X‐linked lysosome‐associated membrane protein‐2 ( LAMP2 ) gene can cause a multisystem Danon disease or a primary cardiomyopathy characterized by massive hypertrophy, conduction system abnormalities, and malignant ventricular arrhythmias. We introduced an in‐frame LAMP2 gene exon 6 deletion mutation (denoted L2 Δ6 ) causing human cardiomyopathy, into mouse LAMP2 gene, to elucidate its consequences on cardiomyocyte biology. This mutation results in in‐frame deletion of 41 amino acids, compatible with presence of some defective LAMP2 protein. Methods and Results Left ventricular tissues from L2 Δ6 and wild‐type mice had equivalent amounts of LAMP2 RNA, but a significantly lower level of LAMP2 protein. By 20 weeks of age male mutant mice developed left ventricular hypertrophy which was followed by left ventricular dilatation and reduced systolic function. Cardiac electrophysiology and isolated cardiomyocyte studies demonstrated ventricular arrhythmia, conduction disturbances, abnormal calcium transients and increased sensitivity to catecholamines. Myocardial fibrosis was strikingly increased in 40‐week‐old L2 Δ6 mice, recapitulating findings of human LAMP2 cardiomyopathy. Immunofluorescence and transmission electron microscopy identified mislocalization of lysosomes and accumulation of autophagosomes between sarcomeres, causing profound morphological changes disrupting the cellular ultrastructure. Transcription profile and protein expression analyses of L2 Δ6 hearts showed significantly increased expression of genes encoding activators and protein components of autophagy, hypertrophy, and apoptosis. Conclusions We suggest that impaired autophagy results in cardiac hypertrophy and profound transcriptional reactions that impacted metabolism, calcium homeostasis, and cell survival. These responses define the molecular pathways that underlie the pathology and aberrant electrophysiology in cardiomyopathy of Danon disease.

The application of next-generation sequencing to study congenital heart disease (CHD) is increasingly providing new insights into the causes and mechanisms of this prevalent birth anomaly. Whole-exome sequencing analysis identifies damaging gene variants altering single or contiguous nucleotides that are assigned pathogenicity based on statistical analyses of families and cohorts with CHD, high expression in the developing heart and depletion of damaging protein-coding variants in the general population. Gene classes fulfilling these criteria are enriched in patients with CHD and extracardiac abnormalities, evidencing shared pathways in organogenesis. Developmental single-cell transcriptomic data demonstrate the expression of CHD-associated genes in particular cell lineages, and emerging insights indicate that genetic variants perturb multicellular interactions that are crucial for cardiogenesis. Whole-genome sequencing analyses extend these observations, identifying non-coding variants that influence the expression of genes associated with CHD and contribute to the estimated ~55% of unexplained cases of CHD. These approaches combined with the assessment of common and mosaic genetic variants have provided a more complete knowledge of the causes and mechanisms of CHD. Such advances provide knowledge to inform the clinical care of patients with CHD or other birth defects and deepen our understanding of the complexity of human development. In this Review, we highlight known and candidate CHD-associated human genes and discuss how the integration of advances in developmental biology research can provide new insights into the genetic contributions to CHD.

Rationale: Pathogenic variations in the lamin gene ( LMNA ) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA -related DCM. Objective: To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 ( Yy1 ) in suppressing Lmna DCM. Methods and Results: We developed a Lmna DCM mouse model induced by cardiac-specific Lmna short hairpin RNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf . Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGF-β (transforming growth factor-β)/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGFβ/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts. Conclusions: Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are common heart muscle disorders that are caused by pathogenic variants in sarcomere protein genes. HCM is characterized by unexplained cardiac hypertrophy (increased chamber wall thickness) that is accompanied by enhanced cardiac contractility and impaired relaxation. DCM is defined as increased ventricular chamber volume with contractile impairment. In this review, we discuss recent analyses that provide new insights into the molecular mechanisms that cause these conditions. HCM studies have uncovered the critical importance of conformational changes that occur during relaxation and enable energy conservation, which are frequently disturbed by HCM mutations. DCM studies have demonstrated the considerable prevalence of truncating variants in titin and have discerned that these variants reduce contractile function by impairing sarcomerogenesis. These new pathophysiologic mechanisms open exciting opportunities to identify new pharmacological targets and develop future cardioprotective strategies.

Aims Our objective was to better understand the genetic bases of dilated cardiomyopathy (DCM), a leading cause of systolic heart failure. Methods and results We conducted the largest genome-wide association study performed so far in DCM, with 2719 cases and 4440 controls in the discovery population. We identified and replicated two new DCM-associated loci on chromosome 3p25.1 [lead single-nucleotide polymorphism (SNP) rs62232870, P = 8.7 × 10−11 and 7.7 × 10−4 in the discovery and replication steps, respectively] and chromosome 22q11.23 (lead SNP rs7284877, P = 3.3 × 10−8 and 1.4 × 10−3 in the discovery and replication steps, respectively), while confirming two previously identified DCM loci on chromosomes 10 and 1, BAG3 and HSPB7. A genetic risk score constructed from the number of risk alleles at these four DCM loci revealed a 3-fold increased risk of DCM for individuals with 8 risk alleles compared to individuals with 5 risk alleles (median of the referral population). In silico annotation and functional 4C-sequencing analyses on iPSC-derived cardiomyocytes identify SLC6A6 as the most likely DCM gene at the 3p25.1 locus. This gene encodes a taurine transporter whose involvement in myocardial dysfunction and DCM is supported by numerous observations in humans and animals. At the 22q11.23 locus, in silico and data mining annotations, and to a lesser extent functional analysis, strongly suggest SMARCB1 as the candidate culprit gene. Conclusion This study provides a better understanding of the genetic architecture of DCM and sheds light on novel biological pathways underlying heart failure.

Background: Peripartum cardiomyopathy (PPCM) occurs in ≈1:2000 deliveries in the United States and worldwide. The genetic underpinnings of PPCM remain poorly defined. Approximately 10% of women with PPCM harbor truncating variants in TTN (TTNtvs). Whether mutations in other genes can predispose to PPCM is not known. It is also not known if the presence of TTNtvs predicts clinical presentation or outcomes. Nor is it known if the prevalence of TTNtvs differs in women with PPCM and preeclampsia, the strongest risk factor for PPCM. Methods: Women with PPCM were retrospectively identified from several US and international academic centers, and clinical information and DNA samples were acquired. Next-generation sequencing was performed on 67 genes, including TTN , and evaluated for burden of truncating and missense variants. The impact of TTNtvs on the severity of clinical presentation, and on clinical outcomes, was evaluated. Results: Four hundred sixty-nine women met inclusion criteria. Of the women with PPCM, 10.4% bore TTNtvs (odds ratio=9.4 compared with 1.2% in the reference population; Bonferroni-corrected P [ P *]=1.2×10 –46 ). We additionally identified overrepresentation of truncating variants in FLNC (odds ratio=24.8, P *=7.0×10 –8 ), DSP (odds ratio=14.9, P *=1.0×10 –8 ), and BAG3 (odds ratio=53.1, P *=0.02), genes not previously associated with PPCM. This profile is highly similar to that found in nonischemic dilated cardiomyopathy. Women with TTNtvs had lower left ventricular ejection fraction on presentation than did women without TTNtvs (23.5% versus 29%, P =2.5×10 –4 ), but did not differ significantly in timing of presentation after delivery, in prevalence of preeclampsia, or in rates of clinical recovery. Conclusions: This study provides the first extensive genetic and phenotypic landscape of PPCM and demonstrates that predisposition to heart failure is an important risk factor for PPCM. The work reveals a degree of genetic similarity between PPCM and dilated cardiomyopathy, suggesting that gene-specific therapeutic approaches being developed for dilated cardiomyopathy may also apply to PPCM, and that approaches to genetic testing in PPCM should mirror those taken in dilated cardiomyopathy. Last, the clarification of genotype/phenotype associations has important implications for genetic counseling.

