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(Circulation Research. 1996;78:829-838.)
© 1996 American Heart Association, Inc.

Articles

Passive Load and Angiotensin II Evoke Differential Responses of Gene Expression and Protein Synthesis in Cardiac Myocytes

Robert L. Kent, Paul J. McDermott

From the Gazes Cardiac Research Institute, Cardiology Division of the Department of Medicine; the Departments of Pharmacology and Anatomy and Cell Biology, Medical University of South Carolina; and the Ralph H. Johnson Department of Veterans Affairs Medical Center, Charleston, SC.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract This study introduced an improved model of loaded adult cardiocytes to address a proposed requirement for angiotensin II (Ang II) in the transduction pathway between load on the cardiac myocyte and its early anabolic responses of gene expression and acceleration of protein synthesis. The isolated cardiocytes were subjected to passive load by step increments of stretch and responded with proportional acceleration of protein synthesis in both adult and neonatal cardiocytes; this response was unaltered by 1 µmol/L [Sar¹,Ile⁸]Ang II, an antagonist peptide to Ang II. Ang II from 1 nmol/L to 10 µmol/L did not increase protein synthesis after 4 hours in adult cardiocytes nor at 100 nmol/L in neonatal cardiocytes. However, 100 nmol/L Ang II did increase [³H]phenylalanine incorporation into neonatal cardiocyte protein over a 24-hour period by 10%, whereas passive load increased [³H]phenylalanine incorporation into protein by 30%, which was not blocked by [Sar¹,Ile⁸]Ang II. Thus, the anabolic effect of load does not require Ang II to increase either 4-hour protein synthesis in both adult and neonatal cardiocytes or 24-hour [³H]phenylalanine incorporation into protein in neonatal cardiocytes. The genetic response of the cardiocyte to load was examined by assessing c-fos and Na⁺-Ca²⁺ exchanger mRNA levels, because these are rapidly expressed at the onset of cardiac pressure overload. The c-fos mRNA was increased fourfold within 1 hour after 100 nmol/L Ang II treatment of either adult or neonatal cardiocytes. This c-fos induction was blocked by [Sar¹,Ile⁸]Ang II. One hour after loading of adult cardiocytes, induction of c-fos expression was increased threefold; this was also blocked by [Sar¹,Ile⁸]Ang II. Thus, load-induced c-fos expression was Ang II dependent in adult cardiocytes. In contrast, exchanger mRNA levels were increased threefold 1 hour after loading of adult cardiocytes, but this increased expression was not blocked by [Sar¹,Ile⁸]Ang II. For additional comparison, c-fos expression was induced by Ang II and phorbol myristate acetate, which did not induce exchanger expression; conversely, exchanger expression was induced by veratridine, which did not increase c-fos expression. Thus, separate c-fos and exchanger expression pathways can be differentiated in adult cardiocytes. This study demonstrated that Ang II is not required for load to initiate the anabolic processes of accelerated protein synthesis or enhanced Na⁺-Ca²⁺ exchanger gene expression in cardiocytes; however, load induced c-fos expression is Ang II dependent.

Key Words: angiotensin II • gene expression • protein synthesis • cardiac myocytes • cell culture

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chronic hemodynamic overload on the heart induces hypertrophic growth to reduce wall stress on the myocardium.¹ However, hypertrophy may be accompanied by a loss of mechanical efficiency that can ultimately progress to heart failure.^{2 3} Cardiac hypertrophy can occur as either a direct response or an indirect response to increased afterload. For example, an increased afterload after arterial stenosis is a direct stimulus for cardiac hypertrophy, whereas elevation in vasoconstrictor hormones such as Ang II can indirectly increase afterload and lead to cardiac enlargement.⁴ In addition, a direct action of vasoconstrictor hormones on cardiac growth may be involved, because local Ang II production and receptors have been demonstrated in the myocardium^{5 6} and angiotensin-converting enzyme inhibitors are very effective in inducing regression of cardiac hypertrophy in both animals and humans.^{7 8} One possible explanation is that cardiac-secreted Ang II might feed back on the myocardium in an autocrine manner and regulate myocyte growth.^{5 9} However, as an alternative possibility, the indirect anabolic effect of Ang II can also occur by increased cardiac workload, since Ang II enhances norepinephrine release from sympathetic nerves with subsequent inotropic and chronotropic actions.¹⁰ Therefore, in order to simplify these complex systemic interactions of hemodynamic load and hormones on cardiac growth in vivo, cardiac myocytes in primary culture have been used to investigate the direct effects of load and hormones on myocyte growth.

