Isolation and culture of adult murine cardiac atrial and ventricular fibroblasts and myofibroblasts

• Published in: Methods (San Diego, Calif.) Calif.), 2022-07, Vol.203, p.187-195
• Main Authors: Sahadevan, Pramod, Allen, Bruce G.
• Format: Article
• Language: English
• Subjects: Atria; Fibroblast activation; Fibroblast isolation; Heart; Ventricle
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2021.04.004

•Fibroblasts can be isolated from mouse or rat cardiac ventricles or atria.•Yield sufficient to study the effects of pharmacological or molecular interventions.•High elastic modulus (107 kPa) polystyrene substrates activate fibroblasts in culture.•Low elastic modulus (e.g., 8 kPa) ‘compliant’ substrates are commercially available.•Selecting substrate compliance allows for culture of fibroblasts or myofibroblasts. Cardiac fibroblasts play a critical role in extracellular matrix homeostasis, wound healing, and cardiac interstitial fibrosis: the latter being a pathophysiological response to a chronic increase in afterload. Using a standard protocol to isolate cardiac fibroblasts and maintain them in their quiescent phenotype in vitro will enable a better understanding of cardiac fibroblast biology and their role in the response to profibrotic stimuli. Here, we describe an enzymatic method for isolating cardiac fibroblasts. The resulting cells are maintained on either a collagen-coated hydrogel-bound polystyrene (compliant) substrate or standard polystyrene culture dishes (non-compliant) to obtain quiescent fibroblasts and activated fibroblasts (myofibroblasts), respectively. Fibroblasts maintained on a non-compliant substrate developed a myofibroblast phenotype, in which the αSMA immunoreactivity was markedly elevated and incorporated into the stress fibers. In contrast, ventricular and atrial fibroblasts retain their quiescent phenotype for up to 3 passages when maintained on a compliant substrate. Hence, the methodology described herein provides a simple and reproducible way to isolate adult murine atrial and ventricular cardiac fibroblasts from a single animal and, by selecting a substrate with the appropriate compliance, examine the mediators of fibroblast activation or inactivation.