X-linked Cardiomyopathy Research Articles

Application of Patient-Specific iPSCs for Modelling and Treatment of X-Linked Cardiomyopathies

Inherited cardiomyopathies are among the major causes of heart failure and associated with significant mortality and morbidity. Currently, over 70 genes have been linked to the etiology of various forms of cardiomyopathy, some of which are X-linked. Due to the lack of appropriate cell and animal models, it has been difficult to model these X-linked cardiomyopathies. With the advancement of induced pluripotent stem cell (iPSC) technology, the ability to generate iPSC lines from patients with X-linked cardiomyopathy has facilitated in vitro modelling and drug testing for the condition. Nonetheless, due to the mosaicism of the X-chromosome inactivation, disease phenotypes of X-linked cardiomyopathy in heterozygous females are also usually more heterogeneous, with a broad spectrum of presentation. Recent advancements in iPSC procedures have enabled the isolation of cells with different lyonisation to generate isogenic disease and control cell lines. In this review, we will summarise the current strategies and examples of using an iPSC-based model to study different types of X-linked cardiomyopathy. The potential application of isogenic iPSC lines derived from a female patient with heterozygous Danon disease and drug screening will be demonstrated by our preliminary data. The limitations of an iPSC-derived cardiomyocyte-based platform will also be addressed.

Arginine kinetics are altered in a pilot sample of adolescents and young adults with Barth syndrome

Barth syndrome (BTHS) is a rare, X-linked cardiomyopathy that is characterized by abnormalities in glucose and lipid metabolism, with less known regarding amino acid metabolism. This pilot study characterized whole-body arginine kinetics and found lower arginine rate of appearance into plasma (0.69 ± 0.09 vs. 0.88 ± 0.06 μmol/kgFFM/min, p < 0.01) and arginine non-oxidative disposal rate (0.64 ± 0.11 vs. 0.80 ± 0.03 μmol/kgFFM/min, p < 0.02) in adolescents and young adults with BTHS compared to Controls. This study provides a foundation for more in-depth studies on how arginine and potentially other amino acid abnormalities contribute to the pathology and clinical manifestations of BTHS.

CARDIOMYOPATHY CHARACTERIZATION AND RISK STRATIFICATION BY CARDIAC MAGNETIC RESONANCE IN DANON DISEASE

Danon disease (DD) is a rare and severe X-linked cardiomyopathy caused by LAMP2 deficiency. LAMP2 mutations may account for a significant proportion of hypertrophic cardiomyopathy. There is no data on cardiac magnetic resonance (CMR) association with outcome in DD. CMR scans of confirmed DD were

Dystrophinopathy in a Family Due to a Rare Nonsense Mutation Causing Predominant Behavioral Phenotype

AbstractDystrophinopathies are a group of X-linked neuromuscular disorders resulting from mutations in DMD gene that encodes dystrophin. The clinical spectrum includes Duchenne muscular dystrophy, Becker muscular dystrophy, X-linked cardiomyopathy, and intellectual disability without involvement of skeletal muscle. Cognitive and behavioral problems are commonly seen among patients with dystrophinopathy. DMD gene is the largest human gene, consisting of 79 exons that produce dystrophin protein. Patients with genetic changes involving shorter dystrophin isoforms such as Dp140 and Dp71 are suggested to have higher rates of intellectual disability, attention-deficit/hyperactivity disorder, and other neuropsychiatric comorbidities. We describe three brothers who presented with prominent neurobehavioral deficits of varying degree, mild proximal weakness, and elevated serum creatine kinase due to a rare nonsense mutation, c.1702C &gt; T; p.Gln568X, in exon 14 of DMD gene. Further studies are needed to better understand the effects of this rare mutation.

Decreasing cytosolic translation is beneficial to yeast and human Tafazzin-deficient cells.

