A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.
The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.
Primary cell culture is an integral resource that offers a controlled environment for detailed mechanistic studies of cardiac myocyte function. Due to their more durable nature and ease of isolation, neonatal rat atrial and ventricular myocytes have been the common source of such cell cultures1. However, adult mouse atrial and ventricular myocytes (AMAMs and AMVMs) are highly desirable for in vitro studies, because their molecular and functional characteristics better mimic those of adult heart cells. Thus, they have become relevant for studies related to cardiac pathologies, most of which develop in adults2.
Furthermore, the availability and use of transgenic and disease mouse models expands the utility of isolated adult cardiac myocytes. Protocols for the isolation and culture of mouse AMVMs for short- and long-term studies have been described in numerous previous publications2,3,4,5,6,7,8,9,10,11. In comparison, few protocols have been described for the isolation of AMAMs. Furthermore, those that are described are primarily optimized for acute studies of freshly isolated cells, with no long-term culturing protocol described to date11,12,13. As such, AMAM isolation protocols were not designed to provide the utility and versatility of published protocols for the isolation and culture of AMVMs. Furthermore, while the pioneering studies for the isolation of AMAMs and AMVMs have proven resourceful, there are no protocols for optimal concurrent isolation and culture of both AMAMs and AMVMs, which results in efficient use of the whole heart for each preparation.
Until now, published AMAM and AMVM isolation protocols were not designed for simultaneous isolation of both cell types, because most studies on atrial and ventricular function have a chamber-specific focus. For instance, AMAMs are used predominantly to study atrial myocyte electrophysiology, partly because of the interest in atrial fibrillation (AF), the most common cardiac arrhythmia in the U.S. However, AF is not a disease that affects the atria in isolation, and it has been implicated as having a causative role in mild to severe left ventricular dysfunction14. Furthermore, electrocardiograms from patients with heart failure with preserved ejection fraction (HFpEF) have illustrated that left atrial size is one of the strongest predictors for susceptibility to heart failure15.
In addition to its role in electrophysiology and contractility, the atrium is also an endocrine organ, secreting cardiokines (i.e., atrial natriuretic peptide [ANP]) that homeostatically regulate blood pressure and volume16,17. Moreover, ANP (presumably from atrial myocytes) has a prominent protective and anti-hypertrophic role in ventricular myocytes16,17. While there is a strong implication of neurohormonal communication between atria and ventricles in various disease states, the mechanisms underlying this communication has not been fully explored. This point is further exemplified by the surge in research focusing on 1) the role of non-myocytes (specifically cardiac fibroblasts and immune cells) in the diseased heart and 2) how cardiac remodeling as a function of disease directly affects cardiac myocyte viability and global cardiac function18,19,20,21,22. Thus, studying cardiac cells from both atria and ventricles is a necessary approach to gain a more complete picture of their roles in cardiac pathophysiology.
The following protocol describes the simultaneous isolation of atrial and ventricular myocytes and non-myocytes from a single mouse heart under physiological and pathophysiological conditions. Additionally, this method is the first to describe optimal conditions necessary for maintaining cultures of atrial cardiac myocytes, as conditions for maintaining cultures of ventricular myocytes have already been published.
All research performed on mice reported in this paper has been reviewed and approved by the SDSU Institutional Animal Care and Use Committee and it conforms to the Guide for the Care and Use of Laboratory Animals published by the National Research Council.
1. Preparation of isolation and culture media and plating
2. Isolation apparatus
3. Surgical procedure (non-survival)
4. Atrial cell isolation and culture
5. Ventricular cell isolation and culture
A wildtype 10-week-old C57b6/j mouse heart typically results in between 75,000−150,000 atrial myocytes and 1.0−1.5 x 106 ventricular myocytes, equating to an approximate yield of 30%−50% for atrial and ventricular myocytes18,19. During and immediately after isolations, viable cardiac myocytes should appear rod-shaped and non-contracting. A majority of isolated cardiac myocytes should adapt this morphology, which is an indication of effective perfusion. The rod-shape morphology can also be a predictor of viability. The protocol aims to enhance the yield and viability of myocytes and non-myocytes isolated from a diseased mouse heart. Furthermore, it has been tested in a model of pressure overload-induced heart failure (data not shown).
