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Summary: Impulses generated by a multicellular, bioelectric signaling center termed the sinoatrial node (SAN) stimulate the rhythmic contraction of the heart. The SAN consists of a network of electrochemically oscillating pacemaker cells encased in a heterogeneous connective tissue microenvironment. Although the cellular composition of the SAN has been a point of interest for more than a century, the biological processes that drive the tissue-level assembly of the cells within the SAN are unknown. Here, we demonstrate that the SAN’s structural features result from a developmental process during which mesenchymal cells derived from a multipotent progenitor structure, the proepicardium, integrate with and surround pacemaker myocardium. This process actively remodels the forming SAN and is necessary for sustained electrogenic signal generation and propagation. Collectively, these findings provide experimental evidence for how the microenvironmental architecture of the SAN is patterned and demonstrate that proper cellular arrangement is critical for cardiac pacemaker biorhythmicity. : How the higher order, tissue-level architecture of the heart’s pacemaker region is patterned during development is poorly understood. Bressan et al. demonstrate that a cellular remodeling process, which depends on the integration of pacemaker muscle with proepicardial-derived mesenchymal cells, establishes the microenvironmental conditions required for rhythmic stimulation of the heart. Keywords: sinoatrial node, cardiac pacemaker cell, cardiac morphogenesis, proepicardium

Mosaicism due to somatic mutations can cause multiple diseases including cancer, developmental and overgrowth syndromes, neurodevelopmental disorders, autoinflammatory diseases, and atrial fibrillation. With the increased use of next generation sequencing technology, multiple tools have been developed to identify low-frequency variants, specifically from matched tumor-normal tissues in cancer studies. To investigate whether mosaic variants are implicated in congenital heart disease (CHD), we developed a pipeline using the cancer somatic variant caller MuTect to identify mosaic variants in whole-exome sequencing (WES) data from a cohort of parent/affected child trios (n = 715) and a cohort of healthy individuals (n = 416). This is a novel application of the somatic variant caller designed for cancer to WES trio data. We identified two cases with mosaic KMT2D mutations that are likely pathogenic for CHD, but conclude that, overall, mosaicism detectable in peripheral blood or saliva does not account for a significant portion of CHD etiology.

Congenital heart disease (CHD) is the leading cause of mortality from birth defects. Here, exome sequencing of a single cohort of 2,871 CHD probands, including 2,645 parent-offspring trios, implicated rare inherited mutations in 1.8%, including a recessive founder mutation in GDF1 accounting for ∼5% of severe CHD in Ashkenazim, recessive genotypes in MYH6 accounting for ∼11% of Shone complex, and dominant FLT4 mutations accounting for 2.3% of Tetralogy of Fallot. De novo mutations (DNMs) accounted for 8% of cases, including ∼3% of isolated CHD patients and ∼28% with both neurodevelopmental and extra-cardiac congenital anomalies. Seven genes surpassed thresholds for genome-wide significance, and 12 genes not previously implicated in CHD had >70% probability of being disease related. DNMs in ∼440 genes were inferred to contribute to CHD. Striking overlap between genes with damaging DNMs in probands with CHD and autism was also found.

Genome sequencing has revolutionized the diagnosis of genetic diseases. Close collaborations between basic scientists and clinical genomicists are now needed to link genetic variants with disease causation. To facilitate such collaborations, we recommend prioritizing clinically relevant genes for functional studies, developing reference variant-phenotype databases, adopting phenotype description standards, and promoting data sharing.

Familial hypertrophic cardiomyopathy (HCM), linked to mutations in myosin, myosin-binding proteins and other sarcolemmal proteins, is associated with increased risk of life threatening ventricular arrhythmias, and a number of animal models have been developed to facilitate analysis of disease progression and mechanisms. In the experiments here, we use the αMHC403/+ mouse line in which one αMHC allele harbors a common HCM mutation (in βMHC, Arg403 Gln). Here, we demonstrate marked prolongation of QT intervals in young adult (10–12 week) male αMHC403/+ mice, well in advance of the onset of measurable left ventricular hypertrophy. Electrophysiological recordings from myocytes isolated from the interventricular septum of these animals revealed significantly (P

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.

Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD). However, underlying CHD gene regulatory network (GRN) imbalances are unknown. Here, we define transcriptional consequences of reduced dosage of the CHD-linked transcription factor, TBX5, in individual cells during cardiomyocyte differentiation from human induced pluripotent stem cells (iPSCs). We discovered highly sensitive dysregulation of TBX5-dependent pathways— including lineage decisions and genes associated with cardiomyocyte function and CHD genetics—in discrete subpopulations of cardiomyocytes. GRN analysis identified vulnerable nodes enriched for CHD genes, indicating that cardiac network stability is sensitive to TBX5 dosage. A GRN-predicted genetic interaction betweenTbx5andMef2cwas validated in mouse, manifesting as ventricular septation defects. These results demonstrate exquisite sensitivity to TBX5 dosage by diverse transcriptional responses in heterogeneous subsets of iPSC-derived cardiomyocytes. This predicts candidate GRNs for human CHDs, with implications for quantitative transcriptional regulation in disease.

SUMMARYThere are numerous hallmarks of aging in mammals, but no unifying cause has been identified. In budding yeast, aging is associated with a loss of epigenetic information that occurs in response to genome instability, particularly DNA double-strand breaks (DSBs). Mammals also undergo predictable epigenetic changes with age, including alterations to DNA methylation patterns that serve as epigenetic “age” clocks, but what drives these changes is not known. Using a transgenic mouse system called “ICE” (for induciblechanges to theepigenome), we show that a tissue’s response to non-mutagenic DSBs reorganizes the epigenome and accelerates physiological, cognitive, and molecular changes normally seen in older mice, including advancement of the epigenetic clock. These findings implicate DSB-induced epigenetic drift as a conserved cause of aging from yeast to mammals.One Sentence SummaryDNA breaks induce epigenomic changes that accelerate the aging clock in mammals

The genetic architecture of sporadic congenital heart disease (CHD) is characterized by enrichment in damaging de novo variants in chromatin-modifying genes. To test the hypothesis that gene pathways contributing to de novo forms of CHD are distinct from those for recessive forms, we analyze 2391 whole-exome trios from the Pediatric Cardiac Genomics Consortium. We deploy a permutation-based gene-burden analysis to identify damaging recessive and compound heterozygous genotypes and disease genes, controlling for confounding effects, such as background mutation rate and ancestry. Cilia-related genes are significantly enriched for damaging rare recessive genotypes, but comparatively depleted for de novo variants. The opposite trend is observed for chromatin-modifying genes. Other cardiac developmental gene classes have less stratification by mode of inheritance than cilia and chromatin-modifying gene classes. Our analyses reveal dominant and recessive CHD are associated with distinct gene functions, with cilia-related genes providing a reservoir of rare segregating variation leading to CHD., Large whole-exome sequencing studies have suggested that the genetic architecture of syndromic congenital heart disease (CHD) is different from sporadic forms. Here, Watkins et al. estimate the relative contribution of damaging recessive and de novo genotypes to CHD in 2391 trios and find them to be associated with different gene functions.

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 shRNA. 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 TGFβ/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.

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.

BackgroundThe contribution of somatic mosaicism, or genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined.ResultsWe developed a computational method, Expectation-Maximization-based detection of Mosaicism (EM-mosaic), to analyze mosaicism in exome sequences of 2530 CHD proband-parent trios. EM-mosaic detected 326 mosaic mutations in blood and/or cardiac tissue DNA. Of the 309 detected in blood DNA, 85/97 (88%) tested were independently confirmed, while 7/17 (41%) candidates of 17 detected in cardiac tissue were confirmed. MosaicHunter detected an additional 64 mosaics, of which 23/46 (50%) among 58 candidates from blood and 4/6 (67%) of 6 candidates from cardiac tissue confirmed. Twenty-five mosaic variants altered CHD-risk genes, affecting 1% of our cohort. Of these 25, 22/22 candidates tested were confirmed. Variants predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The frequency of mosaic variants above 10% mosaicism was 0.13/person in blood and 0.14/person in cardiac tissue. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction.ConclusionsWe estimate that ~1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants expressed at higher allele fraction compared to benign variants. Although blood is a readily-available DNA source, cardiac tissues analyzed contributed ~5% of somatic mosaic variants identified, indicating the value of tissue mosaicism analyses.

