Abstract A novel peptide, myotrophin, has been isolated, purified, cloned, and sequenced from the hearts of spontaneously hypertensive rats (SHR) and from dilated cardiomyopathic human heart tissue. Myotrophin accelerates myocyte growth by stimulating protein synthesis (not by altering myocardial cell division). Our successive studies were conducted to evaluate the pathophysiological significance of myotrophin; a solid-phase radioimmunoassay technique was developed for quantifying the protein in hypertrophied and normal hearts. Specific antipeptide antibody was raised in rabbits against a peptide that represents a selected amino acid sequence of a 17–amino acid myotrophin segment by using the multiple antigenic peptide technique. The specificity of the antibody was evaluated by determining the affinity constant after constructing the Scatchard plot obtained from the ratio of bound to free myotrophin against bound myotrophin. The value obtained was 2.61×107 L/mol. The specificity was further demonstrated by Western blot analysis, in which a single protein band was obtained in the region of 12 kD. Pretreatment of the antibody with myotrophin completely blocked the binding sites, because no protein band was detected on the immunoblot. The antibody prevented the myocardial protein synthesis induced by myotrophin as revealed by the blockage of the stimulation of [3H]leucine incorporation into myocyte protein. Quantification of myotrophin from different heart tissues was achieved by Western blot and dot blot analyses. Amounts of myotrophin present in different dots were determined by using a video image analyzer. The level of myotrophin in the embryonic tissue was found to be similar in male normal and SHR hearts. The level of myotrophin concentration then starts increasing by as early as 3 days of age in SHR. In 9-day-old SHR, the change is statistically significant. In 4-, 8-, 11-, and 17-week-old SHR, myotrophin concentration increased linearly and significantly compared with age- and sex-matched normal control rats. No detectable amount of myotrophin was found in kidney or lung. Our data suggest that myotrophin may play an important role in the pathogenesis of cardiac hypertrophy.
- multiple antigenic peptide
- affinity constant
- hypertensive hypertrophy
- spontaneously hypertensive rats
Recently, a protein factor, myotrophin, was isolated, purified, and partially sequenced from the hearts of spontaneously hypertensive rats (SHR)1 and dilated cardiomyopathic human tissue. Myotrophin obtained from SHR heart and human heart tissue appeared to be very similar in structure and function. In these studies, myotrophin was shown to stimulate myocardial protein synthesis and cell growth and to increase the cell surface area in a dose-dependent fashion compared with the control condition.1 Neonatal myocytes treated with myotrophin displayed an accelerated myofibrillar growth and an organization into sarcomeres as well as a maturation of mitochondria.1 We have shown that when neonatal myocytes maintained in culture were incubated with myotrophin, the myocytes showed a fourfold increase in connexin (gap junction protein) and a twofold increase in total myosin transcript levels.2 In addition, myotrophin caused a selective increase in β-myosin heavy chain expression without a reciprocal decrease in α-myosin heavy chain expression.2 Recently, the cDNA clones encoding myotrophin have been isolated3 and expressed in Escherichia coli. The recombinant myotrophin was purified and tested for immunoreactivity and biological activity, which showed that the recombinant myotrophin was fully biologically active and cross-reacted with antibody against natural myotrophin. The present study was undertaken to evaluate the pathophysiological significance of myotrophin by quantifying levels of myotrophin concentration in male SHR and normal rat hearts and the effect of the antibody on the stimulation of protein synthesis induced by myotrophin. For the quantification of myotrophin concentration, a solid-phase radioimmunoassay technique has been developed by using myotrophin-specific antipeptide antibody raised in rabbits. The peptide used for this purpose was a selected segment of myotrophin consisting of 17 amino acids, and the multiple antigenic peptide (MAP) was made with a high density of this peptide around the lysine core matrix, such that an approximate molecular weight of 10 000 was reached. The specificity of the antibody was assessed by determining the affinity constant of the antibody from Scatchard plot and also Western blot analysis.
Materials and Methods
All SHR and normal (Wistar) rats used in the present study were obtained from Taconic Farms, Germantown, NY. Timed pregnant rats were obtained from Hilltop Farm, Scottdale, Pa. The rabbits (New Zealand White) were purchased from Pioneer Animal Supply, Mount Vernon, Ohio. All procedures observed institutional guidelines for experimental use of animals.