To confirm adequate and replicable isolation of myocytes and non-myocytes from atrial and ventricular tissue, cells were observed and photographed at various days in culture (Figure 2). Additionally, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to measure the levels of transcripts that were cell type-specific. Cardiac muscle troponin T (Tnnt2) is a marker of cardiac myocytes and was robustly expressed in both atrial and ventricular cardiac myocyte cultures (Figure 3A). In contrast, atrial natriuretic peptide (Nppa, which is typically expressed exclusively in adult atrial cardiac myocytes under physiological conditions) and myosin light chain 2 (Myl2, which is a ventricular myocyte specific gene) were robustly and specifically expressed in atrial and ventricular cardiac myocyte cultures, respectively (Figure 3B,C).
Fibroblast markers, transcription factor 21 (Tcf21), platelet-derived growth factor receptor A (Pdgfra), and monocyte-derived cell marker cluster of differentiation 68 (Cd68) were exclusively expressed in non-myocyte cultures isolated from both atrial and ventricular chambers (Figure 3D−F). It is estimated that non-myocytes compromise ~65% of all heart cells and that a majority of these originate from a fibroblast or monocyte-derived lineage18,19,23,24. Thus, markers for these two lineages were chosen to be representative, given the interest in these cellular populations in studies of various models and etiologies of cardiac pathology.
Immunostaining of AMAMs and AMVMs for the t-tubule marker dihydropyridine (DHPR, which is a voltage-dependent (L)-type calcium channel) as well as the ryanodine receptor (RYR2) demonstrated intact t-tubules throughout isolation and long-term culture (Figure 4A,B). The abundance of DHPR and localization that was characteristic and unique to atrial and ventricular myocytes indicated the presences of t-tubules. Moreover, colocalization of DHPR with RYR2 immunostaining was an indicator of intact diad structures. Immunostaining for the sarcomeric protein alpha-actinin in atrial and ventricular cardiac myocytes resulted in the expected sarcomeric striation pattern. The sarcomeric striation pattern was used to assess the purity and viability of isolated cardiac myocytes in conjunction with rod-shaped morphological shape and nuclear staining with TOPRO-3 (Figure 4C,D; purple and red). As expected, ventricular cardiac myocytes were large, exhibiting an average length of ~150 mm, whereas atrial cardiac myocytes averaged ~75 mm. Furthermore, upon immunostaining analysis, atrial cardiac myocytes (but not ventricular cardiac myocytes) exhibited robust expression of atrial natriuretic peptide (ANP) in a staining pattern that was characteristic of localization to the endoplasmic reticulum and secretory granules (Figure 4C,D; green).
A characteristic unique to atrial cardiac myocytes is its classification as an endocrine cell in addition to contractile cell. While atrial myocytes secrete ANP under basal conditions, secretion increases in response to secretagogues (i.e., the alpha-adrenergic agonist, phenylephrine [PE]). Moreover, atrial cardiac myocytes secrete ANP and co-secretionally process a portion of the hormone from its precursor state (Pro-ANP, 15 kD) to the product peptide (ANP 3kD)16,17. This secretory ability can be quantified via immunoblot detection of ANP in the media of isolated atrial cardiac myocytes in response to acute PE treatment (Figure 4E). This secretory and processing ability of the atrial cardiac myocyte was found to be sensitive to culturing conditions. Thus, it is imperative that the atrial myocyte plating medium is supplemented with dexamethasone, insulin, transferrin, and selenium.
Figure 1: Schematic overview of retrograde heart perfusion, digestion, and cell isolation. Shown are the main steps involved in cell isolation from both atrial and ventricular chambers simultaneously from a single mouse heart. (A) A single mouse heart is rapidly cannulated via the ascending aorta and perfused in a retrograde manner. (B) The heart is separated into atrial and ventricular tissues for further digestion and physical separation. (C) Following adequate digestion, the cells are separated via gravity filtration into a total of four cellular fractions that are cultured for subsequent experimentation. Please click here to view a larger version of this figure.
Figure 2: Morphological analysis of isolated atrial and ventricular cardiac myocytes and non-myocytes in culture. (A) Isolated adult mouse atrial myocytes (AMAMs), (B) adult mouse atrial non-myocytes (AMANMs), (C) adult mouse ventricular myocytes (AMVMs), or (D) adult mouse ventricular non-myocytes (AMVNMs) were plated at 5 x 105 cells/chamber on four-chamber (1.7 cm2) glass slides in respective plated media. Phase images were obtained at indicated days in culture using a 10x objective under an epifluorescence microscope. Please click here to view a larger version of this figure.
