Stargardt macular dystrophy and therapeutic approaches.

Stargardt macular dystrophy (Stargardt disease; STGD1; OMIM 248200) is the most prevalent inherited macular dystrophy. STGD1 is an autosomal recessive disorder caused by multiple pathogenic sequence variants in the large ABCA4 gene (OMIM 601691). Major advances in understanding both the clinical and molecular features, as well as the underlying pathophysiology, have culminated in many completed, ongoing and planned human clinical trials of novel therapies.The aims of this concise review are to describe (1) the detailed phenotypic and genotypic characteristics of the disease, multimodal imaging findings, natural history of the disease, and pathogenesis, (2) the multiple avenues of research and therapeutic intervention, including pharmacological, cellular therapies and diverse types of genetic therapies that have either been investigated or are under investigation and (3) the exciting novel therapeutic approaches on the translational horizon that aim to treat STGD1 by replacing the entire 6.8 kb ABCA4 open reading frame.

VLDL receptor gene therapy for reducing atherogenic lipoproteins.

Over the past 40 years, there has been considerable research into the management and treatment of atherogenic lipid disorders. Although the majority of treatments and management strategies for cardiovascular disease (CVD) center around targeting low-density lipoprotein cholesterol (LDL-C), there is mounting evidence for the residual CVD risk attributed to high triglyceride (TG) and lipoprotein(a) (Lp(a)) levels despite the presence of lowered LDL-C levels. Among the biological mechanisms for clearing TG-rich lipoproteins, the VLDL receptor (VLDLR) plays a key role in the trafficking and metabolism of lipoprotein particles in multiple tissues, but it is not ordinarily expressed in the liver. Since VLDLR is capable of binding and internalizing apoE-containing TG-rich lipoproteins as well as Lp(a), hepatic VLDLR expression has the potential for promoting clearance of these atherogenic particles from the circulation and managing the residual CVD risk not addressed by current lipid lowering therapies. This review provides an overview of VLDLR function and the potential for developing a genetic medicine based on liver-targeted VLDLR gene expression.

Post-Translational Modifications and Diastolic Calcium Leak Associated to the Novel RyR2-D3638A Mutation Lead to CPVT in Patient-Specific hiPSC-Derived Cardiomyocytes.

BACKGROUND: Sarcoplasmic reticulum Ca2+ leak and post-translational modifications under stress have been implicated in catecholaminergic polymorphic ventricular tachycardia (CPVT), a highly lethal inherited arrhythmogenic disorder. Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. OBJECTIVE: The aims were to obtain functional hiPSC-derived cardiomyocytes from a CPVT patient harboring a novel ryanodine receptor (RyR2) mutation and model the syndrome, drug responses and investigate the molecular mechanisms associated to the CPVT syndrome. METHODS: Patient-specific cardiomyocytes were generated from a young athletic female diagnosed with CPVT. The contractile, intracellular Ca2+ handling and electrophysiological properties as well as the RyR2 macromolecular remodeling were studied. RESULTS: Exercise stress electrocardiography revealed polymorphic ventricular tachycardia when treated with metoprolol and marked improvement with flecainide alone. We found abnormal stress-induced contractile and electrophysiological properties associated with sarcoplasmic reticulum Ca2+ leak in CPVT hiPSC-derived cardiomyocytes. We found inadequate response to metoprolol and a potent response of flecainide. Stabilizing RyR2 with a Rycal compound prevents those abnormalities specifically in CPVT hiPSC-derived cardiomyocytes. The RyR2-D3638A mutation is located in the conformational change inducing-central core domain and leads to RyR2 macromolecular remodeling including depletion of PP2A and Calstabin2. CONCLUSION: We identified a novel RyR2-D3638A mutation causing 3D conformational defects and aberrant biophysical properties associated to RyR2 macromolecular complex post-translational remodeling. The molecular remodeling is for the first time revealed using patient-specific hiPSC-derived cardiomyocytes which may explain the CPVT proband's resistance. Our study promotes hiPSC-derived cardiomyocytes as a suitable model for disease modeling, testing new therapeutic compounds, personalized medicine and deciphering underlying molecular mechanisms.

A mutation abolishing the ZMPSTE24 cleavage site in prelamin A causes a progeroid disorder.