Rationale: Congenital heart disease (CHD) occurs in about 1 in 100 live births, yet known genetic causes explain less than 20% of CHD cases. While variants that cause frameshift, nonsense, start/stop site gain or loss, and canonical splice site alterations are readily categorized as being pathogenic or loss-of-function (LOF), interpreting the clinical significance of variants without obvious functional consequences remains challenging. Here, we aim to improve classification of variants of unknown significance (VUS) in non-canonical RNA splice sites that may be pathogenic for CHD. Methods: We tested candidate LOF de novo (DNV) and rare (p < 2E-5) inherited variants from whole exome sequencing of 4474 CHD probands and their parents in the NHLBI Pediatric Cardiac Genetics Consortium. Briefly, variants underwent computational selection to prioritize VUS in splice regions that are likely to alter splicing (“high-likelihood VUS”). These variants then underwent in vitro analysis including Minigene construction, transfection, RNA isolation, and sequencing to confirm splicing outcomes. Results: Preliminary results limited to DNV variants showed that 163 of 2678 (6.08%) were high-likelihood VUS. Subsequent analysis in vitro assay of high-likelihood VUS yielded 53 as splice-altering (p < 0.05) and thus LOF. Combined with previously identified 366 DNV LOF variants, the addition of these splice-altering variants represents a 15.3% increase in total LOF DNV variant calls. This includes new pathogenic mutations in known CHD genes such as KMT2D . In one case, a CHD proband with features of Kabuki Syndrome without a definitive diagnosis was found to have a splice-altering variant in KMT2D . We have extended this assay to 34518 rare, inherited variants in the same cohort, of which 868 (2.54%) are in genes previously associated with CHD. Conclusion: Consideration of non-canonical RNA splice sites in this assay increased the yield of LOF mutations from traditional sequencing methods by 15.3% in the CHD cohort. Further analysis of splice-altering variants in both known and unknown pathogenic genes will improve diagnostic classification of VUS and gene-based diagnosis of CHD.

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.

The discovery of damaging gene mutations in congenital heart disease (CHD) patients enables identification of regulators of cardiac development. Exome sequencing identified de novo heterozygous loss-of-function (LoF) and missense variants in GATA6 among CHD probands, most with outflow tract malformations. Other subjects with GATA6 LoF mutations developed pancreatic agenesis. To elucidate the molecular basis for the predominance of this heart defect, we modeled GATA6 mutations in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). GATA6 variants were introduced into isogenic hiPSCs using CRISPR/Cas9 genome editing. Genome-wide molecular profiles including chromatin accessibility (ATAC-Seq) and gene expression (single cell and bulk RNA-Seq) were evaluated during hiPSC-CM differentiation. Analyses of GATA6 mutant hiPSC-CMs showed deficits in hiPSC-CM differentiation, chromatin accessibility and transcriptional profiles. Heterozygous GATA6 LoF hiPSCs made hiPSC-CMs but exhibited reduced expression of second heart field genes. Homozygous GATA6 LoF hiPSCs failed to differentiate and adopted fibroblast expression profiles. hiPSCs carrying a homozygous GATA6 missense variant, R456G, which altered a DNA-binding domain residue, showed enhanced capacity to differentiate into neuroepithelial-like cells. Chromatin-accessibility studies confirmed that GATA6 normally binds to genes in the promoter region and other genes at distal enhancers. Human GATA6 haploinsufficiency disrupts developmental transcriptional responses driving cardiac morphogenesis. The HAND2 -dependent genetic program, operant during outflow tract development, is particularly sensitive to GATA6 dosage. The mixed differentiation patterns observed in mutation-carrying hiPSCs likely contributes to vascular phenotypes observed in CHD patients. GATA6 haploinsufficiency preferentially alters binding of distal enhancers to promoters in genes where GATA6 normally binds the enhancer rather than the promoter. We speculate that pathogenicity of GATA6 haploinsufficiency is mediated by weaker binding of GATA6 to distal enhancers than to promoter elements, altering expression of these genes in GATA6 haploinsufficient patients.

Variants that drive HCM and associated adverse patient outcomes are found in the cardiac sarcomere. These variants range from those that are known to be pathogenic to those that are likely pathogenic or even variants of unknown significance (VUS). CRISPR/Cas-9 engineering has accelerated our ability to generate variants in iPSC to probe changes in cellular function and assess cellular pathogenicity in VUSs. However, iPSC-CMs are not as functionally mature as adult cardiomyocytes. For this reason we have developed a platform to assess contractile function directly at the level of the sarcomere. We use a custom built MatLab algorithm to assess sarcomere length, contraction time, relaxation time, and beat rate of individual sarcomeres within iPSC-CMs. Sarcomeres are visualised using reporter lines that have been engineered with an N-terminal TTN-GFP. We assess the contractile function of thick filament variants in MYH7 and MYBPC3. We show the ability to detect changes in key contractile parameters. This platform allows the screening of pharmacological compounds against these reporter lines with engineered variants.

