(Received for publication, February 16, 1995; and in revised form, May 19, 1995)
From the
The purpose of this study was to determine the mechanism by which contraction acutely accelerates the synthesis rate of the contractile protein myosin heavy chain (MHC). Laminin-adherent adult feline cardiocytes were maintained in a serum-free medium and induced to contract at 1 Hz via electrical field stimulation. Electrical stimulation of contraction accelerated rates of MHC synthesis 28%, p < 0.05 by 4 h as determined by incorporation of phenylalanine into MHC. MHC mRNA expression as measured by RNase protection was unchanged after 4 h of electrical stimulation. MHC mRNA levels in messenger ribonucleoprotein complexes and translating polysomes were examined by sucrose gradient fractionation. The relative percentage of polysome-bound MHC mRNA was equal at 47% in both electrically stimulated and control cardiocytes. However, electrical stimulation of contraction resulted in a reproducible shift of MHC mRNA from smaller polysomes into larger polysomes, indicating an increased rate of initiation. This shift resulted in significant increases in MHC mRNA levels in the fractions containing the larger polysomes of electrically stimulated cardiocytes as compared with nonstimulated controls. These data indicate that the rate of MHC synthesis is accelerated in contracting cardiocytes via an increase in translational efficiency.
Hypertrophic growth occurs in terminally differentiated adult cardiocytes by an increase in cellular mass via a relatively coordinate increase in the proteins comprising each of the cellular components (1) . This accumulation of cardiocyte proteins occurs by an increase in rates of protein synthesis relative to rates of protein degradation (2) . The anabolic changes that occur during hypertrophy of the adult myocardium are generally considered to be a compensatory response to an increase in hemodynamic load(3) . As demonstrated in studies employing isolated papillary muscle preparations, both the active tension and passive strain components of load have been identified as mechanical stimuli for accelerating protein synthesis rates in adult myocardium (4, 5) . These findings have been extended to isolated adult cardiocytes in culture. Rates of protein synthesis were accelerated in response to electrically stimulated cardiocyte contraction(6, 7) , in response to a basal load as defined by adherence of the cardiocytes to the culture dish and establishment of a resting length (8) and in response to passive stretch of cardiocytes adherent to a deformable membrane(9) . Thus, the integrity of adult cardiocytes in primary culture is maintained with respect to the ability to transduce changes in mechanical load into an anabolic response such as accelerated protein synthesis rate.
The specific
mechanisms by which cardiocyte protein synthesis is regulated probably
occur at many levels, including transcriptional, post-transcriptional,
and translational processes(10) . It is well established, for
example, that transcriptional and post-transcriptional mechanisms
regulate steady state mRNA levels and are responsible for qualitative
changes in gene expression during cardiac hypertrophy (11, 12) . At the translational level, protein
synthesis rates can be accelerated by changes in efficiency or
capacity(2, 13) . Translational efficiency refers to
the efficiency with which the cardiocyte utilizes translational
machinery such as mRNA, ribosomes, initiation factors, and elongation
factors; whereas, translational capacity refers to the relative
abundance of these translational components in the cardiocyte, in
particular the ribosome and translation factor pools. Protein synthesis
rates are accelerated by an increased translational capacity during
sustained hypertrophic growth of the
myocardium(2, 14, 15) . In contrast, most of
the evidence for changes in translational efficiency is found in acute
studies in which protein synthesis rates increased within hours after a
load was imposed(5, 16, 17, 18) .
These studies suggest that cardiocyte protein synthesis could be
regulated by a temporal sequence in which changes in translational
efficiency are followed by sustained changes in translational capacity.
In skeletal muscle, alterations in load have a marked effect on
translational efficiency, particularly in the acute
phase(19, 20, 21) . For example, unweighting
of the soleus muscle in rats results in a reduction in MHC ()synthesis which has been attributed to a decrease in
translational activity(21) .
