Autonomous and Growth Factor–Induced Hypertrophy in Cultured Neonatal Mouse Cardiac Myocytes
Comparison With Rat
Abstract—Cultured neonatal rat cardiac myocytes have been used extensively to study cellular and molecular mechanisms of cardiac hypertrophy. However, there are only a few studies in cultured mouse myocytes despite the increasing use of genetically engineered mouse models of cardiac hypertrophy. Therefore, we characterized hypertrophic responses in low-density, serum-free cultures of neonatal mouse cardiac myocytes and compared them with rat myocytes. In mouse myocyte cultures, triiodothyronine (T3), norepinephrine (NE) through a β-adrenergic receptor, and leukemia inhibitory factor induced hypertrophy by a 20% to 30% increase in [3H]phenylalanine-labeled protein content. T3 and NE also increased α-myosin heavy chain (MyHC) mRNA and reduced β-MyHC. In contrast, hypertrophic stimuli in rat myocytes, including α1-adrenergic agonists, endothelin-1, prostaglandin F2α, interleukin 1β, and phorbol 12-myristate 13-acetate (PMA), had no effect on mouse myocyte protein content. In further contrast with the rat, none of these agents increased atrial natriuretic factor or β-MyHC mRNAs. Acute PMA signaling was intact by extracellular signal–regulated kinase (ERK1/2) and immediate-early gene (fos/jun) activation. Remarkably, mouse but not rat myocytes had hypertrophy in the absence of added growth factors, with increases in cell area, protein content, and the mRNAs for atrial natriuretic factor and β-MyHC. We conclude that mouse myocytes have a unique autonomous hypertrophy. On this background, T3, NE, and leukemia inhibitory factor activate hypertrophy with different mRNA phenotypes, but certain Gq- and protein kinase C–coupled agonists do not.
In the early 1980s, our laboratory established the neonatal rat cardiac myocyte culture model for hypertrophy.1 2 3 4 The model has since been used extensively to study hypertrophic signaling in response to many growth stimuli and to identify candidate molecules for testing in the intact animal. Indeed, there are now many knockout and transgenic mouse models of cardiac hypertrophy and failure, providing valuable insights into molecular and cellular mechanisms at the tissue and organ levels.5 6 Mouse culture models derived from these knockout and transgenic hearts would be very useful to distinguish effects on myocytes versus fibroblasts, to eliminate hemodynamic and hormonal influences, and to study cell phenotypes and signaling in more homogeneous populations in a defined environment.
Surprisingly, however, there are so far very few studies in cultured mouse myocytes. Some studies in neonatal mouse myocytes find hypertrophic signaling or responses to leukemia inhibitory factor (LIF),7 8 norepinephrine (NE),9 a protease-activated receptor-1 agonist,10 and mechanical stretch,11 12 but there is no detailed characterization of hypertrophic responses in neonatal mouse cultures or direct comparison with rat cultures. In particular, it would be very interesting to know whether the mouse model would yield data similar to those derived from the rat.
In this study, we addressed these issues by characterizing the hypertrophic responses of cultured neonatal mouse cardiac myocytes. We prepared mouse and rat cultures by a similar method and compared them in the same experiments. We also studied mouse cardiac nonmyocytes and late fetal mouse myocytes. Our data reveal major differences in the hypertrophic responses of cultured mouse and rat cardiac myocytes.
Materials and Methods
Cardiac Myocyte and Nonmyocyte Cultures and Conditioned Medium
Neonatal rat and mouse myocytes were cultured as described,1 13 with minor modifications for mouse myocytes. Ventricles from 2 to 3 litters of 1-day-old neonatal FVB/N, C57BL/6, or CD1 mice or 18- to 20-day fetal mice were separated from the atria using scissors and then dissociated in trypsin and DNase II. Cells were preplated for 2 hours into 100-mm dishes in MEM with 10% defined bovine calf serum (HyClone), and then unattached myocytes were plated at 500 cells/mm2 into Falcon 35-mm dishes (for radiolabeled protein, signaling, and cytochemistry) or 60-mm dishes (for RNA). Medium was MEM with 10% bovine calf serum, 0.1 mmol/L bromodeoxyuridine (BrdU), and 20 μmol/L arabinosylcytosine (Ara-C). After 24 hours (on day 1), medium was changed to serum-free MEM with 10 μg/mL transferrin, 10 μg/mL insulin, 1 mg/mL BSA (MEM-TI-BSA), and 0.1 mmol/L BrdU.