All chemicals used in the present study were ACS-certified analytical reagents purchased from EM Science and Fisher Scientific. Solvents used were of high-performance liquid chromatography (HPLC) grade and were purchased from Baxter Healthcare Corp. DVF12 used for the neonatal myocyte cultures was obtained from GIBCO BRL. All other media and reagents used for the preparation of the neonatal myocytes, for their maintenance in culture, and for the bioassays, including fetal bovine serum albumin (BSA), laminin, transferrin, fetuin, hydrocortisone, and insulin, were purchased from Sigma Chemical Co. Collagenase type II was purchased from Worthington Biochemicals. IgG purification kits were obtained from Pierce Chemical Co. Four to twenty percent gradient polyacrylamide gels were obtained from Bio-Rad Laboratories. GeneScreen was purchased from New England Nuclear. 125I-labeled protein A was purchased from ICN Radiochemicals. [3H]Leucine was obtained from Amersham Corp. Quantification of myotrophin was performed with an image analyzer (Fotodyne Inc).
Preparation of Antibody Against Myotrophin
Antibody against myotrophin peptide was raised by following the MAP system.4 5 6 The system uses a simple scaffolding of a low number of sequential levels (n) of a trifunctional amino acid as the core matrix and 2n peptide antigens to form a macromolecule with a high density of peptide antigens, so that a final molecular weight of ≈10 000 is reached. Peptide sequences exposed outside of the native protein molecule are more likely to produce antibody that would cross-react with intact protein molecule.
The MAP chosen was an octabranching MAP consisting of a core matrix made up of three levels of lysine and eight amino terminals for anchoring peptide antigens. The MAP contained both the core matrix and the peptide consisting of 17 amino acid residues, namely, Gly-Pro-Asp-Gly-Leu-Thr-Ala-Leu-Glu-Ala-Thr-Asp-Asn-Gln-Ala-Ile-Asp (obtained from the known sequence of a tryptic peptide fraction of myotrophin). The MAP was obtained in a single synthesis by the solid-phase method using an Applied Biosystem model 431 peptide synthesizer (Applied Biosystems, Inc). The MAP was purified by reverse-phase HPLC on a semipreparative Vydac C18 column (1.0×25 m) in 0.1% trifluoroacetic acid in water. A 10% to 70% gradient of acetonitrile was used over a period of 2 hours. All purified materials were analyzed and found to contain the predicted amino acid sequence.
Immunization of the Rabbits
One milligram of the MAP was dissolved in 1 mL of PBS. This solution was mixed with complete Freund’s adjuvant in a ratio of 1:1 and vortexed for 3 hours to make a homogeneous emulsion. On day 0, rabbits were immunized by subcutaneous injection with 1 mL of this emulsion. On day 21, the rabbits were again injected with the same amount of MAP in incomplete Freund’s adjuvant. Ten days later, they were bled; ≈10 mL of blood was taken from each rabbit. At 3-week intervals, the rabbits were injected with incomplete Freund’s adjuvant and bled 10 days after the last boosting. After each collection, the blood was allowed to clot for 1 hour at 37°C, and the clot was kept at 4°C overnight to allow it to contract. Finally, the serum was collected by low-speed centrifugation for 10 minutes at 4°C and stored at −40°C.
Characterization of Antipeptide Antibodies
Solid-phase radioimmunoassays and Western blot analyses were used for the characterization of the antibody formed against MAP in rabbits.
