ABSTRACT
Long QT syndromes (LQTS) are heritable diseases characterized by prolongation of the QT interval on an electrocardiogram, which often leads to syncope and sudden cardiac death. Here we report the generation of induced pluripotent stems (iPS) cells from two patients with LQTS type 3 carrying a different point mutation in a sodium channel Nav1.5 (p.V240M and p.R535Q) and functional characterization of cardiomyocytes (CM) derived from them. The iPS cells exhibited all characteristic properties of pluripotent stem cells, maintained the disease-specific mutation and readily differentiated to CM. The duration of action potentials at 50% and 90% repolarization was longer in LQTS-3 CM as compared to control CM but this difference did not reach statistical significance due to high variations among cells. Sodium current recordings demonstrated longer time to peak and longer time to 90% of inactivation of the Na(+) channel in the LQTS-3 CM. This hints at a defective Na(+) channel caused by deficiency in open-state inactivation of the Na(+) channel that is characteristic of LQTS-3. These analyses suggest that the effect of channel mutation in the diseased CM is demonstrated in vitro and that the iPS cell-derived CM can serve as a model system for studying the pathophysiology of LQTS-3, toxicity testing and design of novel therapeutics. However, further improvements in the model are still required to reduce cell-to-cell and cell line-to-cell line variability.
Subject(s)
Action Potentials/genetics , Long QT Syndrome , Membrane Potentials/genetics , Myocytes, Cardiac , NAV1.5 Voltage-Gated Sodium Channel , Pluripotent Stem Cells , Point Mutation , Adult , Cell Differentiation/genetics , Cells, Cultured , Female , Humans , Long QT Syndrome/genetics , Long QT Syndrome/metabolism , Long QT Syndrome/pathology , Long QT Syndrome/physiopathology , Male , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , NAV1.5 Voltage-Gated Sodium Channel/genetics , NAV1.5 Voltage-Gated Sodium Channel/metabolism , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/pathologyABSTRACT
Metallothionein (MT) is the protein that has been shown to bind heavy metals, scavenge free radicals, protect DNA from radiation damage, and alleviate disease symptoms. However, only very limited success has been achieved in expression and production of active recombinant metallothionein. In this study, human metallothionein 1A (hMT1A) was transformed into yeast Pichia pastoris for expression with secretion of the protein into the medium. The expression system was optimized to obtain the targeted protein in active form at 335 mg per litre culture. hMT1A showed the character of extreme instability in the experiment. High concentration, aeration and heavy metal ions are the main factors affecting hMT1A stability.