INTRODUCTION

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HYPERTROPHY is the primary mechanism by which the adult myocardium compensates for hemodynamic overload (10). Hypertrophy is thought to be initiated by an increase in myocardial wall stress as defined by the Laplace equation in which wall stress is calculated as (pressure × radius)/(2 × wall thickness) (13). It has been postulated that, in pressure overload, the increase in pressure in the numerator of the equation increases systolic wall stress, thereby initiating the development of concentric hypertrophy and increasing the thickness of the myocardial wall. This increase in wall thickness offsets the increase in systolic pressure and maintains wall stress within the normal range. In volume overload, increased diastolic stress is thought to trigger the development of eccentric hypertrophy in which lengthening of the cardiac muscle cells leads to chamber enlargement.

The myocardial proteins are in a constant dynamic state of synthesis and degradation when the myocardium is in a steady state with respect to growth (31). For hypertrophy to occur, the net balance between protein synthesis and protein degradation must be altered, by either accelerating the rate of protein synthesis or decreasing the rate of protein degradation. In the volume overload of mitral regurgitation, we have shown that the rate of protein synthesis was not accelerated and that hypertrophic growth was attributable to a decrease in the rate of protein degradation (24). The amount of hypertrophy produced by volume overload is generally less than that produced by pressure overload (5). In contrast, our study of acute aortic stenosis (AS) showed a marked acceleration of the rate of protein synthesis, which occurred without an increase in either the ribosomal RNA pool or in mRNA levels (17). This response to acute pressure overload is indicative of an increase in translational efficiency, defined as the utilization of the mRNA pool by the preexisting translational machinery in the adult myocardium. Similar increases in efficiency have been observed in other models of pressure overload (9, 27, 32). Translational mechanisms are also known to have a central role in accelerating protein synthesis rates during sustained pressure overload. During the growth phase of pressure-overload hypertrophy, there is a well-established increase in the capacity for protein synthesis as defined by the amount of translational machinery, including ribosomes, tRNA, and initiation and elongation factors (31). However, the precise roles of translational efficiency and capacity in controlling the hypertrophic process in pressure overload have never been studied in a unified fashion from the initiation of hypertrophy to the achievement of a new steady state in a large animal model of gradual stenosis such as that seen in humans.

We have recently developed a canine model of aortic stenosis in which pressure overload is controlled by an externally controlled aortic band (23). After an increase in stenosis severity, there is a detectable increase in cardiac mass by 24 h, followed by linear growth that reaches a new steady state in 10 days. We hypothesized that the role of translational efficiency is to accelerate the rate of protein synthesis in response to the initial increase in systolic wall stress, whereas the role of capacity is to produce an overall increase in translational machinery and thereby maintain accelerated protein synthesis rates during sustained hypertrophic growth. The rate of myosin heavy chain (MHC) synthesis was used as a specific marker of protein synthesis in vivo. The hypothesis was tested by examining translational regulation in the canine left ventricle (LV) at three phases of pressure overload: the acute phase (6 h), the rapid growth phase (5 days), and the steady-state phase of hypertrophy (10 days). To determine the linkage between increased systolic wall stress and accelerated rates of cardiac protein synthesis, two key mechanisms for increasing translational efficiency and capacity were examined: 1) changes in the activity of eukaryotic initiation factor 4E (eIF4E) as measured by its phosphorylation and 2) changes in the rate of 60S ribosome formation and net accumulation of rRNA.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Models of canine pressure overload. All procedures were done in accordance to institutional guidelines. Five adult mongrel dogs were used as controls. Anesthesia was induced with an infusion of 0.15 mg · kg¹ · min¹ sufentanil supplemented by isoflurane and maintained by a constant infusion of sufentanil and a low dose of isoflurane by inhalation. A thermodilution Swan-Ganz catheter was inserted via the femoral vein for measuring pulmonary capillary wedge pressure, pulmonary artery pressure, and cardiac output. A second catheter was placed in the femoral artery for measuring arterial blood pressure.