In previous studies employing adult feline cardiocytes in primary culture, we demonstrated that electrically stimulated contraction resulted in an acute acceleration of both total protein synthesis and MHC synthesis rates(6) . Two lines of evidence suggested that this acceleration occurred via a mechanism involving an increase in translational activity. First, protein synthesis rates were accelerated by contraction, even when transcription was blocked with actinomycin D. Second, because the effects of electrical stimulation were observed by 1 h, the acceleration of protein synthesis preceded any measurable changes in the amount of translational machinery as reflected by an increase in the ribosomal pool. In this study, MHC was used as a cardiocyte-specific marker to determine whether translational mechanisms are involved in the acute acceleration of the rate of protein synthesis in response to electrically stimulated contraction of adult cardiocytes. Comparisons were made between quiescent cardiocytes and cardiocytes electrically stimulated to contract by measuring the synthesis rate of MHC protein, the size of the MHC mRNA pool, and the translational efficiency of MHC mRNA. These studies demonstrated that: 1) rates of MHC synthesis are accelerated by electrical stimulation without a corresponding change in steady state mRNA levels, and 2) the mechanism for accelerating MHC synthesis in contracting cardiocytes is an increase in translational efficiency as reflected by a shift of MHC mRNA into larger polysomes.
Figure 1: Schematic diagram of the electrical stimulation apparatus. A physiologic stimulator was employed to initiate contraction via electric field stimulation. Electrical pulses of alternating polarity were delivered to the medium via carbon electrodes positioned at the ends of the culture tray. The arrows denote alternating direction of current.
For the slot-blotting analyses, a 215-base pair cDNA
clone of feline cardiac -MHC mRNA was employed. DNA sequence
analysis revealed that the feline species was greater than 92% similar
to rat
-MHC mRNA in the region between nucleotides 4028 and 4243
and to human
-MHC in the region between 4017 and 4232. The cDNA
insert was subsequently subcloned into a pGEM-3Zf(+) plasmid.
[
P]dCTP-labeled cDNA probes were generated by
the polymerase chain reaction using oligonucleotide primers
complementary to the T7 and SP6 promoter regions flanking either side
of the cDNA insert.
To prepare polysomes for RNase protection assays, post-mitochondrial supernatants were layered onto 15-50% linear sucrose gradients and centrifuged for 95 min at 32,000 rpm in an SW-41 rotor (Beckman Instruments). Gradients were fractionated into seven fractions of 1.2 ml each, starting with the 40 S subunit peak, followed by phenol/chloroform extraction and ethanol precipitation using tRNA as carrier. MHC mRNA levels in each fraction were determined by the RNase protection assay as described above. Prior to hybridization, aliquots were taken to measure 28 S rRNA by slot blotting.
Polysomes were prepared for the slot blotting
method by layering the post-mitochondrial supernatants on sucrose
gradients and centrifuging for 95 min at 35,000 rpm in an SW-41 rotor.
The gradients were fractionated into eight fractions of 1.2 ml each
starting with the top of the gradient. The samples were
phenol/chloroform-extracted and ethanol-precipitated. The RNA was
resuspended in 67% formamide, 7% (v/v) formaldehyde, 25 mM MOPS, pH 7.0, 3 mM EDTA, 0.8 mM sodium acetate
and heated at 60 °C for 10 min. The RNA was immobilized on Hybond-N
membranes (Amersham) by means of a slot blotting apparatus and UV
cross-linked. The blots were hybridized at 42 °C in a buffer
containing 50% formamide, 10 Denhardt's solution, 50
mM Tris, pH 7.5, 0.1%
Na
P
O
, 1% SDS, 100 µg of salmon
sperm DNA/ml and
P-labeled feline MHC cDNA probe in
excess. The blots were washed three times over 1 h with 2
SSC,
0.1% SDS at 42 °C, followed by three more washes over 1 h at 70
°C in 0.1
SSC, 0.1% SDS. The membranes were processed for
autoradiography and the optical density of the hybridization signals
were measured by computer-assisted image analysis. In order to
normalize the MHC signal to the amount of RNA recovered in the polysome
fractions, the blots were stripped and hybridized to the 28 S rDNA
probe. The 28 S rRNA signal was processed for autoradiography and
measured by computer-assisted image analysis.
