© 1995 American Heart Association, Inc.
Articles |
Some Growth Factors Stimulate Cultured Adult Rabbit Ventricular Myocyte Hypertrophy in the Absence of Mechanical Loading
From the Departments of Medicine/Cardiology (R.S.D., M.G.C., M.B.-B., M.L.D.) and Cell and Molecular Biology (R.S.D.), Northwestern University Medical School, Chicago, Ill.
Correspondence to Robert S. Decker, PhD, Department of Medicine/Cardiology S 207, Northwestern University Medical School, 303 E Chicago Ave, Chicago IL 60611.
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Abstract |
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Abstract Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-ß1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with ß-MHC synthesis being elevated and
-MHC synthesis
being suppressed. However, the other growth factors were able to
modestly stimulate the rate of DNA synthesis in this preparation.
Bromodeoxyuridine labeling revealed that these growth factors increased
DNA synthesis in myocytes and nonmyocytes alike, but the heart
cells displayed neither karyokinesis or cytokinesis. In
contrast, cocultures of cardiac myocytes and nonmyocytes and
nonmyocyte-conditioned culture medium failed to enhance the
rate of cardiac MHC synthesis or its accumulation, implying that
quiescent heart cells do not respond to "conditioning" by cardiac
nonmyocytes. These findings demonstrated that insulin and the
IGFs promote passively loaded cultured adult rabbit heart cells to
hypertrophy but suggest that other growth factors tested
may be limited in this regard.
Key Words: cardiac hypertrophy • growth factors • protein turnover • contractile proteins • DNA synthesis
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Introduction |
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Although neurohumoral activation and mechanical loading enhance the expression and turnover of contractile proteins and provoke the hypertrophy of cultured cardiac myocytes,1 2 3 recent observations documenting the presence of a variety of growth factor messages and their cognate peptides within the heart have led investigators to question whether the autocrine and/or paracrine action of these growth factors might provide alternative pathways to modulate cardiac myocyte growth.4 5 6 Components of the renin-angiotensin system have also been identified in cultured neonatal heart cells (ie, within myocytes and fibroblasts), and angiotensin II has been reported to stimulate protein synthesis and growth of some cultured heart cells.7 8 9 Similarly, the endothelium-derived peptide endothelin has also been demonstrated to enhance the expression and synthesis of contractile proteins and induce the growth of neonatal rat heart cultures.10 11 Other growth factor messages, peptides, and their putative receptors have been detected in both developing and adult heart, including IGFs,12 13 14 15 16 FGFs,17 18 19 20 TGF-ß,5 21 22 and a partially characterized growth factor derived from cardiac nonmyocytes.23 Additionally, Long et al24 have recently documented that ß-adrenergic agonists stimulate cardiac nonmyocytes to produce and secrete a peptide factor into culture medium that induces cellular hypertrophy when the "conditioned medium" is added to neonatal ventricular myocyte preparations. All of these "growth factors" have been implicated in regulating cellular hypertrophic growth in cultured neonatal heart cells, yet only insulin and IGFs have been demonstrated to increase protein synthesis in freshly isolated adult cardiac myocytes.25 Whether any of the growth factors mentioned above induce hypertrophic growth in cultured adult myocytes has not been fully explored26 and is the principal goal of this investigation.
The experiments presented extend our previous studies revealing that
physiological levels of insulin increased the fractional rate of
protein synthesis of cultured rabbit heart cells
25%.27 However, it was unclear from this preliminary
study or the observations from Sugden's group25 whether
the insulin-induced synthesis was accompanied by an elevated fractional
growth rate and a concomitant depression in the rate of proteolysis, as
has been proposed by others.28 29 The objectives of the
present study were to determine whether cultured adult cardiac myocytes
respond to recombinant growth factors by altering contractile protein
turnover and whether changes in protein synthesis and/or degradation
promote cellular hypertrophy. The results of this investigation
demonstrated that IGFs modulate protein turnover of cultured rabbit
heart cells, resulting in an accumulation in total and contractile
protein that appears to develop independently of mechanical load.
IGF-1–mediated growth appears to be regulated primarily through the
stimulation of protein synthesis, whereas IGF-2 enhances myocyte growth
by suppressing lysosomally mediated protein degradation. In contrast,
other recombinant growth factors, myocyte-nonmyocyte interactions, and
NMCM failed to stimulate the fractional rate of growth, although some
growth factors stimulated DNA synthesis in these primary cultures of
quiescent rabbit ventricular myocytes.
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Materials and Methods |
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Reagents
L-[4,5-3H]Leucine at 120 Ci/(mmol/L), [methyl-3H]thymidine at 80 Ci/(mmol/L), 2-amino-[1-14C]isobutyric acid at 50 mCi/(mmol/L), and 2-deoxy-D-[3H]glucose at 20 Ci/(mmol/L) were purchased from Amersham Corp. [N-methyl-14C]Dansyl chloride at 112 mCi/(mmol/L) was obtained from Research Products International, and class II collagenase was from Worthington Biochemical. Eagle's MEM and FBS were supplied by the Northwestern University Media Center, and partially purified laminin was obtained from Dr Thomas K. Borg, University of South Carolina. NU serum, insulin, and insulin/transferrin/sodium selenite were acquired from Collaborative Research, Inc. aFGF and bFGF, EGF, IGF-1, IGF-2, monoclonal anti–IGF-1 and –IGF-2 antibodies, IGF binding proteins 1 and 3, TGF-ß1, and PDGF-AB were purchased from UBI.
IR3 was supplied by Oncogene
Sciences. Anti–TGF-ß1 antibody was obtained from R&D Systems.
Nonimmune rabbit IgG, hyaluronidase (type 1-S), ara-C, and all other
culture grade reagents were obtained from Sigma Chemical Co.
Isolation and Culture of Ventricular Rabbit Myocytes
and Nonmyocytes
Male New Zealand White rabbits weighing 1.6 to 1.8 kg (7 to 8
weeks of age) were used in all the experiments described below. The
animals were purchased from an approved vendor (Lessers Rabbitry, Union
Grove, Wis) and housed, handled, and fed according to the protocols
outlined in the "Guide for Care and Use of Laboratory Animals"
(Department of Health, Education, and Welfare publication No. 85-23,
revised 1985, Office of Science and Health Reports, National Institutes
of Health, Bethesda, Md). The protocol for isolating and culturing
heart cells has been approved by the Northwestern University Animal
Care and Use Committee.
