RESUMO
Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac ß-myosin heavy chain (ß-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.
Assuntos
Cadeias Pesadas de Miosina , Transfecção , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Humanos , Transfecção/métodos , Linhagem Celular , Animais , Camundongos , Miosinas CardíacasRESUMO
Aortic aneurysms (AAs) are pathological dilatations of the aorta. Pathogenic variants in genes encoding for proteins of the contractile machinery of vascular smooth muscle cells (VSMCs), genes encoding proteins of the transforming growth factor beta signaling pathway and extracellular matrix (ECM) homeostasis play a role in the weakening of the aortic wall. These variants affect the functioning of VSMC, the predominant cell type in the aorta. Many variants have unknown clinical significance, with unknown consequences on VSMC function and AA development. Our goal was to develop functional assays that show the effects of pathogenic variants in aneurysm-related genes. We used a previously developed fibroblast transdifferentiation protocol to induce VSMC-like cells, which are used for all assays. We compared transdifferentiated VSMC-like cells of patients with a pathogenic variant in genes encoding for components of VSMC contraction (ACTA2, MYH11), transforming growth factor beta (TGFß) signaling (SMAD3) and a dominant negative (DN) and two haploinsufficient variants in the ECM elastic laminae (FBN1) to those of healthy controls. The transdifferentiation efficiency, structural integrity of the cytoskeleton, TGFß signaling profile, migration velocity and maximum contraction were measured. Transdifferentiation efficiency was strongly reduced in SMAD3 and FBN1 DN patients. ACTA2 and FBN1 DN cells showed a decrease in SMAD2 phosphorylation. Migration velocity was impaired for ACTA2 and MYH11 cells. ACTA2 cells showed reduced contractility. In conclusion, these assays for showing effects of pathogenic variants may be promising tools to help reclassification of variants of unknown clinical significance in AA-related genes.
Assuntos
Actinas/genética , Aneurisma Aórtico/etiologia , Fibrilina-1/genética , Cadeias Pesadas de Miosina/genética , Proteína Smad3/genética , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/patologia , Diferenciação Celular/genética , Transdiferenciação Celular/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Modelos Biológicos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Proteína Smad2/metabolismoRESUMO
BACKGROUND: While stress and the absence of social support during pregnancy have been linked to poor health outcomes, the underlying biological mechanisms are unclear. METHODS: We examined whether adverse experiences during pregnancy alter DNA methylation (DNAm) in maternal epigenomes. Analyses included 250 African-American mothers from the Boston Birth Cohort. Genome-wide DNAm profiling was performed in maternal blood collected after delivery, using the Infinium HumanMethylation450 Beadchip. Linear regression models, with adjustment of pertinent covariates, were applied. RESULTS: While self-reported maternal psychosocial lifetime stress and stress during pregnancy was not associated with DNAm alterations, we found that absence of support from the baby's father was significantly associated with maternal DNAm changes in TOR3A, IQCB1, C7orf36, and MYH7B and that lack of support from family and friends was associated with maternal DNA hypermethylation on multiple genes, including PRDM16 and BANKL. CONCLUSIONS: This study provides intriguing results suggesting biological embedding of social support during pregnancy on maternal DNAm, warranting additional investigation, and replication.
