CSAHi study-2: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes: Assessment of reference compounds and comparison with non-clinical studies and clinical information.

With the aim of reconsidering ICH S7B and E14 guidelines, a new in vitro assay system has been subjected to worldwide validation to establish a better prediction platform for potential drug-induced QT prolongation and the consequent TdP in clinical practice. In Japan, CSAHi HEART team has been working on hiPS-CMs in the MEA (hiPS-CMs/MEA) under a standardized protocol and found no inter-facility or lot-to-lot variability for proarrhythmic risk assessment of 7 reference compounds. In this study, we evaluated the responses of hiPS-CMs/MEA to another 31 reference compounds associated with cardiac toxicities, and gene expression to further clarify the electrophysiological characteristics over the course of culture period. The hiPS-CMs/MEA assay accurately predicted reference compounds potential for arrhythmogenesis, and yielded results that showed better correlation with target concentrations of QTc prolongation or TdP in clinical setting than other current in vitro and in vivo assays. Gene expression analyses revealed consistent profiles in all samples within and among the testing facilities. This report would provide CiPA with informative guidance on the use of the hiPS-CMs/MEA assay, and promote the establishment of a new paradigm, beyond conventional in vitro and in vivo assays for cardiac safety assessment of new drugs.

CSAHi study: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes -assessment of inter-facility and cells lot-to-lot-variability.

In vitro screening of hERG channels are recommended under ICH S7B guidelines to predict drug-induced QT prolongation and Torsade de Pointes (TdP), whereas proarrhythmia is known to be evoked by blockage of other ion channels involved in cardiac contraction and compensation mechanisms. A consortium for drug safety assessment using human iPS cells-derived cardiomyocytes (hiPS-CMs), CSAHi, has been organized to establish a novel in vitro test system that would enable better prediction of drug-induced proarrhythmia and QT prolongation. Here we report the inter-facility and cells lot-to-lot variability evaluated with FPDc (corrected field potential duration), FPDc10 (10% FPDc change concentration), beat rate and incidence of arrhythmia-like waveform or arrest on hiPS-CMs in a multi-electrode array system. Arrhythmia-like waveforms were evident for all test compounds, other than chromanol 293B, that evoked FPDc prolongation in this system and are reported to induce TdP in clinical practice. There was no apparent cells lot-to-lot variability, while inter-facility variabilities were limited within ranges from 3.9- to 20-folds for FPDc10 and about 10-folds for the minimum concentration inducing arrhythmia-like waveform or arrests. In conclusion, the new assay model reported here would enable accurate prediction of a drug potential for proarrhythmia.

Voltage-dependent N-type Ca2+ channels in endothelial cells contribute to oxidative stress-related endothelial dysfunction induced by angiotensin II in mice.

N-type voltage-dependent Ca(2+)channels (VDCCs), expressed predominantly in the nervous system, play pivotal roles in sympathetic regulation of the circulatory system. Although N-type VDCCs are also reportedly expressed in the vasculature, their pathophysiological role is obscure. We demonstrated that oxidative stress-related endothelial dysfunction induced by angiotensin (Ang) II is suppressed in mice lacking the N-type VDCC α1B subunit (Cav 2.2). Impairment of endothelium-dependent relaxation of the thoracic aorta observed following Ang II treatment in wild-type (WT) mice was significantly attenuated in the Ang II-treated Cav 2.2-deficient mice, despite the comparable increase of the blood pressure in the two groups of mice. The thoracic aorta of the Cav 2.2-deficient mice showed a smaller positive area of oxidative stress markers as compared to the WT mice. The Ang II-induced endothelial dysfunction was also suppressed by cilnidipine, an L/N-type VDCC blocker, but not by amlodipine, an L-type VDCC blocker; however, this unique effect of cilnidipine was completely abolished in the Cav 2.2-deficient mice. Furthermore, selective inhibition of N-type VDCCs by ω-conotoxin GVIA dramatically suppressed the production of reactive oxygen species (ROS) as well as agonist-induced Ca(2+) influx in the vascular endothelial cells. These results suggest that N-type VDCCs expressed in the vascular endothelial cells contribute to ROS production and endothelial dysfunction observed in Ang II-treated hypertensive mice.

Cilostazol suppresses angiotensin II-induced vasoconstriction via protein kinase A-mediated phosphorylation of the transient receptor potential canonical 6 channel.

OBJECTIVE: The goal of this study was to determine whether inhibition of transient receptor potential canonical (TRPC) channels underlies attenuation of angiotensin II (Ang II)-induced vasoconstriction by phosphodiesterase (PDE) 3 inhibition. METHODS AND RESULTS: Pretreatment of rat thoracic aorta with cilostazol, a selective PDE3 inhibitor, suppressed vasoconstriction induced by Ang II but not that induced by KCl. The Ang II-induced contraction was largely dependent on Ca(2+) influx via receptor-operated cation channels. Cilostazol specifically suppressed diacylglycerol-activated TRPC channels (TRPC3/TRPC6/TRPC7) through protein kinase A (PKA)-dependent phosphorylation of TRPC channels in HEK293 cells. In contrast, we found that phosphorylation of TRPC6 at Thr69 was essential for the suppression of Ang II-induced Ca(2+) influx by PDE3 inhibition in rat aortic smooth muscle cells (RAoSMCs). Cilostazol specifically induced phosphorylation of endogenous TRPC6 at Thr69. The endogenous TRPC6, but not TRPC3, formed a ternary complex with PDE3 and PKA in RAoSMCs, suggesting the specificity of TRPC6 phosphorylation by PDE3 inhibition. Furthermore, inhibition of PDE3 suppressed the Ang II-induced contraction of reconstituted ring with RAoSMCs, which were abolished by the expression of a phosphorylation-deficient mutant of TRPC6. CONCLUSIONS: PKA-mediated phosphorylation of TRPC6 at Thr69 is essential for the vasorelaxant effects of PDE3 inhibition against the vasoconstrictive actions of Ang II.