The mechanism by which genetic variants of the thin filament cause hypertrophic cardiomyopathy (HCM) has not been fully elucidated. Induced pluripotent stem cell (iPSC)-derived cardiomyocytes can provide a rapidly generatable disease modeling tool to study HCM in these variants. While variants in the thick and thin filament both cause HCM, cardiomyocytes that harbor pathogenic variants in the thin filament genes troponin I ( TNNI3 ) and troponin T ( TNNT2 ) display a distinct molecular phenotype, which we hypothesize differs from the thick filament HCM phenotype. Both thin and thick filament mutant cardiomyocytes display increased measured oxygen consumption rate assessed by metabolic assays, however we hypothesize that the mechanism that drives metabolic change is not common between thick and thin filament variants. Mutations in the thick filament typically shift myosin conformations during relaxion towards a higher energy utilizing a disordered relaxed state (DRX) that enables ATP hydrolysis with more free myosin heads, and this likely accounts for their increased oxygen consumption. However thin filament mutations cause myosin heads to preferentially adopt a conformation in which the myosin heads are sequestered and unable to bind actin. This super relaxed state (SRX) is associated with energy conservation, which would predict reduced contractility. Yet contractility data from thin filament mutants replicate the classic HCM phenotype of hypercontractility and disturbed sarcomere relaxation. To further probe the mechanism by which thin filament variants drive HCM pathophysiology we have employed methodologies to assess calcium transients in iPSC-derived cardiomyocytes harboring thin filament variants. TNNT2 variants recapitulate this disrupted calcium handling.

Cardiac microproteins are an emerging class of important regulators of cellular homeostasis, often encoded by presumed long noncoding RNAs. With only few described so far, their prevalence and biological significance largely remains unclear. To create an atlas of cardiac-expressed microproteins, we performed ribosome profiling on 80 human heart samples. This revealed the translation of hundreds of previously undetected microproteins, which we validated in vivo by targeted mass spectrometry. A single heart-specific microprotein caught our attention as it showed strong expression coregulation with components of mitochondrial OXPHOS pathway. We next deleted this microprotein's lncRNA host gene in hiPSCs using CRISPR/Cas9, and, upon differentiation into 30-d old cardiomyocytes, find a strong expression upregulation of genes involved in oxidative phosphorylation and calcium handling. To investigate the impact of the microprotein KO on cardiomyocyte mechanical function, we assessed sarcomere dynamics using an endogenous TTN-GFP tag, enabling live cell imaging analysis with the SarcTrack algorithm. Preliminary analysis sowes a putative increase in sarcomere contraction and relaxation times, suggesting a repressive regulatory function of the eliminated microprotein encoding lncRNA.

Dilated cardiomyopathy (DCM) is a leading cause for heart failure and is associated with a rate of mortality of 20% within 5 years of diagnosis. The most common genetic causes for DCM are mutations of the sarcomere protein titin (encoded by TTN ), which occurs in 10-20% of DCM cases. Dominant DCM mutations truncate titin (TTNtv) and result in haploinsufficiency. Thus, strategies to increase the expression of the wild type TTN allele could attenuate damaging effects of TTNtv. Utilizing bioinformatic tools, we identified a putative enhancer for TTN in its intron 1. We deleted a 658 bp region from intron 1 which encompasses the region of interest in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome editing to validate its function. Utilizing RNA sequencing and qPCR of RNA harvested from hiPSC-derived cardiomyocytes (hiPSC-CMs), we demonstrated that a homozygous deletion in this region leads to a drop in TTN expression compared to the wild type (WT) control (0.344-fold change, p < 0.001). To further characterize this region, we subdivided it into three parts which we called E1 (296 bp), E2 (206 bp), and E3 (139 bp). E1 includes a highly conserved region and a region of open chromatin as identified by the Assay for Transposase-Accessible Chromatin Sequencing (ATAC-Seq) performed on hiPSC-CMs. A homozygous E1 deletion resulted in a decreased TTN expression of 0.63-fold compared to the WT control (p < 0.001) when performing RNA sequencing on hiPSC-CMs. Both homozygous E2 and E3 deletions resulted in an increased TTN expression (1.56-fold change, p < 0.001; 1.19 fold change, p < 0.001). Utilizing a published sarcomere tracking platform, SarcTrack, to investigate hiPSC-CM physiology, we saw a decreased contractility of 6.6% in hiPSC-CMs carrying a homozygous E1 deletion compared to 10.1% in the WT control (p < 0.001). Cells carrying homozygous E2 or E3 deletions were hypercontractile (13.8%, p < 0.001; 13.7%, p < 0.001). Given our results, we hypothesize that TTN expression depends on the E1 region. If confirmed, we expect that increasing the activity of this enhancer using small molecules may provide a novel therapeutic target for DCM caused by TTNtv.

