Gene Silencing in Adult Rat Cardiac Myocytes in Vitro by Adenovirus-mediated RNA Interference

Overview

Journal J Muscle Res Cell Motil

Specialties Cell Biology
Physiology

Date 2006 Jul 29

PMID 16874447

Citations 10

Authors

Andreas Rinne

Christoph Littwitz

Marie-Cecile Kienitz

Andreas Gmerek

Leif I Bosche

Lutz Pott

Kirsten Bender

Affiliations

Soon will be listed here.

Abstract

RNA interference (RNAi) by short double stranded RNA (siRNA) represents an efficient and frequently used tool for gene silencing to study gene function. Whereas efficient ablation of genes has been demonstrated in neonatal cardiac myocytes, thus far information on successful application of this technique in adult cardiac myocytes (ACM), a standard experimental model in cardiac physiology and pathophysiology, is sparse. Here we demonstrate efficient ablation of a transgene encoding for enhanced green fluorescent protein (EGFP) and a cell specific endogenous gene encoding for an inward-rectifier channel subunit (Kir2.1) in ACM in vitro using adenovirus driven transcription of siRNA hairpins. EGFP fluorescence and density of background inward rectifier current (IK1) were reduced by > 90% within about 6-8 days after transformation with the corresponding virus. In Kir2.1-silenced myocytes resting membrane potential was significantly reduced. Survival of these cells in culture was compromised, presumably due to Ca2+ -overload caused by the depolarization. The sequence-specific knockdowns of EGFP and Kir2.1 were confirmed on the RNA level using real-time RT-PCR. In Kir2.1-silenced myocytes density of transient outward current, carried predominantly by Kv4.x subunits remained unaffected. This communication for the first time demonstrates proof of principle of efficient RNA interference using adenovirus-based vectors and demonstrates its large potential in phenotyping of ACM.

Citing Articles

Inward Rectifier K Currents Contribute to the Proarrhythmic Electrical Phenotype of Atria Overexpressing Cyclic Adenosine Monophosphate Response Element Modulator Isoform CREM-IbΔC-X.

Pluteanu F, Seidl M, Hamer S, Scholz B, Muller F J Am Heart Assoc. 2020; 9(23):e016144.

PMID: 33191843 PMC: 7763782. DOI: 10.1161/JAHA.119.016144.

Knockdown of cardiac Kir3.1 gene with siRNA can improve bradycardia in an experimental sinus bradycardia rat model.

Li Y, Fu X, Zhang Z, Yu B Mol Cell Biochem. 2017; 429(1-2):103-111.

PMID: 28205094 DOI: 10.1007/s11010-017-2939-7.

Type 10 soluble adenylyl cyclase is overexpressed in prostate carcinoma and controls proliferation of prostate cancer cells.

Flacke J, Flacke H, Appukuttan A, Palisaar R, Noldus J, Robinson B J Biol Chem. 2012; 288(5):3126-35.

PMID: 23255611 PMC: 3561535. DOI: 10.1074/jbc.M112.403279.

A novel dual-fluorescence strategy for functionally validating microRNA targets in 3' untranslated regions: regulation of the inward rectifier potassium channel K(ir)2.1 by miR-212.

Goldoni D, Yarham J, McGahon M, OConnor A, Guduric-Fuchs J, Edgar K Biochem J. 2012; 448(1):103-13.

PMID: 22880819 PMC: 3475433. DOI: 10.1042/BJ20120578.

Autocrine signaling via A(1) adenosine receptors causes downregulation of M(2) receptors in adult rat atrial myocytes in vitro.

Littwitz C, Timpert M, Bender K, Pott L, Kienitz M Pflugers Arch. 2010; 461(1):165-76.

PMID: 21061016 DOI: 10.1007/s00424-010-0897-y.

