(Received for publication, February 13, 1996; and in revised form, March 8, 1996)
From the
Electrical stimulation of neonatal rat cardiac myocytes in
culture produces increases in myocyte size (hypertrophy) and
organization of actin into myofibrillar arrays. The maturation of the
cells is associated with enhanced contractile parameters and cellular
calcium content. The numbers and intensity of cellular mitochondrial
profiles increase, as measured by scanning laser confocal microscopy.
Consistent with the hypertrophic response is increased cellular content
of -myosin heavy chain and cytochrome oxidase subunit Va messages,
as well as increases in cytochrome oxidase activity in the stimulated
cardiac myocytes. Myocyte contractile capacity is associated with
increased expression of the muscle carnitine palmitoyltransferase
(CPT-I) isoform as measured by Northern analysis, immunoblotting, and
altered sensitivity of CPT-I activity to malonyl-CoA in the stimulated
cells. The data suggest that a switch from the liver isoform of CPT-I,
prominent in the neonatal rat heart, to the muscle CPT-I which
predominates in adult rat heart, takes place in the neonatal cardiac
myocytes over the same time period as the hypertrophic-mediated changes
in myofibrillar assembly and increased contractile activity.
Cardiac myocytes adapt to hemodynamic stress by compensatory
hypertrophy which increases cell mass, but not cell number. As a result
of increased energy demand due either to pathological or physiological
sequellae, gene expression was altered, mediating the expression of the
adaptive process. The signaling mechanisms by which the cardiac
myocytes translate the increase in hemodynamic stress to activate
intermediate early gene and contractile protein gene expression are not
well understood, but may involve production of growth factors expressed
not only by myocytes but also by non-myocytic cells in the intact
heart. Demonstration that some of these growth factors, e.g. transforming growth factor-, insulin-like growth factor,
endothelin, and angiotensin, are present in cardiac myocytes (see (1) for review) suggests that autocrine pathways may mediate,
in part, the hypertrophic response. The ability of mechanical stimuli
applied to cultured neonatal cardiac myocytes in the absence of serum
to enhance c-fos expression (2, 3) and to
stimulate myofibrillar growth and organization in vitro(4) suggests that increases in contractile activity of the
cardiac myocyte in culture may lead to the hypertrophic response by an
autocrine pathway. Although the alterations in contractile protein
content and expression and their role in ventricular remodelling have
been extensively studied in cultured neonatal rat cardiac myocytes, the
role of contractile stimulation of cardiac myocytes in the nuclear
transcription of mitochondrial-specific proteins has not been
described. It is reasonable to anticipate that alterations in energy
supply and demand would require compensatory proliferation of
mitochondrial proteins involved in energy transduction.
Following
electrical stimulation of primary ventricular myocytes in culture,
increased myofibrillar content and organization is accompanied by
increased expression of cardiac myosin light chain-2 and atrial
natriuretic factor(4, 5) . To date, this valuable
model has not been explored for effects of electrical stimulation on
cardiac mitochondrial gene expression. Mitochondrial protein expression
is known to increase in skeletal muscle with exercise training and
chronic electrical stimulation of intact muscle (see (6) for
review). In multicellular organisms, encoding of nuclear respiratory
genes is under both developmental and metabolic control as demonstrated
by coordinate expression of several proteins of the electron transport
chain(7) . Moreover, tissue specific isoforms representing
different mechanisms of regulation or catalysis within an organ have
been described for the adenine nucleotide translocase (8) as
well as for cytochrome c and cytochrome oxidase
subunits(9, 10) . The level of mRNA encoding for these
isoforms is likely growth-regulated as shown by stimulation of mRNA
levels by the presence of serum and growth factors(11) . An
enzyme system crucial to the energy metabolism of the heart via
oxidation of long-chain fatty acids is the mitochondrial carnitine
palmitoyltransferase-I (CPT-I)()(12, 13) .