Figure 3: Representative qRT-PCR analysis of isolated cell cultures. RNA was extracted from freshly isolated cardiac myocytes and non-myocytes, and mRNA levels for cell-specific gene markers were determined by qRT-PCR4. (A) Tnnt2, cardiac muscle troponin T (cardiac myocyte marker); (B) Nppa, atrial natriuretic peptide (atrial myocyte marker); (C) Myl2, myosin light chain 2 (ventricular myocyte marker); (D) Tcf21, transcription factor 21 (fibroblast marker); (E) Pdgfra, platelet-derived growth factor receptor A (fibroblast marker); (F) Cd68, cluster of differentiation 68 (monocyte-derived cell marker). Data represent mean ± SEM (*p ≤ 0.05 different from all other values, as determined by ANOVA followed by Newman Keul’s post-hoc analysis). Please click here to view a larger version of this figure.
Figure 4: Representative morphological and functional analysis of isolated atrial and ventricular cardiac myoyctes. (A) AMAMs or (B) AMVMs were plated at 5 x 105 cells/chamber on four-chamber (1.7 cm2) glass slides in respective plating media for 1 h to allow for adhesion. This was followed by either refeeding atrial myocyte plating media or changing to ventricular myocyte maintaining media supplemented with blebbistatin for an additional 16 h. Cultures were subsequently fixed then immunostained for RYR2 (purple), DHPR (green), and nuclear stain TOPRO-3 (red). (C) AMAMs or (D) AMVMs were isolated and plated, then immunostained for a-actinin (purple), ANP (green), and TOPRO-3 (red). Shown are two representative images for each cell type. (E) AMAMs were plated at 5 x 105 cells/well on a 12 well culture dish for 16 h in atrial myocyte plating media. AMAMs were subsequently treated for 0.5 h with vehicle or the ANP secretagogue (phenylephrine, 50 mM) before media were collected and subjected to immunoblot analysis for ANP. Prior to immunoblot analysis, media samples were centrifuged at 500 x g for 5 min to remove cellular debris and ensure that the observed ANP was the result of active secretion from AMAMs. Please click here to view a larger version of this figure.
The quality of the cells isolated using the procedure described here, as determined by the cell yield and overall health of the cells in culture, depends on numerous controllable factors. Starting with the mouse itself, it has been documented that stress imposed on the animal can negatively affect cell yield and viability in culture, presumably due to excess systemic cortisol, catecholamines, and the hypercontractile state of cardiac tissue2,5,7. For these reasons, measures should be taken to avoid alarming the animal prior to sacrifice. Such measures include covering the animal’s cage and limiting time outside of vivarium prior to sacrifice. Heparin and many barbiturates commonly used for euthanasia can affect signaling pathways; thus, the optimal method of euthanasia should be customized accordingly. The age of the animal has a considerable impact on the quality and viability of isolated cells, most likely due to progressive accumulation of interstitial fibrosis occurring concurrently with the aging process, which can affect tissue digestion25. In data not presented here, while the method described above works in mice of up to 78 weeks old, the quality of the cells was lower in these older animals.
The most critical step in the isolation process described, as well as other protocols featuring a Langendorff apparatus for retrograde perfusion, is the cannulation and initial perfusion of the heart. For optimum results, the time from cardiac explantation to cannulation of the ascending aorta and initiation of perfusion should take no more than 90 s. In addition to time, two additional important factors are the depth of cannula and possibility of introducing air emboli from the perfusion apparatus. Accordingly, the cannula should be advanced into the ascending aorta so as not to enter the aortic root and obstruct the aortic valve, which would impair perfusion of the coronary vessels.
During the digestion process, it is important to regularly test the rigidity of the heart to avoid prolonged exposure to the digestive enzyme collagenase, which reduces cardiac myocyte calcium tolerance. The protocol described above for calcium reintroduction into the isolated ventricular myocyte cultures was designed to limit cardiac myocyte death via inappropriate calcium influx via store operated calcium channels. It should be noted that the stepwise calcium reintroduction should not be performed for isolated atrial myocyte cultures, as this will promote cell death during short- and long-term culture12. For further precaution, the perfusion and digestion buffers used here include the cardiac muscle contraction inhibitor butanedione monoxime (BDM) to avoid hypercontraction of isolated myocytes, as well as the calcium paradox, both of which impact myocyte viability26. However, the switch from BDM to blebbistatin should be noted, as it is the preferred anti-contractile agent in maintaining media for isolated cardiac myocytes. In data not shown, blebbistatin confers greater viability for long-term culture of isolated cardiac myocytes.
