Dissociation of Left Ventricular Hypertrophy, β-Myosin Heavy Chain Gene Expression, and Myosin Isoform Switch in Rats After Ascending Aortic Stenosis
Background Reexpression of the fetal β-myosin heavy chain (β-MHC) gene was reported to be a marker for phenotypic reprogramming and cardiac hypertrophy in rats. Recent in vitro studies strongly suggested a role of angiotensin II for phenotypic reprogramming. In the present investigation, β-MHC gene expression was studied in an experimental model of pressure-overload hypertrophy that is not associated with a concurrent activation of the circulating renin-angiotensin system.
Methods and Results Hypertrophy was induced in rats by ascending aortic banding (n=40). After 7 days, myosin contained 31% (P<.05) of the β-MHC isoform in banded but <5% in sham-operated animals. However, no specific elevation of β-MHC mRNA levels was found in banded animals. In contrast, hearts of rats with abdominal aortic banding displayed a marked increase in β-MHC mRNA levels (3-fold to 5-fold, P<.05). Both the left ventricular weight and left ventricular peak systolic pressure were significantly elevated compared with sham-operated animals (abdominal aortic banding, +13% and 164±7 mm Hg; ascending aortic banding, +27% and 191±9 mm Hg). Plasma renin activity was elevated in rats with abdominal aortic banding (2.5-fold, P<.05) but not in rats with ascending aortic banding.
Conclusions The results of the present work do not support the concept that increased β-MHC gene expression is a general “stable late marker” of myocardial hypertrophy in rats. Our results suggest that the stimulation of the renin-angiotensin system is crucial for the activation of the β-MHC gene.
Pressure overload leads to compensatory growth of the heart. In addition, it is accompanied by phenotypic changes of cardiac muscle cells due to differential gene activation. The latter process, giving rise to myocytes equipped with a new repertory of contractile proteins and ion channels, was formerly also thought to be an adaptive one.1 In the past few years, however, it has been realized that such changes on the myocyte level may contribute to the contractile failure commonly observed in the hypertrophied heart.2 3 4 An understanding of the mechanisms leading to this reprogramming process may be of clinical importance for the understanding of the different pathways responsible for cardiac growth and for the development of successful therapeutic strategies aimed at inducing regression of hypertrophy toward a “normal” state.5
Typically, fetal genes that were repressed or shut off during development are reactivated in the process of hypertrophy. The most thoroughly analyzed examples are the genes for α-skeletal actin, myosin light chain-2, atrial natriuretic peptide, and β-MHC.6 7 Whereas α-skeletal actin is reactivated only transiently, high levels of expression of β-MHC and atrial natriuretic peptide were reported to be maintained over extended time periods in the hypertrophied heart. These two genes have therefore been dubbed “stable late markers” of hypertrophy.8
Recently, it became clear that not only mechanical stress imposed by the increased left ventricular pressure but also Ang II is able to induce phenotypic reprogramming. In cultured neonatal myocytes, Ang II induced a similar pattern of gene activation and a similar increase of cell dimension, respectively, as if such cells were stretched mechanically.8 9 10 Abdominal aortic stenosis, which has repeatedly been used as an experimental model to induce pressure-overload hypertrophy and fetal reprogramming,11 12 13 induces a stimulation of the RAS.14 Therefore, it remains to be established whether myocardial hypertrophy and phenotypic reprogramming are mediated by the increase in mechanical stress, elevated plasma levels of Ang II, or an interaction of the two factors. In the present study, we investigated the development of cardiac hypertrophy and fetal reprogramming after ascending aortic stenosis that causes a large increase in left ventricular pressure in the absence of an activation of the RAS.15 16
Ascending Aortic Banding: Series 1
Cardiac hypertrophy was induced in 40 female Sprague-Dawley rats (180 to 200 g, 9 weeks old) under anesthesia with ketamine HCl/xylazine HCl (100 and 4 mg/kg, respectively) and artificial respiration. The ascending aorta was partially occluded with stainless steel hemostatic clips (Weck & Co, Inc).17 After closure, the opening of the oval clip had a length of 3 mm and a maximal width of 1.1 mm. The mortality was ≈15% after ascending aortic banding; most of these animals died within 1 day after surgery, showing severe pulmonary edema. Sham-operated controls (n=30) underwent the same surgical procedure but without insertion of the clip. At various time points up to 7 days after surgery, animals were killed by cervical dislocation under carbon dioxide anesthesia and weighed. The hearts were removed, and the left ventricles were isolated, weighed, and stored at −80°C for later analysis.
