title: Culture and adenoviral infection of sinoatrial node myocytes from adult mice.
abstract: Pacemaker myocytes in the sinoatrial node of the heart initiate each heartbeat by firing spontaneous action potentials. However, the molecular processes that underlie pacemaking are incompletely understood, in part because of our limited ability to manipulate protein expression within the native cellular context of sinoatrial node myocytes (SAMs). Here we describe a new method for the culture of fully differentiated SAMs from adult mice, and we demonstrate that robust expression of introduced proteins can be achieved within 24-48 h in vitro via adenoviral gene transfer. Comparison of morphological and electrophysiological characteristics of 48 h-cultured versus acutely isolated SAMs revealed only minor changes in vitro. Specifically, we found that cells tended to flatten in culture but retained an overall normal morphology, with no significant changes in cellular dimensions or membrane capacitance. Cultured cells beat spontaneously and, in patch-clamp recordings, the spontaneous action potential firing rate did not differ between cultured and acutely isolated cells, despite modest changes in a subset of action potential waveform parameters. The biophysical properties of two membrane currents that are critical for pacemaker activity in SAMs, the "funny current" (If) and voltage-gated Ca(2+) currents (ICa), were also indistinguishable between cultured and acutely isolated cells. This new method for culture and adenoviral infection of fully-differentiated SAMs from the adult mouse heart expands the range of experimental techniques that can be applied to study the molecular physiology of cardiac pacemaking because it will enable studies in which protein expression levels can be modified or genetically encoded reporter molecules expressed within SAMs.
authors.affiliation: Department of Physiology and Biophysics, University of Colorado - Anschutz Medical Campus, Denver, Colorado; and.
authors.forename: Joshua R
authors.initials: JR
authors.lastname: St Clair
authors.affiliation: Department of Physiology and Biophysics, University of Colorado - Anschutz Medical Campus, Denver, Colorado; and.
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Sharpe
authors.affiliation: Department of Physiology and Biophysics, University of Colorado - Anschutz Medical Campus, Denver, Colorado; and Department of Medicine, Division of Cardiology - Anschutz Medical Campus, Denver, Colorado catherine.proenza@ucdenver.edu.
authors.forename: Catherine
authors.initials: C
authors.lastname: Proenza
mesh_terms: D000200:Action Potentials; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D061251:Primary Cell Culture / Q000379:methods*; D012849:Sinoatrial Node / Q000166:cytology*; D019076:Transgenes
keywords: cell culture; pacemaker cell; sinoatrial node