Pressure- and Volume-Induced Left Ventricular Hypertrophies Are Associated With Distinct Myocyte Phenotypes and Differential Induction of Peptide Growth Factor mRNAs
Background Chronic pressure and volume overload (PO and VO) result in morphologically and functionally distinct forms of myocardial hypertrophy. We tested the hypothesis that PO- and VO-induced left ventricular (LV) hypertrophies are associated with distinct molecular phenotypes and patterns of peptide growth factor induction.
Methods and Results mRNA levels were quantified in LV myocardium from rats with LV hypertrophy due to PO or VO caused by suprarenal aortic constriction or an abdominal aortocaval fistula, respectively, for 1 week. Although PO and VO caused comparable increases in LV weight and prepro–atrial natriuretic factor mRNA, PO but not VO increased mRNA levels for the fetal genes β-myosin heavy chain and skeletal α-actin and reduced the mRNA level of sarcoplasmic reticulum Ca2+ATPase. In a myocyte-enriched myocardial fraction, transforming growth factor-β3 and insulin-like growth factor-1 mRNA levels were increased with PO but not VO; acidic fibroblast growth factor mRNA was unchanged with PO but decreased with VO. In a nonmyocyte-enriched myocardial fraction, transforming growth factor-β3 and insulin-like growth factor-1 mRNA levels were decreased with VO but unchanged with PO.
Conclusions PO- and VO-induced LV hypertrophies are associated with distinct molecular phenotypes and patterns of peptide growth factor induction. Stimulus-specific heterogeneity in the signaling events and peptide growth factors coupled to gene expression could play a role in determining the type of hypertrophy that is caused by various forms of hemodynamic overload.
Chronic PO and VO result in morphologically distinct forms of myocardial hypertrophy.1 2 PO (eg, secondary to aortic constriction) results in “concentric” hypertrophy, which is characterized by an increase in ventricular wall thickness, little or no chamber dilation, and the parallel addition of sarcomeres.3 4 VO (eg, secondary to arteriovenous shunting) results in “eccentric” hypertrophy, which is characterized by relatively little increase in wall thickness, a disproportionately large increase in chamber volume, and the serial addition of sarcomeres.5 6 Cooper and colleagues7 8 9 10 further suggested that the morphological differences in these forms of hypertrophy have functional correlates.
The molecular basis for the structural and functional differences between pressure- and volume-overloaded myocardium is not known. However, it has been shown that PO hypertrophy is associated with the reexpression of a fetal gene program characterized by induction of prepro-ANF, skeletal α-actin, smooth muscle α-actin, and β-MHC.11 12 13 14 Likewise, in PO hypertrophy, several15 16 17 but not all18 studies have shown a reciprocal decrease in the expression of mRNA for SERCA2. By comparison, relatively little is known about the molecular changes associated with VO hypertrophy. In ventricular myocardium from rats with VO due to an arteriovenous fistula, there was reexpression of prepro-ANF19 and increased levels of the V3 myosin isoform,20 suggesting that VO hypertrophy may also be associated with the induction of a fetal gene program. However, the myocardial expression of mRNAs for skeletal α-actin, β-MHC, and SERCA2 has not been characterized in VO hypertrophy.
In vitro, several peptide growth factors cause myocyte hypertrophy and the induction of fetal genes,21 and stimuli that induce myocyte hypertrophy (eg, α1-adrenergic agonists, angiotensin, and mechanical stretch) have been shown to induce peptide growth factor mRNA expression in myocardial cells,22 23 24 suggesting that peptide growth factors might play an autocrine/paracrine role in mediating myocardial hypertrophy.
To determine whether PO- and VO-induced myocardial hypertrophies are associated with distinct molecular and cellular events, we tested two hypotheses. The first hypothesis was that PO- and VO-induced hypertrophies are associated with distinct molecular phenotypes. To test this hypothesis, the mRNA levels for prepro-ANF, skeletal α-actin, β-MHC, and SERCA2 were quantified in LV myocardium from rats with matched degrees of LV hypertrophy due to PO or VO that was caused by suprarenal aortic constriction or an abdominal aortocaval fistula, respectively. The second hypothesis was that PO- and VO-induced hypertrophies are associated with different patterns of peptide growth factor expression. To test this hypothesis, the mRNA levels of TGF-β1, TGF-β3, IGF-1, and aFGF were determined in myocyte- and nonmyocyte-enriched fractions obtained from enzyme-dissociated hearts with PO- and VO-induced hypertrophies.
