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Hypertrophic cardiomyopathy (HCM) is a genetic disease of the myocardium characterized by a hypertrophic left ventricle with a preserved or increased ejection fraction. Cardiac hypertrophy is often asymmetrical, which is associated with left ventricular outflow tract obstruction. Myocyte hypertrophy, disarray, and myocardial fibrosis constitute the histological features of HCM. HCM is a relatively benign disease but an important cause of sudden cardiac death in the young and heart failure in the elderly. Pathogenic variants (PVs) in genes encoding protein constituents of the sarcomeres are the main causes of HCM. PVs exhibit a gradient of effect sizes, as reflected in their penetrance and variable phenotypic expression of HCM. MYH7 and MYBPC3 , encoding β-myosin heavy chain and myosin binding protein C, respectively, are the two most common causal genes and responsible for ≈40% of all HCM cases but a higher percentage of HCM in large families. PVs in genes encoding protein components of the thin filaments are responsible for ≈5% of the HCM cases. Whereas pathogenicity of the genetic variants in large families has been firmly established, ascertainment causality of the PVs in small families and sporadic cases is challenging. In the latter category, PVs are best considered as probabilistic determinants of HCM. Deciphering the genetic basis of HCM has enabled routine genetic testing and has partially elucidated the underpinning mechanism of HCM as increased number of the myosin molecules that are strongly bound to actin. The discoveries have led to the development of mavacamten that targets binding of the myosin molecule to actin filaments and imparts beneficial clinical effects. In the coming years, the yield of the genetic testing is expected to be improved and the so-called missing causal gene be identified. The advances are also expected to enable development of additional specific therapies and editing of the mutations in HCM.

Rationale: Hypertrophic cardiomyopathy (HCM) is a prototypic single-gene disease caused mainly by mutations in genes encoding sarcomere proteins. Despite the remarkable advances, the causal genes in ≈40% of the HCM cases remain unknown, typically in small families and sporadic cases, wherein cosegregation could not be established. Objective: To test the hypothesis that the missing causal genes in HCM is, in part, because of an oligogenic cause, wherein the pathogenic variants do not cosegregate with the phenotype. Methods and Results: A clinically affected trio with HCM underwent clinical evaluation, electrocardiography, echocardiography, magnetic resonance imaging, and whole exome sequencing. Pathogenic variants in the whole exome sequencing data were identified using established algorithms. Family members were genotyped by Sanger sequencing and cosegregation was analyzed. The siblings had a severe course, whereas the mother had a mild course. Variant analysis showed that the trio shared 145 heterozygous pathogenic variants in 139 genes, including 2 in cardiomyopathy genes TTN and ALPK3 . The siblings also had the pathogenic variant p.Ala13Thr variant in MYL2 , a known gene for HCM. The sibling’s father also carried the p.Ala13Thr variant, in whom an unambiguous diagnosis of HCM could not be made because of concomitant severe aortic stenosis. The TTN variant segregated with HCM, except in a 7-year-old boy, who had a normal phenotype. The ALPK3 variant, shared by the affected trio, did not segregate with the phenotype. Conclusions: We posit that a subset of HCM might be oligogenic caused by multiple pathogenic variants that do not perfectly cosegregate with the phenotype.

Rationale: Mutations in the LMNA gene, encoding nuclear inner membrane protein lamin A/C, cause distinct phenotypes, collectively referred to as laminopathies. Heart failure, conduction defects, and arrhythmias are the common causes of death in laminopathies. Objective: The objective of this study was to identify and therapeutically target the responsible mechanism(s) for cardiac phenotype in laminopathies. Methods and Results: Whole-heart RNA sequencing was performed before the onset of cardiac dysfunction in the Lmna −/− and matched control mice. Differentially expressed transcripts and their upstream regulators were identified, validated, and targeted by adeno-associated virus serotype 9-short hairpin RNA constructs. A total of 576 transcripts were upregulated and 233 were downregulated in the Lmna −/− mouse hearts ( q Lmna −/− cardiac myocytes and in the myocardium of human heart failure patients. Nuclear localization of FOXO1 and 3 was increased, whereas phosphorylated (inactive) FOXO1 and 3 levels were reduced in the Lmna −/− hearts. Gene set enrichment analysis and gene ontology showed activation of apoptosis and inflammation and suppression of cell cycle, adipogenesis, and oxidative phosphorylation in the Lmna −/− hearts. Adeno-associated virus serotype 9-short hairpin RNA–mediated suppression of FOXO TFs rescued selected molecular signatures, improved apoptosis, and prolonged survival by ≈2-fold. Conclusions: FOXO TFs are activated and contribute to the pathogenesis of cardiac phenotype in laminopathies. Suppression of the FOXO TFs in cardiac myocytes partially rescues the phenotype and prolongs survival. The findings identify FOXO TFs as potential therapeutic targets for cardiac phenotype in laminopathies.