In 1994 in the Journal of Cell Science, Hennekes and Nigg reported that changing valine to arginine at the endoproteolytic cleavage site in chicken prelamin A abolishes its conversion to lamin A. The consequences of this mutation in an organism have remained unknown. We now report that the corresponding mutation in a human subject leads to accumulation of prelamin A and causes a progeroid disorder. Next generation sequencing of the subject and her parents' exomes identified a de novo mutation in the lamin A/C gene (LMNA) that resulted in a leucine to arginine amino acid substitution at residue 647 in prelamin A. The subject's fibroblasts accumulated prelamin A, a farnesylated protein, which led to an increased percentage of cultured cells with morphologically abnormal nuclei. Treatment with a protein farnesyltransferase inhibitor improved abnormal nuclear morphology. This case demonstrates that accumulation of prelamin A, independent of the loss of function of ZMPSTE24 metallopeptidase that catalyzes processing of prelamin A, can cause a progeroid disorder and that a cell biology assay could be used in precision medicine to identify a potential therapy.

An LQTS6 MiRP1 mutation suppresses pacemaker current and is associated with sinus bradycardia.

BACKGROUND: Sinus node (SN) dysfunction is observed in some long-QT syndrome (LQTS) patients, but has not been studied as a function of LQTS genotype. LQTS6 involves mutations in the hERG ß-subunit MiRP1, which also interacts with hyperpolarization-activated, cyclic nucleotide gated (HCN) channels-the molecular correlate of SN pacemaker current (If ). An LQTS registry search identified a 55-year male with M54T MiRP1 mutation, history of sinus bradycardia (39-56 bpm), and prolonged QTc. OBJECTIVE: We tested if LQTS6 incorporates sinus bradycardia due to abnormal If . METHODS: We transiently co-transfected neonatal rat ventricular myocytes (to study currents in a myocyte background) with human HCN4 (hHCN4, primary SN isoform) or human HCN2 (hHCN2) and one of the following: empty vector, wild-type hMiRP1 (WT), M54T hMiRP1 (M54T). Current amplitude, voltage dependence, and kinetics were measured by whole cell patch clamp. RESULTS: M54T co-expression decreased HCN4 current density by 80% compared to hHCN4 alone or with WT, and also slowed HCN4 activation at physiologically relevant voltages. Neither WT nor M54T altered HCN4 voltage dependence. A computer simulation predicts that these changes in HCN4 current would decrease rate and be additive with published effects of M54T mutation on hERG kinetics on rate. CONCLUSIONS: We conclude that M54T LQTS6 mutation can cause sinus bradycardia through effects on both hERG and HCN currents. Patients with other LQTS6 mutations should be examined for SN dysfunction, and the effect on HCN current determined.

Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics.

Understanding the basis for differential responses to drug therapies remains a challenge despite advances in genetics and genomics. Induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to investigate the pharmacology of disease processes in therapeutically and genetically relevant primary cell types in vitro and to interweave clinical and basic molecular data. We report here the derivation of iPSCs from a long QT syndrome patient with complex genetics. The proband was found to have a de novo SCN5A LQT-3 mutation (F1473C) and a polymorphism (K897T) in KCNH2, the gene for LQT-2. Analysis of the biophysics and molecular pharmacology of ion channels expressed in cardiomyocytes (CMs) differentiated from these iPSCs (iPSC-CMs) demonstrates a primary LQT-3 (Na(+) channel) defect responsible for the arrhythmias not influenced by the KCNH2 polymorphism. The F1473C mutation occurs in the channel inactivation gate and enhances late Na(+) channel current (I(NaL)) that is carried by channels that fail to inactivate completely and conduct increased inward current during prolonged depolarization, resulting in delayed repolarization, a prolonged QT interval, and increased risk of fatal arrhythmia. We find a very pronounced rate dependence of I(NaL) such that increasing the pacing rate markedly reduces I(NaL) and, in addition, increases its inhibition by the Na(+) channel blocker mexiletine. These rate-dependent properties and drug interactions, unique to the proband's iPSC-CMs, correlate with improved management of arrhythmias in the patient and provide support for this approach in developing patient-specific clinical regimens.

LMNA cardiomyopathy: cell biology and genetics meet clinical medicine.