Male Sprague-Dawley rats weighing 200 to 250 g were obtained from Charles River. An arteriovenous fistula was established between the abdominal aorta and the inferior vena cava with an 18-gauge needle as described by Garcia and Diebold.25 Sham-operated animals served as controls and were subjected to the same surgical procedure with the exception of a puncture. Aortic constriction was created as described by Morkin and Ashford.26 Sham-operated animals served as controls, were subjected to the same surgical procedure, and had placement of a loosely tied ligature at the same location. Animals were killed 3 and 7 days after the procedure.
Myocyte and Nonmyocyte Cell Fractions
Myocyte- and nonmyocyte-enriched fractions of adult ventricular myocardium were isolated by a modification of the technique of Claycomb and Palazzo,27 as described in detail previously.22 The myocyte- and nonmyocyte-enriched fractions were resuspended in denaturing solution for mRNA preparation (see below).
The rats were anesthetized with ether, and the heart was rapidly excised and separated into the right and left ventricles (including septum) and atria. mRNA was isolated by a modification of the technique of Chomczynski and Sacchi,28 as previously described.29
Full-length cDNAs for rat prepro-ANF (courtesy of Dr Christine Seidman), rabbit SERCA2 (courtesy of Dr Jonathan Lytton), rat TGF-β1 and mouse TGF-β3 (courtesy of Dr Micheal Sporn), a 0.5-kb fragment of rat IGF-1 (courtesy of Dr Graeme Bell), a 0.47-kb fragment of human aFGF (courtesy of Dr Judith Abraham), and a 136-bp ECO121 fragment of human skeletal α-actin 3′-untranslated region pHMαA-3′UT-TNU (courtesy of Dr James L. Lessard) were labeled with 32P-dCTP (Dupont) to a specific activity of 1×106 to 2×106 cpm/ng cDNA by the random hexamer priming method and hybridized to nylon blots for 18 to 24 hours at 42°C, as previously described.30 Blots hybridized with prepro-ANF, SERCA2, TGF-β1, TGF-β3, and IGF-1 cDNAs were washed twice (15 minutes, room temperature) with 300 mmol/L NaCl/30 mmol/L trisodium citrate (pH 7.0) and 0.1% SDS and twice (15 minutes, 60°C) with 30 mmol/L NaCl/3 mmol/L trisodium citrate (pH 7.0) and 0.1% SDS. Blots hybridized with aFGF and skeletal α-actin cDNA were washed twice (15 minutes, room temperature) with 300 mmol/L NaCl/30 mmol/L trisodium citrate (pH 7.0) and 0.1% SDS and twice (10 minutes, 42°C) with 75 mmol/L NaCl/7.5 mmol/L trisodium citrate (pH 7.0) and 0.1% SDS.
A synthetic oligonucleotide probe 20 bases in length was constructed complementary to the 3′ untranslated region of the rat β-MHC mRNA (GGTCTCAGGGCTTCACAGGC).31 The probe was labeled with 32P-ATP (Dupont) at the 5′ end to a specific activity of 2×107 cpm/μg by use of T4 polynucleotide kinase. Blots hybridized for skeletal α-actin and β-MHC were washed twice (15 minutes, room temperature) with 700 mmol/L NaCl/30 mmol/L trisodium citrate (pH 7.0) and 0.1% SDS and twice (10 minutes, 42°C) with 700 mmol/L NaCl/30 mmol/L trisodium citrate (pH 7.0) and 0.1% SDS.
Blots were exposed to Kodak XAR film with an intensifying screen at −70°C, and films were scanned with a laser densitometer (Ultrascan 2202, LKB). The sizes of the hybridized messages were estimated with the 18S and 28S ribosomal RNA bands as standards. All blots were reprobed with a 32P-labeled oligonucleotide complementary to 18S rRNA as previously described,30 washed, and autoradiographed. All levels of mRNA reported in this article are normalized to the level of 18S rRNA to correct for potential differences in the amount of RNA loaded and/or transferred.
