INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CARDIAC HYPERTROPHY IN RESPONSE to hemodynamic overload is an established risk factor for cardiovascular morbidity and mortality (23). Pressure overload hypertrophy is characterized by a period of compensation, followed by transition to cardiac failure. The mechanisms that are involved in functional decompensation of the hypertrophied heart remain elusive. On the molecular level, ventricular hypertrophy encompasses specific changes in cardiac gene expression, including the induction of "immediate-early" genes and hypertrophy-associated proteins such as atrial natriuretic factor (ANF), leading to a "fetal" phenotype (36, 47). Furthermore, hypertrophied myocardium is characterized by enhanced extracellular matrix (ECM) deposition, increased expression of peptide growth factors, and specific changes of other mediator systems such as the renin-angiotensin system (RAS) and the -adrenergic system (5, 36, 38, 47).

The expression of transforming growth factor-₁ (TGF-₁) mRNA is increased in the left ventricular myocardium of patients with idiopathic hypertrophic cardiomyopathy and dilated cardiomyopathy (27, 37) and in animal models of myocardial infarction, progressive coronary artery occlusion, and pressure overload (10, 19, 50, 51). TGF-₁ is particularly expressed in hypertrophic myocardium during the transition from stable hypertrophy to heart failure (7, 46), and recent in vitro studies (21, 24, 33, 42, 43) suggest a link between this cytokine and -adrenergic signaling. TGF-₁ regulates the proliferation, differentiation, migration, and survival of numerous cell types. Its biological responses are elicited through a heteromeric receptor complex comprising two serine-threonine kinase receptors, termed TGF- receptor type 1 and 2 (TR1 and TR2) (29). Both TGF-₁ ligand and the TR₁ and TR2 receptors are present in the heart, and all are expressed in cardiac myocytes as well as in nonmyocytes (8). In vitro, TGF-₁ induces the production of ECM components, including fibrillar collagen, fibronectin, and proteoglycans in cardiac fibroblasts (14, 20, 49), and promotes fetal gene expression in cultured neonatal cardiac myocytes (8, 35). Thus the in vitro effects of TGF-₁ mimic the characteristic alterations that are found in the hypertrophied and failing heart. The involvement of TGF-₁ signaling is further supported by two recent in vivo studies, in which enhanced TGF-₁ signaling led to atrial cardiac fibrosis (32) and heterozygous TGF-₁ (+/)-deficient mice revealed decreased fibrosis of the aging heart (9).

In addition to the direct effects of TGF-₁, there is substantial evidence for interactions between TGF- signaling and other mediator systems that are involved with cardiac hypertrophy and failure. Several agonists that provoke hypertrophic changes, including angiotensin II and norepinephrine, induce TGF-₁ expression in both cardiac myocytes and nonmuscle cells (2, 16, 26, 39). Excessive stimulation of the sympathetic nerve system plays a pivotal role in the pathogenesis of cardiac hypertrophy and failure, and hypertrophy of cardiomyocytes appears to be critical in this process (5). Whereas the hypertrophic effect of catecholamines on cardiac myocytes in vitro is solely mediated via stimulation of -adrenoceptors (ARs), myocardial hypertrophy in vivo may also be induced by selective stimulation of -ARs (3, 45). It is therefore postulated that autocrine and/or paracrine mechanisms alter the responsiveness to -AR stimulation in vivo. Several in vitro studies (21, 24, 33) have shown that TGF-₁ may alter -adrenergic signaling by modulating the number and function of -ARs in various cell types. More recent studies (42, 43) revealed that TGF-₁ induces hypertrophic responsiveness to -adrenergic stimulation in cardiac myocytes. These studies suggest that TGF-₁ may modulate -adrenergic signaling in vivo, and this phenomenon may be relevant during the transition from hypertrophy to failure.