Cardiac myocytes were obtained from rabbit hearts that were perfused
retrogradely with collagenase and hyaluronidase as
described previously.27 Myocytes were cultured on
laminin-coated 60-mm Petri dishes at low density (2x105
cells per plate) in MEM supplemented with 5% fetal calf serum, 5% NU
serum, and 10 µmol/L ara-C for 1 week before exposing the cell
cultures to growth factors and/or anti–growth factor antibodies or
NMCM. Some cultures were not treated with ara-C to promote the
proliferation of interstitial cells27 ; such
2-week-old cultures were trypsinized, and the nonmyocytes were
collected and cultured in T-75 flasks in standard serum-supplemented
medium. When the fibroblast cultures reached confluence,30
the preparations were rinsed in serum-free medium (three times) and
then cultured in MEM plus 0.2% BSA to produce NMCM. NMCM was collected
at daily intervals for 1 week and frozen at -80°C before use.
Stimulation of Myocyte Growth
One-week-old myocyte cultures were rinsed in serum-free MEM
(three times) and exposed to growth factor–supplemented MEM containing
0.2% BSA for 4 days. Culture medium was changed on a daily basis, and
paired 60-mm Petri dishes were sampled each day for total protein and
DNA content and the fractional rate of protein synthesis as outlined
previously.31 Growth factor concentrations used in these
experiments were based on dose-response curves in which changes in the
value of P* was monitored at varying growth factor concentrations
ranging between 10-11 and 10-6 mol/L. The
specificity of growth factor responses was tested with neutralizing
antibodies or anti-receptor antibodies when such reagents were
available. To test its influence on cardiac myocyte growth, NMCM was
diluted into MEM plus 0.2% BSA in proportions ranging from 20% to
80% by volume.23 Myocyte preparations were exposed to
NMCM for a period of 4 days with NMCM/MEM medium being replaced at
daily intervals. BSA level was maintained at 0.2% in all the growth
factor/conditioned medium–supplemented myocyte cultures.
Derivation of the Fractional Rates of Protein Synthesis,
Accumulation, and Degradation
Heart cells were radiolabeled with 10 µCi/mL of
[3H]leucine during the last 4 hours of culture. The
medium was removed, frozen (-80°C), and assayed at a later date for
the value of F* (in disintegrations per minute per nanomole). The
cells were rinsed three times in serum-free MEM, extracted in 2 mL of
LSB (40 mmol/L NaCl, 1 mmol/L dithiothreitol, 0.1 mmol/L EGTA, and
0.1%/[wt/vol] Triton X-100, pH 7.2), and disrupted with a Polytron
PC1 homogenizer equipped with a microgenerator.
Aliquots were removed to measure total protein, DNA, and the fractional
rate of total protein synthesis as described in detail
elsewhere.30 31 The remainder of the extract (0.5 mL) was
centrifuged at 11 000g for 10 minutes, the
insoluble myofibrillar protein fraction was rinsed in LSB (four times)
and solubilized in running buffer, and equal amounts of contractile
protein were partially separated from one another on a 4% to 12%
vertical gradient SDS-PAGE slab gel. Actin, MHC, and desmin were
identified by Western blot, and corresponding gel bands were cut and
solubilized as reported previously.31 32 The washed
myofibril preparation was also used to partially purify -MHC from
ß-MHC on 4% SDS-PAGE gels. The specific radioactivity (in
disintegrations per minute per nanomole leucine) of dried
myofibrillar proteins was determined by derivatizing leucine with
[14C]dansyl chloride after hydrolyzing the protein(s)
with 6N HCl at 105°C for 24 hours.30 31 32 Fractional
synthesis rates were calculated for each protein fraction from the
measurements of P* and the precursor (F*tRNA) after
correcting for the ratio of leucyl-tRNA to free leucine in the heart
cells.31
The fractional rate of protein accumulation was obtained from cultures labeled continuously with 0.2 µCi/mL [3H]leucine. Total leucine content (in nanomoles) was quantified after dansylating either total protein or specific contractile proteins partially purified on SDS-PAGE gels as described above.32 The amount of protein-bound leucine that accumulated in response to growth factor treatment was derived by dividing the total [3H]leucine incorporated (disintegrations per minute per dish) by the leucine specific activity (in disintegrations per minute per nanomole) of the protein(s), yielding the amount of protein (ie, nanomoles of leucine) synthesized per culture.30 32 These values were plotted against time, and the fractional rate of protein accumulation was obtained by either linear or nonlinear regression analysis.30 31 33 The fractional rate of protein degradation was calculated indirectly from the difference between the rates of protein synthesis and accumulation.30 31 33 All fractional rates (synthesis, accumulation, and degradation) were expressed as percentage per day (±SEM).
Rates of protein degradation also were measured directly by using a double-label pulse-chase protocol.32 34 Rabbit cultures were labeled for 1 week in 0.1 µCi/mL [14C]leucine, and then 4 hours before termination of the continuous isotope-labeling period, the myocytes were pulsed with 2.5 µCi/mL [3H]leucine. Cultures were rinsed for 2 hours to remove unincorporated isotope and eliminate rapidly degraded proteins and then were chased for 120 hours in medium supplemented with growth factors and 2 mmol/L leucine to prevent reincorporation of the 3H- or 14C-labeled isotopes into new protein. The change in the [3H]:[14C]leucine ratio in cell protein(s) was plotted against time, and the half-life of total and specific protein was calculated as described by Clark.34
DNA Synthesis and Nuclear Labeling Index
DNA synthesis was assayed in six-well plates of myocytes or
confluent fibroblasts by determining the amount of
[3H]thymidine incorporated into DNA after a 24-hour
exposure of the cultures to various growth factors or
NMCM.26 Cultures were then labeled with 2 µCi/mL
[3H-methyl]thymidine for an additional 24
hours in growth factor–supplemented medium and rinsed (three times) in
ice-cold HBSS, and the cells were lysed in LSB buffer. An aliquot was
used to quantify DNA,31 and another was counted for
thymidine incorporation into TCA-precipitable material. DNA synthesis
was expressed in disintegrations per minute of
[3H]thymidine per microgram DNA per day (±SEM). The
number of myocytes or nonmyocytes that entered the synthetic
(S) phase of the cell cycle in response to growth factors was monitored
by incubating the cultures with BrdU (10 µmol/L, Sigma) and
respective growth factors for 24 hours at 37°C. Cultures were rinsed
with HBSS, fixed in 4% paraformaldehyde for 10
minutes, rinsed in HBSS (three times), and incubated in 5 µg/mL of
mouse monoclonal anti-BrdU antibody (Sigma). The distribution of
labeled nuclei was assessed with a peroxidase-labeled second antibody.