Background: The impact of sex on phenotypic expression in hypertrophic cardiomyopathy (HCM) has not been well characterized in genotyped cohorts. Methods: Retrospective cohort study from an international registry of patients receiving care at experienced HCM centers. Sex-based differences in baseline characteristics and clinical outcomes were assessed. Results: Of 5873 patients (3788 genotyped), 2226 (37.9%) were women. At baseline, women were older (49.0±19.9 versus 42.9±18.4 years, P P P MYH7 variants where age at diagnosis was comparable for women and men (n=492; 34.8±19.2 versus 33.3±16.8 years, P =0.39). Over 7.7 median years of follow-up, New York Heart Association III-IV heart failure was more common in women (hazard ratio, 1.87 [CI, 1.48–2.36], P P Conclusions: In HCM, women are older at diagnosis, partly modified by genetic substrate. Regardless of genotype, women were at higher risk of mortality and developing severe heart failure symptoms. This points to a sex-effect on long-term myocardial performance in HCM, which should be investigated further.

Key Points Question Do damaging gene variants account for increased cancer risk in patients with congenital heart disease (CHD)? Findings In this case-control study, loss-of-function variants in cancer risk genes were increased approximately 1.3-fold in 4443 patients with CHD compared with 9808 control participants. This burden was highest in cancer risk genes previously associated with CHD (7.2-fold) or that regulate gene expression (1.9-fold); patients with CHD and extracardiac anomalies and/or neurodevelopmental delay had the highest rates of damaging variants in cancer risk genes. Meaning Genetic analyses of patients with CHD may identify precise causes of heart malformations and also patients with CHD and increased cancer risks., This case-control study compares the frequency of damaging cancer risk gene variants in patients with congenital heart disease vs control participants and identifies associated clinical variables., Importance Patients with congenital heart disease (CHD), the most common birth defect, have increased risks for cancer. Identification of the variables that contribute to cancer risk is essential for recognizing patients with CHD who warrant longitudinal surveillance and early interventions. Objective To compare the frequency of damaging variants in cancer risk genes among patients with CHD and control participants and identify associated clinical variables in patients with CHD who have cancer risk variants. Design, Setting, and Participants This multicenter case-control study included participants with CHD who had previously been recruited to the Pediatric Cardiac Genomics Consortium based on presence of structural cardiac anomaly without genetic diagnosis at the time of enrollment. Permission to use published sequencing data from unaffected adult participants was obtained from 2 parent studies. Data were collected for this study from December 2010 to April 2019. Exposures Presence of rare (allele frequency

Supplemental Digital Content is available in the text., Background: Cardiac troponin I (TNNI3) gene mutations account for 3% of hypertrophic cardiomyopathy and carriers have a heterogeneous phenotype, with increased risk of sudden cardiac death (SCD). Only one mutation (p.Arg21Cys) has been reported in the N terminus of the protein. In model organisms, it impairs PKA (protein kinase A) phosphorylation, increases calcium sensitivity, and causes diastolic dysfunction. The phenotype of this unique mutation in patients with hypertrophic cardiomyopathy remains unknown. Methods: We sequenced 29 families with hypertrophic cardiomyopathy enriched for pediatric-onset disease and identified 5 families with the TNNI3 p.Arg21Cys mutation. Using cascade screening, we studied the clinical phenotype of 57 individuals from the 5 families with TNNI3 p.Arg21Cys-related cardiomyopathy. We performed survival analysis investigating the age at first SCD in carriers of the mutation. Results: All 5 families with TNNI3 p.Arg21Cys were from South Lebanon. TNNI3 p.Arg21Cys-related cardiomyopathy manifested a malignant phenotype—SCD occurred in 30 (53%) of 57 affected individuals at a median age of 22.5 years. In select carriers without left ventricular hypertrophy on echocardiogram, SCD occurred, myocyte disarray was found on autopsy heart, and tissue Doppler and cardiac magnetic resonance imaging identified subclinical disease features such as diastolic dysfunction and late gadolinium enhancement. Conclusions: The TNNI3 p.Arg21Cys mutation has a founder effect in South Lebanon and causes malignant hypertrophic cardiomyopathy with early SCD even in the absence of hypertrophy. Genetic diagnosis with this mutation may be sufficient for risk stratification for SCD.

Supplemental Digital Content is available in the text., Background: De novo genic and copy number variants are enriched in patients with congenital heart disease, particularly those with extra-cardiac anomalies. The impact of de novo damaging variants on outcomes following cardiac repair is unknown. Methods: We studied 2517 patients with congenital heart disease who had undergone whole-exome sequencing as part of the CHD GENES study (Congenital Heart Disease Genetic Network). Results: Two hundred ninety-four patients (11.7%) had clinically significant de novo variants. Patients with de novo damaging variants were 2.4 times more likely to have extra-cardiac anomalies (P=5.63×10−12). In 1268 patients (50.4%) who had surgical data available and underwent open-heart surgery exclusive of heart transplantation as their first operation, we analyzed transplant-free survival following the first operation. Median follow-up was 2.65 years. De novo variants were associated with worse transplant-free survival (hazard ratio, 3.51; P=5.33×10−04) and longer times to final extubation (hazard ratio, 0.74; P=0.005). As de novo variants had a significant interaction with extra-cardiac anomalies for transplant-free survival (P=0.003), de novo variants conveyed no additional risk for transplant-free survival for patients with these anomalies (adjusted hazard ratio, 1.96; P=0.06). By contrast, de novo variants in patients without extra-cardiac anomalies were associated with worse transplant-free survival during follow-up (hazard ratio, 11.21; P=1.61×10−05) than that of patients with no de novo variants. Using agnostic machine-learning algorithms, we identified de novo copy number variants at 15q25.2 and 15q11.2 as being associated with worse transplant-free survival and 15q25.2, 22q11.21, and 3p25.2 as being associated with prolonged time to final extubation. Conclusions: In patients with congenital heart disease undergoing open-heart surgery, de novo variants were associated with worse transplant-free survival and longer times on the ventilator. De novo variants were most strongly associated with adverse outcomes among patients without extra-cardiac anomalies, suggesting a benefit for preoperative genetic testing even when genetic abnormalities are not suspected during routine clinical practice. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01196182.