Therefore, we sought to relate the known in vitro effects of TGF-₁ to the in vivo situation. In transgenic mice overexpressing mature TGF-₁, we evaluated the pathophysiological consequences of elevated TGF-₁ levels on cardiac phenotype and the myocardial -adrenergic system. We observed significant cardiac hypertrophy, enhanced -adrenergic signaling, and altered contractile function in transgenic mice compared with age-matched nontransgenic controls.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Alb/TGF-₁ (Cys^223,225Ser) transgenic mice were prepared as previously described (41) and were kindly provided by Dr. S. S. Thorgeirsson and Dr. N. Sanderson (National Cancer Institute, Bethesda, MD). In brief, the 4.7-kb transgene is composed of the murine albumine promoter and enhancer linked to a porcine TGF-₁ construct and the 3' region of the human growth hormone gene, which contains a polyadenylation signal. The TGF-₁ cDNA encodes cysteine/serine substitutions at amino acid residues 223 and 225, resulting in preferential secretion of mature TGF-₁ (40). Fertilized eggs from F1 mice (C57B1/6J × CBA; Jackson Laboratories; Bar Harbor, ME) were microinjected with this cDNA construct and multiple founder animals were obtained. In the present study, we used mice from line 25, which represented the line with the most abundant hepatic transgene expression. In this line, all transgenic mice are male and all normal mice are female, suggesting that the transgene is integrated on the Y chromosome. The line has been maintained by continued backcrosses to F1 mice (C57B1/6J × CBA; Harlan and Winkelmann). Male offspring of F1 × F1 (C57B1/6J × CBA) crosses were used as normal control mice. In line 25, the expression of the transgene in the liver resulted in a >10-fold increase in plasma levels of TGF-₁ compared with age-matched nontransgenic controls (41). All investigations were performed at the age of 8 wk. All animal studies were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Guidelines.

Morphometric and stereological investigations. For tissue fixation, retrograde perfusion fixation was performed as described (48). Animals were perfused with either ice-cold NaCl 0.9% (immunohistochemistry) or 3% glutaraldehyde (morphometric and stereological analysis). After weight and volume was determined, tissue sampling and section staining were performed according to the orientator method (30). Uniformly random sampling was achieved by preparing a set of equidistant slices of the left ventricle and the interventricular septum with a random start. Three slices were selected by area-weighted sampling and processed by using the orientator method (30). Semithin sections (1 µm) were prepared and stained with methylene blue and basic fuchsin. For electron microscopy, ultrathin (0.8 µm) sections were prepared from several animals and stained with uranyl acetate and lead citrate. All investigations were performed in a blinded manner, i.e., the observer was unaware of the protocol. Fractional areas of myocytes, cardiac fibroblasts, and nonvascular interstitium were measured on 12 differentially orientated semithin sections per animal using the point-counting method, as described by Törnig et al. (48). Myocyte diameters were measured on longitudinal sections with a semiautomatic image analyzing system and corrected for sarcomere length as an index of contraction. Wall thickness of the left and right ventricle, the interventricular septum, as well as lumen diameter and area were determined on transmural hematoxylin and eosin (HE)-stained paraffin sections (see below) using an image analyzing system (AnalysisPro, Soft-Imaging; Münster, Germany).

Immunohistochemistry and routine tissue stains. The hearts were cut tranversally and were either fixed in 4% buffered formalin or snap-frozen in liquid nitrogen. After the paraffin sections (4 µm) were embedded, they were prepared and immunostained for collagen types I, III, and IV, as described by Amann et al. (1). The volume percentage of myocardial collagen type III was quantified using an image analyzing system (AnalysisPro, Soft-Imaging). In addition, HE, periodic acid Schiff, and fibrous tissue stains (Sirius red) were routinely performed.

Reverse transcriptase-polymerase chain reaction. Total RNA from ventricular tissue was extracted with RNA-Clean (AGS; Heidelberg, Germany) according to the manufacturers protocol. Reverse transcription (RT) reactions were performed as previously described (42). The following oligonucleotide primers (GIBCO-BRL) were used: ANF sense 5'-ATGGGCTCCTTCTCCATCAC-3' and antisense 5'-TCTTCGGTACCGGA-AGCT-3', for amplification between bp 64 and 520 (17); c-fos sense 5'-TGCCAGATGTGG-ACCTGTCTG-3' and antisense 5'-CCACAGCTTGGTGTGTTTCAC-3', for amplification between bp 999 and 1,390 (13); -actin sense 5'-GAAGTGTGACGTTGACATCCG-3' and antisense 5'-TGCTGATCCACATCTGCTGGA-3', for amplification between bp 2,731 and 3,081 of rat -actin gene (34). After amplification reaction, the products were separated on 5% polyacrylamide gels, stained with ethidium bromide, and photographed under ultraviolet illumination. For quantification, the density of the fragments was determined with the use of ImageQuant software (Molecular Dynamics; Krefeld, Germany). The results for ANF and c-fos expression were normalized for equal loading by -actin amplification.

