Mechanisms of Development, 38 (1992) 85-98 © 1992 Elsevier Scientific Publishers Ireland, Ltd. 0925-4773/92/$05.00
Transforming growth factor-beta 1 in heart development Gary L. Engelmann a,b, Keith D. Boehm c, Maria C. Birchenall-Roberts d and Francis W. Ruscetti d a Department of Medicine, Loyola University of Chicago, Maywood, IL, USA, b Department of Heart & Hypertension, Research Institute of the Cleveland Clinic Foundation, Cleveland, OH, USA, c Department of Reproductive Biology, Case Western Reserve University, Cleveland, OH, USA and a Biological Response Modifiers Program, NC1, Frederick, MD USA (Received 27 February 1992; revision received 8 April 1992; accepted 13 April 1992)
Defined biochemical stimuli regulating neonatal ventricular myocyte (cardiomyocyte) development have not been established. Since cardiomyocytes stop proliferating during the first 3-5 days of age in the rodent, locally generated 'anti-proliferative' a n d / o r differentiation signals can be hypothesized. The transforming growth factor-beta (TGF-~) family of peptides are multifunctional regulators of proliferation and differentiation of many different cell types. We have determined in neonatal and maturing rat hearts that TGF-/31 gene expression occurs in pups of both normotensive (Wistar Kyoto, WKY) and hypertrophy-prone rats (spontaneously hypertensive, SHR). TGF-/3l transcript levels were readily apparent in total ventricular RNA from SHR pups within 1 day of age and elevated in 3-7 day old WKY and SHR hearts when cardiomyocyte proliferation indices are diminished. TGF-~II transcript levels remain at a 'relatively' high level throughout maturation and into adulthood in both strains. Further, TGF-~II transcripts were localized to cardiomyocytes of neonatal rat ventricular tissue sections by in situ hybridization. Immunoreactive TGF-/3 was co-localized to the intracellular compartment of neonatal cardiomyocytes at the light and electron microscopic level. In vitro analysis using primary cultures of fetal and neonatal cardiomyocytes indicated that TGF-/3s inhibit mitogen stimulated DNA synthesis and thymidine incorporation. From these data, we propose that locally generated TGF-~s may act as autocrine a n d / o r paracrine regulators of cardiomyocyte proliferation and differentiation as intrinsic components of a multifaceted biochemical regulatory process governing heart development. Transforming growth factor-beta; Gene expression; Myocyte
Growth and development of the mammalian ventricle during the fetal and neonatal periods is, to a large extent, dependent upon underlying changes occurring in the cardiomyocyte population. A collection of interactive cellular events that incorporate cellular differentiation, migration, replication and enlargement ultimately result in a defined number of ventricular myocytes in the mature animal, that are incapable of further proliferation (Zak, 1984). Although only 25% of the total ventricular cellular population in the ma-
Correspondence to: G.L. Engelmann, Department of Medicine, Rm 0370, Loyola University, Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153, USA.
ture rodent, is composed of cardiomyocytes, they may occupy up to 90% of the total volume of the ventricle (Engelmann et al., 1987; Zak, 1984). Growth of the cardiomyocyte during the fetal/ neonatal periods in rats has been grouped into three general phases: hyperplastic, transitional and hypertrophic (Clubb and Bishop, 1984). Cardiomyocyte hyperplasia occurs primarily in the fetal and early neonatal ( < 5-7 days of age) periods. The transitional 'phase' of cardiomyocyte growth represents a brief 'window' of time wherein hyperplastic growth is negligible or absent and the cardiomyocytes exit from the complete mitotic cycle. This results in incomplete mitosis (karyokinesis without cytokinesis) yielding binuclear/ polyploid ceils a n d / o r polyploid nuclei (Clubb et al., 1987; Engelmann and Gerrity, 1988; Engelmann et al., 1986; Oberpriller and OberpriUer, 1985). The final
86 phase of cardiomyocyte growth, cellular hypertrophy, represents a nearly life-long event whereby additional sarcomeres and intracellular organelles are added to a non-proliferative muscle cell population contained within an expanding ventricular mass of connective tissue and non-myocyte populations (Zak, 1984). Therefore, as with the cells of the brain, the fetal and early neonatal cardiomyocyte growth periods represent the only replicative phases of this long-lived cellular population. Although many potential molecular signals may influence fetal and neonatal cardiomyocyte growth, a particularly attractive signal would be one that stopped cellular replication and facilitated cellular differentiation and maturation. Recent evidence has shown the transforming growth factor-beta (TGF-/3) family as a 'multifunctional' regulator of cellular activity (Roberts and Sporn, 1988; Sporn and Roberts, 1990). The term 'multifunctional' implies that TGF-~ may either stimulate cell proliferation and growth, or inhibit cell proliferation and growth, or have numerous other actions having little or no relationship to either of these two processes (Sporn et al., 1987). TGF-/3 has been implicated in the regulation of both epithelial and mesenchymal (i.e., bone, muscle, blood vessels and blood cells) proliferation and differentiation (Sporn and Roberts, 1990). Further, TGF-fl has been found to exist in several distinct isoforms (Sporn and Roberts, 1990), multiple TGF-/3 genes have been identified (Massagu6, 1990) and TGF-/3 is secreted by virtually all cell types of the body in a biologically inactive form that appears to require proteolytic processing for functional activation (Kanzaki et al., 1990; Lyons et al., 1990; Lyons et al., 1988). TGF-fls bind to a specific family of nearly ubiquitous target cell TGF-/3 receptors (Massagu6, 1990) wherein post-receptor mechanisms of action are, at present, not completely understood, although expression of the type 1 TGF-/3 receptor is strongly associated with the antiproliferative actions of TGF-/3 (Cheifetz et al., 1990; Laiho et al., 1990). The antiproliferative effects of TGF-/3 are well described for epithelial cells; yet mesenchymal cells such as skeletal muscle cells, fibroblasts, hepatocytes and endothelial cells are also sensitive to TGF-/3 inhibition (Sporn and Roberts, 1990). Elevated levels of TGF-/31 mRNA are found in regenerating liver at a time when DNA synthesis begins to diminish, possibly acting as a locally generated regulatory signal involved in the inhibition of further hepatocyte replication (Mead and Fausto, 1989; Strain et al., 1987). There is a paucity of information on the physiological role of TGF-/3 in adult tissues such as brain, heart and kidney, in which there is little mitotic activity yet relatively high levels of TGF-fl RNA and peptide immunoreactivity (Sporn et al., 1986). Cardiomyocytes, which are also of mesenchymal origin, have only recently been studied for the