Both natural myotrophin and MAP were used as antigens to evaluate the binding capacities of the antibody toward these antigens.7 In brief, for the solid-phase radioimmunoassays, 100 ng myotrophin (or MAP) in 100 μL coating buffer (100 mmol/L carbonate buffer, pH 9.6) was added in each well of a 96-well microtiter plate and incubated for 2 hours at room temperature. The solution was then removed, and the wells were washed three times with 100 μL washing buffer (0.05% Tween 20 in PBS) per well each time. One hundred microliters of blocking solution (1% bovine serum albumin [BSA] in PBS) was then added in each well, and the wells were incubated for 1 hour at room temperature. After this solution was removed and the wells were washed, appropriately diluted antiserum in diluting buffer (PBS containing 0.05% Tween 20, 0.25% BSA, and 0.1% sodium azide) was added in each well, and the wells were incubated for 2 hours at room temperature. The solution was then removed, and 125I-labeled protein A (1 to 2×105 cpm) was added per well; the wells were incubated for 5 hours at room temperature. The wells were then washed thoroughly until they were free of radioactivity and dried in air for half an hour; the counts per minute were taken in a gamma counter. The percent bound for each dilution of antiserum was calculated, and a curve was drawn plotting the percent bound of the antibody with the antigen against the dilution of antiserum.
Determination of Affinity Constant
Competitive binding radioimmunoassay techniques were used for the construction of a Scatchard plot to determine the affinity constant of the myotrophin antibody as described by Berson and Yalow.8 Briefly, aliquots of the fixed antibody were saturated with an increasing amount of myotrophin. The bound-to-free ratio of myotrophin was then obtained and plotted on the y axis against the concentration of bound myotrophin on the x axis. If it is assumed that all binding sites of the myotrophin antibody possess identical affinity for myotrophin, the plot is linear and the slope equals −K, the affinity constant.
Western Blot Analysis
Western blot analysis was performed by following the procedure described by Towbin et al9 and Tsang et al,10 with some modifications. Briefly, this procedure includes the electrophoretic transfer of proteins from SDS-polyacrylamide gels to GeneScreen and the identification of different protein bands that cross-react with specific antibodies; this procedure was followed by a second reagent step using 125I-labeled protein A. SDS-PAGE was performed for different protein samples in 4% to 20% acrylamide gels for 40 minutes at a constant voltage of 200 V at room temperature according to the method of Laemmli.11 Before the transfer of the different proteins to the GeneScreen, the gel was equilibrated for 90 minutes in 25 mmol/L Tris and 192 mmol/L glycine buffer, pH 8.3. A piece of GeneScreen slightly larger than the gel was also equilibrated in the same buffer for half an hour. A sandwich was then made with the gel, the membrane, two pieces of filter paper, and two sheets of Scotch-Brite pad. Electrophoretic transfer was then continued for 6 hours at 4°C at a constant voltage of 40 V. The membrane was taken out of the apparatus, rinsed with 10 mmol/L sodium phosphate buffer, pH 7.4, containing 1% NaCl and 1% Tween 20, and dried in air for half an hour. The dry membrane was then immersed in an excess of 10% Carnation instant nonfat dry milk in PBS containing 1% Tween 20 and incubated for 90 minutes. After that, this solution was removed, and an excess of diluted (1:500) antiserum in the milk solution (mentioned above) was added; the solution was incubated for 90 minutes. The solution was removed, and the membrane was rinsed three times with an excess of 20 mmol/L PBS containing 1% Tween 20. The membrane was then immersed in an excess of the previously mentioned milk solution containing 125I-labeled protein A (1 to 2×105 cpm/mL) and incubated for 4 hours at room temperature. The membrane was then rinsed with an excess of PBS containing 1% Tween 20 until the unbound radioactivity was removed. Finally, the membrane was air-dried at room temperature and autoradiographed.
To evaluate the specificity, a parallel Western blot experiment was conducted to show whether myotrophin could block the binding sites of the antibody raised against MAP. The antibody was pretreated with myotrophin for 2 hours. The GeneScreen (in which all the rat heart proteins were transferred from the SDS gel electrophoretically) was then treated with the antibody solution after blocking with milk solution. The rest of the experiment was performed as described above. Later, the same membrane was treated with the antibody solution (without pretreatment with myotrophin), and the autoradiograph was taken as described above.