Data are presented as the mean±SEM. Statistical analysis was performed by Student’s unpaired t tests (two-tailed), and a value of P<.05 was considered significant.
LV Hypertrophy in PO and VO Rats
After aortic banding (PO rats), the LV–to–body weight ratio (mg/g) increased by 15±3% and 33±4% at 3 and 7 days, respectively, relative to sham-operated controls (P<.02 for each, n=5 to 7 for each) (Fig 1A⇓). Likewise, after creation of the arteriovenous fistula (VO rats), the LV–to–body weight ratio increased by 16±2% and 34±3% at 3 and 7 days, respectively, relative to sham-operated controls (P<.01 for each, n=6 to 8 for each) (Fig 1A⇓). Thus, the degrees of LV hypertrophy at both 3 and 7 days were comparable in the two models. Body, heart, and LV weights for PO, VO, and sham-operated rats are shown in the Table⇓.
Induction of Prepro-ANF mRNA
In PO rats, prepro-ANF mRNA was increased 4.2±1.0-fold (P<.01 versus sham; n=5) and 8.6±1.2-fold (P<.01 versus sham; n=5) at 3 and 7 days, respectively (Figs 1B⇑ and 2A⇓). In VO rats, the level of prepro-ANF mRNA was increased 2.3±0.4-fold (P<.01 versus sham; n=6) at 3 days and 8.1±1.2-fold (P<.001 versus sham; n=6) at 7 days (Figs 1B⇑ and 2A⇓). The increases in prepro-ANF mRNA levels were not different between the two models at 3 or 7 days. Furthermore, the fold increase in LV prepro-ANF mRNA correlated with the increase in LV–to–body weight ratio for each model separately (PO, r=.68, P=.03, n=10; VO, r=.56, P=.046, n=12) and for the two models combined (r=.61, P=.002, n=22).
Skeletal α-Actin mRNA
In PO rats, LV skeletal α-actin mRNA was increased 2.9±0.5-fold and 4.6±1.2-fold at 3 and 7 days, respectively (Figs 1C⇑ and 2A⇑). In VO rats, there was no change in the level of skeletal α-actin mRNA at 3 or 7 days, and the increases with PO (versus VO) were significantly greater at both 3 and 7 days (Figs 1C⇑ and 2A⇑).
In PO rats, LV β-MHC mRNA was increased by 2.6±0.4-fold and 6.5±2.9-fold at 3 and 7 days, respectively (Figs 1D⇑ and 2B⇑). In VO rats, there was no change in the level of β-MHC mRNA at 3 or 7 days. The increase with PO (versus VO) was significantly greater at 3 but not at 7 days (Figs 1D⇑ and 2B⇑).
In PO rats, the level of LV SERCA2 mRNA decreased by 31±15% and 31±2% at 3 and 7 days, respectively (Figs 1E⇑ and 2C⇑). The decrease was significant at 7 days (sham, 0.97±0.04-fold; PO, 0.69±0.02-fold; P=.001; n=5) but not at 3 days. In VO rats, the level of SERCA2 mRNA was unchanged at 3 or 7 days (Figs 1E⇑ and 2C⇑).
Distribution of Peptide Growth Factor mRNA Expression in Adult Rat Myocardium
Dissociation of the normal adult rat heart into myocyte- and nonmyocyte-enriched fractions revealed a heterogeneous distribution of peptide growth factor mRNAs. The preponderance of aFGF mRNA was detected in the myocyte fraction, whereas the preponderance of TGF-β1, TGF-β3, and IGF-1 mRNAs was detected in the nonmyocyte-enriched fractions (Fig 3⇓).