Several studies have used cardiocytes in culture to examine the potential anabolic effects of both load and Ang II in regulating anabolic processes that mimic those also induced at the onset of cardiac pressure-overload hypertrophy. Neonatal rat cardiocytes subjected to load in the form of passive stretch increased protein synthesis within 24 hours and the expression of the immediate early gene c-fos within 30 minutes. Both of these responses were blocked by antagonists for the Ang II receptor subtype AT₁.^{9 11} Ang II was secreted by the neonatal cardiocytes in response to passive stretch,⁹ and Ang II has been reported to increase protein synthesis in embryonic chick cardiocytes¹² and rat neonatal cardiocytes¹¹ and to induce immediate-early gene expression.^{13 14} These findings have led to a conclusion that the early anabolic responses of the cardiocytes to load are mediated through a mechanism involving autocrine Ang II receptor activation. In contrast, isolated adult cardiocytes demonstrated no acute effect on protein synthesis in response to Ang II at 1 nmol/L to 10 µmol/L in rat cardiocytes¹⁵ and at 10 nmol/L in feline cardiocytes.¹⁶ Yet adult feline cardiocytes subjected to load on day 3 in culture had increased protein and RNA synthesis within several hours.¹⁷ Furthermore, accelerated protein synthesis and cell growth occurred in adult feline cardiocytes subjected to a sustained workload of electrically stimulated contractions, and neither of these anabolic effects were blocked by an AT₁ receptor antagonist.¹⁶ Thus, dissimilar findings with adult cardiocytes suggest that the anabolic responses to load do not require activation of an angiotensin receptor.

In the present study, an adult cardiocyte model of passive load was developed to address a proposed requirement for Ang II in the transduction pathway between load on the cardiocyte and its early anabolic responses of gene expression and acceleration of protein synthesis. A loaded cardiocyte model was used in which adult feline cardiocyte populations can be passively stretched within 24 hours of isolation without serum or other mitogens. For comparison, the passively stretched neonatal model was also used in order to exclude potential differences in responses between adult feline cardiocytes and rat neonatal cardiocytes. Passive stretch and Ang II, in the presence and absence of the Ang II antagonist [Sar¹,Ile⁸]Ang II, were applied to the cardiocytes, whose subsequent anabolic responses of accelerated protein synthesis and increased gene expression were measured. The specific mRNAs examined were c-fos, an immediate-early gene product critical for cell proliferation, and the Na⁺-Ca²⁺ exchanger, a membrane enzyme critical for ion balance across the sarcolemma. Both c-fos mRNA^{18 19 20} and exchanger mRNA²¹ are rapidly expressed at the onset of cardiac pressure overload and thus are markers for the hypertrophic response. The findings of the present study demonstrate that the hormone Ang II is not obligatorily required for load to initiate the anabolic processes of accelerated protein synthesis or increased Na⁺-Ca²⁺ exchanger gene expression. Yet load on the cardiocyte did initiate an Ang II–dependent expression of c-fos, which was also delineated as a pathway separate from that involving Na⁺-Ca²⁺ exchanger gene expression.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cardiocyte Preparations
The adult feline cardiocytes used in this proposal have been characterized previously in terms of their metabolic, structural, electrophysiological, and functional properties.²² Cats weighing 1.5 to 3.5 kg were anesthetized with ketamine HCl (50 mg/kg IM), acepromazine maleate (1 mg/kg IM), and meperidine (2.2 mg/kg IM) and submitted to rapid cardiectomy under sterile conditions. The aorta was cannulated, and the coronary arteries were perfused retrogradely, first with a Krebs' buffer containing (mmol/L) NaCl 130.0, KCl 4.8, MgSO₄ 1.2, NaH₂PO₄ 1.2, NaHCO₃ 4, HEPES 10, and D-glucose 12.5 and then with the same buffer supplemented with 155 U/mL of type II collagenase. The Ca²⁺ in these solutions ranged from 60 to 80 µmol/L; the osmolarity, from 290 to 310 mOsm, at 37°C and pH 7.4, with continuous equilibration by 100% O₂. The perfusion was terminated when the heart was flaccid. The atria, great vessels, and annulus were removed, and the ventricular tissue was minced in buffer with collagenase and incubated for 5 minutes in a shaking water bath at 36°C. After incubation, the cells were filtered through a 210-µm nylon mesh, rinsed with additional buffer, and then gently centrifuged at 100g for 2 minutes. The pelleted cells were resuspended in buffer supplemented with 1% bovine serum albumin, allowed to resediment, and then resuspended in buffer supplemented with 50 µmol/L Ca²⁺. The resedimented cardiocytes were dispersed in a serum-free, chemically defined culture medium, M199, with Earle's salts and 100 mg/L L-glutamine, which was supplemented with 100 U/mL penicillin. Cardiocytes were kept in a 37°C incubator in which the atmosphere was humidified and equilibrated with 5% CO₂ to achieve a final medium pH of 7.35 to 7.40. Fibroblast contamination was minimized by the use of conventional selective adhesion techniques in which freshly isolated cardiocytes were dispersed in M199 and incubated for 2 hours in polystyrene flasks to which only nonmyocytes attached; the cells that did not attach to this substrate were used for plating. Reproducible yields of 60% to 70% rod-shaped, Ca²⁺-tolerant adult feline cardiocytes were obtained. A 2-mL aliquot of cells was plated onto a laminin-coated synthetic elastic membrane at a final density of 50 000 rod-shaped cells per milliliter. The next day, adherent cells were rinsed to yield a density of 2000 rod-shaped quiescent cardiocytes per square centimeter, with a plating efficiency of 30%.