presence of TGF-/3 peptide/gene transcripts (Casscelles et al., 1990; Flanders et al., 1989; Heine et al., 1988; Qian et al., 1991). Although general conclusions regarding TGF-/31 and heart development can be made from the data presented, we have examined the expression of TGF-/3~ in two rat strains commonly used to evaluate myocyte hypertrophy, the spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rat. When mature, the SHR strain is utilized as a human model of essential hypertension-mediated cardiac hypertrophy and the WKY as its normotensive control. Nevertheless, underlying fetal and neonatal developmental abnormalities detected biochemically or ultrastructurally in the SHR prior to established hypertension have been shown (Clubb et al., 1987; Engelmann et al., 1989; Engelmann and Gerrity, 1988; Engelmann et al., 1986). The relationship of these 'pre-hypertensivc' developmental variations to the eventual pathology associated with longstanding cardiac hypertrophy and eventual heart failure in the SHR can now be evaluated at the molecular level. There is a paucity of information regarding in vitro responsiveness of cardiomyocytes to TGF-/3, particularily TGF-/31 (Schneider and Parker, 1990). Because TGF-fl is known to regulate skeletal myoblast to myotube differentiation, a process wherein multinuclear, non-replicating myotubes are formed (Florini and Magri, 1989), aspects of TGF-fl gene expression during the fetal-to-neonatal cardiomyocyte transition to a terminally differentiated, post-mitotic cell were examined and documented in this report. In addition, direct effects of TGF-fls on DNA synthesis of cultured fetal and neonatal myocytes was evaluated to provide evidence for an in vivo response of this cell type to this growth factor family.
Ventricular expression patterns Initial studies of TGF-fll gene expression in neonatal tissues indicated that ventricular expression was relatively abundant in comparison to neonatal liver (Fig. 1). We detected a 2.4 kilobase (kb) TGF-fll mRNA in the poly (A +) enriched RNA population of both neonatal (14-days of age) rat tissues from both strains as well as human placenta (a positive control tissue) (Derynck et al., 1985). This transcript size has been reported for many tissues and cell lines and the relative abundance of the TGF-/31 mRNA was greater in the neonatal ventricle as compared with that of the neonatal liver. This may reflect the proliferative status of the two organs since neonatal hepatocytes actively prolifer-
1 2 3 4 e
Fig. 1. Northern blot analysis of TGF-/31 transcripts in neonatal rat heart and liver. Poly (A) + enriched RNA ( 1 0 / z g / l a n e ) isolated from multiple (10-15), 14-day-old hearts, livers, or human placenta (3 ~g) were separated under denaturing conditions and hybridized to human TGF-/31 cDNA as described. A 2.4 kb transcript was detected in all samples. Exposure time was 48 h. Lane 1: WKY liver; Lane 2: WKY heart; Lane 3: SHR liver; Lane 4: SHR heart; Lane 5: human placenta.
ate for several weeks, while cardiomyocytes are virtually non-proliferative by this age. Additional studies have been performed with total RNA from multiple age groups utilizing recently developed rodent cDNAs for TGF-/3 t and TGF-fl2 (Fig. 2). Expression of TGF-/3~ was very limited-to-undetectable (not shown) in the fetal myocardium of both strains and was only readily detected after 1-3 days of birth. In the SHR strain, the expression of TGF-/31 occurred earlier (day 1) and at a slightly higher level than in the normotensive control strain (Fig. 2). Ventricular expression of TGF-/31 reached a near adult level within 1-3 weeks in both rat strains and was sustained through 9 weeks of age. The adult myocardium of these two rat strains contains abundant levels of TGF-/31 transcripts, although age-related variations can not be discounted. Transcripts for TGF-/32 were undetectable, at the same ages examined for TGF-/3~ expression, in both strains (not shown).
Therefore, localization of TGF-/3 transcripts to ventricular cardiomyocytes was confirmed by tissue in situ hybridization (Fig. 3). Using neonatal tissue obtained at various ages, evidence of TGF-/31 hybridization was readily apparent and localized predominantly to the cardiomyocytes (Fig. 3A-C). Fig. 3A illustrates the relative level of TGF-/31 hybridization to the cardiomyocytes seen at 1- (both SHR and WKY, not shown) and 3-days (WKY, as shown) of age and the minimal hybridization to endothelial ceils lining the left ventricle. TGF-/31 hybridization to the cardiomyocytes was easily detected using 7-day-old SHR ventricular tissue and was localized primarily over the cardiomyocytes (Fig. 3B). Specificity of the TGF-/3~ hybridization was established by 'cold', probe dilution which reduced (Fig. 3C) and nearly eliminated (Fig. 3D) the hybridization signal at a 10 or 15-fold excess, respectively. Non-specificity of the TGF-/31 hybridization was assessed using chicken RBC and was found to be minimal (Fig. 3E). At 14 days of age, the TGF-/31 hybridization signal was similar in both strains and again localized over the mature cardiomyocytes (Fig. 3F). Our in situ hybridization studies suggest that the cardiomyocyte is the major, but not sole, ventricular cell type primarily responsible for the TGF-/31 mRNA detected in the RNA populations obtained from the entire ventricle and analyzed by Northern blot hybridizations. Further support of the cellular localization of TGF-/3 to the ventricular cardiomyocyte is shown in Fig. 4. Immunoreactive TGF-/3 peptides, as well as TGF-/31 transcripts, were colocalized to the cardiomyocytes using a polyclonal antiserum (LC) specific for intracellu-
Localization of centricular TGF-fl] gene expression The neonatal and mature ventricle contains multiple non-myocyte cell types that comprise 30-50% of the total cellular population of the ventricle and occupy 10-20% of the area of the tissue (Engelmann et al., 1987; Zak, 1984). These different cell types would contribute to the RNA transcripts isolated from the total ventricular homogenate and make ascribing a specific gene transcript to a distinct cell type tenuous.
