ER stress modulates Kv1.5 channels via PERK branch in HL-1 atrial myocytes: Relevance to atrial arrhythmogenesis and the effect of tetramethylpyrazine

Figures

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  Fig. 1. ER stress shortens action potential duration (APD) and upregulates Kv1.5 channel expression in HL-1 atrial myocytes. (A) Representative recording traces of action potential and grouped data of APD in HL-1 atrial myocytes treated or not treated with tunicamycin (TM). (B) Representative blots and grouped data of the expression level of Kv1.5 channels in atrial myocytes treated with or without TM. Full blots are shown in Supplementary Figs. 3–1B. ∗∗P < 0.01 vs. control. n indicates the number of independent experiments performed with cells from different culture batches.

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  Fig. 2. Inhibition of ER stress prevents action potential duration (APD) shortening and Kv1.5 channel upregulation in HL-1 atrial myocytes. (A&B) Representative blots and expression levels of ER stress molecules in cells from different treatment groups. TM: tunicamycin, ER stress inducer; 4-PBA and TUDCA: ER stress inhibitors. Full blots are shown in Supplementary Figs. 3–2A&B. (C) Representative recording traces of action potential and grouped data of APD, showing TM-induced shortening of APD and the restoration of APD90 by 4-PBA and TUDCA. (D) Inhibition of ER stress by 4-PBA or TUDCA prevents TM-induced upregulation of Kv1.5 channels. Full blots are shown in Supplementary Figs. 3–2D. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs.TM. n indicates the number of independent experiments performed with cells from different culture batches.

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  Fig. 3. Inhibition of ER stress suppresses the enhancement of Kv1.5 channel current in HL-1 atrial myocytes. Treatment of HL-1 cells with ER stress inhibitor 4-PBA (A) or TUDCA (B) prevents Kv1.5 channels from tunicamycin (TM)-induced activation. Representative traces and I-V curves of whole-cell K+ currents of cells from different treatment groups before and after application of Kv1.5 channel blocker trimethylapigenin (TMA) (A&B, upper panel). Summarized data of whole-cell K+ current (A&B, lower panel-left) and Kv1.5 channel current (IKv1.5 = IBasal-ITMA) (A&B, lower panel-right) from different treatment groups (n = 9). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs.TM. n indicates the number of independent experiments performed with cells from different culture batches.

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  Fig. 4. PERK branch of unfolded protein response mediates ER stress-induced upregulation of Kv1.5 channels in HL-1 atrial myocytes. (A) The PERK inhibitor GSK2606414 and the IRE1 inhibitor GSK2850163 respectively suppressed the phosphorylation of PERK and IRE1, and the ATF6 inhibitor Ceapin A7 lowered ATF6 protein level, showing the effectiveness of each inhibitor. (B) Inhibition of PERK with GSK2606414 antagonized tunicamycin (TM)-induced Kv1.5 upregulation, but inhibition of IRE1 and ATF6 showed no reversal effect. Corresponding full blots are shown in Supplementary Figs. 3–4A&B. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control; ##P < 0.01, ###P < 0.001 vs.TM. n indicates the number of independent experiments performed with cells from different culture batches.

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  Fig. 5. Inhibition of the PERK branch of ER stress normalizes Kv1.5 channel current and action potential duration (APD). (A) Representative traces and I-V curves of whole-cell K+ currents of cells from different treatment groups before and after application of Kv1.5 channel blocker trimethylapigenin (TMA) (A, upper panel). Summarized data of whole-cell K+ current (A, lower panel-left) and the Kv1.5 channel current (IKv1.5 = IBasal-ITMA) (A, lower panel-right) of cells from different treatment groups (n = 9). (B) Representative traces of action potential and grouped data of APD (n = 7), showing the restoration of APD90 by the PERK inhibitor GSK2606414 in tunicamycin (TM)-exposed HL-1 atrial myocytes. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control; #P < 0.05, ##P < 0.01 vs.TM. n indicates the number of independent experiments performed with cells from different culture batches.

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  Fig. 6. Inhibition of PERK activation triggered by thapsigargin (TG) prevents the increase of Kv1.5 protein and Kv1.5 channel current. (A) TG induced ER stress in HL-1 cells, involving activation of the PERK branch. (B) TG upregulated the protein expression of Kv1.5 channels. (C) Inhibition of PERK by GSK2606414 prevented the upregulation of Kv1.5 protein induced by TG. Corresponding full blots are shown in Supplementary Fig.3-6A, 6B and 6C. (D) Inhibition of PERK by GSK2606414 prevented TG-induced enhancement of Kv1.5 channel current. Representative traces and I-V curves of whole-cell K+ currents of cells from different treatment groups before and after application of Kv1.5 channel blocker trimethylapigenin (TMA) (D, upper panel). Summarized data of whole-cell K+ current (D, lower panel-left) and the Kv1.5 channel current (IKv1.5 = IBasal-ITMA) (D, lower panel-right) of cells from different treatment groups (n = 5). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control; #P < 0.05, ##P < 0.01 vs.TG. n indicates the number of independent experiments performed with cells from different culture batches.

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  Fig. 7. Tetramethylpyrazine (TMP) prevents Kv1.5-associated APD shortening through antagonizing ER stress in HL-1 atrial myocytes. (A) TMP exhibits strong anti-ER stress activity against tunicamycin (TM) in HL-1 atrial myocytes. Full blots are shown in Supplementary Figs. 3–7A. (B) TMP suppresses Kv1.5 channel current in HL-1 atrial myocytes exposed to TM. Representative traces and I-V curves of whole-cell K+ current from different treatment groups before and after application of Kv1.5 channel blocker trimethylapigenin (TMA) (B, upper panel). Summarized data of whole-cell K+ current (B, lower panel-left) and the Kv1.5 channel current (IKv1.5 = IBasal-ITMA) (B, lower panel-right) of cells from different treatment groups (n = 9). (C) TMP inhibits TM-induced expression of Kv1.5 channels (n = 6). Full blots are shown in Supplementary Figs. 3–7C. (D) TMP prevents TM-induced APD shortening. Representative action potential traces from different treatment groups (D, upper panel). Summarized data showing the restoration of APD90 by TMP (D, lower panel, n = 7). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs.TM. n indicates the number of independent…