Differential Protein Expression and Subcellular Distribution of TGFβ1,β2andβ3in Cardiomyocytes During Pressure Overload-induced Hypertrophy

J Mol Cell Cardiol 29, 2213–2224 (1997)

Differential Protein Expression and Subcellular Distribution of TGFb1, b2 and b3 in Cardiomyocytes During Pressure Overload-induced Hypertrophy Jian-Mei Li and Gavin Brooks Cardiovascular Cellular and Molecular Biology, Cardiovascular Research, The Rayne Institute, St Thomas’ Hospital, London SE1 7EH, UK (Received 13 January 1997, accepted in revised form 10 April 1997) J.-M. L  G. B. Differential Protein Expression and Subcellular Distribution of TGFb1, b2 and b3 in Cardiomyocytes During Pressure Overload-induced Hypertrophy. Journal of Molecular and Cellular Cardiology (1997) 29, 2213–2224. The transforming growth factor b (TGFb) superfamily plays an important role in the myocardial response to hypertrophy. We have investigated the protein expression of TGFb1, b2 and b3 in left ventricular tissue, and determined their subcellular distribution in myocytes by immunoblotting and immunocytochemistry during the development of left ventricular hypertrophy (LVH), using isoform specific antibodies to TGFb1, b2 and b3. LVH was produced in rats by aortic constriction (AC) and LV tissue was obtained at days (d)0, 1, 3, 7, 14, 21 and 42 following operation. Compared with age matched sham-operated controls (SH), TGFb1 levels in LV tissue of AC rats increased significantly from d1–d14 (P<0.03) concomitant with the adaptive growth of LV tissue. In contrast, TGFb3 levels decreased in LV tissue of AC rats from d3 post-operation (significant from d14–d42, P<0.03). No significant difference in TGFb2 levels were observed from SH and AC rats after operation. Antibodies to TGFb1 stained intercalated disks, sarcolemmal membranes and cytoplasm, but not nuclei, of cardiomyocytes on LV sections from untreated and SH rats. However, a trans-localisation of TGFb1 to the nuclei of cardiomyocytes was observed in AC hearts. Antibodies to TGFb3 stained T tubules, cytoplasm and the nuclei of cardiomyocytes from untreated and SH rats. However, by d7 post-AC operation, TGFb3 expression was lost rapidly from nuclei of cardiomyocytes followed by a reduction in total TGFb3 immunofluorescence in myocytes. Antibodies to TGFb2 stained sarcolemmal membranes of cardiomyocytes from both SH and AC rats without significant difference between groups. Thus, the differential pattern of protein expression and subcellular distribution of TGFb1, b2 and b3 in myocytes during the development of LVH suggests that these molecules play different roles in the response of cardiomyocytes to LVH.  1997 Academic Press Limited K W: Cardiomyocytes; Hypertrophy; Immunocytochemistry; Pressure overload; TGFb.

Introduction Left ventricular hypertrophy (LVH) has been shown to be a major risk factor for cardiovascular morbidity and mortality and is a well accepted prognostic indicator for subsequent cardiac dysfunction (Weber and Brilla, 1991; Frohlich et al., 1992). Most

commonly, LVH is induced by pressure-overload, as a consequence of systemic hypertension or valvular stenosis, which initially permits the LV to adapt to an increased working load. However, if the increase in LV pressure persists, various alterations in the myocytes may lead to congestive heart failure (Morgan et al., 1987; Weber and Brilla, 1991). In

Please address all correspondence to: Dr Gavin Brooks, Cardiovascular Research, The Rayne Institute, St Thomas’ Hospital, London SE1 7EH, UK.