Assuntos
Metilação de DNA , Apoio Social , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Trifosfatases/genética , Adulto , Negro ou Afro-Americano , Boston , Proteínas de Ligação a Calmodulina/genética , Miosinas Cardíacas/genética , Ilhas de CpG , Proteínas de Ligação a DNA/genética , Epigenoma , Epigenômica , Pai , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Proteínas de Membrana/genética , Antígenos de Histocompatibilidade Menor/genética , Chaperonas Moleculares/genética , Mães , Cadeias Pesadas de Miosina/genética , Avaliação de Resultados em Cuidados de Saúde , Gravidez , Nascimento Prematuro/epidemiologia , Nascimento Prematuro/etnologia , Estudos Retrospectivos , Classe Social , Fatores de Transcrição/genética , População Urbana , Adulto JovemAssuntos
Cardiomiopatias/genética , Adulto , Miosinas Cardíacas/genética , Cardiomiopatias/economia , Custos de Saúde para o Empregador , Feminino , Aconselhamento Genético/economia , Aconselhamento Genético/ética , Genômica , Humanos , Cadeias Pesadas de Miosina/genética , Neoplasias/economia , Neoplasias/genética , Medicina de Precisão/economia , Medicina de Precisão/ética , Adulto JovemRESUMO
Cardiomyopathies represent a well-known cause of heart failure and sudden death. Although cardiomyopathies are generally categorized in distinct nosographic entities, characterized by single gene-to-disease causal relationships, recently, oligogenic mutations have also been associated to relevant cardiac clinical features. We report the case of a master athlete carrying trigenic mutations in desmoglein-2 (DSG2), desmocollin-2 (DSC2) and heavy chain myosin 6 (MYH6), which determine a mild hypertrophic phenotype associated both to ventricular tachyarrhythmias and atrio-ventricular block. We discuss the differential diagnosis and prognostic approach in patient affected by complex cardiomyopathy phenotype, along with the importance of sport restriction and sudden death prevention.
Assuntos
Atletas , Cardiomiopatia Hipertrófica/genética , Morte Súbita Cardíaca/etiologia , Bloqueio Atrioventricular/complicações , Bloqueio Atrioventricular/genética , Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/complicações , Desmocolinas/genética , Desmogleína 2/genética , Diagnóstico Diferencial , Eletrocardiografia , Humanos , Pessoa de Meia-Idade , Mutação , Cadeias Pesadas de Miosina/genética , Marca-Passo Artificial , Fenótipo , Prognóstico , Taquicardia Ventricular/complicações , Taquicardia Ventricular/genéticaRESUMO
The zebrafish pronephros is a well-established model to study glomerular development, structure, and function. A few methods have been described to evaluate glomerular barrier function in zebrafish larvae so far. However, there is a need to assess glomerular filtration as well. In the present study, we extended the available methods by simultaneously measuring the intravascular clearances of Alexa fluor 647-conjugated 10-kDa dextran and FITC-conjugated 500-kDa dextran as indicators of glomerular filtration and barrier function, respectively. After intravascular injection of the dextrans, mean fluorescence intensities of both dextrans were measured in the cardinal vein of living zebrafish (4 days postfertilization) by confocal microscopy over time. We demonstrated that injected 10-kDa dextran was rapidly cleared from the circulation, became visible in the lumen of the pronephric tubule, quickly accumulated in tubular cells, and was detectably excreted at the cloaca. In contrast, 500-kDa dextran could not be visualized in the tubule at any time point. To check whether alterations in glomerular function can be quantified by our method, we injected morpholino oligonucleotides (MOs) against zebrafish nonmuscle myosin heavy chain IIA (zMyh9) or apolipoprotein L1 (zApol1). While glomerular filtration was reduced in zebrafish nonmuscle myosin heavy chain IIA MO-injected larvae, glomerular barrier function remained intact. In contrast, in zebrafish apolipoprotein L1 MO-injected larvae, glomerular barrier function was compromised as 500-kDa dextran disappeared from the circulation and became visible in tubular cells. In summary, we present a novel method that allows to simultaneously assess glomerular filtration and barrier function in live zebrafish.
Assuntos
Barreira de Filtração Glomerular/metabolismo , Taxa de Filtração Glomerular , Peixe-Zebra/metabolismo , Animais , Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Carbocianinas/metabolismo , Dextranos/sangue , Fluoresceína-5-Isotiocianato/análogos & derivados , Corantes Fluorescentes/metabolismo , Larva/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Morfolinos/administração & dosagem , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Eliminação Renal , Fatores de Tempo , Peixe-Zebra/sangue , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of >50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift toward comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. METHODS AND RESULTS: Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused on 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1 to 14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and segregation analysis, where available. Three of 3 previously identified primary mutations were detected by this analysis. In 6 subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and had additional pathological correlation to provide evidence for causality. For 2 subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. CONCLUSIONS: These pilot data demonstrate that ≈30 to 40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.