Cardiac nonmyocytes in the preplates were passaged twice, grown to near confluence in MEM with 10% FCS, and then for experiments changed to serum-free MEM-TI-BSA.13
Serum-free conditioned medium from rat and mouse myocytes and nonmyocytes was collected after 72 hours,13 mixed 1:1 with fresh medium, and tested on rat myocytes.
The content of radiolabeled protein (RLP) was assayed by [3H]phenylalanine incorporation, and absolute protein per cell was quantified by Bradford assay and by counting attached cells in the dishes.2 4 Radiolabeled DNA content in nonmyocytes was estimated using [3H]thymidine instead of phenylalanine. Myocyte myosin was stained with MF20. The mRNAs for α- and β-myosin heavy chain (MyHC), atrial natriuretic factor (ANF), and β-actin in 4 μg total RNA (isolated in Trizol, Gibco) were detected by ribonuclease protection assay (RPA III kit, Ambion) with rat probes and quantified by densitometry.14 A multiple-probe template set was used in RPA for the immediate-early genes (PharMingen). Growth factors are detailed in the online expanded Materials and Methods (available at http://www.circresaha.org).
Activated extracellular signal–regulated kinase (ERK1/2) in total protein from equal numbers of myocytes was quantified by Western blot with a monoclonal antibody specific for dually phosphorylated ERK1/2 and an ECL kit (both from New England BioLabs). Nuclear translocation of activated ERK1/2 in myocytes was assayed by immunocytochemistry with the same antibody. Phospholipase C activation was quantified by release of total [3H]inositol phosphates ([3H]IPs).15
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
Isolation and Culture of Neonatal Mouse Cardiac Myocytes
We prepared a total of ≈100 neonatal mouse myocyte cultures and in many cases made rat cultures at the same time. We used the culture method developed for rat, with minor modifications, including reduced trypsin (1 mg/mL versus 1.25) and dissociation time (3 to 5 minutes versus 5 to 8), prolonged preplating (2 hours versus 0.5 hours), and both Ara-C and BrdU (versus BrdU alone) to reduce nonmyocytes. Mouse myocyte yield was 0.6×106 per heart (n=32), ≈10% of rat heart yield (5×106, n=40), which was in good agreement with the relative weights of mouse and rat hearts (1:10). By counting 17 random, high-power (final ×400) phase-contrast fields in each dish, myocyte plating efficiencies (fraction of plated myocytes that attach) were similar for mouse (26%) and rat (30%). Also similar were low final cell densities (mouse, 130 cells per mm2 or 19 per field; rat, 150 cells per mm2 or 25 per field) and percentage cardiac nonmyocytes (mouse, 12±1%; rat, 10±1%). Neonatal mouse myocytes early in culture appeared generally similar to rat myocytes, except that more mouse cells had elongated cell bodies (see Figures 2⇓ and 3⇓).
