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Background:HeterozygousTTNtruncating variants cause 10% to 20% of idiopathic dilated cardiomyopathy (DCM). Although variants which disrupt canonical splice signals (ie, invariant dinucleotide of the splice donor site, invariant dinucleotide of the splice acceptor site) at exon-intron junctions are readily recognized asTTNtruncating variants, the effects of other nearby sequence variations on splicing and their contribution to disease is uncertain.Methods:Rare variants of unknown significance located in the splice regions of highly expressedTTNexons from 203 DCM cases, 3329 normal subjects, and clinical variant databases were identified. The effects of these variants on splicing were assessed using an in vitro splice assay.Results:Splice-altering variants of unknown significance were enriched in DCM cases over controls and present in 2% of DCM patients (P=0.002). Application of this method to clinical variant databases demonstrated 20% of similar variants of unknown significance inTTNsplice regions affect splicing. Noncanonical splice-altering variants were most frequently located at position +5 of the donor site (P=4.4×107) and position -3 of the acceptor site (P=0.002). SpliceAI, an emerging in silico prediction tool, had a high positive predictive value (86%–95%) but poor sensitivity (15%–50%) for the detection of splice-altering variants. Alternate exons spliced out of mostTTNtranscripts frequently lacked the consensus base at +5 donor and −3 acceptor positions.Conclusions:Noncanonical splice-altering variants inTTNexplain 1-2% of DCM and offer a 10-20% increase in the diagnostic power ofTTNsequencing in this disease. These data suggest rules that may improve efforts to detect splice-altering variants in other genes and may explain the low percent splicing observed for many alternateTTNexons.

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.

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.

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.

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.

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.

Background— Atrial fibrillation (AF) is the most common adverse event following coronary artery bypass graft surgery. A recent study identified chromosome 4q25 variants associated with AF in ambulatory populations. However, their role in postoperative AF is unknown. We hypothesized that genetic variants in the 4q25 chromosomal region are independently associated with postoperative AF after coronary artery bypass graft surgery. Methods and Results— Two prospectively collected cohorts of patients undergoing coronary artery bypass graft surgery, with or without concurrent valve surgery, at 3 US centers. From a discovery cohort of 959 patients, clinical and genomic multivariate predictors of postoperative AF were identified by genotyping 45 single-nucleotide polymorphisms (SNPs) encompassing the 4q25 locus. Three SNPs were then assessed in a separately collected validation cohort of 494 patients. After adjustment for clinical predictors of postoperative AF and multiple comparisons, rs2200733, rs13143308, and 5 other linked SNPs independently predicted postoperative AF in the discovery cohort. Additive odds ratios for the 7 associated 4q25 SNPs ranged between 1.57 and 2.17 ( P =8.0�10 −4 to 3.4�10 −6 ). Association with postoperative AF were measured and replicated for rs2200733 and rs13143308 in the validation cohort. Conclusions— In 2 independently collected cardiac surgery cohorts, noncoding SNPs within the chromosome 4q25 region are independently associated with postoperative AF after coronary artery bypass graft surgery after adjusting for clinical covariates and multiple comparisons.

Background— Cardiac myosin binding protein-C (cMyBP-C) is a thick-filament protein whose presence and phosphorylation by protein kinase A (PKA) regulates cross-bridge formation and kinetics in isolated myocardium. We tested the influence of cMyBP-C and its PKA-phosphorylation on contraction/relaxation kinetics in intact hearts and revealed its essential role in several classic properties of cardiac function. Methods and Results— Comprehensive in situ cardiac pressure–volume analysis was performed in mice harboring a truncation mutation of cMyBP-C (cMyBP-C (t/t) ) that resulted in nondetectable protein versus hearts re-expressing solely wild-type (cMyBP-C WT:(t/t) ) or mutated protein in which known PKA-phosphorylation sites were constitutively suppressed (cMyBP-C AllP-:(t/t) ). Hearts lacking cMyBP-C had faster early systolic activation, which then terminated prematurely, limiting ejection. Systole remained short at faster heart rates; thus, cMyBP-C (t/t) hearts displayed minimal rate-dependent decline in diastolic time and cardiac preload. Furthermore, prolongation of pressure relaxation by afterload was markedly blunted in cMyBP-C (t/t) hearts. All 3 properties were similarly restored to normal in cMyBP-C WT:(t/t) and cMyBP-C AllP-:(t/t) hearts, which supports independence of PKA-phosphorylation. However, the dependence of peak rate of pressure rise on preload was specifically depressed in cMyBP-C AllP-:(t/t) hearts, whereas cMyBP-C (t/t) and cMyBP-C AllP-:(t/t) hearts had similar blunted adrenergic and rate-dependent contractile reserve, which supports linkage of these behaviors to PKA-cMyBP-C modification. Conclusions— cMyBP-C is essential for major properties of cardiac function, including sustaining systole during ejection, the heart-rate dependence of the diastolic time period, and relaxation delay from increased arterial afterload. These are independent of its phosphorylation by PKA, which more specifically modulates early pressure rise rate and adrenergic/heart rate reserve.