In cardiac muscle, this protein exists in two isoforms recently
described as the liver and muscle isoforms(14) . These proteins
differ from each other in their affinities for both substrate and for
the metabolic inhibitor, malonyl-CoA. The liver isoform contributes
25% to total CPT-I activity in the neonatal heart, this level then
decreasing to comprise only 2-3% of CPT-I activity in the
adult(15) . Therefore, the growth related expression of
mitochondrial protein and of the heart isoforms of CPT-I was
investigated in this model of electrical stimulation where hypertrophic
growth and maturation of the cardiac myocyte occur after prolonged
increases in mechanical activity.
After four days in culture (24 h + serum, 72 h - serum), three wells containing myocytes were stimulated for 72 h using the method of Brevet et al.(17) as modified by McDonough and Glembotski (4) , using a GRASS stimulator, Ag:AgCl electrodes, and agarose gel cross-bridges that link the wells in parallel. Stimuli are 80 V with a pulse duration of 10 ms at a pacing frequency of 5 Hz. A cyclic polarity reversal, installed in the circuit by Dr. Craig Hartley, Baylor College of Medicine, reverses the output from the stimulator every 20 s. Three additional wells in the dish were maintained in the same medium as the stimulated cells, but in the absence of contractile stimulation. During the experimental period, the medium bathing the control and stimulated cells was changed after 48 h to fresh Dulbecco's modified Eagle's medium + 1% bovine serum albumin.
Primers were designed from published sequences. DNA and protein
homology searches were performed with BLAST, using GenEMBL and/or
SWISS-PROT data bases. The probes for CPT-I, -myosin, and
cyclophilin were obtained using total RNA from neonatal liver (CPT-I
liver isoform, 738 bp), neonatal skeletal muscle (CPT-I muscle isoform,
428 bp), and neonatal rat heart (
-myosin, 408 bp and cyclophilin,
402 bp) by reverse transcriptase-polymerase chain reaction
(PCT-100
, Watertown, MA) and the reverse
transcriptase-polymerase chain reaction kit from Promega Corp. The cDNA
probes were sequenced by the dideoxy-mediated chain termination method
and sequence analysis was performed using the Genetics Computer Group
sequence analysis package, version 7.2 (University of Wisconsin,
Madison, WI). The 650-bp cDNA fragment of cytochrome oxidase subunit Va
was purified from the vector pOC 1318/Eco (provided by Dr. E.
A. Schon, Columbia University) after digestion with EcoRI. 28
S rRNA cDNA was a gift from Dr. K. Chien. 18 S rRNA cDNA was provided
by Dr. Sue-Hwa Lin (M.D. Anderson Cancer Center) and purchased from
Ambion, Inc. The cDNA was purified from pT7 RNA 18 S using EcoRI and HindIII resulting in a 150-bp cDNA fragment
for use with cyclophilin as internal standards of RNA loading. The cDNA
probes were purified by 1% agarose gel electrophoresis where the uv
detected bands were cut from the gels and isolated by electrical
elution. The cDNA probes were labeled to high specific activity using
random primer labeling (Promega Corp.).
To confirm the hypertrophic effects of electrical
stimulation, cardiac myocytes were stained with the immunofluorescent
probe, BODIPY phallacidin (data not shown). Dramatic differences
between the control and the stimulated myocytes were seen, not only in
cell size, as described previously(4) , but also in the
organization of actin from punctate cores of actin characteristic of
the immature cardiac myocyte (29) into striated arrays of well
organized myofilaments. The effects of electrical stimulation were
assessed using planimetry, for changes in cell area, and for cellular
protein and RNA content. Cell size increases almost 4-fold, from 253
± 45 µm to 970 ± 88.5 µm
(p < 0.005). The alteration in size was accompanied
by significant increases in both protein (124 ± 3.2 to 162
µg ± 7.4/4
10
cells, p <
0.01) and RNA content (1.57 ± 0.2 to 2.5 µg ± 0.8/4
10
cells, p < 0.01) in the control versus stimulated cells, respectively. The increases observed
were consistent with the magnitude of changes observed in other models
of neonatal cardiac myocyte hypertrophy(30) .