Cardiolipin (CL) optimizes diverse mitochondrial processes, including oxidative phosphorylation (OXPHOS). To function properly, CL needs to be unsaturated, which requires the acyltransferase Tafazzin (TAZ). Loss-of-function mutations in the TAZ gene are responsible for the Barth syndrome (BTHS), a rare X-linked cardiomyopathy, presumably because of a diminished OXPHOS capacity. Herein we show that a partial inhibition of cytosolic protein synthesis, either chemically with the use of cycloheximide or by specific genetic mutations, fully restores biogenesis and the activity of the oxidative phosphorylation system in a yeast BTHS model (taz1Δ). Interestingly, the defaults in CL were not suppressed, indicating that they are not primarily responsible for the OXPHOS deficiency in taz1Δ yeast. Low concentrations of cycloheximide in the picomolar range were beneficial to TAZ-deficient HeLa cells, as evidenced by the recovery of a good proliferative capacity. These findings reveal that a diminished capacity of CL remodeling deficient cells to preserve protein homeostasis is likely an important factor contributing to the pathogenesis of BTHS. This in turn, identifies cytosolic translation as a potential therapeutic target for the treatment of this disease.

Unclassifiable arrhythmic cardiomyopathy associated with Emery-Dreifuss caused by a mutation in FHL1.

Emery-Dreifuss muscular dystrophy (EDMD) is a heterogeneous genetic disorder characterized by peripheral muscular weakness often associated with dilated cardiomyopathy. We characterize clinically a large family with a mutation in FHL1 gene (p.Cys255Ser). Penetrance was 44%, 100% for males and 18% for females. The heart was the main organ involved. Affected adult males had mild hypertrophy, systolic dysfunction and restriction with non-dilated ventricles. Carriers had significant QTc prolongation. The proband presented with resuscitated cardiac arrest. There were two transplants. Pathological study of explanted heart showed fibrofatty replacement and scarring consistent with arrhythmogenic cardiomyopathy and prominent left ventricular trabeculations. Myopathic involvement was evident in all males. Females had no significant neuromuscular disease. Mutations in FHL1 cause unclassifiable cardiomyopathy with coexisting EDMD. Prognosis is poor and systolic impairment and arrhythmias are frequent. Thrombopenia and raised creatine phosphokinase should raise suspicion of an FHL-1 disorder in X-linked cardiomyopathy.

A Role for Peroxisome Proliferator-activated Receptor γ Coactivator 1 (PGC-1) in the Regulation of Cardiac Mitochondrial Phospholipid Biosynthesis

The energy demands of the adult mammalian heart are met largely by ATP generated via oxidation of fatty acids in a high capacity mitochondrial system. Peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1)-α and -β serve as inducible transcriptional coregulators of genes involved in mitochondrial biogenesis and metabolism. Whether PGC-1 plays a role in the regulation of mitochondrial structure is unknown. In this study, mice with combined deficiency of PGC-1α and PGC-1β (PGC-1αβ(-/-)) in adult heart were analyzed. PGC-1αβ(-/-) hearts exhibited a distinctive mitochondrial cristae-stacking abnormality suggestive of a phospholipid abnormality as has been described in humans with genetic defects in cardiolipin (CL) synthesis (Barth syndrome). A subset of molecular species, containing n-3 polyunsaturated species in the CL, phosphatidylcholine, and phosphatidylethanolamine profiles, was reduced in PGC-1αβ-deficient hearts. Gene expression profiling of PGC-1αβ(-/-) hearts revealed reduced expression of the gene encoding CDP-diacylglycerol synthase 1 (Cds1), an enzyme that catalyzes the proximal step in CL biosynthesis. Cds1 gene promoter-reporter cotransfection experiments and chromatin immunoprecipitation studies demonstrated that PGC-1α coregulates estrogen-related receptors to activate the transcription of the Cds1 gene. We conclude that the PGC-1/estrogen-related receptor axis coordinately regulates metabolic and membrane structural programs relevant to the maintenance of high capacity mitochondrial function in heart.

