RESUMO
For inherited cardiomyopathies, abnormal sensitivity to intracellular calcium (Ca(2+) ), incurred from genetic mutations, initiates subsequent molecular events leading to pathological remodeling. Here, we characterized the effect of ß-adrenergic stress in familial dilated cardiomyopathy (DCM) using human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) from a patient with RBM20 DCM. Our findings suggest that ß-adrenergic stimulation accelerated defective Ca(2+) homeostasis, apoptotic changes, and sarcomeric disarray in familial DCM hiPSC-CMs. Furthermore, pharmacological modulation of abnormal Ca(2+) handling by pretreatment with ß-blocker, carvedilol, or Ca(2+) -channel blocker, verapamil, significantly decreased the area under curve, reduced percentage of disorganized cells, and decreased terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive apoptotic loci in familial DCM hiPSC-CMs after ß-adrenergic stimulation. These translational data provide patient-based in vitro analysis of ß-adrenergic stress in RBM20-deficient familial DCM hiPSC-CMs and evaluation of therapeutic interventions to modify heart disease progression, which may be personalized, but more importantly generalized in the clinic.
Assuntos
Cálcio/metabolismo , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Homeostase , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Proteínas de Ligação a RNA/metabolismo , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Carbazóis/farmacologia , Carvedilol , Homeostase/efeitos dos fármacos , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Norepinefrina/farmacologia , Propanolaminas/farmacologia , Receptores Adrenérgicos beta/metabolismo , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Estresse Fisiológico , Verapamil/farmacologiaRESUMO
We identified a unique family with autosomal dominant heart disease variably expressed as restrictive cardiomyopathy (RCM), hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM), and sought to identify the molecular defect that triggered divergent remodeling pathways. Polymorphic DNA markers for nine sarcomeric genes for DCM and/or HCM were tested for segregation with disease. Linkage to eight genes was excluded, but a cardiac troponin T (TNNT2) marker cosegregated with the disease phenotype. Sequencing of TNNT2 identified a heterozygous missense mutation resulting in an I79N substitution, inherited by all nine affected family members but by none of the six unaffected relatives. Mutation carriers were diagnosed with RCM (n = 2), non-obstructive HCM (n = 3), DCM (n = 2), mixed cardiomyopathy (n = 1), and mild concentric left ventricular hypertrophy (n = 1). Endomyocardial biopsy in the proband revealed non-specific fibrosis, myocyte hypertrophy, and no myofibrillar disarray. Restrictive Doppler filling patterns, atrial enlargement, and pulmonary hypertension were observed among family members regardless of cardiomyopathy subtype. Mutation of a sarcomeric protein gene can cause RCM, HCM, and DCM within the same family, underscoring the necessity of comprehensive morphological and physiological cardiac assessment in familial cardiomyopathy screening.
Assuntos
Cardiomiopatia Dilatada/genética , Cardiomiopatia Hipertrófica Familiar/genética , Cardiomiopatia Restritiva/genética , Mutação , Troponina T/genética , Adulto , Idoso , Cardiomiopatia Dilatada/fisiopatologia , Cardiomiopatia Restritiva/fisiopatologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
The cardiac sodium channel (SCN5A) is a target for the treatment of arrhythmias. We hypothesized that vulnerability to atrial fibrillation (AF) could be caused by genetic variation in SCN5A. We recruited 157 patients with early-onset AF who lacked traditional risk factors, and 314 matched controls. SCN5A was subject to targeted genotyping of a common loss-of-function H558R polymorphism and comprehensive mutation scanning. Genotype frequencies in the AF cohort vs controls were as follows: HH, 50 vs 63%; HR, 40 vs 33%; and RR, 10 vs 4% (P=0.008). Additional coding sequence mutations were ruled out. The R558 allele was more common in patients than in controls (30 vs 21%, P=0.002), conferring an odds ratios for AF of 1.6 (95% confidence interval 1.2-2.2). The SCN5A R558 allele, present in one-third of the population, thus constitutes a risk factor for lone AF and may increase susceptibility to sodium channel blocker-induced proarrhythmia.