We have demonstrated previously that contraction induced by electrical stimulation accelerated rates of total protein synthesis and fractional rates of MHC synthesis. In the present studies, relatively large numbers of cardiocytes were needed for the preparation of polysomes and RNA. The system was adapted to electrically stimulate large trays of cardiocytes with the use of carbon electrodes (Fig. 1). In order to validate that the same acute anabolic response as observed before (6) was elicited in cardiocytes using this system, rates of MHC synthesis were measured over 4 h of electrically stimulated contraction and compared with nonstimulated controls. In agreement with previous studies, contraction acutely accelerated the rate of MHC synthesis as compared with nonstimulated controls (Fig. 2).
Figure 2: Effect of contraction on MHC synthesis rates. Rates of MHC synthesis were determined after 4 h of labeling in electrically stimulated and quiescent cardiocytes. Asterisk, significant difference, p < 0.05 as determined by a paired Student's t test. Values are mean ± S.E., n = 8 cardiocyte preparations.
In order to determine whether the
acceleration of MHC synthesis was due to an increase in MHC mRNA
levels, MHC mRNA expression was quantified using an RNase protection
assay. In Fig. 3, two controls for the RNase protection assay
are shown. In Fig. 3A (lane 1), it is
demonstrated that the antisense cRNA probe derived from rat -MHC
cDNA hybridized specifically to feline
-MHC mRNA, the isoform
expressed in adult feline cardiocytes. As indicated under
``Materials and Methods,'' this reflects the fact that the
probe is derived from a region that is highly conserved between
-
and
-MHC isoforms. In lane 2, the specificity of the
feline
-MHC cDNA probe used for slot-blot analysis is shown. In Fig. 3B, the linearity of the RNase protection assay is
demonstrated by plotting the hybridization signal of the protected MHC
band as a function of the amount of total feline cardiocyte RNA added
to the assay. These data demonstrate the sensitivity of the assay for
detecting changes in MHC mRNA levels in feline cardiocyte mRNA.
Figure 3:
Specificity of probes for feline cardiac
MHC mRNA. A, Northern blots showing the specificity of the rat
-MHC cRNA probe (lane 1) and the feline
-MHC cDNA
probe (lane 2). 15 µg of total RNA extracted from adult
cat ventricle was run per lane. B, linearity of the RNase
protection assay for MHC mRNA levels in adult feline cardiocytes. Total
RNA was extracted from quiescent cardiocytes and added to the assay in
the indicated amounts. The optical density of the protected MHC band on
the autoradiogram was measured by computer-assisted digital image
analysis. R
=
0.99.
In Fig. 4, MHC mRNA levels were compared between electrically stimulated (S) and nonstimulated controls (C) in each of six experiments. Fig. 4A is an autoradiogram showing the MHC bands following RNase protection. In order to normalize for the amount of total RNA that was recovered and added to the assay, aliquots of each diluted sample were slot-blotted and probed for 28 S rRNA. The summary data of MHC mRNA normalized to 28 S rRNA are shown in Fig. 4B. These data demonstrate that electrically stimulated contraction did not significantly alter MHC mRNA levels. A difference in the MHC signal, such as that observed in experiment number 2, was the result of a differential recovery of total RNA extracted from the cardiocytes. There was not a difference in MHC levels in this particular experiment when corrected for the amount of 28 S rRNA added to the assay. Thus, the acceleration of MHC synthesis measured after 4 h of electrical stimulation occurred without any significant changes in MHC mRNA levels.