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this regard, a thorough understanding of the mechanisms that regulate the adaptive growth of cardiomyocytes during hypertrophy may lead to strategies for improving the prognosis of this disease. The transforming growth factor-bs (TGFbs) represent a large family of cytokines with diverse activities, including the regulation of cell growth, differentiation, and cell function (Massague, 1990; Moses et al., 1990). TGFbs are produced by many different cell types, including cardiomyocytes, and act on a broad spectrum of target cells (Brand and Schneider, 1995). Three isoforms of TGFb, viz. TGFb1, b2 and b3, with similar, but not identical, biological activities have been found in mammals. In the heart, TGFbs have been detected, both during myocardial development and in the adult (MacLellan et al., 1993). During pressure-overload–induced hypertrophy, TGFb1 mRNA expression is upregulated three–four-fold and is associated with an increase in TGFb1 bioactivity in adult rat cardiomyocytes, but not in non-myocytes, (Takahashi et al., 1994). In cell culture, TGFb1 induces the expression of a fetal gene program indicative of cardiomyocyte hypertrophy (Parker et al., 1991; Komuro and Yazaki, 1993). Although these data implicate an important role for TGFbs in mediating the growth potential of cardiomyocytes during hypertrophy, the majority of the previous observations are based on mRNA expression studies and are almost exclusively related to the expression of TGFb1. It is known that at least three isoforms of TGFb exist in the heart (MacLellan et al., 1993) and they have been suggested to function in a similar and interchangeable way, since TGFb1-knockout mice developed a normal cardiovascular system (Kulkarni et al., 1993; MacLellan et al., 1993). However, TGFb1, b2 and b3 are expressed differentially, at both the mRNA and protein levels, during the development of the mammalian heart (Millan et al., 1991; Pelton et al., 1991; Pott et al., 1991), which may suggest different roles of these TGFb isoforms in vivo. Although the expression of these molecules is known during normal development, it is currently unclear whether the protein expression and subcellular distribution of these three isoforms alters during the development of LVH. In the present study, we have examined, by immunoblotting, the changes in protein expression of TGFb1, b2 and b3 in LV tissue of rats during the development of pressure-overloadinduced LVH. In addition, we have shown, by immunocytochemistry, that significant alterations in the subcellular distribution of these TGFbs occur in cardiomyocytes at different stages of LVH.

Materials and Methods Materials ECL Western Blotting reagents were purchased from Amersham International PLC, UK; bovine serum albumin (BSA) type V was purchased from Sigma, UK; isoform specific rabbit polyclonal antibodies against TGFb1 and TGFb2 (recognising both precursor and active forms), corresponding peptides for competition studies and goat anti-rabbit IgG horseradish peroxidase-conjugated antibody were purchased from Santa Cruz Biotechnology Ltd, USA. These antibodies have previously been used by other investigators to demonstrate expression of TGFb1 and TGFb2 in rat tissue both by immunoblotting and immunocytochemistry (Gao et al., 1996). Rabbit polyclonal antibodies to cardiac troponin I and TGFb3 (raised against latency-associated protein, amino acid residues 282–298, and characterised according to the studies by Olofsson et al., 1992; Waltenberger et al., 1993) were gifts from Dr P. Cummins, University of Birmingham, UK. All other chemicals used were of the highest grade available commercially.

Animals Adult male Wistar rats (starting weight: 166±3 g) were obtained from Binton and Kingman, Hull, UK. Animals were killed by an approved method, in accordance with the UK Home Office Animals (Scientific Procedures) Act, 1986.

Induction of LVH and cardiac tissue preparation Chronic pressure overload was produced in rats by subtotal suprarenal constriction of the abdominal aorta (AC) as described previously (Levy et al., 1996). Briefly, under anaesthesia with hypnorm (0.3 ml/kg) and diazepam (2.5 mg/kg) intraperitoneally, the aorta was carefully exposed through an abdominal incision. A uniform degree (0.45 mm in diameter) of constriction was achieved by fixing a titanium clip around the aorta. Agematched sham-operated control rats (SH) underwent the same operation but without aortic constriction. Both AC and SH rats were housed and fed under identical conditions. Six rats from each group were killed at d1, 3, 7, 14, 21 and 42 after surgery. In addition, six rats that did not undergo any operation were used as d0 controls. Body

Expression and Distribution of TGFbs During Hypertrophy

weight was recorded from each rat, both on the day of operation and on the day of killing, when the heart was excised and weighed. The atria were carefully dissected from the ventricles and LV tissue was separated from RV tissue and weighed. Each sample of LV tissue was split into two portions and used for protein preparation and immunohistochemistry, respectively. Right ventricular tissues were retained for control purposes. Heart tissue was frozen immediately in liquid N2 until required for analysis. At least three different hearts from AC and SH rats were examined at each time point following operation and each heart was analysed both by immunoblotting and immunocytochemical analyses as described below.