Neonatal Cardiomyocyte Culture and Bioassay System
The neonatal rat cardiomyocytes were isolated, purified, and maintained in culture following the protocol as described by Sen et al.1 Briefly, the myocytes were isolated by digesting the ventricles from the hearts of 3-day-old rat pups in a small amount of Joklik medium in the presence of collagenase (type II, 84 U/mL) for 20 minutes in a water bath at 37°C. The released cells were aspirated, and the remaining tissue was redigested until almost all the cells were released. The cells were then suspended in DVF12 medium supplemented with 5% fetal bovine serum and plated in laminin-precoated 35-mm wells (20 μg per well) with a density of 106 cells per well. On day 2, the old medium was aspirated, and 2 mL fresh DVF12 medium containing transferrin (1 mg/mL), fetuin (10 mg/mL), and hydrocortisone (2.5 μg/mL) was added per well. On day 3, this supplemented medium was removed, and fresh DVF12 medium was added per well. The factor to be assayed or the buffer was then added, and the wells were preincubated for 24 hours at 37°C. [3H]Leucine (272.2×109 disintegrations per minute per millimole) was added to each well, and the cells were incubated in the presence or absence of the factor for 2 hours at 37°C. The medium was removed, and the cells were lysed by using 1 mL of 0.1% SDS solution. The plates were kept for 30 minutes at room temperature with occasional shaking. A 50 μL aliquot was taken from each well and used for DNA measurement. The remaining lysate was brought to 1N with NaOH solution and kept for half an hour. One milliliter of BSA solution (0.5 mg) was added per well. Protein molecules were precipitated by using 1 mL of 20% trichloroacetic acid (TCA) and collected on individual filter paper in a cell harvester. The filter papers were washed exhaustively with 5% TCA until they were free of radioactive count. Counts were taken in a Beckman β-scintillation counter after the addition of 5 mL scintillation fluid in each tube containing one piece of filter paper. Data were expressed as disintegrations per minute per nanogram DNA.
Determination of DNA
DNA determinations were performed by the fluorometric method described by Labarca and Paigen.12 The standard curve was prepared with calf thymus DNA. Bisbenzimide solutions (Hoechst 33258) were used as fluorescence dye; a Perkin-Elmer LS-5B luminescence spectrometer at excitation 356 nm was used to read the samples.
Measurement of Protein
Protein measurements were carried out by the protein microassay procedure described by Bradford.13 Standard curves were drawn using BSA at different concentrations ranging from 5 to 25 μg. Absorbance was monitored in a Beckman spectrophotometer at 595 nm.
Quantification of Myotrophin
The amount of myotrophin present in the different samples was quantified by dot blot analyses. The dot blot analysis, similar to the Western blot analysis procedure, was carried out by applying the known and unknown protein samples (in PBS, 1 to 5 μL) directly on GeneScreen instead of transferring the samples from SDS-polyacrylamide gel, the technique required for Western blot analysis. Antibody was then allowed to react with myotrophin after being blocked with milk solution. The bound antibody was detected by using 125I-labeled protein A, followed by autoradiography. The quantification of the autoradiographs (the intensities of the various dots or bands) was achieved by using an image analyzer (Fotodyne Inc). In this system, the photographic image from a video camera was transferred to a Mac II computer in which the accurate quantification of each band (or dot) was performed by using a software package (image 1.33, supplied by the National Institutes of Health) after the background was subtracted. Each band was analyzed at least six times. A standard curve was drawn by using the density of the band against the amount of protein. The amount of myotrophin present in the unknown samples was then quantified directly from the standard curve and calculated as density per 5 μg protein.
For the statistical analysis of the quantification of myotrophin in various age groups in normal and SHR hearts, a two-way ANOVA was performed to find any significant variation in myotrophin levels (for each density at each amount of protein) between rat types and different age groups. Then the data for all groups were analyzed by one-way ANOVA. The pairwise comparisons were then performed by using Tukey’s test. Variability of the data was determined by finding the coefficients of variation and the median of the coefficients of variation (for each amount of myotrophin). For protein synthesis studies, values for myotrophin-treated groups (in the presence and absence of immune and preimmune IgG) were normalized to the control value (vehicle treated) in each experiment, consisting of three culture plates (six wells per plate), by using Student’s t test and ANOVA when appropriate. Statistical significance was defined as P<.05.
