Diabetes mellitus is a complex metabolic disease in which insulin secretion or sensitivity of peripheral tissues to insulin is not sufficient to maintain glucose control. Congenital malformations are at least three times more common in the offspring of diabetics and are the leading cause of perinatal mortality in this group of infants (Kucera, 1971). Cardiovascular defects are among the most common and lethal malformations in diabetic offspring (Aberg et al., 2001). The severity of maternal hyperglycemia during early gestation is directly correlated with the occurrence and severity of congenital malformations (Miller et al., 1981). The critical exposure period for diabetes-induced birth defects is thought to be during organogenesis and before the seventh week of human gestation (Mills et al., 1979), which corresponds approximately to embryonic days (E) 8.5 to 12.5 in the mouse. However, the etiology and pathogenesis of diabetes-induced congenital heart defects have yet to be elucidated.
Animal models have been used to examine the role of hyperglycemia in diabetes-induced congenital defects. Hyperglycemia due to chemically induced diabetes in pregnant rats produces offspring with brain and heart abnormalities similar to those seen in humans (Eriksson, 1984). The incidence of these defects is normalized by insulin treatment (Horii et al., 1966), supporting the role of hyperglycemia in their pathogenesis. In vitro rodent models, using culture medium supplemented with excess glucose, produce concentration- and time-dependent increases in embryonic defects (Cockroft and Coppola, 1977; Sadler, 1980; Garnham et al., 1993). Approximately 50% of rat embryos are malformed by in vitro exposure to 600 mg/dl glucose for 2 hr or longer during organogenesis (Reece et al., 1996).
Diabetes mellitus is characterized by increased basement membrane thickness and the deposition of extracellular matrix (ECM) proteins in many adult tissues (Chen et al., 2000). This effect occurs early in diabetes and is directly correlated with severity of the disease (Mauer et al., 1981). The ECM is important in maintaining tissue architecture and contributing to a variety of developmental processes and is composed mainly of collagens, proteoglycans, and glycoproteins. In response to diabetes or hyperglycemia, ECM proteins, especially fibronectin (FN) and collagen, accumulate in the adult kidney, skin, heart, and blood vessels (Brownlee and Spiro, 1979; Mauer et al., 1981; Falk et al., 1983; Osterby, 1983; Spiro et al., 1995), suggesting a generalized abnormality in ECM metabolism associated with diabetes (Nahman et al., 1992). Hyperglycemia has emerged as the factor most responsible for complications of chronic diabetes (DCCT Research Group, 1993; King and Brownlee, 1996). Diabetes-induced ECM accumulation is best characterized in the kidney, where FN is increased in glomerular basement membranes and mesangium (Mauer et al., 1981). The adult diabetic heart demonstrates myocardial hypertrophy, perivascular fibrosis, and thickened basement membranes (Shehadeh and Regan, 1995; Chen et al., 2000). The consequences of hyperglycemia on the developing embryo are poorly understood, and the embryonic heart has not been studied previously for ECM changes resulting from hyperglycemia or maternal diabetes.
Transforming growth factor beta (TGFβ) is a 25-kDa dimeric protein that influences cell growth and differentiation and exists as three isoforms that change expression patterns as development progresses. TGFβ acts through cell-surface receptors (serine/threonine kinases) that exist in at least three forms, TβR-I, -II, and -III. The three mammalian TGFβ isoforms interact with different affinities to the TGFβ receptors. It is reported that TGFβ-responsive cells express TβR-I and -II, whereas TGFβ-resistant cells lack one or both receptors (Mummery, 2001). Both TβR-I and -II receptors are detected in myocytes of embryonic mouse heart (Mariano et al., 1998). Ligand binding causes TβR-II to phosphorylate and activate TβR-I, which phosphorylates Smad proteins that move to the nucleus and alter gene transcription (Massague, 1998).
TGFβ1 likely mediates the increased production and decreased degradation of ECM proteins, especially FN and collagen, in many adult tissues. TGFβ1 promotes the characteristic lesions of diabetic nephropathy, including renal cell hypertrophy and ECM accumulation, and its up-regulation has been demonstrated recently in the kidneys of human diabetics (Chen et al., 2001). Cardiac remodeling in response to disease is characterized by TGFβ1-induced collagen deposition (Booz and Baker, 1995), and TGFβ1 and endothelin-1 released by cardiac fibroblasts mediate cardiomyocyte hypertrophy (Gray et al., 1998). In rat myocardium, anti-TGFβ1 antibodies prevent induction of ECM protein expression and myocardial fibrosis (Lijnen et al., 2000).
