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• W2055962649 abstract "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. 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. INTRODUCTIONHypertrophic 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 (1Marino T.A. Kent R.L. Uboh C.E. Fernandez E. Thompson E.W. Cooper G. Am. J. Physiol. 1985; 249: H371-H379PubMed Google Scholar). This accumulation of cardiocyte proteins occurs by an increase in rates of protein synthesis relative to rates of protein degradation (2Morgan H.E. Gordon E.E. Kira Y. Chua B.H.L. Russo L.A. Peterson C.J. McDermott P.J. Watson P.A. Annu. Rev. Physiol. 1987; 49: 533-543Crossref PubMed Google Scholar). 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(3Cooper IV, G. Annu. Rev. Physiol. 1987; 49: 501-508Crossref PubMed Google Scholar). 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 (4Peterson M.B. Lesch M. Circ. Res. 1972; 31: 317-327Crossref PubMed Scopus (66) Google Scholar, 5Kent R.L. Hoober J.K. Cooper IV, G. Circ. Res. 1989; 64: 74-85Crossref PubMed Scopus (130) Google Scholar). These findings have been extended to isolated adult cardiocytes in culture. Rates of protein synthesis were accelerated in response to electrically stimulated cardiocyte contraction(6Ivester C.T. Kent R.L. Tagawa H. Tsutsui H. Imamura T. Cooper IV, G. McDermott P.J. Am. J. Physiol. 1993; 265: H666-H674Crossref PubMed Google Scholar, 7Kato S. Ivester C.T. Cooper IV, G. Zile M.R. McDermott P.J. Am. J. Physiol. 1995; 268: H2495-H2504PubMed Google Scholar), in response to a basal load as defined by adherence of the cardiocytes to the culture dish and establishment of a resting length (8Cooper IV, G. Mercer W.E. Hoober J.K. Gordon P.R. Kent R.L. Lauva I.K. Marino T.A. Circ. Res. 1986; 58: 692-705Crossref PubMed Scopus (72) Google Scholar) and in response to passive stretch of cardiocytes adherent to a deformable membrane(9Mann D.L. Kent R.L. Cooper IV, G. Circ. Res. 1989; 64: 1079-1090Crossref PubMed Scopus (158) Google Scholar). 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(10Weisner R.J. Zak R. Am. J. Physiol. 1991; 260: L179-L188PubMed Google Scholar). 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 (11Boheler K.R. Schwartz K. Trends Cardiovasc. Med. 1992; 2: 176-182Crossref PubMed Scopus (64) Google Scholar, 12Ojamaa K. Petrie J.F. Balkman C. Hong C. Klein I.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3468-3472Crossref PubMed Scopus (46) Google Scholar). At the translational level, protein synthesis rates can be accelerated by changes in efficiency or capacity(2Morgan H.E. Gordon E.E. Kira Y. Chua B.H.L. Russo L.A. Peterson C.J. McDermott P.J. Watson P.A. Annu. Rev. Physiol. 1987; 49: 533-543Crossref PubMed Google Scholar, 13Nagai R. Low R.B. Stirewalt W.S. Alpert N.R. Litten R.Z. Am. J. Physiol. 1988; 255: H325-H328PubMed Google Scholar). 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(2Morgan H.E. Gordon E.E. Kira Y. Chua B.H.L. Russo L.A. Peterson C.J. McDermott P.J. Watson P.A. Annu. Rev. Physiol. 1987; 49: 533-543Crossref PubMed Google Scholar, 14Morgan H.E. Siehl D. Chua B.H.L. Lautensack-Belser N. Basic Res. Cardiol. 1985; 80: 115-118PubMed Google Scholar, 15Siehl D. Chua B.H.L. Lautensack-Belser N. Morgan H.E. Am. J. Physiol. 1985; 248: C309-C319Crossref PubMed Google Scholar). 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(5Kent R.L. Hoober J.K. Cooper IV, G. Circ. Res. 1989; 64: 74-85Crossref PubMed Scopus (130) Google Scholar, 16Kira Y. Kochel P.J. Gordon E.E. Morgan H.E. Am. J. Physiol. 1984; 246: C247-C258Crossref PubMed Google Scholar, 17Imamura T. McDermott P.J. Kent R.L. Nagatsu M. Cooper IV, G. Carabello B.A. Circ. Res. 1994; 75: 418-425Crossref PubMed Scopus (65) Google Scholar, 18Delcayre C. Klug D. Nguyen V.T. Mouas C. Swynghedauw B. Am. J. Physiol. 1992; 263: H1537-H1545PubMed Google Scholar). 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(19Booth F.W. Kirby C.R. Biochem. Soc. Trans. 