Fig. 2. Northern blot hybridization analysis of TGF-/31 expression in neonatal rat hearts. Total RNA (30 tzg/lane) was obtained from ventricles of rat fetuses or pups, separated under denaturing conditions, transfered to nylon membranes and hybridized with random prime 32p-labeled cDNA insert to mouse TGF-/3 I. Age of the tissue samples were: in days (D) or weeks (W) after birth. Different strains indicated as: SHR samples indicated by S and WKY samples indicated by W. Ethidium bromide stained nylon filter after RNA transfer indicated equivalency of RNA per lane (data not shown).
Fig. 3. Detection by in situ hybridization of TGF-/3~ transcripts in neonatal rat heart tissue. Ventricular tissue from 3 day W K Y (A), 7 day SHR ( B - D ) and 14 day S H R (F) were processed, sectioned and probed for the presence of TGF-/3 transcripts using h u m a n TGF-/3~ c D N A inserts as described. ( A - C ) The hybridization signal is located primarily over the myocytes (m) with few detectable grains over endothelial cells (arrow). (B) The intense myocyte labeling of the SHR seen at 7-days of age. (C, D) The specificity of the signal by 'cold' probe dilution (1 : 10, 3C and 1 : 15, mD) and near elimination of the 7-day SHR hybridization. (E) The minimal non-specific binding of the probe to cRBC. (F) The hybridization signal over mature 14-day SHR myocytes (m). 3 0 0 × .
Fig. 4. Immunohistochemical detection of TGF-/31 peptides in neonatal rat ventricular myocardium. Ventricular tissue from 1 day SHR (A), 1 day WKY (B), 3 day SHR (C) 1 day WKY (D), 7 day WHY (E) and adult WHY (F) were processed, sectioned and stained for the presence of immunoreactive TGF-/3 using the LC-antiserum of Flanders et al. (1989). Nonspecific staining using normal rabbit serum is shown (D). The general increase in the staining pattern of the cardiomyocytes (m) between 1- (B) and 7- (C) days of age is representative. Lack of staining of the smooth muscle cells of developing muscular artery (C, arrow) and endothelial cells (F, arrow) are indicated. A, B 35 x ; C - F 70 x .
Fig. 5. lmmuno-electron microscopy of subcellutar ventricular myocyte TGF-/3 Z. Ventricular tissue from 18 day fetal ( A - C ) and l-week-old SHR (D F) pups were processed, sectioned and stained for the presence of immunoreactive TGF-/3 using the LC-antiserum of Flanders et al. (1989) at the subcellular level. Low to undetectable immunoreactivity was seen in samples from all ages using either NRS (A, D) or purified lgG. Low, but detectable immunoreactivity was consistently observed using 18-day fetal tissue of either strain, yet predominant staining was in the non-myocyte population or surrounding extracellular matrix (arrow) (B, C). Relatively high levels of immunoreactivity were routinely detected in neonatal (E, F) and adult tissue samples (not shown) with the cardiomyocyte the primary cell type stained (arrow). Subcellular organelles labeled are: nucleus (Nu) and mitochondria (Mt). Bar 0.5 ~ m .
lar TGF-/3s. The relatively low TGF-/31 transcript levels detected at 1 day of age in SHR and WKY ventricular RNA is approximated by the staining intensity detected (Fig. 4A, B). As suggested by the Northern
analysis, intense immunoreactivity was localized to the cardiomyocytes throughout the ventricle after 3 days of age in both strains, particularly the SHR (Fig. 4C). There was minimal staining found in the smooth mus-
91 cle cells of the major arteries of the free wall throughout the developmental period under study (Fig. 4C). Virtually no non-specific staining of any cell type was seen with normal rabbit serum (NRS, Fig. 4D). Ventricular tissue from older animals (7 days of age to adult) of both strains demonstrated similar staining patterns and level of immunoreactivity (Fig. 4E,F). Due to the resolution of paraffin embedded tissue, immunohistochemical localization of TGF-/3 to endothelial cells or other non-myocyte cell types of the neonatal ventricle cannot be discounted at this time, yet appears to be minimal and supports the in situ hybridization data that also indicated low hybridization to endothelial-like cells. The general staining pattern for TGF-/3 using this antiserum persisted in older ventricular tissue (not shown). Subcellular localization of TGF-/31 to the intracellular compartment of the fetal and neonatal myocyte was determined using immuno-electron microscopy (Fig. 5). The staining pattern in fetal tissue from both strains was minimal, yet detectable (Fig. 5C,D). The cell types containing immunoreactive material included the fetal myocyte, yet the most prominent cell type labeled was the adjacent non-myocyte (arrow), which resembled an endothelial cell. The localization to the endothelial cell types is in agreement with those of Lenhart and Akhurst (1989) which indicated by in situ hybridization that the endothelial and epicardial cells of the fetal ventricle were the primary cell types expressing TGF-/3 I. In contrast, the TGF-/3~ staining pattern detected in neonatal (1 week of age) and older tissue samples (not shown) was very high (Fig. 5E,F). Immunoreactivity was found predominantly in the cardiomyocyte population at multiple subcellular sites associated with biosynthesis (perinuclear, Fig. 5F) and eventual secretion a n / o r release (arrow, 5E). Immunoreactivity was occasionally found in and around cellular mitochondria (Fig. 5E). This latter observation is in agreement with those reported by Heine et al. using adult heart and liver mitochondrial isolates (Heine et al., 1991).
