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, greatly increasing their susceptibility to enzymatic and mechanical stress during isolation and resulting in low viability. Within the broader literature, Langendorff perfusion remains the predominant method for adult cardiomyocyte isolation⁷. However, it has notable limitations when applied to damaged myocardium. The method demands specialized hardware, precise aortic cannulation, and considerable technical proficiency; in DOX-treated hearts, the additional mechanical strain introduced during Langendorff perfusion can further exacerbate cell injury, yielding inconsistent and often suboptimal results⁸.

To overcome these challenges, we established a simplified and accessible protocol for isolating calcium-tolerant adult ventricular cardiomyocytes from a mouse model of acute DOX-induced cardiotoxicity⁹. By eliminating the need for conventional Langendorff perfusion and instead implementing a controlled ex vivo antegrade perfusion directly through the left ventricle, this method minimizes ischemic exposure and reduces mechanical manipulation of the myocardium. These features are expected to improve procedural consistency and enhance the survival of vulnerable cardiomyocytes derived from compromised hearts^7,10.

The current approach builds on emerging advances in Langendorff-free cardiomyocyte isolation, extending these concepts to adult ventricular cells, which require preservation of mature contractile architecture and calcium-handling properties, features that are not adequately supported by simplified digestion methods developed primarily for neonatal hearts^11,12. This protocol, therefore, offers a practical and reproducible alternative for laboratories lacking specialized perfusion systems. The isolated cardiomyocytes retain their structural integrity and are well-suited for diverse downstream applications, including sarcomere analysis, immunofluorescence imaging, and apoptosis signaling studies, enabling direct mechanistic investigation of DOX-induced cardiac injury in a physiologically relevant context. Collectively, this protocol addresses a critical unmet need for isolating viable cardiomyocytes from DOX-injured hearts.

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 access to food and water. Randomly assign mice to DOX-treated or saline control groups (n = 3 per group).
2. DOX administration
   1. Prepare a fresh DOX solution at 1.5 mg/mL in sterile saline. Protect the solution from light by wrapping the container with aluminum foil.
   2. Weigh each mouse and calculate the injection volume for a single intraperitoneal (i.p.) dose of 15 mg/kg^13,14.
   3. Restrain the mouse gently and administer the DOX solution via intraperitoneal injection. Return the mouse to its cage and proceed according to the experimental schedule.
      CAUTION: DOX is a cytotoxic agent. Wear appropriate personal protective equipment (lab coat, gloves, eye protection) and handle the reagent in a designated chemical safety area.
3. Timeline and monitoring: Monitor mice daily for general health and behavior. Record body weight every other day. Perform cardiomyocyte isolation 7 days after DOX injection .

2. Preparation of solutions and surgical tools for cardiomyocyte isolation

1. Buffer preparation: See Supplementary Table 1 for detailed preparation methods of these solutions.
   1. Prepare perfusion buffer and EDTA buffer. Adjust the pH of all solutions to 7.4 using NaOH.
   2. Prepare enzyme digestion solution containing collagenase type II, collagenase type IV, and protease XIV with perfusion buffer.
   3. Prepare stopping buffer by supplementing perfusion buffer with 5% fetal bovine serum (FBS).
   4. Prepare a complete culture medium by adding 0.1% bovine serum albumin (BSA), insulin-transferrin-selenium (ITS), 10 mM 2,3-Butanedione monoxime (BDM), lipid concentrate, and penicillin/streptomycin (P/S) to M199 medium.
   5. Prepare calcium reintroduction buffers by adding complete culture medium to perfusion buffer at final calcium concentrations of 0.34 mM, 0.68 mM, and 1.02 mM, respectively.
      NOTE: Prepare all solutions under sterile conditions using 0.22 µm filtration. Store solutions at 4 °C, protected from light, for up to two weeks. For the isolation of one mouse heart, prepare 50 mL EDTA buffer, 100 mL perfusion buffer (60 mL for collagenase solution, 10 mL for stopping buffer, remainder for perfusion and gravitational sedimentation), 60 mL collagenase buffer, and 10 mL stopping buffer.
2. Surgical tool preparation: Sterilize all surgical instruments by autoclaving before the procedure. Arrange the following tools sequentially in the surgical area: fine scissors, curved scissors, forceps, curved hemostatic forceps (Figure 1A), and syringe pump (Figure 1B).

3. Isolation of cardiomyocytes from DOX-treated hearts

NOTE: A schematic overview of the cardiomyocyte isolation procedure is shown in Figure 2. After confirming that all surgical instruments, equipment, and solutions are ready,commence the isolation.