Figure 4: MHC mRNA content in quiescent and electrically stimulated cardiocytes. A, a representative autoradiogram of an RNase protection assay for MHC. Total RNA in stimulated (S) and control (C) cultures from six different experimental preparations was assayed. B, summary data of digital image analysis of the autoradiogram in A. Optical densities were normalized to 28 S rRNA in each sample. There was no significant difference between the two groups as determined by a Student's t test. Values are the mean ± S.E., n = 6 cardiocyte preparations.
Because rates of MHC synthesis were accelerated without a change in MHC mRNA, we examined whether the existing MHC mRNA pool was utilized more efficiently for protein synthesis in electrically stimulated cardiocytes. In order to determine the relative amount of the MHC mRNA pool active in translation, we quantified the amount of MHC mRNA that was located in the bound, polysome fractions and in the free, non-polysome fractions of the gradients. Post-mitochondrial supernatants prepared from whole cell extracts were layered onto 15-50% linear sucrose gradients and centrifuged. The gradients were fractionated into a lighter fraction containing mRNP particles, 40 S and 60 S subunits and 80 S ribosomes, and a heavier fraction containing polysomes (refer to absorbance tracing in Fig. 6A). As demonstrated in Fig. 5A, the amounts of MHC mRNA recovered in the free (F) and polysome-bound (B) fractions were the same in electrically stimulated and nonstimulated cardiocytes. Summary data from four experiments are shown in Fig. 5B, confirming that approximately equal amounts of total MHC mRNA were present in the actively translating polysome region of the gradients in both electrically stimulated and nonstimulated cardiocytes. The same results were obtained when MHC mRNA levels were normalized to recovered 28 S rRNA (data not shown). Thus, the acceleration of MHC synthesis was not accounted for by a significant mobilization of MHC mRNA into polysomes.
Figure 6: Effects of electrically stimulated contraction on the distribution of MHC mRNA in polysome gradients. A, a polysome profile of quiescent cardiocytes demonstrating the resolution of the polysome gradient procedure. Seven gradient fractions of 1.2 ml each were collected as indicated by upward displacement and the absorbance at 254 nm was monitored. B, a representative autoradiogram of an RNase protection assay for MHC mRNA content in the corresponding gradient fractions from electrically stimulated and nonstimulated cardiocytes. C, summary data of RNase protection assays for MHC mRNA distribution in polysomes. The gradients were divided into three regions as described in the text. The percentage of MHC mRNA in the gradient fractions encompassing each of these regions was divided by the percentage of 28 S rRNA in each corresponding region of the gradient. Asterisk, significant difference, p < 0.03 as determined by a paired Student's t test. Values are mean ± S.E., n = 6 cardiocyte preparations.
Figure 5: MHC mRNA in free and polysome-bound cardiocyte fractions. A, a representative autoradiogram of an S1 RNase protection assay for MHC mRNA in the free (F) versus polysome bound (B) fractions following sucrose gradient fractionation. The amount of MHC mRNA in each fraction was determined by the RNase protection assay. B, summary data. The relative amount of MHC mRNA in each fraction was calculated as a percentage of total MHC mRNA. There were no significant differences in MHC mRNA content in the free or polysome fractions as determined by Student's t test. The same results were obtained when MHC mRNA signal was normalized to recovered 28 S rRNA Values are mean ± S.E., n = 4 cardiocyte preparations.
Because the relative amount of the MHC mRNA pool in polysomes was unchanged in electrically stimulated cardiocytes, we investigated whether the distribution of MHC mRNA in the actively translating polysomes was changed. Cardiocyte extracts were centrifuged on 15-50% linear sucrose gradients as before and the gradients fractionated into 1.2-ml fractions starting with the 40 S ribosomal peak. Fig. 6A is a representative gradient profile showing the resolution of the fractionation procedure and the content of each fraction. Fig. 6B shows autoradiograms of an RNase protection assay which demonstrated that in nonstimulated cardiocytes, the majority of MHC mRNA was localized to the first three fractions. There were much smaller amounts in fractions 4 through 6, the region of the gradient that contained the larger polysomes. In contrast, electrical stimulation of contraction resulted in a shift of MHC mRNA into gradient fractions 4 and 5 corresponding to larger polysomes.