It is unknown whether the presence of a sarcomeric mutation alone is sufficient to result in abnormal myocardial force generation, or whether additional changes in myocardial architecture (hypertrophy, disarray, and fibrosis) are required to impair systolic function. Speckle tracking echocardiography allows quantification of global strain/strain rates, twist, and dyssynchrony. In the present study we sought to further elucidate early abnormalities of myocardial mechanics in sarcomeric mutation carriers without evidence of clinical disease. Sixty genotype-positive left ventricular hypertrophy-negative (G+left ventricular hypertrophy [LVH]-) patients and 60 normal controls were studied. Velocity vector imaging was applied retrospectively to echocardiographic images to quantify global longitudinal and circumferential strain/strain rate, and rotation parameters. The G+LVH- group demonstrated both smaller left ventricular diastolic cavity dimensions (4.5 ± 0.6 cm vs 4.8 ± 0.4 cm) and a higher LVEF (66 ± 6% vs 60 ± 5%) compared with controls. An increase in circumferential subendocardial systolic strain (-30 ± 5 vs -27 ± 3%) and both systolic and diastolic subendocardial strain rate was seen in the G+LVH- group. Peak rotation angles were higher at the base and apex, with an increase in total twist (9.0 ± 3.8 vs 6.9 ± 2.9). In the control group, global and average segmental strain were similar, suggesting no/minimal dyssynchrony (global mechanical synchrony index [GMSi] 0.97-0.98). In the G+LVH- group GMSi was significantly lower (subendocardial GMSi 0.95; subepicardial GMSi 0.60), suggesting increasing subendocardial to subepicardial dyssynchrony. In conclusion, utilizing multilayer strain analysis, we demonstrate that G+LVH- subjects have enhanced subendocardial systolic strain rate and twist, as well as mechanical dyssynchrony within the left ventricular myocardium. These results demonstrate that abnormalities in myocardial mechanics precede the development of clinical hypertrophy.

Background: No pharmacological therapy exists for calcific aortic valve disease (CAVD), which confers a dismal prognosis without invasive valve replacement. The search for therapeutics and early diagnostics is challenging because CAVD presents in multiple pathological stages. Moreover, it occurs in the context of a complex, multi-layered tissue architecture; a rich and abundant extracellular matrix phenotype; and a unique, highly plastic, and multipotent resident cell population. Methods: A total of 25 human stenotic aortic valves obtained from valve replacement surgeries were analyzed by multiple modalities, including transcriptomics and global unlabeled and label-based tandem-mass-tagged proteomics. Segmentation of valves into disease stage–specific samples was guided by near-infrared molecular imaging, and anatomic layer-specificity was facilitated by laser capture microdissection. Side-specific cell cultures were subjected to multiple calcifying stimuli, and their calcification potential and basal/stimulated proteomes were evaluated. Molecular (protein–protein) interaction networks were built, and their central proteins and disease associations were identified. Results: Global transcriptional and protein expression signatures differed between the nondiseased, fibrotic, and calcific stages of CAVD. Anatomic aortic valve microlayers exhibited unique proteome profiles that were maintained throughout disease progression and identified glial fibrillary acidic protein as a specific marker of valvular interstitial cells from the spongiosa layer. CAVD disease progression was marked by an emergence of smooth muscle cell activation, inflammation, and calcification-related pathways. Proteins overrepresented in the disease-prone fibrosa are functionally annotated to fibrosis and calcification pathways, and we found that in vitro, fibrosa-derived valvular interstitial cells demonstrated greater calcification potential than those from the ventricularis. These studies confirmed that the microlayer-specific proteome was preserved in cultured valvular interstitial cells, and that valvular interstitial cells exposed to alkaline phosphatase–dependent and alkaline phosphatase–independent calcifying stimuli had distinct proteome profiles, both of which overlapped with that of the whole tissue. Analysis of protein–protein interaction networks found a significant closeness to multiple inflammatory and fibrotic diseases. Conclusions: A spatially and temporally resolved multi-omics, and network and systems biology strategy identifies the first molecular regulatory networks in CAVD, a cardiac condition without a pharmacological cure, and describes a novel means of systematic disease ontology that is broadly applicable to comprehensive omics studies of cardiovascular diseases.

Summary Background There are more than 300 known red blood cell (RBC) antigens and 33 platelet antigens that differ between individuals. Sensitisation to antigens is a serious complication that can occur in prenatal medicine and after blood transfusion, particularly for patients who require multiple transfusions. Although pre-transfusion compatibility testing largely relies on serological methods, reagents are not available for many antigens. Methods based on single-nucleotide polymorphism (SNP) arrays have been used, but typing for ABO and Rh—the most important blood groups—cannot be done with SNP typing alone. We aimed to develop a novel method based on whole-genome sequencing to identify RBC and platelet antigens. Methods This whole-genome sequencing study is a subanalysis of data from patients in the whole-genome sequencing arm of the MedSeq Project randomised controlled trial (NCT01736566) with no measured patient outcomes. We created a database of molecular changes in RBC and platelet antigens and developed an automated antigen-typing algorithm based on whole-genome sequencing (bloodTyper). This algorithm was iteratively improved to address cis–trans haplotype ambiguities and homologous gene alignments. Whole-genome sequencing data from 110 MedSeq participants (30 × depth) were used to initially validate bloodTyper through comparison with conventional serology and SNP methods for typing of 38 RBC antigens in 12 blood-group systems and 22 human platelet antigens. bloodTyper was further validated with whole-genome sequencing data from 200 INTERVAL trial participants (15 × depth) with serological comparisons. Findings We iteratively improved bloodTyper by comparing its typing results with conventional serological and SNP typing in three rounds of testing. The initial whole-genome sequencing typing algorithm was 99·5% concordant across the first 20 MedSeq genomes. Addressing discordances led to development of an improved algorithm that was 99·8% concordant for the remaining 90 MedSeq genomes. Additional modifications led to the final algorithm, which was 99·2% concordant across 200 INTERVAL genomes (or 99·9% after adjustment for the lower depth of coverage). Interpretation By enabling more precise antigen-matching of patients with blood donors, antigen typing based on whole-genome sequencing provides a novel approach to improve transfusion outcomes with the potential to transform the practice of transfusion medicine. Funding National Human Genome Research Institute, Doris Duke Charitable Foundation, National Health Service Blood and Transplant, National Institute for Health Research, and Wellcome Trust.

