One-hour Procedure to Isolate Primary Cardiomyocytes from Neonatal Mouse and Rat Hearts

Easily isolate and culture highly viable, functional primary cardiomyocytes

by Hai-Yan Wu, Ph.D.; Kay Opperman, Ph.D.; Barbara Kaboord, Ph.D. - 04/02/14

Cultured primary cardiomyocytes are widely used to study and understand the mechanisms underlying normal cardiovascular function, cardioprotection, and cardiovascular diseases. Cardiomyocyte cultures provide a homogeneous population of single cells, which are easy to visualize and manipulate. Compared with the whole heart, cardiomyocyte cultures are relatively pure, with limited contaminating cell types such as endothelial cells. In addition, preparations of heart cells isolated from small mammals like mouse and rat enable a large number of quick, relatively low-cost experiments compared to studies conducted in whole animals.

Isolating primary cardiomyocytes from neonatal mouse and rat hearts has typically been a time-consuming, labor-intensive task. A common problem during cardiomyocyte isolation is that the heart is a solid organ with strong intercellular attachments, so the dissociation process is more difficult and time consuming. Current methods to isolate neonatal mouse/rat cardiomyocytes use either a series of trypsin digests (typically five to eight incubations for 10 to 20 minutes each)[1,2,3], or a single, long digestion, usually 16 hours to overnight (Table 1).

In this article, we describe a convenient and time-saving protocol for the isolation and culture of primary cardiomyocytes from neonatal mouse and rat hearts. We compared the efficacy of our new method to existing protocols with respect to enzyme digestion, cell isolation efficiency, and cardiomyocyte function. Our new method, commercialized as the Thermo Scientific Pierce Cardiomyocyte Isolation Kit (Part No. 88281) is a significant improvement over existing isolation protocols, enabling consistently high cell yields exhibiting high viability and improved function as assessed by the spontaneous beating activity of the cardiomyocyte cultures.

Isolating primary cardiomyocytes is a delicate process involving the controlled use of enzymes to disrupt complex protein and intercellular matrix interactions found in heart tissue. The choice of enzyme(s), concentration, and timing of the digestions has a profound effect on cardiomyocyte yield and viability. We screened several different proteases and collagenases individually and in combination to determine the most efficient digestion strategy (data not shown).

We selected our best enzyme combination (papain plus thermolysin at certain concentrations) and protocol (Figure 1, Pierce Kit) and then compared it to a standard literature protocol using trypsin, a commercially-available kit, and a selection of commercially-available enzyme blends recommended for cardiomyocyte isolation. As described in Table 1, the do-it-yourself (DIY) trypsin method uses 5 to 8 repeated incubations, and the Neonatal Cardiomyocyte Isolation System (#LK003300; Worthington Biochemical Corporation) uses an overnight digestion followed by a secondary 35- to 40-minute digestion the following day. For the enzyme blends of Liberase™ DH and TM (Roche Diagnostics), we performed one 35-minute incubation at 37°C to match our method.

Our Pierce Kit method consistently provided a 2- to 10-fold higher cell yield compared to the trypsin protocol, the Worthington Kit procedure, and both Roche enzyme-blend methods (Figure 2). Furthermore, viability of the newly isolated cells was 25 to 50% higher with our isolation protocol than with the other methods (Figure 2). Our isolation procedure worked equally well with mouse and rat neonatal hearts (Table 2).

Viability was determined by trypan blue exclusion.

Cell shape and morphology were used as one means of assessing cell health and function. When mouse/rat myocytes were 24 hours after plating (Day 1), the seeded cells quickly began to flatten from their initial round shape and show formation of short, outstretched pseudopodia (Figure 3A). After Day 3, the cardiomyocytes isolated from both mouse and rat hearts looked elongated and their pseudopodia grew across each other to form a confluent monolayer culture (Figure 3A and B).