The rat neonatal cardiocytes used in the present study have been characterized previously in terms of their metabolic, structural, and functional properties.^{23 24 25} Primary cell cultures were prepared from minced ventricular myocardium of 2- to 3-day-old newborn rats, which were anesthetized with the gaseous anesthetic methoxyflurane and submitted to rapid cardiectomy under sterile conditions. The atria, great vessels, and annulus were removed, and then the rats' hearts were minced in Ca²⁺-Mg²⁺–free Hanks' salt solution buffered with HEPES. The cells were dissociated in a water-jacketed Celstir apparatus at 37°C with a mixture of the same buffer supplemented with 155 U/mL of type II collagenase. After each of six successive 20-minute incubations, the isolated cells were pooled, enriched for cardiocytes by differential adhesion, and dispersed in MEM (GIBCO) containing 10% newborn calf serum, 1 µg/mL bovine insulin, 0.1 mmol/L BrdU, amino acids, vitamins, 50 U/mL penicillin, 2 mmol/L glutamine, 10 µg/mL human transferrin, and 0.25 mmol/L ascorbic acid. A 2-mL aliquot of cells was plated onto a laminin-coated synthetic elastic membrane at a final density of 250 000 rod-shaped cells per milliliter. Cardiocytes were kept in a 37°C incubator in which the atmosphere was humidified and equilibrated with 5% CO₂ to achieve a final medium pH of 7.35 to 7.40. The next day and day 3 in culture, adherent cells were rinsed and maintained in this same medium without serum to yield a density of 8000 cardiocytes per square centimeter, with a plating efficiency of 25%. These neonatal cardiocytes formed a nonconfluent weblike syncytium and demonstrated intermittent spontaneous contractions.

Experimental Interventions
Passive load was applied by stretching a laminin-coated elastic membrane to which the isolated cardiocytes were adhered. This 2-mil-thick elastic membrane was made of semitransparent polyurethane and was fixed to the bottom and sides of a culture well of a stretch frame, which was constructed of fluorocarbon plastic as previously described.^{17 26} This polyurethane membrane has a greater adsorptive capacity for proteins than does the polycarbonate silicone membrane used previously, so that many cardiocytes firmly adhere to the membrane after it is coated with 20 µg/mL of laminin, a basement membrane protein that binds to sarcolemmal proteins for adult and neonatal cardiocytes.²⁷ The cardiocytes were cultured in the stretch frames enclosed in Petri dishes and incubated overnight for adult cardiocytes and over 3 days for neonatal cardiocytes. The stretch frame was expanded by turning a pair of opposing thumbscrews mounted horizontally at opposite ends of the frame, such that a 10% change in the diameter of the stretch frame caused a 10% change in the length of the membrane to which the cardiocytes were adhered. Passive load was applied to the cardiocytes by stretching the elastic membrane in three successive steps. After each incremental step increase in membrane stretch, the stretch frames were returned to the incubator for 8 minutes, so that the entire loading procedure was completed within 20 minutes. Adult cardiocytes were stretched to 10% beyond their initial resting length, and neonatal cardiocytes were stretched to 20% beyond their initial resting length. Stretched adult cardiocytes remained quiescent, and stretched neonatal cardiocytes continued to contract spontaneously.

Ang II (100 nmol/L), PMA (1 µmol/L), or veratridine (2 µmol/L) was applied directly to the cultured nonloaded cardiocytes, unless the concentration is noted otherwise. [Sar¹,Ile⁸]Ang II (1 µmol/L), a receptor antagonist for Ang II, was applied to loaded or nonloaded cardiocytes for at least 1 hour. Before dilution by >100-fold in the culture medium, the peptides and veratridine were dissolved in sterile water, and the PMA was dissolved in sterile dimethyl sulfoxide.

Protein Synthesis Measurements
Protein synthesis measurements were initiated in cardiocytes, 15 minutes after either passive stretch or Ang II treatment, by the addition of 10 µCi/mL of [³H]PHE to the cardiocyte culture medium described above. The medium contained 0.4 mmol/L unlabeled L-PHE to facilitate equilibration of the specific radioactivities of the medium PHE and phenylalanyl-tRNA pools.²⁸ Since these precursor pools of PHE were found to be equally equilibrated in quiescent and contracting feline adult²⁹ and rat neonatal²⁴ cardiocytes, the specific radioactivity of the PHE in the medium was used to calculate rates of protein synthesis. Protein synthesis rates were measured after 4 hours of pulse labeling, and total protein incorporation measurements were obtained over a 24-hour labeling period. At the completion of the labeling period, the cells were rinsed in culture medium containing 10 mmol/L PHE. The cells were then scraped from their surface with a solution containing 2% SDS, 10 mmol/L Tris at pH 7.4, and 1.5 mmol/L phenylmethylsulfonyl fluoride, a protease inhibitor. The cell proteins were precipitated in 6% HClO₄ and centrifuged. The protein pellet was then washed three more times with cold HClO₄, incubated in HClO₄ at 80°C for 20 minutes, and washed with HClO₄ twice more. The protein pellet was dissolved in 0.3N NaOH, incorporation of [³H]PHE into protein was counted by liquid scintillation, and rates of protein synthesis were calculated as described previously.^{20 29} The total cardiocyte proteins were not significantly altered by membrane stretch or by the agents applied to either adult cardiocytes within a 4-hour period or neonatal cardiocytes within a 24-hour period. The protein synthesis results are expressed as nanomoles of PHE incorporated per gram of protein per hour. The 24-hour total protein incorporation results are expressed as nanomoles of PHE incorporated per gram of protein per 24 hours.