The BrdU labeling index was expressed as the number of BrdU-positive
nuclei tabulated per 100 myocytes/nonmyocytes (±SEM) and was
derived by counting five 1-mm2 random fields from each
stained culture with a Leitz Orthoplan microscope equipped with an
ocular micrometer.27 The total number of
labeled cells could be obtained by multiplying the labeling index by
the mean number of myocytes/nonmyocytes per culture.
Metabolite Uptake
[3H]2-Deoxyglucose and
2-amino-[1-14C]isobutyric acid were used to measure
hexose and amino acid uptake, respectively.35 Cultures
were treated with insulin, IGF-1, or IGF-2 for 4 hours and then pulsed
for 10 minutes in serum-free MEM supplemented with 5 µCi/mL
[3H]2-deoxyglucose at a final concentration of 0.2 mmol/L
2-deoxyglucose and 0.2 µCi/mL 2-amino-[1-14C]isobutyric
acid at a final concentration of 0.2 mmol/L -aminoisobutyric acid.
Cells were washed rapidly in ice-cold HBSS (twice) and lysed with cold
10% TCA. TCA-soluble radioactivity was counted in a liquid
scintillation spectrometer (model LS6000IC, Beckman, Inc), and uptake
of both analogues was expressed in disintegrations per minute per
microgram protein per hour (±SEM). Hexose and amino acid analogue
uptake experiments were conducted in media lacking
glucose.35
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Serum Regulation of Ventricular Myocyte Growth
Adult rabbit cardiac myocytes displayed enhanced rates of growth (ie, increased protein-bound [3H]leucine content per culture) in response to culture medium supplemented with FBS. Serum dose-response curves revealed that FBS levels above 10% (vol/vol) accelerated the fractional growth rate (rate of accumulation) maximally in these heart cell cultures (Fig 1
). In such
experiments, the fractional rate of growth ranged from 0.6% per day in
medium supplemented with 5% FBS to nearly 5% per day in heart cells
cultured in the presence of 20% FBS. Conversely, a negative nitrogen
balance rapidly developed in myocyte cultures maintained in the absence
of FBS; this negative balance resulted in significant myocyte atrophy
after 4 days of culture (Fig 1). In the absence of FBS, myocytes
displayed a negative growth rate of -6.4% per day, and supplementing
serum-free media with 0.2% BSA only reduced the rate (ie, fractional
growth rate of -3.2% per day) of protein loss somewhat (Fig 1). The
atrophy that evolved in these serum-free cultures resulted from a
reduction in the rate of protein synthesis and an acceleration of
protolysis; eg, the rate of protein synthesis was reduced 20% and
the rate of protein degradation was increased 30% in such
preparations when compared with heart cells cultured in the presence of
our standard 5% FBS (Table 1). When equal amounts of
protein were loaded on SDS-PAGE gels, the atrophic heart cells
displayed a marked reduction in MHC content, whereas actin and
intermediate filament protein (ie, desmin) content fluctuated only
modestly in response to serum deprivation (Fig 2). Moreover, high concentrations of FBS (20%) were
able to stabilize contractile protein content in these myocyte
preparations (Fig 2), but in such nonbeating heart cells, significant
cell spreading developed, and arrays of well-ordered myofibrils were
seldom visualized in these myocytes. Such observations led us to
question whether specific growth factors present in FBS were
responsible for modulating the growth of cultured adult cardiac
myocytes in vitro. The experiments reviewed below document the effects
of a variety of recombinant growth factors, potential
myocyte-nonmyocyte interactions, and cardiac NMCM on the
turnover of contractile proteins and the growth of adult rabbit heart
cells in vitro.
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Insulin and IGF Regulation of Protein
Metabolism
Insulin and IGF-1 accelerated the rate of protein synthesis in a
dose-dependent manner, whereas IGF-2 promoted only a modest increase in
the incorporation of leucine in paired heart cell preparations when the
hormone/growth factors were used at equivalent concentrations (Fig 3A). Insulin (10 nmol/L) provoked a 36% rise in the
value of P* in cultures depleted of serum, and IGF-1 (10 nmol/L)
stimulated a 42% rise in the leucine-specific activity of total
protein in paired cultures (Fig 3A). The EC50 for insulin
was 6x10-10 mol/L, and for IGF-1 the EC50 was
8x10-10 mol/L; in contrast, IGF-2 elevated P* 27%, but
the EC50 for the IGF-2 response was two orders of magnitude
greater (6x10-8 mol/L) than that displayed by either
insulin or IGF-1. If IGF-2 was administered at the concentration
required for IGF-1 to significantly increase protein synthesis
(10-8 mol/L), then IGF-2 induced only a 14.5% rise in P*.