In vitro analysis of TGF-[3 effects on neonatal myocyte DNA synthesis To ascribe a specific, direct effect of TGF-/3~ to the regulation of cardiomyocyte development in vivo, we have utilized primary cultures of neonatal ventricular myocytes as an in vitro model. Although neonatal myocytes are minimally proliferative, they remain capable of synthesizing DNA under control conditions as well as after stimulation by purified molecules like the insulin-like growth factor family of peptides. Because > 90% of the cultured cells express myosin heavy chain or desmin antigenicity (not shown), the effects of exogenous growth factors on myocyte growth and proliferation is assumed, although not without minimal non-
IGF-I I G F-II
Fig. 6. Effects of TGF-/31 on neonatal cardiomyocyte D N A synthesis. Neonatal cardiomyocytes were isolated and cultured as described and thymidine incorporation into acid precipitable material in response to recombinant h u m a n insulin-like growth factor-I or insulinlike growth factor-ll (20 n g / m l ) stimulation in the presence of increasing concentrations of TGF-/3 E was determined during a 4 h incubation. Results represent the data from multiple experiments (5-7) and are presented as the mean_+SEM of the percentage change relative to untreated control cultures. IGF stimulation of control cultures resulted in a 2 0 - 4 0 % increase in thymidine incorporation. Basal levels of thymidine incorporation are 5900+_500 d p m / / ~ g DNA, approximately 8 - 1 0 fold less than fetal cardiomyocyte cultures over a 48 h period as shown in Fig. 7.
myocyte responsiveness. To examine the effects of TGF-fll on cardiomyocyte DNA synthesis, primary neonatal cardiomyocyte cultures were treated with insulin-like growth factor-I or IGF-II (20 ng/ml) in the presence or absence of increasing amounts of TGF-fll (Fig. 6). Both autoradiographic (not shown) and biochemical indices of IGF stimulated cardiomyocyte DNA synthetic properties were inhibited by TGF-/31. The sensitivity of the cultures suggests that the maximal effects are attained at or near 500 pg/ml. When neonatal cardiomyocytes were treated with TGF-/31 alone, no effect on DNA or protein syntheses was seen (not shown). Because fetal cardiomyocytes are 4-7 times more active in thymidine incorporation and cellular proliferation than neonatal myocyte cultures, fetal primary myocyte cultures were used to examine the direct effects TGF-/3s may have on this cell population. When fetal cardiomyocytes were treated with either TGF-fll or TGF-fl2 (1 ng/ml), the stimulation of DNA synthesis by LR-IGF-I (50 p.g/ml, not shown) or basal medium insulin (3 /.~g/ml) under control conditions were significantly (P < 0.01) blunted (Fig. 7). LR-IGF-I was used in these studies since recent data indicates that these modified peptides have increased cell culture activity due to marginal binding by secreted IGF binding proteins (Cascieri et al., 1988). There was no direct effect of exogenous TGF-/3~ or /32 on cellular viability as determined morphologically, yet thymidine incorporation (indicated) and effects on expression of proliferation-associated genes and contractile proteins gene families in both fetal and neonatal cultures was modulated (data not shown). Similar results have been found with fetal myocyte cultures using acidic or basic
Fig. 7. Effect of TGF-/31 and TGF-/32 on fetal cardiomyocyteDNA synthesis. Fetal (day 16 gestation) cardiomyocyteswere isolated and cultured as described. Thymidine incorporation into acid precipitable material under control conditions and in response to Long R 3 IGF-I (LR-IGF-I, 50 ng/ml), TGF-/31 (1 ng/ml), and TGF-/32 (1 ng/ml) alter 24 and 48 h of incubation was determined. The results (mean_+SEM) from 3-5 sets of paired experiments are shown. Concomitant incubation of cardiomyocyteswith LR-IGF-I and TGF/3's were similar to that seen with TGF-/3's alone. Basal levels of thymidine incorporation over the 24-48 h period of culture were 48,595_+6,622 dpm//xg DNA. Significant differences (P < 0.01) are evident by both TGF-/3~and TGF-/32treatment.
fibroblast growth factors as mitogenic agents in the presence or absence of TGF-/3's (data not shown).