Determination of Antibody Titer
Rabbits in which antipeptide antibody was raised against the MAP were bled to obtain the antiserum; the titers of the antiserum were determined by solid-phase radioimmunoassays, with the MAP and natural myotrophin used as separate antigens. The antibody recognized natural myotrophin as the antigen. The binding curves have been drawn by using the percentage of the antibody bound against dilution of the antiserum. Since the nature of these two curves is similar, it showed that the antibody raised against the synthetic oligopeptide, MAP, cross-reacted with natural myotrophin.
Determination of the Specificity of the Antibody
To define the specificity of the polyclonal antibody raised against MAP, the affinity constant of that antibody was calculated from the Scatchard plot, which was constructed by plotting the ratio of bound to free myotrophin against bound myotrophin, as shown in Fig 1⇓. Since the graph is not a perfect straight line, the theoretical best-fitting straight line was drawn with a regression coefficient value of .979. The slope of the graph was calculated as −2.61×107 L/mol. Since the affinity constant equals (negative) slope, the affinity constant of the myotrophin antibody was found to be 2.61×107 L/mol. This value of the affinity constant of myotrophin antibody is in good agreement with the reported values of the affinity constants of a number of known antibodies in the literature. The specificity of the antibody was further demonstrated by Western blot analysis. The result of a typical Western blot analysis is shown in Fig 2⇓, left. We applied crude proteins from the heart homogenates of normal rats (16-week-old Wistar rats) and SHR (16 weeks old). As indicated in Fig 2⇓, left, in each case, only one protein band appeared in the region of 12 kD. The molecular weight of myotrophin is 12 kD. Thus, the result demonstrated that the antibody formed against MAP cross-reacted with myotrophin. The effect of pretreatment of the antibody with myotrophin is shown in Fig 2⇓, right (lane I). No protein band was detected on the autoradiograph. However, when the same membrane was treated with the antibody solution without pretreatment with myotrophin, a single protein band appeared in the region of 12 kD (lane II). These results showed that there are no other proteins present in the heart with a molecular weight similar to that of myotrophin that cross-reacted with the antibody and that only myotrophin could block the binding sites of the antibody.
A number of other growth factors, namely acidic and basic fibroblast growth factor, nerve growth factor, insulin-like growth factor, norepinephrine, and insulin, are known to stimulate cellular growth.14 15 16 17 18 To determine whether or not these growth factors have any affinity for the antibodies raised against MAP, solid-phase radioimmunoassays were performed in which the microtiter wells of the 96-well microtiter plate were separately precoated with myotrophin and each of the growth factors mentioned (Fig 3⇓). The percentage of antibodies bound to myotrophin was plotted against the dilution of the antipeptide antiserum. No significant binding of those growth factors and the antiserum was found, indicating that antibodies are specific only for myotrophin.
Effect of Antibody on Myotrophin-Induced Stimulation of Protein Synthesis in Neonatal Myocytes
To determine whether the antibody could neutralize myotrophin-induced stimulation of protein synthesis in neonatal myocytes, we treated neonatal myocytes with myotrophin in the presence of preimmune and immune IgG. The amount of [3H]leucine incorporation into the myocyte protein was then measured (Fig 4⇓, left). The percent stimulation of [3H]leucine incorporation (disintegrations per minute per nanogram DNA) over the control value was then plotted against different factors studied. Myotrophin alone and separately in the presence of preimmune IgG showed that there was ≈70% stimulation of [3H]leucine incorporation over the control value and that stimulation of protein synthesis by myotrophin was almost completely blocked in the presence of immune IgG. Preimmune IgG had no reducing effect on that activity of myotrophin. In a set of parallel experiments, insulin rather than myotrophin was used. In these instances, immune IgG showed no blocking effect on the stimulatory activity of insulin. Fig 4⇓, right, shows the result.