Expression of TGFβ1 and ECM proteins in response to hyperglycemia has not been examined previously in the embryonic heart. In the current study, E9.5–E13.5 mouse embryos were evaluated by IHC for expression of the three TGFβ isoforms. Embryos were exposed in vitro on E9.5 to normal or hyperglycemic medium for 24 hr or 48 hr and examined for cardiac expression of TGFβ and FN. The method of whole-embryo culture was used to control the precise stage, level, and duration of hyperglycemic exposure. Embryos were also microinjected with TGFβ1 into the pericardial cavity on E9.5 and evaluated for cardiac expression of FN after 24 hr. The results of this work demonstrate predominant expression of the TGFβ1 isoform in the organogenesis-stage heart and increased embryonic expression of TGFβ1 and FN in response to hyperglycemia, suggesting these factors as potential contributors to diabetes-induced cardiac dysmorphogenesis.
TGFβ1 Is Predominant Isoform in Organogenesis-Stage Heart
Mouse embryos were evaluated by section immunohistochemistry (IHC) for cardiac protein expression of TGFβ1, -2, and -3 on E9.5, E10.5, E11.5, E12.5, and E13.5. TGFβ1 protein expression was evident in the heart throughout this period, with the strongest staining between E10.5 and E12.5. TGFβ1 protein was prominently expressed in the myocardial layer of the ventricular and atrial regions of the heart and was generally absent in endocardial cushion tissue (Fig. 1). Protein expression of TGFβ2 and TGFβ3 in the embryonic heart was minimal throughout this period and was increased slightly on E13.5 (Fig. 1). Negative controls treated with no primary antibody demonstrated no staining (Fig. 1).
Considering the prominent expression of TGFβ1 in the organogenesis-stage heart, this isoform became the focus of remaining studies. The E9.5–E11.5 embryonic mouse was used here because it represents a particularly sensitive period of cardiac development. During this stage of organogenesis, the heart undergoes the critical morphogenic process of looping and is metabolically dependent on glucose and glycolysis for normal development.
Hyperglycemia Selectively Increases TGFβ1 Isoform in Embryonic Heart
Mouse embryos were exposed on E9.5 to control (150 mg/dl glucose) or hyperglycemic (600 mg/dl glucose) medium for 24 hr or 48 hr in whole-embryo culture. At the end of culture, embryos were evaluated for TGFβ1, -2, and -3 by IHC, which detects mainly the bound form of TGFβ. Embryos exposed to hyperglycemia for 24 hr demonstrated TGFβ1 immunostaining within the embryonic heart that was similar in distribution but appeared to be increased in intensity compared with controls (Fig. 2). Cardiac staining for TGFβ1 was not increased in embryos exposed to hyperglycemia for 48 hr compared with controls (Fig. 2). Immunostaining for TGFβ2 and TGFβ3 in the heart was not increased after embryonic exposure to hyperglycemia for 24 hr (data not shown) or 48 hr (Fig. 2). Negative controls treated with no primary antibody demonstrated no staining.
Hyperglycemia Increases Total TGFβ1 in Embryonic Heart
Hearts were removed from embryos exposed to hyperglycemic or control medium, as described above, and evaluated individually by enzyme-linked immunosorbent assay (ELISA) for total TGFβ1 concentration. Acid treatment of the isolated heart releases the bound form of TGFβ1, allowing measurement of total (bound plus secreted) TGFβ1 in the solution of heart tissue that is evaluated by ELISA. Total TGFβ1 was increased in hearts of embryos exposed to hyperglycemia for 24 hr but not 48 hr compared with controls (Fig. 3A).
Hyperglycemia Increases TGFβ1 in Embryonic Fluid
After whole-embryo culture, fluid was withdrawn from individual embryos and evaluated by ELISA for TGFβ1. This method evaluates TGFβ1 concentration in the yolk sac and amniotic fluid surrounding the embryo, thus measuring primarily the secreted form of this factor. The concentration of TGFβ1 in embryonic fluid was elevated in embryos exposed to hyperglycemia for 24 hr but not 48 hr compared with controls (Fig. 3B).
Hyperglycemia Increases TGFβ1 mRNA Expression in Embryonic Heart
Hearts exposed to hyperglycemic or control medium, as described above, were pooled and evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) for TGFβ1 and cyclophilin (internal standard) mRNA. Bands were produced at 215 bp for TGFβ1 mRNA and 212 bp for cyclophilin mRNA (Fig. 4A) and evaluated by densitometry. The average ratio of TGFβ1:cyclophilin band densities was approximately 40% higher in hearts of embryos exposed to hyperglycemia than those exposed to control medium for 24 hr (Fig. 4B) but not different after 48 hr (data not shown). These results demonstrate an increase in TGFβ1 mRNA expression in the embryonic heart in response to 24 hr hyperglycemia.
