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 NAA10^R4S variant reduced enzymatic activity, decreased NAA10-NAA15 complex formation, and destabilized the enzymatic complex N-terminal acetyltransferase A. In NAA10^R4S/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 NAA10^R4S/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 NAA10^R4S/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.

Whole-cell patch clamp recordings

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)^50,51. Perforated patch recordings were performed for action potential (AP) analysis and the L-type Ca²⁺ current (I_CaL). Perforated patch was applied in order to prevent run-down in I_CaL recording⁵². The ruptured patch technique was used for I_Na, I_Ks, and I_Kr recordings. Series resistance and cell capacitance were compensated to ~ 60 % for all the voltage clamp experiments. To measure I_Na, 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_Na 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_Ks, 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_Kr, 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_CaL, 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_CaL 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 type⁵³. I_NaL, I_Ks, and I_Kr 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_CaL, and from thin-walled capillaries (TW150-4; World Precision Instruments) for I_Na, I_Ks, and I_Kr. The resistance of the pipettes for I_Na 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 configuration⁵⁴. Access resistance was 10 - 25 MΩ for perforated patch recording and < 5 MΩ for ruptured patch recording. dPatch^®, and SutterPatch^® (Sutter Instrument, CA, USA) were used for data acquisition. All the data were acquired from at least three independent experiments using different biological replicates.

Multi-electrode array with optogenetics

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 described⁵³. Cell suspensions (3 × 10⁴ 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 stimulation⁵⁵. 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.