A. Mouse cardiomyocytes

B. Rat cardiomyocytes

To assess the health of the cultures over time, we evaluated the viability and purity of mouse cardiomyocytes at Day 1 and Day 7 in culture. Propidium Iodide (PI, red), a nuclear fluorescent dye normally excluded from viable cells, was used to reveal dead cells in cultures (Figure 3A, lower panel). Despite the presence of some PI-labeled cells at Day 1 in cultures prepared by each isolation method, the ratio of PI-labeled cells versus total cells in cultures prepared by our method (Figure 4A) was similar to that obtained from cultures prepared using the Worthington kit (19% at Day 1 and 8% at Day 7) but much lower than the ratio obtained in cultures prepared by the trypsin homebrew method (34% at Day 1 and 16% at Day 7) and using the Roche enzyme blends (55% at Day 1, and over 50% at Day 7).

To assess the purity of the cardiomyocyte cultures, we immunostained cells with an antibody specific to the cardiomyocyte marker protein troponin T cardiac isoform (green in Figure 3A, lower panel). Cardiomyocyte purity was calculated as the ratio of total troponin T cardiac isoform stained cells to total cells (determined by nuclear staining). At Day 1, cultures prepared by our method were 61% pure cardiomyocytes, similar to cultures prepared with the Worthington kit (Figure 4B). Cultures prepared using trypsin or Roche enzyme blends were significantly less pure (51% and 45%, respectively). However, once the Cardiomyocyte Growth Supplement, a reagent supplied in our kit that reduces fibroblast contamination, was included in the growth media for all of the cultures the overall culture purity at Day 7 increased by 20-30% except for the culture prepared with the Roche blend (8%)(Figure 4B).

Figure 4. Cardiomyocyte viability and purity in culture. A. Cell viability at Day 1 and Day 7 was calculated as the ratio of total PI negative cells to total cells indicated by Hoechst nuclear staining. B. Cell purity at Day 1 and Day 7 was calculated as the ratio of total troponin T cardiac isoform stained cells to total cells indicated by Hoechst nuclear staining. A total of 200 cells were analyzed from two independent experiments. Data represent the mean ± SD.

Some of the isolated cardiomyocytes exhibited spontaneous beating activity on Day 1 (Figure 5A), with the beating becoming synchronized upon monolayer formation (Day 3). The strong synchronous contractions can be visualized by phase-contrast microscopy after 3 to 6 days in culture (Figure 5A) with an average rate of 100-115 beats/minutes (Figure 5B). Cardiomyocyte cultures prepared using the Pierce kit showed strong, rapid beating rates, an indication of good cell health and function.

B. Beating rates

To demonstrate that our primary cardiomyocyte cultures can serve as good physiological model systems, we looked at the activation of two cell signaling pathways. First, we determined whether we could induce cell death in cultured primary cardiomyocytes by serum deprivation and to assess whether insulin-like growth factor I (IGF-I) might suppress the cell death through activation of a serine/threonine protein kinase Akt [4]. In Day 5 cardiomyocyte cultures, cells were incubated with serum-free medium in the presence or absence of IGF-I for 24 hours, and cell viability was determined by PI staining. The surviving cell number is expressed relative to the untreated cell culture. In the absence of IGF-I, cardiomyocyte viability decreased by 25% after 24 hours of serum withdrawal (Figure 6A). However, cardiomyocyte viability was maintained in the presence of IGF-I. IGF-I-mediated cardiomyocyte protection was abrogated by treatment with 10µM Triciribine, an Akt inhibitor V [4], suggesting that the survival effects of IGF-I are regulated by proteins downstream of Akt. These data support the theory that IGF-I protects against serum deprivation-induced cardiomyocyte death through activation of the Akt pathway.