Rationale: Neural crest cells (NCCs) play a critical role in normal cardiac development, and defects in NCCs likely cause congenital heart disease (CHD). NCCs are transient and multipotent migratory stem cells that give rise to diverse tissues, including cardiac structures such as the smooth muscles of the great arteries and semilunar valves. Induced pluripotent stem cells (iPSC) can be differentiated into NCCs, as demonstrated by expression of several marker proteins including NGFR and HNK1. To better define iPSC-NCCs and to better understand the progression of iPSCs to NCCs, we have compared the transcriptomes of iPSC and iPSC-NCCs. We have also begun to investigate the consequences of loss-of-function mutations in genes implicated in CHD on the differentiation of iPSC-NCCs. Methods and Results: PGP1 iPSCs were differentiated to NCCs and RNA was collected at 0, 5, 10, and 15 days of differentiation. RNAseq analysis showed that by day 15, 6483 genes were upregulated in NCC vs iPSCs, 6406 downregulated, and 6715 unchanged (FDR 5%). Enrichment analysis for the top 500 upregulated genes showed 4 cardiac gene ontology (GO) terms in the top 10, including ‘endocardial cushion morphogenesis’ (fold enrichment 11.85, p = 0.012). Notably, of 45 genes under GO term ‘endocardial cushion development’, 38 (84%) differentially expressed in NCCs by day 15 (FDR 5%). Next, we compared this RNAseq data with that of iPSC-derived cardiomyocyte (CM) differentiation in a subset of 253 genes previously implicated in CHD. Of these, 143 genes were differentially expressed in both iPSC-CMs and iPSC-NCCs. However, 27 genes (10.6%) including MYH6, PITX2, and TBX5 were uniquely upregulated during CM differentiation, while 65 genes (25.7%) including CHD4 and NOTCH1 were uniquely upregulated during NCC differentiation. Conclusion: Transcriptomic changes during iPSC-NCC differentiation, assessed by both bulk and single cell RNAseq, reveal an upregulation of genes involved in cardiac development, particularly endocardial cushions and the outflow tract. Importantly, a subset of genes implicated in CHD are altered during iPSC-NCC differentiation but not in iPSC-CM differentiation. Thus, iPSC-NCCs offer a new model with which to investigate the pathogenesis and mechanisms of CHD.

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.

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.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.

The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin-binding protein C; cMyBPC) or myosin missense mutations cause hypercontractility and poor relaxation in hypertrophic cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches, we explored how depletion of cMyBPC altered sarcomere function. We demonstrated that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM), normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation, enhancing the myosin state that enables ATP hydrolysis and thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with MYBPC3 mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across the cardiac cycle as the pathophysiologic mechanisms by which MYBPC3 truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in patients with DCM, whereas inhibiting myosin by MYK-461 should benefit the substantial proportion of patients with HCM with MYBPC3 mutations.

Activin type II receptor (ActRII) ligands have been implicated in muscle wasting in aging and disease. However, the role of these ligands and ActRII signaling in the heart remains unclear. Here, we investigated this catabolic pathway in human aging and heart failure (HF) using circulating follistatin-like 3 (FSTL3) as a potential indicator of systemic ActRII activity. FSTL3 is a downstream regulator of ActRII signaling, whose expression is up-regulated by the major ActRII ligands, activin A, circulating growth differentiation factor-8 (GDF8), and GDF11. In humans, we found that circulating FSTL3 increased with aging, frailty, and HF severity, correlating with an increase in circulating activins. In mice, increasing circulating activin A increased cardiac ActRII signaling and FSTL3 expression, as well as impaired cardiac function. Conversely, ActRII blockade with either clinical-stage inhibitors or genetic ablation reduced cardiac ActRII signaling while restoring or preserving cardiac function in multiple models of HF induced by aging, sarcomere mutation, or pressure overload. Using unbiased RNA sequencing, we show that activin A, GDF8, and GDF11 all induce a similar pathologic profile associated with up-regulation of the proteasome pathway in mammalian cardiomyocytes. The E3 ubiquitin ligase, Smurf1, was identified as a key downstream effector of activin-mediated ActRII signaling, which increased proteasome-dependent degradation of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a), a critical determinant of cardiomyocyte function. Together, our findings suggest that increased activin/ActRII signaling links aging and HF pathobiology and that targeted inhibition of this catabolic pathway holds promise as a therapeutic strategy for multiple forms of HF.