The progress in congenital heart disease (CHD), since the first anatomic depiction of an anomalous pulmonary venous return by Leonardo da Vinci in 1513, has been unabated. The course has been decorated with occasional sparks stemming largely from the ingenuity of pioneering physicians and surgeons who departed from the prevailing notions and brought forth new brave ideas to diagnosis and management of patients with CHD (Table). The progress, however, was not without frequent disappointments. The extremely high mortality rate in the early days of open-heart surgery with the use of extracorporeal circulatory machine disappointed John Gibbons, the inventor of the first heart–lung machine, to the point that he called for a long-term moratorium on the use of his invention. Yet, the convictions of pioneering surgeons such as C. Walton Lillehei and John Kirklin led them to forge ahead with improved versions of the pump, ultimately changing the treatment of CHD. Advances in diagnostic methods and surgical treatment during the past several decades have transformed CHD malformations from being “hopeless futilities” in neonates, infants, and children to treatable conditions with >90% of patients surviving into the adult years. View this table: Table. Timeline of Some of the Scientific Discoveries and Developments in Congenital Heart Disease Of the ≈125 million children born every year (www.unicef.org), it is estimated that about 1 000 000 to 1 500 000 are born with a CHD. Because of the scientific and technological advances during the past several decades, the vast majority of these children survive to adulthood, and those with less complicated lesions expect a life expectancy similar to that of the general population. Consequently, it is estimated that worldwide ≈50 million adults live with CHD, including ≈1.5 million in the United States alone, more than the number of children with CHD. As a result of an excellent survival rate, the landscape of CHD …

Lamin A/C (LMNA) is a major component of the nuclear membrane, involved in regulation of gene expression. Mutations in the LMNA gene cause a heterogeneous group of diseases, collectively referred to as laminopathies. Cardiac involvement is one of the most prominent feature of the laminopathies, presenting with dilated cardiomyopathy (DCM), conduction defects, and sudden death. Molecular mechanisms involved in the pathogenesis of cardiac phenotypes in laminopathies are not well known. To gain insights into molecular pathogenesis of DCM caused by LMNA deficiency, Lmna gene was deleted in cardiac myocytes ( Myh6-Cre:Lmna Flox ). Homozygous ( Myh6-Cre:Lmna F/F ) had a normal cardiac function and histology at 2 weeks of age. However, the mice showed progressive cardiac dilatation and dysfunction, interstitial fibrosis, increased apoptosis, and died by 4 weeks of age. To identify transcriptomic changes that precede cardiac dysfunction, cardiomyocytes were isolated from 2-week old wild type (WT) and Myh6-Cre:Lmna F/F mice and analyzed by RNA-Seq. A total of 1,420 genes were differentially expressed between WT and Myh6-Cre:Lmna F/F . Pathways analysis of differentially expressed genes (DEGs) showed enrichment of inflammatory, epithelial to mesenchymal transition, and cell death pathways, whereas mitochondrial oxidative function, adipogenesis, and fatty acid metabolism pathways were depressed in Myh6-Cre:Lmna F/F . Consistently, DEGs were enriched for the binding motif of RELA, the main transcription factor of the NFκB1 pathway. Activation of the NFκB1 pathway was validated by western blot showing increased nuclear localization of RELA. To determine therapeutic benefits of targeting the NFκB1 pathway, mice are being treated a specific inhibitor of NFκB (IKK-16). Effects on survival, cardiac function, apoptosis, and fibrosis are being analyzed. In conclusion, LMNA deficiency results in dysregulation of a large number genes, including NFκB1 pathway target genes that precede the onset of cardiac dysfunction and fibrosis. The findings point to the prominent role of inflammation in the development of cardiac dysfunction in myocyte-specific LMNA deficiency and identify the NFκB1 pathway as a therapeutic target in DCM caused by LMNA deficiency.

Rationale: Lamin A/C (LMNA) is a nuclear inner membrane protein that interacts with genome through Lamin-Associated Domains (LADs) and regulates gene expression. Mutations in the LMNA gene causes a diverse array of diseases that are collectively referred to as laminopathies. Dilated cardiomyopathy (DCM) is a major component of laminopathies and the leading cause of premature death. Objective: To identify and characterize LADs in cardiac myocytes, cardiac DNA methylation status, and transcriptomes and integrate the findings to find the pathogenic pathways in DCM Methods: We performed ChIP-Seq, reduced representative bisulfite sequencing (RRBS), and RNA-Seq in 5 control and 5 DCM hearts, the later with defined pathogenic variants. ChIP-Seq was performed using an anti-LMNA antibody on protein extracts from FACS-isolated myocyte nuclei, identified by expression of PCM1. RRBS was performed on whole heart DNA extracts to identify methylation state of ~ 500,000 CpG sites. RNA-Seq was performed on whole heart ribosome-depleted RNA extracts. LADs were identified using Enriched Domain Detector (EDD). Results: We identified 370±50 LADs with an average size of ~ 1Mbp (range: 0.5 to 10 Mb), collectively covering about 12,000 genes and 12% of the genomic regions in the control myocytes. The number and size of the LADs were unchanged in DCM. However, genomic locations of LADs had shifted, leading to loss of 3,302 genes from the LADs. Integration of LADs, CpG methylation, and transcript levels showed CpG hypermethylation and suppressed transcript levels in the LADs both in control and in DCM hearts. However, the effects were differential and LADs were associated with increased hypermethylation and further suppression of gene expression in DCM hearts. Pathway analysis of integrated omic datasets identified TGFβ1 and cell cycle pathways as the most activated and MPAK and glycolytic pathways as the most repressed pathways in DCM hearts. Conclusions: LADs, CpG methylation and transcriptomes are dysregulated in cardiac myocytes in patients with DCM caused by LMNA pathogenic variants. The dysregulated biological pathways provide potential targets for interventions in DCM caused by LMNA mutations.