Hyperglycemia Increases FN Protein in Embryonic Heart
Embryos exposed to hyperglycemic or control medium for 24 hr in vitro were evaluated by section or whole-mount IHC for FN protein expression. Sections of embryos exposed to control medium demonstrated faint FN immunostaining in the cranial region of the neural tube and prominent staining in the heart. Cardiac staining in controls appeared within the myocardium, especially in the walls of the developing ventricles (Fig. 5A). Sections of embryos exposed to hyperglycemia demonstrated a similar pattern of FN staining, but the staining intensity appeared to be greater than in controls, especially in the heart. FN staining was particularly prominent in the myocardium of the developing ventricles in embryos exposed to hyperglycemia (Fig. 5B). Negative controls treated with no primary antibody showed no staining for FN (not shown).
Embryos exposed to hyperglycemia for 24 hr demonstrated increased whole-mount immunostaining for FN compared with controls, and this finding was most prominent in the heart. The developing ventricles appeared to have the strongest FN immunostaining in embryos exposed to hyperglycemia (Fig. 5D) compared with controls (Fig. 5C).
Hearts pooled from cultured embryos and evaluated by Western analysis demonstrated doublet bands for FN at approximately 200–220 kDa that corresponded to those obtained using a FN standard (Fig. 6A). A band for actin (internal standard) appeared at approximately 43 kDa (Fig. 6A). Bands for FN and actin were evaluated by densitometry and demonstrated a FN:actin density ratio of more than twofold higher in hearts of embryos exposed to hyperglycemia compared with controls (Fig. 6B).
Hyperglycemia Increases FN mRNA in Embryonic Heart
Hearts from embryos exposed to hyperglycemic or control medium, as described above, were pooled and evaluated by RT-PCR for FN and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, internal standard) mRNA. Bands were produced at 300 bp for FN mRNA and 225 bp for GAPDH mRNA (Fig. 6C) and evaluated by densitometry. The average ratio of FN:GAPDH band densities was approximately 35% higher in hearts of embryos exposed to hyperglycemia for 24 hr compared with controls (Fig. 6D). There was no difference in FN expression in hearts of embryos exposed to hyperglycemia for 48 hr compared with controls (data not shown).
TGFβ1 Induces FN mRNA Expression in Embryonic Heart
Embryos were exposed to control medium in vitro and microinjected with 0.2 ng of TGFβ1 into the pericardial cavity to allow direct exposure of the heart to this factor. After 24 hr in culture, hearts were pooled and evaluated by RT-PCR for FN and GAPDH mRNA. Bands were produced at 300 bp for FN mRNA and 225 bp for GAPDH mRNA (Fig. 7A) and evaluated by densitometry. The average ratio of FN:GAPDH band densities was approximately threefold higher in hearts of embryos exposed to pericardial microinjection of TGFβ1 compared with controls (Fig. 7B).
Diabetes mellitus is characterized by alterations in carbohydrate, fat, and protein metabolism, with resulting hyperglycemia, hyperketonemia, and increased fatty acids, amino acids, and other compounds (Hall et al., 1984; Taylor and Agius, 1988). Thus, many factors potentially play a role in the diabetic embryopathy, but hyperglycemia represents an important and predictable metabolic derangement of this disease.
Congenital heart malformations are an important cause of neonatal lethality in the offspring of diabetics, but the underlying mechanisms are largely unknown. Heart defects associated with the diabetic embryopathy include ventricular and atrial septal defects, transposition of the great vessels, coarctation of the aorta, tetralogy of Fallot, double outlet right ventricle, and patent ductus arteriosus (Ferencz et al., 1990). This wide variety of defects suggests a complex pathogenesis, although several of these abnormalities may result from perturbed endothelial-to-mesenchymal transformation or abnormal development or migration of neural crest cells.
Within the developing atrioventricular (AV) canal, TGFβ stimulates transformation of endothelial cells to mesenchymal cells, which will form the AV valves and septa (Wunsch et al., 1994). Defects of the AV valves and septa are among the most commonly diagnosed neonatal heart malformations and are thought to result from abnormal interactions between mesenchymal cushion cells and the ECM (Srivastava, 2001). Increased TGFβ activity may increase ECM expression and result in cardiovascular defects, such as those produced in diabetic offspring.
The objective of this work was to characterize TGFβ expression in the organogenesis-stage mouse heart, and to examine the effect of hyperglycemia on the expression of TGFβ1 and FN in the embryonic heart. The in vitro method of whole-embryo culture was used to control the precise stage, level, and duration of hyperglycemic exposure. Hyperglycemia of 600 mg/dl glucose was used, because this level produces significant malformations in rodents (Reece et al., 1996) and it represents a severe, but attainable, level of hyperglycemia in rodent and human diabetics. The E9.5–E11.5 stage was used in this work because it encompasses the period of cardiac looping and represents a stage of particular metabolic sensitivity in the developing heart.