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.

Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD); however, the underlying CHD gene regulatory network (GRN) imbalances are unknown. Here, we define transcriptional consequences of reduced dosage of the CHD transcription factor, TBX5, in individual cells during cardiomyocyte differentiation from human induced pluripotent stem cells (iPSCs). We discovered highly sensitive dysregulation of TBX5-dependent pathways-including lineage decisions and genes associated with heart development, cardiomyocyte function, and CHD genetics-in discrete subpopulations of cardiomyocytes. Spatial transcriptomic mapping revealed chamber-restricted expression for many TBX5-sensitive transcripts. GRN analysis indicated that cardiac network stability, including vulnerable CHD-linked nodes, is sensitive to TBX5 dosage. A GRN-predicted genetic interaction between Tbx5 and Mef2c, manifesting as ventricular septation defects, was validated in mice. These results demonstrate exquisite and diverse sensitivity to TBX5 dosage in heterogeneous subsets of iPSC-derived cardiomyocytes and predicts candidate GRNs for human CHDs, with implications for quantitative transcriptional regulation in disease.

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.).

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

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.

Powering down yields a healthier heart In hypertrophic cardiomyopathy (HCM), the heart muscle enlarges and becomes progressively less efficient at pumping blood. HCM can be caused by mutations in components of the sarcomere (the heart's contractile unit), most notably myosin. Hypercontractility is among the earliest heart disturbances seen in mice carrying these myosin mutations, implying that the mutations inflict their damage by increasing myosin's power production. Green et al. identified a small molecule that binds to myosin and inhibits its activity (see the Perspective by Warshaw). When orally administered to young mice, the molecule prevented the development of several hallmark features of HCM without adversely affecting skeletal muscle. Science , this issue p. 617 ; see also p. 556

SummaryHeterozygous mice (αMHC403/+) expressing the human disease-causing mutation Arg403Gln exhibit cardinal features of hypertrophic cardiomyopathy (HCM) including hypertrophy, myocyte disarray, and increased myocardial fibrosis. Treatment of αMHC403/+mice with the L-type calcium channel (ICa-L) antagonist diltiazem has been shown to decrease left ventricular anterior wall thickness, cardiac myocyte hypertrophy, disarray, and fibrosis. However, the role of the ICa-L in the development of HCM is not known. In addition to maintaining cardiac excitation and contraction in myocytes, the ICa-L also regulates mitochondrial function through transmission of movement of ICa-L via cytoskeletal proteins to mitochondrial voltage-dependent anion channel. Here, the authors investigated the role of ICa-L in regulating mitochondrial function in αMHC403/+mice. Whole-cell patch clamp studies showed that ICa-L current inactivation kinetics were significantly increased in αMHC403/+cardiac myocytes, but that current density and channel expression were similar to wild-type cardiac myocytes. Activation of ICa-L caused a significantly greater increase in mitochondrial membrane potential and metabolic activity in αMHC403/+. These increases were attenuated with ICa-L antagonists and following F-actin or β-tubulin depolymerization. The authors observed increased levels of fibroblast growth factor-21 in αMHC403/+mice, and altered mitochondrial DNA copy number consistent with altered mitochondrial activity and the development of cardiomyopathy. These studies suggest that the Arg403Gln mutation leads to altered functional communication between ICa-L and mitochondria that is associated with increased metabolic activity, which may contribute to the development of cardiomyopathy. ICa-L antagonists may be effective in reducing the cardiomyopathy in HCM by altering metabolic activity.

Congenital heart disease (CHD) patients have an increased prevalence of extracardiac congenital anomalies (CAs) and risk of neurodevelopmental disabilities (NDDs). Exome sequencing of 1213 CHD parent-offspring trios identified an excess of protein-damaging de novo mutations, especially in genes highly expressed in the developing heart and brain. These mutations accounted for 20% of patients with CHD, NDD, and CA but only 2% of patients with isolated CHD. Mutations altered genes involved in morphogenesis, chromatin modification, and transcriptional regulation, including multiple mutations in RBFOX2, a regulator of mRNA splicing. Genes mutated in other cohorts examined for NDD were enriched in CHD cases, particularly those with coexisting NDD. These findings reveal shared genetic contributions to CHD, NDD, and CA and provide opportunities for improved prognostic assessment and early therapeutic intervention in CHD patients.

