Norepinephrine-Induced Changes in Cardiac Transforming Growth Factor-β Isoform Expression Pattern of Female and Male Rats
Transforming growth factor-β (TGF-β) is a ubiquitous growth-regulating protein with an essential role in tissue repair and formation of extracellular matrix (ECM). To better understand the role of different isoforms of TGF-β in the cardiac remodeling process induced by norepinephrine (NE), the expression of TGF-β1, TGF-β2, and TGF-β3 was studied and compared with the expression of collagen. NE (0.1 mg/kg · h) was intravenously infused in female and male Sprague-Dawley rats for several time periods, and freshly obtained ventricular myocardium after 1 day was dissociated into myocyte and nonmyocyte fractions. Prazosin (0.1 mg/kg · h) and metoprolol (1 mg/kg · h) were used to block α- and β-adrenoceptors, respectively. After NE infusion, the three isoforms of TGF-β were differentially induced as far as the magnitude and the time course is concerned. The increased expression of TGF-β2 started earlier with a maximum after 12 hours and was more pronounced (10-fold elevation) than that of the other two isoforms, with a clear specificity for the left ventricle in female hearts. This specificity was also seen in male rats with 16-fold elevation of TGF-β2 after 1 day of NE-stimulation. The increase of TGF-β2 was significant only in the myocyte fraction obtained from female as well as from male hearts. The expression of the mRNA of all TGF-β isoforms of collagen type I and type III, and of the matrix metalloproteinase (MMP)-2 and its inhibitor TIMP-2 was reduced predominantly by α-adrenoceptor blockade with prazosin. The increase in TGF-β isoforms correlated with that of the mRNA expression of collagens, MMP-2 and TIMP-2.
Norepinephrine (NE) has been shown to induce left ventricular (LV) hypertrophy in rats, as a result of an enlargement of myocytes.1–3 This enlargement requires remodeling of the extracellular matrix (ECM), with degradation and increased synthesis of collagen for scaffold reorganization. NE disturbs the balance between formation and degradation of ECM. It elevated the expression of collagen I and III with consecutive fibrosis after 14 days.2 These changes are important in the modulation of cardiac performance and the eventual development of heart failure.
Since its original identification as a factor capable of transforming rat fibroblasts, numerous studies have established that transforming growth factor-β (TGF-β) exerts a wide spectrum of regulatory activities in both normal and malignant cells, in particular as a negative regulator of cell growth. TGF-β is now considered to play a fundamental role in the regulation of cells of the immune system, in epithelia, and in connective tissue.4 It is produced by different cell types, including fibroblasts, and is secreted in an inactive form that is thought to become activated by the action of exogenous proteases. There are five homodimeric isoforms (TGF-β1- β5). Only TGF-β1, TGF-β2, and TGF-β3 are produced by mammalian cells.5
The functions of the TGF-β isoforms include the stimulation of the formation of ECM.6 Its effects on the ECM result from an intricate balance of positive and negative gene regulations: TGF-β enhances the synthesis of collagen and fibronectin by fibroblasts,7 whereas it inhibits the degradation of ECM by stimulating the synthesis of protease inhibitors8 and decreasing the production of proteases,9 thereby favoring the accumulation of matrix proteins. In the heart, the TGF-β isoforms have been shown to be expressed at high levels during cardiac development10 and pathological processes.11,12
The isoforms of TGF-β are also known to be molecules that act contextually, because their action often depends on environmental factors, ie, the cell type, the state of cell differentiation, and the presence of other growth factors. This is best exemplified by their capacity to either stimulate or inhibit proliferation.13 Therefore, we have analyzed the kinetic of NE-induced cardiac expression of all 3 isoforms of TGF-β, as well as the expression in myocytes and nonmyocytes isolated from hearts of rats after in vivo NE-treatment.
It was shown previously that NE induced cardiac hypertrophy in rats via α- and β-adrenoceptor stimulation.14 To investigate the signaling process for ECM remodeling, the mRNA of collagen I and III, the main components of the ECM, and the mRNA of matrix metalloproteinase (MMP)-2 and their inhibitor (TIMP-2) were analyzed after NE treatment and in combination with α- and β-adrenoceptor blockade. This was compared with TGF-β isoform expression. A further aim of this study was to decide whether elevation of collagen synthesis is accompanied only by an increase of TGF-β1 expression or whether the other isoforms of TGF-β, TGF-β2, and TGF-β3 are also involved.
An expanded Methods section can be found in an online supplement available at http://www.hypertensionaha.org.