Heterologous down-regulation of angiotensin type 1 receptors by purinergic P2Y2 receptor stimulation through S-nitrosylation of NF-kappaB.

Cross-talk between G protein-coupled receptor (GPCR) signaling pathways serves to fine tune cellular responsiveness by neurohumoral factors. Accumulating evidence has implicated nitric oxide (NO)-based signaling downstream of GPCRs, but the molecular details are unknown. Here, we show that adenosine triphosphate (ATP) decreases angiotensin type 1 receptor (AT(1)R) density through NO-mediated S-nitrosylation of nuclear factor κB (NF-κB) in rat cardiac fibroblasts. Stimulation of purinergic P2Y(2) receptor by ATP increased expression of inducible NO synthase (iNOS) through activation of nuclear factor of activated T cells, NFATc1 and NFATc3. The ATP-induced iNOS interacted with p65 subunit of NF-κB in the cytosol through flavin-binding domain, which was indispensable for the locally generated NO-mediated S-nitrosylation of p65 at Cys38. ß-Arrestins anchored the formation of p65/IκBα/ß-arrestins/iNOS quaternary complex. The S-nitrosylated p65 resulted in decreases in NF-κB transcriptional activity and AT(1)R density. In pressure-overloaded mouse hearts, ATP released from cardiomyocytes led to decrease in AT(1)R density through iNOS-mediated S-nitrosylation of p65. These results show a unique regulatory mechanism of heterologous regulation of GPCRs in which cysteine modification of transcriptional factor rather than protein phosphorylation plays essential roles.

Regulation of Angiotensin II receptor signaling by cysteine modification of NF-κB.

Angiotensin II (Ang II) is a major vasoactive peptide of the renin-angiotensin system. Ang II is originally found as one of potent vasoconstrictors, but is now attracted attention as an essential mediator of many cardiovascular problems, including endothelial dysfunction, arrhythmia and structural remodeling of cardiovascular systems. Most of the known pathophysiological effects of Ang II are mediated through Ang type1 receptors (AT(1)Rs), and the up-regulation of AT(1)Rs is one of important causes by which Ang II can contribute to cardiovascular diseases. A growing body of evidence has suggested that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in the regulation of AT(1)R signaling. In cardiac fibroblasts, stimulation with cytokines or bacterial toxins induces AT(1)R up-regulation through NADPH oxidase-dependent ROS production. In contrast, nitric oxide (NO) decreases AT(1)R density through cysteine modification (S-nitrosylation) of a transcriptional factor, nuclear factor κB (NF-κB). The difference between the effects of ROS and NO on AT(1)R expression may be caused by the difference between intracellular location of ROS signaling and that of NO signaling, as the agonist-induced S-nitrosylation of NF-κB requires a local interaction between NO synthase (NOS) and NF-κB in the perinuclear region. Thus, the spatial and temporal regulation of cysteine modification by ROS or RNS may underlie the resultant changes of AT(1)R signaling induced by agonist stimulation.

[Regulation of cardiovascular functions by the phosphorylation of TRPC channels].

Calcium ions (Ca²(+)) play an essential role in homeostasis and the activity of cardiovascular cells. Ca²(+) influx across the plasma membrane induced by neurohumoral factors or mechanical stress elicits physiologically relevant timing and spatial patterns of Ca²(+) signaling, which leads to the activation of various cardiovascular functions, such as muscle contraction, gene expression, and hypertrophic growth of myocytes. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca²(+) signaling pathway required for pathological hypertrophy. We have recently found that the inhibition of cGMP-selective phosphodiesterase 5 (PDE5) suppresses agonist- and mechanical stretch-induced hypertrophy through inhibition of Ca²(+) influx in rat cardiomyocytes. The inhibition of PDE5 suppressed the increase in frequency of Ca²(+) spikes induced by receptor stimulation or mechanical stretch. Activation of protein kinase G by PDE5 inhibition phosphorylated TRPC6 proteins at Thr69 and prevented TRPC6-mediated Ca²(+) influx. Substitution of Ala for Thr69 in TRPC6 abolished the antihypertrophic effects of PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlies the prevention of cardiac hypertrophy by PDE5 inhibition. As TRPC6 proteins are also expressed in vascular smooth muscle cells and reportedly participate in vascular remodeling, TRPC6 blockade may be an effective therapeutic strategy for preventing pathologic cardiovascular remodeling.