Functional culture and in vitro genetic and small-molecule manipulation of adult mouse cardiomyocytes

Neal I Callaghan,Shin-Haw Lee,Xavier A Lee,M Ahsan Siraj,Mansoor Husain,Craig A Simmons,Anthony O Gramolini,Sina Hadipour-Lakmehsari,Amine Driouchi,Christopher M Yip

doi:10.1038/s42003-020-0946-9

Abstract

Primary adult cardiomyocyte (aCM) represent the mature form of myocytes found in the adult heart. However, culture of aCMs in particular is challenged by poor survival and loss of phenotype, rendering extended in vitro experiments unfeasible. Here, we establish murine aCM culture methods that enhance survival and maintain sarcomeric structure and Ca2+ cycling to enable physiologically relevant contractile force measurements. We also demonstrate genetic and small-molecule manipulations that probe mechanisms underlying myocyte functional performance. Together, these refinements to aCM culture present a toolbox with which to advance our understanding of myocardial physiology.

Highlights

Primary adult cardiomyocyte represent the mature form of myocytes found in the adult heart
We hypothesized that altering the culture conditions would profoundly extend the utility of isolated myocytes, and we focused on enhancing the matrix with Geltrex together with inhibiting cellular contraction using blebbistatin
CMs isolated from adult mice and cultured using our modified protocol combining Geltrex and blebbistatin demonstrated high adhesion, survivability, and continued functionality in sustained culture (Fig. 1). adult cardiomyocyte (aCM) retained α-actinin-positive sarcomeres and an ordered sarcoplasmic reticulum (SR; visualized by confocal imaging for SERCA2, RyR2, DHPR αsubunit, and STIM1) with both longitudinal and cisternae components observed up to 7 days post isolation (Fig. 1a)

Summary

Introduction

Primary adult cardiomyocyte (aCM) represent the mature form of myocytes found in the adult heart. Neonatal and pluripotent stem cell-derived cardiomyocytes have high survival rates in culture, they do not fully replicate the adult phenotype in terms of morphology, mature protein isoform expression, action potential component currents, Ca2+ transient dynamics, contractile force production, or metabolic substrate preference[1,2]. These immature cells fall short of replicating the physiology of mature CMs required for detailed mechanistic study[1]. We demonstrate the combinatorial benefit of both Geltrex and blebbistatin in aCM culture

Methods

Results

Conclusion