Analysis of mRNA Expression
The mRNA levels for the Na⁺-Ca²⁺ exchanger and c-fos were measured after 1 hour of experimental treatment. Total RNA was extracted from cardiocytes using a mixture of guanidinium thiocyanate, phenol, and chloroform.³⁰ The extracted RNA was electrophoresed in 1% agarose-formaldehyde gels, transferred to nylon membranes, cross-linked to the membranes by UV irradiation, and hybridized with the [³²P]cDNA probe specific for the Na⁺-Ca²⁺ exchanger. This [³²P]cDNA probe was made in a 30-cycle polymerase chain reaction using [³²P]dCTP and a 289-bp feline cDNA clone for the Na⁺-Ca²⁺ exchanger as a template. This feline cDNA has a 93% sequence homology with bases 2293 to 2581 of the dog Na⁺-Ca²⁺ exchanger cDNA sequence,³¹ as previously reported.²¹ The blots were hybridized for 24 hours at 42.5°C and washed at 60°C three times each in 2x SSC (0.3 mol/L NaCl and 0.03 mol/L sodium citrate) with 0.1% SDS and in 0.2x SSC with 0.1% SDS. Autoradiographs of the Northern blots of cardiocyte RNA exhibited a band of 7.2 kb, equivalent to the size of exchanger mRNA.²¹ After hybridization for exchanger mRNA, each Northern blot was hybridized with a second probe specific for c-fos,^{20 32} which resulted in an autoradiographic band of 2.5 kb, consistent with the size of c-fos mRNA. The blots were hybridized a third time with a probe specific for 28S rRNA,³³ which was end-labeled with [³²P]dCTP in a nick-translation reaction. rRNA is 90% of total cellular RNA and served as an RNA baseline, since RNA³³ relative to DNA³⁴ was unchanged after 4 hours of load or drug treatment. The grain density of the autoradiographic bands of mRNA were quantified by computer-assisted digital image analysis. The autoradiographic grain density from the 28S rRNA probe was obtained within a few hours and was used to normalize for the amount of applied RNA to allow a quantitative comparison of exchanger and c-fos mRNA levels. The value for exchanger grain density relative to rRNA grain density from each RNA sample was then expressed as a percentage of the nontreated control value for each experiment.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, a new model was developed in which a large population of adult cardiocytes could be loaded by passive stretch. The cardiocytes were loaded within 24 hours of isolation without requiring serum or mitogens. This cardiocyte model was used to investigate its early anabolic responses to load in terms of protein synthesis and gene expression. Furthermore, this simplified cardiocyte model was used to examine a proposed requirement for activation of the Ang II receptor in eliciting the load-induced anabolic responses from the cardiac myocyte.

Protein Synthesis
An increase in passive load on the cardiocytes was applied by stretching the elastic membrane to which the cardiocytes were firmly adhered in step increments of 3%, 6%, and 10% for adult cardiocytes and 10% and 20% for neonatal cardiocytes. As demonstrated in Fig 1, 4 hours of passive stretch elicited a proportional acceleration of protein synthesis in both adult (r=.98) and neonatal (r=.98) cardiocytes. These data demonstrate that the cardiocytes are capable of responding to small changes in load with proportional changes in protein synthesis. Even though protein synthesis rates were nearly one order of magnitude greater in neonatal rat cardiocytes than in adult feline cardiocytes, protein synthesis rates were accelerated in both cardiocyte preparations in response to stretch.

View larger version (50K): [in this window] [in a new window]   Figure 1. Effects of passive stretch on protein synthesis rates in adult (A) and neonatal (B) cardiocytes. Passive stretch was applied to the cardiocytes by stretching the elastic membrane to which the cardiocytes were firmly adhered in step increments as indicated. Rates of protein synthesis were accelerated in proportion to the static stretch within 4 hours. Each bar height is the average rate of protein synthesis (±SEM) for seven to nine populations of cardiocytes derived from three experiments. *Significant differences in protein synthesis for stretched cardiocytes compared with nonstretched (0%) cardiocytes (P<.05 by a post-ANOVA Newman-Keuls t test).

To examine whether Ang II was involved in facilitating this load-induced acceleration of protein synthesis, the cardiocytes were pretreated for 1 hour before stretch with 1 µmol/L [Sar¹,Ile⁸]Ang II, the sarcosine-isoleucine competitive peptide to Ang II. As shown in Fig 2, the load-induced acceleration of protein synthesis in adult and neonatal cardiocytes was unaltered in the presence of [Sar¹,Ile⁸]Ang II after 4 hours of passive load. Although the Ang II receptor blocker [Sar¹,Ile⁸]Ang II did not alter load-increased protein synthesis, it effectively blocked the induction of c-fos expression by Ang II, as described below. These findings demonstrated that Ang II is not required for load to initiate the anabolic process of accelerated protein synthesis in cardiocytes.

View larger version (38K): [in this window] [in a new window]   Figure 2. Failure of Ang II receptor antagonism to prevent stretch-induced acceleration of cardiocyte protein synthesis in adult cardiocytes (A) and neonatal cardiocytes (B). Cardiocytes were pretreated for 1 hour with 1 µmol/L [Sar1,Ile8]Ang II (SAR-ANG II), an Ang II receptor antagonist, before static stretch of either 10% in adult cardiocytes or 20% in neonatal cardiocytes. Protein synthesis rates were measured over 4 hours of stretch. Each bar height is the average rate of protein synthesis (±SEM) for 9 to 13 populations of neonatal cardiocytes and 10 or 11 populations of adult cardiocytes, respectively, derived from three experiments. *Significant differences in protein synthesis for stretched cardiocytes compared with nonstretched cardiocytes (P<.05 by a post-ANOVA Newman-Keuls t test).