Our results demonstrate that TGFβ1 is the predominant isoform in the E9.5–E13.5 mouse heart, in agreement with previous findings. The three mammalian TGFβ isoforms have distinct but overlapping expression patterns in the mouse embryo that change as development progresses (Gatherer et al., 1990; Pelton et al., 1991). TGFβ1 mRNA is expressed in the cardiac mesoderm and endocardial tube of the presomite mouse embryo (Ankhurst et al., 1990; Dickson et al., 1993) and becomes restricted with development to cells overlying the endocardial cushions (Ankhurst et al., 1990), where it is thought to induce endothelial-to-mesenchymal transformation (Potts and Runyan, 1989). TGFβ1 protein is not present in presomite embryos but is expressed in the mouse heart on E8.5–E16.5 (Montengua et al., 1998), and it declines to barely detectable levels by E17.5 (Pelton et al., 1991). The importance of TGFβ1 to normal cardiogenesis is illustrated by mutant mice lacking TGFβ1, which die early postnatally with severe ventricular and valvular defects (Letterio et al., 1994).
Our finding that TGFβ2 and TGFβ3 protein expression is minimal in the embryonic heart during organogenesis is supported by results of previous studies. Expression of TGFβ2 mRNA is present in cardiac precursor cells in early-somite mouse embryos and prominent in the sinus venosus by E8.5, then declines to low levels by E11.5 (Dickson et al., 1993). By contrast, TGFβ2 protein is not expressed in the early embryonic heart but appears in the myocardium of the AV canal, ventricle, atrium, and outflow tract on E12.5–E16.5 and subsequently declines (Pelton et al., 1991). TGFβ3 mRNA is not present in the early-somite embryo but is expressed around the outflow tract at E8.5, and it is prominent in AV cushions but low in ventricles on E11.5. TGFβ3 protein expression is prominent in atria and ventricles but low in AV cushions on E12.5 and prominent throughout the heart on E17.5 (Pelton et al., 1991).
Our IHC results suggest an increase in TGFβ1 in the embryonic heart in response to hyperglycemia for 24 hr but not 48 hr. By contrast, the TGFβ2 and TGFβ3 isoforms are not increased in response to hyperglycemia at either time point. The TGFβ1 increase observed here in the embryonic heart is consistent with results produced in cultured cells and observed in diabetic tissues. Cultured rodent and human mesangial cells exposed to 450–540 mg/dl glucose demonstrate increased TGFβ1 secretion, bioactivity, and mRNA expression (Wahab et al., 1996; Hoffman et al., 1998; Oh et al., 1998). A similar up-regulation of TGFβ1 is produced in cultured renal tubule cells (Rocco et al., 1992) and mesothelial cells exposed to hyperglycemia (Ha et al., 2001) and in the kidneys of human diabetics (Chen et al., 2001).
Our ELISA results demonstrate an increase in total (secreted plus bound) TGFβ1 protein in hearts isolated from embryos exposed to hyperglycemia for 24 hr but not 48 hr, as demonstrated by IHC. Similar results have been produced in other cell types but in different temporal patterns. For example, cultured mesangial cells demonstrated an increase in total TGFβ1 protein at 24–96 hr after hyperglycemic exposure (Hoffman et al., 1998). The time course of TGFβ1 response to hyperglycemia may be unique to individual tissues and developmental states.
Our ELISA results on embryonic fluid samples demonstrate elevated TGFβ1 protein in the yolk sac and amniotic fluid of embryos exposed to 600 mg/dl glucose for 24 hr, suggesting an increase in secreted TGFβ1 by embryonic tissues in response to hyperglycemia. Noncardiac tissues may contribute TGFβ1 to the fluid sampled by this method, but the paucity of IHC staining for TGFβ1 in noncardiac tissues suggests that the heart is likely the primary source of TGFβ1 at this stage of development. The response and timing of the TGFβ1 increase in embryonic fluid is similar to our results obtained by IHC, as well as by ELISA on isolated hearts, supporting the heart as a primary source of secreted TGFβ1 in response to 24 hr hyperglycemia.
