Readthrough-Promoting Drugs Gentamicin and PTC124 Fail to Rescue Nav1.5 Function of Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes Carrying Nonsense Mutations in the Sodium Channel Gene SCN5A

Background—Several compounds have been reported to induce translational readthrough of premature stop codons resulting in the production of full-length protein by interfering with ribosomal proofreading. Here we examined the effect of 2 of these compounds, gentamicin and PTC124, in human-induced pluripotent stem cell (hiPSC)–derived cardiomyocytes bearing nonsense mutations in the sodium channel gene SCN5A, which are associated with conduction disease and potential lethal arrhythmias. Methods and Results—We generated hiPSC from 2 patients carrying the mutations R1638X and W156X. hiPSC-derived cardiomyocytes from both patients recapitulated the expected electrophysiological phenotype, as evidenced by reduced Na+ currents and action potential upstroke velocities compared with hiPSC-derived cardiomyocytes from 2 unrelated control individuals. While we were able to confirm the readthrough efficacy of the 2 drugs in Human Embryonic Kidney 293 cells, we did not observe rescue of the electrophysiological phenotype in hiPSC-derived cardiomyocytes from the patients. Conclusions—We conclude that these drugs are unlikely to present an effective treatment for patients carrying the loss-of-function SCN5A gene mutations examined in this study.

[1]  E. Behr,et al.  Fibrosis, Connexin-43, and Conduction Abnormalities in the Brugada Syndrome , 2015, Journal of the American College of Cardiology.

[2]  S. Priori,et al.  Genetics of sudden cardiac death. , 2015, Circulation research.

[3]  H. Boukari,et al.  XBP1 mitigates aminoglycoside-induced endoplasmic reticulum stress and neuronal cell death , 2015, Cell Death and Disease.

[4]  Simona Casini,et al.  Immaturity of human stem-cell-derived cardiomyocytes in culture: fatal flaw or soluble problem? , 2015, Stem cells and development.

[5]  Milena Bellin,et al.  Recessive cardiac phenotypes in induced pluripotent stem cell models of Jervell and Lange-Nielsen syndrome: Disease mechanisms and pharmacological rescue , 2014, Proceedings of the National Academy of Sciences.

[6]  D. Atsma,et al.  Strategies for rapidly mapping proviral integration sites and assessing cardiogenic potential of nascent human induced pluripotent stem cell clones. , 2014, Experimental cell research.

[7]  D. Bedwell,et al.  Therapeutics based on stop codon readthrough. , 2014, Annual review of genomics and human genetics.

[8]  E. Adler,et al.  Induced pluripotent stem cells for the study of cardiovascular disease. , 2014, Journal of the American College of Cardiology.

[9]  J. Pu,et al.  Comparison of read-through effects of aminoglycosides and PTC124 on rescuing nonsense mutations of HERG gene associated with long QT syndrome. , 2014, International journal of molecular medicine.

[10]  A. Di Leonardo,et al.  Toward a Rationale for the PTC124 (Ataluren) Promoted Readthrough of Premature Stop Codons: A Computational Approach and GFP-Reporter Cell-Based Assay , 2014, Molecular pharmaceutics.

[11]  Sikandar G. Khan,et al.  Repair of UV photolesions in xeroderma pigmentosum group C cells induced by translational readthrough of premature termination codons , 2013, Proceedings of the National Academy of Sciences.

[12]  Simona Casini,et al.  Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome , 2013, The EMBO journal.

[13]  C. Remme.  Cardiac sodium channelopathy associated with SCN5A mutations: electrophysiological, molecular and genetic aspects , 2013, The Journal of physiology.

[14]  O. Mühlemann,et al.  Nonsense-mediated mRNA decay - mechanisms of substrate mRNA recognition and degradation in mammalian cells. , 2013, Biochimica et biophysica acta.

[15]  S. Peltz,et al.  Ataluren as an agent for therapeutic nonsense suppression. , 2013, Annual review of medicine.

[16]  A. Bonvin,et al.  Gentamicin Binds to the Megalin Receptor as a Competitive Inhibitor Using the Common Ligand Binding Motif of Complement Type Repeats , 2012, The Journal of Biological Chemistry.

[17]  O. Namy,et al.  Sense from nonsense: therapies for premature stop codon diseases. , 2012, Trends in molecular medicine.

[18]  J. Dougherty,et al.  Pharmaceutical therapies to recode nonsense mutations in inherited diseases. , 2012, Pharmacology & therapeutics.

[19]  Christine L Mummery,et al.  Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. , 2012, Circulation research.

[20]  C. Mummery,et al.  Cardiomyocytes Derived From Pluripotent Stem Cells Recapitulate Electrophysiological Characteristics of an Overlap Syndrome of Cardiac Sodium Channel Disease , 2012, Circulation.

[21]  S. Harmer,et al.  Readthrough of long-QT syndrome type 1 nonsense mutations rescues function but alters the biophysical properties of the channel. , 2012, The Biochemical journal.

[22]  T. Morio,et al.  Functional characterization and targeted correction of ATM mutations identified in Japanese patients with ataxia‐telangiectasia , 2012, Human mutation.

[23]  Bernhard M. Schuldt,et al.  A bioinformatic assay for pluripotency in human cells , 2011, Nature Methods.

[24]  Ronald Wilders,et al.  Re-Evaluation of the Action Potential Upstroke Velocity as a Measure of the Na+ Current in Cardiac Myocytes at Physiological Conditions , 2010, PloS one.

[25]  H. Nakauchi,et al.  Development of Defective and Persistent Sendai Virus Vector , 2010, The Journal of Biological Chemistry.

[26]  Mark Potse,et al.  UvA-DARE ( Digital Academic Repository ) Pathophysiological mechanisms of arrhythmogenic right ventricular disorders , 2013 .

[27]  J. Pu,et al.  Readthrough of nonsense mutation W822X in the SCN5A gene can effectively restore expression of cardiac Na+ channels. , 2009, Cardiovascular research.

[28]  J. Pu,et al.  Aminoglycoside antibiotics restore functional expression of truncated HERG channels produced by nonsense mutations. , 2009, Heart rhythm.

[29]  Eitan Kerem,et al.  Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial , 2008, The Lancet.

[30]  Meenal Patel,et al.  PTC124 targets genetic disorders caused by nonsense mutations , 2007, Nature.

[31]  S. Peltz,et al.  Safety, Tolerability, and Pharmacokinetics of PTC124, a Nonaminoglycoside Nonsense Mutation Suppressor, Following Single‐ and Multiple‐Dose Administration to Healthy Male and Female Adult Volunteers , 2007, Journal of clinical pharmacology.

[32]  B. Kerem,et al.  Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin. , 2007, The Journal of clinical investigation.

[33]  H. Dietz,et al.  A strategy for disease gene identification through nonsense-mediated mRNA decay inhibition , 2001, Nature Biotechnology.

[34]  K. Flanigan,et al.  Sequence specificity of aminoglycoside‐induced stop codon readthrough: Potential implications for treatment of Duchenne muscular dystrophy , 2000, Annals of neurology.

[35]  T. Kanda,et al.  Histone–GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells , 1998, Current Biology.

[36]  Arthur A M Wilde,et al.  Cardiac ion channels in health and disease. , 2010, Heart rhythm.