Rationale: Hypertrophic cardiomyopathy (HCM) is a genetic paradigm of cardiac hypertrophy. Cardiac hypertrophy and interstitial fibrosis are important risk factors for sudden death and morbidity in HCM. Oxidative stress is implicated in the pathogenesis of cardiac hypertrophy and fibrosis. Treatment with antioxidant N-acetylcysteine (NAC) reverses cardiac hypertrophy and fibrosis in animal models of HCM. Objective: To determine effect sizes of NAC on indices of cardiac hypertrophy and fibrosis in patients with established HCM. Methods and Results: HALT-HCM (Hypertrophy Regression With N-Acetylcysteine in Hypertrophic Cardiomyopathy) is a double-blind, randomized, sex-matched, placebo-controlled single-center pilot study in patients with HCM. Patients with HCM, who had a left ventricular wall thickness of ≥15 mm, were randomized either to a placebo or to NAC (1:2 ratio, respectively). NAC was titrated ≤2.4 g per day. Clinical evaluation, blood chemistry, and 6-minute walk test were performed every 3 months, and electrocardiography, echocardiography, and cardiac magnetic resonance imaging, the latter whenever not contraindicated, before and after 12 months of treatment. Eighty-five of 232 screened patients met the eligibility criteria, 42 agreed to participate; 29 were randomized to NAC and 13 to placebo groups. Demographic, echocardiographic, and cardiac magnetic resonance imaging phenotypes at the baseline between the 2 groups were similar. WSE in 38 patients identified a spectrum of 42 pathogenic variants in genes implicated in HCM in 26 participants. Twenty-four patients in the NAC group and 11 in the placebo group completed the study. Six severe adverse events occurred in the NAC group but were considered unrelated to NAC. The effect sizes of NAC on the clinical phenotype, echocardiographic, and cardiac magnetic resonance imaging indices of cardiac hypertrophy, function, and extent of late gadolinium enhancement—a surrogate for fibrosis—were small. Conclusions: Treatment with NAC for 12 months had small effect sizes on indices of cardiac hypertrophy or fibrosis. The small sample size of the HALT-HCM study hinders from making firm conclusions about efficacy of NAC in HCM. Clinical Trial Registration: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01537926.

Rationale: PKP2 , encoding plakophilin 2 (PKP2), is the most common causal gene for arrhythmogenic cardiomyopathy. Objective: To characterize miRNA expression profile in PKP2-deficient cells. Methods and Results: Control and PKP2-knockdown HL-1 (HL-1 Pkp2-shRNA ) cells were screened for 750 miRNAs using low-density microfluidic panels. Fifty-nine miRNAs were differentially expressed. MiR-184 was the most downregulated miRNA. Expression of miR-184 in the heart and cardiac myocyte was developmentally downregulated and was low in mature myocytes. MicroRNA-184 was predominantly expressed in cardiac mesenchymal progenitor cells. Knockdown of Pkp2 in cardiac mesenchymal progenitor cells also reduced miR-184 levels. Expression of miR-184 was transcriptionally regulated by the E2F1 pathway, which was suppressed in PKP2-deficient cells. Activation of E2F1, on overexpression of its activator CCND1 (cyclin D1) or knockdown of its inhibitor retinoblastoma 1, partially rescued miR-184 levels. In addition, DNA methyltransferase-1 was recruited to the promoter region of miR-184, and the CpG sites at the upstream region of miR-184 were hypermethylated. Treatment with 5-aza-2′-deoxycytidine, a demethylation agent, and knockdown of DNA methyltransferase-1 partially rescued miR-184 level. Pathway analysis of paired miR-184:mRNA targets identified cell proliferation, differentiation, and death as the main affected biological processes. Knockdown of miR-184 in HL-1 cells and mesenchymal progenitor cells induced and, conversely, its overexpression attenuated adipogenesis. Conclusions: PKP2 deficiency leads to suppression of the E2F1 pathway and hypermethylation of the CpG sites at miR-184 promoter, resulting in downregulation of miR-184 levels. Suppression of miR-184 enhances and its activation attenuates adipogenesis in vitro. Thus, miR-184 contributes to the pathogenesis of adipogenesis in PKP2-deficient cells.

