DOI: 10.5061/dryad.280gb5mvz
title: Electrophysiology data for NAA10-R4S-induced pluripotent-derived cardiomyocytes

description: N-terminal acetyltransferases including NAA10 catalyze N-terminal
 acetylation, an evolutionarily conserved co- and post-translational
 modification. However, little is known about the role of N-terminal
 acetylation in cardiac homeostasis. To gain insight into cardiac-dependent
 NAA10 function, we studied a previously unidentified NAA10 variant
 p.(Arg4Ser) segregating with QT-prolongation, cardiomyopathy, and
 developmental delay in a large kindred. Here, we show that the NAA10R4S
 variant reduced enzymatic activity, decreased NAA10-NAA15 complex
 formation, and destabilized the enzymatic complex N-terminal
 acetyltransferase A. In NAA10R4S/Y-induced pluripotent stem-cell-derived
 cardiomyocytes (iPSC-CMs), dysregulation of the late sodium and slow
 delayed rectifier potassium currents caused severe repolarization
 abnormalities, consistent with clinical QT prolongation. Engineered heart
 tissues generated from NAA10R4S/Y-iPSC-CMs had significantly decreased
 contractile force and sarcomeric disorganization, consistent with the
 pedigree’s cardiomyopathic phenotype. Proteomic studies revealed
 dysregulation of metabolic pathways and cardiac structural proteins. We
 identified small molecule and genetic therapies that normalized the
 phenotype of NAA10R4S/Y-iPSC-CMs. Our study defines the roles of
 N-terminal acetylation in cardiac regulation and delineates mechanisms
 underlying QT prolongation, arrhythmia, and cardiomyopathy caused by NAA10
 dysfunction.
description: <strong>Whole-cell patch clamp
 recordings</strong> Cultured
 iPSC-CMs were dissociated with Accutase and plated sparsely onto
 Geltrex-coated 11mm coverslips. Single iPSC-CMs were analyzed 3 to 6 days
 after dissociating. Single iPSC-CMs were recorded under different
 conditions to acquire each parameter (Supplemental Table 1)<sup>50,51</sup>. Perforated patch recordings were performed for action potential (AP) analysis and the L-type Ca<sup>2+</sup> current (I<sub>CaL</sub>). Perforated patch was applied in order to prevent run-down in I<sub>CaL </sub>recording<sup>52</sup>. The ruptured patch technique was used for I<sub>Na</sub>, I<sub>Ks</sub>, and I<sub>Kr </sub>recordings. Series resistance and cell capacitance were compensated to ~ 60 % for all the voltage clamp experiments. To measure I<sub>Na</sub>, starting from a holding potential of -100 mV, 40 ms of depolarizing pulses from -100 mV to 90 mV were applied in 10-mV increments. For the I<sub>Na </sub>steady-state inactivation, following 400 ms of prepulses with 10-mV increments from -110 mV to -20 mV, 40 ms of 0 mV pulse was applied.  For I<sub>Ks</sub>, test pulses were applied for 5 s with 20 mV increments from -20 mV to 40 mV from a holding potential of -40 mV. For I<sub>Kr</sub>, test pulses were applied for 4 s with 5 mV increments from -35 mV to 10 mV from a holding potential of -40 mV. To measure I<sub>CaL</sub>, starting from a holding potential of -80 mV, a 3-s long -50mV prepulse was applied and then 300 ms of depolarizing pulses from -50 mV to 50 mV were applied in 10-mV increments. For the I<sub>CaL </sub>steady-state inactivation, from -40mV holding potential, test pulses were applied for 2s with 10 mV increments ranging from -80mV to 10mV followed by 10-ms-long -40mV pulse, and then 0mV pulse was applied for 250ms. For AP analysis, iPSC-CMs exhibiting an APD90 / APD50 ratio of less than 1.4 were defined as ventricular type<sup>53</sup>. I<sub>NaL</sub>, I<sub>Ks</sub>, and I<sub>Kr</sub> were defined as currents specifically sensitive to 30 µM TTX, 1 µM HMR1556, and 1 µM E4031 respectively. The current traces were subtracted before and after the drug administration to elicit those specific currents. Pipettes were pulled from thick-walled borosilicate glass capillaries (1B150F-4; World Precision Instruments, FL, USA) for AP and I<sub>CaL</sub>, and from thin-walled capillaries (TW150-4; World Precision Instruments) for I<sub>Na</sub>, I<sub>Ks</sub>, and I<sub>Kr</sub>. The resistance of the pipettes for I<sub>Na</sub> recording was 1 - 2 MΩ. For the other recordings, the pipettes with 3 - 5 MΩ were used. β-escin (25 μM) was applied in the recording solution to create the perforated patch configuration<sup>54</sup>. Access resistance was 10 - 25 MΩ for perforated patch recording and &lt; 5 MΩ for ruptured patch recording. dPatch<sup>®</sup>, and SutterPatch<sup>®</sup> (Sutter Instrument, CA, USA) were used for data acquisition. All the data were acquired from at least three independent experiments using different biological replicates. <strong>Multi-electrode array with optogenetics</strong> Single iPSC-CMs were isolated by incubating collagenase-B (Roche, Roswell, GA, USA, 1mg/mL) for 15 minutes and thereafter 0.25% Trypsin or Accutmax (Innova Cell Technologies, San Diego, CA, USA) for 5 minutes for the dissociation as previously described<sup>53</sup>. Cell suspensions (3 × 10<sup>4</sup> cells in 5 µL) were placed onto fibronectin-coated multi-electrode array (MEA) plates (CytoView MEA; Axion BioSystems, Atlanta, GA, USA). After 3 days, the CMs were infected with the crude adenovirus expressing Channelrhodopsin-2 fused to green fluorescent protein (GFP) (Ad-ChR2-GFP) to enable optical stimulation<sup>55</sup>. Four or more days after the infection, field potentials (FP) were recorded using Maestro Edge (Axion BioSystems). FP signals were digitally sampled at 12.5 kHz and the system bandwidth is 0.01 Hz – 5 kHz. iPSC-CMs were stimulated by using a multi-well light stimulation system (Lumos 24; Axion BioSystems). Specifically, CMs were stimulated at the rate of interest (1 Hz, 2 Hz, or 3 Hz) for 40 beats and the final 30 beats were averaged. FP duration (FPD) was defined as the interval between a spike and a subsequent positive deviation. This parameter was automatically measured with Cardiac Software Module on the system. All the data were acquired from at least three independent biological replicates.