Assuntos
Cardiomiopatias/diagnóstico , Genoma Humano , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Algoritmos , Cardiomiopatias/economia , Cardiomiopatias/genética , Criança , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas com Domínio LIM/genética , Pessoa de Meia-Idade , Cadeias Pesadas de Miosina/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Linhagem , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Adulto JovemRESUMO
The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding ß-myosin heavy chain (ß-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCMsmn). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P < 0.0001) than in HCMsmn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s(-1), respectively). Moreover, compared to HCMsmn, tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 µmol l(-1) s(-1) kN(-1) m(-2), respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCMsmn. Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation.
Assuntos
Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/fisiopatologia , Relaxamento Muscular , Contração Miocárdica , Cadeias Pesadas de Miosina/genética , Sarcômeros/fisiologia , Trifosfato de Adenosina/metabolismo , Adulto , Idoso , Miosinas Cardíacas/metabolismo , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/metabolismo , Sarcômeros/metabolismoRESUMO
AIMS: Disease mechanisms regarding hypertrophic cardiomyopathy (HCM) are largely unknown and disease onset varies. Sarcomere mutations might induce energy depletion for which until now there is no direct evidence at sarcomere level in human HCM. This study investigated if mutations in genes encoding myosin-binding protein C (MYBPC3) and myosin heavy chain (MYH7) underlie changes in the energetic cost of contraction in the development of human HCM disease. METHODS AND RESULTS: Energetic cost of contraction was studied in vitro by measurements of force development and ATPase activity in cardiac muscle strips from 26 manifest HCM patients (11 MYBPC3mut, 9 MYH7mut, and 6 sarcomere mutation-negative, HCMsmn). In addition, in vivo, the ratio between external work (EW) and myocardial oxygen consumption (MVO2) to obtain myocardial external efficiency (MEE) was determined in 28 pre-hypertrophic mutation carriers (14 MYBPC3mut and 14 MYH7mut) and 14 healthy controls using [(11)C]-acetate positron emission tomography and cardiovascular magnetic resonance imaging. Tension cost (TC), i.e. ATPase activity during force development, was higher in MYBPC3mut and MYH7mut compared with HCMsmn at saturating [Ca(2+)]. TC was also significantly higher in MYH7mut at submaximal, more physiological [Ca(2+)]. EW was significantly lower in both mutation carrier groups, while MVO2 did not differ. MEE was significantly lower in both mutation carrier groups compared with controls, showing the lowest efficiency in MYH7 mutation carriers. CONCLUSION: We provide direct evidence that sarcomere mutations perturb the energetic cost of cardiac contraction. Gene-specific severity of cardiac abnormalities may underlie differences in disease onset and suggests that early initiation of metabolic treatment may be beneficial, in particular, in MYH7 mutation carriers.
Assuntos
Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Mutação/genética , Contração Miocárdica/genética , Cadeias Pesadas de Miosina/genética , Citoesqueleto de Actina/genética , Adulto , Idoso , Miosinas Cardíacas/metabolismo , Feminino , Regulação da Expressão Gênica , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Contração Miocárdica/fisiologia , Sarcômeros/genética , Sarcômeros/patologiaRESUMO
Hypertrophic cardiomyopathy (HCM) is a cardiac disease, characterized by marked hypertrophy and genetic variability. HCM has been associated with sarcomere protein mutations, being cardiac b-myosin (coded by the MYH7 gene) and myosin binding protein C (coded by the MYBPC3 gene) the most frequently affected proteins. As in Venezuela only the clinical analysis are performed in HCM patients, we decided to search for genetic variations in the MYH7 gene. Coding regions, including the junction exon-intron of the MYH7 gene, were studied in 58 HCM patients, whose samples were collected at the ASCARDIO Hospital (Barquisimeto, Lara state, Venezuela) and 106 control subjects from the ASCARDIO Hospital and the IVIC (Barquisimeto Lara state and Miranda, Venezuela, respectively). The blood samples were analyzed by genomic DNA isolation, followed by polymerase chain reaction and sequence analysis. The screening of the MYH7 gene revealed eight already reported polymorphic variants, as well as two intronic variations in these HCM patients. Neither any missense mutations nor other pathological mutations in the MYH7 gene were found in the HCM patients.