Protein extraction and immunoblotting Tissue (200 mg/ml) was homogenised using an Ystral C10/25 homogeniser in an ice-cold extraction buffer containing 0.05 mol/l Tris, 0.15 mol/ l NaCl, 0.002 mol/l ethylenediaminetetraacetic acid, 0.002 mol/l phenylmethylsulphonylfluoride, 2 lg/ml leupetin and 2 ll/ml aprotinin, pH 7.4. After sonication, Triton X-100 was added to a final concentration of 0.5%. The lysate was extracted on ice for 15 min and centrifuged at 12 000×g for 20 min. Immunoblotting was performed as described previously (Brooks et al., 1993). Briefly, proteins (40 lg) were separated on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in phosphate buffered saline/0.2% Tween 20 (PBST), followed by incubation with isoform specific rabbit polyclonal antibodies to TGFb1, b2 and b3 diluted (1:1000) in PBST containing 1% non-fat milk for 1 h at room temperature. After washing with PBST, membranes were incubated with goat anti-rabbit IgG conjugated to horse-radish peroxidase for 30 min at room temperature. Bands were visualised by enhanced chemiluminescence and quantified using densitometry. Each protein sample was analysed twice and at least three different hearts from AC and SH rats were examined at each time point following operation.

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carried out as previously described (Li et al., 1995). To avoid possible contamination by TGFbs in the goat serum, sections were blocked with 2% BSA in PBS before applying the primary antibodies, and slides were washed three times with 0.05%BSA/ PBS for 5 min between each staining step. Sections were stained using isoform specific rabbit polyclonal antibodies against TGFb1, b2, b3 and cardiac troponin I (5 lg/ml). Normal rabbit IgG (5 lg/ml) was used in each experiment as a negative control. For competitive inhibition assays, antibodies to TGFb1 and TGFb2 were pre-incubated for 30 min at room temperature with 5 lg/ml of synthesised peptide for TGFb1 and b2, respectively, prior to incubation with sections. Primary antibodies were incubated with the relevant section for 30 min at room temperature and antibody binding was detected using FITCconjugated goat anti-rabbit IgG. Specific fluorescent staining of cardiac tissue was identified under uv light with an Olympus BH2-RFCA microscope using an oil-immersion lens (magnification ×40 or ×100) and photographed using Kodak film. To identify the nuclear staining of antibodies, sections were double stained with the nucleic acid dye, propidium iodide (1.5 lg/ml). The number of nuclei positively stained with antibodies to TGFb1 and TGFb3 (green colour) were counted within the microscopic field and compared with the total number of nuclei stained with propidium iodide (red colour) on the same section, and within the same microscopic field. Three sections from each heart were analysed by two independent assessors and three different hearts were used for each time point after operation.

Protein determination Protein concentrations in cell preparations were determined according to the method of Bradford (1976), using BSA type V as a standard.

Statistical analysis Results were analysed statistically by ANOVA and Bonferroni-t-test.

