leupeptins and Long-QT-Syndrome

Mutations that induce loss of function (LOF) or dysfunction of the human KCNQ1 channel are responsible for susceptibility to a life-threatening heart rhythm disorder, the congenital long QT syndrome (LQTS). Hundreds of

Topics: Cell Membrane; HEK293 Cells; Humans; KCNQ1 Potassium Channel; Leupeptins; Long QT Syndrome; Loss of Function Mutation; Magnetic Resonance Spectroscopy; Mutant Proteins; Mutation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Domains; Protein Folding; Protein Structure, Secondary; Proteolysis

Topics: Calnexin; Calreticulin; ERG1 Potassium Channel; HEK293 Cells; Humans; Leupeptins; Long QT Syndrome; Molecular Chaperones; Mutation; Patch-Clamp Techniques; Propranolol; Protein Transport

Long QT syndrome 2 (LQTS2) caused by missense mutations in hERG channel is clinically associated with abnormally prolonged ventricular repolarization and sudden cardiac deaths. Modelling monogenic arrhythmogenic diseases using human-induced pluripotent stem cells (hiPSCs) offers unprecedented mechanistic insights into disease pathogenesis. We utilized LQTS2-hiPSC-derived cardiomyocytes (CMs) to elucidate pathological changes and to demonstrate reversal of LQTS2 phenotype in a therapeutic intervention using a pharmacological agent, (N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine) (ALLN).. We generated LQTS2-specific CMs (A561V missense mutation in KCNH2) from iPSCs using the virus-free reprogramming method. These CMs recapitulate dysfunction of hERG potassium channel with diminished IKr currents, prolonged repolarization durations, and elevated arrhythmogenesis due to reduced membrane localization of glycosylated/mature hERG. Dysregulated expression of folding chaperones and processing proteasomes coupled with sequestered hERG in the endoplasmic reticulum confirmed trafficking-induced disease manifestation. Treatment with ALLN, not only increased membrane localization of mature hERG but also reduced repolarization, increased IKr currents and reduced arrhythmogenic events. Diverged from biophysical interference of hERG channel, our results show that modulation of chaperone proteins could be therapeutic in LQTS2 treatment.. Our in vitro study shows an alternative approach to rescue diseased LQTS2 phenotype via corrective re-trafficking therapy using a small chemical molecule, such as ALLN. This potentially novel approach may have ramifications in other clinically relevant trafficking disorders.

Topics: Animals; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Humans; Induced Pluripotent Stem Cells; Leupeptins; Long QT Syndrome; Male; Mice; Mutation; Myocytes, Cardiac; Phenotype; Protein Transport

Mutations in the human ether-a-go-go-related gene (hERG) cause chromosome 7-linked long QT syndrome type II (LQT2). We have shown previously that LQT2 mutations lead to endoplasmic reticulum (ER) retention and rapid degradation of mutant hERG proteins. In this study we examined the role of the ubiquitin-proteasome pathway in the degradation of the LQT2 mutation Y611H. We showed that proteasome inhibitors N-acetyl-L-leucyl-L-leucyl-L-norleucinal and lactacystin but not lysosome inhibitor leupeptin inhibited the degradation of Y611H mutant channels. In addition, ER mannosidase I inhibitor kifunensine and down-regulation of EDEM (ER degradation-enhancing alpha-mannosidase-like protein) also suppressed the degradation of Y611H mutant channels. Proteasome inhibition but not mannosidase inhibition led to the accumulation of full-length hERG protein in the cytosol. The hERG protein accumulated in the cytosol was deglycosylated. Proteasome inhibition also resulted in the accumulation of polyubiquitinated hERG channels. These results suggest that the degradation of LQT2 mutant channels is mediated by the cytosolic proteasome in a process that involves mannose trimming, polyubiquitination, and deglycosylation of mutant channels.

Topics: Acetylcysteine; Alkaloids; Blotting, Western; Cell Line; Cell Membrane; Cysteine Proteinase Inhibitors; Cytosol; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Enzyme Inhibitors; Glycosylation; Green Fluorescent Proteins; Humans; Immunoprecipitation; Leupeptins; Long QT Syndrome; Mutation; Potassium Channels; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Ribonucleases; Subcellular Fractions; Time Factors; Transfection; Ubiquitin