1. Aortic clamping and heart excision
   1. Anesthetize the mouse with 3% isoflurane and confirm anesthesia by checking for lack of response to a toe pinch¹⁵.
   2. Secure the mouse in a supine position on the dissection board.
   3. Make a midline incision through the skin and thoracic wall to expose the heart, and cut the descending aorta.
   4. Perfuse 7 mL of EDTA buffer at a controlled, steady rate into the base of the right ventricle over approximately 1 min to rapidly remove blood from the ventricles while minimizing ischemic exposure and preserving cardiac structural integrity, particularly in fragile DOX-injured hearts (Figure 2A).
   5. Clamp the ascending aorta quickly (Figure 2B), excise the heart, and immediately transfer it to a 100 mm dish containing ice-cold EDTA buffer.
      NOTE: The total time from thoracotomy to heart transfer is ideally kept under 2 min to minimize ischemia.
2. Perfusion setup
   1. Trim away the surrounding tissue carefully.
   2. Place the heart in a dissection dish containing ice-cold perfusion buffer.
   3. Perfuse 10 mL of EDTA buffer steadily through the apex of the left ventricle using an indwelling needle over 6 min to destabilize extracellular connections.
   4. Transfer the heart to another 100 mm dish and remove residual EDTA from the cardiac chambers by perfusing 3 mL of perfusion buffer through the same catheter over 2 min.
      NOTE: Observe cardiac swelling during perfusion and secure the aorta with curved hemostatic forceps to establish a closed perfusion system. Carefully control the perfusion rate and pressure to avoid excessive hydrostatic force and minimize the risk of left atrial rupture.
3. Enzymatic digestion
   1. Transfer the excised heart to a 100 mm dish containing 5 mL of enzyme digestion solution.
   2. Connect the indwelling needle to a 20 mL syringe filled with the enzyme digestion solution and securely fix the needle to the biosafety cabinet work surface using sterilization indicator tape.
   3. Place the syringe in a microinjection pump and initiate perfusion at a controlled rate of 3 min/mL (Figure 2C).
   4. Perfuse the heart through the left ventricle with the enzyme solution for 60-90 min untilcomplete digestion is achieved, indicated by a total loss of tissue elasticity and pale coloration of the ventricular surface (Figure 2D). If incomplete digestion is observed after the initial perfusion, administer an additional 15-20 mL of enzyme solution for continued digestion.
      NOTE: Prevent needle dislodgement during syringe changes; if dislodged, reinsert into the original left ventricle site to preserve the perfusion path.
4. Tissue dissociation
   1. Transfer the digested heart to a 60 mm dish containing 5 mL of stopping buffer.
   2. Tear the ventricular tissue into small pieces using fine forceps (Figure 2E).
   3. Gently triturate the ventricular tissue suspension with a 5 mL pipette to dissociate individual cardiomyocytes (Figure 2F).
   4. Filter the cell suspension through a 100 µm cell strainer to remove large tissue fragments.
   5. Rinse the strainer with an additional 5 mL of stopping buffer to maximize cell yield.
      NOTE:Perform all trituration steps gently and limit pipetting to no more than 50 repetitions to avoid mechanical damage to the cardiomyocytes.
5. Calcium reintroduction
   NOTE: Allow the cardiomyocytes to undergo 3-4 sequential 15-min periods of gravitational sedimentation in 15 mL centrifuge tubes (Figure 2G). After each sedimentation, carefully remove 80% of the supernatant and resuspend the pellet in 3 mL of the subsequent calcium reintroduction buffer. Progressively increase the calcium concentration using the prepared gradient solutions at each step¹⁶.
6. Cell counting and plating
   1. Quantify cell density using a hemocytometer and calculate the proportion of rod-shaped versus rounded cardiomyocytes^1,9 (Figure 2H).
   2. Plate 1-5 × 10⁵ cells in plating medium into 6-well plates pre-coated with laminin (10 µg/mL) for 1 h at 37 °C and washed with phosphate-buffered saline (PBS). Prepare plates one day in advance and store at 4 °C if needed. Change media to fresh, prewarmed culture medium after 1 h and every 48 h thereafter.
      NOTE: The protocol can be paused after step 3.6. Cells can be maintained in short-term culture for up to 6 h for immediate experiments.

4. Assessment of cardiomyocyte structural integrity by immunostaining

1. Fix cells with 4% paraformaldehyde for 15 min at room temperature.
2. Wash cells three times with PBS, 5 min each.
3. Permeabilize cells with 0.1% Triton X-100 in PBS for 10 min and wash three times with PBS, 5 min each.
4. Block non-specific binding with 1% BSA in PBS for 1 h.
5. Incubate cells with primary antibody against sarcomeric α-actinin overnight at 4 °C.
6. Wash gently three times with PBS, then incubate with the appropriate fluorescent secondary antibody for 1 h at room temperature.
7. Wash gently three times with PBS, then counterstain nuclei with DAPI for 5 min.
8. Mount coverslips on glass slides using antifade mounting medium.
9. Acquire images using a fluorescence microscope to visualize staining patterns.

5. Analysis of apoptotic signaling in isolated cardiomyocytes

1. Protein extraction and quantification
   1. Lyse cardiomyocytes in RIPA-based buffer supplemented with protease and phosphatase inhibitors.
   2. Centrifuge the homogenates at 12,000 × g for 15 min at 4 °C. Collect the supernatant for subsequent analysis.
   3. Determine protein concentration using a bicinchoninic acid colorimetric assay.
2. Western blot analysis
   1. Separate 30 µg of protein on SDS-polyacrylamide gels and transfer the resolved proteins onto PVDF membranes.
   2. Block the membranes with 5% non-fat milk in TBS containing 1% Tween-20, then incubate them overnight at 4 °C with primary antibodies against Bax, Bcl-2, and cleaved caspase-3, key protein markers commonly assessed in DOX-cardiotoxicity studies¹⁷.
   3. Incubate with appropriate horseradish peroxidase-conjugated secondary antibodies. Detect signals using enhanced chemiluminescence reagent and acquire images with a digital imaging system¹⁸.
3. Data analysis
   1. Present quantitative data as mean ± SEM from three independent biological replicates.
   2. Perform statistical analyses using GraphPad Prism, applying two-way ANOVA for comparisons across multiple groups and time points, or an unpaired two-tailed t-test for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

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