Method Article

Isolating Ventricular Cardiomyocytes from a Mouse Model of Doxorubicin Cardiotoxicity

DOI:

10.3791/70411

February 27th, 2026

 ,  ,  , 

Corresponding Authors: Lei Xiao <xiaolei0122@xjtu.edu.cn>, Nanping Wang <npwang@hsc.ecnu.edu.cn>

In This Article

Summary

Loading...
$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

This protocol describes a reproducible, Langendorff-free method for isolating viable adult mouse ventricular cardiomyocytes from a doxorubicin-induced cardiotoxicity model. It employs direct left-ventricular antegrade perfusion followed by controlled enzymatic digestion to yield calcium-tolerant, rod-shaped myocytes suitable for structural, molecular, and functional analyses.

Abstract

Loading...
$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Doxorubicin (DOX) is an effective chemotherapeutic drug whose clinical application is limited by progressive, dose-dependent cardiotoxicity that predominantly affects the ventricular myocardium. Studying cellular injury mechanisms in this context requires reliable isolation of adult cardiomyocytes, a process that is technically challenging in diseased or structurally fragile hearts. Here, we describe a simplified and reproducible Langendorff-free protocol for isolating functional ventricular cardiomyocytes from mice with DOX-induced cardiotoxicity. After establishing the DOX model in C57BL/6 mice, the heart is rapidly excised, and the aorta is clamped ex vivo. Non-recirculating antegrade perfusion is performed directly through the left ventricle using calcium-free and enzyme-containing buffers. Gentle mechanical dissociation of digested ventricular tissue yields individual cardiomyocytes, followed by controlled calcium reintroduction to restore calcium tolerance. This method provides high-quality, viable, rod-shaped cardiomyocytes that are compatible with downstream assays, including immunofluorescence, sarcomere organization analysis, apoptosis detection, and molecular signaling studies. By eliminating the need for specialized Langendorff perfusion equipment, this protocol offers an accessible, high-yield solution for laboratories investigating mechanisms of DOX-induced cardiac injury.

Introduction

Loading...
$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Doxorubicin (DOX) is a potent anthracycline chemotherapeutic agent, yet its clinical utility is restricted by cumulative cardiotoxicity that can progress to dilated cardiomyopathy and heart failure1,2. Cardiomyocyte apoptosis is a key pathogenic process in this toxicity3,4,5, and studying these cellular events requires reliable access to functional adult cardiomyocytes derived from DOX-injured hearts. However, DOX exposure renders cardiomyocytes structurally fragile and metabolically compromised6

Access restricted. Please log in or start a trial to view this content.

Protocol

Loading...
$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

All animal procedures were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals and were reviewed and approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University (Approval No. XJTUAE2022-1441). The consumables, reagents, and equipment used are listed in the Table of Materials.

1. Establishing the DOX-induced acute cardiotoxicity model in C57BL/6 mice

  1. Animal preparation: Use 8-12-week-old male C57BL/6 mice (24-28 g). House animals under standard conditions with a 12 h light/dark cycle and ad libitum....

Access restricted. Please log in or start a trial to view this content.

Results

Loading...
$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Assessment of cell yield and viability

Mice receiving DOX exhibited a marked decline in body weight relative to the saline-treated group (Supplementary Figure 1A). The successful establishment of the acute DOX-induced cardiotoxicity model was further confirmed by marked increases in serum cardiac injury markers, including cTnT and CK (Supplementary Figure 1B,C), as well as a pronounced elevation in cardiomyocyte apoptosis, as shown by terminal.......

Access restricted. Please log in or start a trial to view this content.

Discussion

Loading...
$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Successful isolation of high-quality, calcium-tolerant adult cardiomyocytes is essential for cellular-level studies of cardiac pathophysiology. This protocol reliably yields functional cardiomyocytes from both healthy and pathological mouse hearts, with two steps being particularly crucial for preserving cell viability. First, carefully optimize and monitor the duration of enzymatic perfusion. Insufficient digestion produces poor tissue dissociation and low yield, whereas over-digestion compromises structural integrity, .......

Access restricted. Please log in or start a trial to view this content.

Disclosures

Loading...
$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Acknowledgements

Loading...
$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

This work was supported by the Natural Science Foundation of China (grant 82230012 and 81830015 to N. Wang, grant 82570478 to L. Xiao).

....

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
100 mm cell cluture dishesJet Bio-Filtration TCD000100
100 μm cell strainerCorning431752
15 mL centrifuge tubesJet Bio-Filtration CFT011150
50 mL centrifuge tubesJet Bio-Filtration CFT011500
60 mm cell culture dishesJet Bio-Filtration TCD100060
Bovine Serum Albumin (BSA)Sigma-AldrichA1470 
Collagenase IISigma-AldrichC6885
Collagenase IVSigma-AldrichC5138
Curved hemostatic forcepsRWD Life ScienceF22003-10
Curved-end scissorsRWD Life ScienceS12001-09
Fine scissorsRWD Life ScienceS12000-09
ForcepsRWD Life ScienceF13014-12
Indwelling cannula (26G)Becton, Dickinson and Company381312
Insulin-Transferrin-Selenium (ITS)MedChemExpressHY-150287
Isoflurane  RWD Life ScienceR510-22-10
Laminin (murine)Thermo Scientific23017015
Lipid Concentrate (Chemically defined)MedChemExpressHY-K3019
M199 MediumSigma-AldrichM4530
Penicillin-StreptomycinMedChemExpress HY-K1006
Protease XIVSigma-AldrichP5147
Sterilization Indicator TapeMinnesota Mining and Manufacturing (3M)CT060934701
Syringe Pump (ISPLab01)Baoding Shenchen Precision PumpSC-49464A

References

Loading...
$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Singal, P. K., Iliskovic, N. Doxorubicin-induced cardiomyopathy. N Engl J Med. 339 (13), 900-905 (1998).
  2. Zamorano, J. L., et al.

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Cardiomyocyte IsolationMouse Heart ModelCalcium Free BufferMechanical DissociationCardiac Injury MechanismsApoptosis DetectionSarcomere OrganizationImmunofluorescence Assay

Related Articles