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Cited by 236Chemotherapy-induced cardiotoxicity and mitigationCircular RNAs in diseasesCardiac Fibrosis and Remodeling

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Quaking Inhibits Doxorubicin-Mediated Cardiotoxicity Through Regulation of Cardiac Circular RNA Expression

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Gupta, Shashi Kumar, Garg, Ankita, Bär, Christian, Chatterjee, Shambhabi, Foinquinos, Ariana, Milting, Hendrik, Streckfuß-Bömeke, Katrin, Fiedler, Jan, Thum, Thomas·2018·Circulation Research
DOI 10.1161/circresaha.117.311335PMC PMC5771684
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Abstract

Rationale: RBPs (RNA-binding proteins) have been described to be expressed and regulated in various organs including the heart. Little is known about the role of RBPs in heart failure induced by the chemotherapy drug doxorubicin and their interaction with circular RNAs. Objective: We aimed to identify key RBPs involved in doxorubicin-mediated heart failure and to elucidate their function. Methods and Results: Global transcriptome profiling from murine myocardium exposed to doxorubicin identified 5 differentially expressed RBPs. Expression of the RBP QKI (Quaking) in response to doxorubicin was strongly downregulated in rodent cardiomyocytes and human induced pluripotent stem cell–derived cardiomyocytes in vitro and in vivo in mice. Knockdown of Qki in primary cardiomyocytes increased apoptosis and atrophy after treatment with doxorubicin, whereas lentiviral mediated overexpression of Qki5 inhibited the doxorubicin-induced apoptosis in cardiomyocytes. In vivo, AAV9 (adeno-associated virus serotype 9)–mediated cardiac overexpression of Qki5 prevented cardiac apoptosis and cardiac atrophy induced by doxorubicin and improved cardiac function. Mechanistically, by lentiviral-based overexpression and CRISPR/Cas9-mediated silencing of Qki5 , we identified regulated expression of specific circular RNAs derived from Ttn (Titin), Fhod3 (Formin homology 2 domain containing 3), and Strn3 (Striatin, calmodulin-binding protein 3). Moreover, inhibition of Ttn -derived circular RNA increased the susceptibility of cardiomyocytes to doxorubicin. Conclusions: We here show that overexpression of Qki5 strongly attenuates the toxic effect of doxorubicin via regulating a set of circular RNAs. Qki5 is, thus, an interesting target molecule to combat doxorubicin-induced cardiotoxicity.