Quantification of Myotrophin
The quantification of myotrophin concentration in the hearts of different age groups of SHR and in other organs was performed by using dot blot analyses. The arbitrary density units of the image analyzer was plotted against the amount of protein applied on GeneScreen. For a certain range of the protein and density units, a straight line could be obtained. The protein was applied within this linear region. The results of a typical dot blot analysis, for which we applied 1 to 5 μg of crude protein from the 17-week-old normal Wistar and 17-week-old SHR hearts, are shown in Fig 5⇓. A detectable amount of myotrophin was present even in 1 μg of crude protein present in SHR heart homogenate, but no detectable amount of myotrophin was found in 1 μg of crude proteins in normal rat heart homogenate. When 4 μg of the crude protein was applied, myotrophin was detectable in normal hearts as well (Fig 5⇓). The interassay variability of six different experiments performed with 5 μg of crude proteins obtained from 17-week-old male SHR and normal Wistar rat hearts showed no significant difference in the density units. The results are summarized in the Table⇓. Proteins from embryo, 1-day-, 3-day-, 9-day-, 4-week-, 8-week-, 11-week-, and 17-week-old normal Wistar rats and SHR hearts were used for the present study. Data are summarized in Fig 6⇓. In the embryonic stage, the myotrophin concentration remains similar in both normal rat and SHR hearts. It clearly showed that myotrophin levels started increasing as early as 3 days of age in SHR hearts. At 9 days, the increase in myotrophin concentration was statistically significant. Myotrophin concentration then increased linearly up to 17 weeks of age in SHR hearts. On the other hand, in normal rat hearts, the myotrophin level did not change significantly in any age groups. The 17-week-old SHR heart contains approximately sevenfold more myotrophin than does the corresponding age- and sex-matched normal rat heart. No detectable amount of myotrophin was found in lung, kidney, or liver.
In the present study, we have quantified levels of myotrophin concentration in the hearts of SHR and normal rats of different age groups. Myotrophin was quantified in normal and hypertrophied rat hearts for various age groups by using the specific antibody raised against MAP. The antibody was characterized by solid-phase radioimmunoassays and Western blot analysis. To define the specificity of the polyclonal antibody raised against myotrophin, the affinity constant was calculated from the Scatchard plot. The accuracy of the quantification depends on the specificity of the antibody raised against MAP and its affinity toward the antigen (myotrophin). The specificity of the antibody has been established by three methods. First, we determined the affinity constant of the myotrophin antibody from the Scatchard plot as described in “Materials and Methods.” The Scatchard plot was constructed by plotting the ratio of bound to free myotrophin against the concentration of bound myotrophin. The best-fitting graph was drawn with a regression coefficient value of .979. We calculated the slope of the graph as −2.61×107 L/mol. From the equation, the affinity constant equals (negative) slope. Hence, the affinity constant of the myotrophin antibody was found to be 2.61×107 L/mol. Typical values of affinity constants of various known antibodies range from 105 to 1011 L/mol. The binding of the antibody to the antigen is considered to be nonspecific if the value of the affinity constant is <104 L/mol.19 Therefore, our data showed that the antibody raised against MAP was specific and of high affinity toward natural myotrophin. Second, we determined the effect of the antibody on the biological activity of myotrophin (as determined by its effect on the stimulation of protein synthesis in myocytes), which showed complete blocking of the stimulation of protein synthesis when preincubated with antibody, whereas preincubation with the preimmune IgG (Fig 4⇑, left) did not show any inhibitory effect. Third, the specificity of the antibody was further confirmed by Western blot analysis as shown in Fig 2⇑, left, where only one protein band was found in the region of 12 kD (molecular mass of myotrophin is 12 kD). To rule out the possibility that no other proteins with molecular weight or biological activity similar to that of myotrophin are present in the heart and cross-react with the antibody, we performed another Western blot experiment in which myotrophin blocked the binding sites of this antibody (when the antibody was pretreated with myotrophin). As shown in Fig 2⇑, right (lane I), no protein band appeared on the autoradiograph as a result of the pretreatment of the antibody with myotrophin, whereas a single protein band appeared in the region of 12 kD when the same membrane was treated with the antibody solution without pretreatment with myotrophin (lane II). Finally, no other known growth factors (as shown in Fig 3⇑) showed any affinity for this antibody. Combining all these data together, our results showed that the antibody raised against MAP was specific for myotrophin and suitable for quantifying myotrophin from different tissues.