Assuntos
Fibrilação Atrial/genética , Predisposição Genética para Doença , Canais de Sódio/genética , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.5 , Polimorfismo GenéticoRESUMO
A third of inherited diseases result from premature termination codon mutations. Aminoglycosides have emerged as vanguard pharmacogenetic agents in treating human genetic disorders due to their unique ability to suppress gene translation termination induced by nonsense mutations. In preclinical and pilot clinical studies, this therapeutic approach shows promise in phenotype correction by promoting otherwise defective protein synthesis. The challenge ahead is to maximize efficacy while preventing interaction with normal protein production and function.
Assuntos
Aminoglicosídeos/farmacologia , Códon sem Sentido , Doenças Genéticas Inatas/tratamento farmacológico , Farmacogenética/métodos , Modificação Traducional de Proteínas/efeitos dos fármacos , Aminoglicosídeos/uso terapêutico , Animais , Doenças Genéticas Inatas/genética , HumanosRESUMO
BACKGROUND: It is unknown whether progression of familial idiopathic dilated cardiomyopathy differs from progression in the non-familial form. It has been suggested that familial disease indicates a worse prognosis, and that this should be considered when planning the timing of heart transplantation. OBJECTIVE: To compare five year survival or time to heart transplantation in an unselected series of patients with dilated cardiomyopathy who had been evaluated for familial v non-familial disease through the echocardiographic investigation of first degree relatives. DESIGN: Medical records were reviewed and questionnaires were mailed to all patients who had previously participated in a family based study of dilated cardiomyopathy. Information was gathered about survival, heart transplantation, and left ventricular ejection fraction (LVEF) measurements. Survival data were censored at the time of cardiac transplantation. RESULTS: Follow up data were obtained for 99 of 101 patients (69 with non-familial and 30 with familial disease). Five year survival was 55% for non-familial and 51% for familial patients (NS). The main predictor of mortality was an LVEF of < 30%. Familial status did not predict mortality. There was no significant difference in follow up LVEF values between the groups. CONCLUSIONS: Five year survival is not significantly different in the familial and non-familial forms of dilated cardiomyopathy.
Assuntos
Cardiomiopatia Dilatada/mortalidade , Adolescente , Adulto , Idoso , Cardiomiopatia Dilatada/genética , Criança , Pré-Escolar , Progressão da Doença , Ecocardiografia , Feminino , Seguimentos , Transplante de Coração , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Linhagem , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/mortalidadeRESUMO
Filtration is commonly employed in water and wastewater treatment to remove particles and reduce the concentration of microbial pathogens. All physical models of packed-bed filtration are based on a proportional relationship between particle removal per unit depth of bed and the local particle concentration, dC/dz = -C/l, where l is the filtration length scale. Although l is known to vary with time and filter depth for heterogeneous suspensions or "dirty" beds, this paper demonstrates that the filtration rates of even seemingly monodisperse particle suspensions under clean-bed filtration conditions cannot be described with a single filtration length scale. A new model is derived for particle filtration that accounts for heterogeneity at two different spatial scales. Heterogeneity at the scale of the pathogen and/or collector (microscale heterogeneity) leads to a slow power-law decay of contaminant concentration with distance, instead of the fast exponential decay predicted by the standard model. Heterogeneity at the filter scale (macroscale heterogeneity) provides another level of complexity that can be evaluated once microscale heterogeneity effects are characterized. This model for microscale and macroscale heterogeneous particle filtration is verified by filtration experiments on a recombinant analogue of the waterborne pathogen Norwalk virus.
Assuntos
Eliminação de Resíduos Líquidos/métodos , Microbiologia da Água , Purificação da Água/métodos , Abastecimento de Água , Filtração , Modelos Teóricos , Vírus Norwalk/isolamento & purificação , Tamanho da PartículaRESUMO
Proteins in cardiac myocytes assemble into contractile units known as sarcomeres. Contractile force is generated by interaction between sarcomeric thick and thin filaments. Thin filaments also transmit force within and between myocytes. Mutations in genes encoding the thin filament proteins actin and tropomyosin cause hypertrophic cardiomyopathy. Mutations affecting functionally distinct domains of actin also cause dilated cardiomyopathy (DCM). We used a non-positional candidate gene approach to test further the hypothesis that dysfunction of sarcomeric thin filaments, due to different mutations in the same gene, can lead to either hypertrophic or dilated cardiomyopathy. Mutational analyses of alpha-tropomyosin 1 were performed in patients with idiopathic DCM. We identified two mutations that alter highly conserved residues and that, unlike hypertrophic cardiomyopathy-associated mutations, cause localized charge reversal on the surface of tropomyosin. Therefore, substitution of different amino acid residues in the same thin filament proteins is associated with the distinct phenotypes of cardiac hypertrophy or congestive heart failure.