Myocyte Hypertrophy by Content of RLP
To test for an overall hypertrophic response in neonatal mouse myocytes, we treated them with different growth factors and quantified an index of myocyte size, RLP by [3H]phenylalanine incorporation.2 4 We used doses that are maximum in rat cultures,2 4 13 15 and as a control, we tested rat myocyte cultures simultaneously. As shown in Table 1⇓, rat myocytes had a robust response to all factors tested, with a 34% to 201% increase in RLP. The result was very different in neonatal mouse myocytes (Table 1⇓). NE, a combined α- and β-AR agonist, increased RLP by 21%, and this was entirely a β-AR response, because it was blocked by the nonselective β-AR antagonist timolol and reproduced by the β-AR agonist isoproterenol. α1-AR stimulation (the agonists PE, A6, or NE in the presence of timolol), ET-1, PGF2α, PMA, and IL-1β all had no effect. TNF-α (11%), LIF (27%), T3 (29%), and FCS (77%) all increased protein, although only T3 was as efficacious in the mouse as in the rat (Table 1⇓). Similarly, in no case was the hypertrophic response sufficient to give an easily detectable increase in myocyte area as seen through the microscope (not shown), in further marked contrast with the rat.2 3 4
Insulin in the serum-free medium did not alter the effects of growth factors in mouse myocytes, because RLP with PE, NE, T3, and PMA was identical in MEM with 6 concentrations of insulin from 0 to 10 μg/mL (not shown). Similarly, omission of BSA from the medium had no effect (not shown). Serum-free culture for 72 hours before growth factor addition, rather than 24 hours, did not change the RLP with NE or T3 or uncover a response to PMA (not shown). Reduced responses in neonatal mouse myocytes were not due to developmental stage, because results were similar in fetal mouse myocytes (Table 1⇑). Ara-C, which was used in mouse cultures but not rat, had no effect on the RLP responses to PE or T3 in rat cultures (not shown). Thus, the absent or blunted hypertrophic responses in mouse myocytes versus rat were not due to culture conditions.
Growth in Neonatal Mouse Cardiac Nonmyocyte Cultures
The myocyte cultures were contaminated by 12% nonmyocytes, which could confound a cell-nonspecific assay such as RLP. To test this possibility, we assayed RLP and radiolabeled DNA content in neonatal mouse nonmyocytes. In nonmyocyte cultures, NE, ISO, PE, PMA, and T3 had no significant effect on protein or DNA contents; LIF decreased DNA; and FCS stimulated both DNA and protein. Thus, FCS stimulation of RLP in myocyte cultures (Table 1⇑) might in part be due to contaminating nonmyocytes, but this was not likely for the other growth factors.
MyHC and ANF mRNAs in Mouse Myocytes
We tested for transcriptional signaling and different phenotypes in the mouse model using RNase protection assay (RPA) for 4 target mRNAs, α- and β-MyHC, ANF, and β-actin (Figure 1⇓). Induction of α-MyHC is a marker for physiological hypertrophy, whereas β-MyHC is more nonspecific.16 17 18 19 By RPA we could quantify both MyHCs in the same lane with a single small total RNA sample, along with β-actin, which increases in hypertrophy in proportion to total RNA.20 ANF is induced nonspecifically by hypertrophic and other stimuli and serves as a general marker for myocyte-specific signaling16 19
In the cultured mouse myocytes, α-MyHC was increased significantly by T3 (≈2-fold) and NE (≈1.5-fold), and there was a nonsignificant trend with FCS (≈1.25-fold) (Figures 1A⇑ and 1B⇑). Interestingly, α-MyHC induction by NE appeared to require α1-AR activation along with β-AR, because it was seen with NE but not with ISO or PE alone (Figure 1B⇑). None of the other growth factors changed α-MyHC (Figure 1B⇑). Surprisingly, β-MyHC mRNA either was unchanged (with PE, PMA, and ET-1), decreased mildly (≈75% of vehicle with TNF-α and LIF), or decreased markedly (less than half of vehicle with NE, ISO, T3, and FCS) (Figures 1A⇑ and 1B⇑). Further surprisingly, ANF mRNA was not changed with any growth factor (Figures 1A⇑ and 1C⇑). In a parallel control experiment with rat myocytes that confirmed prior results,17 21 β-MyHC was induced by PMA (3.2-fold)>NE (2.1-fold)=PE plus timolol (2-fold)>ISO (1.4-fold) and reduced to one-third of vehicle by T3; α-MyHC was changed only by T3 (2.8-fold increase).
Signaling in Neonatal Mouse Myocytes
The absent hypertrophic responses to growth factors coupled to Gq (α1-AR, ET-1, and PGF2α) or protein kinase C (PKC) (PMA) might have been due to absent receptors or signal transducers. To test this, we focused on PMA, a potent activator of PKC. PMA is a very robust hypertrophic agonist in the rat (Table 1⇑ and Rokosh et al,15 Kariya et al,21 and Henrich and Simpson22 ) but was without effect on protein or mRNAs in the mouse (Table 1⇑; Figure 1⇑). Nevertheless, in mouse myocytes, as shown in Figure 2⇓, PMA had a very strong effect to activate ERK1/2, as measured by nuclear translocation of activated (phospho)-ERK and increased phospho-ERK on immunoblot (Figure 2A⇓). Also, PMA strongly increased immediate-early gene mRNA levels (eg, fos and jun) (Figure 2B⇓).