La miocardiopatía hipertrófica (MH) es una enfermedad cardiaca primaria, caracterizada por una marcada hipertrofia y variabilidad genética. MH ha sido asociada con mutaciones en las proteínas del sarcómero, siendo la beta miosina cardiaca, codificada por el gen MYH7 y la proteína de unión a miosina C, codificada por el gen MYBPC3, las principalmente afectadas. En Venezuela únicamente se realiza el diagnóstico clínico de MH, por lo cual el objetivo principal de este trabajo fue realizar el análisis genético en los pacientes, iniciando con el gen MYH7. Para ello, se estudió la región codificante, incluyendo la región de unión exón-intron del gen MYH7 en 58 pacientes provenientes de ASCARDIO (Barquisimeto, estado Lara) y 106 controles provenientes de ASCARDIO e IVIC (estados Lara y Miranda, Venezuela). Se colectaron las muestras de sangre para el aislamiento del ADN genómico, se realizó la técnica de PCR, seguido del análisis de secuencias para la detección de mutaciones en pacientes y controles. Se encontraron 8 polimorfismos previamente reportados, y 2 variaciones intrónicas. No se encontraron mutaciones que involucraran un cambio de aminoácido en ninguno de los exones del gen MYH7 de la beta miosina cardiaca.
Assuntos
Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/genética , Variação Genética , Cadeias Pesadas de Miosina/genética , Cardiomiopatia Hipertrófica/epidemiologia , DNA , Análise Mutacional de DNA , Éxons/genética , Frequência do Gene , Testes Genéticos , Hipertrofia Ventricular Esquerda/epidemiologia , Hipertrofia Ventricular Esquerda/genética , Íntrons/genética , Polimorfismo de Nucleotídeo Único , Venezuela/epidemiologiaRESUMO
In the present study, different reference genes were isolated, and their stability in the skeletal muscle of fine flounder subjected to different nutritional states was assessed using geNorm and NormFinder. The combinations between 18S and ActB; Fau and 18S; and Fau and Tubb were chosen as the most stable gene combinations in feeding, long-term fasting and refeeding, and short-term refeeding conditions, respectively. In all periods, ActB was identified as the single least stable gene. Subsequently, the expression of the myosin heavy chain (MYH) and the insulin-like growth factor-I receptor (IGF-IR) was assessed. A large variation in MYH and IGF-IR expression was found depending on the reference gene that was chosen for normalizing the expression of both genes. Using the most stable reference genes, mRNA levels of MYH decreased and IGF-IR increased during fasting, with both returning to basal levels during refeeding. However, the drop in mRNA levels for IGF-IR occurred during short-term refeeding, in contrast with the observed events in the expression of MYH, which occurred during long-term refeeding. The present study highlights the vast differences incurred when using unsuitable versus suitable reference genes for normalizing gene expression, pointing out that normalization without proper validation could result in a bias of gene expression.