Continuous infusion of radiolabeled leucine. Hearts were radiolabeled by continuous infusion with L-[3,4,5-³H(N)]leucine over 6 h as described previously (24). In the acute AS experiments, infusion was begun as soon as the hemodynamic conditions were stable. Blood samples were withdrawn from the ascending aorta at regular intervals for measurement of plasma leucine-specific radioactivity. When the infusion period was completed, the hearts were rapidly removed and rinsed in ice-cold saline, and the great vessels were perfused retrogradely with ice-cold saline. For measurements of the rate of ribosome formation, portions of the LV free wall were minced and homogenized immediately. The remaining heart tissue was frozen in liquid nitrogen and stored. Samples of frozen LV and RV free wall were used to measure the rate of MHC synthesis, MHC mRNA levels, MHC content, and total RNA content.

Determination of plasma leucine-specific radioactivity. Blood samples were centrifuged, and perchloric acid (PCA) was added to a final concentration of 6%. The plasma proteins were pelleted by centrifugation. The supernatant was neutralized by addition of KOH, centrifuged, and dried by vacuum centrifugation. The sample was resuspended in 0.1 M NaHCO₃-Na₂CO₃ buffer, pH 9.5, and reacted with an equal volume of 5 mM [¹⁴C-methyl]dansyl chloride (110-120 mCi/mmol) at 37°C for 1 h. The dansylated amino acids were purified by two-dimensional thin-layer chromatography on micropolyamide plates as described previously (1). The ³H-to-¹⁴C ratio of the dansyl-leucine spot was used to calculate the plasma leucine-specific radioactivity.

Determination of MHC synthesis rates. The fractional rate of MHC synthesis (K_s) was determined by the continuous infusion method using the following formula
where P* is the specific radioactivity of leucine in MHC and F*dt is the plasma leucine-specific radioactivity time curve. MHC was purified as described previously (17) and the specific radioactivity of leucine in MHC was measured by the dansyl-chloride method.

Measurement of rate of ribosome formation. The rate of 60S ribosome formation was determined by measuring the rate of incorporation of [³H]leucine into protein of purified 60S ribosomes and dividing by the specific radioactivity of the plasma leucine pool (24, 37). Rates of 60S ribosome formation were calculated using the following formula

Measurement of RNA content, total protein content, and MHC content. Total RNA content in the supernatant was determined spectrophotometrically at a wavelength of 260 nm as described previously (37). For measuring total protein content, the residual pellet was washed three times with 0.2 N PCA, resuspended with 0.3 N NaOH, and incubated at 37°C overnight. The concentration of total protein content was measured by the bicinchoninic acid method (Pierce, Rockford, IL). MHC content was measured as described previously (24).

Determination of MHC mRNA levels. RNA was extracted using the guanidinium thiocyanate method (8). Slot-blot hybridization analysis was carried out using a ³²P-labeled feline MHC cDNA probe as described previously (24). The autoradiograms were analyzed by digital image analysis using NIH Image software. The optical densities of the hybridization signals were in the linear range.

Measurement of eIF4E phosphorylation. Portions of frozen LV (100 mg) were homogenized in lysis cell buffer (LCB) [20 mM HEPES, pH 7.5, 100 mM KCl, 0.2 mM EDTA, 7 mM -mercaptoethanol, 10% (vol/vol) glycerol, 0.5% (vol/vol) Triton X-100, 80 mM 2-glycerophosphate, 50 mM NaF, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.5 mM sodium orthovanadate, 1 µM okadaic acid, 1 µM microcystin LR, 25 µg/ml leupeptin, 2 U/ml aprotinin, and 20 µg/ml chymostatin] and centrifuged at 12,000 g for 10 min. Twenty microliters of washed m⁷GTP-Sepharose 4B (Pharmacia) were added to the supernatant and incubated for 1 h at 4°C. The m⁷GTP-Sepharose was pelleted and washed three times with LCB. The phosphorylation state of eIF4E was determined by vertical slab gel isoelectric focusing and Western blotting as described previously (40). The percentage of phosphorylated eIF4E was quantified by digital image analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Hemodynamics of experimental pressure overload. In Table 1, hemodynamic data for the control and AS dogs are shown. Hemodynamic measurements were taken at hourly intervals during continuous infusion and averaged. Heart rate was increased as a result of pacemaker implementation to ~100 beats/min and was maintained over the duration of AS. Systolic and diastolic blood pressure were significantly lower during the acute AS procedure but were normal as measured after 5 and 10 days of sustained AS. The severity of pressure overload was demonstrated by a significant 60% increase in LV systolic pressure in the acute AS model and increases of 75% and 72% after 5 and 10 days of AS, respectively. Pulmonary capillary wedge pressure was significantly increased but remained within the normal range for our laboratory. Pulmonary artery systolic pressure was increased by an average of 17%, but these increases were not statistically different compared with controls. Stroke volume was decreased significantly at 5 days of AS but increased toward the control value by 10 days of AS, indicating that the LV compensated for the sustained increase in afterload. Table 1 also shows that there were no significant differences in body weight between control and AS dogs at any experimental time point.

