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Doxorubicin Induces Caspase-Mediated Proteolysis of KV7.1

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Doxorubicin Induces Caspase-Mediated Proteolysis of KV7.1

Anne Strigli et al. Commun Biol.

Abstract

Kv7.1 (KCNQ1) coassembles with KCNE1 to generate the cardiac IKs -channel. Gain- and loss-of-function mutations in KCNQ1 are associated with cardiac arrhthymias, highlighting the importance of modulating IKs activity for cardiac function. Here, we report proteolysis of Kv7.1 as an irreversible posttranslational modification. The identification of two C-terminal fragments of Kv7.1 led us to identify an aspartate critical for the generation of one of the fragments and caspases as responsible for mediating proteolysis. Activating caspases reduces Kv7.1/KCNE1 currents, which is abrogated in cells expressing caspase-resistant channels. Enhanced cleavage of Kv7.1 can be detected for the LQT mutation G460S, which is located adjacent to the cleavage site, whereas a calmodulin-binding-deficient mutation impairs cleavage. Application of apoptotic stimuli or doxorubicin-induced cardiotoxicity provokes caspase-mediated cleavage of endogenous IKs in human cardiomyocytes. In summary, caspases are novel regulatory components of IKs channels that may have important implications for the molecular mechanism of doxorubicin-induced cardiotoxicity.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Kv7.1 is cleaved at aspartate 459. a Western blot analysis of HeLa cell lysates overexpressing human and murine Kv7.1 constructs. Untransfected cells (Ø) served as negative control. Densitometric analysis of four independent experiments of the CTF1 or CTF2 band intensity normalized to the full-length Kv7.1 band intensity. b Immunoblot analysis of lysates derived from HeLa cells overexpressing indicated constructs. Untransfected cells (Ø) served as negative control. c Schematic illustration of different deletion constructs in the linker region between helices A and B. HeLa cell lysates overexpressing indicated constructs analyzed by western blot. Untransfected cells (Ø) served as negative control. d Alanine scan of position 457 to 462. Lysates from HeLa cells overexpressing indicated constructs were used for western blot analysis. Untransfected cells (Ø) served as negative control. Densitometric analysis of six independent experiments of CTF2 band intensity normalized to Kv7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. e Cleavage site sequence logo of Kv7.1 derived from 32 different species. Logo was created with weblogo.berkley.edu and adapted accordingly. The arrow indicates the cleavage site. a Anti-Kv7.1 antibody. bd Anti-MYC antibody. All dot blots are shown as mean and error bars as SEM
Fig. 2
Fig. 2
Cleavage of Kv7.1 occurs during apoptosis. a Western blot analysis of Cos7 cells overexpressing Kv7.1-MYC treated for 12 h with 450 nmol per L Q-VD-OPh. Untransfected (Ø), nontreated and vehicle-treated cells served as negative controls. Densitometric analysis of five independent experiments of CTF2 band intensity normalized to Kv7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. b Lysates derived from HeLa cells expressing Kv7.1-MYC and Kv7.1-D459A-MYC constructs treated with 1 µmol per L staurosporine for 8 h analyzed by immunoblotting. Untransfected (Ø) and vehicle-treated cells served as negative controls. Densitometric analysis of eight independent experiments of the CTF2 band intensity normalized to Kv7.1 full-length band intensity. c Western blot analysis of HeLa cell lysates overexpressing CT-MYC construct treated for 8 h with 1 µmol per L staurosporine. Vehicle-treated cells served as negative controls. ac Anti-MYC antibody, a, c anti-β-actin antibody. All dot blots are shown as mean and error bars as SEM
Fig. 3
Fig. 3
Caspases are responsible for the generation of CTF2. a Western blot analysis of lysates derived from HEK 293T cells stably expressing Kv7.1-MYC treated with either 1 µmol per L staurosporine for 6 h or treated with 1 µmol per L staurosporine for 6 h and pretreated for 2 h with 20 or 50 µmol per L of a caspase-8 inhibitor II. Untransfected (Ø) and vehicle-treated cells served as negative controls. Densitometric analysis of 4–6 independent experiments of the CTF2 band intensity normalized to Kv7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. # indicates nonspecific binding of the antibody. b MCF-7 lysates expressing Kv7.1-MYC and caspase-3 or caspase-3-D28A-D175A analyzed by immunoblot. Untransfected (Ø) and eGFP-transfected cells served as negative controls. # indicates nonspecific binding of the antibody. c Lysates of HEK 293T cells stably expressing Kv7.1 and coexpressing indicated caspases analyzed by immunoblot. Untransfected (Ø) and eGFP-transfected cells served as negative controls. d Lysates of HL-1 cells treated for 6.5 h with 0.5, 1, 1.5 and 2 µmol per L of staurosporine analyzed by immunoblot. Vehicle-treated cell lysates served as negative control. Densitometric analysis of CTF2 band intensity normalized to Kv7.1 full-length band intensity of five independent experiments. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. ac Anti-MYC antibody. Anti-β-actin antibody. a, b, d Anti-caspase-3 antibody. d Anti-Kv7.1 antibody. All dot blots are shown as mean and error bars as SEM
Fig. 4
Fig. 4
Cleavage of Kv7.1 in physiology and pathophysiology. a Representative current traces for Kv7.1-MYC and Kv7.1-D459A-MYC, both coexpressed with KCNE1. b Mean currents amplitude was plotted versus voltage to obtain current−voltage (I−V) relationships in cells expressing Kv7.1-MYC (n = 39 for vehicle, n = 16 for staurosporine treatment) or Kv7.1-D459A-MYC (n = 27 for vehicle, n = 17 for staurosporine treatment) and KCNE1 treated with 500 nmol per L staurosporine for 10–12 h. Statistics were tested with two-way ANOVA followed by Bonferroni post-tests. c Immunoblot analysis of HeLa cells coexpressing Kv7.1 with KCNE1-MYC treated with 1 µM staurosporine for 4.5 h. Untransfected (Ø) and vehicle-treated cells served as negative controls. d Biotinylating study analyzed by immunoblots of Hek 293 cells coexpressing Kv7.1 and KCNE1-MYC treated with 1 µM staurosporine for 3 h. Untransfected (Ø) cells as well as cells not treated with biotin served as negative controls. IP Immunoprecipitation. TL total lysate. e Schematic illustration to highlight the position of G460 and A372 and calmodulin binding site in helix A. Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and Kv7.1-D459A-transfected cells served as negative controls. Densitometric analysis of four independent experiments of CTF2 band intensity normalized to Kv7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. f Coimmunoprecipitation study analyzed by immunoblots of HeLa cells overexpressing wild-type Kv7.1 and the A372D mutant with endogenous calmodulin. IP Immunoprecipitation with anti-Kv7.1 antibody, IB Immunoblot, TL total lysate. Untransfected cells (Ø) served as negative control. c Anti-KCNE1 antibody, anti-caspase 3 antibody. c, d Anti-Kv7.1 antibody, anti-GAPDH antibody. e Anti-β-actin antibody. e, f Anti-MYC antibody. f Anti-calmodulin antibody. All graphs are shown as mean and error bars as SEM
Fig. 5
Fig. 5
Doxorubicin induces cleavage of Kv7.1 to CTF2. a Immunoblot analysis of human-induced pluripotent stem cell-derived cardiomyocytes treated either with vehicle, staurosporine (2 µM) for 4 h or doxorubicin (10 µM) overnight. # indicates nonspecific binding of the Kv7.1 antibody. b Densitometric analysis of 3–6 independent experiments of band intensities of CTF2 normalized to GAPDH band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. a Anti-Kv7.1 antibody, anti-caspase-3 antibody, anti-GAPDH antibody. All dot blots are shown as mean and error bars as SEM