We tested for Gq-coupled phospholipase C activation using [3H]IP release. PE for 60 minutes increased [3H]IPs by 3±0.5-fold (n=9, P<0.01), and this response was eliminated by the α1-AR antagonist prazosin (200 nmol/L, n=4). ET-1 increased [3H]IPs by 2±0.2-fold (n=3, P<0.05). ET also increased fos/jun mRNAs by 2-fold (n=3, P<0.05). Thus, for PE, ET-1, and PMA, aspects of acute signaling were intact in mouse myocytes.
Autonomous Hypertrophy of Mouse Myocytes
A potential basis for the absent or blunted hypertrophic responses in mouse myocyte cultures was revealed by detailed study of the myocytes under basal, serum-free conditions. Myosin staining showed an increase in myocyte area over time in the absence of any added growth factor (Figure 3A⇓, left 2 panels). This was not seen in rat cultures (Figure 3A⇓, right panel), in agreement with earlier results.2 3 4 Similarly, serial phase-contrast micrographs of the same field showed an increase in area of living myocytes (Figure 3B⇓). Mouse myocyte total protein per cell increased by 56% from ≈900 pg/cell on day 2 to ≈1400 pg/cell on day 5 (Figure 3C⇓), an increase not seen in rat cultures2 4 (Figure 3C⇓). In mouse cultures from day 2 to day 12, with medium change at day 7, hypertrophy reached a plateau at day 7; there was no change in myocyte numbers (not shown). Omission of insulin, transferrin, and BSA from the serum-free medium had no effect on hypertrophy (3 cultures, not shown). Hypertrophy tended to be greatest in C57BL/6>FVB/N>CD1 mouse myocytes (not shown).
Hypertrophy in basal, serum-free mouse cultures was also evident in mRNA assays (Figure 4⇓). ANF mRNA was just detectable in both mouse and rat myocytes on culture day 2 and then increased markedly (6-fold) in mouse myocytes by day 5 but did not change in rat cultures (Figure 4A⇓). Similarly, the predominant MyHC mRNA on mouse culture day 2 was α-MyHC, with β-MyHC just detectable, similar to the intact neonatal mouse heart (Figure 4B⇓) and to neonatal rat cultures.17 β-MyHC mRNA then increased greatly (6-fold) by mouse culture day 5, whereas α-MyHC mRNA decreased slightly (0.6±0.1-fold of day 2). Thus, cultured neonatal mouse myocytes had “autonomous” hypertrophy, with increases in myocyte area, protein, and ANF and β-MyHC mRNAs in the absence of added growth factors.
Effects of Conditioned Medium and Growth Factor Antagonists
To test whether autonomous hypertrophy of mouse myocytes might be due to some paracrine or autocrine growth factor not made by rat cells under the identical conditions, we tested conditioned medium from myocyte and nonmyocyte cultures of both species.13 Rat myocytes were used as a target because of the autonomous hypertrophy of mouse myocytes. Conditioned medium from cardiac nonmyocytes increased RLP markedly in rat myocytes, as seen previously,13 but medium from rat nonmyocytes was actually more potent than medium from mouse (≈75% increase versus ≈50%, Figure 5⇓). Conditioned medium from cardiac myocytes had a small but significant effect on rat myocytes (15 to 20%), but mouse and rat myocyte conditioned media were similar (Figure 5⇓). Thus, there was no evidence that autonomous hypertrophy of mouse cells was caused by paracrine or autocrine factors different from rat.13
We also tested antagonists of known hypertrophic agonists. There was no inhibition of autonomous hypertrophy by the β-AR antagonist timolol (2 μmol/L, RLP 1.0±0.02-fold of vehicle, n=4) or the angiotensin II receptor antagonists (Sar, Thr)-ATII (200 nmol/L, 1.03±0.02-fold, n=2) and (Sar, Ala, Val)-ATII (200 nmol/L, 1.00±0.00-fold, n=2). An ET-1A and ET-1B antagonist (PD 142893, 1 μmol/L) inhibited hypertrophy by only ≈10% (0.9±0.02-fold of vehicle, n=2). Thus, catecholamines, angiotensin II, or ET probably did not play a major role in autonomous hypertrophy.