Development of pressure-overload hypertrophy. Changes in LV mass were determined by ventriculography. The baseline value for LV mass was measured in each dog before the increase in pressure gradient. As shown in Fig. 1, LV mass was significantly increased by 11.1% and 26.4% after 5 and 10 days of AS, respectively. There were corresponding increases in the LV mass-to-body weight ratio of 15.1% and 28.1%, confirming that compensatory LV hypertrophy occurred in response to sustained pressure overload. Body weight did not change from baseline values during experimental AS, indicating that the adult dogs were in a steady state with respect to growth (23.1 ± 1.0 kg at baseline vs. 22.9 ± 1.3 kg after 5 days of AS; 21.2 ± 1.0 kg at baseline vs. 21.5 ± 1.1 kg after 10 days of AS).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Cardiac hypertrophy in dogs in response to experimental pressure overload. A: for both groups of dogs, left ventricular (LV) mass was determined by ventriculography at baseline and after 5 (5-day AS) and 10 days of aortic stenosis (10-day AS). B: LV mass normalized to body weight in same dogs. Values are means ± SE. * P < 0.05;  P < 0.07 vs. corresponding baseline value as determined by a paired t-test.

Specific radioactivity of precursor pool during continuous infusion. Plasma leucine-specific radioactivity equilibrates with leucyl-tRNA-specific radioactivity in the canine LV during continuous infusion (17) and was therefore used as the precursor pool for calculating the rate of MHC synthesis and the rate of 60S ribosome formation. Figure 2 shows the plasma leucine-specific radioactivity time curve during 6 h of continuous infusion with [³H]leucine in control and AS dogs. In each experimental group, the plasma leucine-specific radioactivity rose rapidly during the first 30 min and plateaued to a constant value over the remainder of the infusion period. There were no significant differences in plasma leucine-specific radioactivity in the control dogs compared with any of the AS groups.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Plasma leucine-specific radioactivity during continuous infusion with [3H]leucine. Values are means ± SE for each experimental group of dogs during continuous infusion.

Effect of pressure overload on rate of protein synthesis. The anabolic effects of AS on cardiac protein synthesis were determined by measuring K_s. Figure 3A shows that MHC K_s was significantly increased by 29% in response to acute pressure overload compared with control. MHC K_s decreased after 5 days of pressure overload and slowed to the control value by 10 days. In Fig. 3B, the rate of total MHC synthesis after 5 and 10 days of AS was calculated by multiplying K_s (%/day) by the corresponding value for total MHC content per LV [(mg MHC/g LV protein) × g LV]. This calculation takes into account the increase in LV mass that occurs during pressure-overload hypertrophy. Accordingly, the rate of total MHC synthesis was increased by 87% and 63% after 5 and 10 days of AS, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Rates of myosin heavy chain (MHC) synthesis during pressure-overload hypertrophy. A: MHC fractional rate of synthesis (Ks) values were measured in each experimental group of dogs by continuous infusion method. B: rates of total MHC synthesis and MHC degradation were calculated in control dogs and in dogs after 5 and 10 days of AS as described in text. Values are means ± SE. * P < 0.05, significantly greater than control as determined by ANOVA followed by Dunnett's test.