Immunocytochemistry

Results

LV tissues from AC and SH rats at d0, 7, 14, 21 and 42 post-operation were snap-frozen and cryosectioned into 6 lm sequential sections. Pretreatment of sections and immunofluorescence was

Effect of AC on ventricular mass Six rats from either AC or SH groups were killed at 1, 3, 7, 14, 21 and 42 days after surgery. Body

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J.-M. Li and G. Brooks Table 1 Changes in ventricular mass following operation LV/body weight ratio (mg/g) Days 1 3 7 14 21 42

RV/body weight ratio (mg/g)

SH

AC

Increase†

SH

AC

2.5±0.06 2.5±0.04 2.3±0.16 2.3±0.04 2.2±0.06 2.2±0.08

2.8±0.10 2.9±0.71∗ 3.0±0.15∗ 3.2±0.10∗ 3.3±0.12∗ 3.3±0.16∗

12% 16% 30% 39% 50% 50%

0.6±0.04 0.7±0.03 0.7±0.04 0.6±0.02 0.6±0.02 0.5±0.04

0.6±0.06 0.7±0.02 0.8±0.05 0.7±0.03 0.6±0.08 0.6±0.05

Values are means ±... from six animals/group. ∗P<0.01 compared with SH controls. †Percentage of increase in LV weight/body weight ratio of AC rats relative to age matched SH controls.

Sham-operation

TGF

1

TGF

2

TGF

3

Aortic constriction

Troponin I Days

0

1

3

7

14

21

42

0

1

3

7

14

21

42

Figure 1 Representative autoradiograph of an immunoblot showing the expression of TGFb1, b2 and b3 proteins in LV tissues following sham and aortic constriction operation. Equal amounts of protein (40 lg) from each sample were separated by 12% SDS/PAGE and transferred to nitrocellulose filters. Filters were probed with isoform-specific antibodies to TGFb1, b2, b3 and cardiac troponin I as described in Materials and Methods.

weight and ventricular weight were measured, and results expressed as the ratio of ventricular weight to body weight. Table 1 shows that, relative to agematched SH controls, LV weight/body weight ratio in AC rats increased progressively with time from d1 to d21 after operation (P<0.01). After 21 days, LV mass in AC rats stabilised such that no further increase in LV weight/body weight ratio was observed up to 6 weeks following operation. In contrast to the changes in LV weight to body weight ratio during AC, no significant difference was observed in RV weight/body weight ratio following operation between AC and SH rats.

Changes in TGFb1, b2 and b3 protein expression following operation In order to determine the expression of TGFb proteins during the development of LV hypertrophy (LVH), we measured the amount of TGFb1, b2 and b3 proteins in LV tissues from AC and SH rats

by immunoblotting. Equal protein loading in each experiment was confirmed by Coomassie blue staining of the gels and by probing a duplicate transfer membrane with a rabbit polyclonal antibody to cardiac troponin I (Fig. 1). Each experiment was repeated at least twice to confirm the reproducibility of the results. TGFb1 and b2 were expressed predominantly as their full length precursors in LV tissues of both AC and SH rats and detected by immunoblot as a single band of approximately 55 kD for TGFb1 and 50 kD for TGFb2. TGFb3 was detected as its latency associated protein which migrated as a single band of 28 kD in LV tissues of both AC and SH rats. The lack of detection of the mature, active forms of TGFb isoforms may reflect the adhesive nature of these forms resulting in loss during the isolation procedure (Grainger D., personal communication). As shown in Figure 1, the three isoforms of TGFb were expressed differentially in LV tissues of rats after AC or SH operation. Thus, expression of TGFb1 protein in LV tissue of AC rats increased rapidly from d1 to d14 post-operation and then began to decrease, such

Expression and Distribution of TGFbs During Hypertrophy 2.5

Densitometry index

*

*

*

7

14

(a)

2.0 1.5

*

1.0 0.5

0

21

28

35

42

2.5

Densitometry index

(b) 2.0 1.5 1.0 0.5

0

7

14

21

28

35

42

2.5

Densitometry index

(c) 2.0 1.5 1.0 0.5

0

* * 7

*

*

14 21 28 Days post-operation

35

42

Figure 2 Quantitative changes in TGFb1 (a), b2 (b) and b3 (c) protein levels during the development of LVH. (Χ) Aortic constriction (AC) group; (Β) sham operation (SH) group. Protein samples from three different hearts at each time point and from each treatment group (SH and AC) were examined by immunoblotting, and the resultant autoradiograms scanned densitometrically. Each protein sample was analysed at least twice. Results were normalised to the expression of the same protein in a normal sample without operation applied on every gel and expressed as a ratio to the normal control. Results show means±...; ∗P<0.03.