The direct application of Ang II to the adult feline cardiocytes over a range of 1 nmol/L to 10 µmol/L did not alter protein synthesis rates, as shown in Fig 3A. In addition, 100 nmol/L Ang II also failed to alter protein synthesis after 4 hours in neonatal rat cardiocytes (Fig 3B). However, previous studies reported that [³H]PHE incorporation into protein of neonatal rat cardiocytes was increased during a 48-hour labeling period with 10 nmol/L Ang II¹¹ and that [Sar¹,Ile⁸]Ang II prevented a similar stretch-induced increase during a 24-hour labeling period.⁹ These apparent discrepancies with results from both adult and neonatal cardiocytes during a 4-hour labeling period, as shown in Figs 1, 2, and 3, led to a comparison between the effects of Ang II and stretch on neonatal cardiocytes, as measured by the incorporation of [³H]PHE into protein over a 24-hour labeling period. Unfortunately, continuous long-term labeling in culture is intrinsically nonlinear,³⁵ such that labeled amino acid accumulates asymptotically into protein, as shown in Fig 4 in nonstretched neonatal cardiocytes. This significantly alters the corresponding rate of amino acid incorporation to a point where it no longer reflects primarily the rate of protein synthesis, which occurred by 16 hours in Fig 4. With such limitations for this measurement in mind, the 24-hour incorporation of [³H]PHE into protein in neonatal cardiocytes was significantly increased by 33% in response to stretch and was not blocked by pretreatment with 1 µmol/L [Sar¹,Ile⁸]Ang II, an Ang II receptor antagonist, as shown in Fig 5. In contrast, there was only a 10% increase after 100 nmol/L Ang II treatment. Thus, the effect of load on the incorporation of [³H]PHE into protein was threefold greater than that with Ang II.

View larger version (46K): [in this window] [in a new window]   Figure 3. Effects of Ang II on protein synthesis rates in adult (A) and neonatal (B) cardiocytes. Ang II was added to the culture medium, and protein synthesis rates in cardiocytes were measured after 4 hours. Each bar height is the average rate of protein synthesis (±SEM) for 7 to 12 populations of cardiocytes derived from three experiments. No significant differences in protein synthesis rates were found for Ang II–treated cardiocytes compared with nontreated cardiocytes (P>.05 by an ANOVA F test).

View larger version (20K): [in this window] [in a new window]   Figure 4. [3H]PHE accumulation into neonatal cardiocyte protein over 24 hours compared with its corresponding rate of amino acid incorporation. The initial amino acid accumulation into protein was proportional to the [3H]PHE labeling period but became nonlinear with longer incorporation periods. After 12 hours, this significantly altered the corresponding rate of amino acid incorporation from its initial rate. Each circular symbol is the average [3H]PHE incorporation rate (±SEM) for five or six populations of cardiocytes. *Significant differences in the rate of incorporation of [3H]PHE into protein compared with the 4-hour initial rate (P<.05 by a post-ANOVA Newman-Keuls t test).

View larger version (47K): [in this window] [in a new window]   Figure 5. Effects of passive stretch and Ang II on amino acid incorporation into neonatal cardiocyte protein over 24 hours. A, After a 1-hour pretreatment with 1 µmol/L [Sar1,Ile8]Ang II (SAR-ANG II), neonatal cardiocytes were statically stretched by 20% and were then continuously labeled with [3H]PHE over 24 hours. The Ang II receptor antagonist did not prevent stretch-induced amino incorporation into total cell protein. Each bar height is the average [3H]PHE incorporation into protein over 24 hours (±SEM) for 13 to 17 populations of stretched cardiocytes derived from three experiments. B, Ang II treatment of neonatal cardiocytes increased [3H]PHE incorporation into protein by 10% at 100 nmol/L, which was prevented by 1 µmol/L SAR-ANG II. Each bar height is the average [3H]PHE incorporation into protein over 24 hours (±SEM) for six to eight populations of cardiocytes derived from three experiments. *Significant differences in incorporation of [3H]PHE into cardiocyte proteins compared with nonstretched or nontreated cardiocytes (P<.05 by a post-ANOVA Newman-Keuls t test).

In summary, the anabolic effects of load to accelerate protein synthesis and to increase 24-hour [³H]PHE incorporation into protein in neonatal cardiocytes were produced without an obligatory requirement for Ang II. Similarly, the acceleration of protein synthesis in adult cardiocytes in response to load did not require Ang II.