Contractile Activity and Sarcomere Organization in Mouse Myocytes
About 20% to 40% of mouse myocytes in serum-free medium contracted spontaneously (beat), at greatly varying rates, with the higher fractions of beating cells observed at later times in culture (days 4 to 5). The proportion of beating mouse myocytes was much higher than in rat cultures (<10%). 2,3-Butanedione monoxime (2 mmol/L) inhibited myocyte beating but was toxic (≈50% myocyte death after 72 hours), and timolol (2 μmol/L) did not inhibit beating. However, verapamil (10 μmol/L) inhibited beating completely but inhibited hypertrophy by only ≈10% (RLP 0.9±0.02-fold of vehicle, n=3). Thus, contractile activity was largely dispensable for autonomous hypertrophy.
We also looked for myocyte cross-striations as evidence for sarcomere organization, which some consider a feature of the hypertrophic response. However, cross-striations were not obvious in mouse myocytes by phase contrast or MF20 staining (see Figure 3⇑). We did not use phalloidin.
Here we provide the first detailed characterization of a neonatal mouse culture model to study hypertrophy, and compare it with the rat model. The approach was almost identical to that validated previously for the rat, and replicate experiments were feasible despite the small size of the mouse heart. We show that the model will be useful to investigate hypertrophic signaling by catecholamines, thyroid hormone, and cytokines such as LIF. We also show that, under identical culture conditions, the mouse model differs from the rat model in an intriguing autonomous hypertrophy of mouse myocytes, a basal hypertrophy that appears to obscure or eliminate any responses to certain Gq- and PKC-coupled growth factors. Figure 6⇓ summarizes the main findings and compares the mouse and rat models.
We call “autonomous hypertrophy” the increases over time in mouse myocyte area, protein, and mRNAs (β-MyHC and ANF) in serum-free medium without added growth factors. Autonomous hypertrophy was robust, with increases comparable to those seen with α1-AR agonists in neonatal rat cultures.3 17 It is very curious why this basal hypertrophy is not seen with rat myocytes under identical conditions (present study and Simpson et al2 and Simpson4 ). Autonomous hypertrophy was accompanied by greater contractile activity than seen in rat cultures, but verapamil experiments suggested that beating was not a major stimulus for autonomous hypertrophy. Possibly the mouse cells make autocrine or paracrine factors different from those of rat cells,13 but conditioned medium experiments provided no evidence for this, and hypertrophy was not prevented by adrenergic, angiotensin, or ET antagonists. Certainly we cannot exclude the possibility that cultured mouse heart cells make some other short-lived and/or low-abundance factor(s) not made by rat, and indeed the very low cell densities (see Figure 3B⇑) suggest that any autocrine or paracrine factor must be quite potent. Mouse myocytes might also express growth factor receptors that rat myocytes do not. Alternatively, mouse myocytes might have constitutively active hypertrophic signaling. It will be very interesting to see, for example, whether the culture process in the mouse reduces some critical intracellular inhibitor(s) of hypertrophy or increases some activator(s).
Autonomous hypertrophy might share pathway(s) with α1-AR agonists, ET-1, PGF2α, and PMA. These and other agonists stimulate hypertrophy in the rat culture model that is dose related, with a maximum increase in myocyte size with any given growth factor.2 3 4 13 However, it is possible to detect additive hypertrophy with different factors2 (see below). Thus, the fact that these agonists were not additive with autonomous hypertrophy might suggest that autonomous hypertrophy activates maximally some common pathway(s). We tested whether absent hypertrophy with α1-AR agonists, ET-1, PGF2α, and PMA was caused alternatively by absent receptors on mouse myocytes, given that neonatal mouse myocytes do not express the α1C-AR subtype (D.G. Rokosh, L.D. Snyder, P.C. Simpson, unpublished data, 1999) that mediates hypertrophy in neonatal rat myocytes.15 However, we saw significant phospholipase C activation by PE and by ET-1, and ET-1 increased immediate-early genes. PMA signaling in mouse myocytes was robust by mitogen-activated protein kinase activation and fos/jun induction (Figure 2⇑); in addition, mouse myocytes express a complement of PKC isoforms similar to those of rat, and these are activated by PMA.23 Thus certain aspects of α1-AR, ET-1, and PMA signaling were intact, and it will be important to identify major pathway(s) common to these agonists and autonomous hypertrophy.