Myotrophin, a protein molecule present in both SHR and human dilated cardiomyopathic hearts, has been found to stimulate the incorporation of [3H]leucine and [14C]phenylalanine into myocyte protein in vitro.1 Myotrophin was purified to homogeneity and partially sequenced from SHR hearts and separately sequenced from dilated cardiomyopathic human heart tissue. Structurally and functionally, the rat and human myotrophin appeared to be very similar. Myotrophin was found to increase the specific activity of leucyl tRNA without significantly changing the intracellular leucine pool.20 Myotrophin has also been shown to be specific for myocytes only, because it has no effect on fibroblasts, endothelial cells, or aortic smooth muscle cells. It has no mitogenic activity, as evidenced by the absence of any change in tritiated thymidine uptake in neonatal myocytes when compared with that in untreated control cells.1 This novel peptide has the unique property of stimulating the myocytes to grow but not to divide, suggesting that it plays a role in cell differentiation and hypertrophy.
More recent studies have shown that neonatal myocytes exposed to myotrophin have a 4-fold increase in total myosin transcript levels. This increase was accompanied by a selective increase in the β-myosin heavy chain expression without affecting the α-myosin heavy chain expression.2 Neonatal myocytes maintained in culture and treated with myotrophin for 30 minutes were also shown to have a marked increase (20-fold) in c-myc, c-fos, and c-jun mRNA levels. Myocytes treated with myotrophin for 24 hours showed 6-, 3-, and 4-fold increases in atrial natriuretic factor, skeletal α-actin, and connexin transcript levels, respectively,21 indicating that myotrophin may play an important role in the pathogenesis of myocardial hypertrophy.
Recently, the cDNA clones coding for the myotrophin gene have been isolated, and the deduced amino acid composition was determined.3 We have also expressed myotrophin in E coli and purified it, and the biological activity of the recombinant myotrophin was determined by bioassay following the usual procedure as described in “Materials and Methods”; a significant increase in protein synthesis was found. Our recent data confirmed that myotrophin is a novel molecule that stimulates protein synthesis in vitro. Therefore, the quantification of this protein appears to be a valuable marker for hypertrophy in hypertension.
A dissociation between blood pressure and development of hypertrophy has been demonstrated by many investigators.22 23 24 25 26 27 28 These data suggested the existence of one or more factors other than blood pressure control in the initiation of myocardial hypertrophy in hypertension. It is still not known how external loads (mechanical) play a critical role in increase in cardiac mass.
Autocrine or paracrine factors have been suggested as potential candidates for the initiation of cardiac hypertrophy in several studies.29 Hammond et al30 first showed the existence of a soluble factor that stimulated protein synthesis in the heart. It is known that some growth factors can affect the level of expression of other growth factor genes, as reported by Schneider and Parker31 and Dzau and Pratt.32 Sadoshima et al33 also reported that a 6-hour treatment with angiotensin II increases the level of expression of transforming growth factor-β1 as well as angiotensinogen genes in cardiac myocytes. Our data suggest that myotrophin is a novel and unique factor that has been identified, isolated, and purified from SHR heart and that selectively stimulates protein synthesis both in adult and neonatal myocytes. Although myotrophin stimulated myocyte growth, its role under pathological conditions such as cardiac hypertrophy has not been demonstrated. The data in the present study further support the role of myotrophin in the development and especially the initiation of hypertrophy. As shown here, in neonatal rats, the myotrophin level was found to be high; as the animals grew older, the myotrophin level remained elevated in SHR (up to 17 weeks) but remained unchanged in normal rats. However, the mechanism by which myotrophin exerts its effect in increased protein synthesis in the neonates and in older SHR has yet to be determined. Currently, work is in progress to define the intracellular mechanism by which myotrophin stimulates myocyte growth. Further studies are necessary to define the pathophysiological effect of myotrophin, especially the mechanism by which it translates cardiac load and myocardial stress (eg, hypertension) into biochemical messages leading to protein synthesis.
This study was supported in part by National Institutes of Health grants HL-33713 and HL-27838. We are grateful to David Young for technical assistance, to Laura Battista for typing the manuscript, and to Christine Kassuba and Suzanne Hazan for editorial assistance.
- Received April 15, 1994.
- Accepted February 8, 1995.
- © 1995 American Heart Association, Inc.
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