Rationale: Mutations in desmosome proteins cause arrhythmogenic cardiomyopathy (AC), a disease characterized by excess myocardial fibroadipocytes. Cellular origin(s) of fibroadipocytes in AC is unknown. Objective: To identify the cellular origin of adipocytes in AC. Methods and Results: Human and mouse cardiac cells were depleted from myocytes and flow sorted to isolate cells expressing platelet-derived growth factor receptor-α and exclude those expressing other lineage and fibroblast markers (CD32, CD11B, CD45, Lys76, Ly −6c and Ly 6c , thymocyte differentiation antigen 1, and discoidin domain receptor 2). The PDGFRA pos :Lin neg :THY1 neg :DDR2 neg cells were bipotential as the majority expressed collagen 1 α-1, a fibroblast marker, and a subset CCAAT/enhancer-binding protein α, a major adipogenic transcription factor, and therefore, they were referred to as fibroadipocyte progenitors (FAPs). FAPs expressed desmosome proteins, including desmoplakin, predominantly in the adipogenic but not fibrogenic subsets. Conditional heterozygous deletion of Dsp in mice using Pdgfra-Cre deleter led to increased fibroadipogenesis in the heart and mild cardiac dysfunction. Genetic fate mapping tagged 41.4±4.1% of the cardiac adipocytes in the Pdgfra-Cre : Eyfp:Dsp W /F mice, indicating an origin from FAPs. FAPs isolated from the Pdgfra-Cre : Eyfp:Dsp W /F mouse hearts showed enhanced differentiation to adipocytes. Mechanistically, deletion of Dsp was associated with suppressed canonical Wnt signaling and enhanced adipogenesis. In contrast, activation of the canonical Wnt signaling rescued adipogenesis in a dose-dependent manner. Conclusions: A subset of cardiac FAPs, identified by the PDGFRA pos :Lin neg :THY1 neg :DDR2 neg signature, expresses desmosome proteins and differentiates to adipocytes in AC through a Wnt-dependent mechanism. The findings expand the cellular spectrum of AC, commonly recognized as a disease of cardiac myocytes, to include nonmyocyte cells in the heart.

Rationale: Arrhythmogenic cardiomyopathy is caused primarily by mutations in genes encoding desmosome proteins. Ventricular arrhythmias are the cardinal and typically early manifestations, whereas myocardial fibroadiposis is the pathological hallmark. Homozygous DSP (desmoplakin) and JUP (junction protein plakoglobin) mutations are responsible for a subset of patients with arrhythmogenic cardiomyopathy who exhibit cardiac arrhythmias and dysfunction, palmoplanter keratosis, and hair abnormalities (cardiocutaneous syndromes). Objective: To determine phenotypic consequences of deletion of Dsp in a subset of cells common to the heart and skin. Methods and Results: Expression of CSPG4 (chondroitin sulfate proteoglycan 4) was detected in epidermal keratinocytes and the cardiac conduction system. CSPG4 pos cells constituted ≈5.6±3.3% of the nonmyocyte cells in the mouse heart. Inducible postnatal deletion of Dsp under the transcriptional control of the Cspg4 locus led to ventricular arrhythmias, atrial fibrillation, atrioventricular conduction defects, and death by 4 months of age. Cardiac arrhythmias occurred early and in the absence of cardiac dysfunction and excess cardiac fibroadipocytes, as in human arrhythmogenic cardiomyopathy. The mice exhibited palmoplantar keratosis and progressive alopecia, leading to alopecia totalis, associated with accelerated proliferation and impaired terminal differentiation of keratinocytes. The phenotype is similar to human cardiocutaneous syndromes caused by homozygous mutations in DSP . Conclusions: Deletion of Dsp under the transcriptional regulation of the CSPG4 locus led to lethal cardiac arrhythmias in the absence of cardiac dysfunction or fibroadiposis, palmoplantar keratosis, and alopecia, resembling the human cardiocutaneous syndromes. The findings offer a cellular basis for early cardiac arrhythmias in patients with arrhythmogenic cardiomyopathy and cardiocutaneous syndromes.

Progress in science is a continuum but not uniform. Occasional sparks, typically ignited by the so-called disruptive discoveries, decorate the continuum. Relevant to this editorial on advances in the genetic testing technologies, is the disruptive technology of capillary sequencing, also known as Sanger sequencing.1 It enabled the initial sequencing of the human genome2,3 and led to the second Noble prize for the late Dr Fred Sanger in 1980 (http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1980/). The simple and elegant capillary sequencing technology ushered in the dawning of the most successful years in cardiovascular genetics and deciphering the genetic basis of single gene cardiovascular disorders, such as hereditary cardiomyopathies, ion channel disorders, and autosomal-dominant hypercholesterolemia, among others.4–8 The advances also enabled genetic testing based on the candidate gene approach, albeit the approach was time consuming, labor intensive, and relatively expensive. Hence, genetic testing based on the Sanger sequencing technique did not make the transition from the research laboratories to the bedside. Nevertheless, the genetic discoveries based on Sanger sequencing had an enormous impact on genetics as well as in medicine. This is exemplified by discovery of PCSK9 as a causal gene for autosomal-dominant hypercholesterolemia in 2003.5 The pace of subsequent mechanistic discoveries and advances toward the clinical applications were blazingly fast. 9 Within a decade of the discovery of the gene, its function was partially unraveled, and monoclonal antibodies to inhibit PCSK9 were developed, and shown to be clinically effective in lowering low-density lipoprotein cholesterol and improving clinical outcomes.10,11 Subsequently, Food and Drug Administration recently approved 2 PCSK9 inhibitors for clinical use in a subset of patients with high low-density lipoprotein cholesterol. Article, see p 603 The elegant Sanger sequencing technique, however, was no exception to unrelenting technological advances, and soon it was all but replaced …