In Fig. 6C, summary data are presented for six separate experiments using the RNase protection assay to measure MHC mRNA in the gradient fractions. Using absorbance traces such as that in Fig. 6A as a reference, the gradients were divided into the ribosomal subunit and 80 S ribosome region (Gradient Fraction 1), the small polysome region (Gradient Fractions 2 and 3) and the large polysome region (Gradient Fractions 4-6). It should be noted that there was some overlap of the 80 S ribosome peak between gradient fractions 1 and 2 and of the peak containing four polysomes between gradient fractions 3 and 4. An MHC mRNA signal above background was not detected in gradient fraction 7. The percentage of MHC mRNA in the gradient fractions encompassing each of these regions was calculated from the sum total of MHC mRNA in all the gradient fractions. In order to normalize for rRNA recovery in the fractions, the values were divided by the percentages of 28 S rRNA in each corresponding region of the gradient as determined from slot blots. Therefore, summary data were corrected for differences between primary cell preparations, RNA loading of the gradients, and variations in specific radioactivities of the probes used for hybridization.
The data in Fig. 6C demonstrate that MHC mRNA relative to 28 S rRNA was highest in the region of the gradient containing smaller polysomes (approximately one to four polysomes) in both nonstimulated and electrically stimulated cardiocytes. There was also a large fraction of MHC mRNA in the region containing the ribosomal subunits and 80 S ribosomes, consistent with the data in Fig. 5. In the region containing larger polysomes (approximately five polysomes and greater), MHC mRNA normalized to the amount of recovered 28 S rRNA increased from 0.1 ± 0.05 to 0.34 ± 0.07 in electrically stimulated cardiocytes, a significant increase of 240%. Although the decreases in MHC mRNA in the ribosomal subunit, 80 S, and small polysome fractions of electrically stimulated cardiocytes were not statistically significant, the total decrease of 0.21 accounted for the corresponding increase of 0.24 in the heavy polysome fractions. Thus, a small shift of MHC mRNA from the lighter regions of the gradient resulted in a relatively big increase of MHC mRNA in the larger polysome fractions of electrically stimulated cardiocytes.
In these experiments, recovery of ribosomes and polysomes was accounted for by hybridization to a 28 S rRNA probe. There were no significant differences in the percentage of 28 S rRNA in each region of the gradient between electrically stimulated and nonstimulated gradient fractions (data not shown).
In order to
confirm the results obtained by the RNase protection method, the
experiments were repeated and MHC mRNA levels in the gradient fractions
were measured by slot blotting using a feline -MHC cDNA probe. The
specificity of the cDNA probe is demonstrated in Fig. 3A (lane 2). In the slot-blot method, the entire RNA sample
recovered from each gradient fraction was immobilized directly onto the
hybridization membrane. Furthermore, the amount of recovered RNA in
each fraction was measured by stripping the blot and hybridizing to the
28 S rRNA probe. These experiments also differed from the RNase
protection experiments in that the gradients were centrifuged at a
faster speed to improve the resolution of the polysome region of the
gradients and enhance the signal intensity in the larger polysome
fractions.