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Figure 1. Doxorubicin treatment reduced Qki (Quaking) expression. A, Heatmap showing differentially expressed mRNAs in mouse myocardium treated with vehicle control or doxorubicin (n=3 each). Qki protein (B) and mRNA (C) levels in the myocardium of mice that had received doxorubicin compared with vehicle control ([B] n=10 vehicle and 15 doxorubicin and [C] n=9 vehicle and 9 doxorubicin). D, Expression of QKI in human failing heart compared with nonfailing heart (n=10 nonfailing and 20 failing). Doxorubicin (0.1 µmol/L) treatment for 72 h in HL-1 (E), neonatal rat cardiomyocytes (F), and human induced pluripotent stem cell–derived cardiomyocytes (G) significantly decreases Qki protein levels. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001.
Figure 1. Doxorubicin treatment reduced Qki (Quaking) expression. A, Heatmap showing differentially expressed mRNAs in mouse myocardium treated with vehicle control or doxorubicin (n=3 each). Qki protein (B) and mRNA (C) levels in the myocardium of mice that had received doxorubicin compared with vehicle control ([B] n=10 vehicle and 15 doxorubicin and [C] n=9 vehicle and 9 doxorubicin). D, Expression of QKI in human failing heart compared with nonfailing heart (n=10 nonfailing and 20 failing). Doxorubicin (0.1 µmol/L) treatment for 72 h in HL-1 (E), neonatal rat cardiomyocytes (F), and human induced pluripotent stem cell–derived cardiomyocytes (G) significantly decreases Qki protein levels. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001.CC BY · PubMed Central
Figure 2. Quaking exerts protective effects on cardiomyocyte exposed to doxorubicin. Apoptosis measured by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling; green) staining (A and C) and cell size measurement (B and C) of positive primary cardiomyocytes (red—α-actinin sarcomeric) transfected with either siRNA control or siRNA Qki5, in presence or absence of doxorubicin. D, Caspase 3/7 activity in primary cardiomyocytes exposed to doxorubicin after inhibition of Qki or control. E, Western blot confirming lentiviral overexpression of Qki isoforms 5, 6, and 7 in HL-1 cells. F, Luminescence readings showing caspase 3/7 activity in different HL-1 pLV (plasmid Lentivirus) overexpression cell lines. G–H, Percentage of TUNEL (green)-positive nuclei in pLV empty and pLV Qki5 overexpression cells with or without doxorubicin treatment. I, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay shows survival in HL-1 cells with Qki5 overexpression compared with controls after doxorubicin treatment. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001; #P=0.12. Bar=100 µm. a.u. indicates arbitrary unit; and RLU, relative luminescence unit.
Figure 2. Quaking exerts protective effects on cardiomyocyte exposed to doxorubicin. Apoptosis measured by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling; green) staining (A and C) and cell size measurement (B and C) of positive primary cardiomyocytes (red—α-actinin sarcomeric) transfected with either siRNA control or siRNA Qki5, in presence or absence of doxorubicin. D, Caspase 3/7 activity in primary cardiomyocytes exposed to doxorubicin after inhibition of Qki or control. E, Western blot confirming lentiviral overexpression of Qki isoforms 5, 6, and 7 in HL-1 cells. F, Luminescence readings showing caspase 3/7 activity in different HL-1 pLV (plasmid Lentivirus) overexpression cell lines. G–H, Percentage of TUNEL (green)-positive nuclei in pLV empty and pLV Qki5 overexpression cells with or without doxorubicin treatment. I, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay shows survival in HL-1 cells with Qki5 overexpression compared with controls after doxorubicin treatment. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001; #P=0.12. Bar=100 µm. a.u. indicates arbitrary unit; and RLU, relative luminescence unit.CC BY · PubMed Central
Figure 3. Cardiac circular RNAs are regulated by Quaking. A, Filtering strategy for screening of circular RNAs. B, Significantly changed expression levels of Ttn (Titin)-, Fhod3 (Formin homology 2 domain containing 3)-, Strn3 (Striatin, calmodulin-binding protein 3)-, and Arhgap32-derived circular RNAs in pLV (plasmid Lentivirus) empty, pLV Qki5, CRi empty, CRi 3+4 Qki, and CRi 5+6 Qki HL-1 cell lines. C, Circular RNAs derived from Ttn, Fhod3, Strn3, and Arhgap32 expression in presence of doxorubicin. D, Expression of circular RNAs in murine myocardium exposed to doxorubicin (n=9 each). E, Circular RNAs and linear HPRT (hypoxanthine guanine phosphoribosyl transferase) polymerase chain reaction products from RNA treated with or without RNase R. F, Circular RNA expression levels in presence of doxorubicin are maintained with Qki5 overexpression in HL-1 cells. G, siRNA-mediated knockdown of Ttn 104 to 110 circular RNA. H, Caspase 3/7 activity in response to doxorubicin on inhibition of Ttn 104 to 110 in HL-1 cells compared with control siRNA. I, Caspase 3/7 activity in pLV Qki5 cells after inhibition of circular RNA Ttn 104 to 111. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001. RLU indicates relative luminescence unit.
Figure 3. Cardiac circular RNAs are regulated by Quaking. A, Filtering strategy for screening of circular RNAs. B, Significantly changed expression levels of Ttn (Titin)-, Fhod3 (Formin homology 2 domain containing 3)-, Strn3 (Striatin, calmodulin-binding protein 3)-, and Arhgap32-derived circular RNAs in pLV (plasmid Lentivirus) empty, pLV Qki5, CRi empty, CRi 3+4 Qki, and CRi 5+6 Qki HL-1 cell lines. C, Circular RNAs derived from Ttn, Fhod3, Strn3, and Arhgap32 expression in presence of doxorubicin. D, Expression of circular RNAs in murine myocardium exposed to doxorubicin (n=9 each). E, Circular RNAs and linear HPRT (hypoxanthine guanine phosphoribosyl transferase) polymerase chain reaction products from RNA treated with or without RNase R. F, Circular RNA expression levels in presence of doxorubicin are maintained with Qki5 overexpression in HL-1 cells. G, siRNA-mediated knockdown of Ttn 104 to 110 circular RNA. H, Caspase 3/7 activity in response to doxorubicin on inhibition of Ttn 104 to 110 in HL-1 cells compared with control siRNA. I, Caspase 3/7 activity in pLV Qki5 cells after inhibition of circular RNA Ttn 104 to 111. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001. RLU indicates relative luminescence unit.CC BY · PubMed Central
Figure 4. Qki5 overexpression reverses doxorubicin-induced cardiotoxicity. A, Schematic representation of the in vivo animal model and treatment scheme. B, Percentage body weight change during the experiment (n=5 each). Ejection fraction (C) and interventricular septum thickness (D) in AAV9 (adeno-associated virus serotype 9) control and AAV9 Qki5-treated mice with or without doxorubicin (n=4–5 each). Apoptosis measured by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining in AAV9 control and AAV9 Qki5 heart sections (E and F; n=5 each). Cardiomyocyte cell size measurement by Wheat germ agglutinin staining of heart (G and H; n=5 each). Bar=100 µm. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001. a.u. indicates arbitrary unit; BW, body weight; EF, ejection fraction; and IVS s, interventricular septum thickness systole.
Figure 4. Qki5 overexpression reverses doxorubicin-induced cardiotoxicity. A, Schematic representation of the in vivo animal model and treatment scheme. B, Percentage body weight change during the experiment (n=5 each). Ejection fraction (C) and interventricular septum thickness (D) in AAV9 (adeno-associated virus serotype 9) control and AAV9 Qki5-treated mice with or without doxorubicin (n=4–5 each). Apoptosis measured by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining in AAV9 control and AAV9 Qki5 heart sections (E and F; n=5 each). Cardiomyocyte cell size measurement by Wheat germ agglutinin staining of heart (G and H; n=5 each). Bar=100 µm. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001. a.u. indicates arbitrary unit; BW, body weight; EF, ejection fraction; and IVS s, interventricular septum thickness systole.CC BY · PubMed Central