Several reports of growth factors, growth factor-related proto-oncogenes and proto-oncogene R N A transcripts during select periods of cardiac muscle development have been published (Schneider and Parker, 1990). Expression of TGF-/3's, principally TGF-/3 l, have been determined in the developing heart of the fetus (Akhurst et al., 1990; Lenhart and Akhurst, 1988; Potts and Runyan, 1989; Wilcox and Derynck, 1988), neonate (Thompson et al., 1989b) or the adult rat (Casscelles et al., 1990; Thompson et al., 1989a,b). It has been suggested that TGF-/3 t may play some role in the response of the adult heart to injury after acute myocardial infarction or ischemia (Casscelles et al., 1990; Lefer et al., 1990; Thompson et al., 1989a). In contrast, the role of TGF-/3s, particularly TGF-/33, in early embryonic chick heart development may be related to mesenchymal induction and heart morphogenesis (Potts et al., 1991; Potts and Runyan, 1989). In addition, the localization of immunoreactive TGF-/3 peptide in the fetal heart has been shown to be primarily to the endocardial cushion region, with little TGF-/3 detected in the ventricular myocyte until after birth. Because of the pronounced effects TGF-/3 has on cellular proliferation and differentiation and the prominent expression of TGF-/3 in the myocardium, we have examined TGF-/3
expression during neonatal heart development. Because this 'transition' period of heart development is so critical in establishing myocyte cell number, the potential role TGF-/3 may play in modulation of cardiomyocyte growth and maturation during this specific time period was evaluated. Numerous studies of fetal rodent heart development have indicated that the ventricular myocyte has limited to no expression of TGF-/3 t (Akhurst et al., 1990; Lenhart and Akhurst, 1988; Wilcox and Derynck, 1988; Schmid et al., 1991). TGF-/3~ transcripts are primarily localized to the endocardial cushion region of the early fetal mouse heart as well as the endocardial cells lining the ventricle and pericardial cells surrounding the growing myocardium. Limited embryonic and fetal mouse heart (days 9.5-16.5 gestation) expression of TGF-/31 and -/33 has been reproduced, yet TGF-/32 expression was found primarily in regions of cardiac septation and valve formation (Millan et al., 1991). Recently, Pelton et al. have shown that the fetal mouse myocardium is avidly stained using a TGF-/33 antisera (Pelton et al., 1991). Jones et al. (1991) have localized by in situ hybridization Vgr-1 transcripts to the atrioventricular cushions and truncus arteriosus of the developing mouse heart, as was previously shown for TGF-/3~. Therefore, in embryonic and fetal rodent myocardium, the general expression patterns appear to suggest that limited and focal sites of TGF-/3t biosynthesis can be anticipated. In avian heart development, Choy et al. have also localized TGF-/31 peptide immunoreactivity to the endocardial cells of the developing chick heart, with little or no myocyte staining detected as the embryonic myocardium develops (Choy et al., 1990, 1991). A recent report by Jakowlew et al. (1991) have examined the expression of TGF-/3 l, -/32,-/33 and "/34 during chick heart embryogenesis and have also shown that /31 has limited expression in heart tissue and cultured cardiomyocytes. In select neonatal stages of heart development, Thompson et al. (1989b) have reported both TGF-/3 immunohistochemical cardiomyocyte localization and TGF-/3~ transcripts in neonatal mouse ventricular RNA. Lyons et al. (1989) have also reported that Vgr-1 transcripts (a member of the TGF-/3 family) increase in the mouse heart from fetal to 3-day-old neonates. Our data are in general agreement with the aforementioned studies of fetal and neonatal expression of TGF-/3 during heart development. We have not examined embryonic (days 8-12 of gestation) rat heart development for relative TGF-/3 expression levels, yet we feel that a coordinate, limited, and localized expression pattern would be expected. We have extended these previous studies by focusing on multiple time points during a critical, yet brief period of heart development, the term fetal-early neonatal 'transition' period. Since this pe-
93 riod of time represents 'transition' from proliferative cardiomyocyte to permanently postmitotic cardiomyocyte, this is a unique window of development wherein regulatory stimuli appear to be necessary to specifically, and selectively terminate the expansion of the myocyte population. Although a slightly accelerated level of expression of TGF/3's was noted" in the SHR strain, pronounced variations in the relative level of ventricular expression of TGF-/3s were not seen in the two strains analyzed. When mature, the SHR strain is utilized as a human model of essential hypertension-mediated cardiac hypertrophy and the WKY as its normotensive control. Underlying fetal and neonatal developmental abnormalities detected biochemically or ultrastructurally in the SHR have been shown (Clubb et al., 1987; Engelmann et al., 1989; Engelmann and Gerrity, 1988; Engelmann et al., 1986). We have provided evidence in support of a possible cardiomyocyte proliferative deficiency during neonatal development in the SHR (Engelmann et al., 1989; Engelmann and Gerrity, 1988; Engelmann et aI., 1986). Our present results show TGF-/31 transcript levels are modestly yet prematurely elevated, relative to the WKY, shortly after birth. This correlates with the developmental period wherein other biochemical indices of ventricular growth in the SHR (i.e., DNA synthesis, thymidine incorporation) are decreased (Engelmann and Gerrity, 1988). The relative TGF-/3 transcript abundance, as well as biological activity, in the 1- to 7-day-old SHR may be associated with the rapid decrease in mitotic activity of the newborn ventricular cardiomyocytes (Clubb et al., 1987; Engelmann and Gerrity, 1988). Further, within the ages studied, the relative level of TGF-/31 mRNA is near maximal at 7-14 days of age in ventricles of both strains, a time when normal cardiomyocyte proliferation ceases (Clubb et al., 1987; Engelmann and Gerrity, 1988; Rakusan, 1984). Adult ventricular levels of TGF/31 remain high in both strains. Therefore, TGF-/31 produced from transcripts present in the developing myocyte may be an important biochemical signal regulating cardiomyocyte proliferation in the fetal and neonatal rat ventricle. Premature elevated expression of TGF-/31 in the developing SHR ventricle may be responsible, at least in part, for the cardiomyocyte proliferative deficiency proposed for this strain (Engelmann and Gerrity, 1988). Because immunohistochemical localization of TGF-/3 peptides and molecular transcripts for TGF-/31 are still evident in the adult heart, this suggests that the maintenance of the non-proliferative status of the cardiomyocyte may be partially maintained by locally generated TGF-/3 peptide(s). We demonstrate that TGF-/31 gene transcripts are present in relatively high abundance in the neonatal rat heart and their level of expression varies during the first 7-14 days of age in the two strains examined. The
