Fig 2: PKC inhibitors abolish PMA-induced reduction in Kv1.5 expression and IKv1.5in HEK293 cells.A, western blots result showing effects of 10 nM PMA treatment for 3 h on Kv1.5 expression with or without 10 µM BIM-1. Representative western blot images are displayed along with summarized data (n = 4). **p< 0.01 versus CTL; ##p < 0.01 versus PMA. B, confocal images showing effects of 10 nM PMA treatment for 3 h on Kv1.5 expression with or without 10 µM BIM-1. Kv1.5 was labeled with an anti-Kv1.5 primary antibody and Alexa Fluor 488-conjugated (green) secondary antibody. The cell membrane was labeled using a Texas Red X-conjugated WGA (red) (n = 4). C, effects of 10 nM PMA treatment for 3 h on IKv1.5 with or without 10 µM BIM-1. Representative current traces are shown along with summarized I-V relationship. **p < 0.01 versus CTL; ##p < 0.01 versus PMA+BIM-1 for current amplitudes upon >10 mV depolarizing steps. n = 6–13 cells. D, western blots image showing the effects of 200 nM Sotrastaurin or 200 nM Staurosporine on Kv1.5 expression of Kv1.5-HEK cells treated with or without 10 nM PMA for 3 h (n = 3). **p< 0.01 versus CTL; ##p < 0.01 versus PMA. For western blots, data are presented as box plots and mean ± SD. For patch clamp, data are presented as mean + SD. Rel, relative values.

Fig 3: Western blotting of proteins encoded by the Kv4.3a, KvLQT1, Kv1.5, L-Caa1c and NCX genes. (A) Total protein was harvested, separated by SDS/PAGE and subjected to immunoblot analyses. The primary antibodies against Kv4.3a, KvLQT1, Kv1.5, L-Caa1c, NCX and actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Bound antibodies were detected using the ECL system (Pierce Biotechnology, Inc., Rockford, IL, USA). (B) The immunoblot experiments were repeated at least 3 times. The mean normalized OD of detected Kv4.3a, KvLQT1, Kv1.5, L-Caa1c or NCX protein bands relative to the OD of the actin band from the same individual was calculated, respectively. The mean ± SD was calculated. OD, optical density; HF, heart failure.

Fig 4: Generation of KCNA5 knockout model. (A) Illustration of polycistronic self-replicating RNA used for reprogramming (Yoshioka et al., 2013). Reading frames for OCT4, KLF4, SOX2, GLIS1, and puromycin resistance are separated by 2A peptide-encoding or IRES sequences. (B) Phase contrast morphology (5 x) and alkaline phosphatase staining of the F1 wild-type hiPSC line generated using the RNA vector shown in (A). (C) Representative F1 karyogram indicating a normal male genotype. (D) The RNA vector is undetectable in established F1 hiPSCs following puromycin withdrawal, whereas freshly transfected parental fibroblasts display robust transgene expression (RT-qPCR data, n = 2). (E) Bisulfite sequencing of OCT4 and NANOG promoter regions of the indicated samples showing an hESC-like hypomethylated state in F1 cells. (F) RT-qPCR analysis of F1 hiPSCs in comparison to the original fibroblasts and a hESC control (n = 2). (G) Immunostains showing robust pluripotency factor expression in F1 hiPSCs at the protein level. (H) Illustration of the Kv1.5 channel. The voltage sensor in transmembrane helix S4 is indicated by symbols. The ion pore region is in-between segments S5 and S6. (I) KCNA5 knockout strategy based on simultaneously transfecting 4 CRISPR/Cas9 nickase vectors targeting the 5' end of the 1-exon gene body. A mutant clone generated this way displayed homozygous deletions causing a frame shift and premature stop as illustrated at the bottom. (J) PCR spanning the mutation site validates the sequencing-verified frame shift-causing deletion in KCNA5fs/fs cells at the DNA and RNA levels.

Fig 5: PKC activation by PMA treatment decreases currents of Kv1.5 channels expressed in neonatal rat ventricular myocytes. Representative current traces (A) and summarized current–voltage relationships (B) of IKv1.5 recorded from Kv1.5-transfected neonatal rat ventricular myocytes treated with PMA (10 nM) for 3 h in the absence and presence of BIM-1 (10 µM) or Baf (1 µM) are shown. **p < 0.01 versus CTL; ##p < 0.01 versus PMA for current amplitudes upon >0 mV depolarizing steps. n = 9–12 cells in each group. Data are presented as mean + SD.