that levels were not significantly different from SH controls by d21 post-operation (Fig. 2). In contrast to AC hearts, TGFb1 protein expression was not significantly changed in LV tissue of SH rats throughout the d1–d42 post-operative period (Figs

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1 and 2). TGFb2 protein expression remained constant in LV tissues of both AC and SH rats throughout the post-operative time course (Figs 1 and 2). In contrast to TGFb1, expression of TGFb3 protein in LV tissues of AC rats decreased gradually with time, whereas no significant change was observed for TGFb3 in LV tissues from SH rats throughout the 42 day post-operative period (Figs 1 and 2). The changes in TGFb1, b2 and b3 protein expression in AC rats was not due to variations in the amount of protein loaded as duplicate gels probed with an antibody to troponin I exhibited a band at 28 kD, whose level altered very little in samples from both AC and SH controls throughout the 6 week post-operative period (Fig. 1). The specificity of antibody binding was confirmed in each case by competitive inhibition with synthetic peptides for TGFbs (1 lg/ml) (data not shown). Figure 2 shows the quantitative changes in the expression of TGFb1, b2 and b3 proteins following AC and SH operation following densitometric scanning of autoradiographs obtained from three separate hearts with two determinations per tissue sample. Results were normalised individually to the density of the control d0 sample applied onto every gel, and the results expressed as the ratio to the normal control. Compared with SH controls, a significant increase in TGFb1 protein expression was observed in LV tissues from AC rats from d1–d14 following operation (P<0.03) [Fig. 2(a)]. In contrast, a significant reduction in TGFb3 protein expression was observed in LV tissue from AC rats from d7–d42 following operation (P<0.05) [Fig. 2(c)]. Although there was a slight rise in the levels of TGFb2 protein expression in LV tissues from both SH and AC rats during the first 7 days following operation, this was not significant [Fig. 2(b)]. Immunocytochemical detection of TGFb1, b2 and b3 protein in cardiomyocytes In order to determine the subcellular distribution of TGFb1, b2 and b3 proteins in cardiomyocytes, we performed immunofluorescent staining on cardiac cryosections from the same series of LV tissues as used for protein measurements. Cardiomyocytes were identified by staining with an antibody to the cardiomyocyte-specific protein, cardiac troponin I, on sequential sections in each experiment (data not shown). The antibody to TGFb1 stained intercalated disks, cytoplasm and sarcolemmal membranes of cardiomyocytes on sections from normal and SH rats [Fig. 3(a)]. However, TGFb1 failed to stain nuclei of cardiomyocytes from SH rats, which were

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Figure 3 Immunofluorescent detection of TGFb1 in cardiomyocytes on LV sections from normal and hypertrophic hearts. A cryosection from a normal heart was double stained with antibody to TGFb1 (a) and with propidium iodide (b) and shows TGFb1 staining of intercalated disks and cytoplasm, but not nuclei, of cardiomyocytes. Sections from aortic constriction rats at d7 [(c) and (d)] and d21 [(e) and (f)] following operation were double stained with antibody to TGFb1 [(c) and (e)] and with propidium iodide [(d) and (f)] and show the appearance of nuclear staining of TGFb1 during the development of LVH (magnification: 400×). The results are representative of those obtained from three separate hearts per treatment group taken at each time point.

labelled on the same section by propidium iodide [Fig. 3(b)]. During the development of LVH, a distinct myocyte nuclear staining was observed from d7–d42 following AC operation and this was accompanied by an overall increase in TGFb1 immunofluorescence intensity in myocytes at d7 [Fig. 3(c)] and d21 [Fig. 3(e)] post-operation. Compared with nuclei labelled by propidium iodide on the same sections at d7 [Fig. 3(d)] and d21 [Fig. 3(f)] we found that approximately 60% of nuclei

were positively stained for TGFb1 at d7 post-operation [Fig. 3(c)], increasing to approximately 80% of nuclear staining at d21 post-operation [Fig. 3(e)] for AC rats, respectively. The antibody to TGFb2 stained predominantly myocyte cell membranes [Fig. 4(a)], but not nuclei, which were labelled by propidium iodide on the same section [Fig. 4(b)], from normal rat LV sections. In contrast to TGFb1, no significant difference was seen for TGFb2 immunostaining in cardiomyocytes on LV sections

