Search

SummaryCardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and strategies to improve therapeutic opportunities require deeper understanding of the molecular processes of the normal heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavor. Here, using large-scale single cell and nuclei transcriptomic profiling together with state-of-the-art analytical techniques, we characterise the adult human heart cellular landscape covering six anatomical cardiac regions (left and right atria and ventricles, apex and interventricular septum). Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, revealing distinct subsets in the atria and ventricles indicative of diverse developmental origins and specialized properties. Further we define the complexity of the cardiac vascular network which includes clusters of arterial, capillary, venous, lymphatic endothelial cells and an atrial-enriched population. By comparing cardiac cells to skeletal muscle and kidney, we identify cardiac tissue resident macrophage subsets with transcriptional signatures indicative of both inflammatory and reparative phenotypes. Further, inference of cell-cell interactions highlight a macrophage-fibroblast-cardiomyocyte network that differs between atria and ventricles, and compared to skeletal muscle. We expect this reference human cardiac cell atlas to advance mechanistic studies of heart homeostasis and disease.

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.

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.

Rationale : Approximately 40% of hypertrophic cardiomyopathy (HCM) is caused by heterozygous missense mutations in β-cardiac myosin heavy chain (β-MHC). Associating disease phenotype with mutation is confounded by extensive background genetic and lifestyle/environmental differences between subjects even from the same family. Objective : To characterize disease caused by β-cardiac myosin heavy chain Val606Met substitution (VM) that has been identified in several HCM families with wide variation of clinical outcomes, in mice. Methods and Results : Unlike 2 mouse lines bearing the malignant myosin mutations Arg453Cys (RC/+) or Arg719Trp (RW/+), VM/+ mice with an identical inbred genetic background lacked hallmarks of HCM such as left ventricular hypertrophy, disarray of myofibers, and interstitial fibrosis. Even homozygous VM/VM mice were indistinguishable from wild-type animals, whereas RC/RC- and RW/RW-mutant mice died within 9 days after birth. However, hypertrophic effects of the VM mutation were observed both in mice treated with cyclosporine, a known stimulator of the HCM response, and compound VM/RC heterozygous mice, which developed a severe HCM phenotype. In contrast to all heterozygous mutants, both systolic and diastolic function of VM/RC hearts was severely impaired already before the onset of cardiac remodeling. Conclusions : The VM mutation per se causes mild HCM-related phenotypes; however, in combination with other HCM activators it exacerbates the HCM phenotype. Double-mutant mice are suitable for assessing the severity of benign mutations.

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a “second hit,” that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome., Author Summary Children with cartilage tumor syndromes form multiple tumors of cartilage next to joints. These tumors can occur inside the bones, as with Ollier disease and Maffuci syndrome, or on the surface of bones, as in the Multiple Osteochondroma syndrome (MO). In a hybrid syndrome, called metachondromatosis (MC), patients develop tumors both on and within bones. Only the genes causing MO are known. Since MC is inherited, we studied genetic markers in an affected family and found a region of the genome, encompassing 100 genes, always passed on to affected members. Using a recently developed method, we captured and sequenced all 100 genes in multiple families and found mutations in one gene, PTPN11, in 11 of 17 families. Patients with MC have one mutant copy of PTPN11 from their affected parent and one normal copy from their unaffected parent in all cells. We found that the normal copy is additionally lost in cartilage cells that form tumors, giving rise to cells without PTPN11. Mutations in PTPN11 were not found in other cartilage tumor syndromes, including Ollier disease and Maffucci syndrome. We are currently working to understand how loss of PTPN11 in cartilage cells causes tumors to form.

We sought to further define the role of sarcomere mutations in dilated cardiomyopathy (DCM) and associated clinical phenotypes.Mutations in several contractile proteins contribute to DCM, but definitive evidence for the roles of most sarcomere genes remains limited by the lack of robust genetic support.Direct sequencing of 6 sarcomere genes was performed on 334 probands with DCM. A novel D230N missense mutation in the gene encoding alpha-tropomyosin (TPM1) was identified. Functional assessment was performed by the use of an in vitro reconstituted sarcomere complex to evaluate ATPase regulation and Ca(2+) affinity as correlates of contractility.TPM1 D230N segregated with DCM in 2 large unrelated families. This mutation altered an evolutionarily conserved residue and was absent in1,000 control chromosomes. In vitro studies demonstrated major inhibitory effects on sarcomere function with reduced Ca(2+) sensitivity, maximum activation, and Ca(2+) affinity compared with wild-type TPM1. Clinical manifestations ranged from decompensated heart failure or sudden death in those presenting early in life to asymptomatic left ventricular dysfunction in those diagnosed during adulthood. Notably, several affected infants had remarkable improvement.Genetic segregation in 2 unrelated families and functional analyses conclusively establish a pathogenic role for TPM1 mutations in DCM. In vitro results demonstrate contrasting effects of DCM and hypertrophic cardiomyopathy mutations in TPM1, suggesting that specific functional consequences shape cardiac remodeling. Along with previous reports, our data support a distinctive, age-dependent phenotype with sarcomere-associated DCM where presentation early in life is associated with severe, sometimes lethal, disease. These observations have implications for the management of familial DCM.