In Fig. 7A, the distribution of MHC mRNA in gradients of nonstimulated and electrically stimulated cardiocytes are compared. The percentage of MHC mRNA in each gradient fraction was calculated from the sum total of MHC mRNA in all the gradient fractions. The values were divided by the percentage of 28 S rRNA in each corresponding fraction, thereby correcting for any differences in recovery of ribosomes and polysomes. For comparison to the gradients in Fig. 6C, fractions 2 and 3 correspond to the ribosomal subunit and 80 S fraction, fractions 4 through 6 correspond to the small polysome fraction, and fractions 7 and 8 correspond to the large polysome fraction. In nonstimulated cardiocytes, MHC mRNA relative to 28 S rRNA was highest in fractions 4 and 5 and subsequently declined in the fractions containing the larger polysomes. By comparison, there was significantly more MHC mRNA relative to 28 S rRNA in fraction 8 of electrically stimulated cardiocytes. MHC mRNA in this fraction increased significantly from 0.74 ± 0.26 in nonstimulated to 1.33 ± 0.24 in electrically stimulated cardiocytes. Because recovery of 28 S rRNA was the same (Fig. 7B), these data demonstrate that there was a reproducible shift of MHC mRNA from the smaller polysomes into the larger polysomes contained in fraction 8 of electrically stimulated cardiocytes. These data are consistent with the results shown in Fig. 6, demonstrating that small shifts of MHC mRNA from the smaller polysome fractions result in substantial increases in MHC mRNA located in the larger polysomes of electrically stimulated cardiocytes. Furthermore, the absolute amount of MHC mRNA in fraction 8 of electrically stimulated cardiocytes, normalized to 28 S rRNA, increased 215% ± 51%, p < 0.03 over nonstimulated controls (mean ± S.E., n = 4 paired experiments).
Figure 7:
A, effects of electrically stimulated
contraction on the distribution of MHC mRNA in polysome gradients.
Eight fractions of 1.2 ml each were collected by upward displacement
beginning with the top of the gradients and the absorbance at 254 nm
was monitored. MHC mRNA was measured by slot blotting using a feline
-MHC cDNA probe. The blots were stripped and probed for 28 S rRNA.
The percentage of MHC mRNA in each gradient fraction was calculated and
corrected for rRNA recovery by dividing by the percentage of 28 S rRNA
in each corresponding fraction. B, comparison of 28 S rRNA
recovery in the gradients of electrically stimulated and quiescent
cardiocytes. The relative amount of 28 S rRNA per fraction was plotted
as a percentage of the total amount of recovered 28 S rRNA. Asterisk, significant difference, p < 0.03 as
determined by a paired Student's t test. Values are mean
± S.E., n = 4 cardiocyte
preparations.
In Fig. 7B, it is demonstrated that the changes in the distribution of MHC mRNA in the gradients of electrically stimulated cardiocytes were not due to differential recovery of ribosomes and polysomes. For each gradient, the relative amount of 28 S rRNA per fraction was plotted as a percentage of the total amount of recovered 28 S rRNA. There were no significant differences in the recovery of 28 S rRNA throughout the gradients of quiescent and electrically stimulated cardiocytes. Furthermore, there were no significant differences in absolute amounts of recovered 28 S rRNA in the gradient fractions between paired samples of electrically stimulated and nonstimulated cardiocytes (data not shown).
The increase in translational efficiency could have resulted from an overall increase in translational initiation in which free ribosomal subunits were recruited into polysomes. In order to test this possibility, the percentage of total RNA localized to 40 and 60 S ribosomal subunits was quantified. The percentage of RNA in the subunit region of the gradient was 34% ± 10% in electrically stimulated and 33 ± 11% (mean ± S.E.) in nonstimulated cardiocytes. Thus, similar to the MHC mRNA pool, electrical stimulation did not mobilize a significant amount of the free ribosome pool into polysomes.
