Isolating Ventricular Cardiomyocytes from a Mouse Model of Doxorubicin Cardiotoxicity

Summary

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

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

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 failure^1,2. Cardiomyocyte apoptosis is a key pathogenic process in this toxicity^3,4,5, 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

Protocol

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....

Results

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.......

Discussion

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, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 mm cell cluture dishes	Jet Bio-Filtration	TCD000100
100 μm cell strainer	Corning	431752
15 mL centrifuge tubes	Jet Bio-Filtration	CFT011150
50 mL centrifuge tubes	Jet Bio-Filtration	CFT011500
60 mm cell culture dishes	Jet Bio-Filtration	TCD100060
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A1470
Collagenase II	Sigma-Aldrich	C6885
Collagenase IV	Sigma-Aldrich	C5138
Curved hemostatic forceps	RWD Life Science	F22003-10
Curved-end scissors	RWD Life Science	S12001-09
Fine scissors	RWD Life Science	S12000-09
Forceps	RWD Life Science	F13014-12
Indwelling cannula (26G)	Becton, Dickinson and Company	381312
Insulin-Transferrin-Selenium (ITS)	MedChemExpress	HY-150287
Isoflurane	RWD Life Science	R510-22-10
Laminin (murine)	Thermo Scientific	23017015
Lipid Concentrate (Chemically defined)	MedChemExpress	HY-K3019
M199 Medium	Sigma-Aldrich	M4530
Penicillin-Streptomycin	MedChemExpress	HY-K1006
Protease XIV	Sigma-Aldrich	P5147
Sterilization Indicator Tape	Minnesota Mining and Manufacturing (3M)	CT060934701
Syringe Pump (ISPLab01)	Baoding Shenchen Precision Pump	SC-49464A