For years, many reasoned, perhaps not so naively, that genetics of congenital heart diseases (CHD) would be the last frontier in efforts to elucidate the genetic causes of cardiovascular disease. This impression was based on 2 main reasons. First, CHD seldom exhibits a clear familial inheritance pattern, as opposed to many single gene disorders. This is despite the strong evidence for familial aggregation of CHD and a higher risk of recurrence in the offspring, which denote a clear genetic cause.1,2 CHD are often sporadic or part of the aneuploidy syndromes with pleomorphic noncardiac phenotypes.1,3 Thus, an approach distinct from the genetic linkage analysis in large families, which was commonly applied to delineate the genetic basis of hereditary cardiovascular diseases such as cardiomyopathies and arrhythmia syndromes,4–8 was needed to define the genetic cause of CHD. Second, the extreme phenotypic assortment of CHD,3,9–11 which is far beyond phenotypic variability of single gene disorders, as well as the diversity of common complex diseases, such as coronary artery disease and systemic arterial hypertension. Consequently, it was difficult envisioning how apparently the simple phenotype of atrial septal defects and the complex phenotype of tetralogy of Fallot, which share no anatomic and physiological similarities, would causally share a common class of genetic networks, let alone arise from mutations in a single gene. Article, see p 884 Recent discoveries are transforming the landscape of molecular genetics of CHD and diluting the aforementioned antediluvian impression. The pioneering work of Basson et al12 and Schott et al13 in late 1990s led to the identification of loss-of-function mutations in TBX5 and NKX2-5 as causes of Holt–Oran syndrome and atrial septal defect, respectively. An intriguing finding was the assortment of clinical phenotypes in the mutation …

The discovery of the ACE2 gene broadened the scope of the regulatory mechanisms that govern the biological effects of the renin–angiotensin system in the cardiovascular system and beyond The late Daniel E. Koshland Jr. (1920–2007), a former editor of Science (1985–1995), characterized scientific discoveries into 3 categories of Charge, Challenge, and Chance, which he dubbed as The Cha-Cha-Cha Theory of Scientific Discoveries.1 Citing Nobel laureate Albert Szent-Gyorgyi, Dr Koshland described the first Cha, as to see what everyone else has seen and think what no one else has thought before. Charge solves the obvious problems, such as the laws of heredity, which were delineated by Gregory Mendel and the theory of gravity, which was developed by Sir Isaac Newton. The second Cha refers to discovery of a new concept that pulls together the accumulated facts that had remained unexplained, such as the description of base pairing in double helix by Watson and Crick.2 The third Cha is for the serendipitous discoveries, which per Louis Pasteur favor the prepared mind.1 Sir Alexander Fleming observed clear spots on petri dish and went on to discover penicillin from the mold Penicillin notatum . Scientific discoveries are typically incremental. The magnitude of increments follows a gradient from small to large. Major discoveries are not made in a scientific vacuum or through the Eureka moments but rather are based on a series of small discoveries that pave the path for the major breakthroughs. Thus, contributions of all steps in the ladder of scientific discoveries, whether small or large, are important. Nevertheless, certain steps epitomize the breakthroughs and apt to Dr Koshland’s Cha-Cha-Cha Theory of Scientific Discoveries. The progress in cardiovascular science is no exception. Hence, the Editors of Circulation Research have invited the audience to identify and highlight seminal discoveries that have …

See related article, pages 862–870 Evolution of biological processes is fascinating and more so the evolutionary reduction of mitochondrial genome. A proteobacterium that invaded the nucleus-containing host cell ≈1.5 billion years ago, as would the endosymbiotic model surmises,1 ended up enslaved and yet became the essence of life of the invaded cell. And, if so, the invader could not have had a genome that was comprised of only 16 569 base pairs (bp), coding for only 13 proteins, 22 transfer (t)RNAs, and 2 ribosomal (r)RNAs (NC_012920).2,3 Such a small size would seem to be incompatible with survival of a free-living organism as the smallest known bacterial genome is that of Carsonella Ruddi, which is 159 662 bp, ie, ≈10 times the size of mitochondrial genome, and codes for 182 proteins. Yet, C Ruddi is not a free-living organism but rather an endosymbiont.4 On the contrary, obligate intracellular lifestyle can exist with as little as 5386 bp, as for ϕX174, which is smallest known genome.5 If indeed this organelle, ie, the mitochondrion, was one time a free-living organism with a much larger genome, then, how did it shrink its genome size to the current size and manage to survive? The generation of ATP through oxidative phosphorylation, one of the several mitochondrial functions, alone involves 89 genes and requires a much larger genome to sustain that the mitochondrial (mt)DNA affords.6 Apparently, the invader could not escape from the evolutionary pressure imposed by the host environment and a symbiotic life. Consequently, mitochondria gradually streamlined its genome by relaxed selection of the genes that were superfluous to its survival. Through yet-to-be defined mechanisms the invader seems to have transferred thousands of its genes, particularly those that mutated during mtDNA replication, to nuclear (n)DNA wherein they reside …