Mutations in the LMNA gene, which encodes A-type nuclear lamins (intermediate filament proteins expressed in most differentiated somatic cells), cause a diverse range of diseases, called laminopathies, that selectively affect different tissues and organ systems. The most prevalent laminopathy is cardiomyopathy with or without different types of skeletal muscular dystrophy. LMNA cardiomyopathy has an aggressive clinical course with higher rates of deadly arrhythmias and heart failure than most other heart diseases. As awareness among physicians increases, and advances in DNA sequencing methods make the genetic diagnosis of LMNA cardiomyopathy more common, cardiologists are being faced with difficult questions regarding patient management. These questions concern the optimal use of intracardiac cardioverter defibrillators to prevent sudden death from arrhythmias, and medical interventions to prevent heart damage and ameliorate heart failure symptoms. Data from a mouse model of LMNA cardiomyopathy suggest that inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways are beneficial in preventing and treating cardiac dysfunction; this basic research discovery needs to be translated to human patients.

Ablation of Nkx2-5 at mid-embryonic stage results in premature lethality and cardiac malformation.

AIMS: Human congenital heart disease linked to mutations in the homeobox transcription factor, NKX2-5, is characterized by cardiac anomalies, including atrial and ventricular septal defects as well as conduction and occasional defects in contractility. In the mouse, homozygous germline deletion of Nkx2-5 gene results in death around E10.5. It is, however, not established whether Nkx2-5 is necessary for cardiac development beyond this embryonic stage. Because human NKX2-5 mutations are related to septum secundum type atrial septal defects (ASD), we hypothesized that Nkx2-5 deficiency during the processes of septum secundum formation may cause cardiac anomalies; thus, we analysed mice with tamoxifen-inducible Nkx2-5 ablation beginning at E12.5 when the septum secundum starts to develop. METHODS AND RESULTS: Using tamoxifen-inducible Nkx2-5 gene-targeted mice, this study demonstrates that Nkx2-5 ablation beginning at E12.5 results in embryonic death by E17.5. Analysis of mutant embryos at E16.5 shows arrhythmias, contraction defects, and cardiac malformations, including ASD. Quantitative measurements using serial section histology and three-dimensional reconstruction demonstrate growth retardation of the septum secundum and enlarged foramen ovale in Nkx2-5-ablated embryos. Functional cardiac defects may be attributed to abnormal expression of transcripts critical for conduction and contraction, including cardiac voltage-gated Na(+) channel pore-forming α-subunit (Na(v)1.5-α), gap junction protein connexin40, cardiac myosin light chain kinase, and sarcolipin within 4 days after tamoxifen injection. CONCLUSION: Nkx2-5 is necessary for survival after the mid-embryonic stage for cardiac function and formation by regulating the expression of its downstream target genes.

Calcium-triggered short-coupled polymorphous ventricular tachycardia.

We describe a case of an 18-year-old man presenting with syncope found to have short-coupled premature ventricular complexes (PVCs) with subsequent nonsustained polymorphous ventricular tachycardia (PVT). Electrophysiology testing revealed premature PVCs and PVT provoked by calcium but not isoproterenol. It was noted that the earliest triggered event appeared to arise from ventricular muscle with subsequent involvement of the fascicles and these areas were ablated. The potential mechanisms for calcium triggering of these arrhythmias are discussed.

Recent progress in congenital long QT syndrome.

PURPOSE OF REVIEW: As genetic testing for long QT syndrome (LQTS) has become readily available, important advances are being made in understanding the exact link between ion channel mutation and observed phenotype. This paper reviews recent findings in the literature. RECENT FINDINGS: Congenital LQTS is an important cause of sudden cardiac death. To date, 12 genes have been identified as the cause of congenital LQTS. With increasing availability of genetic testing, subtype-specific management of LQTS has become the standard of care. Detailed correlative studies between LQTS mutations and clinical phenotypes are leading the field towards 'mutation-specific' management within LQTS subtypes. A clear link between the distinct functional/biophysical defect in each LQT mutation and disease phenotype is complicated by the variable penetrance and pleiotropic expression of clinical phenotype. This is especially evident with the overlap syndrome now documented for several sodium channel (SCN5A) mutations. SUMMARY: The management of LQTS has become subtype-specific due to the availability of genotype information. Review of recent literature suggests that 'mutation-specific' management is possible based upon distinct functional/biophysical characteristics of mutations within each LQT gene. Further research is required to clearly delineate the molecular and cellular mechanisms underlying variable penetrance, and pleiotropic expression of LQTS mutations.

Slow progressive conduction and contraction defects in loss of Nkx2-5 mice after cardiomyocyte terminal differentiation.