Assuntos
Cardiomiopatia Dilatada/genética , Proteínas de Drosophila , Tropomiosina/genética , Adulto , Sequência de Aminoácidos , Animais , Cardiomiopatia Dilatada/patologia , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Linhagem , Conformação Proteica , Sarcômeros/ultraestrutura , Tropomiosina/químicaRESUMO
The molecular mechanisms by which different mutations in actin lead to distinct cardiomyopathies are unknown. Here, actin mutants corresponding to alpha-cardiac actin mutations causing hypertrophic cardiomyopathy [(HCM) P164A and A331P] and dilated cardiomyopathy [(DCM) R312H and E361G] were expressed in yeast and purified for in vitro functional studies. While P164A appeared unaltered compared to wild-type (WT) actin, A331P function was impaired. A331P showed reduced stability in circular dichroism melting experiments; its monomer unfolding transition was 10 degrees C lower compared to WT actin. Additionally, in vitro filament formation was hampered, and yeast cell cultures were temperature sensitive, implying perturbations in actin-actin interactions. Filament instability of the A331P mutant actin could lead to actomyosin dysfunction observed in HCM. Yeast strains harboring the R312H mutation did not grow well in culture, suggesting that cell viability is compromised. The E361G substitution is located at an alpha-actinin binding region where the actin filament is anchored. The mutant actin, though unaltered in the in vitro motility and standard actomyosin functions, had a threefold reduction in alpha-actinin binding. This could result in impairment of force-transduction in muscle fibers, and a DCM phenotype.
Assuntos
Actinas/genética , Actinas/metabolismo , Cardiomiopatias/genética , Saccharomyces cerevisiae/genética , Actinina/metabolismo , Actinas/química , Adenosina Trifosfatases/metabolismo , Sítios de Ligação/fisiologia , Humanos , Mutagênese/fisiologia , Mutação de Sentido Incorreto/fisiologia , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Fenótipo , Polímeros/metabolismo , Estrutura Terciária de ProteínaRESUMO
Mutations in genes encoding sarcomeric proteins cause hypertrophic cardiomyopathy (HCM). The sarcomeric protein actin plays a central, dual role in cardiac myocytes, generating contractile force by interacting with myosin and also transmitting force within and between cells. Two missense mutations in the cardiac actin gene (ACTC), postulated to impair force transmission, have been associated with familial dilated cardiomyopathy (DCM). Recently, a missense mutation in ACTC was found to cosegregate with familial HCM. To further test the hypothesis that mutations within functionally distinct domains of ACTC cause either DCM or HCM, we performed mutational analyses in 368 unrelated patients with familial or sporadic HCM. Single strand conformation polymorphism and sequence analyses of genomic DNA were performed. De novo mutations in ACTC were identified in two patients with sporadic HCM who presented with syncope in early childhood. Patients were heterozygous for missense mutations resulting in Pro164Ala and Ala331Pro amino acid substitutions, adjacent to regions of actin-actin and actin-myosin interaction, respectively. A mutation that cosegregated with familial HCM was also found, causing a Glu99Lys substitution in a weak actomyosin binding domain. The cardiac phenotype in many affected patients was characterized by an apical form of HCM. These findings support the hypothesis that a single amino acid substitution in actin causes either congestive heart failure or maladaptive cardiac hypertrophy, depending on its effect on actin structure and function.
Assuntos
Actinas/genética , Cardiomiopatia Hipertrófica/etiologia , Cardiomiopatia Hipertrófica/genética , Predisposição Genética para Doença , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-IdadeRESUMO
We have isolated genomic DNA containing the human tissue inhibitor of metalloproteinases-4 gene (TIMP4) and determined the structure of the exons comprising the gene. Like other members of the TIMP family, the TIMP-4 protein is encoded by five exons. These span 6 kb of genomic DNA, so that TIMP4 is similar in size to Timp1 but considerably smaller than TIMP2 and TIMP3. The exon-intron boundaries of TIMP4 are at locations very similar to those of the other TIMP genes, demonstrating the high degree of conservation of gene structure in this family. The human and mouse TIMP-4 genes map to comparable locations in the respective genomes, localizing to human chromosome 3p25 and mouse chromosome 6.