P.13.11 Proteomic analysis of cardiomyopathic tissue from the aged mdx model of Duchenne muscular dystrophy

Senescent heart tissue from the mdx mouse model of Duchenne muscular dystrophy exhibits distinct pathological aspects of X-linked cardiomyopathy. In order to establish proteome-wide alterations in dystrophin-deficient hearts during aging, cardiomyopathic tissue from young versus aged mdx mice was examined by label-free LC–MS/MS analysis. Significant age-dependent alterations were established for 67 proteins, of which 28 proteins were found to be decreased and 39 proteins were shown to be increased in their expression levels. Drastic changes were demonstrated for proteins involved in ion transportation, the immune response, the cellular stress response, muscle contraction, metabolic regulation and the extracellular matrix. An immunoblotting survey of young and old wild type versus mdx hearts confirmed these proteomic findings and illustrated the effects of natural aging versus dystrophin deficiency. These proteome-wide alterations suggest a disintegration of the basal lamina structure and cytoskeletal network in dystrophin-deficient cardiac fibres, increased levels of antibodies in a potential autoimmune reaction of the degenerating heart, compensatory binding of excess iron and a general perturbation of metabolic pathways in dystrophy-associated cardiomyopathy. The proteomic technologies and analytical tools used in this study to identify significant proteins of interest have the potential to be brought forward and provide new therapeutic and diagnostic biomarkers for dystrophinopathy-related cardiomyopathy.

Deacylation on the matrix side of the mitochondrial inner membrane regulates cardiolipin remodeling

The mitochondrial-specific lipid cardiolipin (CL) is required for numerous processes therein. After its synthesis on the matrix-facing leaflet of the inner membrane (IM), CL undergoes acyl chain remodeling to achieve its final form. In yeast, this process is completed by the transacylase tafazzin, which associates with intermembrane space (IMS)-facing membrane leaflets. Mutations in TAZ1 result in the X-linked cardiomyopathy Barth syndrome. Amazingly, despite this clear pathophysiological association, the physiological importance of CL remodeling is unresolved. In this paper, we show that the lipase initiating CL remodeling, Cld1p, is associated with the matrix-facing leaflet of the mitochondrial IM. Thus monolysocardiolipin generated by Cld1p must be transported to IMS-facing membrane leaflets to gain access to tafazzin, identifying a previously unknown step required for CL remodeling. Additionally, we show that Cld1p is the major site of regulation in CL remodeling; and that, like CL biosynthesis, CL remodeling is augmented in growth conditions requiring mitochondrially produced energy. However, unlike CL biosynthesis, dissipation of the mitochondrial membrane potential stimulates CL remodeling, identifying a novel feedback mechanism linking CL remodeling to oxidative phosphorylation capacity.

Abstract 63: Missense Mutation Lys18Asn in Dystrophin that Triggers X-Linked Cardiomyopathy Affects Protein Structure, Decreases Protein Stability and Increases Protein Unfolding

A genetic mutation in dystrophin that replaces lysine at amino acid position 18 by an asparagine (K18N) results in X-linked cardiomyopathy (XCM), however, the underlying molecular mechanisms are not known. Such knowledge might help in developing effective therapies to treat XCM. The first step in understanding disease-triggering mechanisms is to probe how mutation affects protein structure and function. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD) of dystrophin. Here, we examined the effect of K18N on the structure, stability, and function of N-ABD. Fig. 1A shows the 15 N- 1 H HSQC NMR spectrum before and after the mutation. Each crosspeak in the spectrum represents the structure around an amino acid. Mutation affects the position of nearly all the crosspeaks. These changes are not just located at the mutation site, but were observed in regions far away from the mutation site and also in the three actin binding regions with which N-ABD interacts with actin. In addition, the mutation makes the protein more dynamic. These structural changes result in decreased actin binding function of N-ABD. Also, the K18N mutation decreases the stability by ΔΔG = 5.3 kcal/mol (Fig. 1B) and increases the rate of unfolding by 3.2 times (Fig. 1C), which exponentially increase the relative population of unfolded state that is more prone to proteolysis and hence will result in decreased levels of the functional protein. These results indicate that the physical mechanism by which the K18N mutation triggers the disease might be by affecting protein structure leading to decreased actin binding and by decreasing the net protein levels leading to decreased net function.