Background and Purpose— A ruptured intracranial aneurysm (IA) is the leading cause of a subarachnoid hemorrhage. This study seeks to define a specific gene whose mutation leads to disease. Methods— More than 500 IA probands and 100 affected families were enrolled and clinically characterized. Whole exome sequencing was performed on a large family, revealing a segregating THSD1 (thrombospondin type 1 domain containing protein 1) mutation. THSD1 was sequenced in other probands and controls. Thsd1 loss-of-function studies in zebrafish and mice were used for in vivo analyses and functional studies performed using an in vitro endothelial cell model. Results— A nonsense mutation in THSD1 was identified that segregated with the 9 affected (3 suffered subarachnoid hemorrhage and 6 had unruptured IA) and was absent in 13 unaffected family members (LOD score 4.69). Targeted THSD1 sequencing identified mutations in 8 of 507 unrelated IA probands, including 3 who had suffered subarachnoid hemorrhage (1.6% [95% confidence interval, 0.8%–3.1%]). These THSD1 mutations/rare variants were highly enriched in our IA patient cohort relative to 89 040 chromosomes in Exome Aggregation Consortium (ExAC) database ( P THSD1 loss impaired endothelial cell focal adhesion to the basement membrane. These adhesion defects could be rescued by expression of wild-type THSD1 but not THSD1 mutants identified in IA patients. Conclusions— This report identifies THSD1 mutations in familial and sporadic IA patients and shows that THSD1 loss results in cerebral bleeding in 2 animal models. This finding provides new insight into IA and subarachnoid hemorrhage pathogenesis and provides new understanding of THSD1 function, which includes endothelial cell to extracellular matrix adhesion.

Over the last quarter-century, there has been tremendous progress in genetics research that has defined molecular causes for cardiomyopathies. More than a thousand mutations have been identified in many genes with varying ontologies, therein indicating the diverse molecules and pathways that cause hypertrophic, dilated, restrictive, and arrhythmogenic cardiomyopathies. Translation of this research to the clinic via genetic testing can precisely group affected patients according to molecular etiology, and identify individuals without evidence of disease who are at high risk for developing cardiomyopathy. These advances provide insights into the earliest manifestations of cardiomyopathy and help to define the molecular pathophysiological basis for cardiac remodeling. Although these efforts remain incomplete, new genomic technologies and analytic strategies provide unparalleled opportunities to fully explore the genetic architecture of cardiomyopathies. Such data hold the promise that mutation-specific pathophysiology will uncover novel therapeutic targets, and herald the beginning of precision therapy for cardiomyopathy patients.

Many common illnesses differentially affect men and women for unknown reasons. The autoimmune diseases lupus and Sjögren’s syndrome affect nine times more women than men1,2, whereas schizophrenia affects men more frequently and severely3–5. All three illnesses have their strongest common-genetic associations in the Major Histocompatibility Complex (MHC) locus, an association that in lupus and Sjögren’s syndrome has long been thought to arise from HLA alleles6–13. Here we show that the complement component 4 (C4) genes in the MHC locus, recently found to increase risk for schizophrenia14, generate 7-fold variation in risk for lupus (95% CI: 5.88-8.61; p < 10−117 in total) and 16-fold variation in risk for Sjögren’s syndrome (95% CI: 8.59-30.89; p < 10−23 in total), with C4A protecting more strongly than C4B in both illnesses. The same alleles that increase risk for schizophrenia, greatly reduced risk for lupus and Sjögren’s syndrome. In all three illnesses, C4 alleles acted more strongly in men than in women: common combinations of C4A and C4B generated 14-fold variation in risk for lupus and 31-fold variation in risk for Sjögren’s syndrome in men (vs. 6-fold and 15-fold among women respectively) and affected schizophrenia risk about twice as strongly in men as in women. At a protein level, both C4 and its effector (C3) were present at greater levels in men than women in cerebrospinal fluid (p < 10−5 for both C4 and C3) and plasma among adults ages 20-5015–17, corresponding to the ages of differential disease vulnerability. Sex differences in complement protein levels may help explain the larger effects of C4 alleles in men, women’s greater risk of SLE and Sjögren’s, and men’s greater vulnerability in schizophrenia. These results nominate the complement system as a source of sexual dimorphism in vulnerability to diverse illnesses.

Background Individuals with hypertrophic cardiomyopathy (HCM), even when asymptomatic, are at‐risk for sudden cardiac death and stroke from arrhythmias, making it imperative to identify individuals affected by this familial disorder. Consensus guidelines recommend that first‐degree relatives (FDRs) of a person with HCM undergo serial cardiovascular evaluations. Methods We determined the uptake of family screening in patients with HCM and developed an online video intervention to facilitate family communication and screening. Family screening and genetic testing data were collected through a prospective quality improvement initiative, a standardized clinical assessment and management plan (SCAMP), utilized in an established cardiovascular genetics clinic. Patients were prescribed an online video if screening of their FDRs was incomplete and a pilot study on video utilization and family communication was conducted. Results Two‐hundred and sixteen probands with HCM were enrolled in SCAMP Phase I and 190 were enrolled in SCAMP Phase II. In both phases, probands reported that 51% of FDRs had been screened (382/749 in Phase I, 258/504 in Phase II). Twenty patients participated in a pilot study on video utilization and family communication. Nine participants reported watching the video and six participants reported sharing the video with relatives; however only one participant reported sharing the video with relatives who were not yet aware of the diagnosis of HCM in the family. Conclusion Despite care in a specialized cardiovascular genetics clinic, approximately one half of FDRs of patients with HCM remained unscreened. Online interventions and videos may serve as supplemental tools for patients communicating genetic risk information to relatives.

Arrhythmogenic cardiomyopathy (ACM), also known as arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), is a leading cause of sudden death among young adults. Over half of ACM cases are associated with inherited desmosome gene mutations, most commonly in the gene PKP2 which encodes plakophilin-2. One of the obstacles to better understanding ACM pathogenesis is the lack of appropriate models which encompass the early stages of disease development; this imposes significant constraints on the advancement of clinical therapies. The recent advent of human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) has enabled the development of models for studying human cardiac cell biology and pathology. In this work, we combine hiPSC-CMs, CRISPR/Cas9 genome editing, and engineered cardiac tissue platforms to develop human in vitro systems for investigating the molecular mechanisms of ACM pathogenesis. Isogenic control and mutant cell lines of hiPSC-CMs harboring ACM-associated PKP2 mutations were generated using CRISPR/Cas9 technology. Specifically, the PKP2tv cell line has an early truncation in plakophilin-2 that mimics PKP2 c.235C>T found in multiple family lineages. The effects of the PKP2tv mutation on cardiac tissue contractility were characterized using a 3D cardiac micro-tissue (CMT) platform. CMTs composed of PKP2tv cardiomyocytes were shown to have significantly decreased contractile forces compared to the control, which recapitulates the reduced ventricular systolic function seen in ACM patients. This result demonstrated the feasibility of using the hiPSC-derived tissue-engineering model to recapitulate ACM disease phenotype and allow for future investigation into the disease mechanisms.