1991; 19: 374-378Crossref PubMed Scopus (4) Google Scholar, 20Booth F.W. Kirby C.R. Am. J. Physiol. 1992; 262: R329-R332PubMed Google Scholar, 21Thomason D.B. Biggs R.B. Booth F.W. Am. J. Physiol. 1989; 257: R300-R305PubMed Google Scholar). For example, unweighting of the soleus muscle in rats results in a reduction in MHC 1The abbreviations used are: MHCmyosin heavy chainPipes1,4-piperazinediethanesulfonic acidMOPS4-morpholinepropanesulfonic acid. synthesis which has been attributed to a decrease in translational activity(21Thomason D.B. Biggs R.B. Booth F.W. Am. J. Physiol. 1989; 257: R300-R305PubMed Google Scholar).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(6Ivester C.T. Kent R.L. Tagawa H. Tsutsui H. Imamura T. Cooper IV, G. McDermott P.J. Am. J. Physiol. 1993; 265: H666-H674Crossref PubMed Google Scholar). 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.MATERIALS AND METHODSElectrical Stimulation ModelAdult feline cardiocytes were obtained by collagenase digestion in combination with mechanical agitation as described previously(6Ivester C.T. Kent R.L. Tagawa H. Tsutsui H. Imamura T. Cooper IV, G. McDermott P.J. Am. J. Physiol. 1993; 265: H666-H674Crossref PubMed Google Scholar, 8Cooper IV, G. Mercer W.E. Hoober J.K. Gordon P.R. Kent R.L. Lauva I.K. Marino T.A. Circ. Res. 1986; 58: 692-705Crossref PubMed Scopus (72) Google Scholar, 9Mann D.L. Kent R.L. Cooper IV, G. Circ. Res. 1989; 64: 1079-1090Crossref PubMed Scopus (158) Google Scholar). Following cell isolation, the Ca2+-tolerant cardiocytes were placed in Life Technologies, Inc. M199 serum-free medium with Earle's salts at a concentration of 50,000 rod-shaped cells/ml. The cells were plated on 245 × 245-mm culture trays (Nunclone) coated with 20 μg/ml laminin (Upstate Biotechnology, Inc.) to facilitate cell adhesion. After an overnight incubation, the medium was changed to remove nonadherent cells. The M199 medium was modified as before, and adult feline cardiocytes, which are normally quiescent in culture, were induced to contract synchronously via electrical field stimulation as described previously with modification(6Ivester C.T. Kent R.L. Tagawa H. Tsutsui H. Imamura T. Cooper IV, G. McDermott P.J. Am. J. Physiol. 1993; 265: H666-H674Crossref PubMed Google Scholar). In order to stimulate a larger number of cardiocytes, the system was adapted according to the method of Berger et al.(22Berger H.J. Prasad S.K. Davidoff A.J. Pimental D. Ellingsen O. Marsh J.D. Smith T.W. Kelly R.A. Am. J. Physiol. 1994; 266: H341-H349Crossref PubMed Google Scholar) for use with large culture trays in which electrical pulses were delivered to the medium via carbon electrodes submersed at the opposite ends of each tray (Fig. 1). The distance between electrodes was 23 cm. The polarity of the electrodes was alternated with each electrical pulse to minimize electrolysis at the electrodes. In order to set the threshold voltage for contraction, the lowest voltage required to stimulate greater than 50% of the cells was determined and then exceeded by 10%, resulting in an approximate response ratio of 70%. The resultant pulses of approximately 6 volts/cm between electrodes were delivered as a square wave for a duration of 5 ms at 1 Hz for 4 h.Measurement of Myosin Heavy Chain Synthesis RatesRates of MHC synthesis were determined by measuring incorporation of [3H]Phe into electrophoretically purified MHC. Cells were radiolabeled with 0.4 mM [3H]Phe (20 μCi/ml, Amersham Corp.) over the entire 4 h of electrical stimulation. Total cell protein was scraped in a lysis buffer consisting of: 0.4 M Trizma (Tris base) glycine, 5% sodium dodecyl sulfate (SDS), 20% glycerol, 0.5%β-mercaptoethanol, and 1.5 mM phenylmethylsulfonyl fluoride. The lysates were boiled for 5 min and cooled to room temperature. The samples were electrophoresed on 3-mm polyacrylamide-N,N‘-diallyltartardiamide 10-13% gradient gels for 24 h at 10 V/cm. The MHC band was identified by Coomassie Brilliant Blue R-250 (Bio-Rad) staining