Abstract 9635: A Novel in vitro Model of Danon Disease Confirms the Critical Role of LAMP2 in Regulating Autophagy

Introduction: Danon disease is an X-linked cardiomyopathy caused by mutations in the lysosomal associated membrane protein 2 (LAMP2). LAMP2 plays a critical role in autophagy, but the mechanisms in which mutations lead to myocyte dysfunction are poorly understood. We identified a novel frame shift mutation that introduces a premature termination codon (PTC) in the LAMP2 gene in a patient with Danon disease. Degradation of PTC-containing mRNA transcripts by nonsense mediated decay (NMD) has been shown in a variety of heart diseases. We hypothesized that 1) the mechanism of LAMP2 dysfunction in this patient is NMD mediated, and 2) induced Pluripotent Stem (iPS) cell-derived myocytes generated from these fibroblasts would show evidence of impaired autophagy. Methods: Dermal fibroblast lines were established from a patient with Danon disease and used for RNA and protein assays. Fibroblasts were subsequently used to create iPS cell-derived cardiomyocytes using previously described techniques. Immunohistochemistry and protein gel electrophoresis of the autophagosome associated protein, LC3-II, were used to assay for autophagic function. Ultrastructural studies were performed using electron microscopy. Results: Patient fibroblasts were shown to completely lack the LAMP2 protein. Treatment with the protein synthesis inhibitor cyclohexamide stabilized LAMP2 mRNA levels, suggesting this LAMP2 mutation is targeted by NMD. Compared to wild type cells, Danon disease iPS-derived cells demonstrated reduced basal levels of the autophagosome-associated protein LC3-II, suggestive of impaired autophagy. Electron microscopy of iPS cells showed excessive intracytoplasmic vacuoles consistent with the disease phenotype. Conclusions: We have successfully created the first in vitro human model of Danon disease. Using this system, we identified NMD as a novel mechanism responsible for LAMP2 dysfunction in a patient with Danon disease. Furthermore, we confirmed the critical role of LAMP2 in the regulation of autophagy. This study highlights the utility of this in vitro system for elucidating the pathophysiology of inherited cardiovascular disease.

Barth Syndrome: An X-linked Cardiomyopathy with a Novel Mutation

The authors report a 6yr old boy with Barth syndrome who presented with cardiomyopathy, neutropenia and hypotonia. Urine gas chromatography showed high level of 3-methylglutaconic acid. The DNA of both the patient and the mother showed a heterozygous 3bp deletion in exon 8 of the tafazzin gene. This abnormality involves the deletion of the bases TGA starting at cDNA nucleotide 891 (c891_893delTGA), resulting in the absence of glutamic acid at codon 202 from a highly conserved area of the tafazzin protein, consistent with the diagnosis of Barth syndrome. This is the first case report of Barth syndrome in Arab population emphasizing the importance of detailed investigations in cases of hereditary cardiomyopathy.

Disease-causing missense mutations in actin binding domain 1 of dystrophin induce thermodynamic instability and protein aggregation

Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD) most commonly through loss of protein expression. In a small subpopulation of patients, missense mutations can cause DMD, Becker muscular dystrophy, or X-linked cardiomyopathy. Nearly one-half of disease-causing missense mutations are located in actin-binding domain 1 (ABD1) of dystrophin. To test the hypothesis that ABD1 missense mutations cause disease by impairing actin-binding activity, we engineered the K18N, L54R, D165V, A168D, L172H, and Y231N mutations into the full-length dystrophin cDNA and characterized the biochemical properties of each mutant protein. The K18N and L54R mutations are associated with the most severe diseases in humans and each caused a small but significant 4-fold decrease in actin-binding affinity, while the affinities of the other four mutant proteins were not significantly different from WT dystrophin. More interestingly, WT dystrophin was observed to unfold in a single-step, highly cooperative manner. In contrast, all six mutant proteins were significantly more prone to thermal denaturation and aggregation. Our results suggest that missense mutations in ABD1 may all cause loss of dystrophin function via protein instability and aggregation rather than through loss of ligand binding function. However, more severe disease progressions may be due to the combinatorial effects of some mutations on both protein aggregation and impaired actin-binding activity.