Assuntos
Linguado/genética , Músculo Esquelético/metabolismo , Estado Nutricional , Animais , Linguado/crescimento & desenvolvimento , Linguado/metabolismo , Expressão Gênica , Cadeias Pesadas de Miosina/biossíntese , Cadeias Pesadas de Miosina/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor IGF Tipo 1/biossíntese , Receptor IGF Tipo 1/genéticaRESUMO
We carried out analyses with the goal of identifying rare variants in exome sequence data that contribute to disease risk for a complex trait. We analyzed a large, 47-member, multigenerational pedigree with 11 cases of autism spectrum disorder, using genotypes from 3 technologies representing increasing resolution: a multiallelic linkage marker panel, a dense diallelic marker panel, and variants from exome sequencing. Genome-scan marker genotypes were available on most subjects, and exome sequence data was available on 5 subjects. We used genome-scan linkage analysis to identify and prioritize the chromosome 22 region of interest, and to select subjects for exome sequencing. Inheritance vectors (IVs) generated by Markov chain Monte Carlo analysis of multilocus marker data were the foundation of most analyses. Genotype imputation used IVs to determine which sequence variants reside on the haplotype that co-segregates with the autism diagnosis. Together with a rare-allele frequency filter, we identified only one rare variant on the risk haplotype, illustrating the potential of this approach to prioritize variants. The associated gene, MYH9, is biologically unlikely, and we speculate that for this complex trait, the key variants may lie outside the exome.
Assuntos
Transtorno Autístico/genética , Cromossomos Humanos Par 22/genética , Variação Genética , Proteínas Motores Moleculares/genética , Cadeias Pesadas de Miosina/genética , Exoma , Feminino , Ligação Genética , Haplótipos , Humanos , Masculino , Modelos Genéticos , Método de Monte Carlo , Linhagem , Análise de Sequência de DNARESUMO
Synonymous codon usage bias is a broadly observed phenomenon in bacteria, plants, and invertebrates and may result from selection. However, the role of selective pressures in shaping codon bias is still controversial in vertebrates, particularly for mammals. The myosin heavy-chain (MyHC) gene family comprises multiple isoforms of the major force-producing contractile protein in cardiac and skeletal muscles. Slow and fast genes are tandemly arrayed on separate chromosomes, and have distinct patterns of functionality and expression in muscle. We analyze both full-length MyHC genes (~5400 bp) and a larger collection of partial sequences at the 3' end (~500 bp). The MyHC isoforms are an interesting system in which to study codon usage bias because of their length, expression, and critical importance to organismal mobility. Codon bias and GC content differs among MyHC genes with regards to functional type, isoform, and position within the gene. Codon bias even varies by isoform within a species. We find evidence in favor of both chromosomal influences on nucleotide composition and selection against nonsense errors (SANE) acting on codon usage in MyHC genes. Intragenic variation in codon bias and elongation rate is significant, with a strong trend for increasing codon bias and elongation rate towards the 3' end of the gene, although the trend is dependent upon the degeneracy class of the codons. Therefore, patterns of codon usage in MyHC genes are consistent with models supporting SANE as a major force shaping codon usage.
Assuntos
Código Genético , Cadeias Pesadas de Miosina/genética , Algoritmos , Animais , Códon sem Sentido , Simulação por Computador , Evolução Molecular , Dosagem de Genes , Variação Genética , Humanos , Modelos Genéticos , Método de Monte Carlo , Cadeias Pesadas de Miosina/metabolismo , Biossíntese de Proteínas , Isoformas de Proteínas/genética , RNA de Transferência/genética , Seleção Genética , Análise de Sequência de DNA , Vertebrados/genéticaRESUMO
BACKGROUND: Hypertensive nephrosclerosis is a chronic kidney disease (CKD) associated with essential hypertension. The lack of correlation between hypertension control and progression to end-stage CKD suggests an intrinsic and primitive disease. New evidence suggests that MYH9 gene alterations are associated with polymorphisms in African Americans. The aim of this study is to investigate whether a polymorphism of MYH9 in Caucasians is linked to essential hypertension and nephrosclerosis. The secondary objective is to identify the clinical risk factors of progression to end-stage CKD. This is a retrospective study that will compare patients with nephrosclerosis and essential hypertensives without renal disease, and also patients with nephrosclerosis and impaired renal function with those that are stable. METHOD: Between October 2009 and October 2010, 500 patients with stages 3-5 CKD attributed to nephrosclerosis according to usual clinical criteria, and 300 essential hypertensives (eGFR>60 mL/min/1.73 m2; microalbuminuria <300 mg/g) are to be recruited. A total of 200 healthy controls from the general population are also to be included for the genetic study. There are two study sections, being the first and final visits to the clinic (for stage 5 cases, the start of replacement therapy will be the end of follow-up). Clinical and laboratory data will be recorded, and blood samples will be collected. DISCUSSION: Our study will aim to determine if there is a relationship between the diagnosis of nephrosclerosis and the MYH9 gene in Caucasians, and to study possible risk factors for progression to end-stage CKD, on both clinical and genetic bases.