Gene Expression for Na⁺-Ca²⁺ Exchanger and c-fos mRNA
The ability of load to alter gene expression in adult cardiocytes was examined by measuring changes in c-fos and Na⁺-Ca²⁺ exchanger mRNA levels. Expression of both of these genes is rapidly induced at the onset of cardiac pressure overload.^{18 19 20 21} As demonstrated in Northern blots in Fig 6A, c-fos expression was increased threefold after 1 hour of cardiocyte stretch. Pretreatment with 1 µmol/L [Sar¹,Ile⁸]Ang II blocked c-fos mRNA induction. The changes in c-fos mRNA levels in cardiocytes subjected to load with or without [Sar¹,Ile⁸]Ang II are summarized in Fig 7B. The levels of c-fos mRNA were normalized for the amount of total RNA applied to the blot by rehybridizing with a probe specific for 28S rRNA, and the c-fos mRNA–to–28S rRNA ratio was obtained for comparison with that for nontreated cardiocytes. In Fig 6B, it is demonstrated that expression of Na⁺-Ca²⁺ exchanger mRNA levels was increased threefold in response to passive stretch. The induction of Na⁺-Ca²⁺ exchanger mRNA was not blocked by pretreatment with [Sar¹,Ile⁸]Ang II. Summary data comparing exchanger mRNA–to–28S rRNA ratios in stretched and control cardiocytes are shown in Fig 7A. Thus, these findings demonstrated that Ang II is not required for load to induce Na⁺-Ca²⁺ exchanger gene expression. However, load-induced c-fos expression is Ang II dependent.

View larger version (29K): [in this window] [in a new window]   Figure 6. Effects of passive stretch (STR) on gene expression in adult cardiocytes. Autoradiographs of Northern blots demonstrated greater c-fos mRNA (A) and Na+-Ca2+ exchanger mRNA (B) after 1 hour of STR of adult cardiocytes compared with nonstretched control (CON) cardiocytes.

View larger version (39K): [in this window] [in a new window]   Figure 7. Differential gene expression for Na+-Ca2+ exchanger (A) and c-fos (B) after 1 hour of various treatments applied to adult cardiocytes. The RNA was extracted from each treatment group, subjected to Northern blotting, and hybridized with a [32P]cDNA probe specific for either Na+-Ca2+ exchanger mRNA or c-fos mRNA. Treatments applied to cardiocytes are indicated as follows: CON, control nontreated; STR, stretched; STR & SAR, stretched with [Sar1,Ile8]Ang II pretreatment; SAR, [Sar1,Ile8]Ang II pretreatment only; PMA, PMA treatment; VERA, veratridine treatment; ANG, Ang II treatment; and Ang & SAR, Ang II and [Sar1,Ile8]Ang II pretreatment. After autoradiography, the blots were probed again for 28S rRNA to account for applied RNA. The grain density of the autoradiographic bands of mRNA was normalized relative to the corresponding 28S rRNA grain density and then expressed as a percentage of the nontreated cardiocyte value (CON) in each experiment. Each bar height is the average value (±SEM) from five to eight experiments. *Significant differences in mRNA levels for STR or treated cardiocytes compared with nonstretched and nontreated CON cardiocytes (P<.05 by a nonparametric Kruskal-Wallis test).

As shown in Fig 8A, Ang II directly induced c-fos mRNA expression after 1 hour in both adult and neonatal cardiocytes, an effect reported by others.^{13 14} This Ang II–dependent expression of c-fos was blocked by a 1-hour pretreatment with 1 µmol/L [Sar¹,Ile⁸]Ang II. Thus, although Ang II did not accelerate protein synthesis rates in either adult or neonatal cardiocytes, as described above, the hormone elicited a change in immediate-early gene expression. Yet Ang II did not affect the expression of Na⁺-Ca²⁺ exchanger mRNA, as shown in Fig 7A. These data indicated that separate signaling pathways were involved in inducing the expression of c-fos mRNA and Na⁺-Ca²⁺ exchanger mRNA. To identify these separate pathways, the cells were treated with either the phorbol ester PMA, an activator of protein kinase C, or veratridine, a Na⁺ and Ca²⁺ influx stimulator in excitable cells. As demonstrated in Fig 7, PMA elicited a marked expression of c-fos mRNA, but it caused a small decrease in Na⁺-Ca²⁺ exchanger mRNA compared with nontreated cardiocytes. Veratridine induced exchanger expression in cardiocytes, as previously reported,²¹ but it failed to induce c-fos expression across a range of 1 to 10 µmol/L (see Figs 7B and 8B). In summary, these findings suggest that there are separate signaling pathways involved in regulating the expression of c-fos mRNA and Na⁺-Ca²⁺ exchanger mRNA in adult cardiocytes. These differences may account for the differential changes in gene expression that occur in response to load and Ang II treatment.