Our RT-PCR results demonstrate an increase in TGFβ1 mRNA expression in hearts of embryos exposed to hyperglycemia, consistent with that in cultured cells and observed in diabetic tissues, but in different temporal patterns. In cultured mesangial cells, hyperglycemia produced a slight increase in TGFβ1 mRNA at 24 hr and a twofold increase at 48 hr, which persisted until 72 hr, with no change in message stability (Hoffman et al., 1998). Recent work in this laboratory demonstrates increased TGFβ1 protein and mRNA in embryonic hearts after maternal streptozotocin-induced diabetes for 24 hr but not 48 hr (Joyner and Smoak, unpublished data). Thus, the temporal pattern of hyperglycemia-induced TGFβ1 expression in the embryonic heart is the same after in vivo and in vitro exposures but differs from that of adult tissues. Previous work demonstrated that SM22α promoter-driven overexpression of TGFβ1 in the heart and vasculature of organogenesis-stage mice caused yolk sac vascular defects and embryonic death by E11.5 (Agah et al., 2000). Taken together, these results suggest that a TGFβ1 increase for 24 hr during the critical developmental period of organogenesis is sufficient to induce deleterious effects in the embryonic heart that include the deposition of ECM proteins.
Fibronectin is a high molecular weight ECM glycoprotein that forms a scaffold to which other ECM components attach. FN interacts with cells by means of integrin cell surface receptors and is widely distributed in connective tissue, basement membranes, and on cell surfaces. FN functions in cell–cell and cell–ECM interactions, embryonic differentiation, wound healing, and neoplastic transformation (McDonagh, 1981; Dufour et al., 1988). In the rat embryo, FN mRNA expression is elevated in the endocardium of the early heart tube, and FN protein later becomes uniformly distributed in the ECM (cardiac jelly) between the myocardium and endocardium of the heart tube (Suzuki et al., 1995). FN is important in the migration of precardiac cells and the development of cardiac cushion cells (Linask and Lash, 1986; Icardo et al., 1992). Inhibiting the activity of matrix metalloproteinases, enzymes that digest ECM proteins, blocks effective migration of precardiac neural crest cells (Cai and Brauer, 2002), supporting a role for increased ECM proteins in the pathogenesis of cardiac defects.
The IHC, Western analysis, and RT-PCR results presented here demonstrate an increase in FN protein and mRNA expression in the embryonic heart in response to a 24 hr in vitro exposure to hyperglycemia of 600 mg/dl glucose compared with controls. These findings in the embryonic heart are consistent with those produced in cultured cells, in which exposure to high levels of glucose results in the proliferation of ECM proteins. Cultured rodent or human endothelial cells exposed to hyperglycemia (360–540 mg/dl glucose) demonstrate increased ECM (collagen and FN) protein or mRNA expression (Cagliero et al., 1988; Spiro et al., 1995; Mueller et al., 1997). Cultured renal mesangial cells exposed to hyperglycemia demonstrate increased expression of ECM components, including FN, collagen, laminin, and others (Ayo et al., 1991; Nahman et al., 1992; Wahab et al., 1996). Diabetic mice have increased TGFβ1 mRNA and ECM deposition in renal glomeruli and myocardium (Yang et al., 1995; Chen et al., 2001), and ECM proteins accumulate in the kidney, skin, heart, and blood vessels of human diabetic patients (Mauer et al., 1981; Osterby, 1983). Recent work in this laboratory demonstrates increased FN protein and mRNA in embryonic hearts exposed to maternal streptozotocin-induced diabetes for 24 hr and increased FN after 48 hr (Joyner and Smoak, unpublished data). Thus, it appears that the response of the embryonic heart to hyperglycemia is similar to that of adult cells in producing increased expression of FN. We have found that a similar response occurs with laminin (unpublished data) and may be generalized to the production of other ECM proteins. This response in the embryonic heart is significant for its potential role in the pathogenesis of diabetes-induced cardiac defects.
The RT-PCR data presented here suggest that direct exposure of the E9.5 embryonic heart to TGFβ1, by microinjection into the pericardial cavity, induces increased FN mRNA expression within the heart. These results further support the role of TGFβ1 in ECM deposition in the embryonic heart, similar to findings in other tissues. For example, diabetic rodents demonstrate increased TGFβ1 mRNA and ECM deposition in renal glomeruli and myocardium, effects that are neutralized by inhibiting TGFβ1 activity (Yang et al., 1995; Lijnen et al., 2000; Chen et al., 2001; Goldfarb and Ziyadeh, 2001).
There are several potential mechanisms by which hyperglycemia may activate TGFβ1 and FN expression. A putative glucose response element has been identified in the TGFβ1 promoter that may be directly stimulated by hyperglycemia (Hoffman et al., 1998). Protein kinase C (PKC) is also activated by hyperglycemia and has been shown to induce the expression of TGFβ1 and FN in cultured mesothelial cells (Ha et al., 2001). Thrombospondin (TSP-1) is increased in cultured mesangial cells exposed to hyperglycemia (Poczatek et al., 2000; Tada et al., 2001; Yevdokimova et al., 2001) and may act upstream of TGFβ1 in hyperglycemia-induced fibrosis. Mesangial cells cultured in high glucose and treated with a PKC inhibitor demonstrated attenuated increases of TSP-1 and active TGFβ1 (Tada et al., 2001), suggesting that PKC acts upstream of TSP-1. The activity of PKC, TSP-1, or other factors potentially stimulating TGFβ1 and FN expression in response to hyperglycemia has not been examined in the embryonic heart. Such factors will be the focus of future investigations.