Assuntos
Cromossomos Humanos Par 3/genética , Camundongos/genética , Inibidores Teciduais de Metaloproteinases/genética , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Evolução Molecular , Éxons , Humanos , Hibridização in Situ Fluorescente , Íntrons , Metaloendopeptidases/antagonistas & inibidores , Dados de Sequência Molecular , Família Multigênica , Mapeamento por Restrição , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
To test the hypothesis that actin dysfunction leads to heart failure, patients with hereditary idiopathic dilated cardiomyopathy (IDC) were examined for mutations in the cardiac actin gene (ACTC). Missense mutations in ACTC that cosegregate with IDC were identified in two unrelated families. Both mutations affect universally conserved amino acids in domains of actin that attach to Z bands and intercalated discs. Coupled with previous data showing that dystrophin mutations also cause dilated cardiomyopathy, these results raise the possibility that defective transmission of force in cardiac myocytes is a mechanism underlying heart failure.
Assuntos
Actinas/genética , Cardiomiopatia Dilatada/genética , Mutação , Actinas/química , Actinas/fisiologia , Adolescente , Adulto , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Criança , Pré-Escolar , Cromossomos Humanos Par 15 , Éxons , Feminino , Coração/fisiopatologia , Humanos , Masculino , Miocárdio/química , Miocárdio/patologia , Linhagem , Fenótipo , Polimorfismo Conformacional de Fita Simples , Conformação Proteica , Sarcômeros/fisiologiaRESUMO
Dilated cardiomyopathy (DCM) is a significant health care problem. The etiology is idiopathic in approximately half of the patients. Recognition that 20%-25% of idiopathic DCM cases are familial has advanced the hypothesis that single gene defects are important in the disease's pathogenesis. General linkage analyses in rare, large DCM families have determined the chromosome location of five idiopathic DCM genes. Candidate-gene mutational analyses in more typical, small pedigrees represent an alternative strategy for DCM gene identification. Human molecular genetics will play a fundamental role in defining pathogenic mechanisms for DCM with the prospect of new, molecular-based diagnostic and therapeutic approaches. (Trends Cardiovasc Med 1997;7:60-63). © 1997, Elsevier Science Inc.
RESUMO
Dilated cardiomyopathy (DCM) is a common disorder characterized by cardiac dilation and reduced systolic function. To identify a cardiomyopathy gene, we studied a family with DCM associated with sinus node dysfunction, supraventricular tachyarrhythmias, conduction delay, and stroke. A general linkage approach was used to localize the disease gene in this family. Linkage to D3S2303 was identified with a two-point lod score of 6.09 at a recombination fraction of 0.00. Haplotype analyses mapped this locus to a 30 cM region of chromosome 3p22-p25, excluding candidate genes encoding a G-protein (GNAI2), calcium channel (CACNL1A2), sodium channel (SCN5A), and inositol triphosphate receptor (ITPR1). These data indicate that a gene causing DCM associated with rhythm and conduction abnormalities is located on chromosome 3p, and represent the first step toward disease gene identification.
Assuntos
Cardiomiopatia Dilatada/genética , Cromossomos Humanos Par 3 , Feminino , Ligação Genética , Marcadores Genéticos , Humanos , Masculino , Linhagem , FenótipoRESUMO
Case control studies have reported associations between specific HLA class II antigens and idiopathic dilated cardiomyopathy (DCM), suggesting that genetically regulated immune response factors may be involved in the pathogenesis of this disease. In this study, families with DCM were used to test the hypothesis that a heritable gene defect in the HLA region is the primary genetic determinant for a subset of cases. Twelve families with DCM were identified. By formal segregation analysis, the inheritance of the disease was most consistent with an autosomal dominant gene defect with incomplete penetrance. Genotyping was performed with five highly polymorphic linked dinucleotide repeat markers that span the HLA locus. Linkage analysis was used to determine whether or not these genetic markers cosegregated with the disease phenotype. Genetic linkage between the disease phenotype and a 21 cM region spanning the HLA was excluded (lod score < or = -2) in at least 60% of our families. These results indicate that a gene defect in the HLA locus region is not the primary genetic determinant of DCM in a series of familial cases. However, our data do not exclude the possibility that HLA regulated immune response factors may have a modifying effect on disease penetrance and expression.