When neutralizing antibody directed against IGF-1 was included with the
growth factor at a saturating concentration (100 µg/mL), the antibody
blocked the anticipated rise in P* (Fig 3B), whereas the antibody
itself or the addition of autologous IgG (100 µg/mL) induced no
change in P* when used in the absence of the growth factors (data not
illustrated). Likewise, a cross-reactive monoclonal anti–IGF-1
receptor antibody (IR3, 10 µg/mL36 ) significantly
inhibited the incorporation of [3H]leucine into total
protein when cultures were preincubated with the antibody for 2 hours
before and during the pulse labeling of the cultures in the presence of
IGF-1 (Fig 3B). The IR3 antibody failed to block the insulin-induced
rise in P* in paired myocyte cultures (ie, P*=922±37 versus 903±48
dpm/nmol leucine, n=5). In addition, IGFBP3 also markedly suppressed
leucine labeling of total protein in a dose-dependent fashion when
culture medium was supplemented with IGFBP3 and IGF-1 (Fig 3B). P*
values could be markedly elevated in myocyte cultures when they were
exposed to high concentrations of IGF-2 (
10-7 mol/L)
(Fig 3A); however, the rise in P* could be blocked by preincubating
cultures with the anti–IGF-1 receptor antibody (ie, P*=854±57 versus
695±47 dpm/nmol leucine, n=7). Both the anti–IGF-2 antibody (data not
shown) and IGFBP3 (P*=873±64 versus 722±47 dpm/nmol leucine, n=7)
also suppressed the increase in P* in IGF-2–treated (10-7
mol/L) preparations. Moreover, the rise in P* induced by supplementing
cultures with IGF-1 and IGF-2 (10-7 mol/L) was not
additive, suggesting that both growth factors may be activating the
same receptor.
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), IGF-1
(
), or IGF-2 (
). EC50 for protein synthesis
in response to insulin is 6x10-10 mol/L; for IGF-1,
the EC50 is 8x10-10 mol/L; and for IGF-2,
it is 6x10-8 mol/L (A). B, Effects of anti–IGF-1
antibodies (
Modulation of Myocyte Growth by Insulin and IGFs
Since the growth-promoting properties of insulin and the
IGFs are believed to be mediated by increasing the fractional rate of
protein synthesis while simultaneously suppressing the
fractional rate of protein degradation, changes in the rates of protein
synthesis, accumulation, and degradation were monitored in response to
insulin and the IGFs to determine whether fluctuations in the
fractional rate of myocyte protein accumulation were regulated
predominantly through anabolic or catabolic pathways. When changes in
the fractional rate of protein accumulation were derived from
preparations exposed to insulin or IGF-1 and compared with values
obtained from cultures maintained in our standard culture medium (MEM
plus 5% FBS), insulin (10-8 mol/L) enhanced the rate of
protein accumulation to approximately the same value as that derived
from serum-supplemented medium, whereas IGF-1 significantly
(P<.05) elevated myocyte growth above that induced by
either insulin, IGF-2, or 5% FBS. (Table 1
). Using the indirect
approach30 31 33 to monitor changes in the fractional rate
of growth, it became evident that the negative rate of myocyte growth
(ie, atrophy) that developed after serum deprivation
resulted from a combined action of simultaneously
depressing protein synthesis and enhancing proteolysis (Table 1). In
addition to the anabolic effects of insulin, the hormone also depressed
the fractional rate of degradation 20%, resulting in a growth rate
of 0.5% per day, a value similar to that reported for rabbit heart
cells that were cultured in the presence of 5% FBS (Table 1 and
Reference 3131 ). IGF-1 accelerated protein accumulation at approximately
a threefold greater rate than did insulin, and when the degradation
rate was derived, IGF-1 appeared to suppress protein degradation
somewhat more effectively than did insulin (Table 1). Conversely, IGF-2
accelerated growth only 0.3% per day, a sixfold slower rate than
IGF-1 when the growth factors were used at identical concentrations.
Although IGF-2 did not stimulate the rate of protein synthesis to the
same degree as did insulin or IGF-1, IGF-2 was a potent suppressor of
the rate of degradation, inhibiting proteolysis approximately one third
(Table 1). IGFBP3 (10-7 mol/L) blocked both IGF-1– and
IGF-2–dependent increases in the synthesis rate and prevented
reductions in the degradation rate, thereby maintaining the catabolic
state of the cultures (Table 1 and Fig 3). To independently verify that
the actions of insulin and the IGFs on protein turnover paralleled
other well-characterized metabolic events known to be
regulated by the hormone/growth factor(s),35 37 glucose
transport, amino acid uptake, and DNA synthesis were monitored in
paired myocyte preparations. In the present investigation, insulin
and IGF-1 were equally effective at enhancing 2-deoxyglucose and
-aminoisobutyric acid transport, but only IGF-1 stimulated
[3H]thymidine incorporation into DNA (Fig 4). When the BrdU-labeling indices were derived from
these cultures, it was evident that the nonmyocytes displayed a
significantly higher labeling index than did the myocytes (18.7±3.1%
versus 2.6±0.4%), implying that the nonmyocyte proliferative
potential was enhanced in response to IGF-1. IGF-2 used at a
concentration (10-8 mol/L) that inhibited
"accelerated" proteolysis had virtually no influence on
metabolite uptake and no affect on DNA synthesis in cultured rabbit
heart cells (Fig 4). Cultures exposed to insulin and the anti–IGF-1
receptor antibody (10 µg/mL) exhibited enhanced uptake of the hexose
and amino acid analogues, whereas the antibody completely blocked the
rise in metabolite transport and DNA synthesis induced by IGF-1 alone
(Fig 4). The present observations have revealed that insulin and
IGF-1 apparently modulate amino acid and glucose uptake through unique
receptor pathways and indicate that the atrophy induced in rabbit heart
cells cultured in the absence of serum could be reversed by insulin and
the IGFs, with IGF-1 inducing a greater rate of growth than either
insulin or IGF-2 in this regard. Furthermore, these results also
suggested that insulin and the IGFs exert their influence on myocyte
growth via their respective receptors.
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Contractile Protein Turnover in Response to Insulin and
IGFs
When equal amounts of myocyte protein were electrophoresed on 4%
to 12% linear gradient SDS-PAGE gels, low-salt extracts derived from
cultures depleted of serum for 4 days revealed a disproportionate loss
of MHC compared with changes in actin and desmin content (Fig 2).
