Culture and adenoviral infection of sinoatrial node myocytes from adult mice

2016 Computing in Cardiology Conference (CinC)

et al. 2016

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Section: Methodsmentioning

confidence: 99%

“…AP waveform parameters (defined as illustrated in Fig. 1B) were determined for each cell from average waveforms from 5 s recording windows [6,7].…”

Section: Methodsmentioning

confidence: 99%

Dynamic Regulation of Pacemaker Activity by the Na+:K+ Pump

Morotti

Clair

2016 Computing in Cardiology Conference (CinC)

et al. 2016

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“…NOTE: The contractile inhibitor 2,3-butanedione monoxime (BDM) is included in the plating and culture media to prevent contraction, which causes attrition of cells 9 .…”

Section: Plating and Culture Of Sinoatrial Myocytes (Skip For Acutmentioning

confidence: 99%

“…NOTE: The following protocol is an example of functional assessments of isolated SAMs using the amphotericin perforated-patch technique to record both spontaneous APs and I f from the same cell (see reference 9 ).…”

Section: Functional Evaluation Of Acutely Isolated or Cultured Samsmentioning

confidence: 99%

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Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sharpe

Clair

2016

JoVE

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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.

ParamAP: Standardized Parameterization of Sinoatrial Node Myocyte Action Potentials

Rickert

2017

Biophysical Journal

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Sinoatrial node myocytes act as cardiac pacemaker cells by generating spontaneous action potentials (APs). Much information is encoded in sinoatrial AP waveforms, but both the analysis and the comparison of AP parameters between studies is hindered by the lack of standardized parameter definitions and the absence of automated analysis tools. Here we introduce ParamAP, a standalone cross-platform computational tool that uses a template-free detection algorithm to automatically identify and parameterize APs from text input files. ParamAP employs a graphic user interface with automatic and user-customizable input modes, and it outputs data files in text and PDF formats. ParamAP returns a total of 16 AP waveform parameters including time intervals such as the AP duration, membrane potentials such as the maximum diastolic potential, and rates of change of the membrane potential such as the diastolic depolarization rate. ParamAP provides a robust AP detection algorithm in combination with a standardized AP parameter analysis over a wide range of AP waveforms and firing rates, owing in part to the use of an iterative algorithm for the determination of the threshold potential and the diastolic depolarization rate that is independent of the maximum upstroke velocity, a parameter that can vary significantly among sinoatrial APs. Because ParamAP is implemented in Python 3, it is also highly customizable and extensible. In conclusion, ParamAP is a powerful computational tool that facilitates quantitative analysis and enables comparison of sinoatrial APs by standardizing parameter definitions and providing an automated work flow.