Evolution of mathematical models of cardiomyocyte electrophysiology.

Advanced computational techniques and mathematical modeling have become more and more important to the study of cardiac electrophysiology. In this review, we provide a brief history of the evolution of cardiomyocyte electrophysiology models and highlight some of the most important ones that had a major impact on our understanding of the electrical activity of the myocardium and associated transmembrane ion fluxes in normal and pathological states. We also present the use of these models in the study of various arrhythmogenesis mechanisms, particularly the integration of experimental pharmacology data into advanced humanized models for in silico proarrhythmogenic risk prediction as an essential component of the Comprehensive in vitro Proarrhythmia Assay (CiPA) drug safety paradigm.

Senescence-induced immunophenotype, gene expression and electrophysiology changes in human amniocytes.

The aim of the study was to evidence replicative senescence-induced changes in human amniocytes via flow cytometry, quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) and automated/manual patch-clamp. Both cryopreserved and senescent amniocytes cultured in BIO-AMF-2 medium featured high percentages of pluripotency cell surface antigens SSEA-1, SSEA-4, TRA1-60, TRA1-81 (assessed by flow cytometry) and expression of pluripotency markers Oct4 (Pou5f1) and Nanog (by qRT-PCR). We demonstrated in senescent vs cryopreserved amniocytes decreases in mesenchymal stem cell surface markers. Senescence-associated ß-galactosidase stained only senescent amniocytes, and they showed no deoxyuridine incorporation. The gene expression profile revealed a secretory phenotype of senescent amniocytes (increased interleukin (IL)-1α, IL-6, IL-8, transforming growth factor ß, nuclear factor κB p65 expression), increases for cell cycle-regulating genes (p16INK4A ), cytoskeletal elements (ß-actin); HMGB1, c-Myc, Bcl-2 showed reduced changes and p21, MDM2 decreased. Via patch-clamp we identified five ion current components: outward rectifier K+ current, an inactivatable component, big conductance Ca2+ -dependent K+ channels (BK) current fluctuations, Na+ current, and inward rectifier K+ current. Iberiotoxin 100 nmol/L blocked 71% of BK fluctuations, and lidocaine 200 µmol/L exerted use-dependent Na+ current block. Transient receptor potential (TRP)M7-like current density at -120 mV was significantly increased in senescent amniocytes. The proinflammatory profile acquired by senescent amniocytes in vitro may prevent their use in clinical therapies for immunosuppression, antiapoptotic and healing effects.

Recording of multiple ion current components and action potentials in human induced pluripotent stem cell-derived cardiomyocytes via automated patch-clamp.

INTRODUCTION: The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative proposes a three-step approach to evaluate proarrhythmogenic liability of drug candidates: effects on individual ion channels in heterologous expression systems, integrating these data into in-silico models of the electrical activity of human cardiomyocytes, and comparison with experiments on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Here we introduce patch-clamp electrophysiology techniques on hiPSC-CM to combine two of the CiPA steps in one assay. METHODS: We performed automated patch-clamp experiments on hiPSC-CM (Cor.4U®, Ncardia) using the CytoPatch™2 platform in ruptured whole-cell and ß-escin-perforated-patch configurations. A combination of three voltage-clamp protocols allowed recording of five distinct ion current components (voltage-gated Na+ current, L-type Ca2+ current, transient outward K+ current, delayed rectifier K+ current, and "funny" hyperpolarization-activated current) from the same cell. We proved their molecular identity by either Na+ replacement with choline or by applying specific blockers: nifedipine, cisapride, chromanol 293B, phrixotoxin-1, ZD7288. We developed a C++ script for automated analysis of voltage-clamp recordings and computation of ion current/conductance surface density for these five cardiac ion currents. RESULTS: The distributions from n = 54 hiPSC-CM in "ruptured" patch-clamp vs. n = 35 hiPSC-CM in ß-escin-perforated patch-clamp were similar for membrane capacitance, access resistance, and ion current/conductance surface densities. The ß-escin-perforated configuration resulted in improved stability of action potential (AP) shape and duration over a 10-min interval, with APD90 decay rate 0.7 ±â€¯1.6%/min (mean ±â€¯SD, n = 4) vs. 4.6 ±â€¯1.1%/min. (n = 3) for "ruptured" approach (p = 0.0286, one-tailed Mann-Whitney test). DISCUSSION: The improved stability obtained here will allow development of CiPA-compliant automated patch-clamp assays on hiPSC-CM. Future applications include the study of multi ion-channel blocking properties of drugs using dynamic-clamp protocols, adding a valuable new tool to the arsenal of safety-pharmacology.