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 …

Although DNA methylation is the best characterized epigenetic mark, the mechanism by which it is targeted to specific regions in the genome remains unclear. Recent studies have revealed that local DNA methylation profiles might be dictated by cis-regulatory DNA sequences that mainly operate via DNA-binding factors. Consistent with this finding, we have recently shown that disruption of CTCF-binding sites by rare single nucleotide variants (SNVs) can underlie cis-linked DNA methylation changes in patients with congenital anomalies. These data raise the hypothesis that rare genetic variation at transcription factor binding sites (TFBSs) might contribute to local DNA methylation patterning. In this work, by combining blood genome-wide DNA methylation profiles, whole genome sequencing-derived SNVs from 247 unrelated individuals along with 133 predicted TFBS motifs derived from ENCODE ChIP-Seq data, we observed an association between the disruption of binding sites for multiple TFs by rare SNVs and extreme DNA methylation values at both local and, to a lesser extent, distant CpGs. While the majority of these changes affected only single CpGs, 24% were associated with multiple outlier CpGs within ±1kb of the disrupted TFBS. Interestingly, disruption of functionally constrained sites within TF motifs lead to larger DNA methylation changes at nearby CpG sites. Altogether, these findings suggest that rare SNVs at TFBS negatively influence TF-DNA binding, which can lead to an altered local DNA methylation profile. Furthermore, subsequent integration of DNA methylation and RNA-Seq profiles from cardiac tissues enabled us to observe an association between rare SNV-directed DNA methylation and outlier expression of nearby genes. In conclusion, our findings not only provide insights into the effect of rare genetic variation at TFBS on shaping local DNA methylation and its consequences on genome regulation, but also provide a rationale to incorporate DNA methylation data to interpret the functional role of rare variants., Author summary One of the major challenges for human genetics in the post-genomic era is to interpret the functional relevance of genetic variation. Quantitative trait locus (QTL) analyses have associated an important fraction of genetic variants with a wide range of molecular phenotypes including gene expression (eQTL) and DNA methylation (meQTL), providing insights into the mechanisms by which genetic variation can contribute to health and disease. Although QTL mapping represents an excellent approach to identify biologically relevant functional variants, these studies have been mainly focused on common variants and do not include low-frequency and rare variants. Here, we observed that rare regulatory variants, i.e, single nucleotide variants (SNVs) that disrupt transcription factor binding sites (TFBSs), are associated with changes in DNA methylation at both local and, to a lesser extent, broader locations, most likely, by altering the DNA-binding affinity of transcription factors (TFs). Interestingly, we have also shown that this change in DNA methylation can alter expression levels of nearby genes. Overall, these data suggest a role of rare regulatory SNVs in shaping DNA methylation, and suggest that the incorporation of DNA methylation data may help to interpret the functional consequences of human genetic variation.

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.

Dilated cardiomyopathy (DCM), a disorder that occurs in 1:250 individuals, is associated with rates of mortality of 20% within 5 years of diagnosis and is a leading cause for heart failure and cardiac transplantation. Mutations in the massive sarcomere protein titin (encoded by TTN ) are the most common genetic cause of DCM, occurring in 10-20% of cases. As dominant DCM mutations truncate titin (TTNtv) and result in haploinsufficiency, we predict that strategies to increase the expression of the wild type (WT) TTN allele might attenuate the damaging effects of TTNtv. We used bioinformatic analyses to identify a putative TTN enhancer within intron 1. To confirm its function, we deleted 658 bp from intron 1 that encompasses the putative TTN enhancer in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome editing. We used qPCR and RNA sequencing of RNA harvested from hiPSC-derived cardiomyocytes (hiPSC-CMs) and demonstrated that a homozygous deletion in this region leads to decreased TTN gene expression compared to the WT control (0.344 fold change, p < 0.001), and also to decreased expression of other sarcomeric genes such as TNNT2 (0.074 fold change, p < 0.001), MYH6 (0.18 fold change, p < 0.001), MYH7 (0.008 fold change, p < 0.001), and ACTN2 (0.118 fold change, p < 0.001). The expression of transcription factors (TF) that have binding sites in this region is also affected, such as MEIS2 (0.4 fold change, p < 0.001) and KLF6 (0.33 fold change, p < 0.001), both of which are known to be involved in cardiogenesis. These TF may act as a link between the deletion in TTN Intron 1 and a decreased transcription of other cardiac-relevant genes. We also utilized Assay for Transposase-Accessible Chromatin Sequencing (ATAC-Seq) on WT hiPSC-CMs to identify open regions of chromatin that are accessible for TF binding. This provided additional evidence that this 658 bp region in TTN intron 1 has enhancer activity. Ongoing studies are aiming to refine the TTN Intron 1 enhancer by further studies using CRISPR/Cas9, CRISPR droplet sequencing (CROP-Seq), and luciferase-based enhancer activity assays. If confirmed, we expect that increasing the activity of this 658 bp region with small molecules may provide a novel therapeutic target for DCM caused by TTNtv.