Background and Purpose— Familial aggregation of intracranial aneurysms (IA) strongly suggests a genetic contribution to pathogenesis. However, genetic risk factors have yet to be defined. For families affected by aortic aneurysms, specific gene variants have been identified, many affecting the receptors to transforming growth factor-beta (TGF-β). In recent work, we found that aortic and intracranial aneurysms may share a common genetic basis in some families. We hypothesized, therefore, that mutations in TGF-β receptors might also play a role in IA pathogenesis. Methods— To identify genetic variants in TGF-β and its receptors, TGFB1 , TGFBR1 , TGFBR2 , ACVR1 , TGFBR3 , and ENG were directly sequenced in 44 unrelated patients with familial IA. Novel variants were confirmed by restriction digestion analyses, and allele frequencies were analyzed in cases versus individuals without known intracranial disease. Similarly, allele frequencies of a subset of known SNPs in each gene were also analyzed for association with IA. Results— No mutations were found in TGFB1 , TGFBR1 , TGFBR2 , or ACVR1 . Novel variants identified in ENG (p.A60E) and TGFBR3 (p.W112R) were not detected in at least 892 reference chromosomes. ENG p.A60E showed significant association with familial IA in case-control studies ( P =0.0080). No association with IA could be found for any of the known polymorphisms tested. Conclusions— Mutations in TGF-β receptor genes are not a major cause of IA. However, we identified rare variants in ENG and TGFBR3 that may be important for IA pathogenesis in a subset of families.

The hedgehog genes encode signaling molecules that play a role in regulating embryonic morphogenesis. We have cloned and sequenced human cDNA copies of two of these genes, SHH and IHH. The SHH clone includes the full coding sequence and encodes a protein 92.4% identical to its murine homologue. The IHH clone is 89% complete and encodes a protein 94.6% identical to its murine homologue. IHH is expressed in adult kidney and liver. SHH expression was not detected in adult tissues examined; however, it is expressed in fetal intestine, liver, lung, and kidney. SHH mapped to chromosome 7q and IHH to chromosome 2 by PCR with DNA from a panel of rodent-human somatic cell hybrids. To identify the chromosomal location of SHH more precisely, a P1 genomic clone of SHH was isolated. This phage contained a CA repeat sequence tagged site that was used to map SHH relative to a polysyndactyly disease locus, using DNA prepared from affected and unaffected members of a large pedigree. SHH is closely linked, but distinct from the polysyndactyly disease locus at 7q36 (maximum lod score = 4.82, theta = 0.05) tightly linked to the EN2 locus. The murine homologues Shh, Ihh, and Dhh were mapped using (C57BL/6J x Mus spretus)F1 x C57BL/6J interspecific backcross. Shh mapped to a position 0.6 cM distal to En2 and 1.9 cM proximal to Il6 on mouse chromosome 5. This location is closely linked but distinct from the murine limb mutation Hx and syntenic to human chromosome 7q36.

OBJECTIVES We sought to determine whether the development of left ventricular hypertrophy (LVH) can be demonstrated during adulthood in genetically affected relatives with hypertrophic cardiomyopathy (HCM). BACKGROUND Hypertrophic cardiomyopathy is a heterogeneous cardiac disease caused by mutations in nine genes that encode proteins of the sarcomere. Mutations in cardiac myosin-binding protein C (MyBPC) gene have been associated with age-related penetrance. METHODS To further analyze dormancy of LVH in patients with HCM, we studied, using echocardiography and 12-lead electrocardiography, the phenotypic expression caused by MyBPC mutations in seven genotyped pedigrees. RESULTS Of 119 family members studied, 61 were identified with a MyBPC mutation, including 21 genetically affected relatives (34%) who did not express the HCM morphologic phenotype (by virtue of showing normal left ventricular wall thickness). Of these 21 phenotype-negative individuals, 9 were children, presumably in the prehypertrophic phase, and 12 were adults. Of the 12 adults with normal wall thickness ≤12 mm (7 also with normal electrocardiograms), 5 subsequently underwent serial echocardiography prospectively over four to six years. Of note, three of these five adults showed development of LVH in mid-life, appearing for the first time at 33, 34 and 42 years of age, respectively, not associated with outflow obstruction or significant symptoms. CONCLUSIONS In adults with HCM, disease-causing MyBPC mutations are not uncommonly associated with absence of LVH on echocardiogram. Delayed remodeling with the development of LVH appearing de novo in adulthood, demonstrated here for the first time in individual patients with prospectively obtained serial echocardiograms, substantiates the principle of age-related penetrance for MyBPC mutations in HCM. These observations alter prevailing perceptions regarding the HCM clinical spectrum and family screening strategies and further characterize the evolution of LVH in this disease.