Assuntos
Cardiomiopatia Dilatada/genética , Antígenos HLA/genética , Cardiomiopatia Dilatada/imunologia , Feminino , Marcadores Genéticos , Humanos , Escore Lod , Masculino , LinhagemRESUMO
We reviewed medical records from eight patients (4 to 29 years of age) with a functional single ventricle and pulmonary microthrombosis as observed on open lung biopsy specimens. Hemodynamic assessment before biopsy revealed pulmonary hypertension (mean pulmonary artery pressure 19 to 53 mm Hg) in the seven patients tested and severely increased pulmonary arteriolar resistance (6 to 13 U.m2) in three of the five patients in whom these measurements were performed. Pulmonary blood flow varied (< 4 L/min per square meter in two patients and > 5 L/min per square meter in four patients), and the pulmonary/systemic blood flow ratio was low or normal (0.6 to 1.1) in five of six cases. Hemoglobin values were increased (16.4 to 22 gm/dl) in seven of eight patients. Lung biopsy specimens revealed thrombotic obstructive lesions in 56% (range 28% to 96%) of muscular pulmonary arteries. Coexistent medial hypertrophy was absent or mild in all but one specimen, and none had features of plexogenic pulmonary arteriopathy. Microscopic pulmonary thrombi accounted for unexpected pulmonary hypertension, precluding the Fontan operation, in five patients and unsuccessful Fontan operation in two. Clinically significant thrombi should be considered in patients with severe polycythemia (hemoglobin value > 20 gm/dl) and unobtainable or unreliable pulmonary blood flow or pulmonary resistance determinations.
Assuntos
Cardiopatias Congênitas/complicações , Cardiopatias Congênitas/cirurgia , Embolia Pulmonar/complicações , Adolescente , Biópsia , Criança , Pré-Escolar , Feminino , Cardiopatias Congênitas/fisiopatologia , Hemodinâmica , Humanos , Pulmão/patologia , Masculino , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/fisiopatologia , Estudos Retrospectivos , Resistência VascularRESUMO
Supravalvular aortic stenosis (SVAS) is a localized or diffuse congenital narrowing of the ascending aorta which may occur sporadically, as a familial defect, or in association with Williams syndrome. Familial cases suggest an autosomal dominant gene defect but the underlying molecular basis of SVAS is unknown. In this study, we sought to localize the genetic defect in familial SVAS by linkage analysis in a large three generation family. A total of 44 polymorphic markers were examined for linkage, including 17 Southern blot-based RFLPs, 2 PCR-based RFLPs, and 25 microsatellites, primarily of the (CA)n repeat type. We report linkage of the disease phenotype to a highly informative (CA)n repeat marker, Mfd 50, at locus D7S440 which has been localized to chromosome arm 7q. Using a 100% penetrance model, which was more conservative than lower values of penetrance, a peak LOD score of 4.66 at a recombination frequency of 0.043 was found. A number of candidate genes have been localized to this region, including collagen 1A2, laminin B1, and elastin. Based on our preliminary linkage data, the abnormal microscopic appearance of aortic elastic fibers in SVAS, and analogous animal and human diseases associated with elastic fiber and vascular abnormalities, there is indirect evidence suggesting elastin as a possible candidate gene for this disorder.
Assuntos
Estenose da Valva Aórtica/genética , Cromossomos Humanos Par 7 , Mapeamento Cromossômico , Elastina/genética , Feminino , Genes Dominantes , Ligação Genética , Marcadores Genéticos , Humanos , Masculino , Oligodesoxirribonucleotídeos/genética , Linhagem , Polimorfismo Genético , Sequências Repetitivas de Ácido NucleicoRESUMO
The kinetics and mechanism of Fe(III) reduction to Fe(II) were studied in pure batch cultures of Pseudomonas sp. 200. The rate of iron reduction has been mechanistically related to aqueous phase iron speciation. In the absence of microbial activity the iron reduction rate was negligible. Initial rates of microbial iron reduction were accelerated more than 20-fold by the addition of equimolar quantities of nitrilotriacetic acid (NTA) to media initially containing 1.86 x 10(-3)M total Fe(III). Numerical techniques were utilized to quantify relationships between the observed rate of Fe(II) production and the calculated (equilibrium) aqueous phase speciation. These results indicate that soluble ferric iron species are not equivalent in terms of their susceptibility to bacterial (dissimilative) iron reduction. The concentration of Fe(NTA)(OH)(2) (2-) correlated strongly with observed iron reduction rates. Ferrous iron species appeared to inhibit the reduction process.