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References

    1. Yu FH, Yarov-Yarovoy V, Gutman GA, Catterall WA. Overview of molecular relationships in the voltage-gated ion channel superfamily. Pharmacol. Rev. 2005;57:387–395. doi: 10.1124/pr.57.4.13. - DOI - PubMed
    1. Jentsch TJ, Schroeder BC, Kubisch C, Friedrich T, Stein V. Pathophysiology of KCNQ channels: neonatal epilepsy and progressive deafness. Epilepsia. 2000;41:1068–1069. doi: 10.1111/j.1528-1157.2000.tb00302.x. - DOI - PubMed
    1. Lehman A, et al. Loss-of-function and gain-of-function mutations in KCNQ5 cause intellectual disability or epileptic encephalopathy. Am. J. Hum. Genet. 2017;101:65–74. doi: 10.1016/j.ajhg.2017.05.016. - DOI - PMC - PubMed
    1. Wang Q, et al. Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias. Nat. Genet. 1996;12:17–23. doi: 10.1038/ng0196-17. - DOI - PubMed
    1. Neyroud N, et al. A novel mutation in the potassium channel gene KVLQT1 causes the Jervell and Lange-Nielsen cardioauditory syndrome. Nat. Genet. 1997;15:186–189. doi: 10.1038/ng0297-186. - DOI - PubMed

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