Real-time quantitative polymerase chain reaction. cDNA was transcribed from left and right ventricle RNA templates from TGF- mice and NTG controls. RT was performed using Sensiscript Reverse Transcriptase (Qiagen) and the following oligo-dt primers (GIBCO-BRL): ANF sense 5'-AGAGTGGGCAGAGACAGCAAA-3' and antisense 5'-ATGGAGAAGGAGCCCATGC-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense 5'-CCTGGACCACCCAGCCCAGCA-3' and antisense 5'-TGTTATGGGGTCTGGGATGGA-3'. Polymerase chain reaction (PCR) was carried out in 25-µl-containing PCR Master Mix buffer (Sybr Green, AmpliTaqGold DNA polymerase, dNTPs), 1-µl template DNA and 200 nM of sense and antisense primers according to a two-step PCR protocol (5 min at 95°C, 40 cycles for 15 s at 95°C and 1 min at 59°C). The ABI PRISM Sequence Detection System 7700 was used to detect the fluorescent signal. Threshold cycle values were determined using the Sequence Detector 1.7 software (Applied Biosystems). ANF signals were normalized for GAPDH and are expressed as fold increase over NTG (means ± SE, *P < 0.05).

Membrane preparation and -adrenoceptor binding studies. Membranes from left ventricular tissue were prepared as previously described (4). -Adrenoceptors in cardiac tissue were investigated using [¹²⁵I]iodocyanopindolol (ICYP) as the radiolabeled ligand (specific activity of 2,000 Ci/mmol). For estimation of total -adrenoceptor density of the maximal number of binding sites (B_max) and dissociation constant (K_d), ICYP saturation curves with eight increasing concentrations of ICYP between 3 and 300 pmol/l and 3 µmol/l of propranolol for determination of nonspecific binding were used. Cold ligand-binding affinity was measured by ligand-ICYP competition curves using 25 pmol/l of ICYP to maintain the radioligand concentration at approximate K_d. The assay was performed in a total volume of 250 µl. The total amount of protein used per assay was 20-30 µg. The incubation at 25°C for 120 min allowed complete equilibration of the -adrenoceptors with the radioligand. The reaction was terminated by rapid vacuum filtration through filters (model GF/C, Whatman; Clifton, NJ). The filters were washed immediately three times with 6 ml of ice-cold incubation buffer. All experiments were performed in triplicate.

Tissue homogenization and Western blot analysis. Left ventricular tissue was homogenized by incubation in extraction buffer (10 mM cacodylicacid, 150 mM NaCl, 1 µM ZnCl₂, 20 mM CaCl₂, 1.5 mM NaN₃, 0.01% Triton X-100, pH 5.0) for 12 h at 4°C and subsequent centrifugation for 10 min at 1,200 g. For separation of membrane and cytosolic fractions, the tissue was incubated in a 20 mM Tris · HCl pH 8.0, 1 mM EDTA, and 1 mM dithiothreitol (TED) buffer and centrifuged for 15 min at 480 g. The supernatant was diluted in a similar volume of 1 M KCl and centrifuged for 30 min at 100,000 g. After centrifugation, the supernatant represented the cytosolic fraction, and the pellet was resuspended in TED buffer and represented the membrane fraction. Similar amounts of protein were resolved on a 10% SDS-polyacrylamide electrophoresis gel, and the proteins were transferred to Immobilon and subjected to Western blot analysis.

Materials and antibodies. The TGF-₁-antibody was purchased from R&D (MAB 240) and used at a 1:500 dilution. The antibodies against collagens were purchased from Biogenesis (type I, 1:100 and III, 1:500) and Southern Biotechnology (type IV, 1:500). The inhibitory G protein (G_i) antibody (MB₁) was raised against the COOH-terminus of retinal transducin (KENLKDCGLF). It recognizes G_i1 and G_i2 and was used at a 1:5,000 dilution. The G_s antibody was purchased from DuPont-NEN (NEI 805), the -adrenoceptor kinase-1 (ARK-1) antibody was from Santa Cruz (GRK2, sc-562), and the sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA2) antibody was purchased from ABR (MA3-919). The GTPase-activating protein of Ras (RAS/GAP) antibody was a generous gift from Dr. A. Kazlauskas (Harvard Medical School, Boston, MA) and was used at a 1:5,000 dilution.