All experiments were conducted in accordance with institutional guidelines and the Guide for the Care and Use of Laboratory Animals put forth by the US Department of Health and Human Services, NIH publication 86-23, and approved by local authorities (Az 37-9185.81/066/92 and 37-9185.81/55/93).
Abdominal Aortic Banding: Series 2
In a total of 12 animals, the effects of ascending aortic banding (series 1) were compared with abdominal aortic banding. In 3 animals, the abdominal aorta was banded between the renal arteries through an abdominal incision. The opening of the clip was 3 mm long by 0.2-mm maximal width. Three sham-operated animals were treated in the same manner except for insertion of the clip. No animals died after abdominal aortic banding. In 3 animals, ascending aortic banding was performed as described above and another 3 animals served as sham-operated controls. All animals together with 3 untreated controls were killed 7 days after surgery, and the left ventricles were isolated and stored at −80°C for later analysis by Northern blotting and exponential RT-PCR.
Hemodynamic Measurements and Blood Sampling: Series 3
In a final series of experiments, in 16 rats, ascending or abdominal aortic banding was compared with sham-operated controls. Five days after surgery, a permanent catheter was implanted into the abdominal aorta and led to an incision in the neck. Six days after surgery, animals were put into individual cages, and the catheter was connected to a syringe. Thirty minutes later, a 1-mL blood sample was withdrawn from the awake, unstressed animal and replaced by an equal amount of saline. Seven days after surgery, animals were anesthetized with thiopental sodium (75 mg/kg), and respiration was maintained by tracheal intubation. The thorax was opened through the diaphragm, and left ventricular pressure was measured by puncture of the left ventricular wall with a steel cannula connected to a Statham pressure transducer.
RNA was extracted from tissue samples pulverized under liquid nitrogen.18 Northern blots were prepared with formaldehyde-agarose gels and nitrocellulose, loading 2 μg RNA per well.19 For quantification, RNA was blotted to nitrocellulose in serial dilutions (8, 4, 2, and 1 μg RNA per slot) with a slot blot apparatus. Blots were probed consecutively with cDNAs specific for α-MHC mRNA or β-MHC mRNA (plasmids were kindly donated by Dr S. Schiaffino, Padova, Italy).20 The isolated cDNA was labeled by the random-priming method. Prehybridization (2 hours; 40% formamide, 5×SSC, 50 mmol/L phosphate buffer [pH 7.4], 10×Denhardt's solution, 0.2% SDS, 100 μg yeast tRNA) and hybridization (16 hours; 50% formamide, 3×SSC, 10 mmol/L phosphate buffer [pH 7.4], 2×Denhardt's solution, 0.2% SDS, 50 μg yeast tRNA) were carried out at 42°C.19 Blots were washed 2×15 minutes with 2×SSC/0.1% SDS at 42°C and exposed. After these measurements, it was confirmed that equal amounts of RNA had been blotted by hybridization of the blots to a cDNA specific for cytosolic 28S rRNA. Autoradiographs of the slot blots or Northern blots were scanned densitometrically, and tissue levels of mRNAs were expressed as arbitrary densitometric units per 28S densitometric unit, taking care that the signal was in the linear range for all measurements.
The actual number of MHC mRNA molecules per microgram of tissue RNA was measured by a quantitative RT-PCR method described previously.21 The oligonucleotides used for reverse transcription were complementary to the indicated positions of the 3′ untranslated sequence22 : α-MHC, positions 292 to 273; β-MHC, positions 226 to 207. The oligonucleotides used for amplification of the cDNAs generated by reverse transcription matched the sequences of the coding strands at the indicated positions: α-MHC, positions 216 to 235; β-MHC, positions 128 to 147.
Briefly, the two oligonucleotides specific for and complementary to α-MHC and β-MHC mRNA were simultaneously reverse-transcribed into cDNA in a single reaction, with 1 μg ventricular RNA used as template. Then, the two additional primers specific for and complementary to α-MHC and β-MHC cDNA were added, and the cDNAs were amplified by PCR in the presence of [α-32P]dCTP. Aliquots of the reaction were withdrawn after consecutive cycles, and the two PCR products, which differed slightly in length, were separated by agarose gel electrophoresis. Product accumulation can be quantified in molar concentrations from the radioactivity incorporated into the product DNAs, the specific radioactivity of the precursor, and the number of cytidines incorporated into the newly synthesized product. The molar concentration of template at cycle zero, ie, MHC mRNA, was calculated by plotting product accumulation (log moles produced) versus cycle number and analyzing these data by linear regression. The numbers of α-MHC and β-MHC mRNA per microgram of substrate RNA were obtained by use of Avogadro's number.