Although these one-dimensional gels will not completely separate MHC,
actin, or desmin from other minor proteins of corresponding molecular
weights, previous two-dimensional gel patterns have provided evidence
that these myofibrillar/cytoskeletal proteins appear to
represent the predominant species present in their
respective molecular mass range.38 Therefore,
laser-scanning densitometry of these Coomassie blue–stained
gels31 provided an opportunity to quantify relative
changes of contractile protein content in growth factor–supplemented
and serum-depleted myocyte cultures. A 29% reduction in MHC content
developed after 4 days of exposure to a serum-free environment, the
amount of actin declined 17%, and desmin content was reduced only
10% after serum deprivation (Fig 2, lane 2 versus lane 4).
The removal of serum from the culture medium also depressed the
fractional rate of actin and MHC synthesis by 25% to 40% and that
of desmin by 7%, respectively (Table 2). Supplementing
the culture medium with IGF-2 (10 nmol/L) failed to enhance the
synthesis and/or accumulation of these proteins (Table 2 and Fig 2),
but IGF-1 (10 nmol/L) and, to a lesser extent, insulin increased the
rate of synthesis for all three myofibrillar/cytoskeletal proteins
(Table 2). When the fractional synthesis rates of the MHC isoforms were
measured in response to IGF-1, the results indicated that IGF-1
significantly elevated the synthesis rate for ß-MHC (1.8±0.5% per
day versus 3.2±0.4% per day, P<.05, n=6) while not
apparently affecting the rate of -MHC synthesis (1.2±0.4% per day
versus 1.5±0.4% per day, n=6). When the separated myosin isoforms
were quantified by laser-scanning densitometry, the relative ß-MHC
content of the treated myocytes increased from 62% of total MHC to
>70%, whereas -MHC content declined to 30% (n=4). The total MHC
content of these IGF-1–treated cultures compared favorably with paired
serum-supplemented cultures, and only minor reductions in actin or
desmin content could be documented in cultures maintained in the
presence of the growth factor (Fig 2, lane 2 versus lane 6).
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Cardiocyte cultures double-labeled with
[14C]leucine and [3H]leucine and then
chased for 120 hours in the presence or absence of serum, insulin, or
IGFs were used to directly monitor changes in the degradation of actin,
desmin, and MHC. Pulse-chase experiments revealed that the breakdown of
total protein (Fig 5) and of contractile (Table 3) and cytoskeletal proteins was biphasic, suggesting
the existence of at least two pools of protein that display unique
decay kinetics.31 32 34 Such experiments demonstrated that
over the first 48 hours of the chase period, 65% of the nascent
labeled protein turned over at a rapid rate (ie, half-life, 24
hours). However, if the half-life of this myofibrillar protein was
derived from the slope of the pulse/chase curve beyond 48 hours of
chase (Fig 5), then it became evident that the half-life of this same
protein pool lengthened significantly (half-life, 
12.3±0.8 days,
n=5). When such cultures were deprived of serum, degradation of nascent
protein was accelerated such that its apparent half-life was reduced
from 24 hours to 18 hours, while the half-life of long-lived
proteins remained essentially unchanged at 11.8 days. Moreover, the
rate of degradation of this long-lived pool of myofibrillar protein
appeared to be independent of the presence or absence of FBS in the
culture medium (Fig 5). Insulin and the IGFs appeared to selectively
inhibit the degradation of the nascent pool of protein when cultures
were maintained in a serum-free state (Table 3). Since serum and/or
insulin deprivation has been documented to accelerate
lysosomal proteolysis in perfused hearts39 and cultured
neonatal cardiac myocytes,40 radiolabeled cultures were
chased in the presence or absence of lysosomotropic agents that are
believed to selectively inhibit endosomal/lysosomal proteolysis. The
cysteine protease inhibitor E64 (Table 3),
leupeptin, and chloroquine (data not illustrated) inhibited accelerated
proteolysis 40%, with newly labeled protein being preferentially
spared during the first 48 hours of the chase period. Nevertheless,
myofibrillar/cytoskeletal protein breakdown (eg, MHC) was not altered
significantly by any of the growth factors or lysosomotropic compounds
used in these experiments (Table 3). The interventions discussed above
also failed to alter the degradation of long-lived proteins, including
the contractile proteins. The half-life of this long-lived pool
remained 12 days regardless of the presence or absence of insulin or
the IGFs.
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Influence of Other Growth Factors, Cell-Cell Interactions, and
Cardiac NMCM on Cardiocyte Growth
Cultured neonatal heart cells respond to a wide variety of growth
factors by increases in cell size, protein accumulation, and RNA
content.4 5 14 18 23 24 Dose-response curves obtained from
low-density rabbit heart cell cultures have revealed that of the growth
factors used in the present investigation, only TGF-ß1 was
capable of enhancing the fractional rate of protein synthesis (Table 4). Moreover, the modest change in the synthesis rate
induced by TGF-ß1 was of the magnitude observed with IGF-2 (Table 1),
but unlike IGF-2, the growth factor had no impact on the rate of
protein degradation; therefore, the myocytes remained in negative
nitrogen balance. TGF-ß1 also stimulated the fractional rate of MHC
synthesis (Fig 6), and neutralizing anti–TGF-ß1
antibodies (1 µg/mL) blocked the rise in the MHC synthesis rate
induced by the growth factor (2.5±0.3% per day versus 1.3±0.4% per
day, P<.05, n=5), demonstrating the specificity of the
response. Measuring the synthesis rates of - and ß-MHC isoforms
partially separated on 4% SDS-PAGE gels further revealed that the
growth factor (TGF-ß1) stimulated ß-MHC synthesis and suppressed
the synthesis of -MHC (Fig 6). Paired myocyte preparations exposed
for the same interval (ie, 4 days) to triiodothyronine
(10-7 mol/L) predominantly synthesized -MHC, confirming
the responsiveness of the cultured rabbit myocytes to thyroid
hormone31 ; nevertheless, triiodothyronine was unable to
elevate the fractional rate of total MHC synthesis under the
present culture conditions (Fig 6). Conversely, of aFGF, bFGF, EGF,
and PDGF-AB, none had any demonstrable affect on protein turnover in
this preparation (Table 4). In an attempt to determine whether the lack
of response to these growth factors could be correlated with the
suppression of other signal transduction pathways known to be
activated by these growth factors, DNA synthesis was measured
in separate cultures. All of the recombinant growth factors used in the
present investigation, with the exception of TGF-ß1, modestly
stimulated [3H]thymidine incorporation into the DNA
(Table 4). Since 3% (3.1±2.4%) of the cells in these rabbit
myocyte cultures were nonmyocytes that survived exposure to
ara-C for 1 week, the question arose whether the myocytes,
nonmyocytes, or both cell populations were incorporating
[3H]thymidine into nuclear DNA in response to the growth
factors. BrdU was used as a probe to resolve this issue. The number of
BrdU-labeled myocyte and nonmyocyte nuclei was tabulated, and
the results are illustrated in Table 4. The number of BrdU-positive
myocyte nuclei increased approximately twofold and the
nonmyocyte labeling index rose approximately fourfold to
fivefold after a 24-hour exposure to either aFGF, bFGF, EGF, or PDGF-AB
(Table 4). Although the BrdU labeling indices varied somewhat from one
growth factor to another, nonmyocytes consistently
displayed a strikingly higher labeling index than did the heart cells.
