Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Abstract

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology. Video LinkThe video component of this article can be found at http://www.jove.com/video/54012/ IntroductionCultured cardiomyocytes are frequently used to monitor cell behavior in a well-controlled environment in vitro. For example, morphological, electrical, biochemical, or mechanical cell properties can be studied on engineered substrates, 1,2 in defined media, and in response to small molecule drugs, peptides, gene regulation, 3 or electrical stimulation. 4 The cellular content can also be controlled using defined co-cultures. 5These in vitro experiments are useful in large drug or genetic screens and complement in vivo methods for various types of investigations involving cardiomyocyte biology.Long-term culture enables experimental avenues that require extended periods of time to achieve phenotypic change. A timely example is that of adult mammalian cardiomyocyte proliferation, where dedifferentiation, cell cycle re-entry, and cell division is typically studied over several days to weeks. 6,7 Here, the extended culture time facilitates genetic manipulation, 7,8 functional dedifferentiation (e.g., sarcomeric disassembly) 9 and potentially transcriptional dedifferentiation.6 Subsequent cell cycle re-entry and cell division requires even longer culture periods to observe, especially if multiple rounds of division are the experimental goal. The importance of the cardiomyocyte cell-cycle is central to several recent key scientific works in heart regeneration, where the dedifferentiation and proliferation of pre-existing cardiomyocytes has been shown responsible for heart regeneration in ...

Myocardial Rev-erb–Mediated Diurnal Metabolic Rhythm and Obesity Paradox

“…The isolation and culture of adult mouse cardiomyocytes follow the established procedures. 23,24 The pcDNA3.1-E4bp4 plasmid (Addgene 34572) 25 was cotransfected with a green fluorescent protein plasmid. Radioactive isotope tracer studies were as described previously.…”

Clonal Hematopoiesis of Indeterminate Potential With Loss of Tet2 Enhances Risk for Atrial Fibrillation Through Nlrp3 Inflammasome Activation

“…Atrial cardiomyocytes isolated from lethally irradiated Ldlr −/− mice with hematopoietic-specific inactivation of Tet2 or WT controls were incubated with a fluorescent calcium sensitive indicator, Fluo-3 AM, to visualize and quantitatively characterize calcium transients (Figure 5 D), as previously described. 48 , 49 Atrial cardiomyocytes from mice with hematopoietic-specific inactivation of Tet2 had dysfunctional intracellular calcium handling resulting in a trend ( P =0.055) toward a prolonged transient time to peak, and an increased frequency of spontaneous calcium release events after removal of depolarizing stimuli (Figure 5 E through 5 G). Release of calcium from the SR is predominantly mediated by RyR2; thus, SR calcium stores can provide an approximation of RyR2 activity.…”

Mesoscopic optical imaging of whole mouse heart

“…In particular, the cannulation of the heart through the proximal aorta can be quite difficult, but it is an essential step to wash and fix the organ properly. Judd et al 17 , showed how to perform this step effectively. Moreover, the degassing procedure needed by the CLARITY protocol is quite complex too, but it is essential for tissue preservation; if this step is not performed properly, the tissue could encounter damages and decay during the incubation in clearing solution.…”