| 2. |
- Smolina, N, et al.
(författare)
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Primary Murine Myotubes as a Model for Investigating Muscular Dystrophy
- 2015
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Ingår i: BioMed research international. - : Hindawi Limited. - 2314-6141 .- 2314-6133. ; 2015, s. 594751-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Mikhailova, VB, et al.
(författare)
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Functional Analysis of SCN5A Genetic Variants Associated with Brugada Syndrome
- 2022
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Ingår i: Cardiology. - : S. Karger AG. - 1421-9751 .- 0008-6312. ; 147:1, s. 35-46
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Tidskriftsartikel (refereegranskat)abstract
- <b><i>Background:</i></b> Brugada syndrome (BrS) is a rare inherited cardiac arrhythmia with increased risk of sudden cardiac death. Mutations in gene <i>SCN5A</i>, which encodes the α-subunit of cardiac voltage-gated sodium channel Na<sub>V</sub>1.5, have been identified in over 20% of patients with BrS. However, only a small fraction of Na<sub>V</sub>1.5 variants, which are associated with BrS, are characterized in electrophysiological experiments. <b><i>Results:</i></b> Here we explored variants V281A and L1582P, which were found in our patients with BrS, and variants F543L and K1419E, which are reportedly associated with BrS. Heterologous expression of the variants in CHO-K1 cells and the Western blot analysis demonstrated that each variant appeared at the cell surface. We further measured sodium current in the whole-cell voltage clamp configuration. Variant F543L produced robust sodium current with a hyperpolarizing shift in the voltage dependence of steady-state fast inactivation. Other variants did not produce detectable sodium currents, indicating a complete loss of function. In a recent cryoEM structure of the hNa<sub>V</sub>1.5 channel, residues V281, K1419, and L1582 are in close contacts with residues whose mutations are reportedly associated with BrS, indicating functional importance of respective contacts. <b><i>Conclusions:</i></b> Our results support the notion that loss of function of Na<sub>V</sub>1.5 or decrease of the channel activity is involved in the pathogenesis of BrS.
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