Protective function of the voltage-gated potassium channel Kv11.3 in a mouse model of cardiac ischemia/reperfusion injury

Figures

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  Fig 1. Generation of Kv11.3 KO mice. (A) The guide strand of the CRISPR/Cas9 system was designed to target the mouse Kcnh7 exon 4 (NCBI accession number NM_133207). DNA sequencing of the targeted region in knockout (KO) mice. (B) Schematic protein structure of Kv11.3. The mutant N-terminally truncated Kv11.3 proteins generated in KO mice lose their functional domains include a pore-forming domain. S1–S6, transmembrane helices. H5, pore-forming helix. (C) Western blotting for Kv11.3 in the whole heart and brain lysates. In KO brain, the bands of full-length Kv11.3 (including dimer) and Kv11.3 isoform X6 (NCBI accession number XP_017171420) were not detected. Original blots are presented in S4 Fig.

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  Fig 2. Kv11.3 deficiency in the mouse model of cardiac ischemia/reperfusion (I/R). (A) The mortality rate during 2-h reperfusion: wild-type mice (WT), n = 13 mice. Kv11.3 knockout mice (KO), n = 12 mice. (B) 2,3,5-triphenyltetrazolium chloride (TTC) staining of the hearts exposed to 1.5-h reperfusion. Scale bars, 2 mm. (C) The proportion of infarct area (IA) to all areas in the hearts exposed to 1.5-h reperfusion, n = 5 mice per genotype. (D, E) The heart rate variability frequency domain metrics and the corrected QT interval (QTc) before and after I/R. LF, low frequency. HF, high frequency. n = 5 mice per genotype. (F) The proportion of IA to all area in the Langendorff-perfused hearts. WT, n = 11 mice. KO, n = 8 mice. P-values, *** < 0.001, ** < 0.01, * < 0.05. Statistical significance was determined by Fisher’s exact test (A), unpaired Student’s t-test (C, F), paired Student’s t-test (D) and Tukey-Kramer test (E).

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  Fig 3. Effect of NS-1643 in a murine cardiac ischemia/reperfusion (I/R) model. (A, B) Ratio of the corrected QT interval (QTc) and the heart rate variability (HRV) frequency domain metrics in sham-operated wild-type mice treated with NS-1643. Arrows represent injections. LF, low frequency. HF, high frequency. n = 3 mice. (C) 2,3,5-triphenyltetrazolium chloride (TTC) staining of the hearts that were treated with high concentrations of NS-1643 and exposed to 2-h reperfusion. Scale bars, 2 mm. (D) The proportion of infarct area (IA) to all areas in the reperfused hearts treated with NS-1643. DMSO, dimethyl sulfoxide; HD, high-dose NS-1643 at 2 or 6 mg/kg; LD, low-dose NS-1643 at 0.1 or 0.5 mg/kg. DMSO, n = 6 mice. HD, n = 6 mice. LD, n = 7 mice. (E) Heart rate variability frequency domain metrics before and after I/R. LD (0.1 mg/kg), n = 4 mice. HD (6 mg/kg), n = 3 mice. (F) The mortality rate during 2-h reperfusion. DMSO, n = 13 mice. HD, n = 6 mice. LD, n = 13 mice. P-values, ** < 0.01, * < 0.05. Statistical significance was determined by paired Student’s t-test (A, B), Dunnett’s test (D), paired Student’s t-test (E), and Fisher’s exact test (F).

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  Fig 4. The predicted interactions of the Kv channel proteins (Kv11.1, Kv11.3, Kv4.3, and Kv4.2) with NS-1643. (A) Theoretical binding poses of Kv channel proteins with NS-1643 obtained by molecular dynamic (MD) trajectory clustering. The most frequent structure that occurred during the simulation is shown. The protein is illustrated in ribbon representation with the representative binding sites in ball and stick representation. (B) Schematic representation of the interactions between Kv channel proteins and NS-1643 that occur over more than 5.0% of the 50.0 ns simulation time are depicted as arrows.