Natalia Smolina

Variants in the NOTCH1 Gene in Patients with Aortic Coarctation

BACKGROUND AND OBJECTIVE Malformations of the left ventricular outflow tract are one of the most common forms of congenital heart disorders. Recently, it has been shown that mutations in the NOTCH1 gene can lead to bicuspid aortic valve, aortic aneurysm, and hypoplastic left heart syndrome. The aim of our study was to estimate the frequency of NOTCH1 gene mutations/substitutions in patients with aortic coarctation, isolated or combined with bicuspid aortic valve. DESIGN AND PATIENTS The study included 51 children with coarctation. Detailed family history was obtained for every study subject, and echocardiographic data were obtained for the relatives when available. We applied a strategy of targeted mutation screening for 10 out of 34 exons of the NOTCH1 gene by direct sequencing. Control DNA was obtained from 200 healthy donors. RESULTS In more than half of the cases, coarctation was combined with bicuspid aortic valve, and in approximately half of the cases, it was combined with hypoplasia of the aortic arch or descending aorta. Familial history of congenital heart disease was observed in 34.3% of the cases. In total, 29 variants of the NOTCH1 gene were identified in the patient group and in the control subjects. Four of those variants led to amino acid exchange, of which only one, R1279H, was identified in both the patient group and in the controls. This variant was significantly overrepresented in the patients with aortic coarctation compared with those in the control group (P < .05). We conclude that the R1279H substitution in the NOTCH1 gene is significantly overrepresented in patients with aortic coarctation and, therefore, may represent a disease-susceptibility allele.

Seventy years after the siege of Leningrad: does early life famine still affect cardiovascular risk and aging?

Objective: To assess the cardiovascular health, markers of cardiovascular aging and telomere length in survivors of the siege of Leningrad, who were either born during the siege or lived in the besieged city in their early childhood. Methods: Survivors of the Leningrad siege (n = 305, 64–81 years) and a control group of age and sex-matched individuals (n = 51, 67–82 years) were examined in terms of a observational retrospective cohort study. All participants were interviewed regarding risk factors, cardiovascular diseases, and therapy. Blood pressure measurement, anthropometry, echocardiography, and electrocardiography were performed according to standard guidelines. Fasting lipids and glucose were measured. Relative telomere length was measured by quantitative PCR, and the ratio of telomere repeat copy number to single gene copy number (T/S) was calculated for each DNA sample. Results: Survivors had lower anthropometric parameters (height, weight, and BMI) and higher high-density lipoprotein level. There were no significant differences in the prevalence of cardiovascular diseases and target organ damage between groups. However, survivors had shorter telomere length: T/S ratio 0.44 (0.25; 0.64) vs. controls 0.91 (0.47; 1.13) (P < 0.0001), both in men and women, with clear association with the period of famine in early life. Exposure to famine in childhood and intrauterine period of life was associated with a higher prevalence of hypertension and shorter telomere length. Conclusion: Early-life famine, especially started in the intrauterine period and late childhood, may contribute to accelerated aging with telomere shortening in both sexes, but has no direct effect on the prevalence of cardiovascular diseases and risk factors after seven decades since exposure.

Genetic Spectrum of Idiopathic Restrictive Cardiomyopathy Uncovered by Next-Generation Sequencing.

Background Cardiomyopathies represent a rare group of disorders often of genetic origin. While approximately 50% of genetic causes are known for other types of cardiomyopathies, the genetic spectrum of restrictive cardiomyopathy (RCM) is largely unknown. The aim of the present study was to identify the genetic background of idiopathic RCM and to compile the obtained genetic variants to the novel signalling pathways using in silico protein network analysis. Patients and Methods We used Illumina MiSeq setup to screen for 108 cardiomyopathy and arrhythmia-associated genes in 24 patients with idiopathic RCM. Pathogenicity of genetic variants was classified according to American College of Medical Genetics and Genomics classification. Results Pathogenic and likely-pathogenic variants were detected in 13 of 24 patients resulting in an overall genotype-positive rate of 54%. Half of the genotype-positive patients carried a combination of pathogenic, likely-pathogenic variants and variants of unknown significance. The most frequent combination included mutations in sarcomeric and cytoskeletal genes (38%). A bioinformatics approach underlined the mechanotransducing protein networks important for RCM pathogenesis. Conclusions Multiple gene mutations were detected in half of the RCM cases, with a combination of sarcomeric and cytoskeletal gene mutations being the most common. Mutations of genes encoding sarcomeric, cytoskeletal, and Z-line-associated proteins appear to have a predominant role in the development of RCM.