The rapid pace of genetic and genomic discoveries in cardiovascular medicine remains unabated. During the past few years, several new causal genes and susceptibility variants for several cardiovascular disease have been identified.1–7 Likewise, genomic and epigenetic discoveries, encompassing noncoding RNAs, DNA methylation, and covalent histone modifications, have shed significant light onto the mechanisms that regulate expression of the cardiovascular phenotypes.5,8–13 Although the main impact of the genetic and genomic discoveries has been in elucidation of the molecular pathogenesis of cardiovascular diseases, recent findings, particularly in single gene discoveries, are also clinically impactful and are becoming increasingly incorporated into the practice of cardiovascular medicine.14–24 In addition, these discoveries have paved the way for genetic-based interventions and gene therapy.25–28 The discoveries have also enabled generation and mechanistic characterization of induced pluripotent stem cell models of genetic cardiovascular diseases.29–35 This progress has been driven in part by the technological advances, most notably the massively parallel nucleic acid sequencing techniques, which have enabled sequencing of the entire human exome, if not the genome, and the whole transcriptome of an individual within a reasonable cost.36,37 The ability to decode an individual exome has also posed a daunting challenge to clinicians and researchers as well as the patients in an accurate identification of the causal or risk variants from the myriad of variants that are present in each exome and in the population.38–40 The plethora of variants in each exome and genome underscores the necessity of incorporating family information in increasing the use of the genetic data and garnering clinically robust and meaningful genetic information. Approaches to the elucidation of genetic causes of cardiovascular diseases have evolved along with the technological advances. Conventional …

See Research Commentary, p e2 Genetics provide a robust scientific platform for establishing a relationship between a cause, for example, a genetic variant, and an effect, such as a disease phenotype. Yet, causality in genetics is a probabilistic and rarely a deterministic certainty. The causal relationship between a genetic variant and a phenotype is provisional to the conditions and the environment, such as the genetic backgrounds, in which the causal variants and the phenotype operate. The degree of probabilistic causality is, in part, determined by the effect sizes of the genetic variants, which typically follow a gradient ranging from minimal to large.1,2 Genetic variants (single nucleotide variants, small insertion/deletions, and structural variations) with large effect sizes are highly penetrant, as exemplified by variants that are responsible for the single gene disorders with Mendelian patterns of inheritance. In such circumstances, the cause-and-effect relationship is best analyzed through cosegregation and linkage analyses, whereby a logarithm of odds score of ≥3 is considered strong evidence of a genetic linkage but not necessarily a definitive indicator of causality. On the opposite end of the spectrum are the variants that exert modest effect sizes and, hence, are less penetrant. In such cases, establishing a cause-and-effect relationship is more challenging, because such low-penetrant variants typically do not show a clear cosegregation with the phenotype and are often found in the general population as well. Establishing the causal role of the variants is more challenging in small families, and even more so in a single individual. Further compounding the ascertainment of causality is the influence of the genetic backgrounds (modifier genetic variants) and the environmental factors, which are expected to exert greater influence when the effect sizes of the causal genetic variants are rather small. Hence, a genetic variant with a small or moderate …

Abstract —Identification of mutations in the ATP binding cassette transporter ( ABCA1 ) gene in patients with Tangier disease, who exhibit reduced HDL cholesterol (HDL-C) and apolipoprotein A1 (apoA1) levels and premature coronary atherosclerosis, has led to the hypothesis that common polymorphisms in the ABCA1 gene could determine HDL-C and apoA1 levels and the risk of coronary atherosclerosis in the general population. We sequenced a 660-bp 5′ fragment of the ABCA1 gene in 24 subjects and identified 3 novel polymorphisms: −477C/T, −419A/C, and −320G/C. We developed assays, genotyped 372 participants in the prospective Lipoprotein Coronary Atherosclerosis Study (LCAS), and determined the association of the variants with fasting plasma lipids and indices of quantitative coronary angiograms obtained at baseline and 2.5 years after randomization to fluvastatin or placebo. Distribution of −477C/T and −320G/C genotypes were 127 CC, 171 CT, and 74 TT and 130 GG, 168 GC, and 75 CC, respectively, and were in complete linkage disequilibrium ( P P =0.09) and apoA1 ( P =0.05) levels and a lesser response of apoA1 to treatment with fluvastatin ( P =0.04). The mean number of coronary lesions causing 30% to 75% diameter stenosis was greater in subjects with the TT genotype (3.1±2.1) or CT genotype (2.9±1.9) than in subjects with the CC genotype (2.2±1.8) ( P =0.002). Similarly, compared with subjects with the CC genotype , greater numbers of subjects with the TT or CT genotype had ≥1 coronary lesion ( P =0.001). No association between the genotypes and progression of coronary atherosclerosis or clinical events was detected. We conclude that ABCA1 genotypes are potential risk factors for coronary atherosclerosis in the general population.

