Adenovirally delivered shRNA strongly inhibits Na+-Ca 2+ exchanger expression but does not prevent contraction of neonatal cardiomyocytes

Cecilia Hurtado, Bradley P. Ander, Thane G. Maddaford, Anton Lukas, Larry V. Hryshko, Grant N. Pierce

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

The cardiac Na+-Ca2+ exchanger (NCX1) is the main mechanism for Ca2+ efflux in the heart and is thought to serve an essential role in cardiac excitation-contraction (E-C) coupling. The demonstration that an NCX1 gene knock-out is embryonic lethal provides further support for this essential role. However, a recent report employing the Cre/loxP technique for cardiac specific knock-out of NCX1 has revealed that cardiac function is remarkably preserved in these mice, which survived to adulthood. This controversy highlights the necessity for further investigation of NCX1 function in the heart. In this study, we report on a novel approach for depletion of NCX1 in postnatal rat myocytes that utilizes RNA interference (RNAi), administered with high efficiency via adenoviral transfection. Depletion of NCX1 was confirmed by immunocytochemical detection, Western blots and radioisotopic assays of Na+-Ca2+ exchange activity. Exchanger expression was inhibited by up to ∼94%. Surprisingly, spontaneous beating of these cardiomyocytes was still maintained, although at a lower frequency. Electrical stimulation could elicit a normal beating rhythm, although NCX depleted cells exhibited a depressed Ca2+ transient amplitude, a depressed rate of Ca2+ rise and decline, elevated diastolic [Ca 2+], and shorter action potentials. We also observed a compensatory increase in sarcolemmal Ca2+ pump expression. Our data support an important, though non-essential, role for the NCX1 in E-C coupling in these neonatal heart cells. Furthermore, this approach provides a valuable means for assessing the role of NCX1 and could be utilized to examine other cardiac proteins in physiological and pathological studies.

Original languageEnglish (US)
Pages (from-to)647-654
Number of pages8
JournalJournal of Molecular and Cellular Cardiology
Volume38
Issue number4
DOIs
StatePublished - Apr 2005
Externally publishedYes

Keywords

  • Action potential
  • Adenovirus
  • Ca transport
  • Excitation-contraction coupling
  • Heart function
  • NCX1
  • RNA interference (RNAi)
  • Short hairpin RNA (shRNA)

ASJC Scopus subject areas

  • Molecular Biology
  • Cardiology and Cardiovascular Medicine

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