Hypertrophic Cardiomyopathy (HCM) occurs in 1 of every 500 people and has a wide phenotypic variability. In the majority of cases, HCM is caused by known mutations in genes that code for sarcomere proteins. Although gene testing is widely available for HCM, knowing the phenotype caused by different gene mutations remains a challenging task. We recruited 28 families with HCM, of which 19 (67.8%) have at least one patient with pediatric onset. Index patients from 20 families received targeted sequencing for a panel of genes including TNNI3 , and 7 families received Sanger sequencing for the TNNI3 . We identified a missense mutation p.R21C in TNNI3 segregating with HCM in four families from South Lebanon. Through cascade screening, we identified 30 patients from the four families; twenty of them (67%) had a clinical diagnosis of HCM with a median age of 37 years, while 9 (30%), with a median age 21 years, had no evidence of HCM on echocardiography. An additional 27 members of the families had evidence of HCM, including 22 with SCD in the setting of no past medical history, and their carrier status for p.R21C was implied from the pedigrees. Survival analysis for 57 HCM patients with the mutation revealed a markedly decreased age at first adverse event as compared to 47 HCM patients with the MYBPC3 p.R502W mutation. Founder mutations in HCM that cause a severe phenotype are uncommon. The p.R21C mutation in TNNI3 is the first HCM mutation described in the Lebanese population and has a founder effect in South Lebanon. Early and more frequent screening with different imaging modalities as well as tailored management might be warranted for carriers of this mutation.

Background: Hypertrophic cardiomyopathy (HCM) affects at least 1 in 500 people worldwide, and results in the thickening of the ventricular walls and reduced cardiac function. Mutations in MYBPC3 , encoding cardiac myosin binding protein-C, are the most common cause of HCM. Previously, a highly prevalent 25bp deletion within intron 32 of MYBPC3 was described in the South Asian population. The MYBPC3 d25bp variant is present in approximately 100 million people, and encompasses a splicing branch point predicted to result in abnormal splicing of exon 33. Thus, there is a critical need to understand the mechanism by which MYBPC3 d25bp may cause cardiomyopathy. Methods: To determine the role of the 25bp deletion in vivo , knock-in humanized mice were created in which intron 32 was replaced with the human intron 32, with or without the MYBPC3 d25bp mutation. Mice were characterized at 3- and 6-months of age by echocardiography, histological, and protein analysis. The presence of aberrant exon splicing was also determined in mice carrying the MYBPC3 d25bp variant through RT-PCR and mini-gene assays. Finally, exon trapping experiments were performed to understand the mechanism behind exon skipping. Results: Under baseline conditions, MYBPC3 d25bp displayed no changes in cardiac function or morphology as measured by echocardiography (FS (%): NTG 35.3%, WT 32.8%, Het 33.7%), heart weight to body weight ratio, or histology. While exon 33 skipping was not detected by RT-PCR, the presence of an alternative splice site within exon 33 was identified in MYBPC3 d25bp mice. However, this did not affect the protein levels of cMyBP-C. Furthermore, mini-gene experiments demonstrated that the MYBPC3 d25bp mutation significantly reduced the percentage of correctly spliced transcripts (86.2% vs. 77.5%). Conclusions: These data demonstrate that the presence of the highly prevalent 25bp deletion is not sufficient to cause disease under baseline conditions. However, it is possible that the increased levels of aberrant splicing may increase the risk for developing HCM.

There are established associations between advanced paternal age and offspring risk for psychiatric and developmental disorders. These are commonly attributed to genetic mutations, especially de novo single nucleotide variants (dnSNVs), that accumulate with increasing paternal age. However, the actual magnitude of risk from such mutations in the male germline is unknown. Quantifying this risk would clarify the clinical significance of delayed paternity. Using parent-child trio whole-exome-sequencing data, we estimate the relationship between paternal-age-related dnSNVs and risk for five disorders: autism spectrum disorder (ASD), congenital heart disease, neurodevelopmental disorders with epilepsy, intellectual disability and schizophrenia (SCZ). Using Danish registry data, we investigate whether epidemiologic associations between each disorder and older fatherhood are consistent with the estimated role of dnSNVs. We find that paternal-age-related dnSNVs confer a small amount of risk for these disorders. For ASD and SCZ, epidemiologic associations with delayed paternity reflect factors that may not increase with age., Advanced paternal age associates with increased risk for psychiatric and developmental disorders in offspring. Here, Taylor et al. utilize parent-child trio exome sequencing data sets to estimate the contribution of paternal age-related de novo mutations to multiple disorders, including heart disease and schizophrenia.

Immune checkpoint inhibitors have improved clinical outcomes associated with numerous cancers, but high-grade, immune-related adverse events can occur, particularly with combination immunotherapy. We report the cases of two patients with melanoma in whom fatal myocarditis developed after treatment with ipilimumab and nivolumab. In both patients, there was development of myositis with rhabdomyolysis, early progressive and refractory cardiac electrical instability, and myocarditis with a robust presence of T-cell and macrophage infiltrates. Selective clonal T-cell populations infiltrating the myocardium were identical to those present in tumors and skeletal muscle. Pharmacovigilance studies show that myocarditis occurred in 0.27% of patients treated with a combination of ipilimumab and nivolumab, which suggests that our patients were having a rare, potentially fatal, T-cell-driven drug reaction. (Funded by Vanderbilt-Ingram Cancer Center Ambassadors and others.).

Truncating mutations in the TTN gene are the most common genetic cause of dilated cardiomyopathy in adults but their role in young patients is unknown. We studied 82 young dilated cardiomyopathy subjects and found that the prevalence of truncating TTN mutations in adolescents was similar to adults, but surprisingly few truncating TTN mutations were identified in affected children, including one confirmed de novo variant. In several cases, truncating TTN mutations in children with dilated cardiomyopathy had evidence of additional clinical or genetic risk factors. These findings have implications for genetic testing and suggest that single truncating TTN mutations are insufficient alone to cause pediatric-onset dilated cardiomyopathy.

Article, see p 1477 A core paradigm in precision genomic medicine is the ability to identify the underlying molecular mechanism of a disease in the individual patient and to match the specific therapeutic interventions deployed to this mechanism. In neoplastic disorders, where much of the fundamental disease biology can itself be efficiently studied ex vivo, genomic sequencing has enabled physicians to combine the identification of specific driver mutations, arising de novo in a tissue and inciting tumor formation.1,2 Empirical testing of specific targeted therapies and even parallel coclinical modeling of the interaction between tumor, host, and therapy have accelerated therapeutic innovation3 to significantly improve outcomes in several major neoplasms.4 Although this paradigm has succeeded in oncology, for germline disorders that contribute to prevalent chronic cardiovascular disease, attribution of molecular mechanisms at the level of the individual patient has proven considerably more difficult. Although genetic and genomic studies have allowed discovery of numerous disease genes for cardiomyopathies,5 arrhythmias,6 aortopathies,7 and other disorders, recent studies demonstrate more complex relationships between genotype and phenotype in these syndromes. Genomic sequencing …