Diagnostic Challenge in Desmin Cardiomyopathy With Transformation of Clinical Phenotypes

Desmin cardiomyopathy is a rare cause of congestive heart failure. Its clinical manifestation in adulthood often is associated with conduction disorders and a neuromuscular phenotype. Only a few cases have been reported, with early manifestation in childhood mostly due to severe cardiomyopathy dilationand conduction abnormalities. However, the disease can result in the variety of clinical phenotypes, including hypertrophic, restrictive, and arrthythmogenic cardiomyopathy. This report describes the first case of desmin cardiomyopathy with early manifestation in adolescence and transformation of several clinical phenotypes over time, representing sufficient difficulties for the correct clinical diagnosis and treatment of the disease at an early stage.

Various lamin A/C mutations alter expression profile of mesenchymal stem cells in mutation specific manner

Various mutations in LMNA gene, encoding for nuclear lamin A/C protein, lead to laminopathies and contribute to over ten human disorders, mostly affecting tissues of mesenchymal origin such as fat tissue, muscle tissue, and bones. Recently it was demonstrated that lamins not only play a structural role providing communication between extra-nuclear structures and components of cell nucleus but also control cell fate and differentiation. In our study we assessed the effect of various LMNA mutations on the expression profile of mesenchymal multipotent stem cells (MMSC) during adipogenic and osteogenic differentiation. We used lentiviral approach to modify human MMSC with LMNA-constructs bearing mutations associated with different laminopathies--G465D, R482L, G232E, R527C, and R471C. The impact of various mutations on MMSC differentiation properties and expression profile was assessed by colony-forming unit analysis, histological staining, expression of the key differentiation markers promoting adipogenesis and osteogenesis followed by the analysis of the whole set of genes involved in lineage-specific differentiation using PCR expression arrays. We demonstrate that various LMNA mutations influence the differentiation efficacy of MMSC in mutation-specific manner. Each LMNA mutation promotes a unique expression pattern of genes involved in a lineage-specific differentiation and this pattern is shared by the phenotype-specific mutations.

Aggregate-prone desmin mutations impair mitochondrial calcium uptake in primary myotubes

Desmin, being a major intermediate filament of mature muscle cell, interacts with mitochondria within the cell and participates in mitochondria proper localization. The goal of the present study was to assess the effect of aggregate-prone and non-aggregate-prone desmin mutations on mitochondrial calcium uptake. Primary murine satellite cells were transduced with lentiviruses carrying desmin in wild type or mutant form, and were induced to differentiate into myotubes. Four mutations resulting in different degree of desmin aggregates formation were analyzed. Tail domain mutation Asp399Tyr has the mildest impact on desmin filament polymerization, rod domain mutation Ala357Pro causes formation of large aggregates composed of filamentous material, and Leu345Pro and Leu370Pro are considered to be the most severest in their impact on desmin polymerization and structure. For mitochondrial calcium measurement cells were loaded with rhod 2-AM. We found that aggregate-prone mutations significantly decreased [Ca(2+)]mit, whereas non-aggregate-prone mutations did not decrease [Ca(2+)]mit. Moreover aggregate-prone desmin mutations resulted in increased resting cytosolic [Ca(2+)]. However this increase was not accompanied by any alterations in sarcoplasmic reticulum calcium release. We suggest that the observed decline in [Ca(2+)]mit was due to desmin aggregate accumulation resulting in the loss of desmin mitochondria interactions.

Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness

Mitochondrial respiration is the most important generator of cellular energy under most circumstances. It is a process of energy conversion of substrates into ATP. The Seahorse equipment allows measuring oxygen consumption rate (OCR) in living cells and estimates key parameters of mitochondrial respiration in real-time mode. Through use of mitochondrial inhibitors, four key mitochondrial respiration parameters can be measured: basal, ATP production-linked, maximal, and proton leak-linked OCR. This approach requires application of mitochondrial inhibitors-oligomycin to block ATP synthase, FCCP-to make the inner mitochondrial membrane permeable for protons and allow maximum electron flux through the electron transport chain, and rotenone and antimycin A-to inhibit complexes I and III, respectively. This chapter describes the protocol of OCR assessment in the culture of primary myotubes obtained upon satellite cell fusion.