In this study, we have demonstrated an increased expression of TGFβ1 and FN protein and mRNA in the embryonic heart in response to 24 hr in vitro exposure to hyperglycemia of 600 mg/dl glucose compared with normoglycemia of 150 mg/dl glucose. The increase in TGFβ1 at 24 hr may induce the expression of ECM proteins in the embryonic heart that persists until at least 48 hr. This study represents the first to evaluate the TGFβ1 and ECM response to hyperglycemia in the embryonic heart. Our results suggest that the embryonic heart may be susceptible to a similar cascade of signaling events that occurs in adult cells exposed to hyperglycemia. Glucose extremes experienced by poorly controlled pregnant diabetics, thus, may have profound effects on the developing heart and may induce signaling mechanisms that enhance TGFβ1 and FN expression. These factors potentially play a role in the pathogenesis of diabetes-induced cardiac dysmorphogenesis.
Mice of the CD-1 strain were mated overnight and checked for vaginal plugs the next morning, which was designated E0.5. Pregnant mice were killed by cervical dislocation on the appropriate day (E9.5–E13.5), uteri were removed, and embryos were dissected in cold Tyrode's buffer to remove decidua, parietal yolk sac, and Reichert's membrane. Principles of laboratory animal care, as described by the National Institutes of Health, and as approved by the NC State University Institutional Animal Care and Use Committee, were followed in these studies.
Section IHC for TGFβ1, -2, and -3
Distribution and levels of TGFβ1, -2, and -3 in the developing mouse heart were qualitatively evaluated by using section IHC. Embryos were isolated on E9.5, E10.5, E11.5, E12.5, and E13.5, dissected free of surrounding membranes, rinsed in cold Tyrode's buffer, and fixed in 4% paraformaldehyde for 24 hr or longer. Embryos were dehydrated in increasing ethanol and xylene, embedded in paraffin, sectioned sagittally at 5 μm, dehydrated, deparaffinized, and rehydrated. Sections were blocked for endogenous peroxidase activity with 3% H2O2, then blocked with serum for 30 min at 37°C. Sections were probed with goat polyclonal primary antibody for TGFβ1 or rabbit polyclonal primary antibodies for TGFβ2 or -3 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100 in 1× phosphate-buffered saline (PBS). Negative controls were treated with 1× PBS or preimmune control serum of the appropriate isotype. Sections were then treated with the Biogenex (San Roman, CA) kit components, Link (biotinylated secondary antibody) and Label (peroxidase-conjugated streptavidin). Sections were stained with 3-amino-9-ethyl-carbazole (AEC) chromogen (Biogenex), counterstained with Mayer's hematoxylin, cover-slipped, and imaged.
Embryos were isolated on E9.5, and only those with full rotation, regular heartbeat, and intact visceral yolk sac and ectoplacental cone were used for whole-embryo culture. Embryos were randomly assigned to culture medium that contained either 150 mg/dl glucose (control) or 600 mg/dl glucose (hyperglycemia) and consisted of 75% rat serum and 25% Tyrode's buffer, supplemented as needed with concentrated (50 mg/ml) D-glucose. Glucose levels were monitored with a Beckman Glucose Analyzer II (Beckman Coulter, Fullerton, CA). Embryos were placed in 30-ml culture vials in 2 ml of medium per embryo. Vials were rotated at 30 rpm within a 37°C incubator and supplemented with glucose every 12 hr to maintain normal and hyperglycemic levels. Vials were aerated with 20% O2, 5% CO2, and 75% N2 at 0 hr and 12 hr and 95% O2 and 75% N2 at 24 hr and 36 hr. Blood was obtained from the descending aorta of ether-anesthetized Sprague-Dawley retired-breeder male rats and centrifuged immediately for 5 min at 25°C, then 30 min at 4°C. The resulting serum was heat inactivated at 56°C for 30 min and treated with 100 U/ml penicillin and 100 μg/ml streptomycin, and stored at −20°C. At the end of culture, embryonic fluid was sampled from yolk sac and amniotic cavities and evaluated for TGFβ1 by ELISA, as described below. Embryos were removed from surrounding membranes and prepared for IHC, or hearts were removed and pooled for Western analysis or RT-PCR, as described below.