Mutations in homeoprotein NKX2-5 are linked to human congenital heart disease, resulting in various cardiac anomalies, as well as in postnatal progressive conduction defects and occasional left ventricular dysfunction; yet the function of Nkx2-5 in the postnatal period is largely unexplored. In the heart, the majority of cardiomyocytes are believed to complete cell-cycle withdrawal shortly after birth, which is generally accompanied by a re-organization of chromatin structure shown in other tissues. We reasoned that the effects of the loss of Nkx2-5 in mice may be different after cell-cycle withdrawal compared with those of the perinatal loss of Nkx2-5, which results in rapid conduction and contraction defects within 4 days after the deletion of Nkx2-5 alleles (Circ Res. 2008;103:580). In this study, floxed-Nkx2-5 alleles were deleted using tamoxifen-inducible Cre transgene (Cre-ER) beginning at 2 weeks of age. The loss of Nkx2-5 beginning at 2 weeks of age resulted in conduction and contraction defects similar to the perinatal loss of Nkx2-5, however, with a substantially slower disease progression shown by 1 degrees atrioventricular block at 6 weeks of age (4 weeks after tamoxifen injections) and heart enlargement after 12 weeks of age (10 weeks after tamoxifen injections). The phenotypes were accompanied by a slower and smaller degree of reduction of several critical Nkx2-5 downstream targets that were observed in mice with a perinatal loss of Nkx2-5. These results suggest that Nkx2-5 is necessary for proper conduction and contraction after 2 weeks of age, but with a substantially distinct level of necessity at 2 weeks of age compared with that in the perinatal period.

Perinatal loss of Nkx2-5 results in rapid conduction and contraction defects.

Homeobox transcription factor Nkx2-5, highly expressed in heart, is a critical factor during early embryonic cardiac development. In this study, using tamoxifen-inducible Nkx2-5 knockout mice, we demonstrate the role of Nkx2-5 in conduction and contraction in neonates within 4 days after perinatal tamoxifen injection. Conduction defect was accompanied by reduction in ventricular expression of the cardiac voltage-gated Na+ channel pore-forming alpha-subunit (Na(v)1.5-alpha), the largest ion channel in the heart responsive for rapid depolarization of the action potential, which leads to increased intracellular Ca2+ for contraction (conduction-contraction coupling). In addition, expression of ryanodine receptor 2, through which Ca2+ is released from sarcoplasmic reticulum, was substantially reduced in Nkx2-5 knockout mice. These results indicate that Nkx2-5 function is critical not only during cardiac development but also in perinatal hearts, by regulating expression of several important gene products involved in conduction and contraction.

Identification of cardiac-specific myosin light chain kinase.

Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca(2+)/calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction.

Distinct roles of HF-1b/Sp4 in ventricular and neural crest cells lineages affect cardiac conduction system development.

The heterogeneous cell types of the cardiac conduction system are responsible for coordinating and maintaining rhythmic contractions of the heart. While it has been shown that the cells of the conduction system are derived from myocytes, additional cell types, including neural crest cells, may play a role in the development and maturation of these specialized cell lineages. Previous work has shown that the expression of the hf-1b gene is required for specification of the cardiac conduction system. Using Cre-Lox technology, we conditionally mutated the hf-1b gene in the ventricular and the neural crest cell lineages. Cx40 immunohistochemistry on HF-1b tissue-restricted knockouts revealed a requirement for HF-1b in the cardiomyogenic lineage. Electrophysiological studies identified a second requirement for HF-1b in the neural crest-derived cells. Absence of HF-1b in the neural crest led to atrial and atrioventricular dysfunction resulting from deficiencies in the neurotrophin receptor trkC. Therefore, in this study, we document that a single transcription factor, HF-1b, acts through two separate cell types to direct distinct functions of the cardiac conduction system.

Predominant fusion of bone marrow-derived cardiomyocytes.

OBJECTIVES: Here we address the capacity of bone marrow-derived cells (BMDCs) to trans-differentiate into mature myocytes under the physiological stimulus of exercise training. METHODS: For this purpose, we have transplanted bone marrow from mice ubiquitously expressing enhanced green fluorescence protein (eGFP) into host mice that have been subjected to a prolonged program of exercise. RESULTS: In all successful bone marrow reconstitutions (greater than 80%), we observed rare but consistent events of bone marrow-derived cardiomyocytes, the frequency of which was unchanged upon exercise training. We have further determined whether these recruited myocytes are a product of trans-differentiation or fusion by the use of a genetic system that distinguishes cell fusion from trans-differentiation in a single-cell assay. CONCLUSIONS: We concluded that both in the unchallenged mouse and in the trained specimens, fusion is the most prominent mechanism by which bone marrow-derived cells are observed in the myocyte compartment.