The genome continues to fascinate the enthusiasts, and the captivation seems unabating because of the continuous stream of new discoveries. What was once considered a junk DNA has now emerged to contain a large number of important regulatory elements.1 As an example of such discoveries is the recent finding that the genome contains ≈6200 enhancer elements that are operational in the human fetal and adult hearts.2 Genes occupy only ≈1.5% and coding exons only ≈1% of the genome, which make up only ≈60 million nucleotides of the 6.4 billion nucleotides (3.2 billion base pairs) in the genome.3 Yet, ≈5% of the human genome has undergone purifying selection and hence are likely functional.4 It is intriguing that about two third of the evolutionary constrained genomic elements are located in introns and the intergenic regions, suggestive of their regulatory roles in the genome.4 These conserved regions are enriched in loci that have been found to be associated with clinical phenotypes in the genome-wide association studies.4 The initial findings of the ENCODE (Encyclopedia of DNA Elements) project, although preliminary, illustrate the presence of numerous regulatory elements in the genome, including the enhancers.1 The discoveries largely made possible by the recent advances in the high-throughput RNA sequencing point to enormous RNA splicing diversity and the plethora of alternative splicing variants. Approximately 95% of the multiexon genes undergo alternative splicing, resulting in ≈100 000 abundant splice variants in various tissues.5 Moreover, it seems that almost all genomic regions in 1 form or shape are transcribed, which seems perplexing as only ≈1% of the genome codes for proteins, as has been understood to date. Only recently we have started to appreciate the diverse biological functions of these nonprotein coding transcripts, which are referred to as noncoding RNAs (ncRNAs). …

Abstract —A series of pro-oxidant and antioxidant enzymes, such as the NADPH oxidase system, maintain the redox state in the vessel wall. A major component of NADPH oxidase is p22 phox , which is implicated in atherosclerosis. We prospectively studied the association of the histidine (H) 72 →tyrosine (Y) mutation in p22 phox with the severity and progression/regression of coronary artery disease (CAD), plasma lipid levels, clinical events, and response to treatment with fluvastatin in a well-characterized population. Genotypes were determined by polymerase chain reaction and restriction digestion with Rsa I enzyme in 368 subjects in the Lipoprotein and Coronary Atherosclerosis Study (LCAS). Fasting plasma lipids and quantitative coronary angiograms were obtained at baseline and 2.5 years after randomization to fluvastatin or placebo. Subjects with CC genotype (n=157) were identified by the presence of 396-bp and 113-bp products on gel electrophoresis. Those with TT (n=39) were identified by the presence of 316-bp, 113-bp, and 80-bp products, and those with CT (n=172) by the presence of 396-bp, 316-bp, 113-bp, and 80-bp products. Baseline and final plasma levels of lipids and the baseline severity of CAD were not significantly different among the genotypes. In the placebo group, subjects with the mutation had a 3- to 5-fold greater loss in mean minimum lumen diameter (MLD) (TT: −0.15±0.15; CT: −0.17±0.26; and CC: −0.03±0.22 mm; P =0.006) and lesion-specific MLD (TT: −0.15±0.06; CT: −0.18±0.03; and CC: −0.06±0.03 mm; P =0.038) than those without. Progression was also more (TT: 8/17 [47%]; CT: 35/73 [48%]; and CC: 17/62 [27%]) and regression less (TT: 0/17 [0%]; CT: 1/73 [1%]; and CC: 11/72 [18%]) common in those with the mutation ( P =0.002). The C 242 T mutation in p22 phox , involved in maintaining the redox state in the vessel wall, is associated with progression of coronary atherosclerosis in the LCAS population.

Abstract Mutations in the β-myosin heavy chain (βMyHC) induce hypertrophic cardiomyopathy (HCM), cardiac hypertrophy, and sarcomere disarray, with the latter being the characteristic hallmark. Thus, we sought to determine whether expression of mutant βMyHC in adult feline cardiac myocytes, a species known to develop HCM with a phenotype identical to that in humans, induces sarcomere disarray. A full-length βMyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg 403 Gln) was induced in the βMyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant βMyHC cDNAs were cloned into pΔE1spIB shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/βMyHC-N and Ad5/CMV/βMyHC-403) were generated through homologous recombination of pΔE1spIB/CMV/βMyHC-N or Ad5/CMV/βMyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-1 cells with the βMyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was >95%. Expression of the βMyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription–PCR. The net total protein and β-myosin synthesis were determined by using the amount of incorporation of [ 3 H]phenylalanine into total protein and β-myosin, respectively. Although the total amount of protein synthesis was equal among experimental groups, the net myosin synthesis at 48 hours was greater in cardiac myocytes infected with normal or mutant βMyHC constructs than control myocytes or those infected with vector alone ( P

The ever-increasing number of publications poses a formidable challenge for many investigators: how can one keep up with the advances in science? Inherent in the accelerating pace of discoveries and in the expanding number of platforms for their dissemination is the danger that major scientific leaps may be overlooked or not placed in proper perspective by some segments of the community of scholars. The issue here is not just to be aware of new publications, but also to be able to form a critical opinion regarding their limitations, context, significance, and impact. It is practically impossible to read all new important articles that appear every week, let alone assess their significance. Recognizing this conundrum, the editors of Circulation Research …