Expression and Distribution of TGFbs During Hypertrophy

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Figure 4 Immunofluorescent detection of TGFb2 in cardiomyocytes on LV sections from normal, SH and AC rats. A cryosection from a normal heart was double stained with antibody to TGFb2 (a) and with propidium iodide (b) and shows staining of intercalated disks and sarcolemmal membranes of cardiomyocytes. Sections from sham (SH) rats at d7 (c) and d21 (e), and from aortic constriction (AC) rats at d7 (d) and d21 (f) post-operation were stained with antibodies to TGFb2. Note that no significant difference was observed between SH and AC groups, and no nuclear staining of TGFb2 was observed in cardiomyocytes for both groups (magnification: 400×). The results are representative of those obtained from three separate hearts per treatment group taken at each time point.

between SH and AC rats at d14 [Figs 4(c) and 4(d)] or d42 [Figs 4(e) and 4(f)] post-operation. Antibodies to TGFb3 produced a strong immunostaining of the T-tubules, cytoplasm and nuclei of cardiomyocytes on LV sections of normal [Fig. 5(a)] and SH rats (data not shown). Although Figure 5(a) suggests that TGFb3 may be expressed at significant levels in the sarcolemmal membranes of cardiac myocytes, we have shown in additional experiments that such levels are very low. Thus, comparison of

Figure 5(a) with a sequential section stained with an antibody to laminin [which stains cardiac myocyte membranes specifically (Price et al., 1992)] showed that any staining of cardiac myocyte sarcolemmal membranes with TGFb3 is low compared with elsewhere in the cell (data not shown). In addition, immunoblot analysis of subcellular fractions prepared from adult rat LV tissue demonstrated that the majority of TGFb3 protein was expressed in the cytoskeletal/nuclear fraction (Triton X-100

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Figure 5 Immunofluorescent detection of TGFb3 in cardiomyocytes on LV sections from normal and hypertrophic hearts. A cryosection from a normal heart was double stained with antibody to TGFb3 (a) and with propidium iodide (b) and shows TGFb3 staining of nuclei, T tubules and cytoplasm of cardiomyocytes. Sections from aortic constriction rats at d14 [(c) and (d)] and d42 [(e) and (f)] following operation were double stained with antibody to TGFb3 [(c) and (e)] and with propidium iodide [(d) and (f)] and show a significant reduction in total fluorescent staining of TGFb3 during the development of LVH (magnification: 385×). The results are representative of those obtained from three separate hearts per treatment group taken at each time point.

insoluble), with lower levels in the cytosolic fraction. Minimal levels of TGFb3 were found in the sarcolemmal protein fraction (Triton X-100 soluble) (data not shown). TGFb3 was found in approximately 80% of myocyte nuclei which were labelled by propidium iodide on the same section of normal [Fig. 5(b)] and SH rats. Compared with normal and SH controls, the fluorescent intensity of TGFb3 was significantly reduced in the cytoplasm of myocytes from AC rats at d14 [Fig. 5(c)] and d42 [Fig. 5(e)] post-operation, and only 40% of

myocyte nuclei were stained positively for TGFb3 at d14 and about 60% at d42, as indicated by double staining of the same sections with propidium iodide [Figs 5(d) and 5(f)], respectively. Interestingly, the down-regulation of TGFb3 was evident in the nuclei before it became apparent in the cytoplasm (Fig. 6). At d7 post-operation, compared with approximately 80% of nuclear staining of TGFb3 in myocytes from SH hearts [Figs 6(a) and 6(b)], TGFb3 was not detectable (<5% of nuclear staining) in the nuclei of cardiomyocytes of AC rats

