Long-term functional culture and in vitro manipulation of primary adult cardiomyocytes

Callaghan, Neal (1, 2), Hadipour-Lakmehsari, Sina (2, 3), Lee, Shin-Haw (2, 3), Simmons, Craig (1, 2, 4), Gramolini, Anthony (2, 3, 5)

(1) Institute of Biomaterials and Biomedical Engineering, Faculty of Applied Science and Engineering, University of Toronto

(2) Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research

(3) Department of Physiology, Faculty of Medicine, University of Toronto

(4) Department of Mechanical and Industrial Engineering

(5) Toronto General Research Institute, University Health Network

The benefit of cell culture is its ability to maintain the cell environment to a fine degree, such that factors of interest can be manipulated independently using drugs, genetic tools, or other stimuli. In the field of cardiac physiology, cell models are vital to provide mechanistic descriptions of drug cardiotoxicity and genetic mutation leading to reduced or changed function of key proteins. However, adult primary cardiomyocyte (CM) culture and in vitro experimentation is hampered by rapid mortality and dedifferentiation. As the cell dies or loses its contractility, morphology, and electrophysiological character, it no longer responds physiologically to experimental stimuli. Here we introduce murine adult CM culture conditions that enhance both survival and the maintenance of sarcomeric structure and calcium cycling function in vitro. Furthermore, we introduce a traction force microscopy workflow for adult CMs that allows for physiologically-relevant contractile force measurement in real-time, sensitive to its chemical and physical environment. Most importantly, through the enhanced timescales and experimental flexibility afforded by these methods, we demonstrate the ability to quantitate adult CM responses to in vitro treatments. Proof-of-concept of pharmaceutical (antibiotic) and genetic (shRNA and cDNA overexpression) manipulations upstream of signature physiological parameters are assessed to demonstrate partial loss-of-function. These techniques provide a basis for a new level of experimentation on physiologically-relevant cells, and suggest the adult CM as a viable model for mechanistic physiology in vitro.