Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice.
Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.
Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus.
Emily J
EJ
Sharpe
Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus; Department of Bioengineering, University of Colorado Anschutz Medical Campus.
Joshua R
JR
St Clair
Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus; Department of Medicine, Division of Cardiology, University of Colorado Anschutz Medical Campus; Catherine.Proenza@ucdenver.edu.
Catherine
C
Proenza
D000200:Action Potentials; D000818:Animals; D002118:Calcium; D018929:Cell Culture Techniques; D006339:Heart Rate; D051379:Mice; D032342:Muscle Cells; D012849:Sinoatrial Node
D002118:Calcium