Journal: iScience

Article Title: Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2

doi: 10.1016/j.isci.2022.104969

Figure Lengend Snippet: Acute hypoxia inhibits I K1 via SUMOylation I K1 in rat ventricular cardiomyocytes (RVCMs) was studied by the whole-cell patch-clamp. Currents were evoked in a high external K + recording buffer and measured at −120 mV, as described in the . Hypoxia was a drop in O 2 from ambient levels to 2% (red), measured at the cell. The time-course of hypoxic inhibition was studied by recording the magnitude of I K1 every second. Cells were studied with the following pipette solutions: control (blue), SUMO1 (1 μM, orange), or SENP1 (2 μM, magenta). CRY2-SENP1 was targeted to the cell membrane by the activation of a 460 nm LED during the time period indicated. I K1 currents were blocked by the addition of 3 mM Ba 2+ (green). (A) Exposure to acute hypoxia (2% O 2 ) inhibits ∼40% of I K1 in RVCMs. Left , representative current sweeps; right , a representative time course showing the kinetics of hypoxic inhibition in RVCMs studied with a control pipette solution. The inhibition is precluded by including SENP1 (2 μM) in the recording pipette. The reverse ramp recording protocol is inset. (B) Including SUMO1 (1 μM) in the recording pipette inhibits I K1 and precludes the effects of hypoxia. SENP1 augments I K1 and protects the current from hypoxia. Data are from 8 to 10 RVCMs per group, ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test. (C) Hypoxic inhibition of I K1 was reversed by the activation of CRY2-SENP1 with 460 nm light (cyan box) when RVCMs are studied with a control pipette solution. Right , CRY2-SENP1 does not alter current when 2 μM SENP1 is included in the recording pipette. Data are from 6 RVCMs, ∗p < 0 . 05 , ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test.

Article Snippet: Rat Cardiomyocyte Growth Medium (rCGM) BulletKit , Lonza Biosciences , Cat# CC-4515.

Techniques: Patch Clamp, Inhibition, Transferring, Control, Membrane, Activation Assay, Two Tailed Test

Journal: iScience

Article Title: Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2

doi: 10.1016/j.isci.2022.104969

Figure Lengend Snippet: PIP 2 opposed hypoxic inhibition of I K1 I K1 was studied in rat ventricular cardiomyocytes (RVCMs) using a whole-cell patch-clamp recording. To the knockdown Kir2.1 expression, RVCMs were transduced with lentiviral particles carrying eGFP and shRNA targeting KCNJ2. Kir2.1 knockdown cells (Kir2.1 kd -CMs) were identified by the expression of eGFP. Cells were studied with a control pipette solution (blue), or with pipette solutions containing purified SUMO1 (1 μM, orange) or SENP1 (2 μM, magenta). Where indicated, the control pipette solution contained diC8 PIP 2 . Paired patch-clamp data were analyzed by Students t -test; ∗∗∗, p < 0.01. The proximity ligation assay (PLA) for native Kir2.1-SUMO1 interactions was performed as described in the and analyzed using an unpaired Mann-Whitney rank test, ∗∗∗∗p < 0.001. See also Figures S7 and . (A) Left , Example sweeps show that I K1 is diminished in Kir2.1 kd -CMs and the remaining current is insensitive to acute hypoxia. Kir2.1 kd -CMs are identified by expression of GFP ( inset , scale bar = 10 μm). Right , summary data from 8 to 10 cells per group show that the regulation of I K1 by hypoxia, SUMO1, and SENP1 is lost in Kir2.1 kd -CMs. (B) The magnitude and the regulation of I K1 by hypoxia, SUMO1, and SENP1 are unaltered when cells are treated with a control, scrambled shRNA; 7-8 cells per group. (C) Left , Example traces to show that including diC8-PIP 2 in the pipette solution reduces the hypoxic inhibition of I K1 in RVCMs in a concentration-dependent manner. The arrow indicates the change in current magnitude between exposure to ambient O 2 and 2% O 2 in the same cell. Right , Concentration-response curve showing that including diC8-PIP 2 in the pipette opposes hypoxic inhibition of I K1 in control RVCMs and RVCMs expressing the scrambled shRNA. The hypoxic-response is diminished in Kir2.1 kd -CMs. Data are mean ± s.d. for 7-8 cells per condition. (D) DiC8-PIP 2 decreases hypoxic inhibition of Kir2.1 channels expressed in HEK293T cells (control) in a concentration-dependent manner using the experimental paradigm described in C, above. Current inhibition is diminished when SENP1 is included in the recording pipette and is not observed when Kir2.1-K49Q channels are studied. Data are mean ± s.d. for 6 cells per condition. (E) PLA shows that native Kir2.1 colocalizes with SUMO1 in RVCMs and that Kir2.1-SUMO interactions are increased by acute hypoxia. Kir2.2 and Kir2.3 do not associate with SUMO1. Representative data are shown with DAPI-labeled nuclei in red and PLA interactions in green for ease of visualization. Summary data are obtained from multiple experiments each with multiple fields of view containing 20-30 nuclei. The scale bar is 10 μm.

Article Snippet: Rat Cardiomyocyte Growth Medium (rCGM) BulletKit , Lonza Biosciences , Cat# CC-4515.

Techniques: Inhibition, Patch Clamp, Knockdown, Expressing, Transduction, shRNA, Control, Transferring, Purification, Proximity Ligation Assay, MANN-WHITNEY, Concentration Assay, Labeling

Journal: iScience

Article Title: Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2

doi: 10.1016/j.isci.2022.104969

Figure Lengend Snippet:

Article Snippet: Rat Cardiomyocyte Growth Medium (rCGM) BulletKit , Lonza Biosciences , Cat# CC-4515.

Techniques: Recombinant, Virus, shRNA, Control, In Situ, Software, Microscopy