description: # **Electrophysiology data for NAA10-R4S-induced pluripotent-derived
 cardiomyocytes**
 [https://doi.org/10.5061/dryad.280gb5mvz](https://doi.org/10.5061/dryad.280gb5mvz) The enclosed dataset contains electrophysiology data from patient-derived and genome-edited induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with dysfunctional NAA10. Data includes multi-electrode array (MEA) recordings of monolayer cultures and patch-clamp electrophysiology of single cells. ### **Description of the data and file structure** Files are in the GraphPad Prism format version 9 and version 10. Data is organized by related experiment and separated by patient-derived, genome-editing and wild type (WT) lines. All files are also uploaded in .csv format. Filenames are the same for both file formats. Any empty cells are replaced with “null” in the .csv files. These empty cells were left in the prism files to facilitate statistical calculations.  Description of individual files and corresponding data * **MEA_1Hz_baseline:** Field potential duration (FPD) measurements of plated iPSC-CMs from different genotypes. Measurements are in msec. * **MEA_GS967_recuse**: Field potential duration (FPD) measurements of plated iPSC-CMs with and without the sodium channel blocker GS967. Measurements are in msec. * **INa-IV_curve:** Current measurements with varying membrane potentials for the inward sodium current. Current measurements are in pA and voltage in mV. * **INa-activation_inactivation_comparison**: Steady state voltage dependent inactivation and activation curves for iPSC-CMs for three genotypes. Data is averaged from multiple traces with corresponding statistics contained in the files. Number of traces per trial and standard error of the mean for the set of points are included. * **INa-Late_current:** The ratio of late sodium currents measured at the end of the stimulating pulse compared to the peak inward sodium current in the same cell. Measurements as % of peak currents. * **IKs:** Current measurements with varying membrane potentials for the slow activating inward potassium current IKs. Current measurements are in pA and voltage in mV. * **IKr:** Current measurements with varying membrane potentials for the rapid activating inward potassium current IKs. Measurements were made as instantaneous recordings after a common voltage step. Current measurements are in pA and voltage in mV. * **Action_potential_statistics:** Measurements of induced action potentials from each of three iPSC-CM genotypes. Definitions of each statistic: * APD90 – The action potential duration measured at 90% of activation and inactivation (msec) * APD50 – The action potential duration measured at 50% of activation and inactivation (msec) * APD20 – The action potential duration measured at 20% of activation and inactivation (msec) * APA – Peak action potential amplitude (mV) * MDP – Mean diastolic membrane potential (mV) * Vmax – Maximum upstroke action potential velocity, dV/dT (mV/msec)  * **ICaL-IV:** Current measurements with varying membrane potentials for the inward calcium current. Current measurements are in pA and voltage in mV. * **MEA_NAA10_adeno_treatment:** Field potential duration (FPD) measurements of plated iPSC-CMs from different genotypes with and without adenovirus expression of NAA10. Measurements are in msec. ### **Sharing/Access information** For clinical information or gene sequencing information, please contact Dr. Vassilios Bezzerides at [vassilios.bezzerides@childrens.harvard.edu](mailto:vassilios.bezzerides@childrens.harvard.edu) * Please expect one week for responses. An approved IRB may be needed to receive more detailed clinical data, but collaborations are very welcome. ### **Code/Software** Single-cell electrophysiology data was obtained using Sutter Patch v2.3.1 and analyzed by standard scripts in Sutter Patch and pClamp v11 (Axon Instruments). MEA data was obtained using the Meastro (Axion Biosystems) and analyzed using the Cardiac package.