Analysis of Protein Isoforms of Myosin
Days 1 and 7 of experiment 1 were chosen for extensive myosin isoform analysis. Native myosin was extracted from small pieces (10 mg) of the ventricles23 from banded and sham-operated hearts from these 2 days (n=5 in each group). The tissue levels of α-MHC and β-MHC protein were assayed by separation of the three myosin isoforms (V1, V2, and V3) by native pyrophosphate polyacrylamide gel electrophoresis.23 Isoform fractions were quantified by densitometric scanning of the wet gels stained with Coomassie brilliant blue.
Determination of PRA
For the measurement of PRA, plasma samples were incubated in the presence of 6 mmol/L EDTA, 1.6 mmol/L dimercaptopropanolol, and 100 mmol/L N-tris(hydroxymethyl)-aminoethanesulfonate at pH 7.30 and 37°C for 60 minutes. The amount of angiotensin I formed was determined by radioimmunoassay.
Data obtained for banded animals were compared with those for sham-operated animals killed at the same time after surgery by a two-tailed Student's t test. Multiple comparisons were tested by ANOVA followed by the Bonferroni procedure. A confidence level of P<.05 was considered to be indicative of a statistically significant difference.
Ascending Aortic Banding
The development of cardiac hypertrophy and of changes in gene expression was investigated thoroughly in a model of ascending aortic banding in adult rats. In the same hearts, mitochondrial gene expression has been analyzed extensively, and some accessory data shown here have been published previously.24 In this experimental series, left ventricular mass increased significantly, by 27%, 24 hours after aortic constriction and was increased by about 30% after 7 days (Table 1⇓). This increase was not due to tissue edema, because the ratios of wet weight to dry weight and the protein content per gram wet weight were the same in all sham-operated and banded animals. Body weights also did not differ between sham-operated and banded animals. Total tissue RNA was increased in hypertrophic hearts by about 30% from 24 hours to 3 days after surgery and returned to initial values at day 7. Total poly-(A)+-RNA, ie, the fraction of total mRNA as determined by hybridization to oligo-dT, was unchanged in all hearts throughout the experiment.24
β-MHC mRNA transiently increased in both groups but returned to initial values at day 7 (Table 1⇑). However, at no time were β-MHC mRNA levels in banded animals significantly different from those in sham-operated animals (Table 1⇑). Levels of α-MHC slowly decreased in hearts of banded animals over the course of the experiment; after 2 days, the values were significantly different from those of the sham-operated group (Table 1⇑). In sham-operated animals, there was a transient increase of α-MHC mRNA at day 3.
Analysis of Myosin Isoform Expression After Ascending Aortic Banding
Since a permanent activation of the β-MHC gene was absent in hypertrophied hearts after ascending aortic banding, the expression of the β-MHC protein, present as native myosin isoforms V2 and V3, was investigated. Measurements were made in hearts from animals 24 hours and 7 days after surgery to estimate early and late changes. After 24 hours, in 2 of 5 hypertrophic hearts, V2 and V3 were detectable in small amounts (about 5% of the total). After 7 days, banded hearts contained considerable amounts of the V2 and V3 myosin isoforms (Fig 1A⇓). Isoform fractions were 58±9% (V1), 22±4% (V2), and 20±5% (V3; n=5). Thus, myosin was composed of 69% of the α-MHC and 31% of the β-MHC isoform. This myosin isoform shift occurred in the absence of any detectable change in β-MHC mRNA levels (Fig 1B⇓). In all sham-operated animals as well as weight-matched, untreated control rats, the V1 isoform represented >95% of the total myosin.
Comparison of β-MHC Gene Expression After Ascending or Abdominal Aortic Banding
The finding that the β-MHC gene was not permanently induced in hypertrophied hearts is in contrast with previous studies in which a model of abdominal aortic banding was used.11 12 Thus, in a second series of experiments, both surgical procedures, ascending as well as abdominal aortic banding, were performed, and MHC gene expression was analyzed in these hearts. In this series, all animals were killed 1 week after surgery. Left ventricular mass had increased significantly (P<.05), by 24%, after ascending but only by 13% (P<.05) after abdominal aortic banding. In a first approach, the levels of MHC mRNA isoforms were analyzed on Northern blots (Fig 2⇓). The results showed a strong activation of the β-MHC gene after abdominal aortic banding, confirming previous reports.11 12 In contrast, banding of the ascending aorta was not associated with increased β-MHC mRNA levels (Fig 2⇓), similar to series 1 (Table 1⇑ and Fig 1⇑), even though left ventricular mass increased considerably more in this model.