View larger version (44K): [in this window] [in a new window]   Figure 8. Effects of Ang II (ANG) and veratridine on c-fos expression in adult cardiocytes. A, Autoradiographs of Northern blots of c-fos mRNA after ANG or PMA treatment of cardiocytes in the presence and absence of [Sar1,Ile8]Ang II (SAR). This ANG receptor antagonist prevented ANG induction of c-fos mRNA but did not prevent PMA induction of c-fos mRNA. B, Autoradiographs of c-fos mRNA blots after treatment of adult cardiocytes with either PMA, a phorbol ester that can directly stimulate protein kinase C, or with veratridine, a stimulant of Na+-Ca2+ exchanger activity. In contrast with PMA induction of c-fos mRNA, veratridine failed to increase c-fos mRNA despite greater amounts of total RNA applied to the blot, as confirmed by reprobing for 28S rRNA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This is the first study to examine protein synthesis in populations of passively loaded adult cardiocytes within 1 day after isolation without serum, mitogens, or antimetabolites, all of which are used in the routine neonatal cardiocyte model. In a previous adult model of passive stretch, cardiocytes required 3 days and an overcoating of proteins to firmly adhere to a distensible polycarbonate silicone membrane,¹⁷ whereas cardiocytes in the new model rapidly adhered to an elastic polyurethane membrane without overcoating proteins and were subject to passive stretch within 24 hours. In the previous adult model, there was a small number of cardiocytes that adhered to the membranes, which limited the analysis to single-cell measurements of protein synthesis and RNA synthesis by optical methods.¹⁷ The improved model allowed for measurements of protein synthesis and mRNA expression in populations of passively loaded adult cardiocytes by standard methods used in the present study. Since the heart is an admixture of myocytes, fibroblasts, and other supporting cells, the isolated myocyte subjected to load in culture provides a means to examine the cellular processes specific for the myocyte without the complexity of hormones, neurohumors, and other factors. Despite their difference in development stages, isolated adult and neonatal cardiocytes responded to incremental passive stretch with proportional acceleration of protein synthesis, as shown in Fig 1. A similar acceleration of protein synthesis in proportion to passive or active loading was observed in isolated papillary muscles.³⁶ Thus, this passively loaded adult cardiocyte model mimicked an acceleration of protein synthesis that is an intrinsic response of the cardiocyte to the onset of greater loading conditions in isolated hearts³⁷ and in intact hearts.^{38 39 40} Therefore, this model of passively loaded adult cardiocytes provides a means to investigate the transduction pathway between load on the cardiocyte and its early anabolic responses of gene expression and acceleration of protein synthesis.

This cardiocyte model was used to examine the direct action of load versus a requirement for Ang II to mediate load-induced initiation of early cardiocyte responses associated with the onset of cardiac hypertrophy. The structure, function, and biochemical composition of the myocardium are continuously remodeled as the cardiocyte responds to its mechanical loading environment.⁴¹ This was demonstrated by surgically unloading and reloading a papillary muscle, which atrophied and then enlarged to normal with concomitant changes in myosin and actin content.⁴² The converse was also demonstrated when pressure overload (and the associated cardiac enlargement) was followed by regression of the heart size upon restoration of normal pressure.⁴³ Angiotensin-converting enzyme inhibitors are also effective in accelerating regression of chronic pressure-overload hypertrophy,⁸ yet they do not prevent its development.^{44 45} This ameliorative action by Ang II antagonists may result from a reduction in cardiac workload caused by local Ang II–induced norepinephrine release from sympathetic nerves and its inotropic and chronotropic actions.^{10 46} Therefore, the direct effects of load and Ang II on the early anabolic processes associated with cardiocyte hypertrophy were examined in the simplified loaded cardiocyte in culture. The present study demonstrated that the load-induced acceleration of protein synthesis was not altered in either the adult or neonatal cardiocyte model by the Ang II antagonist peptide [Sar¹,Ile⁸]Ang II (Fig 2). In addition, 4 hours of Ang II had no direct effect on protein synthesis in either model (Fig 3). A similar lack of Ang II effect on protein synthesis was reported previously for adult cardiocytes from rats¹⁵ and cats¹⁶ after 1 and 24 hours of Ang II treatment, respectively. These findings demonstrate that Ang II is not required for early events of transducing load into protein synthesis.

These results conflict with earlier reports that Ang II can substantially increase protein synthesis in neonatal rat cardiocytes¹¹ and that load-accelerated protein synthesis was mediated by Ang II.⁹ However, these previous reports measured protein synthesis by continuous labeling of [³H]PHE into protein for a 24- or 48-hour period. Unfortunately, continuous long-term labeling in culture is intrinsically nonlinear,³⁵ such that labeled amino acid accumulates asymptotically into protein, as shown in Fig 4 in nonstretched neonatal cardiocytes. This significantly alters the corresponding rate of amino acid incorporation to a point where it no longer reflects primarily the rate of protein synthesis, which occurred by 16 hours in Fig 4. The net incorporation of radiolabeled amino acid into protein represents the difference between protein synthesis and protein degradation. Therefore, changes in amino acid incorporation in response to Ang II could also be accounted for by changes in the rate of protein degradation. In the present study, there was a small increase in radiolabeled protein of neonatal cardiocytes after a 24-hour treatment with 100 nmol/L Ang II, but it was less than a third of the increase measured when load was applied to these cardiocytes (Fig 5). Nonetheless, the load-induced incorporation of [³H]PHE into protein over 24 hours was not prevented by the Ang II antagonist [Sar¹,Ile⁸]Ang II, as shown in Fig 5. Perhaps culture conditions in a previous report⁹ allowed stretch-released Ang II to play a greater role in long-term incorporation of radiolabel into protein over 24 hours. In contrast to the present study, a previous study found that the direct effect of Ang II on amino acid incorporation into protein was prominent when measured after 48 hours of Ang II treatment.¹¹ Such differences between cardiocyte responses may reflect alterations in cellular properties as a result of chronic Ang II treatment. This explanation is supported by findings in adult feline cardiocytes, in which Ang II required 6 days of continuous treatment in order to increase pulse-labeled protein synthesis by 22%.¹⁶ In the present study, the pulse-labeling measurements of load-accelerated protein synthesis were not altered by Ang II antagonists in either adult or neonatal cardiocytes, as demonstrated in Fig 2. Thus, the findings in the present study demonstrate no obligatory requirement for Ang II in the transduction pathway between load on the cardiac myocyte and its early anabolic response of accelerated protein synthesis.

