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Updated: Dec 3, 2025
Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis
Published on: October 7, 2020
Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis
Doğacan Yücel1, Jacob Solinsky2, Jop H van Berlo3
1Department of Integrative Biology and Physiology, University of Minnesota; Lillehei Heart Institute, Department of Medicine, University of Minnesota.
Insights
This study introduces a new method for isolating adult mammalian heart cells. The protocol preserves cardiomyocyte morphology and maximizes yield, enabling accurate DNA content analysis and immunocytochemistry.
Area of Science:
- Cardiovascular Biology
- Cellular Biology
- Histology
Background:
- Identifying cardiomyocyte nuclei in histological sections is challenging.
- Current methods for isolating viable cardiomyocytes require extensive optimization and yield variable results.
- Preserving in vivo morphology is crucial for accurate analysis.
Purpose of the Study:
- To develop a reproducible protocol for isolating adult cardiomyocytes with maximum yield and preserved morphology.
- To establish an automated platform for analyzing cardiomyocyte nuclei and DNA content.
- To enable reliable immunocytochemistry and ploidy assessment of cardiomyocytes.
Main Methods:
- Heart fixation prior to enzymatic digestion (collagenase) and KCl-induced arrest.
- Cardiomyocyte enrichment via differential centrifugation after cell singularization.
- Immunostaining for Troponin T and α-actinin to confirm purity.
- DAPI staining and automated image analysis for nucleation and ploidy assessment.
Main Results:
- The fixation-first protocol yields maximum cardiomyocyte numbers while preserving native morphology.
- Automated analysis platform provides consistent and reproducible results for cardiomyocyte nucleation and ploidy.
- Purity of isolated cardiomyocytes was confirmed using specific markers.
Conclusions:
- The developed protocol offers a reproducible method for high-yield adult cardiomyocyte isolation with preserved morphology.
- The automated analysis platform facilitates accurate assessment of cardiomyocyte nucleation and DNA content.
- This approach enhances the reliability of immunocytochemistry and ploidy studies in cardiac research.
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.