Animals used in this study were maintained in accordance with the Guide for Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH Publication No. 85 to 23, revised 1996). All substances were given as constant intravenous infusion. NE (Sigma, Deisenhofen, Germany) and the α-adrenoceptor blocker prazosin (Pr; Pfizer, Karlsruhe, Germany) were administered at a dose of 0.1 mg/kg · h; 1-mg/kg · h metoprolol (M; Ciba-Geigy, Wehr, Germany) was used to block β1-adrenoceptors. NaCl-infused animals served as controls.
Cell Isolation was done after dissociation of cardiac myocytes and nonmyocytes with collagenase, as previously described.15
TGF-β1 and TGF-β2 ELISA
ELISA (R&D Systems) was applied for the determination of TGF-β1 and TGF-β2 concentration in the myocardium.
MMP-2 Activity and Total Protein Concentration of MMP-2
The Biotrak MMP-2 activity assay system (Amersham Biosciences Europe) provides quantitative determination of MMP-2 activity in the cardiac tissue after homogenization.
RNase Protection Assay
Total RNA of 7.5 μg, isolated using TRIZOL (GibcoBRL, Karlsruhe, Germany), was used for the detection of TGF-β mRNA, and 2.5 μg of total RNA was used for the detection of ECM mRNA in the RNase protection assay (RPA), as previously described.16
Rabbit pan-specific anti-TGF-β antibody (AB-100-NA; R&D Systems, Wiesbaden, Germany) was used for the immunohistochemical analysis.
All data were analyzed and expressed as mean±SEM. A multiple-sample comparison was applied to test the differences between the groups with different modes and time intervals of treatment for significance. A value of P<0.05 was considered to be significant.
Kinetics of Norepinephrine-Induced Expression of TGF-β Isoforms
When female rats were infused with NE in vivo, there was an increase in the mRNA expression of all 3 isoforms of TGF-β (Figure 1). The mRNA of TGF-β2 was elevated first after 6 hours only in the LV (Figure 1B), reached a maximum after 12 hours, and was elevated until 4 days of NE-treatment, predominantly in the LV. The earliest increase of TGF-β1 was seen after 2 days in the LV (Figure 1A). A further increase occurred at 4 days in both ventricles. The expression of TGF-β3 was elevated after 3 days in the LV (Figure 1C), increased further until 4 days, and was more pronounced in the LV. After 14 days of NE-treatment, the expression of all isoforms of TGF-β was not changed from control (Figure 1A through 1C).
In male rats, the expression of TGF-β1 was elevated already after 1 day predominantly in the LV (Figure 1D). TGF-β2 was elevated earlier than TGF-β3, and this increase was more pronounced than in female hearts (Figure 1E). The expression of TGF-β3 was elevated after 3 to 4 days of NE-treatment, also predominantly in the LV, but to a lesser extent than in female hearts (Figure 1F).
The NE-induced time-dependent elevation of TGF-β mRNA expression predominantly of the isoform TGF-β2 in the LV of rats from both sexes was accompanied by an elevation of TGF-β2 protein after 3 to 4 days of NE-treatment (Table 1).
Expression Pattern of TGF-β Isoforms in Cardiac Cells
The content of all TGF-β isoforms was higher in nonmyocytes than in myocytes when the cells had been isolated from female control animals (Figure 2). However, the ratio of the relative mRNA amounts of TGF-β1:TGF-β2:TGF-β3 was comparable (Table 2). There were not large differences between the content of TGF-β isoforms in the heart from control male animals especially in the myocyte fraction (Figure 3A through 3C). An obviously higher content of TGF-β2 was found in myocytes from male hearts (Table 2).
When rats were stimulated with NE in vivo, the expression pattern of TGF-β isoforms was changed differently in the isolated nonmyocytes and myocytes in female and male hearts (Table 2). At first sight, the x-fold stimulation of TGF-β by NE was nearly comparable (Figures 2E through 2G and 3D through 3F). However, there were some significant differences. The expression of TGF-β1 was elevated to the same extent in both cell populations in female hearts (Figure 2E). In male hearts, TGF-β1 was elevated only in myocytes (Figure 3D). The expression of TGF-β2 (Figure 2F) and TGF-β3 (Figure 2G) was elevated only in myocytes in female hearts. In male hearts, TGF-β2 was also elevated only in myocytes (Figure 3E) and the expression of TGF-β3 was not affected in myocytes and reduced in nonmyocytes (Figure 3F).