ELISA for TGFβ1
At the end of culture, embryos were rinsed in cold Tyrode's buffer, wicked dry, and a 25-μl aliquot of embryonic fluid was removed from yolk sac and amniotic cavities using a 1-ml syringe and 25-gauge needle. Hearts were isolated, sonicated in 90 μl of Dulbecco's PBS, treated with 1 μl 1 N HCl for 15 min to release bound TGFβ, then neutralized with 1 μl 1N NaOH. Heart solution and embryonic fluid samples were assayed in duplicate for TGFβ1 by ELISA using the Emax kit (Promega, Madison, WI), following manufacturer's instructions. Specifically, ELISA plates (Microlon; Greiner) were coated overnight at 4°C with TGFβ Coat monoclonal antibody diluted 1:1,000 in Carbonate Coating Buffer (0.025 M sodium bicarbonate, 0.025 M sodium carbonate). Wells were emptied and treated with TGFβ block at 37°C for 35 min, and washed in Tris-buffered saline with Tween 20 (TTBS). Aliquots of 10 μl of embryonic fluid or 25 μl of heart solution were loaded in each well, supplemented with sample buffer for total volume of 100 μl, and incubated at 25°C for 90 min at 600 rpm on a plate shaker. A standard curve (15.6–1,000 pg/ml) was produced by serial dilution of TGFβ standard in TGFβ sample buffer. Wells were washed with TTBS and incubated for 2 hr at 600 rpm on a plate shaker at 25°C with anti-TGFβ polyclonal antibody diluted 1:1,000 in TGFβ sample buffer. Plates were incubated for 2 hr at 600 rpm on a plate shaker at 25°C with TGFβ horseradish peroxidase (HRP) conjugate diluted 1:100 in TGFβ sample buffer. Color was developed with TMB One Solution, stopped with 1 N HCl, and quantified with a spectrophotometer (Tecan, Durham, NC) at 450 nm, using a reference wavelength of 405 nm. Average TGFβ1 concentration (pg/μl) was calculated for duplicates of each sample.
Section IHC for FN
Distribution and levels of FN protein in the embryonic heart after exposure to normoglycemia or hyperglycemia were qualitatively evaluated by using section IHC. Embryos were fixed, embedded, and sectioned as described above. Sections were blocked as described above, and probed with goat polyclonal primary antibody for FN (Santa Cruz Biotechnology) diluted 1:20 in 1× PBS. Sections were treated, as described above, with secondary antibody, peroxidase-conjugated streptavidin, chromogen, and counterstain, and imaged.
Whole-Mount IHC for FN
Expression of FN protein in intact embryos after exposure to normoglycemia or hyperglycemia was evaluated by using whole-mount IHC. Embryos were removed from culture, rinsed, and fixed, as described above. Embryos were dehydrated in increasing methanol concentrations, then blocked for 2 hr at 25°C with 3% H2O2 in methanol. Embryos were stored in 100% methanol at −20°C overnight, then rehydrated in decreasing methanol concentrations to 1× PBS. Embryos were incubated overnight at 4°C in goat anti-FN primary antibody diluted 1:10 in PBS/Tween (PBST) with 1% block. Embryos were washed several times for 30 min each in PBST and exposed overnight at 4°C to anti–goat-HRP secondary antibody diluted 1:200 in PBST in 1% block. Embryos were treated with 4-chloro-1-naphthol and 0.03% H2O2 for 30 min at 25°C, washed in 1× PBS, and imaged.
Hearts were removed from embryos, by severing inflow and outflow tracts, and pooled (6–12 hearts per treatment) in 200 μl of cold 1× PBS containing 1:100 Protease Inhibitor Cocktail (Sigma, St. Louis, MO). Hearts were dispersed by trituration and centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was removed, and the pellet was resuspended in 50 μl of 4% sodium dodecyl sulfate (SDS) and boiled at 100 °C for 5 min. This solution was centrifuged at 1,200 rpm for 5 min at 25°C, and the supernatant (SDS extract) was stored at −70°C.
The SDS extracts from hearts of embryos exposed to normoglycemia or hyperglycemia were boiled in sample buffer (1.5% w/v SDS, 2.5% v/v beta-mercaptoethanol, 5% v/v glycerol, 0.01% w/v bromophenol blue, 31 mM Tris) at 95°C for 5 min, centrifuged for 1 min, and loaded onto a 4–20% gradient SDS gel. Proteins were resolved by electrophoresis at 165 V in Running Buffer (Bio-Rad, Hercules, CA) and transferred to a polyvinylidene difluoride membrane by electrophoresis at 15 V for 20 hr at 4°C in 1× Transfer Buffer (Bio-Rad). The membrane was blocked for 1 hr at 25°C in 5% nonfat dry milk in TTBS and divided horizontally at approximately 100 kDa. The membrane strips were probed for 1 hr at 25°C with goat polyclonal antibodies (Santa Cruz Biotechnology) to FN (top strip) and actin (bottom strip) diluted 1:1,000 in TTBS. The membrane strips were washed in TTBS and exposed for 1 hr at 25°C to donkey anti-goat secondary antibody (Santa Cruz Biotechnology) diluted 1:150,000 in TTBS with 0.5% block. Chemiluminescence was produced by using SuperSignal WestDura (Pierce, Rockford, IL). Bands were imaged by using an Epi Chemi II (UVP, Upland, CA) image analysis system, and band densities were calculated by using LabWorks 4.0 (UVP) software. The ratio of FN to actin band densities was calculated for each sample.