and excised. The band was solubilized in 2% periodic acid, 4% lactic acid, pH adjusted to 3.0 with NH4OH. The protein was precipitated by adding 70% HClO4 to a final concentration of 6% and centrifuged 15 min at 10,000 × g. The pellet was washed twice with 6% HClO4 and dried by vacuum centrifugation. The pellet was solubilized in 0.3 N NaOH by heating to 60°C for 1 h. Aliquots were assayed for radioactivity by liquid scintillation counting and for MHC protein by the BCA method (Pierce) using bovine serum albumin as a standard. The specific radioactivity of Phe in the medium was determined by scintillation counting of aliquots of culture medium. Previous studies have shown that the Phe-tRNA pools equilibrate to 80% of medium Phe-specific radioactivity in both electrically stimulated and quiescent cardiocytes(6Ivester C.T. Kent R.L. Tagawa H. Tsutsui H. Imamura T. Cooper IV, G. McDermott P.J. Am. J. Physiol. 1993; 265: H666-H674Crossref PubMed Google Scholar).Probes for MHC mRNATwo probes were used to measure MHC mRNA levels. For RNase protection assays, [32P]UTP-labeled antisense cRNA probes were synthesized by in vitro transcription from a 436-base pair fragment (p-MHC-5) of the rat α-MHC cDNA inserted into a pGEM-3Zf(+) plasmid(23Dillmann W.H. Barrieux A. Shanker R. Endocr. Res. 1989; 15: 565-577Crossref PubMed Scopus (18) Google Scholar). This region of α-MHC mRNA extends from nucleotides 3459 through 3895 of the coding sequence and is highly conserved as indicated by a 90% sequence similarity to rat β-MHC mRNA and an 89% sequence similarity to human β-MHC mRNA. In the ventricle of adult cat, it has been demonstrated that the β-MHC isoform is expressed exclusively(24Wisenbaugh T. Allen P. Cooper IV, G. O'Connor W.N. Mezaros L. Streter F. Bahinski A. Houser S. Spann J.F. Am. J. Physiol. 1984; 247: H146-H154PubMed Google Scholar). In order to confirm that the degree of homology of the probe was the same for feline β-MHC mRNA, a partial cDNA clone for feline β-MHC was isolated from an adult feline cardiocyte cDNA library. The identity of the cDNA clone as the β-MHC isoform was based upon the high degree of sequence similarity to the 3′-untranslated regions of the rat and human β-MHC mRNA isoforms. Sequence analysis of the region extending between nucleotides 3459-3714 revealed that the degree of homology of feline β-MHC to rat α-MHC is 92%, the same as for rat β-MHC. 2W. J. Tuxworth and P. J. McDermott, unpublished data. RNase protection using total RNA extracted from adult cardiocytes yielded a protected band of approximately 83 nucleotides, the same size band observed for rat β-MHC mRNA.For the slot-blotting analyses, a 215-base pair cDNA clone of feline cardiac β-MHC mRNA was employed. DNA sequence analysis revealed that the feline species was greater than 92% similar to rat β-MHC mRNA in the region between nucleotides 4028 and 4243 and to human β-MHC in the region between 4017 and 4232. The cDNA insert was subsequently subcloned into a pGEM-3Zf(+) plasmid. [32P]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.Quantitation of MHC mRNA ExpressionTotal cellular RNA was extracted from cell cultures via the RNAzol method (Biotecx Laboratories Inc.), and MHC mRNA was quantified by an RNase protection assay as follows. The RNA was resuspended in hybridization buffer containing 80% formamide, 40 mM Pipes, pH 6.4, 1 mM EDTA, 400 mM NaCl. Aliquots were taken for determination of 28 S rRNA by slot blotting as described previously(25Johnson T.B. Kent R.L. Bubolz B.A. McDermott P.J. Circ. Res. 1994; 74: 448-459Crossref PubMed Scopus (42) Google Scholar). Antisense cRNA probe was added in excess, and the samples were denatured 15 min at 85°C. RNA was solution hybridized 18 h at 47°C followed by S1 nuclease digestion of nonhybridized RNA for 2 h with 300 μl of digestion buffer (50 mM sodium acetate, pH 4.6, 280 mM NaCl, 4.5 mM ZnSO4, 250 units/ml S1 nuclease (Promega)). Reactions were terminated with 50 μl of stop buffer (4 M ammonium