Assessment of cardiac function. Myocardial tissue was obtained from the left hearts of the animals, as previously described (6). The experiments were performed using isolated electrically driven muscle preparations. Immediately after excision, atrial trabeculae were placed in ice-cold preaerated Tyrode solution composed of (in mmol/l) 119.8 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.05 MgCl₂, 0.42 Na₂HPO₄, 22.6 NaHCO₃, 0.05 Na₂EDTA, 0.28 ascorbic acid, and 5.0 glucose. Muscle strips of uniform size with muscle fibers running approximately parallel to the length of the strips were dissected under microscopic control with the use of scissors in aerated modified Tyrode solution at room temperature. Connective tissue was trimmed away carefully. The muscles were suspended in an organ bath (75 ml) containing a modified Tyrode solution, which was maintained at 37°C and continuously aerated with 95% O₂-5% CO₂. The muscle strips were stimulated with two platinum electrodes (frequency 1 Hz, impulse duration 5 ms, intensity 10-20% greater than threshold) by using field stimulation from an electronic field stimulator (model S88, Grass; Quincy, MA). Each muscle was stretched to the length at which force of contraction was maximal. Isometric force of contraction was measured with an inductive force transducer (Fleck; Mainz, Germany) attached to a recorder (Brush 2400, Gould; Cleveland, OH). All preparations were allowed to equilibrate for at least 90 min, and the bathing solution was changed once after 45 min. Concentration-dependent mechanical effects were obtained. Control strips kept in Tyrode solution with identical composition as original experiments revealed maximally 10% reduction of baseline isometric tension over the period necessary to complete pharmacological testing. Agents were applied cumulatively to the organ bath. Each muscle was used only once to record a concentration-response curve.

Statistical analysis. All data are expressed as means ± SE. Statistical significance was estimated using the Student's t-test for paired and unpaired observations. A P value of <0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Increased myocardial TGF-₁ levels in transgenic mice. To evaluate the in vivo effects of TGF-₁ on myocardial tissue, hearts from Alb/TGF-₁ (Cys^223,225 Ser) transgenic mice were compared with those from age-matched wild-type controls. To confirm that overexpression of TGF-₁ in this model resulted in elevated TGF-₁ levels in cardiac tissue, heart homogenate prepared from 8-wk-old male mice was subjected to Western blot analysis. When normalized for protein loading, there was an approximately eightfold increase of total TGF-₁-levels in transgenic hearts compared with control hearts (Fig. 1). Previous studies (40) have shown that the cysteine-to-serine substitutions in the transgene result in preferential secretion of active TGF-₁.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Cardiac expression levels of transforming growth factor-1 (TGF-1) in hearts from control mice and Alb/TGF-1 (Cys223,225Ser) transgenic mice. A: representative Western blots for TGF-1 (top) and protein loading of GTPase-activating protein of RAG [(RAS)-GAP, bottom] of heart homogenates from 8-wk-old control (C) and transgenic (T) mice. B: densitometric analysis revealed an almost eightfold increase in cardiac TGF-1 levels in transgenic mice (TGF-) compared with nontransgenic mice (NTG) (* P < 0.01).