Such results convey the impression that the cultured
nonmyocytes have a significantly greater proliferative
potential than do the adult heart cells. It should be emphasized,
however, that none of the growth factor–treated myocytes that
disclosed BrdU-positive nuclei were observed in mitosis, and
karyokinesis also could not be documented in this preparation over the
duration of the experiments.
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|
The production of other growth-promoting factors also has been
reported, and two have been isolated and partially characterized from
cultured cardiac nonmyocytes prepared from neonatal rat
hearts.23 24 To determine whether a similar population of
cells isolated from retrogradely perfused adult rabbit hearts could
stimulate protein accumulation in rabbit heart cells,
ventricular myocyte cultures were established and
maintained in the absence of ara-C27 to foster the
proliferation of nonmyocytes. In another set of experiments,
conditioned medium was collected from confluent cardiac
nonmyocyte cultures and applied to 1-week-old rabbit heart cell
cultures exposed continuously to ara-C. In neither paradigm did
cellular hypertrophy (ie, an increase in the leucine
content of total protein or MHC) develop. P* values were elevated
dramatically in the cocultures, but the rise in the fractional rate of
protein synthesis and the increase in total protein-bound leucine
content was associated predominantly with the higher rate of
nonmyocyte protein metabolism in this
preparation.30 In such cocultures, the accumulation of MHC
was monitored as an indicator of hypertrophy because it is
a predominantly myocyte-specific protein. When the fractional rate of
MHC synthesis was derived from these same preparations, it was
depressed to the same degree as that derived from paired serum-depleted
myocyte cultures (Table 5). Nevertheless, the leucine
content of MHC was only slightly lower than the MHC content of heart
cells cultured in the presence of our standard medium supplemented with
5% FBS, implying that some modulation of contractile protein turnover
may have transpired in the coculture paradigm. Since no fine structural
evidence of physical contacts (ie, intercellular junctions) could be
documented between nonmyocytes and heart cells in coculture
(authors' unpublished data, 1994), a paracrine-acting diffusible
factor(s) was hypothesized to be responsible for inhibiting MHC
degradation. To test this hypothesis further, ara-C–treated cultures
were exposed to NMCM for 4 days; however, no changes could be
documented in the leucine content of total protein or MHC in these
"conditioned" cultures (Table 5). Furthermore, the fractional
rate of total protein and MHC synthesis remained depressed in these
preparations when compared with paired cultures maintained in the
presence of serum (Table 5). Therefore, the potential reduction in
proteolysis believed to have developed in coculture could not be
confirmed when ara-C–treated myocyte cultures were supplemented with
high concentrations of NMCM (80% [vol/vol]).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observations derived from this investigation reveal that a battery of recombinant growth factors and NMCM elicit a different repertoire of responses from adult cardiac myocytes than from other cultured heart cells. Only insulin and IGFs promoted significant cellular hypertrophic growth in these quiescent myocytes, with insulin and IGF-1 stimulating protein accumulation by enhancing synthesis and inhibiting proteolysis. In contrast, the small degree of growth obtained with IGF-2 apparently resides in its ability to inhibit protein degradation rather than elevating the fractional rate of protein synthesis. Other recombinant growth factors appeared to have only minimal influence on the turnover of rabbit myocyte protein, although all the growth factors, with the exception of TGF-ß1, induced some DNA synthesis in both myocytes and nonmyocytes alike. Cocultures of rabbit heart cells with myocardial nonmyocytes also failed to elevate the synthesis or accumulation of MHC, suggesting that cell-cell interactions appear to have a minimal role in regulating the growth of adult heart cells in the present in vitro conditions. Furthermore, no paracrine-acting diffusible factors could be identified in culture medium conditioned by cardiac interstitial cells that might modulate the turnover of contractile proteins in adult myocytes exposed to such media. It must be remembered, however, that a cell culture environment is unlikely to completely reflect the in vivo world; therefore, the results derived from this investigation are only indicative of the potential role(s) growth factors may have in modulating myocardial growth. Nevertheless, the experimental data reported here imply that many of the growth factors used in the present study failed to enhance the rate of contractile protein turnover or the fractional rate of growth of cultured adult cardiocytes when compared with the responses elicited from fetal or neonatal heart cells, in which such autocrine/paracrine acting factors are believed to be instrumental in regulating myocyte proliferation as well as growth during cardiogenesis.4 5
Insulin and the IGFs have been identified as members of a family of peptide hormones that possess a broad range of metabolic and mitogenic properties.37 Although cardiac and skeletal muscle have long been recognized as major targets of insulin action, recent reports have documented the presence of specific high-affinity receptors for IGF-1 and IGF-2 in primary cultures of skeletal41 42 and cardiac14 43 44 muscle cells and in a variety of established muscle cell lines.35 45 Both peptides have been implicated in regulating the growth and differentiation of developing muscle cells via autocrine/paracrine pathways42 45 46 ; however, the relative roles of the IGFs in modulating physiological growth in the adult myocardium continue to remain a controversial issue.47 48 Similarly, the enhanced expression of IGF-1 message and the appearance of a cognate peptide in the hearts of several animal models that develop cardiac hypertrophy have also implicated the growth factor as a potential regulator of myocyte growth in pathophysiological circumstances.15 16
That insulin and IGFs elevated the fractional rate of protein synthesis
of cultured adult heart cells confirms previous observations derived
from Sugden's laboratory25 and extends them by
demonstrating that the changes in protein turnover documented in the
present study were indeed translated into cellular hypertrophic
growth, albeit at rates significantly slower than those reported for
cultured neonatal heart cells.14 43 The mechanisms
mediating insulin and IGF-induced protein accumulation appear distinct,
however. Although insulin and IGF-1 appeared equally effective at
stimulating total protein synthesis, IGF-1 enhanced the fractional rate
of protein accumulation at approximately three times the rate of
insulin. Such differences may be explained, in part, because IGF-1 was
a more potent stimulator of contractile protein synthesis than insulin,
a property also shared by cultured skeletal muscle.49
Since direct and indirect measures of protein degradation (Tables 1 and 3) further demonstrated that IGF-1 and insulin suppressed proteolysis
equally, such observations imply that IGF-1 promoted a significantly
greater degree of myocyte growth through its ability to accelerate
contractile protein synthesis more than insulin. Whether IGF-1 is
operating through its receptor or the insulin receptor is an important
issue regarding the specificity of the growth factor responses. The
present study has addressed this concern by demonstrating that
neutralizing antibodies directed against the IGF-1
receptor36 blocked IGF-1 but not insulin-mediated
increases in protein synthesis and metabolite transport (Figs 3 and 4).