In accordance with the findings on the mRNA levels, positive signals for TGF-β were obtained by immunohistochemistry predominantly in myocytes after NE-treatment in female and male hearts (Figure 4). Only in myocytes, there was an elevation of TGF-β2 and TGF-β3 after NE treatment in female hearts (Figure 2F and 2G, respectively) and of TGF-β1 and TGF-β2 in male hearts (Figure 3D and 3E, respectively). Despite the high expression level of all 3 isoforms of TGF-β mRNA in nonmyocytes in both genders (Figures 2B through 2D and 3A through 3) there was rarely a positive signal for TGF-β detectable by immunohistochemistry (Figure 4G) in this cell population.
Comparison of ECM Proteins in Female and Male Myocardium
NE induced a higher increase of collagen I mRNA expression in female LV than in male LV (Figure 5A) after 3 to 4 days of NE-treatment. The expression of collagen III, MMP-2, and TIMP-2 mRNA was elevated to nearly the same level by NE (Figures 5B–D, respectively). Although the MMP-2 concentration was lower in female than in male LV after NE-treatment (Figure 5E), there was a comparable stimulation and a comparable concentration of active MMP-2 (Figure 5F). Only 0.6% of total MMP-2 was active.
Effects of α- and β-Adrenoceptor Blockade
Blockade of α-adrenoceptors by prazosin and of β-adrenoceptors by metoprolol in cardiac control hearts had no significant effect on TGF-β1 (Figure 6A), TGF-β2 (Figure 6B), and TGF-β3 (Figure 6C) expression. The NE-induced elevation of all TGF-β isoforms was blocked only by prazosin.
Blockade of α-adrenoceptors by prazosin and of β-adrenoceptors by metoprolol in cardiac control hearts had no significant effect on collagen I (Figure 7A), collagen III (Figure 7B), MMP-2 (Figure 7C), and TIMP-2 (Figure 7D) expression. The NE-induced elevation of all mRNAs was blocked by prazosin as well as by metoprolol.
NE induced the expression of TGF-β isoform mRNAs differentially and in a time-dependent manner. In control and NE-stimulated hearts, TGF-β1 was the dominant isoform in the myocyte and nonmyocyte fraction and was expressed predominantly in nonmyocytes (Figures 2B and 3⇑A). TGF-β1 has been reported to be present in cardiac myocytes and fibroblasts.17,12 It has been implicated in cardiac myocyte growth,17 fibrosis,18,19 and in the re-expression of the fetal isoforms of myofibrillar protein genes.20 NE induced elevation of TGF-β1 which started 1 hour after stimulation. TGF-β1 decreased to control level after 2 days and was elevated again to the same extent after 3 and up to 6 days.21 In the present study, the mRNA-expression of TGF-β1 started to be elevated only after 2 days, without biphasic characteristics (Figure 1A).
The elevation of cardiac TGF-β1 was the result of an increase of mRNA expression in myocytes and nonmyocytes in female hearts (Figure 2B) and in myocytes in male hearts (Figure 3D). This was also described by Takahashi et al.17 They showed that NE induced an elevation of TGF-β1 only in myocytes after 36 hours of in vivo NE-treatment and cell separation and not in nonmyocytes. The NE-induced elevation of TGF-β1 in the nonmyocyte fraction seems to be characteristic for female hearts. Furthermore, TGF-β1 was stimulated by 1 μmol/L NE, predominantly via α-adrenoceptors, in cultured neonatal rat ventricular myocytes and not in nonmyocytes after 1 day.17 In contrast, 0.2- and 2-μmol/L NE induced secretion of TGF-β in cultured neonatal rat cardiac fibroblasts.22
Little is known about the function of TGF-β2. The TGF-β isoforms bind to TGF-β I and II receptors, although TGF-β2 has an essentially lower affinity than TGF-β1.23 However, different functions are considered, because the TGF-β isoforms show different tissue distributions and also affect different cell types differently. Moreover, it is known that the proportion of TGF-β isoforms influences the biologic effects. Therefore, the shift in the ratio of TGF-β1:TGF-β2:TGF-β3 from 22:1:4 in myocytes from female control animals to 7:1:1 after 1 day of in vivo NE treatment suggests that TGF-β2 may play an important role in myocardial remodeling (Table 1). The importance of TGF-β2 was more pronounced in male hearts. The ratio of the three isoforms changed from 3:1:2 to 1:1:0.2 in male myocyte fraction after NE in vivo treatment. The expression of TGF-β2 increased earlier and to a higher extent than that of the other two isoforms (Figure 1B and 1E), with a clear LV specificity. This elevation was significant only in the myocyte fraction (Figures 2F and 3⇑E). An NE-induced isoform shift from TGF-β1 to TGF-β2 was also detected by Fisher and Absher.22 The expression pattern of TGF-β isoforms and the responsiveness of cardiac cells to increase the TGF-β expression seems to be changed during development.10 Analysis of TGF-β2 null mice has revealed that TGF-β2 is most commonly involved in epithelial-mesenchymal interactions, cell growth, ECM production, and tissue remodeling. Moreover, it plays an essential role in the development of the heart.24