Overexpression of TGF-₁ induces cardiac hypertrophy. Hearts of transgenic mice were characterized by significant cardiac hypertrophy as reflected by an increase in absolute heart weight without differences in total body weight, resulting in a significant increase in heart weight-to-body weight ratio (Fig. 2A). Figure 2B shows representative cross sections of hearts from 8-wk-old transgenic (TGF-) and control (NTG) mice, stained with HE (top) and Sirius red (bottom). There was no significant difference in right ventricular wall thickness (0.57 ± 0.05 vs. 0.65 ± 0.11 mm); however, the septum (1.72 ± 0.10 vs. 1.15 ± 0.26 mm; P < 0.05) and left ventricular free wall (1.99 ± 0.43 vs. 1.51 ± 0.33 mm; P < 0.05) were significantly hypertrophied in hearts from transgenic mice. This hypertrophic response was characterized by a marked increase in connective tissue and interstitial fibrosis in the left ventricle, as the fractional areas of connective tissue and cardiac fibroblasts in hearts from transgenic mice were 2.25 ± 0.27 and 0.68 ± 0.09% compared with 1.64 ± 0.42 and 0.55 ± 0.06% in control mice (P < 0.05), respectively, whereas no difference in the fractional area of cardiac myocytes was found (Fig. 3, A-C). The staining of myocardial cross sections with Sirius red and immunohistochemistry revealed a significant increase in myocardial collagen content (fractional area 9.3 ± 4.6 vs. 3.5 ± 1.9%; P < 0.05) in hearts from Alb/TGF-₁ (Cys^223,225Ser) mice (Fig. 2B). In addition to the effects on cardiac interstitial tissue, TGF-₁ overexpression also led to hypertrophy of cardiac myocytes, as indicated by a marked increase in mean cardiomyocyte diameter from 15.6 ± 0.3 µm in control mice to 17.4 ± 0.5 µm in transgenic mice (P < 0.05) (Fig. 3D). Further evaluation of myocardial tissue by electron microscopy showed frequent activation of cardiac fibroblasts in hearts from transgenic mice (Fig. 2C).

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Cardiac hypertrophy in transgenic mice overexpressing TGF-1 (TGF-). A: mean body weight, heart weight, and heart weight-to-body weight ratio in 8-wk-old NTG (n = 16) and TGF- mice (n = 16), * P < 0.01. B: representative cross sections of hearts from NTG and TGF- mice stained with hematoxylin and eosin (H and E) (top), and Sirius red (bottom). C: electron microscopy of myocardial tissue from NTG and TGF- mice. The arrow points to an activated fibroblast showing an activated nucleus and an enlarged endoplasmatic reticulum, indicating collagen synthesis. C, Capillary.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Quantification of morphological alterations in hearts from TGF- mice. Shown are the fractional areas of connective tissue (A), cardiac fibroblasts (B), cardiac myocytes (C), and mean cardiac myocyte diameter (D) in hearts from NTG (n = 10) and TGF- (n = 10) mice. * P < 0.05.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Alterations of atrial natriuretic factor (ANF) expression in TGF- mice. A: reverse transcriptase-polymerase chain reaction (RT-PCR) of ANF and -actin was performed in ventricular tissue from TGF- mice and NTG controls as described in MATERIALS AND METHODS. B: real-time quantitative PCR (Sybr Green) was performed in myocardial tissue from left (LV) and right ventricle (RV) from TGF- mice and NTG controls. The ABI PRISM Sequence Detection System 7700 was used to detect the fluorescent signal. Threshold cycle values were determined using the Sequence Detector 1.7 software (Applied Biosystems). ANF signals were normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and are expressed as fold increase over NTG (means ± SE; n = 4 in each group). * P < 0.05.

Alterations of -adrenergic signaling in TGF-₁ transgenic mice. Recent in vitro studies (42, 43) revealed that TGF-₁ induces hypertrophic responsiveness to -adrenergic stimulation in cardiac myocytes. Therefore, we were interested whether overexpression of TGF-₁ would lead to alterations of -adrenergic signaling in vivo. To this end, myocardial -AR density was determined by competitive binding experiments using [¹²⁵I]ICYP (Fig. 5, A and B). Overexpression of TGF-₁ led to a moderate but statistically significant increase of myocardial -AR density, as indicated by an increase in B_max from 7.3 ± 0.3 fmol/mg (K_d, 36.9 ± 7.2 pmol/l) in control mice to 11.2 ± 1.1 fmol/mg (K_d, 49.3 ± 6.3 pmol/l) in transgenic mice (Fig. 5B) (P < 0.05).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Alterations of myocardial -adrenoceptor density in TGF-1 overexpressing mice. A: representative saturation binding of [125I]-iodocyanopindolol (ICYP) to membranes prepared from whole heart homogenates from 8-wk-old NTG and TGF-1 overexpressing mice (TGF-). Binding is expressed as fmol/mg. Inset: corresponding Scatchard transformation. B/F, function ratio of bound [125I]ICYP and free [125I]ICYP. Squares, measurements from TGF- mice; triangles, measurements from NTG mice. B: means ± SE for the maximal number of binding sites (Bmax) in NTG (n = 5) and TGF- (n = 5) mouse hearts, as determined from Scatchard blots. * P < 0.05.