Structure Function Analysis of Disease-Causing Missense Mutations in Dystrophin

Duchenne muscular dystrophy (DMD) affects 1 of every 3500 males and results in death during the mid to late twenties. Mutations in the dystrophin gene leading to DMD commonly result in loss of protein expression or expression of a truncated protein lacking essential ligand binding domains. In some cases, point mutations leading to a single amino acid change in the dystrophin protein cause DMD, Becker muscular dystrophy or X-linked cardiomyopathy. Of the known disease causing mutations, 9 are located in the N-terminal actin-binding domain of dystrophin. Examining the effects of these mutations on actin binding activity will lead to a better understanding of key residues for dystrophin function in vivo. With this in mind, we engineered all 9 N-terminal disease-causing mutations into the full-length dystrophin cDNA and have begun to characterize the biochemical properties of each mutant protein expressed in the baculovirus system. We have found that R82P and A172P mutants did not express well enough to enable further biochemical characterization. The 7 remaining mutants were consistently less soluble and more aggregated than WT dystrophin. We have analyzed four mutants K18N, L54R, D165V and L172H for their ability to bind F-actin and found that K18N and L54R decreased the affinity of dystrophin for F-actin by 3–4 fold. The L172H mutation affected solubility but not actin binding properties of the full-length dystrophin protein. These data suggest that disease phenotypes associated with missense dystrophin mutations are caused by either loss of solubility or a combination of insolubility and decreased F-actin affinity. We also found that mutations that cause a more severe disease phenotype (K18N and L54R) bound actin with a lower affinity and were less soluble than WT dystrophin.

A novel mouse model of X-linked cardiac hypertrophy

We recovered a novel mouse mutant exhibiting neonatal lethality associated with severe fetal cardiac hypertrophy and with some adult mice dying suddenly with left ventricular hypertrophic cardiomyopathy. Using Doppler echocardiography, we screened surviving adult mice in this mutant line for cardiac hypertrophy. Cardiac dimensions were obtained either from two-dimensional images collected using a novel ECG-gated ultra-high-frequency ultrasound system or by traditional M-mode imaging on a clinical ultrasound system. These analyses identified, among the littermates, two populations of mice: those with apparent cardiac hypertrophy with hypercontractile function characterized by ejection fraction of 75-80%, and normal littermates with ejection fraction of 53-55%. Analysis of the ECG-gated two-dimensional cines indicated that the hypertrophy was of the nonobstructive type. Further analysis of heart-to-body weight ratio confirmed the ultrasound diagnosis of left ventricular hypertrophic cardiomyopathy. Histopathology showed increased ventricular wall thickness, enlarged myocyte size, and mild myofiber disarray. Ultrastructural analysis by electron microscopy revealed mitochondria hyperproliferation and dilated sarcoplasmic reticulum. Genome scanning using microsatellite DNA markers mapped the mutation to the X chromosome. DNA sequencing showed no mutations in the coding regions of several candidate genes on the X chromosome, including several known to be associated with left ventricular hypertrophic cardiomyopathy. These findings suggest that this mouse line may harbor a mutation in a novel gene causing X-linked cardiomyopathy.

X-linked Cardiomyopathy Presenting as Contracted Endocardial Fibroelastosis

We report a case of familial contracted endocardial fibroelastosis (EFE) in a young boy presenting at 14 months of age with severe heart failure. A previous echocardiogram showed normal left ventricular (LV) size and systolic function. The family history was suggestive of X-linked cardiomyopathy. These findings are assessed in light of earlier reports of contracted EFE.

Familial X-linked cardiomyopathy (Danon disease): diagnostic confirmation by mutation analysis of the LAMP2gene

A boy presented at age 2.5 years with mild left ventricular hypertrophy and mild myopathy. Hypertrophic cardiomyopathy progressed relentlessly, leading to death at age 16 years shortly before planned heart transplantation. During the course of the disease, his mother developed severe dilated cardiomyopathy and died of its complications at 46 years of age. The combination of myopathy and cardiomyopathy, the biochemical and electron microscopy findings in a muscle biopsy, and the pedigree suggested Danon disease (MIM 300257), an X-linked lysosomal storage disorder caused by deficiency of lysosome-associated membrane protein-2 (LAMP2). The diagnosis was confirmed by the identification of a novel mutation, G138A, in the LAMP2gene, leading to the premature stop codon W46X. Early diagnosis of Danon disease is important for genetic counselling and timely cardiac transplantation, the only effective therapeutic option.