Nuclear lamins regulate osteogenic differentiation of mesenchymal stem cells

AbsractNuclear lamins are the main proteins of the nuclear envelope providing nuclear-membrane strength. Recently, it became clear that lamins in cells play not only a structural role, but are also involved in regulation of gene expression. The LMNA gene encodes lamin A or C depending on the synthesizing splicing variant. The best-known LMNA mutation causes severe disorders in development known as progeria (premature aging syndrome). The disease is of rare occurrence. More frequently, point mutations in LMNA gene encoding lamin A/C result in so-called laminopathies, these diseases manifesting as tissue damage, mostly in tissues of mesenchymal origin. The mutations manifest in a tissue-specific manner: particular mutations always display the same disease phenotype. The nature of this phenomenon, as well as the mechanisms by which lamins regulate cell differentiation remain poorly understood. The aim of this study was to investigate the effect of different LMNA mutations on human mesenchimal stem cell (MSC) osteogenic differentiation and explore the possible interaction of lamins and Notch signaling pathway. We modified human MSCs with mutant LMNA bearing known mutations with tissue specific phenotype associated with different laminopathies. Differentiation was evaluated 21 days after its induction by number of differentiated cells, as well as by the expression level of specific osteogenic markers SPP, IBSP, and BGLAP. Some mutations enhance differentiation whereas others decrease its level. These findings support the notion that lamin A/C is involved in the regulation of MMSC differentiation. Introduction of mutant LMNA forms together with the activated Notch domain modified the expression of HEY1, a major target of Notch signaling. Thereby, one of the mechanisms involved in the regulation of MSC differentiation may be the interaction of lamins A/C with components of Notch signaling.

Lamin A/C mutations alter differentiation potential of mesenchymal stem cells

Mutations in the lamin A/C gene (LMNA) lead to severe disorders collectively called laminopathies. The mechanisms by which lamin mutations cause the diseases are not clear. Since the mesenchymal lineages, adipose tissue in particular, are mostly affected in laminopathies, the aim of the study was to estimate the effect of LMNA mutations on differentiation of mesenchymal stem cells, adipose tissue stromal cells (ATSCs), into adipose lineages. ATSCs transduced with lentiviral vectors carrying LMNA gene mutations associated with various syndromes (myodystrophy, cardiomyopathy, lipodystrophy, progeroid syndrome) were induced to adipose differentiate. It was found that introduction of genetic constructions with LMNA gene point mutations G465D, R482L, and R527C promote adipogenic differentiation compared to wild-type lamin gene; mutation R471C reduced the differentiation. Introduction of R471C or R527C lamin mutations profoundly increased the expression of adipogenesis markers PPARG, SREBP1, and adipsin. Mutations in A/C lamin gene strongly and variously affect the differentiation of mesenchymal stem cells that probably underlie the pathogenic changes in patients with laminopathies.

Primary Murine Myotubes as a Model for Investigating Muscular Dystrophy.

Muscular dystrophies caused by defects in various genes are often associated with impairment of calcium homeostasis. Studies of calcium currents are hampered because of the lack of a robust cellular model. Primary murine myotubes, formed upon satellite cell fusion, were examined for their utilization as a model of adult skeletal muscle. We enzymatically isolated satellite cells and induced them to differentiation to myotubes. Myotubes displayed morphological and physiological properties resembling adult muscle fibers. Desmin and myosin heavy chain immunoreactivity in the differentiated myotubes were similar to the mature muscle cross-striated pattern. The myotubes responded to electrical and chemical stimulations with sarcoplasmic reticulum calcium release. Presence of L-type calcium channels in the myotubes sarcolemma was confirmed via whole-cell patch-clamp technique. To assess the use of myotubes for studying functional mutation effects lentiviral transduction was applied. Satellite cells easily underwent transduction and were able to retain a positive expression of lentivirally encoded GFP up to and after the formation of myotubes, without changes in their physiological and morphological properties. Thus, we conclude that murine myotubes may serve as a fruitful cell model for investigating calcium homeostasis in muscular dystrophy and the effects of gene modifications can be assessed due to lentiviral transduction.