Although both the insulin and IGF-1 receptors possess tyrosine kinase
activity,37 they apparently function independently of one
another in cultured adult myocytes. Ito et al14 have
documented that IGF-1–enhanced expression and synthesis of contractile
proteins appeared to be regulated through the IGF-1 receptor in
cultured neonatal heart cells, and Eppenberger's
laboratory44 has reported that IGF-1 stimulated
myofibrillogenesis in cultured adult rat cardiocytes, providing
additional support for the contention that IGF-1 specifically modulates
contractile protein expression and turnover that appear to be
correlated with cardiocyte growth. Other reports that have
documented increases in IGF-1 message and protein in a variety of
animal models used to investigate the regulation of cardiac
hypertrophy further implicate the growth factor as a
putative autocrine/paracrine mediator of myocardial
growth.15 16 50
In contrast to the growth-promoting properties of insulin and IGF-1,
IGF-2 managed to induce only a meager increase in the fractional rate
of myocyte growth in the present study; nevertheless, it was a more
potent inhibitor of protein degradation than either insulin
or IGF-1 (Table 1). Furthermore, at a concentration (10 nmol/L) that
maximally inhibited proteolysis, IGF-2 failed to stimulate amino acid
and hexose transport or DNA and protein synthesis significantly, unlike
IGF-1 (Table 1 and Fig 4). Since many of the metabolic
consequences of IGF-2 are believed to be mediated through its binding
to the IGF-1 receptor in cultured skeletal and cardiac muscle
cells,14 35 41 42 we questioned whether the inhibition of
protein degradation documented in this report required IGF-2 activation
of the IGF-1 receptor. Two separate sets of experiments were conducted
to test this hypothesis. First, the observation that the IGF-2 receptor
shares a high degree of homology with the M-6-P receptor51
implied that IGF-2 effects on protein degradation may be mediated
through the modulation of endosomal-lysosomal pathways.52
Several observations have lent support to this contention. IGF-2 or
antibodies directed against the IGF-2/M-6-P receptor have been
documented to inhibit the uptake of lysosomal enzyme isoforms that
have a high affinity for that receptor.53 These acid
hydrolases are believed to be transported and recycled via this
receptor in cultured cells52 ; therefore, interfering with
this subcellular transport route may depress lysosomal proteolysis.
Anti–IGF-2/M-6-P receptor antibodies have been reported to block the
suppression of proteolysis normally encountered when cultured cells
were exposed to insulin or IGF-1.54 Last, the present
study has demonstrated that IGF-2 inhibits accelerated proteolysis to
the same degree as lysosomotropic agents; moreover, preincubation of
myocyte cultures with E64 or leupeptin eliminated the
ability of exogenous IGF-2 to suppress protein degradation that
normally is accelerated after serum deprivation. The second
set of observations that argue against the assertion that IGF-2 was
activating the IGF-1 receptor revealed that the anti–IGF-1 receptor
antibody blocked IGF-1–induced changes in protein synthesis and
metabolite transport but would not inhibit the IGF-2–mediated
reduction in proteolysis. That this change in protein degradation was
modulated by IGF-2 receptor could be corroborated indirectly by
demonstrating that IGFBP3 significantly increased protein degradation
in cultures incubated in the presence of anti–IGF-1 receptor antibody
and IGF-2. Only when cultured myocytes were treated with concentrations
of IGF-2 of 10-7 mol/L were protein synthesis and
metabolite transport elevated, and such changes could be inhibited by
the anti–IGF-1 receptor antibody. These observations imply that IGF-2
and IGF-1 may regulate protein turnover independently of one another in
cultured adult heart cells and further suggest that IGF-1 and IGF-2
could function in a cooperative fashion to modulate cardiac myocyte
growth.48 In addition, the presence of the IGFBPs provides
another site capable of regulating cardiac myocyte growth in
vivo37 48 ; consequently, a thorough knowledge of the
circulating levels of both IGFs and the IGFBPs is required before the
relative growth-promoting properties of each of the IGFs can be
documented in the heart.