level of expression of TGF-/31 is negligible to non-detectable in the fetal myocardium and increases with myocardial maturation. In addition, we have shown that TGF-/31 transcripts are found predominantly in the cardiomyocyte population by in situ hybridization. Immunohistochemistry and immuno-electron microscopy studies have co-localized TGF-/3~ reactivity to the neonatal and adult ventricular myocyte population. Therefore, neonatal heart growth and myocyte maturation may be modulated by autocrine or paracrine mechanisms of action by endogenous, myocyte generated stimuli such as the TGF-/3s. Nevertheless, a definitive role for TGF-/3s in the developing heart remains speculative. To better evaluate this hypothesis, direct effects of TGF-/3s on neonatal and fetal myocyte cultures were examined. In vivo studies of the effect TGF-/3s may have on DNA synthetic responses of the ventricular myocyte have not been performed. Nevertheless, the most common effect of TGF-/3 on cell growth is to potentiate or suppress the growth stimulatory effects of other growth factors (Rizzino, 1988; Sporn and Roberts, 1990; Sporn et al., 1987). Our data show that acute treatment with TGF-/31 has no direct effect on neonatal myocyte DNA synthesis by itself, yet is capable of near complete inhibition of the 20-40% increase in DNA synthetic response of the myocytes to IGF stimulation (Engelmann et al., 1989). Although neonatal cardiomyocytes are basically non-proliferative, they retain the ability to synthesize DNA which may result in the formation of binuclear a n d / o r polyploid nuclei within a relatively static muscle cell population. The results in both cultures suggest that the basal level of DNA synthesis may represent an indirect insulin via IGF-I receptor-mediated response that is insensitive to TGF-/3 inhibition while the acute IGF-/3 -mediated effects are TGF-/3 sensitive. As recently reviewed (Florini and Ewton, 1988), TGF-/3 may regulate post-receptor growth factor-mediated stimuli that manifest after a critical early 'branch point' is traversed. This suggests that the acute (12-14 h) IGF-I-mediated response (as assessed by DNA synthesis) of the neonatal cardiomyocytes is within this TGF-/3-mediated sensitivity time frame. A response of the cardiomyocytes to TGF-/3 inhibition of IGF-I stimulated DNA synthesis is not without precedent. TGF-/3 is a biphasic mitogen in primary cultures of osteoblast-enriched cultures (Centrella et al., 1988) blocks the proliferative effects of IGF-I on embryonic fibroblasts (Hill et al., 1986), yet by itself is without effect on total myocyte protein or RNA content (Parker et al., 1990b). Utilizing fetal cardiomyocytes in culture as a more proliferative population of cells, the effects of TGF-/3 l and TGF-/32 on stimulated and basal DNA synthesis was more marked. Since commercial TGF-/33 is not available, we utilized TGF-fll and TGF-/32 peptides
94 since they are both reported to function through similar receptor interactions and post-receptor effects on cell proliferation, yet have 10-20 fold differences in affinity constants for the Type I and Type II TGF-/3 receptors (Massagu6, 1990). These results suggest that embryonic and fetal TGF-/3 effects on morphogenesis and induction would require the restrictive, highly localized expression pattern determined by in situ hybridization analysis since ventricular expression and localized activation would potentially restrict the proliferative potential of a cell type that is already severely limited. In contrast, the increased expression of TGF-/3s seen shortly after birth coincides appropriately with an endogenous regulator of cardiomyocyte proliferation and posssibly differentiation. Analysis of the role TGF-/3 may play in the regulation of cell proliferation and development has illustrated that skeletal muscle myogenic differentiation is particularly sensitive to TGF-/3-mediated modulation (Sporn and Roberts, 1990). In contrast to skeletal muscle, TGF-/3 induces differentiation of a variety of other cell types (epithelial, endothelial, chondrocytes) and these effects are modulated by the preexisting cell phenotype and state of differentiation (Florini and Magri, 1989; Rizzino, 1988). TGF-/3 inhibition of skeletal muscle cell differentiation has been well characterized, particularly when added to rapidly proliferating myoblasts. The expression of differentiated functions coincides closely with the loss of the ability to proliferate. Although skeletal muscle cell lines have provided convincing evidence that TGF-/3 can influence myogenesis, formal proof that TGF-/3 regulates cell differentiation in vivo is lacking (Florini and Magri, 1989; Rizzino, 1988). In the absence of a defined cardiomyocyte cell line, primary cell cultures represent the only viable alternative method of analysis of TGF-/3-mediated cardiac-specific regulation of growth and development. Because the cardiomyocyte in the late fetus/ early neonate is a highly differentiated, specialized, nearly postmitotic cell type, extrapolation to the results of muscle and non-muscle cell lines is tenuous at best. Nevertheless, these data provide compelling points of comparison and logical experimental mimicry. Relevant to the current studies, Parker et al. (1990a,b) have presented data that TGF-/3 may modulate cardiomyocyte myosin and actin isozyme protein synthesis, yet they showed no direct effect on myocyte DNA synthesis. Majack (1987) has suggested that TGF-/3 may play an important role in vascular smooth muscle cells (VSM) proliferation and organization during development and these effects are modulated in vivo by cell density. Similar cell density studies remain to be performed with the cardiomyocytes. Owens et al. (1988) have reported that VSM respond to chronic exposure to exogenous TGF-/3 L by an extended cell cycle, cellular hypertrophy and nuclear polyploidy. Similar long-
term responses of the ventricular myocyte to locally generated TGF-/3s may also occur since these cellular characteristics are found in vivo. We have recently shown that IGF gene expression is transiently elevated in 1-3 day-old SHR ventricular tissue relative to the WKY (Engelmann et al., 1989). The expression, synthesis and secretion of IGFs may also function during heart development as locally generated proliferation a n d / o r differentiation stimuli of the cardiomyocytes as they progress toward the postmitotic state in the neonate. Therefore, the coordinate expression of the IGFs and TGF-/3s during the 'transition' period of cardiomyocyte development may represent an intrinsic mechanism of ventricular growth regulation mediated by defined biochemical signals. Although we have used active TGF-/3 peptides in our in vitro studies, it remains to be demonstrated that the TGF-/3's that may be produced in vivo by the developing cardiomyocytes are biologically active or latent. Immunohistochemical localization of TGF-/3 to the cardiomyocytes strongly suggests that the TGF-/3 transcripts are translated into immunoreactive proteins that are contained within the cell. Nevertheless, the antiserum used does not differentiate between active or inactive TGF-/3. In addition, Tsuji et al. (1990) have recently cloned the large subunit of the TGF-/3 masking protein and have shown its expression to parallel TGF-/31 expression in several tissues, including the adult heart. Because activation of the latent form of TGF-/3 represents a critical component of the regulatory mechanism of TGF-/3s action, further studies are underway to assess TGF-/3 latency in heart development. As demonstrated by Ewton and Florini (1990) on the role TGF-/3 binding and action may play in skeletal myoblast to myotube transition, a possible change in TGF-/3 receptor subtypes a n d / o r affinity may also modulate cardiomyocyte responsiveness to locally generated TGF-/3s. Further studies of ventricular TGF-/3 receptor subtype variations are underway to confirm this hypothesis.