This is the first isolated cell study to demonstrate increased Na⁺-Ca²⁺ exchanger mRNA expression as a genetic response of the cardiocyte to its loading environment. As shown in Fig 7A, exchanger mRNA levels were increased by threefold after 1 hour of loading adult cardiocytes, but this greater expression was not blocked by [Sar¹,Ile⁸]Ang II. Thus, induction of cardiocyte exchanger expression in response to load is independent of Ang II but may represent a need for more exchanger proteins to balance load-altered Na⁺ and Ca²⁺ gradients across the sarcolemma, as has been suggested.^{21 36} In an attempt to mimic these load-initiated ion fluxes, pharmacological stimulation of Na⁺ and Ca²⁺ fluxes into isolated cardiocytes was found to also induce exchanger expression and was accompanied by acceleration of general and contractile protein synthesis²¹ —a hallmark of initiating cardiocyte hypertrophy. Induction of Na⁺-Ca²⁺ exchanger mRNA expression serves as a characteristic cardiocyte response to load that accompanies cardiac hypertrophy, since the expression of Na⁺-Ca²⁺ exchanger mRNA is rapidly increased at the onset of pressure overload,²¹ its protein is increased within 2 days of pressure overload,²¹ its activity is increased in chronic pressure-overload hypertrophy,⁴⁷ and its expression remains elevated during the progression into heart failure.⁴⁸ Since the expression of the exchanger mRNA is increased from the onset of pressure-overload hypertrophy to cardiac failure, the cellular signals and processes that control this load response of the cardiocyte may be closely linked to the development of cardiac hypertrophy.

In contrast, induction of c-fos expression after loading of adult cardiocytes was blocked by [Sar¹,Ile⁸]Ang II, and similar results were reported for loaded neonatal¹³ and adult¹⁴ cardiocytes. Although the immediate-early gene c-fos is also rapidly expressed at the onset of cardiac pressure overload,^{18 19 20} its functional role in the adult cardiocyte is uncertain. Induction of c-fos by increased systolic wall stress in isolated perfused rat hearts is followed by increased Fos protein,¹⁴ which combines with Jun protein to form the AP-1 complex, which may in turn regulate expression of other genes.^{49 50} However, expression of c-fos and c-jun is not sufficient to initiate cardiocyte hypertrophy, since extracellular ATP induces expression of these immediate-early genes in neonatal cardiocytes without cellular hypertrophy.⁵¹ Although load induced both c-fos and exchanger expression in adult cardiocytes, as shown in Figs 6 through 8, the Ang II receptor antagonist [Sar¹,Ile⁸]Ang II blocked only the c-fos expression. Furthermore, a separation of the pathways initiating the responses of c-fos and exchanger expression was found by applying known stimulants for these two mRNAs to the adult cardiocytes. Ang II and PMA, a known protein kinase C stimulant, induced only c-fos expression, whereas veratridine, a Na⁺ and Ca²⁺ influx stimulator in excitable cells, induced only exchanger expression (Figs 7 and 8). Thus, various agents can demonstrate differential expression of c-fos and exchanger mRNAs in adult cardiocytes, which indicates that separate pathways are probably involved in stimulating their transcription.

In conclusion, the present study presents an improved model for passively loading adult cardiocytes in culture. Cardiocytes that were passively loaded within 1 day after their isolation demonstrated acceleration of protein synthesis and induction of Na⁺-Ca²⁺ exchanger gene expression. The present study attests to previous work that load induction of the immediate-early gene expression for c-fos in cardiocytes is Ang II dependent. However, there are two major findings in the present study that do not support the hypothesis of an Ang II–dependent initiation of adult cardiocyte hypertrophy. First, acute Ang II treatment did not directly increase the rate of protein synthesis in adult cardiocytes. Second, the Ang II antagonist [Sar¹,Ile⁸]Ang II did not block either load-accelerated protein synthesis or increased exchanger expression. These findings demonstrate no obligatory requirement for Ang II in the transduction pathway between load on the cardiac myocyte and its early anabolic responses of accelerated protein synthesis or enhanced Na⁺-Ca²⁺ exchanger gene expression.

	Selected Abbreviations and Acronyms

 

[3H]PHE = L-[ring-2,3,4,5,6-3H]phenylalanine Ang II = angiotensin II BrdU = 5-bromo-2'-deoxyuridine PHE = phenylalanine PMA = phorbol 12-myristate 13-acetate

	Acknowledgments

 
This study was supported by the Research Service of the Department of Veterans Affairs and by the Medical University of South Carolina Institutional Research Funds of 1994-1995. We appreciate the expert technical assistance provided by Valerie Young, Mary Barnes, and Tom Gallien. The authors are also grateful for the gift of polyurethane plastic by Argotec, Inc, Greenfield, Mass.

	Footnotes

 
Reprint requests to Robert L. Kent, PhD, Cardiology Section, VA Medical Center, 109 Bee St, Charleston, SC 29401.

Received August 11, 1995; accepted February 21, 1996.

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