Background: MYBPC3 encodes cardiac myosin binding protein-C, which regulates sarcomeric stability and contractility. Previous work described a prevalent 25bp deletion in intron 32, MYBPC3 Δ 25bp , in the South Asian population. Occurring in an estimated 100 million carriers, this variant may be associated with cardiomyopathy and heart failure. MYBPC3 Δ25bp encompasses a splicing branch point that could affect splicing of exon 32 to exon 33, potentially resulting in an aberrantly spliced MYBPC3 transcript where exon 33 is skipped entirely. However, the mechanism by which MYBPC3 Δ 25bp associates with disease remains unclear. Methods: To determine the consequence of the 25bp deletion in vivo , mice were engineered that replaced the endogenous Mybpc3 intron 32 with either the normal human intron 32 or human intron 32 containing the 25bp deletion ( Mybpc3 Δ 25bp ) . We characterized Mybpc3 splicing in mice carrying human intron-32 and human iPSC derived cardiomyocytes (hiPSC-CM) from patients heterozygous and homozygous for MYBPC3 Δ 25bp by RT-PCR and using mini-gene assays (across 5 different mouse and human gene segments encompassing exon 32 through exon 34). Results: Echocardiography of mice 3 months of age revealed no changes in cardiac function by fractional shortening (WT: 32.8%, Het: 34.2%, Homo: 36.7%). However, homozygous Mybpc3 Δ 25bp mice had significantly smaller hearts compared to heterozygous and wildtype (3.4 vs 3.71 and 4.25 mg/g p=0.001). Mybpc3 Δ 25bp heterozygous and homozygous mice displayed minimal alternative splicing in exon 33 at baseline. Similarly, transcripts from hiPSC-CM from heterozygous and homozygous MYBPC3 Δ 25bp carriers also show minimal alternative splicing. Mini gene assays suggest that the 25bp deletion does reduce the proportion (77.5% v. 86.2%) of normally spliced transcripts. Conclusions: Taken together, these data suggest that under baseline conditions, MYBPC3 Δ 25bp results in minimal alternative splicing in both human iPSC-CMs and humanized mice harboring the 25bp deletion. While increase aberrant splicing may increase risk of hypertrophic cardiomyopathy in variant-carrying individuals, ongoing studies will determine whether other factors or gene mutations modulate the pathogenicity of the MYBPC3 Δ25bp variant.

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.

We describe a method to transform blood lymphocytes into human-induced pluripotent stem cells by delivering four transcription factors with a non-integrative virus. Using human peripheral blood mononuclear cells (PBMCs) as the source cell type for hiPSC reprogramming is advantageous since blood samples are rapidly and safely obtained from nearly-all subjects. Reprogramming factors needed to make hiPSCs are introduced by infecting the PBMCs with non-integrating Sendai virus vectors. Reprogrammed cells can subsequently be quickly expanded for downstream use. In this unit, we present current protocols for the isolation of PBMCs from a small sample of human blood and subsequent viral reprogramming and expansion of PBMCs into hiPSCs. © 2018 by John Wiley & Sons, Inc.

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.

The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin binding protein-C; cMyBPC) or myosin missense mutations cause hyper-contractility and poor relaxation in hypertrophic cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches we explored how depletion of cMyBPC altered sarcomere function. We demonstrate that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM) normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation - enhancing the myosin state that enables ATP hydrolysis and thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with MYBPC3 mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across all phases of the cardiac cycle as the pathophysiologic mechanisms by which MYBPC3 truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in DCM patients, while inhibiting myosin by MYK-461 should benefit the substantial proportion of HCM patients with MYBPC3 mutations.One Sentence SummaryAnalyses of cardiomyocytes with hypertrophic cardiomyopathy mutations in MYBPC3 reveal that these directly activate myosin contraction by disrupting myosin states of relaxation, and that genetic or pharmacological manipulation of myosin therapeutically abates the effects of MYBPC3 mutations.