In serum-free medium and in the absence of pacing, control cells were often quiescent for prolonged periods. With pacing, the contractility parameters of the control cells were significantly depressed when compared to cells which were exposed to 72 h of electrical stimulation. In the stimulated myocytes, time to peak contraction was abbreviated to 32.3 ± 9% of control values and percent shortening was dramatically increased by 7.85-fold from 6.6 ± 1 to 52.2 ± 2.7% (p < 0.01) in the stimulated cells. The peak calcium fluorescent intensities also increase in parallel with the enhancement in cellular contractility from relative peak fluorescence intensities of 42.6 ± 5.8 to 87.8 ± 9.3 in the stimulated cells.
Subsequent studies were designed to monitor the effect of the hypertrophic response, and accompanying increases in contractile function, on the cellular capacity for oxidative metabolism and energy production. Mitotracker labels mitochondria within living cells where it was oxidized as a fluorescent product(31) . A perinuclear distribution of fluorescence was easily discerned in the control myocyte and the relative fluorescent intensity of the dye was displayed in the ``fishnet'' plot (Fig. 1A). There was a dramatic increase in both mitochondrial content and fluorescent intensity in the cells which were electrically stimulated for 72 h (Fig. 1B).
Figure 1:
Increased
mitochondrial content in neonatal cardiac myocytes following electrical
stimulation in culture. Following stabilization of the cells in
serum-free medium, the control and stimulated cells were maintained for
an additional 72 h in serum-free medium. The stimulated myocytes were
stimulated as described under ``Experimental Procedures.'' At
the end of the 72 h, the cells were stained with Mitotracker (1000
nM) and cell fluorescence was visualized using a Molecular
Dynamics 2001 Scanning Laser Confocal microscope. A: top, fluorescent image of mitochondria from a typical control,
unstimulated neonatal cardiac myocyte; bottom,
three-dimensional-Fishnet plot demonstrating relative fluorescent
intensities of the mitochondrial membranes in the control cell. B: top, fluorescent image of mitochondria from a
representative stimulated neonatal cardiac myocyte; bottom,
three-dimensional-Fishnet plot where both the amount and intensity of
mitochondrial fluorescence were dramatically increased. The
magnification is 400 .
Consistent with the increased
mitochondrial content in the stimulated cells was a 1.7-fold increase
in cytochrome oxidase activity (Fig. 2). This increase in
cytochrome oxidase activity was accompanied by a 2.38-fold
(densitometry units) increase in cytochrome oxidase (subunit Va) mRNA
content in the stimulated cells (Fig. 3). The level of
-myosin heavy chain mRNA was also significantly elevated after 72
h of stimulation (Fig. 3). The re-expression of embryonic
contractile protein isoforms was characteristic of the hypertrophic
growth response in neonatal cardiac myocytes(32) .
Figure 2: Cytochrome oxidase activity in control and stimulated neonatal cardiac myocytes in culture. After stabilization of the cardiac neonatal myocytes in serum-free medium, the control and stimulated cells were maintained in culture for an additional 72 h, after which the myocytes were permeabilized with 1% octyl glucoside. Cytochrome oxidase activity was measured by following oxidation of cytochrome c at 550 nm(21) . The results are the mean ± S.E. of four different cell cultures.
Figure 3:
Increased content of -myosin heavy
chain and cytochrome oxidase (subunit Va) mRNA in control and
stimulated neonatal cardiac myocytes in culture. Following
stabilization of the cells in serum-free medium, control and stimulated
cells were maintained in culture for an additional 72 h. Northern blot
hybridization was carried out using the cDNA probes described under
``Experimental Procedures'' for
-myosin, cytochrome
oxidase, subunit Va, and cyclophilin. The hybridization signals for
-myosin heavy chain and for cytochrome oxidase are shown in the top two panels of total RNA from control (C) and
stimulated (S) neonatal cardiac myocytes. The lower panels are the hybridization signals obtained from cyclophilin, 28 and 18
S RNA, used as internal standards for RNA loading. The Northern blot
shown is representative of blots obtained with RNA preparations from
four different cell cultures.