During the hypertrophic process in adult myocardium, there is a relatively proportional increase in cardiocyte protein such that the terminally differentiated phenotype is essentially maintained(1) . The synthesis of myofibrillar proteins such as actin and myosin are increased in order to facilitate net accumulation of protein and subsequent assembly into nascent myofibrils(27, 28, 29) . In the electrical stimulation model used in this study, there was no measurable accumulation of myosin and/or total cell protein over the 4-h time period. However, consistent with other adult models of hypertrophy, we have recently demonstrated that continuous electrical stimulation of adult feline cardiocytes increased protein content and cell size over 7 days without any significant changes in morphology and myofibrillar architecture(7) . It is well established in several models of hypertrophy that transcription has a role in regulating the synthesis of contractile proteins, particularly qualitative changes in the expression of contractile protein isoforms in rodent species(11, 30) . However, in the ventricles of larger animals, including humans, these changes in isoform expression occur only to a very limited extent for actin and do not occur for myosin(11, 24, 30) . In a quantitative context, changes in contractile protein synthesis rates in response to load do not necessarily correspond to mRNA levels in adult myocardium(18, 31) . In perfused heart preparations, contractile arrest over several hours resulted in a decrease in MHC synthesis rate without a change in MHC mRNA levels(18) . When an additional load was imposed via an elevation of aortic perfusion pressure, MHC synthesis rate was accelerated in arrested hearts without a change in MHC mRNA. Thus, translational mechanisms could play a major role in regulating contractile protein synthesis rates in adult cardiocytes, especially in the early phase after a change in load.
Because cardiac MHC mRNA has and
isoforms, the
possibility existed that there was induction of the
-MHC isoform
and preferential translation after 4 h of electrically stimulated
contractile activity. The two isoforms diverge in sequence in the
3`-untranslated region. We were unsuccessful in our attempts to detect
an
-MHC mRNA signal in adult feline cardiocyte RNA using
oligonucleotide probes directed against the 3`-untranslated region of
either rat or human
-MHC mRNA. Definitive confirmation that the
-MHC isoform was not expressed or induced in adult feline
cardiocytes will require the isolation of a feline
-MHC clone.
However, the possibility that
-MHC mRNA was induced is unlikely
for the following reasons. 1) Adult feline cardiocytes express the
-MHC isoform exclusively, and MHC isoform expression in adult cats
did not change in response to pressure overload
hypertrophy(24) . 2) Contractile activity did not induce
-MHC isoform expression in primary cultures of neonatal rat
cardiocytes nor did it induce
-MHC mRNA in perfused rat heart
preparations(18, 25, 32) . In fact,
contractile activity and/or increased workload induce expression of the
-MHC isoform in rodent models of hypertrophy, the isoform already
expressed in adult feline cardiocytes(11, 32) . 3)
Available sequence data from both rat and human
- and
-MHC
isoforms predict that the size and pattern of the MHC band obtained by
the RNase protection assay would change if
-MHC mRNA was induced
by electrically stimulated contraction. No such changes were observed
as the protected band remained the same size as the rat
-MHC band.
4) The shift of MHC into larger polysomes was detected using either the
rat
-MHC probe or the feline
-MHC probe. Both probes were
specific for the highly conserved rod region of the molecule in which
there was a sequence similarity of at least 90% between the
- and
-MHC isoforms of rat and human species. Both probes detect
-
and
-isoforms with equal affinity so that the total MHC mRNA pool
was actually being measured.
The acute acceleration of MHC synthesis in response to electrically stimulated contraction occurred independent of a change in MHC mRNA abundance, suggesting that the rapid alterations in synthesis rate were due to greater translational efficiency. There are two possible mechanisms to explain the more efficient utilization of the MHC mRNA pool at the level of peptide chain initiation. The first mechanism involves a mobilization of mRNA from translationally inactive mRNP particles to actively translating polysomes, whereas the second mechanism involves an increased rate of ribosome initiation on mRNA in the polysome pool(33) . The data in Fig. 5indicated that MHC synthesis rates were not accelerated by selective mobilization of MHC mRNA into polysomes, even though there was a large percentage of total cellular MHC mRNA localized in the free mRNP pool. The reason for the relatively high percentage of inactive MHC mRNA with respect to translation is not known, although the percentage of MHC in mRNPs in skeletal myotube preparations varied between 35 and 90% (34, 35, 36) . The adult cardiocytes were cultured and maintained in a two-dimensional tissue culture environment and were not mechanically loaded to the same extent as cardiocytes in vivo. In addition, cultures of adult feline cardiocytes in serum-free medium have a slightly decreased rate of protein synthesis as compared with cells maintained in medium supplemented with serum(37) . These lower rates may reflect an overall decreased translational efficiency and in part account for the relatively high percentage of MHC mRNA in the mRNP fraction.