Alternative splicing (AS) of protein isoforms is an integral mechanism for cardiac development. RNA Binding Protein, Fox-1 Homolog (C. Elegans) 2 (RBFOX2) is an RNA binding protein preferentially expressed in muscle and neuronal cells and regulates tissue-specific alternate exon splicing in ~2,100 target genes by binding the conserved RNA sequence motif (U)GCAUG. RBFOX2 was recently implicated in the pathogenesis of abnormal cardiac and cerebral development via loss-of-function studies in zebrafish and mouse. However, convincing evidence remains incomplete, as the full complement of RBFOX2 target genes and differential exon usage (DEU) in human cardiovascular cell lines are incompletely defined. We identified de novo mutations in RBFOX2 from four human cases of congenital heart disease (CHD) with hypoplastic left heart syndrome (HLHS) via whole exome sequencing. To test the hypothesis that RBFOX2 mutations alter DEU in known target genes, we performed RNA-seq on ductus arteriosus tissue from human CHD cases with and without RBFOX2 mutation. Analysis of RNA-seq for DEU was performed with DEXSeq. To limit the effect of differential gene expression, we restricted analysis to subjects with high global gene expression correlation (r2 > 0.9, case=1 vs. control=5). DEU in known RBFOX2 target genes were highly enriched compared to all known genes (115/2,100 vs. 589/26,310, p=5.78e-15). A high percentage of the DEU genes (60.0%, 69/115) have high heart expression (HHE) in the developing mouse (mean expression of four cardiac chambers at e14.5). DEU genes with HHE include VCL, TPM1, FN1, ACTN1, and CALD1. Functional annotation clustering reveals enrichment for several actin, cytoskeletal, and contractile Gene Ontology terms, suggesting a possible role in the epithelial-mesenchymal transition (EMT) developmental processes active during early cardiac formation. We also identified enrichment from genes implicated in CHD by allelic specific expression or de novo mutations (44/1,263 vs. 589/26,310, p=0.007). These results are the first in humans to identify differentially expressed exons associated with RBFOX2 mutations in CHD and suggests RBFOX2-mediated alternate splicing may influence EMT pathways implicated in the pathogenesis of HLHS.

Mutations in the human gene encoding the nucleotide-binding region in the gamma-subunit of AMP-activated protein kinase (AMPK) cause cardiomyopathy with preexcitation syndrome. Mutant AMPK showed reduced binding affinity to nucleotides in vitro raising the possibility that altered regulation of AMPK activity by AMP/ATP could contribute to the disease phenotype. In this study, we determined the sensitivity of AMPK activity to AMP/ATP in the beating hearts using transgenic mice expressing a mutant (N488I, gamma2-mutant) or wild-type gamma2-subunit (gamma2-TG). The [ATP] and [AMP] were unaltered in all hearts but the AMPK activity was increased by 2.5-fold in gamma2-mutant hearts freeze-clamped at normal AMP/ATP compared with nontransgenic (WT) or gamma2-TG. The increased basal AMPK activity was caused by increased Thr-172 phosphorylation of the alpha-subunit (p-AMPK, by 4-fold) at normal [ATP] and was not changed by reducing glycogen content by 60% in the gamma2-mutant hearts. A reversal of AMP/ATP, caused by ATP degradation, increased p-AMPK by 7-fold in WT but caused no change in gamma2-mutant hearts. These results demonstrate that the mutation renders AMPK insensitive to the inhibitory and stimulatory effects of the regulatory nucleotides ATP and AMP, respectively, suggesting that the pathogenesis of the human disease may not be attributable to a simple loss- or gain-of-function.

Background —Inherited gene defects are an important cause of dilated cardiomyopathy. Although the chromosome locations of some defects and 1 disease gene (actin) have been identified, the genetic etiologies of most cases of familial dilated cardiomyopathy remain unknown. Methods and Results —We clinically evaluated 3 generations of a kindred with autosomal dominant transmission of dilated cardiomyopathy. Nine surviving and affected individuals had early-onset disease (ventricular chamber dilation during the teenage years and congestive heart failure during the third decade of life). The disease was nonpenetrant in 2 obligate carriers. To identify the causal gene defect, linkage studies were performed. A new dilated cardiomyopathy locus was identified on chromosome 2 between loci GCG and D2S72 (maximum logarithm of odds [LOD] score=4.86 at θ=0). Because the massive gene encoding titin, a cytoskeletal muscle protein, resides in this disease interval, sequences encoding 900 amino acid residues of the cardiac-specific (N2-B) domain were analyzed. Five sequence variants were identified, but none segregated with disease in this family. Conclusions —A dilated cardiomyopathy locus (designated CMD1G ) is located on chromosome 2q31 and causes early-onset congestive heart failure. Although titin remains an intriguing candidate gene for this disorder, a disease-causing mutation is not present in its cardiac-specific N2-B domain.

Abstract We created a mouse that lacks a functional α-tropomyosin gene using gene targeting in embryonic stem cells and blastocyst-mediated transgenesis. Homozygous α-tropomyosin “knockout” mice die between embryonic day 9.5 and 13.5 and lack α-tropomyosin mRNA. Heterozygous α-tropomyosin knockout mice have ≈50% as much cardiac α-tropomyosin mRNA as wild-type littermates but similar α-tropomyosin protein levels. Cardiac gross morphology, histology, and function (assessed by working heart preparations) of heterozygous α-tropomyosin knockout and wild-type mice were indistinguishable. Mechanical performance of skinned papillary muscle strips derived from mutant and wild-type hearts also revealed no differences. We conclude that haploinsufficiency of the α-tropomyosin gene produces little or no change in cardiac function or structure, whereas total α-tropomyosin deficiency is incompatible with life. These findings imply that in heterozygotes there is a regulatory mechanism that maintains the level of myofibrillar tropomyosin despite the reduction in α-tropomyosin mRNA.