Tissue-specific isoforms of mitochondrial proteins provide regulatory and/or catalytic properties which were uniquely responsive to metabolic signaling pathways of the organ. The heart contains two CPT-I isoforms, the liver isoform which decreases and the muscle isoform which increases in content with cardiac development(15) . To confirm the presence of message for these two proteins in the neonatal heart cells, Northern blots of the total RNA extracted from liver, heart, and muscle were probed with the cDNA sequences for the liver and skeletal muscle CPT-I isoforms. Liver CPT-I cDNA hybridizes with liver and neonatal heart RNA, but no signal was obtained from muscle total RNA (Fig. 4). Conversely, the cDNA probe for the muscle CPT-I hybridizes with RNA from heart and muscle, but not with liver RNA (Fig. 4). After 72 h of stimulation, the mRNA content of the liver isoform appears to decrease in the cardiac myocytes, whereas the mRNA content of the muscle isoform was increased in the stimulated cells (Fig. 4). The relative intensity of the signals from the control and stimulated cells was quantitated by densitometry. The mRNA content for the CPT-I liver isoform in the stimulated myocytes was approximately half the content that was present in the control cells (Fig. 5). Conversely, the mRNA for the muscle isoform increases 2.5-fold in the stimulated cells when compared to control (Fig. 5).
Figure 4:
Northern blot of liver and muscle CPT-I
isoforms: tissue specificity and abundance of mRNA in control and
stimulated neonatal cardiac myocytes. The total RNA is isolated from
liver, skeletal muscle, and cardiac tissue of neonatal rats and from
control and stimulated neonatal cardiac myocytes after 72 h in
serum-free medium in the presence and absence of electrical
stimulation. The cDNA probes representing the muscle and liver isoforms
of CPT-I were hybridized to the total RNA (10 µg/lane) from the
different tissues and cells, where the abbreviations, L, H, M, C, and S, stand for liver, heart, skeletal muscle, and
control and stimulated neonatal cardiac myocytes. In the top
lane, the 4.3-kilobase signal represents the mRNA for the liver
CPT-I isoform (4.7 kilobases, (24) ) and the 2.8-kilobase
signal, the mRNA for the muscle CPT-I isoform(25) . The blot is
representative of blots obtained with five different cell
cultures.
Figure 5: Content of liver and muscle CPT-I mRNA in control and stimulated myocytes. The Northern blot data from the experiments described in the legend to Fig. 4were scanned using densitometry (Imagemaster, Pharmacia) to quantitate the differences in levels of mRNA in the control and stimulated neonatal cardiac myocytes. The autoradiograph scans were normalized for exposure time (n = 5) where: *, p < 0.01 and**, p < 0.005.
To determine whether there was increased
expression of the muscle CPT-I protein, immunoblotting of the control
and stimulated cell lysates was carried out using the antibody against
the etomoxir-binding protein from rat heart. A 2.8-fold increase,
determined by densitometric scanning, in the amount of immunoreactivity
representing the predominant cardiac CPT-I isoform was detected in the
stimulated myocytes (Fig. 6). This increase in the lower
molecular weight isoform of CPT-I, which has been identified as the
muscle isoform (33) was accompanied by a 3-fold increase in
CPT-I activity in the stimulated cells (Fig. 6). The CPT-I
activity in the control and in the stimulated cells display differing
affinities of the enzyme for its inhibitor, malonyl-CoA. The
serum-deprived control cells display a lower CPT-I activity and an
IC for malonyl-CoA which was in the range observed for the
liver isoform of CPT-I, i.e. low µM (Fig. 7). The stimulated cells have a prominent high
affinity inhibitory response to malonyl-CoA with an I
of
approximately 90 nM which resembles the sensitivity of the
adult cardiac muscle with its predominant muscle CPT-I
isoform(14) .