Immediately after isolation, it is important to consider the ramifications of long-term culture of cardiac cells, especially myocytes. The cardiac non-myocyte isolation and culturing protocol described here is based on common methods that take advantage of the different densities and adhesive properties of different cardiac cells. The benefit of non-myocytes is their high expansion potential in culture; thus, unlike cardiac myocytes, they are amenable to passaging for perpetuation. However, it is known that culturing conditions, including medium supplementation with FBS, can affect cardiac myocyte functionality27. The culture media described here were designed to optimize viability and limit functional derangements, especially for the isolated atrial myocytes. While no overt impaired contractile ability was observed in isolated cardiac myocytes after culture in the absence of blebbistatin supplementation, studies that focus on electrophysiology, contractility, and other single-cell in vivo-based molecular signaling should be performed soon after isolation, when the sarcomeric structure and molecular signature still mimics that of the intact heart.
A hallmark feature of the atrial myocyte is its ability to moonlight as an endocrine cell with immense secretory capacity, in addition to their contractile function. Under physiological conditions, atrial myocytes produce large quantities of ANP, which is stored in the endoplasmic reticulum and in large dense-core secretory granules poised for regulated exocytosis upon receiving a stimulus16,17. While many isolated atrial myocyte studies focus on their unique electrophysiological properties, this is the first study to design a culture media. This allows for long-term viability as well as promotion of the maintained functions of endocrine and contractile properties of atrial myocytes. This novel method for culturing, as well as simultaneous isolation of all cell types from atrial and ventricular chambers from a single mouse heart, will be useful and efficacious for studies on the physiological and pathophysiological properties of both atrial and ventricular myocytes.
The authors have nothing to disclose.
E.A.B. was supported by the National Institutes of Health (1F31HL140850), the ARCS Foundation, Inc., San Diego Chapter, and is a Rees-Stealy Research Foundation Phillips Gausewitz, M.D. Scholar of the SDSU Heart Institute. E.A.B. and A.S.B. were supported by the Inamori Foundation. CCG by (NIH) grants R01 HL135893, R01 HL141463 and HL149931.
(-)-Blebbistatin | Sigma-Aldrich | B0560 | |
1 Liter Water Jacketed Reservoir | Radnoti | 120142-1 | |
2,3-Butanedione monoxime | Sigma-Aldrich | B0753 | |
5-0 Silk Suture Thread | Fine Science Tools | 18020-50 | |
Adenosine | Sigma-Aldrich | A9251 | |
Bovine Serum Albumin | Sigma-Aldrich | A6003 | |
Bubble Trap Compliance Chamber | Radnoti | 130149 | |
Calcium Chloride Anhydrous | Fisher Scientific | C614-500 | |
Carnitine hydrochloride | Sigma-Aldrich | C9500 | |
Collagenase type 2 | Worthington Biochemical Corporation | LS004176 | |
Creatine | Sigma-Aldrich | C0780 | |
Dexamethasone | Sigma-Aldrich | D2915 | |
DMEM/F12 (1:1; 1X) | Gibco | 11330-032 | |
Dumont #7 – Fine Forceps | Fine Science Tools | 11274-20 | |
Epifluorescent micropscpe | Olympus X70 | IX70 | |
Fetal Bovine Serum (Heat Inactivated) | Omega Scientific | FB-12 | Lot# 206018 |
Fine Scissors – Sharp | Fine Science Tools | 14060-09 | |
Graefe Forceps | Fine Science Tools | 11051-10 | |
Headband Magnifiers | Fine Science Tools | 28030-04 | |
Hemacytometer (Bright-Line) | Hausser Scientific | 1475 | |
HEPES (1M) | Gibco | 15630-080 | |
Inosine | Sigma-Aldrich | I4125 | |
Insulin-Transferrin-Selenium-X | Gibco | 51500-056 | |
Isotemp 105 Water Bath | Fisher Scientific | NC0858659 | |
Isotemp 3006 | Fisher Scientific | 13-874-182 | |
Joklik Modified Minimum Essential Media | Sigma-Aldrich | M-0518 | |
Laminin (Natural, Mouse) | Gibco | 1795024 | Lot# 1735572 |
L-Glutamine | Sigma-Aldrich | G8540 | |
Masterflex C/L Single-Channel Variable-Speed Compact Pump | Cole-Palmer | EW-77122-24 | |
Minimum Essential Medium (MEM 1X) | Gibco | 12350-039 | |
Molecular Biology Grade Water | Corning | 46-000-CM | |
Pen Strep Glutamine (100X) | Gibco | 10378-016 | |
Spring Scissors – 6mm Cutting Edge | Fine Science Tools | 15020-15 | |
Taurine | Sigma-Aldrich | T-8691 |