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Authors
Gupta, S. K., Garg, A., Bär, C., Chatterjee, S., Foinquinos, A., Milting, H., Streckfuß-Bömeke, K., Fiedler, J., & Thum, T.
Affiliations
  • University Hospitals of the Ruhr-University of Bochum (DE)
  • From the Institute of Molecular and Translational Therapeutic Strategies (S.K.G., A.G., C.B., S.C., A.F., J.F., T.T.) and Excellence Cluster REBIRTH (T.T.), Hannover Medical School, Germany; Herz- und Diabeteszentrum NRW, Universitätsklinikum der Ruhr Universität Bochum, Erich und Hanna Klessmann-Institute for Cardiovascular Research and Development, Bad Oeynhausen, Germany (H.M.); Clinic for Cardiology and Pneumology, Stem Cell Laboratory, University Medical Center, Gottingen, Germany (K.S.-B.);...
Corresponding author
  • Gupta, Shashi Kumar
  • Thum, Thomas
ORCID
Venue
Circulation Research
Publisher
Ovid Technologies (Wolters Kluwer Health)
Published
2018-01-19
Volume
122
Issue
2
Pages
246-254
ISSN
  • 0009-7330 (ISSN-L)
  • 1524-4571
DOI
10.1161/circresaha.117.311335
PMC
PMC5771684
OpenAlex
W2770047542
Cited by
227
References
16
Citations by year
2026: 9 · 2025: 17 · 2024: 26 · 2023: 20 · 2022: 28 · 2021: 42 · 2020: 39 · 2019: 21 · 2018: 25
FWCI
17.96
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Circulation Research (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • GoeScholar The Publication Server of the Georg-August-Universität Göttingen (Georg-August-Universität Göttingen) (submittedVersion, cc-by)
  • GoeScholar The Publication Server of the Georg-August-Universität Göttingen (Georg-August-Universität Göttingen) (submittedVersion, other-oa)
Funders
  • European Commission
  • Deutsche Forschungsgemeinschaft (GU-1664/1-1, 01EO1302)
  • Fondation Leducq
  • Bundesministerium für Bildung und Forschung (01EO1302)
  • Medizinischen Hochschule Hannover
Topics
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Chemotherapy-induced cardiotoxicity and mitigation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Circular RNAs in diseases
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cardiac Fibrosis and Remodeling
Keywords
doxorubicin cardiotoxicity, Quaking (QKI), RNA-binding proteins, circular RNA, heart failure, cardiomyocyte apoptosis, cardiac atrophy, titin, striatin, AAV9 gene therapy, iPSC-derived cardiomyocytes, cardioprotection
MeSH terms
RNA, Circular, Animals, Antibiotics, Antineoplastic, Doxorubicin, Mice, Inbred C57BL, Random Allocation, RNA, Gene Expression, RNA-Binding Proteins, Myocytes, Cardiac, Mice, Cardiotoxicity

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