Materials and Methods
Neonatal animals were obtained from spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats bred in our AAALAC approved facility or obtained from Harlan Sprague-Dawley (Indianapolis, IN). Nucleic acid extraction and isolation materials were from Fluka Chemical Company, Ronkonkoma, NY (guanidine • HC1; guanidine thiocyanate); BRL, Gaithersburg, MD (cesium chloride; oligo-(dT) cellulose; vanadyl ribonucleoside complex, Elu-tip columns, nick translation kit) or Sigma Chemical Co., St. Louis, MO (Antifoam A; fl-mercaptoethanol; Sigma-cote, sodium dodecyl sulfate, SDS). [32p]dCTP, [35S]dCTP and [3H]deoxy
95 nucleotide triphosphates (dNTPs = dCTP, dGTP, dATP, TTP) were obtained from either Amersham or ICN. Ilford K.5D liquid nuclear track emulsion was from Polysciences (Warington, PA). Human recombinant IGF-1 (IGF-1), media grade Long R 3 IGF-I (LRIGF-I) and IGF-II were obtained from Amgen (Thousand Oaks; CA), GroPep (Adelaide, Australia), or a gift from Eli Lilly (Dr. Michele C. Smith, Indianapolis, IN), respectively. All peptides, in lyophilized form, were resuspended in 0.01 M HC1 containing 1 mg/ml bovine serum albumin (BSA), aliquoted and stored at -20°C until used. Porcine transforming growth factor-beta 1 (TGF-/31) was obtained from R & D Systems (Minneapolis, MN) resuspended in 0.1 M acetic acid containing 1.0 mg/ml BSA, aliquoted, and stored at -20°C until used. Recombinant human TGF-/3~ and TGF-/32 were obtained from Dr. Larry Ellingsworth (Celtrix Laboratories, Palo Alto, CA). PC-1 tissue culture medium was obtained from the Ventrex Corporation (Portland, ME); while a 1:1 mix of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 (DMEF~2) was from Gibco (Gaithersburg, MD).
RNA isolation and analysis Ventricles were frozen in liquid nitrogen (N2), stored at -75°C, pulverized into a powder under N 2, and homogenized in guanidine thiocyanate (Engelmann et al., 1989, 1991). Total cellular RNA or Poly-A + enriched RNA was obtained and used directly for Northern blot hybridization analysis of TGF-/3 transcripts using electrophoretically separated RNA transferred by capillary blotting to nylon filters (Duralon, Stratagene), UV crosslinked, and probed with 3zp-labeled cDNA inserts from a human (Derynck et al., 1985) or mouse (Miller et al., 1989a) TGF-/3~ probe, or a mouse TGF-/32 probe (Miller et al., 1989b). The cDNA inserts were excised from their plasmids, purified by separation in a low melt agarose gel, and random prime labeled with [32p]dCTP to a specific activity of 1-5 x 108 cpm//zg DNA. Hybridization conditions were 50% formamide, 6 x SSC, 0.1 M NaPO 4, 0.1% sodium pyrophosphate, 0.1% SDS, 1 x Denhart's solution, 2% dextran sulfate, and 100/xg/ml sonicated salmon sperm DNA at 42°C for 16-20 h. Nylon filters were washed at room temperature in 0.2 x SSC and then in 0.1 x SSC containing 0.1% SDS at 55-60°C. Each washing procedure was for 30 min and was repeated three times. (20 x SSC: 3 M NaC1 and 0.3 M sodium citrate). Northern blots were de-hybridized and re-probed with 24-mer oligonucleotide (5' A-C-G-G-T-A-T-C-T-G-AT-C-G-T-C-T-T-C-G-A-A-C-C 3') specific to 18s rRNA (Chan et al., 1984) to assess the RNA loading and transfer equivalency between samples (data not shown).
In situ hybridization analysis Freshly isolated ventricular tissue was fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) containing 5 mM MgC12 overnight (Engelmann et al., 1989; Wang et al., 1988). Tissue samples were dehydrated through 95% ethanol, infiltrated at 4°C and embedded in LKB Historesin under vacuum at room temperature (RT) overnight. Sections were cut at 1-2 /zm, spread on water containing 10% acetone, placed on poly-L-lysine coated glass microscope slides and air dried. All samples were treated with 0.25% acetic anhydride in 0.1 M tri-ethanolamine (pH 8.0) for 10 min at R.T., washed in 2 x SSC, dehydrated through 100% ethanol and air dried. Hybridizations were done as previously described (Engelmann et al., 1989), under the following conditions: 50% formamide, 300 mM NaC1, 1 ~ g / m l yeast tRNA, 10 mM Tris (pH 7.5), 1 mM EDTA, and 2% dextran sulfate at 50°C in a 6 x SSC-humidified chamber using 2-5 ng/slide of dCTP (S.A. of 1 x 107 cpm//xg DNA) or quadruple-[3H]dNTP (spec. act. of 1-2 x 107 cpm//zg DNA) labeled TGF-/3j probe. Probe storage, hybridizations and washes of 35S-labeled sections were done in the presence of 10 mM DTT (Dithiothreitol). The probes were added in a total volume of 10-20 /zl to each sample, covered with a siliconized glass coverslip, and incubated overnight. As a control for hybridization specificity, 'cold' probe dilution studies were performed by adding excess amounts of unlabeled TGF-/31 cDNA inserts (50-75 ng) to samples containing 2-5 ng of radiolabeled cDNA inserts prior to denaturation and addition to the tissue sections. Slides were washed with 2 x SSC to remove the coverslips, then in 0.1 x SSC with 0.1% SDS at 60-65°C for 30 min three times, dehydrated with ethanol, air dried, coated with Ilford K.5D emulsion prewarmed to 42°C for 1 h, exposed for 7 or 14 days (35S or 3H, respectively) at -20°C followed by routine autoradiographic grain development with Kodak D-19. Samples were counterstained with toluidine blue for adequate nuclear detail with minimal cytoplasmic staining. Clarification of potential non-specific binding of TGF-/31 cDNA to any cellular target was determined by ribonuclease A (RNase) pretreatment (20 mg/ml RNase in PBS at 37°C for 1 h, rinsed in PBS twice, dehydrated, and air dried prior to hybridization) with subsequent reduction of autoradiographic signal to indicate the specificity of the hybridization reactions to cellular RNAs (data not shown).