Myocardial Peptide Growth Factor mRNA Expression With PO
One week after abdominal aortic constriction, TGF-β1 mRNA levels were increased (3.3±0.39-fold; P=.001; n=5) in the myocyte-enriched fraction but were unchanged (1.03±0.17-fold; P=.94; n=5) in the nonmyocyte-enriched fraction compared with sham. In rats with abdominal aortic constriction, TGF-β3 (1.8±0.11-fold; P=.001; n=5) and IGF-1 (2.3±0.2-fold; P=.001; n=3) mRNA levels were also increased in the myocyte-enriched fraction but unchanged in the nonmyocyte-enriched fraction (Figs 4⇓ and 5⇓). By contrast, aFGF mRNA levels were unchanged in the myocyte or nonmyocyte fractions from PO hearts (Figs 4⇓ and 5⇓).
Myocardial Peptide Growth Factor mRNA Expression With VO
One week after the creation of an arteriovenous fistula, TGF-β1 mRNA levels were increased (2.8±0.2-fold; P=.001; n=6) in the myocyte-enriched fraction but unchanged in the nonmyocyte-enriched fraction. Neither TGF-β3 nor IGF-1 mRNA levels were induced in the myocyte fraction of the VO hearts. However, TGF-β3 and IGF-1 mRNA levels were decreased by 45±11% (P=.001; n=4) and 39±7% (P=.001; n=4), respectively, in the nonmyocyte-enriched fractions from volume-overloaded hearts. aFGF mRNA levels were decreased by 32±7% (P=.001; n=6) in the myocyte fraction and unchanged in the nonmyocyte fraction from VO hearts (Figs 4⇑ and 5⇑).
A major new finding of this study is that the patterns of LV myocardial gene expression were different in rats with PO- and VO-induced hypertrophies. As previously observed by several groups,11 12 13 14 15 16 17 18 PO was associated with induction of fetal isoforms of actin and myosin mRNA (skeletal α-actin and β-MHC) and downregulation of SERCA2 mRNA in LV myocardium. In contrast, the steady-state levels of skeletal α-actin, β-MHC, and SERCA2 mRNA were not altered in VO rats. Thus, PO- and VO-induced myocardial hypertrophies are associated with distinct molecular phenotypes.
In contrast to the levels of skeletal α-actin, β-MHC, and SERCA2 mRNA, the levels of prepro-ANF mRNA did not distinguish between the two forms of hemodynamic stimulus. Furthermore, when examined at 3 and 7 days, the level of LV prepro-ANF mRNA correlated with the extent of LV hypertrophy for both PO and VO. Thus, prepro-ANF mRNA appears to be a relatively quantitative marker for the degree of LV hypertrophy with both PO and VO. These findings suggest that whereas the induction of prepro-ANF is a relatively conserved molecular event in the hypertrophic process, the modulation of β-MHC, skeletal α-actin, and SERCA2 mRNA levels by PO (but not VO) may reflect mechanisms that are less conserved and perhaps more stimulus specific.
Prepro-ANF, β-MHC, and skeletal α-actin are generally viewed as components of a fetal gene program. However, our data suggest that, at least at the mRNA level, these genes can be regulated independently in response to different hypertrophic stimuli. In support of this concept, aFGF has been shown to cause directionally opposite changes in prepro-ANF and skeletal α-actin mRNA levels in cultured neonatal rat myocytes.32 Likewise, there is spatial and temporal dissociation of the accumulation of β-MHC and skeletal α-actin mRNAs in the hypertrophied LV of the rat after aortic banding.11
Although aortic constriction has been widely used to study the molecular and cellular consequences of myocardial hypertrophy, relatively little comparable information is available for VO-induced LV hypertrophy, in part reflecting the difficulty of surgically creating VO in small animals. The recent description by Garcia and Diebold25 of a simple and reproducible method to create VO in the rat by use of a needle facilitated our study. This model was used by Ruzicka, Leenen, and colleagues33 34 35 36 to study the role of the renin-angiotensin system in the development and maintenance of LV hypertrophy. These investigators showed that with this technique, at 1 week there is eccentric LV hypertrophy with an ≈25% increase in LV weight, an increase in LV internal diameter, and a decrease in the ratio of LV wall thickness to radius.33
We did not measure hemodynamics in the present study. However, the degree of LV hypertrophy observed at 1 week agrees well with that reported by others for both VO33 and PO.26 Furthermore, by design, we selected conditions that resulted in similar degrees of LV hypertrophy for the two models. In animals with PO, LV and arterial systolic pressures are elevated,37 whereas in animals with VO, LV and arterial systolic pressures are reduced and the LV end-diastolic pressure is elevated.33 Thus, in this study, it is likely that the hemodynamic loads placed on the LV in PO and VO animals differed in important qualitative ways.