To confirm these results by an independent method, α-MHC and β-MHC mRNA levels were quantified by exponential RT-PCR.21 The results of the amplification procedure as well as actual numbers of β-MHC mRNA molecules per microgram of tissue RNA calculated by linear regression analysis are shown in Fig 3⇓. We found that ventricles of control rats contain 1.7×108 and 1.6×107 molecules of α- and β-MHC mRNA per microgram of total RNA, respectively, which is very similar to data reported previously by the same method for older animals.21 Similar values (2×108 and 4×107 molecules of α- and β-MHC mRNA per microgram of total RNA, respectively) were also reported in ventricles of male Wistar weanling rats when RT-PCR and an internal standard were used.25 Again confirming the data obtained by hybridization (Table 1⇑ and Fig 2⇑), no changes of β-MHC mRNA levels were found after ascending aortic banding, but a marked induction of the β-MHC gene was detected after abdominal aortic banding compared with untreated control animals (Fig 3⇓).
Hemodynamic Data and PRA After Ascending and Abdominal Aortic Banding
In a third experimental series, the hemodynamic load imposed on the hearts by the two procedures and the activity of the circulating RAS was assessed. Left ventricular mass corrected for body weight had increased by 27% after ascending and by 21% after abdominal aortic banding in this series (Table 2⇓). Fig 4⇓ shows that 1 week after surgery, left ventricular peak pressure was elevated significantly, by 60 mm Hg in hearts subjected to ascending aortic banding and by 33 mm Hg after abdominal aortic banding. Left ventricular end-diastolic pressure (Fig 4⇓) and lung weight (Table 2⇓) were normal in both groups, demonstrating the absence of cardiac pump failure.
PRA was unchanged after ascending aortic banding compared with sham-operated animals but was increased significantly, by a factor of three, after abdominal aortic banding (Fig 4⇑).
The major findings of the present work are (1) that after ascending aortic banding, β-MHC, which is one of the classic marker genes for phenotypic reprogramming in rats, is not activated and (2) that despite unchanged β-MHC mRNA levels, ascending aortic banding caused a myosin isoform shift toward V2 and V3 containing the β-MHC protein.
β-MHC Gene Expression
Even though β-MHC mRNA levels transiently increased after ascending aortic banding, they returned to initial values within 7 days (Table 1⇑). Moreover, this initial increase in β-MHC mRNA levels was also observed to the same extent in sham-operated animals and therefore does not appear to be a consequence of the stenosis itself. Possible causes may be surgical stress or the manipulation of the hearts in situ. Thus, in the longer term, this “stable late marker” gene of hypertrophy8 was not activated in this model, despite the development of significant hypertrophy in banded hearts (Table 1⇑).
In three previous studies investigating the effects of pressure overload on cardiac hypertrophy,11 12 13 the abdominal aorta was constricted in adult rats above the origin of both renal arteries, resembling a one-kidney, one-clip model.26 This treatment, as well as constricting the abdominal aorta between the renal arteries as in series 2 of the present study, resembling a two-kidney, one-clip model, also activates the renal RAS and thus results in increased plasma concentrations of Ang II (Fig 4⇑).14 27 28 29 30 Renal hypertension develops and causes compensatory pressure-overload hypertrophy. An immediate rise in afterload imposed on the ventricle is attenuated in these models by dissipation of pressure through vessels between the clip and the heart (Reference 16 and our own unpublished observations). In contrast, constriction of the ascending aorta will cause an increase in left ventricular but also left atrial pressure, which prevents the pressure-dependent activation of the renal RAS via a strong activation of cardiopulmonary mechanoreceptors and the release of atrial natriuretic peptide.15 16 31 This lack of a pressure-dependent activation of the circulating RAS by ascending aortic banding was also found in the present study (Fig 4⇑).
Thus, it seems most likely that increased circulating Ang II and not mechanical stress induces β-MHC gene expression after banding of the abdominal aorta (Figs 2 and 4⇑⇑). In the absence of increased plasma levels of Ang II, as in ascending aortic banding, this induction is absent (Table 1⇑ and Figs 1 through 3⇑⇑⇑), even in the presence of higher systolic blood pressure (Fig 4⇑) and a higher degree of hypertrophy. A pivotal role of circulating Ang II for the early activation of the β-MHC gene is also suggested by a recent study showing that low-dose Ang II infusion causes heart hypertrophy as well as β-MHC induction in rats before significant increases of blood pressure.32
Our results do not exclude a possible role of an activation of the cardiac RAS for other phenotypic changes during the development of cardiac hypertrophy. Furthermore, both the cardiac and the circulating RAS may be involved in the regulation of gene expression during long-term ascending aortic stenosis.33 34
Discordant with the results of the present investigation, in three previously published studies an induction of the β-MHC gene was reported in rats subjected to ascending aortic banding from 1 day up to 20 weeks after surgery.20 25 35 These experiments, however, were all done in weanling rats. As shown by Schiaffino et al,20 the “availability for induction” of the β-MHC gene depends inversely on the time that has elapsed since the gene became repressed during postnatal development.36 This may imply that if the β-MHC gene has been repressed recently, its reactivation may be independent of elevated levels of circulating Ang II. Alternatively, in young rats, the local RAS may be induced more rapidly, causing elevated β-MHC mRNA levels.