Isolation and culture of neonatal cardiomyocytes All litters were used at 2 days of age, irrespective of sex. Isolation and culture procedures have been described (Engelmann et al., 1990). In brief, repeated
96 (5-7) ventricular collagenase digests were used to obtain isolated cells (both non-myocyte and myocyte), the cells were maintained in isolation buffer containing 0.1% serum on ice prior to culture, all released cells were isolated by centrifugation (100 x g, 4 min), and the 'enriched' myocyte population ( < 5% non-myocytes as determined by PAS or desmin staining) plated under serum-free conditions on collagen coated glass coverslips or multiwell culture plates using Ventrex ~ PC-1 medium at a concentration of 0.8 x 106/ml. The most common contaminating non-myocyte cell type was endothelial-like in appearance. Fresh medium was added 12-14 h prior to the addition of the IGFs (both MSA and IGF-I, 20 ng/ml) wherein most of the PC-1 contributed insulin would have been degraded. Spontaneously contracting cultures were maintained under these serum-free conditions for 36-48 h prior to analysis of protein and DNA syntheses. Radioactive labeling of cardiomyocyte DNA synthesis used [3H] thymidine (TdR) (spec. act. 6.7 Ci/mmol) at 2 /zCi/ml for 4 h after a 12-14 h pretreatment _+20 ng I G F/ m l . DNA synthesis was determined by incorporation of TdR into cultured cells as previously described (Engelmann and Gerrity, 1988) and quantitated as dpm//zg DNA (performed in triplicate). Autoradiographic analysis of TdR incorporation by the cultured cells was as described to verify that the major cell type labeled was myocyte-like in its morphology (not shown) (Engelmann and Gerrity, 1988; Engelmann et al., 1989).
Tissue immunohistochemistry Ventricular tissue from rat pups of various ages were removed, after decapitation into liquid nitrogen, rinsed in HBSS and fixed overnight in 10% neutral buffered formalin at 4°C. Tissues were dehydrated, embedded in paraffin, sectioned at 6/zm and mounted on glass slides. A detailed protocol of the pretreatment of the sections and complete characterization of the immunohistochemical localization of intracellular TGF-/3 using the LC (1-30-1)S antiserum was described by Flanders et al. (1989). In brief, tissue sections were pre-treated with hyaluronidase for 30 min at 37°C, rinsed with TBS (0.01 M Tris-HC1, pH 7.4; 0.85% NaC1), pre-treated with excess normal serum, and incubated with primary antiserum (LC 1-30-1 S) or normal rabbit serum (negative control) overnight at 4°C. The antiserum was washed off with TBS + 0.1% BSA, incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) and avidin-biotin complex detection system (peroxidase) (ABC) for 1 h at RT for each reaction. The immunocomplex was localized by incubation with 3-amino-9-ethylcarbazole (AEC) dissolved in dimethylformamide and 0.02 M acetate buffer (pH 5.2) for 20 min at RT. Slides were washed
in TBS for 5 min, washed in water, counterstained with hematoxylin for 2 min, covered with Biomeda Crystal mount (Fisher Scientific), and hardened at 80°C for 20 min. Images were taken with an Olympus microscope using Kodak color print, negative film.
Electron microscopy and immunogold localization Ventricular tissue was obtained from fetal (day 1418 Gestation) and neonatal (day 1-21) pups. Tissue samples (< 1 mm square) were immersion fixed in 4% paraformaldehyde containing 0.5% glutaraldehyde for 1 h at 4°C. Fixed specimens were then processed and embedded in either LR White or Lowacryl embedding media according to manufacturers directions. The LR White embedded materials were polymerized for 48-72 h under UV light at 4°C. All samples were 'thin'-sectioned to gold interference thickness, mounted upon formvar-coated zinc 300 mesh grids for subsequent processing. Sections were blocked for non-specific staining by floating the sections on a drop of 10% goat serum for 30 rain at room temperature. Sections were then incubated overnight at 4°C with the primary antisera (1:50-100), non-immune normal rabbit serum (NRS) (1:25-50) or the IgG fraction of non-immune serum in TBS containing fish gelatin (1%). The following day, sections were washed 5-7 times with TBS containing 0.1% Triton X-100 and then twice with TBS (pH 8.2) alone. The sections were then incubated for 2 h at room temperature with a 1:50 dilution of goat anti-rabbit IgG that was labeled with 30 nm colloidal gold particles. Sections were then washed 5-7 times for 5 min each with TBS containing 0.1% Triton X-100, 15 min with MilliQue water, and then counter stained with uranyl acetate (1 min) and lead citrate (30 s). Sections were air dried under vacuum, carbon coated, and examined with a Hitachi H-600 electron microscope at 75 kV.
Acknowledgements Authors thank Dr. Rik Derynck for access to the human TGF-/31 probe, Dr. Hal Moses for access to the mouse TGF-/3t_ 3 probes, and Drs. Flanders and Roberts for access to the LC anti-TGF-/3 antiserum. The technical assistance of J. Yun, M. Peters and Y. Peng is gratefully appreciated. Supported in part by: Kidney Foundation of Ohio (KDB) and NIH HL 42218 and Diabetes Assoc. Greater Cleveland (GLE).
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