Effect of pressure overload on rate of protein degradation. The fractional rate of MHC degradation (K_d) can be calculated indirectly using the formula K_g = K_s  K_d, where K_g is the fractional rate of MHC accumulation. We calculated MHC K_d at 5 days of AS because prior studies using serial echocardiography showed that LV hypertrophy in the AS dogs occurred as a linear function over the first 9 days (23). K_g was determined at the 5-day time point by fitting growth to a linear function and dividing the rate of growth (g LV/day) by the corresponding value for LV mass (g). The K_g value derived from LV mass was substituted for MHC K_g because MHC protein as a fraction of total LV protein remained constant. The calculated MHC K_d after 5 days of AS was 1.14%/day, a value lower than the control value of 2.63%/day. Thus, whereas MHC K_s was increased during the growth phase of AS-induced hypertrophy, MHC K_d was decreased. The rate of total MHC degradation, calculated by multiplying K_d by total MHC content per LV, was decreased after 5 days of AS, but it was not significantly different from that in the control (Fig. 3B). LV growth reached a plateau after 10 days of AS, which is indicative of a new steady state (23). Accordingly, Fig. 3B shows that MHC degradation increased and became equivalent to MHC synthesis after 10 days of AS.

Capacity for protein synthesis during pressure-overload hypertrophy. Total RNA content is a well-described marker for changes in translational capacity of cardiac muscle because ribosomal RNA is ~85% of the total RNA pool. Figure 4A shows that RNA content normalized to total protein increased after 5 days of AS and that the increase was maintained at 10 days of AS. In Fig. 4B, the overall increase in translational capacity during hypertrophic growth was calculated by multiplying RNA content (mg RNA/g LV protein) by the corresponding values for LV mass (g). Pressure overload caused a sustained increase in capacity as indicated by a 64% and 71% increase in RNA content per LV after 5 and 10 days of AS, respectively. To determine the mechanism for rRNA accumulation, the rate of 60S ribosome formation was measured during pressure-overload hypertrophy (Fig. 4C). The rate of ribosome formation was significantly accelerated in response to acute AS but returned to the control value after 5 days. Given that the half-life of the ribosome is ~10-12 days, these data suggest that the early increase in ribosome formation accounted for the accumulation in RNA content that occurred by 5 days of pressure-overload hypertrophy.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of pressure overload on translational capacity for protein synthesis. A: total RNA normalized to total LV protein in each experimental group of dogs. B: translational capacity as calculated by total RNA content per LV in control dogs and in dogs after 5 and 10 days of AS. C: rates of 60S ribosome formation in each experimental group of dogs. Leu, leucine. Values are means ± SE. * P < 0.05, significantly greater than control as determined by ANOVA followed by Dunnett's test.

Efficiency of protein synthesis during pressure-overload hypertrophy. Translational efficiency is defined as the rate of protein synthesis normalized to the amount of translational machinery, in particular, the ribosome pool (31). To calculate translational efficiency, the rate of MHC synthesis at each time point of AS was divided by total RNA content (Fig. 5A). These data show that translational efficiency was increased in response to acute AS because the rate of MHC synthesis was accelerated before an increase in RNA content. Figure 5A also shows that translational efficiency progressively decreased after 5 and 10 days of AS in association with a corresponding increase in translational capacity. These data indicate that once the increase in capacity has occurred, steady-state levels of translational efficiency are sufficient to maintain the accelerated rate of total MHC synthesis in the hypertrophied LV.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effects of pressure overload on translational efficiency for protein synthesis. A: translational efficiency was calculated in each group of dogs by dividing rate of MHC synthesis by total RNA content. B: representative slot-blot experiment showing MHC mRNA and 28S rRNA levels in control and AS dogs. RNA from both right ventricle (RV) and LV was labeled between duplicate samples of each experimental group. Exposure time of autoradiograms was 24 h for MHC mRNA and 1 h for 28S rRNA. Con, control; A-AS, dogs with acute AS; 5D-AS, dogs after 5 days of AS; 10D-AS, dogs after 10 days of AS. C: summary data for all dogs. MHC mRNA-to-28S rRNA ratios in LV were normalized to companion RV. Values are means ± SE. * P < 0.05, significantly greater than control as determined by ANOVA followed by Dunnett's test.