Publisher's version (útgefin grein), Objective The myosin-binding protein C (MYBPC3) c.927-2A>G founder mutation accounts for >90% of sarcomeric hypertrophic cardiomyopathy (HCM) in Iceland. This cross-sectional observational study explored the penetrance and phenotypic burden among carriers of this single, prevalent founder mutation. Methods We studied 60 probands with HCM caused by MYBPC3 c.927-2A>G and 225 first-degree relatives. All participants underwent comprehensive clinical evaluation and relatives were genotyped. Results Genetic and clinical evaluation of relatives identified 49 genotype-positive (G+) relatives with left ventricular hypertrophy (G+/LVH+), 59 G+without LVH (G+/LVH-) and 117 genotype-negative relatives (unaffected). Compared with HCM probands, G+/LVH+ relatives were older at HCM diagnosis, had less LVH, a less prevalent diastolic dysfunction, fewer ECG abnormalities, lower serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin I levels, and fewer symptoms. The penetrance of HCM was influenced by age and sex; specifically, LVH was present in 39% of G+males but only 9% of G+females under age 40 years (p=0.015), versus 86% and 83%, respectively, after age 60 (p=0.89). G+/LVH- subjects had normal wall thicknesses, diastolic function and NT-proBNP levels, but subtle changes in LV geometry and more ECG abnormalities than their unaffected relatives. Conclusions Phenotypic expression of the Icelandic MYBPC3 founder mutation varies by age, sex and proband status. Men are more likely to have LVH at a younger age, and disease manifestations were more prominent in probands than in relatives identified via family screening. G+/LVH- individuals had subtle clinical differences from unaffected relatives well into adulthood, indicating subclinical phenotypic expression of the pathogenic mutation., This study was supported by the Howard Hughes Medical Institute (CES), the National Institutes of Health (5HL084553: CES, JGS; 1P20HL101408: CYH; 1P50HL112349: CYH), Akureyri Hospital Research Fund (GTG), Landspitali–The National University Hospital of Iceland Research Fund (BA), the Icelandic Cardiac Society Research Fund (GTG).

Heterozygous truncating variants in titin (TTNtv) are the major genetic cause of dilated cardiomyopathy (DCM). Though variants which disrupt essential splicing dinucleotides (GT/AG) are readily recognized as TTNtv, the effects of other nearby sequence variations on splicing is uncertain. We

IMPORTANCE: Racial differences are recognized in multiple cardiovascular parameters, including left ventricular hypertrophy and heart failure, which are 2 major manifestations of hypertrophic cardiomyopathy. The association of race with disease expression and outcomes among patients with hypertrophic cardiomyopathy is not well characterized. OBJECTIVE: To assess the association between race, disease expression, care provision, and clinical outcomes among patients with hypertrophic cardiomyopathy. DESIGN, SETTING, AND PARTICIPANTS: This retrospective cohort study included data on black and white patients with hypertrophic cardiomyopathy from the US-based sites of the Sarcomeric Human Cardiomyopathy Registry from 1989 through 2018. EXPOSURES: Self-identified race. MAIN OUTCOMES AND MEASURES: Baseline characteristics; genetic architecture; adverse outcomes, including cardiac arrest, cardiac transplantation or left ventricular assist device implantation, implantable cardioverter-defibrillator therapy, all-cause mortality, atrial fibrillation, stroke, and New York Heart Association (NYHA) functional class III or IV heart failure; and septal reduction therapies. The overall composite outcome consists of the first occurrence of any component of the ventricular arrhythmic composite end point, cardiac transplantation, left ventricular assist device implantation, NYHA class III or IV heart failure, atrial fibrillation, stroke, or all-cause mortality. RESULTS: Of 2467 patients with hypertrophic cardiomyopathy at the time of analysis, 205 (8.3%) were black (130 male [63.4%]; mean [SD] age, 40.0 [18.6] years) and 2262 (91.7%) were white (1351 male [59.7%]; mean [SD] age, 45.5 [20.5] years). Compared with white patients, black patients were younger at the time of diagnosis (mean [SD], 36.5 [18.2] vs 41.9 [20.2] years; P

Genetic testing has become more accessible and is increasingly being incorporated into the care of patients with hypertrophic cardiomyopathy. Genetic test results can help to refine diagnosis and distinguish at-risk relatives from those who are not at risk.

Neurodevelopmental abnormalities are the most common noncardiac complications in patients with congenital heart disease (CHD). Prenatal brain abnormalities may be due to reduced oxygenation, genetic factors, or less commonly, teratogens. Understanding the contribution of these factors is essential to improve outcomes. Because primary sulcal patterns are prenatally determined and under strong genetic control, we hypothesized that they are influenced by genetic variants in CHD. In this study, we reveal significant alterations in sulcal patterns among subjects with single ventricle CHD (n = 115, 14.7 ± 2.9 years [mean ± standard deviation]) compared with controls (n = 45, 15.5 ± 2.4 years) using a graph-based pattern-analysis technique. Among patients with CHD, the left hemisphere demonstrated decreased sulcal pattern similarity to controls in the left temporal and parietal lobes, as well as the bilateral frontal lobes. Temporal and parietal lobes demonstrated an abnormally asymmetric left–right pattern of sulcal basin area in CHD subjects. Sulcal pattern similarity to control was positively correlated with working memory, processing speed, and executive function. Exome analysis identified damaging de novo variants only in CHD subjects with more atypical sulcal patterns. Together, these findings suggest that sulcal pattern analysis may be useful in characterizing genetically influenced, atypical early brain development and neurodevelopmental risk in subjects with CHD.

Background Truncating variants in the TTN gene ( TTNtv) are common in patients with dilated cardiomyopathy (DCM) but also occur in the general population. Whether TTNtv are sufficient to cause DCM or require a second hit for DCM manifestation is an important clinical issue. Methods We generated a zebrafish model of an A-band TTNtv identified in 2 human DCM families in which early-onset disease appeared to be precipitated by ventricular volume overload. Cardiac phenotypes were serially assessed from 0 to 12 months using video microscopy, high-frequency echocardiography, and histopathologic analysis. The effects of sustained hemodynamic stress resulting from an anemia-induced hyperdynamic state were also evaluated. Results Homozygous ttna mutants had severe cardiac dysmorphogenesis and premature death, whereas heterozygous mutants ( ttna

Background During cardiac surgery with cardiopulmonary bypass, delivery of cardioplegia solution to achieve electromechanical cardiac quiescence is obligatory. The addition of lidocaine to cardioplegia has advantages, although its consequences at a molecular level remain unclear. We performed whole-genome RNA sequencing of the human left ventricular (LV) myocardium to elucidate the differences between whole-blood (WB) cardioplegia with and without addition of lidocaine (LC) on gene expression. Methods We prospectively enrolled 130 patients undergoing aortic valve replacement surgery. Patients received high-potassium blood cardioplegia either with (n = 37) or without (n = 93) lidocaine. The LV apex was biopsied at baseline, and after an average of 74 minutes of cold cardioplegic arrest. We performed differential gene expression analysis for 18,258 genes between these 2 groups. Clinical and demographic variables were adjusted in the model. Gene ontology (GO) and network enrichment analysis of the retained genes were performed using g:Profiler and Cytoscape. Results A total of 1,298 genes were differentially expressed between cardioplegic treatments. Compared with the WB group, genes upregulated in the LC group were identified by network enrichment to play a protective role in ischemic injury by inhibiting apoptosis, increasing transferrin endocytosis, and increasing cell viability. Downregulated genes in the LC group were identified to play a role in inflammatory diseases, oxygen transport, and neutrophil aggregation. Conclusions The addition of lidocaine to cardioplegia had pronounced effects on a molecular level with genes responsible for decreased inflammation, reduced intracellular calcium binding, enhanced antiapoptotic protection, augmented oxygen accessibility through transferrins, and increased cell viability showing measurable differences.