Assuntos
Hipertensão/genética , Proteínas Motores Moleculares/genética , Estudos Multicêntricos como Assunto/métodos , Cadeias Pesadas de Miosina/genética , Nefroesclerose/genética , Adulto , Idoso , Comorbidade , Progressão da Doença , Feminino , Predisposição Genética para Doença , Humanos , Hipertensão/complicações , Hipertensão/epidemiologia , Hipertensão/etnologia , Falência Renal Crônica/epidemiologia , Falência Renal Crônica/etiologia , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto/economia , Nefroesclerose/epidemiologia , Nefroesclerose/etnologia , Nefroesclerose/etiologia , Apoio à Pesquisa como Assunto , Estudos Retrospectivos , Fatores de Risco , Espanha/epidemiologia , População Branca/genéticaRESUMO
BACKGROUND: Myosin performs ATP free energy transduction into mechanical work in the motor domain of the myosin heavy chain (MHC). Energy transduction is the definitive systemic feature of the myosin motor performed by coordinating in a time ordered sequence: ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm rotation of the power stroke. These functions are carried out by several conserved sub-domains within the motor domain. Single nucleotide polymorphisms (SNPs) affect the MHC sequence of many isoforms expressed in striated muscle, smooth muscle, and non-muscle tissue. The purpose of this work is to provide a rationale for using SNPs as a functional genomics tool to investigate structurefunction relationships in myosin. In particular, to discover SNP distribution over the conserved sub-domains and surmise what it implies about sub-domain stability and criticality in the energy transduction mechanism. RESULTS: An automated routine identifying human nonsynonymous SNP amino acid missense substitutions for any MHC gene mined the NCBI SNP data base. The routine tested 22 MHC genes coding muscle and non-muscle isoforms and identified 89 missense mutation positions in the motor domain with 10 already implicated in heart disease and another 8 lacking sequence homology with a skeletal MHC isoform for which a crystallographic model is available. The remaining 71 SNP substitutions were found to be distributed over MHC with 22 falling outside identified functional sub-domains and 49 in or very near to myosin sub-domains assigned specific crucial functions in energy transduction. The latter includes the active site, the actin binding site, the rigid lever-arm, and regions facilitating their communication. Most MHC isoforms contained SNPs somewhere in the motor domain. CONCLUSIONS: Several functional-crucial sub-domains are infiltrated by a large number of SNP substitution sites suggesting these domains are engineered by evolution to be too-robust to be disturbed by otherwise intrusive sequence changes. Two functional sub-domains are SNP-free or relatively SNP-deficient but contain many disease implicated mutants. These sub-domains are apparently highly sensitive to any missense substitution suggesting they have failed to evolve a robust sequence paradigm for performing their function.
Assuntos
Genômica/métodos , Cadeias Pesadas de Miosina/genética , Polimorfismo de Nucleotídeo Único , Substituição de Aminoácidos , Sítios de Ligação , Humanos , Modelos Moleculares , Método de Monte Carlo , Mutação de Sentido Incorreto , Isoformas de Proteínas/genética , Estrutura Terciária de ProteínaRESUMO
Distinctions between craniofacial and axial muscles exist from the onset of development and throughout adulthood. The masticatory muscles are a specialized group of craniofacial muscles that retain embryonic fiber properties in the adult, suggesting that the developmental origin of these muscles may govern a pattern of expression that differs from limb muscles. To determine the extent of these differences, expression profiling of total RNA isolated from the masseter and tibialis anterior (TA) muscles of adult female mice was performed, which identified transcriptional changes in unanticipated functional classes of genes in addition to those attributable to fiber type. In particular, the masseters displayed a reduction of transcripts associated with contractile and cytoskeletal load-sensing and anabolic processes, and heightened expression of genes associated with stress. Associated with these observations was a significantly smaller fiber cross-sectional area in masseters, significantly elevated load-sensing signaling (phosphorylated focal adhesion kinase), and increased apoptotic index in masseters compared with TA muscles. Based on these results, we hypothesize that masticatory muscles may have a fundamentally different strategy for muscle design, compared with axial muscles. Specifically there are small diameter fibers that have an attenuated ability to hypertrophy, but an increased propensity to undergo apoptosis. These results may provide insight into the molecular basis for specific muscle-related pathologies associated with masticatory muscles.