Effect of pressure overload on eIF4E activity. eIF4E is involved in controlling the binding of mRNA to the 40S ribosomal subunit, a rate-limiting step during the process of translational initiation. Phosphorylation of eIF4E enhances its affinity for the 7-methylguanosine cap on the mRNA molecules. In Fig. 6, phosphorylation of eIF4E in canine LV was used as a marker to assess changes in the activity of eIF4E. Acute AS increased the extent of eIF4E phosphorylation to 24% compared with 8% in the LV of controls, and this increase was maintained during sustained pressure overload. These findings suggest that eIF4E activity as measured by phosphorylation correlated with the initial increase in translational efficiency and that eIF4E activity remained elevated as translational capacity increased during pressure-overload hypertrophy. The relative amount of eIF4E protein, normalized to total LV protein, did not change during pressure-overload hypertrophy (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Effects of pressure overload on eukaryotic initiation factor 4E (eIF4E) phosphorylation. eIF4E was extracted from LV samples and the nonphosphorylated and phosphorylated (eIF4E-P) isoforms were resolved by isoelectric focusing followed by Western blotting. A: representative immunoblot containing samples from control and AS dogs. B: summary data. Values represent means ± SE of relative percentage of phosphorylated eIF4E as measured by digital image analysis of corresponding autoradiograms. * P < 0.05, significantly greater than control as determined by ANOVA followed by Dunnett's test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

A direct comparison between pressure and volume overload suggests that increased wall stress particular to each type of mechanical overload is the primary determinant of the hypertrophic phenotype (5, 13). In the case of LV pressure-overload hypertrophy, the molecular basis for altering the phenotype can be found in two distinct processes. The first is increased expression of specific mRNAs encoding proteins that directly affect cardiac growth and function. These include diverse classes of proteins such as transcription factors (20), growth factors (3), tubulin (38), and fetal isoforms of the myofibrillar proteins myosin and actin (7). The second process involves acceleration of the rate of protein synthesis to produce an overall increase in LV mass (31). The resultant hypertrophy of the individual cardiac muscle cells is characterized by increases in constitutively expressed proteins such as myosin and actin. The interplay of these two processes during hypertrophy enables plasticity of the cardiac phenotype in the setting of a compensatory increase in LV mass.

With the use of MHC as a specific marker of cardiac protein synthesis in vivo, MHC synthesis was accelerated by pressure overload without any measurable changes in MHC mRNA abundance. This study shows that translational mechanisms coordinately accelerate the rate of protein synthesis during pressure-overload hypertrophy. The role of translational efficiency is to accelerate the rate of protein synthesis in response to the initial increase in systolic wall stress, whereas the role of capacity is to maintain accelerated rates of protein synthesis during hypertrophic growth, thereby producing a new steady state. The efficiency of protein synthesis in adult myocardium is quite high even during steady-state conditions; ~80% of the ribosome pool can be found in the actively translating 80 S and polysome fractions (30, 31). Thus the potential to accelerate the rate of protein synthesis during sustained hypertrophic growth is limited. A new steady-state protein synthesis rate can be maintained in the hypertrophied myocardium via the combination of its normally high levels of efficiency and the increase in translational capacity. This conclusion is supported by the data in Fig. 3B showing that total MHC synthesis rates are maintained at a higher steady-state value during sustained pressure-overload hypertrophy, even though efficiency has returned to the control level.