Different expressions of X-linked cardiomyopathy in monozygotic triplets with Becker's dystrophy.

Cardiac involvement is common in skeletal muscles disorders associated with dystrophin defect. It has been suggested however, that X-linked dilated cardiomyopathies with minimal or absent skeletal disease are distinct entities, resulting from mutations in cardiac-specific regions of dystrophin gene. This study presents a unique observation of phenotypic variability in monozygotic triplets with Becker's muscular dystrophy. The expressions of the disease range from severe peripheral myopathy to severe congestive heart failure. No deletion in dystrophin gene was observed and the mechanisms responsible for selective impairment of morphologically and functionally different muscles in three monozygotic siblings remain unclear.

Mutation analysis of the G4.5 gene in patients with isolated left ventricular noncompaction

Mutations in the gene G4.5, originally associated with Barth syndrome, have been reported to result in a wide spectrum of severe infantile X-linked cardiomyopathies. The purpose of this study was to investigate patients with isolated left ventricular noncompaction (LVNC) for disease-causing mutations in G4.5. In 27 patients including 10 families with isolated LVNC, mutation analysis of G4.5 was performed using single-strand DNA conformation polymorphism (SSCP) analysis and DNA sequencing. A novel splice acceptor site mutation of intron 8 of G4.5 was identified in a family with severe infantile X-linked LVNC without the usual findings of Barth syndrome. This mutation results in deletion of exon 9 from the mRNA, and is predicted to significantly disrupt the protein product. Genotype–phenotype correlation of G4.5 mutations in all 38 cases reported in the literature to date revealed that there was no correlation between location or type of mutation and either cardiac phenotype or disease severity. We suggest that males presenting with cardiomyopathy, particularly during infancy, even in the absence of the typical signs of Barth syndrome, should be evaluated for mutations in G4.5.

Infantile Dilated X-Linked Cardiomyopathy, G4.5 Mutations, Altered Lipids, and Ultrastructural Malformations of Mitochondria in Heart, Liver, and Skeletal Muscle

Mutations in the Xq28 gene G4.5 lead to dilated cardiomyopathy (DCM). Differential splicing of G4.5 results in a family of proteins called “tafazzins” with homology to acyltransferases. These enzymes assemble fatty acids into membrane lipids. We sequenced G4.5 in two kindreds with X-linked DCM and in two unrelated men, one with idiopathic DCM and the other with DCM of arrhythmogenic right ventricular dysplasia. We examined the ultrastructure of heart, liver, and muscle biopsy specimens in these three DCM types; we used gas chromatography to compare fatty acid composition in heart, liver, and muscle autopsy specimens of two patients of kindred 1 with that of controls. In X-linked DCM, G4.5 had a stop codon (E188X), a nonsense mutation, in kindred 1 and an amino acid substitution (G240R), a missense mutation, in kindred 2. In the two men with isolated DCM, G4.5 was not mutated. Ultrastructural mitochondrial malformations were present in the biopsy tissues of the patients with DCM. Cardiac biopsy specimens of both kindreds with X-linked DCM exhibited greatly enlarged mitochondria with large bundles of stacked, compacted, disarrayed cristae that differed from those of the two types of isolated DCM. Autopsy tissue of patients with X-linked DCM had decreased unsaturated and increased saturated fatty acid concentrations. Seven of 13 published G4.5 missense mutations, including the one presented here, occur in acyltransferase motifs. Impaired acyltransferase function could result in increased fatty acid saturation that would decrease membrane fluidity. Mitochondrial membrane proliferation may be an attempt to compensate for impaired function of acyltransferase. Cardiac ultrastructure separates X-linked DCM with G4.5 mutations from the two types of isolated DCM without G4.5 mutations. Electron microscopy of promptly fixed myocardial biopsy specimens has a role in defining the differential diagnosis of DCM. Mutational analysis of the G4.5 gene also serves this purpose.