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View larger version (36K): [in this window] [in a new window]   Fig. 6.   Alterations of -adrenergic signaling in TGF-1 transgenic mice. Shown are Western blot analyses for inhibitory G protein Gi (A), Gs (B), and -adrenoreceptor kinase-1 (ARK-1) (C) in membranes prepared from heart homogenates from 8-wk-old control (C) and transgenic (T) mice. Bottom: controls for protein loading (Ras-GAP). For each protein, quantification by densitometric analysis is shown on the right. * P < 0.05.

Effects of TGF-₁ overexpression on myocardial contractility. To investigate whether the alterations of -adrenergic signaling affect the contractile responsiveness to isoprenaline stimulation, we measured the force of contraction in electrically stimulated isolated atrial trabeculae from transgenic and control mice. Figure 7 shows the cumulative concentration-response-curves for isoprenaline and calcium. In control mice (n = 4), isoprenaline led to a concentration-dependent increase in force of contraction, resulting in a maximal increase of 0.19 ± 0.10 mN at 3 µmol/l. The responsiveness to isoprenaline in hearts from TGF-₁-overexpressing mice was significantly better as the basal force of contraction was increased by 0.43 ± 0.09 mN at 3 µmol/l (n = 12). The resulting EC₅₀ values for isoprenaline in control and transgenic hearts were 0.28 (0.07-1.13) and 0.04 (0.02-0.10) µmol/l, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Contractile reponse to isoprenaline stimulation. Concentration-response curves for the effects of isoprenaline (0.001-3.0 µmol/l) (A) and calcium (1.8-9.0 mmol/l) (B) on force of contraction in the left atria from 8-wk-old NTG (n = 4) and TGF- (n = 12) mice.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study reveals that overexpression of TGF-₁ in transgenic mice induces cardiac hypertrophy, as reflected by a significant increase in absolute heart weight and heart weight-to-body weight ratio. Myocardial hypertrophy in this model was characterized by both interstitial fibrosis and hypertrophy of cardiac myocytes. Furthermore, overexpression of TGF-₁ led to alterations of -adrenergic signaling as the density of myocardial -ARs was increased in transgenic animals, whereas the expression of inhibitory G proteins (G_i) and ARK-1 was decreased. Finally, altered -adrenergic signaling led to enhanced inotropic responsiveness to -adrenergic stimulation.

In humans, myocardial hypertrophy due to hemodynamic overload is characterized by increased deposition of ECM constituents and proliferation of cardiac fibroblasts, frequently accompanied by hypertrophic growth of cardiac myocytes (47). As a consequence of this remodeling process, increased diastolic stiffness and decreased ventricular compliance are likely to contribute to diastolic dysfunction of the heart. Over time, a period of preserved systolic function is followed by the development of progressive contractile dysfunction. TGF-₁ is particularly expressed during the transition from stable hypertrophy to heart failure in experimental models (7) and human heart failure (46) and is thus one of a few markers discriminating between compensated and decompensated cardiac hypertrophy. TGF-₁ may mediate contractile dysfunction either by directly affecting myocardial integrity or by influencing other systems related to cardiac function such as the -adrenergic system.