The second mechanism to increase translational efficiency is by increasing the rate of initiation of ribosomes onto mRNA relative to the rate of peptide chain elongation, resulting in an increased size of MHC mRNA polysomes(33, 38, 39) . The data in Fig. 6and Fig. 7are consistent with this type of mechanism as there was more MHC mRNA per unit mass of recovered 28 S rRNA in larger polysomes. In these experiments, the 28 S rRNA probe was used to correct for recovery of polysomes from the gradient fractions. Because recovery of 28 S rRNA in the gradient fractions was the same between nonstimulated and electrically stimulated cardiocytes, the shift of MHC mRNA into large polysomes of electrically stimulated cardiocytes is indicative of the fact that ribosomes are initiating faster onto MHC mRNA relative to elongation rate. The result is that the MHC mRNA pool would be more efficient as more polypeptide chains are synthesized per mRNA molecule. As an alternative possibility, the shift of mRNA into heavier polysomes could have resulted from a decrease in peptide chain elongation, as occurs during treatment with cycloheximide(38, 39) . In unloaded soleus muscle, a reduction in protein synthesis rate was the result of a slower rate of peptide chain elongation(40) . The possibility of a decreased elongation rate is unlikely, because inhibition of peptide chain elongation would be expected to cause a decrease, rather than an increase, in MHC synthesis rates.
It is not known how selective the increase in translational efficiency was for MHC mRNA. The changes in MHC synthesis rate in contracting cardiocytes may be indicative of a more global increase in translational efficiency. Our previous studies demonstrated that rates of total protein synthesis were acutely accelerated by electrical stimulation, and MHC synthesis rates were accelerated to a similar extent(6) . If there was an overall increase in translational efficiency of total protein synthesis, a greater amount of the ribosome pool would be expected to mobilize into the polysome pool and/or shift into larger polysomes. This was not the case as the percentage of total RNA in subunits remained the same in electrically stimulated cardiocytes, and there were no differences in the amount of recovered rRNA in the larger polysome fractions of electrically stimulated as compared with quiescent cardiocytes. However, an overall shift of ribosomes into larger polysomes could have gone undetected because of the relatively small percentage of total rRNA in the large polysome fractions (16% in Fig. 6and 20-25% in Fig. 7). In a terminally differentiated cell type such as the adult cardiocyte, the synthesis of MHC can increase in proportion to total cell protein because the relative abundance of MHC mRNA is maintained at a constitutively high level. Furthermore, based on steady state synthesis rates, MHC mRNA probably has an intrinsically high rate of initiation. Therefore, relatively rapid changes in MHC synthesis as measured in response to electrically stimulated contraction could occur by the MHC pool more effectively competing for a limited quantity of one of more of the translational components that are involved in accelerating protein synthesis(41) .
It is generally recognized that overall changes in translational efficiency probably involve altering the specific activities of translational initiation factors that are rate-limiting for peptide chain initiation(33, 41) . In the adult cardiocyte, a translational mechanism for accelerating the rate of protein synthesis would serve to coordinate protein synthesis and maintain the differentiated phenotype during the early phase of hypertrophic growth. The present studies show that protein synthesis can be accelerated prior to an increase in the RNA pool, indicating that transcription was not required. Furthermore, a translational mechanism for regulating the efficiency of protein synthesis rates acutely would be followed by an increase in translational capacity, a well characterized mechanism for accelerating protein synthesis during sustained hypertrophic growth of the heart (2) . Thus, a temporal sequence for regulating cardiocyte protein synthesis rates in response to a hypertrophic stimulus could potentially involve altering the activity of initiation factors initially, followed by a sustained increase in the relative amount of translational machinery.