The αB-crystallin protein, the predominant structural protein of the ocular lens, is a member of the small heat shock proteins that is also expressed abundantly in the heart and skeletal muscle.1 The αB-crystallin was initially discovered in the vertebrate ocular lens and was dubbed crystallin because of its role in maintenance of lens transparency.2 It is also essential for maintenance of microtubular integrity in striated muscles.3 In the heart, as in the ocular lens, αB-crystallin forms soluble multimeres that function as chaperone molecules, facilitating protein folding and translocation.4 Thus, the principal function of αB-crystallin protein is to prevent unfolding of cellular proteins damaged by all forms of stress. Ischemia and oxidative stress increase the expression of αB-crystallin in the heart.5 In response to stress, intracellular kinases phosphorylate αB-crystallin,6 leading to its translocation from the cytosolic pool to Z lines and intercalated disks. Translocated αB-crystallin binds to the components of the intermediary filaments and cytoskeletal proteins, such as actin and desmin, and prevents their aggregation.3 7 The protective role of αB-crystallin in the maintenance of cytoskeletal integrity has been confirmed in gene transfer studies in cultured cardiac myocytes8 and in transgenic mice.9 Overexpression of αB-crystallin protects cardiac myocytes against apoptosis and reperfusion injury.8 9 Interest in αB-crystallin has been heightened because of recent elucidation of the genetic basis of desmin-related myopathy (DRM), a familial muscular disorder characterized by skeletal myopathy, heart failure, conduction defect, and arrhythmias. Pathologically, …

June 26, 2010, marked the 10th anniversary of the completion of the draft sequence of the human genome,1,2 an achievement that was the pinnacle of the Human Genome Project, started approximately 11 years earlier and officially completed in 2003.1–3 The Human Genome Project offered the first systematic glimpses into the sequence of a haploid genome.1–3 It facilitated immensely the identification of the genes and mutations responsible for single gene disorders. However, an individual extent of the DNA sequence variations (DSVs) in the human genome remained largely unknown until the publication of Craig Venter’s diploid genome in 2007.4 By revealing the extent and complexity of variations in the human genome, the findings ushered in new insights into the remarkable genetic diversity of our species. Particularly intriguing was the finding that ≈45% of Venter’s genes were polymorphic and that his genome contained ≈4 million DSVs, including >3 million single-nucleotide polymorphisms (SNPs), ≈10 000 nonsynonymous (ns) SNPs, and several hundred thousand structural variations (SVs). Some SVs involved several million DNA base pairs (bps) and multiple genes, resulting in copy number variations (CNVs). Because of their size, often involving large DNA segments, the SVs accounted for three-fourths of the variant nucleotides. The success in sequencing the human genome was achieved using the ingenious DNA sequencing method developed by Frederick Sanger and Walter Gilbert.5 However, this technique (for which Sanger and Gilbert were awarded the Nobel Prize in Chemistry in 1980) was relatively costly and time-consuming and, therefore, not practical for routine whole-genome sequencing. Thus, clinical application of the information content of the DSVs had to await the development of more efficient DNA sequencing methods. The introduction of the array-based 454 pyrosequencing technology in 2005 ushered in the era of Next-Generation or massively parallel DNA sequencing.6 This technique has …

Abstract The mechanism(s) by which mutations in sarcomeric proteins cause hypertrophic cardiomyopathy (HCM) remains unknown. A leading hypothesis proposes that mutant sarcomeric proteins impair cardiac myocyte contractility, providing an impetus for compensatory hypertrophy. To test this hypothesis, we determined the impact of expression of a mutant (Arg 92 Gln) human cardiac troponin T (cTnT), known to cause HCM in humans, on adult cardiac myocyte contractility. A full-length human cTnT cDNA was cloned, and the Arg 92 Gln mutation was induced. Recombinant adenoviruses Ad5/CMV/cTnT-N and Ad5/CMV/cTnT-Arg 92 Gln were generated through homologous recombination. Adult feline cardiac myocytes were infected with recombinant adenoviruses or a control viral vector (Ad5ΔE1) at a multiplicity of infection of 100. Expression levels of the full-length normal and mutant cTnT proteins were equal on Western blots. Expression of the exogenous cTnT proteins in cardiac myocytes was also shown by immunocytochemistry and immunofluorescence, and their incorporation into myofibrils was confirmed by Western blotting on myofibrillar extracts. Electron microscopy showed intact sarcomere structure in rod-shaped cardiac myocytes in all groups. Cell fractional shortening and the peak velocity of shortening were not significantly different among the groups 24 hours after transduction. However, 48 hours after transduction, both fractional shortening and the peak velocity of shortening were significantly reduced (24% [ P P 92 Gln compared with the Ad5/CMV/cTnT-N groups. The magnitude of the reductions was greater at 72 hours after transduction (45% and 39%, respectively; P 92 Gln) cTnT, known to cause HCM in humans, impaired intact adult cardiac myocyte contractility. Our data also show that both normal and mutant cTnT were incorporated into myofibrils. These results provide a potential mechanism by which mutations in sarcomeric proteins cause HCM.