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Production of Spontaneously Beating Neonatal Rat Heart Tissue for Calcium and Contractile Studies

  • Fnu Gerilechaogetu
  • Hao Feng
  • Honey B. Golden
  • Damir Nizamutdinov
  • Donald M. Foster
  • Shannon Glaser
  • David E. Dostal
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1066)

Abstract

Neonatal rat ventricular myocytes (NRVM) and fibroblasts (FB) serve as in vitro models for studying fundamental mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Typically, these cell types are separated using Percoll density gradient procedures. Cells located between the Percoll bands (interband cells [IBCs]), which contain less mature NRVM and a variety of non-myocytes, including coronary vascular smooth muscle cells and endothelial cells (ECs), are routinely discarded. However, we have demonstrated that IBCs readily attach to extracellular matrix-coated coverslips, plastic culture dishes, and deformable membranes to form a 2-dimensional cardiac tissue layer which quickly develops spontaneous contraction within 24 h, providing a robust coculture model for the study of cell-to-cell signaling and contractile studies. Below, we describe methods that provide good cell yield and viability of IBCs during isolation of NRVM and FB obtained from 0- to 3-day-old neonatal rat pups. Basic characterization of IBCs and methods for use in intracellular calcium and contractile experiments are also presented. This method maximizes the use of cells obtained from neonatal rat hearts.

Key words

Cardiac myocytes Fibroblasts Neonatal Density separation Interband cells 

Notes

Acknowledgments

This manuscript is the result of work supported with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas. Funding was provided the National Institutes of Health (5R01-HL068838-06), VA Merit Award (IO1 BX000801-01), VA hospital, and Scott and White Hospital.

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Fnu Gerilechaogetu
    • 1
  • Hao Feng
    • 1
  • Honey B. Golden
    • 1
  • Damir Nizamutdinov
    • 1
  • Donald M. Foster
    • 2
  • Shannon Glaser
    • 2
    • 3
  • David E. Dostal
    • 4
  1. 1.Texas A&M Health Science Center, College of Medicine, Cardiovascular Research InstituteTempleUSA
  2. 2.Central Texas Veterans Health Care SystemTempleUSA
  3. 3.Digestive Disease Research Center, Texas A&M Health Science Center, College of MedicineTempleUSA
  4. 4.Division of Molecular Cardiology, Department of MedicineTexas A&M Health Science Center, Cardiovascular Research InstituteTempleUSA

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