As one would expect from previous in vitro studies and from correlative observations in humans (8, 10, 14, 19, 20, 27, 35, 37, 49-51), we found that increased TGF-₁ activity in transgenic mice results in the development of myocardial fibrosis and hypertrophy in vivo. These morphological alterations are consistent with two other recent in vivo studies. Heterozygous TGF-₁ (+/)-deficient mice, in which the lack of one TGF-₁ allele resulted in a decrease in TGF-₁ mRNA abundance of ~50%, showed markedly decreased age-related fibrosis, a lower myocardial collagen content as well as greater compliance, a more rapid contraction, and lower myocardial stiffness compared with age-matched controls (9). Thus decreased TGF-₁ activity appeared to ameliorate the morphological and functional alterations of the aging heart. Conversely, targeted overexpression of TGF-₁ (Cys³³Ser) using a transgenic model similar to, but differing from, ours led to a significant hypertrophic response in the atria, yet no overt hypertrophy was found in the ventricles (32). Whereas the reasons for the distinct responsiveness of atrial and ventricular myocardium in this study remained unclear, possible explanations for the distinct ventricular phenotype compared with our results include the use of different mutant forms of TGF-₁ harboring Cys-Ser substitutions at either amino acid residue 33 or 223 and 225, and different approaches to overexpress TGF-₁. Nakajima et al. (32) used the -myosin heavy chain promoter to target the expression to myocardial tissue. Although similar transgene activity was found in atria and ventricles, it is important to note that a targeted genetic modification may potentially alter the normal developmental program, and that TGF-₁ has been shown to downregulate -myosin heavy chain in the ventricular myocardium (8). In the present study, we cannot exclude the possibility that the cardiac alterations may at least in part have occurred secondary to other effects induced by markedly elevated circulating levels of TGF-₁. Finally, given the pleiotropic effects of TGF-₁ on cellular responses, the level of overexpression may be critical in determining the type of response. Nakajima et al. (32) also found cadiomyocyte hypertrophy but no overt fibrosis in ventricular myocardium, and fibroblast DNA synthesis was inhibited in transgenic hearts. In contrast, TGF-₁ overexpression in our model led to ventricular fibrosis including an increase in the fractional area of cardiac fibroblasts. Because TGF-₁ can stimulate or inhibit cell proliferation and the type of response appears to be dose dependent, the level of overexpression may indeed be critical. This idea is further supported by the fact that targeted cardiac expression of a constitutively active TGF-₁ receptor ALK5 inhibited cardiac development and resulted in embryonic lethality (11), whereas overexpression of TGF-₁ led to the phenotypes described above. The distinct phenotypes likely reflect the various degrees to which the TGF- signaling cascade is activated. In our model, myocardial fibrosis was associated with increased collagen type I and III (1.9-fold and 1.7-fold), which correlated with a significant decrease in interstitial collagenase mRNA expression (75%) and activity (91%) (44). In contrast, gelatinase matrix metalloproteinase (MMP-2 and MMP-9) expression and activity were not altered. Furthermore, the expression of endogenous MMP inhibitors [tissue inhibitors of metalloproteinases (TIMP-1)] was 4 (twofold each), and 2 (sixfold) was dramatically increased. Thus the morphological findings in TGF-₁ transgenic mice correlate with significant alterations of collagen degrading proteolytic enzymes and their endogenous inhibitors. Therefore, myocardial fibrosis induced by TGF-₁ appears to be mediated not only by stimulation of matrix protein formation but also by a complex regulation of the MMP-TIMP system, which leads to decreased matrix protein degradation (44). Taken together, the described findings and the well-known in vitro effects of TGF-₁ collectively suggest that TGF-₁ contributes to the pathological events that are responsible for myocardial fibrosis and hypertrophy.

In addition to the hypertrophic cardiac phenotype of TGF-₁ transgenic mice, we found significant alterations of -adrenergic signaling. These include a statistically significant increase in -AR density and a marked downregulation of signaling molecules that act as negative regulators downstream of the -AR such as G_i and ARK-1. As a result, we found increased contractile responsiveness to -adrenergic stimulation in hearts from transgenic mice. This is consistent with the finding that chronic stimulation of engineered heart tissue with TGF-₁ results in increased contractility (W. Zimmermann and T. Eschenhagen; personal communication). It is possible that the TGF-₁-dependent increase of -AR number in our model occurs as a consequence of decreased ARK expression, as -AR desensitization in cardiac hypertrophy was shown to result from increased ARK (12). Because the observed differences in -AR density are rather moderate (although statistically significant) they alone are unlikely to be of functional relevance, particularly compared with other studies in which -adrenergic receptors were overexpressed. However, the combination of decreased G_i and ARK and upregulation of -ARs appeared to be functionally relevant. Several recent studies (21, 24, 33) clearly demonstrated that TGF-₁ directly affects -adrenergic signaling in vitro. Interestingly, TGF-₁ has been shown to induce hypertrophic responsiveness to -adrenergic stimulation in cardiac myocytes (43). Furthermore, a recent study (42) showed that TGF-₁ overexpression promotes the isoprenaline-induced cardiac expression of hypertrophy-associated proteins including ANF and c-fos. When the induction of -AR-mediated hypertrophic growth of cardiac myocytes by TGF-₁ was further characterized, it appeared to depend specifically on induction of ornithine decarboxylase, the rate-limiting enzyme of the polyamine metabolism (42). In the present study, hypertrophic growth of cardiac myocytes correlates with enhanced -adrenergic signaling in vivo. Thus TGF-₁ is able to evoke hypertrophic responsiveness to -adrenergic stimulation in cardiac myocytes by inducing ornithine decarboxylase, and this effect appears to be relevant in vivo.