acetate, 100 mM EDTA). The samples were phenol/chloroform-extracted and precipitated in ethanol with 20 μg of Escherichia coli tRNA as carrier. The samples were pelleted by centrifugation, washed in 70% ethanol, and resuspended in 80% formamide, 10 mM EDTA. Samples were denatured for 10 min at 65°C and electrophoresed on 8 M urea, 4% polyacrylamide gels. The gels were washed, dried, and processed for autoradiography. The protected MHC mRNA band was quantified by computer-assisted image analysis of autoradiograms and normalized to the corresponding amount of 28 S rRNA/sample as determined by slot blotting.Quantitation of MHC mRNA in Polysome FractionsCardiocyte cultures were rinsed three times on ice with ice-cold phosphate-buffered saline to which 100 μg/ml cycloheximide was added to arrest polypeptide chain elongation. Cardiocytes were scraped from the culture trays twice in 10 ml of phosphate-buffered saline/cycloheximide, pelleted by centrifugation, and resuspended in 1 ml of resuspension buffer (10 mM Tris, pH 7.5, 250 mM KCl, 2 mM MgCl2, 0.5% (v/v) Triton X-100). The resuspended cells were homogenized with 18 strokes of a glass A pestle Dounce homogenizer and transferred to a chilled 1.5-ml microtube. 150 μl of a solution containing 10% (v/v) Tween 80, 5% (w/v) deoxycholate was added, and the homogenate was vortexed and incubated on ice for 15 min. Homogenates were centrifuged 10 min at 10,000 × g to pellet nuclei, mitochondria, and insoluble proteins. The polysome distribution of MHC mRNA was measured by two methods, RNase protection and slot blotting.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% Na4P2O7, 1% SDS, 100 μg of salmon sperm DNA/ml and 32P-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.Quantitation of Subunit RNAPost-mitochondrial supernatants were prepared from electrically stimulated and quiescent cultures as described above for polysomes. A 150-μl aliquot of the post-mitochondrial supernatant was removed to assay for total RNA and a 900-μl aliquot was layered onto 10 ml of 15-68% exponential sucrose gradients. Gradients were centrifuged 18 h in a Sorvall SW-41 rotor at 37,000 rpm, and 40 and 60 S subunit peaks were collected by gradient fractionation and pooled. Total RNA and RNA in subunits were assayed by the method of Munro and Fleck(26Munro H. Fleck A. Methods Biochem. Anal. 1966; 14: 113-176Crossref PubMed Google Scholar), and RNA in subunits was calculated as a percentage of total RNA(15Siehl D. Chua B.H.L. Lautensack-Belser N. Morgan H.E. Am. J. Physiol. 1985; 248: C309-C319Crossref PubMed Google Scholar, 26Munro H. Fleck A. Methods Biochem. Anal. 1966; 14: 113-176Crossref PubMed Google Scholar).Statistical AnalysisStatistical analysis was performed using Stat-View 4.0 software (Abacus Concepts Inc.). Because the two experimental groups of cardiocytes were derived from the same preparation, differences between nonstimulated and electrically stimulated groups were determined using a paired Student's t test. A value of p < 0.05 was considered to be a significant difference.RESULTSWe 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 (6Ivester C.T. Kent R.L. Tagawa H. Tsutsui H. Imamura T. Cooper IV, G. McDermott P.J. Am. J. Physiol. 1993; 265: H666-H674Crossref PubMed Google Scholar) 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 α-MHC cDNA hybridized specifically to feline β-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 α- and β-MHC isoforms. In lane 2, the specificity of the feline β-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 α-MHC cRNA probe (lane 1) and the feline β-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. R2 = 0.99.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 t" @default.
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• W2055962649 title "Contraction Accelerates Myosin Heavy Chain Synthesis Rates in Adult Cardiocytes by an Increase in the Rate of Translational Initiation" @default.
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