The expression of TGF-β3 was increased later than the other two isoforms after NE treatment (Figure 1C and 1F), with an expression pattern similar to that of TGF-β2 in female hearts. TGF-β3 was the TGF-β isoform which was increased predominantly in the infarct area after myocardial infarction.16 It was proposed that TGF-β3 affects wound healing.25
The NE-induced elevation of expression of TGF-β mRNA isoforms seems to be a result of the stimulation of α-adrenoceptors, because the expression of all 3 isoforms was blocked by prazosin (Figure 5). TGF-β1 was elevated also by α-adrenoceptor stimulation with phenylephrine in cultured human vascular smooth muscle cells.26
The elevation of both type I and type III collagen mRNA expression after NE-treatment (Figure 5A and 5B) was accompanied by consecutive collagen accumulation.2 The NE-induced elevated MMP-2 mRNA expression (Figure 5C) was associated with an increase of total MMP-2 (Figure 5E) in both sexes. TIMP-2 mRNA expression, which occurred parallel to the elevated MMP-2 expression (Figure 5D), seems to be necessary to reduce the MMP-2 activity after an early phase of a higher level of ECM remodeling after NE treatment.2 The elevation of the MMP-2 activity (Figure 5F) is accompanied by remodeling of ECM.3 The NE-induced increased expression of these mRNAs was a result of α- and β-adrenoceptor stimulation, because the expression was reduced by prazosin as well as by metoprolol (Figure 7). The NE-induced increase of collagen protein in cultured human vascular smooth muscle cells was also prevented by prazosin.26 Furthermore, β-adrenoceptor stimulation with isoproterenol induced an elevation of collagen III but not of collagen I mRNA in the LV after 3 days of subcutaneous continuous infusion.27 After 7 days of β-adrenoceptor stimulation with isoproterenol, there was an elevation of collagen I and collagen III mRNA.28,27 Carvedilol, but not metoprolol, attenuated the increase in collagen content in the noninfarcted region after myocardial infarction.29 This result supports the hypothesis that α-adrenoceptor stimulation and not β-adrenoceptor stimulation may induce collagen accumulation with a signal transduction including TGF-β isoforms. The prevention of collagen accumulation after myocardial infarction could also be explained by the antioxidant effects,30 as well as by the antiproliferative effect of carvedilol on vascular smooth muscle.31
All three isoforms of TGF-β showed a positive correlation with the mRNAs of collagen I, collagen III, and TIMP-2 in female hearts, but also in male hearts (data not shown). The best correlation was found between MMP-2 mRNA and TGF-β2 mRNA (Figure 8B) compared with TGF-β1 and TGF-β3 mRNA (Figure 8A and 8C, respectively) in female hearts. A comparable correlation was found between TGF-β2 protein concentration and MMP-2 activity (data not shown).
The increase of TGF-β2 mRNA expression only in the myocyte fraction indicates that it may be part of the reprogramming of fetal gene expression, which is associated with the development of hypertrophy. Moreover, it may be the signal of the myocytes, which becomes transmitted to the surrounding ECM, that a remodeling of the ECM is necessary for the growth of myocytes. The further increase of the other two isoforms, TGF-β1 and TGF-β3, seems also to be necessary for restructuring of the ECM, because the elevation of all isoforms correlated with the NE-induced elevation of both collagen fractions. Furthermore, they may serve as a signal for remodeling of the ECM because all isoforms correlated with the NE-induced elevation of MMP-2 and its inhibitor TIMP-2.
This work was supported by the Deutsche Forschungsgemeinschaft (ZI 199/10-3, ZI 199/10-4), by a grant of the Medical Faculty of the University of Leipzig (formel.1-10), and by a grant of BMBF (NBL-3-support; No 01ZZ0106). The excellent technical assistance of Grit Marx and Brigitte Mix is gratefully appreciated.
- Received March 4, 2004.
- Revision received April 7, 2004.
- Accepted July 12, 2004.
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