Figure 6:
Increased cellular content of muscle CPT-I
isoform and enzyme activity in neonatal heart cells in culture
following electrical stimulation. The immunoblot of total cellular
protein in the control and stimulated cells was incubated with
polyclonal antibodies to a 79-kDa etomoxir-labeled protein from rat
heart and subsequently developed using a chemiluminescent detection
system (upper panel, 100 µg of total protein loaded per
lane). CPT-I activity (lower histogram) is defined as
malonyl-CoA sensitive activity measured in digitonin permeabilized
cells as described previously(16) . The activities represent
the mean ± S.E. for four different cultures where: *, p < 0.005 between control and stimulated cells; and ,
control cells; and
, stimulated
cells.
Figure 7:
Malonyl-CoA sensitivity of CPT-I
activities measured in neonatal cardiac myocytes in culture following
electrical stimulation. CPT-I activities were measured in
digitonin-permeabilized cells from control and electrically stimulated
cell cultures maintained in the absence of serum for 72 h following
stabilization in serum-free medium. CPT-I activity in the electrically
stimulated cells was measured in the absence and presence of a range of
malonyl-CoA concentrations ( = mean ± S.E., n = five separate cultures). The I
value was
determined after subtracting out the contribution of the low affinity
component. The malonyl-CoA sensitivity of the control cells maintained
in serum-free medium was shown in the inset, where activity is
measured over the same range of malonyl-CoA concentrations as for the
stimulated cells (
, mean ± S.E., n = five
separate cultures).
The RNA levels of mitochondrial proteins such as the adenine nucleotide translocase increase in response to serum(11) , similar to other growth-regulated genes, e.g. c-fos and c-myc(34, 35) . The adenine nucleotide translocase mRNA appears to increase in direct response to a growth factor signal(11) . This response of mitochondrial proteins to serum was consistent with the increased energy demands of proliferating cells (11, 36) . Nuclear promoter sequences as well as the cognate nuclear factors for these sequences have been characterized for cytochrome c by Evans and Scarpulla(36) . The physiological affector molecules which interact with the promoter elements were potentially important to the modulation of respiratory gene expression as energy demands of the cell were changed. This is the first report that documents mitochondrial proliferation in response to contractile stimulation in the absence of exogenous growth factors as evidenced by the higher levels of cytochrome oxidase mRNA and enzyme activity as well as the increased numbers of mitochondria observed microscopically in the stimulated cells. In addition, the effects of contractile stimulation on expression of the muscle-specific isoform of carnitine palmitoyltransferase I in the cultured cardiac myocytes is the first model system where a tissue-specific production of a mitochondrial protein can been ``switched on'' in the absence of serum factors.
A change in the expression of CPT-I isoforms has been
documented in the developing rat heart where liver CPT-I was reported
to contribute 25% to total cardiac CPT-I activity at
birth(15) . This value declines to 2-3% as the rat pups
mature to adulthood(15) . Recently, a cDNA clone encoding the
muscle isoform of heart CPT-I has been identified as distinct from the
liver CPT-I cDNA sequence (25) and expressed(26) , thus
providing the molecular basis for the biochemical findings.