On the baseline of autonomous hypertrophy, T3, NE, and LIF stimulated additive hypertrophy with different MyHC mRNA phenotypes (Figure 6⇑). This result suggests that these agonists recruit hypertrophic signaling pathways different from autonomous pathways, at least in part. With T3, the magnitude of hypertrophy and the MyHC phenotype (increased α, reduced β) were identical in mouse and rat, indicating that the 2 species are not entirely different when cultured. The T3 effect in culture is perhaps analogous to the rat heart in vivo, in which treatment with T3 in pressure overload stimulates additional hypertrophy and reverses the MyHC phenotype.18 24 T3 signaling is an important paradigm, because T3 induces a physiological type of hypertrophy and prevents or reverses the transition to failure in pressure overload and other models.18 Thus, it is fortunate that T3 hypertrophic signaling can be studied in cultured mouse genetic models, where it might be easier to identify mechanisms for these beneficial effects.
Surprisingly, in the mouse model the catecholamine NE stimulated hypertrophy through a β-AR, rather than predominantly through an α1-AR as in rat myocytes,3 4 13 17 20 and at variance with other reports in mouse cultures.9 10 Equally surprising, NE induced α-MyHC and repressed β-MyHC, the same pattern as that of T3. Typically in the rat model, NE through an α1-AR induces β-MyHC and has no effect on α-MyHC (present study and Waspe et al17 ). NE likely activated a myocyte β-AR, not a nonmyocyte β-AR, because paracrine factors from nonmyocytes, such as transforming growth factor-β,25 induce β-MyHC and not α-MyHC.19 NE induction of α-MyHC is important as a signature of more physiological hypertrophic signaling18 and suggests pathway(s) alternate to the β-AR signaling that causes pathological hypertrophy and apoptosis in some transgenic and culture studies.26 27 Studies on myocytes from β-adrenergic transgenic and knockout animals in this culture model should help resolve these confusing issues.
The cytokine LIF, and to a lesser extent TNF-α, also caused hypertrophy in the mouse model, in agreement with prior reports.7 8 An effect on contaminating nonmyocytes was more difficult to exclude with LIF, because α-MyHC was not induced and β-MyHC was mildly repressed, which was possibly caused by dilution with nonmyocyte RNA. Against a dilutional artifact, 2 other myocyte-specific mRNAs were not reduced, α-MyHC and ANF; in addition, LIF was the only agonist that inhibited nonmyocytes (Table 2⇓). Effects on myocytes and nonmyocytes from mouse models involving interleukin-6 family cytokines and their gp130 receptor can be studied in this culture model.
In summary, neonatal mouse myocytes have a unique autonomous hypertrophy that might share a pathway(s) with α1-AR agonists, ET-1, PGF2α, and PMA, as well as phenotypically distinct hypertrophic signaling by NE, T3, and LIF (Figure 6⇑). The results emphasize differences in mouse and rat myocytes and underscore the diversity in hypertrophic phenotypes. The mouse model should be a very useful additional tool in studies of cardiac hypertrophy and failure.
P.C.S. is supported by the NIH and the Department of Veterans Affairs. X.-F.D. received a postdoctoral fellowship from the Medical Research Council of Canada. D.G.R. received a postdoctoral fellowship from the Western States Affiliate of the American Heart Association. We thank Marietta Paningbatan for her excellent technical assistance.
- Received May 30, 2000.
- Revision received August 29, 2000.
- Accepted August 29, 2000.
- © 2000 American Heart Association, Inc.
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