Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

Abstract

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and α-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

Stimulation of Cardiomyocyte Proliferation Is Dependent on Species and Level of Maturation

“…To determine if the DNA synthesis stimulatory effects of TT-10 and p21 inhibition would lead to cardiomyocyte proliferation and new cell formation, we used a series of experiments. First, we used a previously published imaging algorithm to measure DAPI intensity to determine the amount of DNA in each cardiomyocyte nucleus ( Yücel et al, 2020 ). As expected, we were able to separate diploid (2n) and polyploid (4n) cardiomyocyte nuclei successfully ( Supplementary Figure S3 ).…”

“…We then measured masked Troponin and EdU images to measure mean intensity of TnnT and EdU within these nuclei to categorize nuclei as cardiomyocyte or non-cardiomyocyte and proliferating or non-proliferating ones. For ploidy analysis, we measured DAPI intensity from background subtracted DAPI images in individual nuclei objects ( Yücel et al, 2020 ).…”

Accelerated Growth, Differentiation, and Ploidy with Reduced Proliferation of Right Ventricular Cardiomyocytes in Children with Congenital Heart Defect Tetralogy of Fallot

“…The nuclei of isolated cells were stained with DNA-specific fluorochrome DAPI (4′-6-diamidino-2phenylindole-HCl). The use of DAPI was based on its ability to bind to phosphate groups of DNA in a stoichiometric ratio, which makes it possible to calculate the relative DNA content [ 74 , 75 ]. Cell nuclei were examined using a Keyence BZ-9000 fluorescence microscope (Japan); the nuclei belonging to the CMCs were established by the cross striation of myofibrils.…”