Hypoxia Produces Pro-arrhythmic Late Sodium Current in Cardiac Myocytes by SUMOylation of NaV1.5 Channels

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  Figure 1.. Acute Hypoxia and SUMO1 Augment the ILATE in Human iPS-CMs INa in human iPS-CMs was studied by whole-cell patch clamp. ILATE was measured at −30 mV and is shown as a mean percentage of the peak current remaining between 50 and 100 ms after the peak current. Hypoxia challenge was a drop in O2 from 21% to 1.5%. The O2 level was measured in real time in the recording chamber and fell from 21% to 1.5% O2 in 11 ± 3 s (not shown). The time course of hypoxic modulation of INa was studied by steps from −100 mV to −30 mV every 10 s. Whole-cell currents were not studied for more than 600 s to maintain consistent voltage clamp control of INa. Cells were studied with control solution (black), 1 nM SUMO1 (red), or 1 nM SENP1 (blue) in the recording pipette. Data are mean ± SEM for 8 to 10 cells per group. Measured values and statistical analyses are listed in Table 1. (A) Left: example traces showing ILATE at −30 mV increased when control perfusate at 21% O2 (black) was exchanged with a hypoxic solution at 1.5% O2 (black dash). Inset: the increase in ILATE shown in response to hypoxia. Right: the time course for changes in ILATE in response to hypoxia and on return to normoxia (21%). Scale…

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  Figure 2.. FRET between NaV1.5 and SUMO1 at the Cell Surface Requires K442 Human NaV1.5 was expressed with NaVβ1 and studied in live CHO-K1 cells per the STAR Methods. FRET was assessed by measuring the time constant (τ) for mTFP photobleaching (donor) in the presence of YFP (acceptor) from 3 regions of 5–7 cells per group. Data are mean τ ± SEM, and significant changes in τ compared to free YFP are indicated (*p < 0.001, two-way ANOVA). (A) Top: a cartoon showing the secondary structure of a mTFP-NaV1.5 subunit and the location of putative SUMOylation sites identified by the SUMOplot algorithm. The NaV1.5 α-subunit has four homologous domains (I–IV), each with six transmembrane segments that fold to form one central ion conduction pore. Domains I and II are critical to opening the pore (activation) (Catterall, 2017; Cestèle et al., 1998, 2001). Fast inactivation results from pore occlusion by an IFM triplet of residues in the linker between domains III and IV(Vassilev et al., 1988; West et al., 1992; Cha et al., 1999). Bottom: fluorescent photomicrographs showing that mTFP-NaV1.5 (blue) and YFP-SUMO1 (yellow) reach the cell surface. Scale bar represents 10 μm. The montage shows…

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  Figure 3.. K442 Is Required for Hypoxia and SUMO1-Mediated ILATE Human NaV1.5 wild-type (WT) or NaV1.5-K442Q was expressed in CHO-K1 cells with NaVβ1 and studied under normoxic (21%) and hypoxic (1.5%) O2 conditions with control solution (black), 1 nM SUMO1 (red), or 1 nM SENP1 (blue) in the recording pipette, as indicated. The late current (ILATE) was studied at −30 mV and is shown as a mean percentage of the peak current remaining between 50 and 100 ms after the peak current. The time course of hypoxic modulation of INa was studied by steps from −100 mV to −30 mV every 10 s. Data are mean ± SEM for 8 to 12 cells per group. Single-channel currents were studied in cell-attached mode and elicited every 5 s by a 50-ms depolarizing pulse to −30 mV from a holding potential of −120 mV. Data were recorded at filter and sampling frequencies of 5 and 50 kHz, respectively, and processed offline using a 1.2-kHz Bessel filter for display. Recording pipettes were filled with extracellular buffer. For each cell, null sweeps (with no channel activity) were identified, averaged offline, and subtracted from the data sweeps before analysis. (A) Example traces from a cell expressing WT NaV1.5 channel…

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  Figure 4.. Hypoxia Recruits One SUMO1 Monomer to Each Cell Surface NaV1.5 Channel Single mTFP-NaV1.5 channels (blue) and SUMO1 tagged with mCherry (m-SUMO1, red) were studied in CHO-K1 cells by TIRFM. Stoichiometric (photobleaching) and pixel-by-pixel analysis for subunit density and co-localization were performed as described in the STAR Methods. Manders’ coefficients were assessed post hoc for 3–5 regions per cell, and co-localization was defined as the presence of both fluorophores at more than 30% of the maximum fluorescence level recorded in that stack (and their overlap is represented in the images as white pixels). The time course of hypoxic modulation of NaV1.5 ILATE was studied with steps from −100 mV to −30 mV every 10 s and normalized to the peak current. Data represent 5–8 cells, and biophysical parameters and single particle statistical analyses are summarized in Tables S1 and S3. (A) Left: single co-localized particles with mCherry and TFP fluorescence were observed at the surface of cells expressing NaV1.5 and SUMO1. The time courses for simultaneous photobleaching of the fluorophores revealed that complexes have one subunit of each type. (B) Histogram of photobleaching steps…

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  Figure 5.. Hypoxia-Induced Increase in APD Mediated by SUMO Modulation of ILATE Spontaneous action potentials from human iPS-CMs were recorded in current-clamp mode with the same sequence of perfusates: normoxic (21% O2, solid line), hypoxic (1.5% O2, dashed line), and then hypoxia with 1 μM ranolazine (purple). Action potentials were studied with control intracellular solution (black) or with 1 nM SUMO1 (red) or SENP1 (blue) in the recording pipette. APD50 was determined by calculating the time required for the membrane potential to return to 50% of the resting value from the peak deflection of the action potential. Hypoxia was induced as described in the STAR Methods. Data are means ± SEM for 6–9 cells studied per group (*p < 0.01, two-way paired t test). The O’Hara-Rudy model for human action potentials was applied as described in the STAR Methods and Table S3. Scale bars represent 25 mV and 250 ms in (A)–(C) and 30 mV and 150 ms in panel (D). (A) Examplar spontaneous action potentials recorded from iPS-CMs studied with control pipette solution under normoxic conditions and hypoxia with and without ranolazine. The APD50 increased with hypoxia by 22%, from 663 ± 2 ms to 810 ± 2 ms, and…