The present study reflects the early phase of myocardial hypertrophy. Since there may be temporal differences in the activation of signaling pathways (eg, renin-angiotensin) that are involved in myocardial remodeling,38 the present results may not be representative of later events. In this regard, it is noteworthy that Mercadier et al20 found that the level of the V3 myosin isoenzyme was increased in 3 of 16 rats late (12 weeks) after establishment of VO. The increased V3 isoenzyme in these animals may reflect the more chronic nature of their VO and possibly the development of heart failure.18
The second major finding of this study is that PO and VO have different effects on the expression of mRNAs for at least three peptide growth factors in the myocardium. In the myocardial fraction enriched with myocytes, TGF-β3 and IGF-1 mRNA levels were increased in PO (versus sham-operated animals) but not VO rats, and aFGF mRNA was unchanged in PO rats but decreased in VO rats. In the myocardial fraction enriched with nonmyocytes, the TGF-β3 and IGF-1 mRNA levels were unchanged in PO but decreased in VO rats, whereas TGF-β1 and aFGF mRNA levels were unchanged by PO or VO.
Peptide growth factors can cause many of the effects that are associated with myocardial hypertrophy, including myocyte hypertrophy, interstitial fibrosis, and increased fetal gene expression.21 39 Furthermore, there is evidence that some peptide growth factors, including TGF-β1 and IGF-1, are induced in cultured myocytes in response to hypertrophic stimuli22 23 24 or in myocardium in response to mechanical or hormonal stimulation.22 40 The mechanism(s) responsible for the distinct patterns of peptide growth factor mRNA expression in both the myocyte and nonmyocyte fractions in these models of hypertrophy remains to be defined but may in part reflect differences in the inherent nature of the initiating hemodynamic load (pressure versus volume) and/or the neurohormonal milieu. In this regard, it is of interest that endothelin-1, which can stimulate peptide growth factor expression in cardiac myocytes (N. Takahashi and W. Colucci, unpublished data), is induced in the myocardium of rats with PO- but not VO-induced hypertrophy.41
In summary, PO- and VO-induced LV hypertrophies are associated with distinct myocyte phenotypes. Furthermore, these differences in myocyte gene expression are associated with distinct patterns of peptide growth factor regulation in both myocytes and nonmyocytes. Although the relation between peptide growth factors and gene expression in these models remains to be demonstrated, these observations raise the possibility that the distinct myocyte phenotypes observed in PO- and VO-induced LV hypertrophies reflect stimulus- and/or model-specific heterogeneity in autocrine and/or paracrine signaling pathways. Elucidating the divergent aspects of these signaling pathways may lead to a better understanding of the mechanisms that govern physiological and pathological myocardial hypertrophy.
Selected Abbreviations and Acronyms
|β-MHC||=||b-myosin heavy chain|
|aFGF||=||acidic fibroblast growth factor|
|IGF-1||=||insulin-like growth factor-1|
|prepro-ANF||=||prepro–atrial natriuretic factor|
|SERCA2||=||sarcoplasmic reticulum Ca2+ATPase|
|TGF||=||transforming growth factor|
This work was supported in part by a grant from the NIH (HL-42539). Dr Calderone was the recipient of postdoctoral fellowships from le Fonds de la Recherche en Santé du Québec (1992 and 1993) and the Medical Research Council of Canada (1993 through 1995). Dr Thaik was the recipient of a National Research Service Award from the NIH. We gratefully acknowledge Olga Smirnova for excellent technical assistance and Paula McColgan for expert typing.
- Received January 26, 1995.
- Revision received August 31, 1995.
- Accepted September 1, 1995.
- Copyright © 1995 by American Heart Association
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