Expression and Distribution of TGFbs During Hypertrophy

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Figure 6 Lack of nuclear expression of TGFb3 in cardiomyocytes in aortic constriction (AC) hearts at d7 post-operation. A section from a sham operated (SH) heart at d7 post-operation was double stained with antibody to TGFb3 (a) and with propidium iodide (b). More than 80% nuclei were stained positively for TGFb3 (magnification 432×). (c) was the same as (a) but under larger magnification (1080×). (d) Sequential section of (a) was stained with normal rabbit IgG as a negative control. A section from an AC heart at d7 post-operation was double stained with an antibody to TGFb3 (e) and with propidium iodide (f) and shows that myocyte nuclei were negative for TGFb3 expression (magnification 432×). [(g) and (h)] were the same as (e) and (f) but under larger magnification (1080×). The results are representative of those obtained from three separate hearts per treatment group taken at each time point.

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[Figs 6(e) and 6(f)]. This rapid loss of TGFb3 in the nuclei of cardiomyocytes from AC rats was confirmed under larger magnification [Fig. 6(c) for SH, Figs 6(g) and 6(h) for AC]. Specificity of TGFb1 and TGFb2 antibody-binding to LV sections was confirmed by competition studies with synthetic TGFb1 and TGFb2 peptides as described in Materials and Methods. In each case, incubation of sequential sections with competing peptide resulted in a complete abolition of specific TGFb1 or TGFb2 staining compared to staining with antiserum alone (data not shown). As a further control, normal rabbit IgG when used at the same concentration as for TGFbs (5 lg/ml) produced no specific staining of cardiomyocytes on sequential sections [Fig. 6(d).

Discussion In this study, we demonstrate for the first time how expression and subcellular localisation of TGFb1, b2 and b3 changes during the development of LVH in rats. TGFb1 mRNA expression in cardiomyocytes has previously been shown to be increased during LVH induced either by pressure overload or by norepinephrine treatment (Takahashi et al., 1994; Brand and Schneider, 1995). Following aortic constriction, TGFb1 mRNA levels were shown to increase significantly as early as 12 h following operation, and decreased to control levels by 14 days (Villarreal and Dillmann, 1992). Our results presented here extend these previous reports, since we have shown a significant increase in the protein levels of TGFb1 in LV tissue from d1–d14 during the development of pressure-overload-induced LVH. The increase in TGFb1 was observed at the precursor protein level (molecular weight about 55 kD), since the activated form of TGFb1 (molecular weight about 18 kD) was almost undetectable in LV tissue lysates by immunoblotting. The functional significance of this result remains unclear, although there is currently no direct evidence to show the presence of a lower, active Mr form of TGFb1 in vivo, despite the fact that it has been demonstrated in in vitro experiments. TGFb1 has previously been reported to be localised intracellularly to both mitochondria and contractile filaments in cardiomyocytes (Heine et al., 1991), and this localisation has been suggested to be critical for maintaining rapid calcium fluxes, and consequently rhythmicity, in these cells (Roberts and Sporn, 1993). Our results demonstrate very clearly that during the development of LVH, a distinct translocation of TGFb1 protein to the nuclei of cardiomyocytes occurs in AC hearts, concomitant with the adaptive growth of