Nkx2-5 pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block.

Human mutations in Nkx2-5 lead to progressive cardiomyopathy and conduction defects via unknown mechanisms. To define these pathways, we generated mice with a ventricular-restricted knockout of Nkx2-5, which display no structural defects but have progressive complete heart block, and massive trabecular muscle overgrowth found in some patients with Nkx2-5 mutations. At birth, mutant mice display a hypoplastic atrioventricular (AV) node and then develop selective dropout of these conduction cells. Transcriptional profiling uncovered the aberrant expression of a unique panel of atrial and conduction system-restricted target genes, as well as the ectopic, high level BMP-10 expression in the adult ventricular myocardium. Further, BMP-10 is shown to be necessary and sufficient for a major component of the ventricular muscle defects. Accordingly, loss of ventricular muscle cell lineage specification into trabecular and conduction system myocytes is a new mechanistic pathway for progressive cardiomyopathy and conduction defects in congenital heart disease.

Defects in cardiac conduction system lineages and malignant arrhythmias: developmental pathways and disease.

To unravel the complex disease phenotype of heart failure, we are utilizing an integrative approach employing genomics, physiology, and mouse genetics to identify nodal pathways for specific physiological end points such as myocyte stretch activation responses, contractility and electrical conduction. A new class of genetic pathways for cardiac sudden death and associated arrhythmias has been based on transcription factors that control conduction system lineages, including HF1b/SP4 and NKX2.5. Previous studies have established that HF1b plays a critical role in conduction system lineage formation and the loss of HF1b leads to a confused electrophysiological identity in Purkinje and ventricular cell lineages, resulting in cardiac sudden death and marked tachy and brady arrhythmias. Utilizing Hf1b and Nkx2.5 floxed alleles, we now have identified the primary pathways which link these transcription factors with cardiac arrythmogenesis. Mice which harbour a neural crest restricted knockout of HF1b display marked arrhythmogenesis and conduction system defects, implicating neural crest cues in conduction system development and disease. Mice which harbour a ventricular-restricted knockout of Nkx2.5 display completely normal conduction at birth, but a hypoplastic atrioventricular (AV) node. During maturation, progressive complete heart block ensues, associated with a selective dropout of distal AV nodal cell lineages at the boundaries of the penetrating His bundle. Single cell analyses examining individual nodal cells within AV node of ventricular restricted Nkx2.5 knockout mice clearly document a cell autonomous requirement for NKX2.5 within AV nodal lineages per se. Micro-electrophysiological AV nodal mapping indicates a selective conduction defect at the boundary of the distal AV node and His bundle. HF1b and NKX2.5 reflect new cardiac cell non-autonomous and autonomous pathways for conduction system lineage defects and associated cardiac arrythmogenesis.

Gene-engineered models for genetic manipulation and functional analysis of the cardiovascular system in mice.

Cardiovascular disease remains a key issue in healthcare. During the last decade, transgenic and gene-targeted mouse technology has provided invaluable insights into cardiovascular molecular biology. Given the similarities between the mouse and human genomes, this study proposes that information experimentally derived using genetically manipulated mice can contribute significantly to the understanding of human cardiovascular pathophysiology. We first introduced the basic principles and methods of genetic manipulation, such as the breeding background in mice and the factors of construct design. Secondly, we reviewed the analyses related to genetic manipulation of the cardiovascular system from embryonic to adult mice. In conclusion, the gene-engineered mouse model is one of the most important tools developed in recent basic and clinical research.

Beyond peripheral arteries in Buerger's disease: angiographic considerations in thromboangiitis obliterans.

Thromboangiitis obliterans is an inflammatory peripheral vascular disease that is strongly associated with smoking. It predominantly affects distal small- and medium-sized blood vessels of both the upper and lower extremities. We present histological evidence of this disease process affecting the internal mammary arteries. This can be of paramount clinical significance for patients with Buerger's disease who present with obstructive coronary artery disease and require coronary artery bypass grafting surgery (CABG). Internal mammary arteries involved with thromboangiitis obliterans cannot be utilized as arterial conduits during CABG and other alternatives have to be used. Therefore, we recommend preoperative angiography of both internal mammary arteries in patients with Buerger's disease requiring CABG to prevent extensive intraoperative dissection of diseased internal mammary arteries.