Background: No pharmacological therapy exists for calcific aortic valve disease (CAVD), which confers a dismal prognosis without valve replacement. The search for therapeutics and early diagnostics is challenging since CAVD presents in multiple pathological stages. Methods: A total of 25 human stenotic aortic valves obtained from valve replacement surgery were analyzed by multiple modalities, including transcriptomics and global unlabeled and tandem-mass-tagged proteomics by liquid chromatography-mass spectrometry. Results: Global transcriptional and protein expression signatures differed between the non-diseased, fibrotic, and calcific stages of CAVD, with consistent trends in gene and protein expression across disease stages. Anatomical aortic valve microlayers exhibited unique proteome profiles that were maintained throughout disease progression, and revealed GFAP as a specific marker of valvular interstitial cells (VICs) from the spongiosa layer. CAVD disease progression was marked by an emergence of smooth muscle cell activation, inflammation, and calcification-related pathways. Proteins overrepresented in the disease-prone fibrosa are functionally annotated to fibrosis and calcification pathways, and we found that, in vitro , fibrosa-derived VICs demonstrated greater calcification potential than those from the ventricularis. These studies confirmed that the microlayer-specific proteome was preserved in cultured VICs, and that VICs exposed to TNAP-dependent and TNAP-independent calcifying stimuli had distinct proteome profiles, both of which overlapped with that of the whole tissue. Network analysis of protein-protein interaction networks found a significant closeness to multiple inflammatory and fibrotic diseases. Conclusions: A spatially- and temporally-resolved multi-omics and systems biology strategy identifies the first molecular regulatory networks in CAVD, a cardiac condition without a pharmacological cure, and describes a strategy for endophenotype characterization that is broadly applicable to comprehensive omics studies of cardiovascular diseases.

and KeywordsBackgroundSex differences exist in the prevalence, presentation, and outcomes of ischemic heart disease. Females have higher risk of heart failure post myocardial infarction relative to males and the female sex is an independent risk factor for hospital and operative mortality after cardiac surgery. However, the mechanisms underlying this sexual dimorphism remain unclear. We examined sex differences in human myocardial gene expression in response to ischemia.MethodsLeft ventricular biopsies from 68 male and 46 female patients undergoing aortic valve replacement surgery were obtained at baseline and after a median 74 minutes of cold cardioplegic arrest/ischemia and respective transcriptomes were quantified by RNA-Seq. Sex-specific responses to ischemia were quantified by differential gene expression, expression quantitative trait loci (eQTL) and pathway and functional analysis. Cell-type enrichment analysis. was used to obtain an estimate of the identity and relative proportions of different cell types present in each sample.ResultsA sex-specific response to ischemia was observed for 271 genes. Functional annotation analysis revealed sex-specific modulation of the oxytocin signaling pathway and common pathway of fibrin clot formation. The eQTL analysis identified variant-by-sex interaction eQTLs at baseline and post-ischemia, indicative of sex differences in the genotypic effects on gene expression, and cell-type enrichment analysis showed sex-bias in proportion of specific cell types.ConclusionIn response to myocardial ischemia, the human left ventricle demonstrates changes in gene expression that differ between the sexes. These differences provide insight into the sexual dimorphism of ischemic heart disease and may aid in the development of sex-specific therapies that reduce myocardial injury.

Sarcomere cardiomyopathies are genetic diseases that perturb contractile function and lead to hypertrophic or dilated myocardial remodeling. Identification of preclinical mutation carriers has yielded insights into the earliest biomechanical defects that link pathogenic variants to cardiac dysfunction. Understanding this early molecular pathophysiology can illuminate modifiable pathways to reduce the emergence of overt cardiomyopathy and curb adverse outcomes. Here, the authors review current understandings of how human hypertrophic cardiomyopathy- and hypertrophic dilated cardiomyopathy-linked mutations disrupt the normal structure and function of the sarcomere.

Background In the second trimester of human fetal development, a tenfold increase in fetal size occurs while cardiac valves grow and retain their function. Patterns of transcription in normally growing human aortic valves are unknown. Methods Discarded human aortic valve samples were collected from the second trimester, 6 from early (14, 15, 17 weeks) and 6 from late (20, 21, 22 weeks) gestation. Network analysis of RNA sequencing data identified subnetworks of significantly increasing and decreasing transcripts. Subsequent cluster analysis identified patterns of transcription through the time course. Pathway enrichment analysis determined the predominant biological processes at each interval. Results We observed phasic transcription over the time course, including an early decrease in cell proliferation and developmental genes (14 to 15 weeks). Pattern specification, shear stress, and adaptive immune genes were induced early. Cell adhesion genes were increased from 14 to 20 weeks. A phase involving cell differentiation and apoptosis (17 to 20 weeks) was followed by downregulation of endothelial-to-mesenchymal transformation genes and then by increased extracellular matrix organization and stabilization (20 to 22 weeks). Conclusions We present a unique data set, comprehensively characterizing human valve development after valve primordia are formed, focusing on key processes displayed by normal aortic valves undergoing significant growth. We build a time course of genes and processes in second trimester fetal valve growth and observe the sequential regulation of gene clusters over time. Critical valve growth genes are potential targets for therapeutic intervention in congenital heart disease and have implications for regenerative medicine and tissue engineering.

Background: A better understanding of the factors that contribute to heterogeneous outcomes and lifetime disease burden in hypertrophic cardiomyopathy (HCM) is critically needed to improve patient management and outcomes. The SHaRe registry (Sarcomeric Human Cardiomyopathy Registry) was established to provide the scale of data required to address these issues, aggregating longitudinal data sets curated by 8 international HCM specialty centers. Methods: Data on 4591 patients with HCM (2763 genotyped) followed up for a mean of 5.4±6.9 years (24 791 patient-years; median, 2.9 years; interquartile range, 0.3–7.9 years) were analyzed for cardiac arrest, cardiac transplantation, appropriate implantable cardioverter-defibrillator therapy, all-cause death, atrial fibrillation, stroke, New York Heart Association functional class III/IV symptoms (all making up the overall composite end point), and left ventricular ejection fraction Results: Median age at diagnosis was 45.8 (interquartile range, 30.9–58.1) years, and 37% of patients were female. Age at diagnosis and sarcomere mutation status were predictive of outcomes. Patients 60 years old. Young patients with HCM (age, 20–29 years) had 4-fold higher mortality than the general US population at a similar age. Patients with pathogenic/likely pathogenic sarcomere mutations had a 2-fold greater risk for adverse outcomes compared with patients without mutations; sarcomere variants of uncertain significance were associated with intermediate risk. Heart failure and atrial fibrillation were the most prevalent adverse events, although typically not emerging for several years after diagnosis. Ventricular arrhythmias occurred in 32% (95% CI, 23–40) of patients 60 years old at diagnosis. Conclusions: The cumulative burden of HCM is substantial and dominated by heart failure and atrial fibrillation occurring many years after diagnosis. Young age at diagnosis and the presence of a sarcomere mutation are powerful predictors of adverse outcomes. These findings highlight the need for close surveillance throughout life and the need to develop disease-modifying therapies.