LV tissue. It is generally accepted that the biological activities of TGFbs are mediated via the binding of these polypeptide growth factors to membrane bound receptors (Massague, 1992); however, it is feasible that these growth factors could mediate direct effects intracellularly also, e.g. by acting as nuclear transcription factors. Although previous studies have not provided evidence for such a direct biological function of TGFb in the nucleus, it is known that the molecular responses of TGFb are related to the transcriptional regulation of other genes, most of which are located in the nuclei (Massague, 1990). Furthermore, TGFb1 has recently been found to regulate the expression of many cell cycle dependent molecules, such as G1 cyclins and cyclin dependent kinases (CDKs) (Ewen et al., 1993; Geng and Weinberg, 1993; Koff et al., 1993), and the CDK inhibitors, p21 and p27 (Datto et al., 1995; Mal et al., 1996), thereby directly implicating this growth factor in cellular growth. Our demonstration of nuclear translocalisation of TGFb1 during the development of LVH suggests a role for this molecule in the control of cardiac hypertrophic growth, possibly via the manipulation of growthrelated gene expression in the nuclei of cardiomyocytes. Indeed, TGFb has previously been implicated in the processes of repair and adaptation of the heart following myocardial injury or stress (Thompson et al., 1988), since it was shown that certain cells surrounding the margins of an infarcted zone stained intensely for TGFb1 approximately 24–48 h after infarction, which persisted for up to 10 weeks following damage. Thus, TGFb1 may play a pivotal role in protecting the heart during infarction and in mediating the hypertrophic response by helping to restore function to the affected myocardium. TGFb2 mRNA has previously been shown to be highly expressed in all cardiac precursor cells during early mouse embryo development (Dickson et al., 1993; Brand and Schneider, 1995). However, in a developmental study, TGFb2 gene expression was not reported in fetal, neonatal, or adult hearts (Engelmann, 1993). In the present study, we have shown by a combination of immunoblotting and immunocytochemistry, that TGFb2 protein is detectable in adult LV tissue, and that the levels did not change significantly in either SH or AC operated rats during the 6-week post-operative period. Subcellular distribution of TGFb2, as revealed by immunocytochemistry, was restricted mainly to sarcolemmal membranes in both SH and AC hearts, and displayed no evidence of translocation following operation. These results suggest that TGFb2 is probably not involved in the hypertrophic growth response of cardiomyocytes induced by pressure-overload.

Expression and Distribution of TGFbs During Hypertrophy

TGFb3 mRNA has previously been found to increase markedly during neonatal rat heart development, with maximum expression occurring at about 5 weeks of age (Engelmann, 1993). However, during the development of pressure-overload-induced cardiac hypertrophy in rats, TGFb3 mRNA expression in LV tissue was shown to remain unchanged compared with control levels (Villarreal and Dillmann, 1992). Interestingly, we found that the protein expression of TGFb3 gradually decreased with time following operation in AC rats. Immunocytochemical analyses confirmed this reduction of TGFb3 in AC hearts, and showed that the loss of TGFb3 protein occurs initially in the nuclei and is followed by a subsequent loss in the cytoplasm of cardiomyocytes. Thus, there is a reciprocal expression of TGFb1 and b3 proteins during the development of LVH, such that TGFb1 levels rise whilst there is a concomitant fall in the expression of TGFb3. Interestingly, the reciprocal expression of TGFb1 and b3 proteins during LVH is opposite to that observed by us during post-natal development (Haworth et al., 1994, 1995). Therefore, it would appear that cardiomyocytes express the fetal TGFb profile during the development of LVH in an analogous manner to that reported for other gene products, e.g. myosin heavy chain and a-skeletal actin (Parker et al., 1991). The differences observed in protein expression and in the subcellular distribution between TGFb1 and TGFb3 in both sham and hypertrophic cardiomyocytes suggests different biological functions of these two isoforms on the growth behaviour of cardiomyocytes. In summary, this study has extended previous reports regarding TGFbs expression in the adult rat heart and during the development of LVH. The majority of previous studies have concentrated on mRNA expression of these cytokines, whereas we have monitored protein levels of individual TGFb isoforms. In addition, we report for the first time the subcellular distribution of these isoforms, in detail, during a time course of pressure-overloadinduced LVH. Our discovery that TGFb1 is upregulated whilst TGFb3 is down-regulated, coupled with the differential subcellular distribution of TGFb1, b2 and b3 during the development of LVH, extends our understanding of the role of TGFbs in modulating cardiac function during hypertrophy.

Acknowledgements This work was supported by the Wellcome Trust (J-ML) and the British Heart Foundation (GB). The

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authors are grateful to Professor D. J. Hearse for his continued support and encouragement and Drs D. Grainger and R. S. Haworth for valuable discussions.

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