Myosin Isoform Shift
The second main finding of the present study is that ascending aortic banding caused a myosin isoform shift in the absence of elevated β-MHC mRNA levels (Fig 1⇑). Several explanations can be postulated to account for this observation. They all must take into consideration that although the β-MHC mRNA is present in significant amounts in hearts of rats of this age (Fig 3⇑), V2 and V3 myosin isoforms containing the β-MHC polypeptide are virtually absent (Fig 1⇑). This can also be concluded from the data reported by Feldman et al25 on β-MHC mRNA levels and from data on the fractions of myosin isozymes analyzed in rats of similar age.37 First, there could be a specific translational repression of the “embryonic” β-MHC mRNA, which may become relieved during hypertrophy. Second, translation of β-MHC mRNA may be specifically activated during hypertrophy, when both the capacity and efficiency for protein synthesis are increased.38 The discovery of an antisense β-MHC mRNA in cardiac nuclei,39 whose abundance could regulate the availability of β-MHC mRNA for translation, supports the possible existence of such mechanisms. Finally, β-MHC mRNA present in equal amounts may be translated at the same rate in all ventricles but in sham-operated hearts may not become assembled into thick filaments, which are composed mainly of α-MHC and prefer to associate with the homologous isoform.37 In the early stages of hypertrophy, however, when the efficiency of protein synthesis increases considerably in the expanding myocyte,40 the β-MHC protein may become assembled into thick filaments and consequently become visible on native protein gels. To prove this hypothesis, it would be necessary to analyze the pool sizes of free (unassembled) and assembled myosins, which, at least to the best of our knowledge, is impossible at this time because of technical constraints.
In rodents, cardiac hypertrophy is associated with a reexpression in cardiac myocytes of genes that are normally expressed only during early stages of development. This fetal reprogramming includes reexpression of β-MHC, atrial natriuretic peptide, myosin light chain-2, and α-skeletal actin in the ventricles. These phenotypic changes may participate in those cellular events that eventually lead to the development of cardiac failure.4 5 6 The results of the present work demonstrate that after ascending aortic banding, β-MHC, a marker gene for phenotypic reprogramming in rodents, is not activated. These findings refute the concept that elevated β-MHC mRNA levels are a general “stable late marker” of myocardial hypertrophy in rats. The observed dissociation of left ventricular hypertrophy and β-MHC mRNA levels implies that mechanical stress alone is not sufficient but that a stimulation of the RAS is crucial for the activation of the β-MHC gene. Thus, hormonal factors appear to be important for the development of a specific hypertrophic phenotype, which may eventually determine whether or not cardiac failure develops. A similar myosin isoform shift toward β-MHC expression has been reported previously in human hypertrophied atria in patients with mitral valve dysfunction.41 It must be emphasized, however, that in humans the β-MHC remains the predominant isoform in the adult ventricle and myosin isoform shifts of this type do not seem to play a role in the development of ventricular failure.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|MHC||=||myosin heavy chain|
|PCR||=||polymerase chain reaction|
|PRA||=||plasma renin activity|
This study was supported by the Deutsche Forschungsgemeinschaft, SFB 320, C5 (Drs Wiesner and Ru¨egg) and NIH grants HL-20592 and HL-45646 (Dr Zak). We wish to thank Dr S. Schiaffino (Padova, Italy) for the gift of plasmids containing α-MHC and β-MHC sequences. The skillful technical assistance of B. Sogl and C. Bletz is gratefully appreciated, as well as the secretarial help of Annelie Ebling. We thank K. Mu¨nter (Knoll AG, Ludwigshafen) for the determination of PRA.
Drs Ehmke and Wiesner contributed equally to this work and are both considered first authors.
- Received May 15, 1996.
- Revision received September 11, 1996.
- Accepted October 23, 1996.
- Copyright © 1997 by American Heart Association
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