Previous studies have established a role for translational efficiency in accelerating protein synthesis in response to pressure overload in vivo (17, 27, 32). An increase in efficiency likewise accelerated the rate of protein synthesis when adult cardiac muscle cells in vitro were subjected to acute increases in load, in the form of either electrically stimulated contraction or passive stretch on a deformable membrane (18, 19, 22). The translational efficiency of protein synthesis is usually determined by the rate-limiting reactions governing peptide chain initiation, and efficiency can be improved by increasing the activity of initiation factors (16). In the process of translational initiation, binding of eIF4E to the 7-methylguanosine cap of mRNA is required for the translationally active eIF4F complex to perform its functions of unwinding mRNA secondary structure and promoting binding of mRNA to the 40S ribosomal subunit. Phosphorylation of eIF4E on the serine-209 residue increases binding affinity for the 7-methylguanosine cap present on virtually all mRNA molecules and stabilizes the translationally active eIF4F complex (2, 21, 28). The positive correlation between pressure overload and eIF4E phosphorylation is a potential mechanism for increasing efficiency of protein synthesis in the cardiac muscle cell. Our previous studies showed that acute pressure overloading of the LV produced by AS increased eIF4E phosphorylation but that acute volume overload of the LV did not stimulate eIF4E phosphorylation (40). The present studies show that the acute increase in eIF4E phosphorylation was sustained during chronic AS and suggest that eIF4E phosphorylation may have several roles in regulating the rate of protein synthesis during pressure-overload hypertrophy. Initially, eIF4E phosphorylation may increase translational efficiency in response to acute pressure overload. The persistent increase in eIF4E phosphorylation during chronic AS may serve the function of maintaining steady-state levels of translational efficiency as the capacity increases during sustained hypertrophic growth.

The acceleration of MHC synthesis in response to pressure overload did not involve selective upregulation of MHC mRNA levels in the canine myocardium. Rather, the MHC mRNA pool increased in proportion to the ribosome pool during hypertrophic growth. These observations underscore an important distinction between changes in the relative expression of - and -MHC mRNA isoforms that occur in rodent models of pressure-overload hypertrophy and actual net increases in the total MHC mRNA levels. It is well established that transcriptional and posttranscriptional mechanisms control expression of MHC isoforms during pressure-overload hypertrophy in rats via alterations in the relative abundance of the corresponding MHC mRNA (6, 15, 29, 34). However, there is no shift in MHC isoform composition during hypertrophy of the canine LV because -MHC is constitutively expressed as the predominant isoform, similar to other large mammalian species, including humans (39). To our knowledge, there are no data employing any large mammalian model of LV hypertrophy proving that the overall rate of MHC synthesis is controlled by selectively increasing the size of the MHC mRNA pool. A recent study (41) showed that banding of the ascending aorta in adult rats produced hypertrophy without an increase in -MHC mRNA levels, yet expression of -MHC protein was increased. This study also revealed that selective increases in -MHC mRNA levels after constriction of the abdominal aorta proximal to the renal arteries may result from activation of the renin-angiotensin system, suggesting that -MHC mRNA expression can be dissociated from the increase in MHC synthesis that occurs in response to pressure overload. Thus the mechanical stimulus of pressure overload produced by constriction of the ascending aorta in either rat or dog is apparently sufficient to produce LV hypertrophy and demonstrates that selective induction of -MHC mRNA is not required to accelerate the rate of MHC synthesis.

Sustained pressure overload triggered an increase in total RNA content, a standard index of the translational capacity in adult myocardium. The accumulation of RNA was attributable to an increase in the rate of ribosome formation that occurred after 6 h of pressure overload. It has been shown that cardiac muscle can increase capacity in response to load by altering one or more of the reactions involved in ribosome formation (9, 37). For example, the onset of pressure overload in vivo increased 28S rRNA synthesis (36) and the activity of RNA polymerase I, the enzyme that transcribes rDNA (12). These observations were confirmed using neonatal rat myocytes in vitro (25, 26). More recent findings (14) suggest that capacity is increased during growth of neonatal rat myocytes via the activity of UBF, a DNA binding protein that regulates transcription of the rDNA genes.

We conclude that translational mechanisms coordinately regulate the rate of protein synthesis during hypertrophy induced by a stepwise increase in LV pressure overload. The rate of protein synthesis is accelerated by an increase in translational efficiency and is maintained at a higher steady state by an adaptive increase in translational capacity during sustained hypertrophic growth.