The obvious question arising from these findings is what the physiological/pathophysiological relevance of enhanced -adrenergic signaling in this context may be. In the infarcted and hypertrophied heart, ECM deposition and fibroblast proliferation in the ventricular wall contribute to an increase in myocardial stiffness and a decrease in ventricular compliance, resulting in diastolic dysfunction, which precedes systolic failure. Thus enhancement of -adrenergic signaling in concert with TGF-₁-induced tissue repair and remodeling may serve as a compensatory mechanism at this stage of the disease in an effort to maintain systolic function. Although increased -adrenergic signaling may lead to functional enhancement and thus appears to have therapeutic potential in heart failure, the detrimental effects of chronic and excessive adrenergic drive have been shown in several experimental studies and in humans (5, 15, 18, 22, 25, 28, 31). In genetically modified mice, overexpression of various members of the -adrenergic signaling pathway, including -ARs, adenylyl cyclase, G_s, or ARK inhibitor affected cardiac morphology and contractile function (15, 18, 22, 25, 28, 31). These studies have also shown that the consequences of enhanced -AR function in the heart are highly dependent on which signaling elements are altered and to what extent. Furthermore, the duration of -adrenergic overdrive appears to be critical. In young animals, enhancement of -AR signaling by overexpression of ₁/₂-ARs, adenylyl cyclase, or G_s, led to enhanced cardiac function (15, 18, 22, 28, 31). Importantly, whereas older transgenic mice moderately overexpressing ₂-ARs revealed only minor morphological abnormalities and preserved contractile function (28, 31), transgenic overexpression of ₁-ARs, G_s, or high levels of ₂-ARs has led to phenotypes of cardiac failure with aging (15, 22, 28). These findings strongly support the concept that chronic and excessive enhancement of -adrenergic signaling is deleterious.

In TGF-₁ transgenic mice, we found that -adrenergic signaling was altered at various levels of the signaling cascade. Although the increase in the number of -adrenergic binding sites was rather moderate, it is likely that the alterations of -AR density, G_i, and ARK-1 act synergistically, and thus lead to enhanced hypertrophic and contractile responsiveness to -adrenergic stimulation. This is in contrast to the above noted transgenic models, in which only one member of the -adrenergic signaling pathway was genetically modified. In human heart failure, however, alterations at various levels of the signaling cascade have been reported (5). Thus the present model may reflect a more physiological situation than the vast overexpression of a single gene encoding only one member of the -adrenergic signaling cascade. The increased contractile responsiveness to -adrenergic stimulation corresponds to the enhanced cardiac function in young transgenic mice overexpressing members of the -adrenergic signaling pathway, which have been shown to develop heart failure with time.

In summary, we report that overexpression of TGF-₁ in transgenic mice leads to cardiac hypertrophy and enhanced -adrenergic signaling, which involves increased -AR density and downregulation of some but not all of the signaling molecules downstream of the receptor. This enhancement of -adrenergic signaling by TGF-₁ may contribute to excessive catecholamine stimulation during the transition from stable hypertrophy to heart failure. Given the phenotype of cardiac hypertrophy in these mice, it is challenging to discriminate between the direct effects of TGF-₁ on myocardial tissue and the ones resulting from induction of the -adrenergic system. Further studies are required to more specifically characterize the functional importance of these findings, and to distinguish between the direct effects of TGF-₁ from the effects occuring secondary to -adrenergic induction. This may be achieved by specific blockade of -adrenergic or TGF- signaling.