References

Dillin A . The specifics of small interfering RNA specificity. Proc Natl Acad Sci U S A. 2003; 100(11):6289-91. PMC: 164437. DOI: 10.1073/pnas.1232238100. View

van de Wetering M, Oving I, Muncan V, Pon Fong M, Brantjes H, van Leenen D . Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 2003; 4(6):609-15. PMC: 1319205. DOI: 10.1038/sj.embor.embor865. View

Elbashir S, Harborth J, Weber K, Tuschl T . Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods. 2002; 26(2):199-213. DOI: 10.1016/S1046-2023(02)00023-3. View

Bender K, Meyer T, Bunemann M, Pott L . Overexpressed A(1) adenosine receptors reduce activation of acetylcholine-sensitive K(+) current by native muscarinic M(2) receptors in rat atrial myocytes. Circ Res. 2000; 86(6):643-8. DOI: 10.1161/01.res.86.6.643. View

Huesken D, Lange J, Mickanin C, Weiler J, Asselbergs F, Warner J . Design of a genome-wide siRNA library using an artificial neural network. Nat Biotechnol. 2005; 23(8):995-1001. DOI: 10.1038/nbt1118. View

Seth M, Sumbilla C, Mullen S, Lewis D, Klein M, Hussain A . Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes. Proc Natl Acad Sci U S A. 2004; 101(47):16683-8. PMC: 528906. DOI: 10.1073/pnas.0407537101. View

Faivre J, Calmels T, Rouanet S, Javre J, Cheval B, Bril A . Characterisation of Kv4.3 in HEK293 cells: comparison with the rat ventricular transient outward potassium current. Cardiovasc Res. 1999; 41(1):188-99. DOI: 10.1016/s0008-6363(98)00215-6. View

Yu J, DeRuiter S, Turner D . RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A. 2002; 99(9):6047-52. PMC: 122899. DOI: 10.1073/pnas.092143499. View

Lipp P, Huser J, Pott L, Niggli E . Spatially non-uniform Ca2+ signals induced by the reduction of transverse tubules in citrate-loaded guinea-pig ventricular myocytes in culture. J Physiol. 1996; 497 ( Pt 3):589-97. PMC: 1160957. DOI: 10.1113/jphysiol.1996.sp021792. View

10.

Montgomery M, Xu S, Fire A . RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1998; 95(26):15502-7. PMC: 28072. DOI: 10.1073/pnas.95.26.15502. View

11.

Dorsett Y, Tuschl T . siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov. 2004; 3(4):318-29. DOI: 10.1038/nrd1345. View

12.

Miyagishi M, Taira K . U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol. 2002; 20(5):497-500. DOI: 10.1038/nbt0502-497. View

13.

Ennis I, Li R, Murphy A, Marban E, Nuss H . Dual gene therapy with SERCA1 and Kir2.1 abbreviates excitation without suppressing contractility. J Clin Invest. 2002; 109(3):393-400. PMC: 150851. DOI: 10.1172/JCI13359. View

14.

Watanabe A, Arai M, Yamazaki M, Koitabashi N, Wuytack F, Kurabayashi M . Phospholamban ablation by RNA interference increases Ca2+ uptake into rat cardiac myocyte sarcoplasmic reticulum. J Mol Cell Cardiol. 2004; 37(3):691-8. DOI: 10.1016/j.yjmcc.2004.06.009. View

15.

Milhavet O, Gary D, Mattson M . RNA interference in biology and medicine. Pharmacol Rev. 2003; 55(4):629-48. DOI: 10.1124/pr.55.4.1. View

16.

Yin C, Xi L, Wang X, Eapen M, Kukreja R . Silencing heat shock factor 1 by small interfering RNA abrogates heat shock-induced cardioprotection against ischemia-reperfusion injury in mice. J Mol Cell Cardiol. 2005; 39(4):681-9. DOI: 10.1016/j.yjmcc.2005.06.005. View

17.

Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T . Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001; 411(6836):494-8. DOI: 10.1038/35078107. View

18.

Chen X, Zhang X, Kubo H, Harris D, Mills G, Moyer J . Ca2+ influx-induced sarcoplasmic reticulum Ca2+ overload causes mitochondrial-dependent apoptosis in ventricular myocytes. Circ Res. 2005; 97(10):1009-17. DOI: 10.1161/01.RES.0000189270.72915.D1. View

19.

Reynolds A, Leake D, Boese Q, Scaringe S, Marshall W, Khvorova A . Rational siRNA design for RNA interference. Nat Biotechnol. 2004; 22(3):326-30. DOI: 10.1038/nbt936. View

20.

Dykxhoorn D, Novina C, Sharp P . Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol. 2003; 4(6):457-67. DOI: 10.1038/nrm1129. View