Confirmation that both the liver and the muscle isoforms for CPT-I may
be present to varying degrees in the neonatal heart myocyte cultures
suggests that both proteins are important to cardiac development and to
the energy metabolism of the contracting heart. The physiological
importance of early expression of the liver CPT-I may relate to its low K
(30 µM) for carnitine, in keeping
with the low cardiac carnitine content at birth (15) . In the
serum-free cell cultures, we have evidence suggesting that the
predominant CPT mRNA expressed is the liver isoform, implying that
serum withdrawal leads to a less active contractile state in which
muscle-specific gene expression is diminished. In response to
contractile stimulation and activation of transcription of the muscle
CPT-I isoform, an increase in CPT-I activity is observed, as well as an
increase in the amount of immunoreactive 79-kDa protein which
corresponds to the dominant adult rat cardiac etomoxir-binding protein
(muscle CPT-I). Enhanced expression of this protein in the stimulated
cells was further supported by a dramatic alteration in the high
affinity binding properties for malonyl-CoA, with an approximate
40-fold decrease in the I
for malonyl-CoA after 72 h of
electrical stimulation. A double reciprocal plot of CPT-I activity versus carnitine concentration was also carried out (data not
presented). Whereas the stimulated cells demonstrated a single slope
with high activity and a K
which was consistent
with cardiac CPT-I activity, the control myocytes exhibit kinetic
behavior consistent with the presence of two different isoforms, i.e. two distinct linear regions with differing affinities for
carnitine. These results were consistent with the presence of both
isoforms of CPT-I in the cardiac myocyte, with the liver isoform being
in high enough concentration in the control neonatal cells to
contribute significantly to the velocities measured.
Tissue-specific
isoforms have been described for other mitochondrial proteins including
cytochrome c(9) , the subunit isoform of
mitochondrial ATP synthase(37) , as well as the adenine
nucleotide translocase(38) . It might be anticipated that the
kinetic properties and metabolic regulation of the isoforms should
reflect the physiological demands of that tissue as has been reported
for mitochondrial ATP synthase(39) . The predominance of the
high K
muscle isoform for CPT-I in skeletal muscle
and heart probably reflects the higher carnitine concentrations
endogenous to these organs(40) . Still unresolved, however, is
the adaptation required by the heart that results in such a profound
difference in the I
values for malonyl-CoA. Since measured
malonyl-CoA concentrations in heart may exceed the I
values for malonyl-CoA in that tissue by as much as 1000-fold,
investigators have hypothesized that the majority of the malonyl-CoA in
heart was compartmentalized away from the inhibitory site for
malonyl-CoA on the outer mitochondrial membrane(40) .
Alternatively, it has been suggested that the presence of the liver
isoform in heart may have a role in facilitating basal rates of fatty
acid oxidation when malonyl-CoA was in an inhibitory range for the
muscle isoform(15) . Unlike the liver CPT-I, the activity and
malonyl-CoA sensitivity of the muscle (cardiac) isoform was not
influenced by diabetes and starvation, conditions which alter the
sensitivity of the liver CPT-I for its inhibitor(41) .
The mechanisms which act to regulate the expression of nuclear genes during mitochondrial proliferation with subsequent differentiation to reflect tissue-specific expression include transcription, message stabilization, and activation of translation rates(42) . The two mRNAs which encode the muscle and liver CPT-I isoforms may be the product of different genes, as has been suggested for the adenine nucleotide translocase(38) . This possibility was strengthened by the observations that the liver and muscle CPT-I proteins were immunologically distinct (43) and that there is tissue specificity exhibited by the two cDNA probes(25) . Alternatively, the liver and muscle CPT-I proteins may be products of the same gene by either different transcriptional or post-transcriptional processing. It is possible that the nuclear encoded CPT-I muscle isoform, like the subunit Va of cytochrome oxidase, responds directly to electrical stimulation as a consequence of growth factor activation of cardiac myocyte calcium channels (44) or to other endogenous intracellular signals which promote cell growth in the absence of serum. In this regard, we have reported a potent mitogenic effect of insulin growth factor-1 (10 ng/ml) on CPT-I and CPT-II expression in the neonatal cardiac myocytes in the absence of serum(45) . Significantly, transient expression of insulin growth factor-1 occurred and increased insulin growth factor-1 mRNA levels were seen with pressure overload hypertrophy(46) . Therefore, the model of electrical stimulation provides an excellent tool to assess the role of mitochondrial proteins and the energy-producing mechanisms of the cell in the development of cardiac myocyte hypertrophy and in the physiological adaptation to increased energy demands as part of the mitogenic response. The role of c-fos oncogene expression in the mediation of this pathway during electrical stimulation is currently under investigation.