Hearts were removed from embryos and pooled (6–12 hearts per treatment) in cold diethylpyrocarbonate (DEPC) –PBS, triturated in 1ml Tri-Reagent (Sigma), and stored at −70°C. Samples were thawed, allowed to sit at 25°C for 10 min, treated with 200 μl of chloroform, and centrifuged at 12,00 rpm for 10 min at 4°C. The top layer was removed and treated with 1 ml of isopropanol to precipitate RNA, centrifuged at 14,000 rpm for 15 min at 4°C, washed with 1 ml of 75% ethanol in DEPC-dH2O, and recentrifuged. Resulting RNA was resuspended in The RNA Solution (Ambion, Austin, TX) and was treated with 2 U of DNA-free (Ambion) and 2 U of Superase-In (Ambion). Total RNA was quantified with spectrophotometry at 260 and 280 nm, and quality was checked with formaldehyde agarose gel electrophoresis. A 50-ng aliquot of total RNA was reverse transcribed with the SuperScript First Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) using oligo d(T) primers.
TGFβ1 and cyclophilin (housekeeping gene) cDNA were amplified in a 50-μl reaction with 1× PCR reaction buffer with MgCl2 (Roche, Indianapolis, IN) with 1× Rediload (Research Genetics, Indianapolis IN), 200 mM of each dNTP, 200 mM of each primer, and 1.5 U of Taq polymerase (Roche). Primers for TGFβ1 were 5′-GCCCTGGACACCAACTATTGCT-3′ (forward) and 5′-GGCTCCAAATGTAGGGGCAGG-3′ (reverse), and primers for cyclophilin were 5′-AATTAGAGC-3′ (forward) and 5′-CTGGGACCAAACACAAACGGTTC-3′ (reverse). A master mix approach was used to minimize variation between reactions. Cycling conditions were 94°C for 2 min, 60°C for 40 sec, 72°C for 1 min, then 30 cycles of 94°C for 30 sec, 60°C for 40 sec, then 94°C for 30 sec, 60°C for 40 sec, 72°C for 10 min. Number of cycles was optimized to be in linear range of the PCR curve.
FN and GAPDH cDNA were amplified as above, with the addition of 1× Q buffer (Qiagen, Valencia, CA) and 400 mM of each primer. Primers for FN were 5′-CTCATCAGCATCCAGCAG-3′ (forward) and 5′-GGGAGCAGGTCAGGAATG-3′ (reverse), and primers for GAPDH were 5′-TGTGAACGGATTTGGCCGTA-3′ (forward) and 5′-ATCGCTCCTGGAAGATGGTGA-3′ (reverse). Cycling conditions were 95°C for 2 min, then 31 cycles of 95°C for 30 sec, 54°C for 1 min, 72°C for 1 min, then 72°C for 10 min.
The PCR products were visualized on a 1.6% agarose gel by using an Epi Chemi II (UVP) image analysis system, and bands were quantified by densitometry using LabWorks 4.0 (UVP) software. Ratios of TGFβ1 to cyclophilin and FN to GAPDH band densities were compared between treatments.
Microinjection of TGFβ1
Embryos were placed in whole-embryo culture on E9.5 in normoglycemic (150 mg/dl glucose) medium, as described above. A 200-nl (0.2 ng) aliquot of TGFβ1 protein (R&D Systems, Minneapolis, MN), diluted in suspension buffer (4 mM HCl with 0.1% BSA) and stained 1:25 with Fast Green (Sigma) for localization, was microinjected (Drummond Nanoject II, Broomall) into the pericardial cavity using a pulled capillary tube and a micromanipulator under a stereozoom microscope. Controls received suspension buffer stained with Fast Green. Only embryos with successful microinjection into the pericardial cavity were returned to culture for 24 hr and evaluated by RT-PCR for cardiac FN mRNA, as described above.
Numerical data were statistically analyzed with an analysis of variance for overall significance and a Tukey's test for between group differences using PROC GLM of SAS software (SAS Institute, Cary, NC) at P < 0.05.