Objectives The prognostic value of genetic studies in cardiomyopathies is still controversial. Our objective was to evaluate the outcome of patients with cardiomyopathy with mutations in the converter domain of β myosin heavy chain (MYH7). Methods Clinical characteristics and survival of 117 affected members with mutations in the converter domain of MYH7 were compared with 409 patients described in the literature with mutations in the same region. Results Twenty-five mutations were evaluated (9 in our families including 3 novel (Ile730Asn, Asp717Gly and Arg719Pro)). Clinical diagnoses were hypertrophic (n=407), dilated (n=15), non-compaction (n=4) and restrictive (n=5) cardiomyopathies, unspecified cardiomyopathy (n=11), sudden death (n=50) and 35 healthy carriers. One hundred eighty-four had events (cardiovascular death or transplant). Median event-free survival was 50±2 years in our patients and 53±3 years in the literature (p=0.27). There were significant differences in the outcome between mutation: Ile736Thr had fewer events than other mutations in the region (p=0.01), while Arg719Gln (p

Background: The exact mechanisms that underlie the pathological processes of myocardial ischemia in humans are unclear. Cardiopulmonary bypass with cardioplegic arrest allows the authors to examine the whole transcriptional profile of human left ventricular myocardium at baseline and after exposure to cold cardioplegia-induced ischemia as a human ischemia model. Methods: The authors obtained biopsies from 45 patients undergoing aortic valve replacement surgery at baseline and after an average of 79 min of cold cardioplegic arrest. Samples were RNA sequenced and analyzed with the Partek® Genomics Suite (Partek Inc., St. Louis, MO) for differential expression. Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA) and Biobase ExPlain (Biobase GmbH, Wolfenbuettel, Germany) systems were used for functional and pathway analyses. Results: Of the 4,098 genes with a mean expression value greater than 5, 90% were down-regulated and 9.1% were up-regulated. Of those, 1,241 were significantly differentially expressed. Gene ontology analysis revealed significant down-regulation in immune inflammatory response and complement activation categories and highly consistent was the down-regulation of intelectin 1, proteoglycan, and secretory leukocyte peptidase inhibitor. Up-regulated genes of interest were FBJ murine osteosarcoma viral oncogene homolog and the hemoglobin genes hemoglobin α1 (HBA1) and hemoglobin β. In addition, analysis of transcription factor–binding sites revealed interesting targets in factors regulating reactive oxygen species production, apoptosis, immunity, cytokine production, and inflammatory response. Conclusions: The authors have shown that the human left ventricle exhibits significant changes in gene expression in response to cold cardioplegia-induced ischemia during cardiopulmonary bypass, which provides great insight into the pathophysiology of ventricular ischemia, and thus, may help guide efforts to reduce myocardial damage during surgery.

Objectives The study sought to assess the safety, feasibility, and effect of diltiazem as disease-modifying therapy for at-risk hypertrophic cardiomyopathy (HCM) mutation carriers. Background HCM is caused by sarcomere mutations and characterized by left ventricular hypertrophy (LVH) with increased risk of heart failure and sudden death. HCM typically cannot be diagnosed early in life, although subtle phenotypes are present. Animal studies indicate that intracellular calcium handling is altered before LVH develops. Furthermore, early treatment with diltiazem appeared to attenuate disease emergence. Methods In a pilot, double-blind trial, we randomly assigned 38 sarcomere mutation carriers without LVH (mean 15.8 years of age) to therapy with diltiazem 360 mg/day (or 5 mg/kg/day) or placebo. Treatment duration ranged from 12 to 42 months (median 25 months). Study procedures included electrocardiography, echocardiography, cardiac magnetic resonance imaging, and serum biomarker measurement. Results Diltiazem was not associated with serious adverse events. Heart rate and blood pressure did not differ significantly between groups. However, mean left ventricular (LV) end-diastolic diameter improved toward normal in the diltiazem group but decreased further in controls (change in z-scores, +0.6 vs. –0.5; p Conclusions Pre-clinical administration of diltiazem is safe and may improve early LV remodeling in HCM. This novel strategy merits further exploration. (Treatment of Preclinical Hypertrophic Cardiomyopathy With Diltiazem; NCT00319982).

Background— The screening of Icelandic patients clinically diagnosed with hypertrophic cardiomyopathy resulted in identification of 8 individuals from 2 families with X-linked Fabry disease (FD) caused by GLA (α-galactosidase A gene) mutations encoding p.D322E (family A) or p.I232T (family B). Methods and Results— Familial screening of at-risk relatives identified mutations in 16 family A members (8 men and 8 heterozygotes) and 25 family B members (10 men and 15 heterozygotes). Clinical assessments, α-galactosidase A (α-GalA) activities, glycosphingolipid substrate levels, and in vitro mutation expression were used to categorize p.D322E as a classic FD mutation and p.I232T as a later-onset FD mutation. In vitro expression revealed that p.D322E and p.I232T had α-GalA activities of 1.4% and 14.9% of the mean wild-type activity, respectively. Family A men had markedly decreased α-GalA activity and childhood-onset classic manifestations, except for angiokeratoma and cornea verticillata. Family B men had residual α-GalA activity and developed FD manifestations in adulthood. Despite these differences, all family A and family B men >30 years of age had left ventricular hypertrophy, which was mainly asymmetrical, and had similar late gadolinium enhancement patterns. Ischemic stroke and severe white matter lesions were more frequent among family A men, but neither family A nor family B men had overt renal disease. Family A and family B heterozygotes had less severe or no clinical manifestations. Conclusions— Men with classic or later-onset FD caused by GLA missense mutations developed prominent and similar cardiovascular disease at similar ages, despite markedly different α-GalA activities.

Recent decades have witnessed robust successes in conquering the acutely lethal manifestations of heart and vascular diseases. Many patients who previously would have died now survive. Lifesaving successes like these provide a tremendous and easily recognized benefit to individuals and society. Although cardiovascular mortality has declined, the devastating impact of chronic heart disease and comorbidities on quality of life and healthcare resources continues unabated. Future strides, extending those made in recent decades, will require continued research into mechanisms underlying disease prevention, pathogenesis, progression, and therapeutic intervention. However, severe financial constraints currently jeopardize these efforts. To chart a path for the future, this report analyzes the challenges and opportunities we face in continuing the battle against cardiovascular disease and highlights the return on societal investment afforded by fundamental cardiovascular research.