Assuntos
Apoptose/genética , Perfilação da Expressão Gênica , Mastigação/genética , Músculos da Mastigação/metabolismo , Fibras Musculares Esqueléticas/citologia , Animais , Feminino , Mastigação/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , RNA/metabolismoRESUMO
More than 200 mutations in the beta-myosin gene (MYH7) that cause clinically distinct cardiac and/or skeletal myopathies have been reported, but to date, no comprehensive statistical analysis of these mutations has been performed. As a part of this review, we developed a new interactive database and research tool called MyoMAPR (Myopathic Mutation Analysis Profiler and Repository). We report that the distribution of mutations along the beta-myosin gene is not homogeneous, and that myosin is a highly constrained molecule with an uncommon sensitivity to amino acid substitutions. Increasing knowledge of the characteristics of MH7 mutations may provide a valuable resource for scientists and clinicians studying diagnosis, risk stratification, and treatment of disease associated with these mutations.
Assuntos
Miosinas Cardíacas/genética , Biologia Computacional , Genômica , Mutação , Cadeias Pesadas de Miosina/genética , Miosinas Cardíacas/química , Cardiomiopatias/genética , Análise Mutacional de DNA , Bases de Dados Genéticas , Predisposição Genética para Doença , Humanos , Doenças Musculares/genética , Cadeias Pesadas de Miosina/química , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin-resistance gene under the control of either a smooth muscle alpha-actin (SMalphaAlpha) or smooth muscle-myosin heavy chain (SM-MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and flow cytometry and were designated APSCs (SMalphaAlpha-puromycin-selected cells) or MPSCs (SM-MHC-puromycin-selected cells), respectively. Both SMC lines displayed agonist-induced Ca(2+) transients, expressed functional Ca(2+) channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin-1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC-derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.
Assuntos
Embrião de Mamíferos/citologia , Indução Embrionária , Contração Muscular/fisiologia , Miócitos de Músculo Liso/fisiologia , Células-Tronco/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Colágeno/metabolismo , Marcadores Genéticos , Camundongos , Morfogênese , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Neoplasias/prevenção & controle , Regiões Promotoras Genéticas , Seleção Genética , Transgenes , Vasoconstritores/farmacologiaRESUMO
Changes in myosin heavy chain (MHC) isoform expression and protein composition occur during cardiac disease and it has been suggested that even a minor shift in MHC composition may exert a considerable effect on myocardial energetics and performance. Here an overview is provided of the cellular basis of the energy utilisation in cardiac tissue and novel data are presented concerning the economy of myocardial contraction in diseased atrial and ventricular human myocardium. ATP utilisation and force development were measured at various Ca(2+) concentrations during isometric contraction in chemically skinned atrial trabeculae from patients in sinus rhythm (SR) or with chronic atrial fibrillation (AF) and in ventricular muscle strips from non-failing donor or end-stage failing hearts. Contractile protein composition was analysed by one-dimensional gel electrophoresis. Atrial fibrillation was accompanied by a significant shift from the fast alpha-MHC isoform to the slow beta-MHC isoform, whereas both donor and failing ventricular tissue contained almost exclusively the beta-MHC isoform. Simultaneous measurements of force and ATP utilisation indicated that economy of contraction is preserved in atrial fibrillation and in end-stage human heart failure.