In the second experiment, we tested if cultured cardiomyocytes can respond to Endothelin-1 properly through activation of ERK. Endothelin-1 has been reported to induce the activation of the cardiomyocyte hypertrophic signaling pathways involving mitogen activated protein (MAP) kinase, also called extracellular regulated kinase (ERK) [5,6]. Day 5 cardiomyocytes were exposed to 1µM Endothelin-1 for 24 hours, cell lysates were prepared, and Western blot analysis was performed to look for ERK phosphorylation. Cardiomyocyte exposure to Endothelin-1 for 24 hours induced phosphorylation of ERK without changing the total protein levels of ERK (Figure 6B). These results indicate that our improved cardiomyocyte isolation protocol produces healthy cardiomyocytes that can be used to study mechanisms underlying cardiomyocyte function and protection.

A. Cardiomyocyte function via effectors of cell death

B. Cardiomyocyte function via activation of ERK

We have described a new technique for rapid isolation of primary cardiomyocytes from neonatal mouse or rat hearts, as well as the successful culture and function of the isolated cells. The reagents and protocol for this new procedure have been commercialized as the Pierce Primary Cardiomyocyte Isolation Kit (Part No. 88281). Unlike existing methods, the procedure takes less than one hour to complete. In addition, the technique provides high cell yield and viability, and produces more than 85% pure cardiomyocytes in Day 7 culture (Figure 5B). The cardiomyocytes are sensitive to serum deprivation-induced cardiomyocyte death, and can respond to Endothelin-1 properly through activation of ERK, demonstrating that cardiomyocytes isolated by our method can be used for a broad spectrum of experiments including ischemia and hypoxia studies.

Primary cardiomyocyte isolation:

For our method (Pierce Kit), freshly dissected whole mouse/rat neonatal hearts at Day 1 to 3 were minced, incubated with Pierce Cardiomyocyte Isolation Enzyme 1 and 2 for 35 minutes and washed twice with Hanks Balanced Salt Solution (HBSS). The tissue was disrupted in Complete DMEM for Primary Cell Isolation by pipetting up and down 25 to 30 times with a pipette fitted with a 1000µL tip to generate a single cell suspension. For do-it-yourself trypsin-based isolation (DIY Trypsin), we used the repeated digestion method described in references 1-3. For isolation using the Neonatal Cardiomyocyte Isolation System (#LK003300) from Worthington Biochemical Corporation (Worthington Kit), we followed the isolation procedure described in the kit instruction booklet. With Liberase DH (#05401054001) and Liberase TM (#05401119001) enzyme blends from Roche Diagnostics (Roche Blends), we followed Roche’s application protocol for isolation of rodent cardiac myocytes [7] with some modification. Briefly, minced heart tissues were incubated with Liberase DH or TM at the recommended concentration for 35 minutes at 37°C, and then tissues were further disrupted to obtain a single cell suspension by pipetting up and down 25 to 30 times with a pipette fitted with a 1000µL tip. After single cell suspensions were obtained from each method, total cell yield was determined using an Invitrogen™ Countess™ Automated Cell Counter and cell viability was determined by trypan blue exclusion assay.

Immunostaining of primary cardiomyocytes:

Cardiomyocytes at the indicated days in culture were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton™ X-100 in HBSS for 10 minutes at room temperature and blocked with 3% BSA in HBSS for 30 minutes at room temperature. Cells were then incubated with primary antibodies overnight at 4°C, with the corresponding secondary antibodies at room temperature for 1 hour, and then washed twice with HBSS.

Western blots:

Equal quantities of total protein (10 to 20μg/lane) were resolved by SDS-PAGE (2-10% gels) and transferred to nitrocellulose membranes. Membranes were blocked with 3% bovine serum albumin and incubated with primary antibody overnight at 4°C. Blots were incubated with goat anti-rabbit (Part No. 31460) or goat anti-mouse (Part No.31430) horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature and then washed. Bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080) and exposed to film.

Animal care:

Time-pregnant CD-1 mice or Sprague Dawley (SD) rats were obtained from Charles River and housed in the University of Illinois College of Medicine at Rockford animal facility. Experiments were performed exactly as approved by the Animal Care and Use Committee at the University of Illinois College Of Medicine in Rockford, IL, and conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

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