The administration of a wide variety of other recombinant growth
factors, including EGF, aFGF, bFGF, and PDGF-AB, previously has been
demonstrated to induce a pattern of fetal/neonatal gene
transcription,18 55 DNA synthesis, and protein
accumulation in cultured neonatal heart cells.5 23 24 In
contrast to the growth-promoting properties of the IGFs documented in
the present study, EGF, aFGF, bFGF, and PDGF-AB stimulated DNA
synthesis modestly but failed to enhance the fractional rate of growth
or the synthesis of contractile proteins in adult rabbit heart cells
(Table 4). The present results also have demonstrated that both
myocytes and nonmyocytes are capable of synthesizing DNA in
response to these growth factors. These results have confirmed previous
reports derived from freshly isolated25 and cultured adult
rat cardiomyocytes19 26 and extended them by
demonstrating that a higher percentage of cardiac nonmyocytes
incorporate thymidine in response to the growth factors than do
myocytes. Even though aFGF and bFGF messages, peptides, and cognate
receptors have been demonstrated in adult heart
cells,19 20 cultured adult myocytes appear to have
retained the ability to synthesize some DNA in response to FGF, for
example, but no longer display a hypertrophic
response.23 24 Perhaps the subdued DNA synthetic capacity
retained by growth factor–stimulated rabbit myocytes results in the
polyploidy that has been reported to exist in some mature cardiac
myocytes.56 Although no attempt was made to investigate
the properties of these growth factor receptors, future studies must
address whether the signal transduction pathways that modulate the
growth of developing heart cells have become downregulated, uncoupled,
or modified in the adult heart cell.
Of the growth factors studied in the present investigation, only
TGF-ß1 stimulated protein synthesis in the rabbit heart cell
cultures. Both total protein (Table 4) and MHC synthesis were enhanced
over the 4-day exposure to the growth factor; furthermore, the
fractional rate of ß-MHC but not -MHC synthesis appeared to be
elevated in these preparations (Fig 6). Although modulation of MHC
synthesis in response to TGF-ß1 has not been reported previously, the
upregulation of ß-MHC gene transcription and the corresponding
inhibition of -MHC expression has been documented in neonatal
myocytes,6 46 55 implying that in this instance, TGF-ß1
may activate similar signal transduction pathways in adult
cardiac myocytes. It must be emphasized, nevertheless, that a net
negative nitrogen balance accompanied TGF-ß1 treatment in this model
system, unlike the results derived from neonatal
preparations.23 24 TGF-ß1 expression also has been
reported to be upregulated in the hearts of aortically banded
rats,21 in infarcted
myocardium,57 and in hamsters displaying a
genetically based cardiomyopathy.58
The ability of TGF-ß1 to elevate the synthesis of ß-MHC while
simultaneously suppressing the synthesis of -MHC in
adult rabbit heart cells sustains previous observations that the growth
factor may be influential in modulating the expression of fetal
contractile protein isoforms that are known to be upregulated during
cardiac hypertrophy.6 The responses induced in
adult rabbit heart cells by the peptide growth factors discussed in
this and the preceding paragraph appear to be divided into two camps.
EGF, aFGF, bFGF, and PDGF-AB stimulated DNA synthesis but failed to
provoke cellular hypertrophic growth. TGF-ß1, on the other hand,
enhanced the synthesis of ß-MHC and suppressed -MHC synthesis
while not inducing DNA synthesis in the rabbit myocyte preparation. The
present observations suggest that adult heart cells apparently
share some but not all of the responses previously reported in cultured
neonatal cardiac myocytes when such preparations were exposed to
exogenous recombinant growth factors.
In addition to the potential autocrine/paracrine effects of
well-characterized growth factors known to be present in the adult
heart,59 others have reported that cell-cell interactions
and the production of diffusible factors from cardiac
nonmyocytes also may regulate myocyte growth and the expression
of contractile proteins.23 24 60 The two experiments
described in the present study were unable to confirm these
observations. Rabbit myocytes cultured in the absence of ara-C become
surrounded by cardiac nonmyocytes such that by 7 days of
culture 85% of cells are of nonmyocyte origin.
Nevertheless, no ultrastructural evidence of cell-cell contacts of the
variety (ie, gap junctions between myocytes and mesothelial cells)
described by Eid et al,60 could be demonstrated in the
adult rabbit preparations (authors' unpublished data, 1994).
Furthermore, the synthesis of MHC in such preparations was depressed
somewhat, although the leucine content of MHC remained higher than in
cultures lacking nonmyocytes, suggesting the potential
existence of cell-cell interactions that may modulate the turnover of
MHC. A second set of experiments supplementing myocyte cultures with
conditioned medium derived from nonmyocyte cultures, however,
failed to elevate leucine incorporation into total protein or MHC even
at the highest (80% [vol/vol]) concentration of conditioned medium
used in the present study (Table 5). Since no significant increase
in MHC synthesis could be documented, these results imply that
coculture may inhibit protein degradation, but additional experiments
will be required to clarify this observation.
In summary, the present experiments have demonstrated that cultured
adult myocytes display cellular hypertrophic growth when exposed to
exogenous insulin or the IGFs but do not accumulate contractile
proteins when treated with several other growth factors that are
thought to regulate the growth of developing heart cells. Like
/ß-adrenergic agonists, which provoke similar changes in the
fractional rate of cardiocyte growth in vitro,31
the IGFs appear to represent another autocrine/paracrine
pathway that regulates adult cardiocyte protein turnover.
Fluctuations in the fractional rates of protein synthesis,
accumulation, and degradation derived from adult rabbit myocyte
cultures also compare favorably with values obtained from the hearts of
rabbits, in which such measurements were made after infusion with
radiolabeled amino acids.31 61 62 63 In these investigations,
neurohumoral,31 61 mechanical,63 and
nutritional62 manipulation of the rabbits generated
changes in the fractional rates of protein turnover that either
provoked cardiac hypertrophy or atrophy. Changes in these
parameters were of the same magnitude as those documented
in the present study. Although it is difficult to extrapolate the
results derived from an in vitro to an in vivo setting, cultured
quiescent adult rabbit cardiac myocytes allow one to directly explore
the pathways and mechanisms that regulate cardiocyte growth in
the absence of significant mechanical loading and will provide future
opportunities to unravel potential neurohumoral and mechanical
synergisms that may exist in the heart. The present results support
the contention that the IGFs represent another major player
with a potential to modulate the growth of the adult
myocardium.
|
Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a US Public Health Service grant (HL-33616) and by funds provided by NASA (NAGW-3615). Dr Decker is a member of the Feinberg Cardiovascular Research Institute, Northwestern University Medical School. The authors thank Dr William A. Clark for his lively interest in this project and critical comments on the report.
Received May 23, 1994; accepted May 17, 1995.
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