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Myostatin Is a Human Placental Product That Regulates Glucose Uptake

Liggins Institute, University of Auckland (M.D.M., C.C.O., K.-C.L., J.J.B.), and National Research Center for Growth and Development (M.D.M.), Auckland 1003, New Zealand; and Functional Muscle Genomics, AgResearch Ltd., Ruakura Agricultural Center (C.D.M.), Hamilton 2001, New Zealand

Address all correspondence and requests for reprints to: Dr. Murray D. Mitchell, Liggins Institute, University of Auckland, Private Bag 92019, Auckland, New Zealand. E-mail: m.mitchell{at}auckland.ac.nz.

Abstract

Context: Myostatin is a member of the TGF-ß superfamily and is primarily known for its ability to inhibit muscle growth. It also has actions on glucose metabolism. We hypothesized that it may act as a paracrine regulator of glucose uptake in the placenta, potentially contributing to fetal and placental growth.

Objectives: The objective of this study was to determine whether myostatin is present in and formed by the human placenta and to evaluate its effects on glucose uptake.

Materials and Methods: Myostatin protein and mRNA were measured using Western immunoblotting and real-time PCR, respectively. Glucose uptake was assessed by uptake of radiolabeled deoxyglucose in vitro. Placental tissues were obtained at term (n = 8), preterm (n = 8; 24–34 wk), and early in pregnancy (n = 6; 9–13 wk).

Results: Human placentas were shown to express myostatin protein, with a significantly lower expression in term samples compared with samples collected in preterm samples. Human placentas express myostatin mRNA throughout gestation, which does not change. Myostatin treatment of human term placental explants resulted in an increase in deoxyglucose uptake compared with controls.

Conclusions: Myostatin is synthesized, released, and acts within the human placenta. It contributes to placental glucose homeostasis and may be a therapeutic target in diseases ranging from placental insufficiency to diabetes in pregnancy.

MYOSTATIN, ALSO KNOWN as growth differentiating factor 8, is a member of the TGF-ß superfamily of proteins (1). Myostatin is synthesized as a precursor protein consisting of two domains: an N-terminal propeptide and a C-terminal mature domain. The precursor protein is believed to be present as a homodimer. Once cleaved from the precursor protein, the propeptide may noncovalently bind to the mature myostatin and function as an inhibitor of mature myostatin activity (2). The mature myostatin is believed to be present as a dimer, and two propeptides are thought to bind to form this mature homodimer.

Myostatin is a negative regulator of muscle growth. Muscle mass is increased in double muscled cattle that have a myostatin mutation (3). It has been shown to negatively regulate hypertrophy and hyperplasia of muscle, regeneration, and metabolic responses in fat deposition (4, 5). Myostatin is also involved in glucose metabolism, because myostatin-null mice show a reduction in hyperglycemia (6). Similar results were found when hyperglycemia in diet-induced obesity was reduced by inactivating myostatin with increased propeptide, indicating that the ratio between the propeptide and mature myostatin is physiologically important (7).

Myostatin knockout mice display a marked increase in muscle mass (1) and are considerably bigger than wild-type mice at birth. The increased birth weight indicates that the fetal nutrient supply via the placenta must increase to allow the extra growth of the larger heavily muscled fetus, and this may relate to the metabolic role myostatin has in controlling glucose uptake. Therefore, myostatin may control placental size, cellular composition of the placenta, and metabolic transfer of nutrients, providing a therapeutic route to treat conditions such as placental insufficiency. Hence, we hypothesized that myostatin may be produced by the placenta and act in a paracrine manner to regulate glucose uptake.

Materials and Methods

Tissue collection

This study was approved by the Auckland University ethics committee. First trimester placentas were collected from elective termination of normal pregnancies with informed signed consent. Tissues were collected from six placentas from pregnancies between 9 and 13 wk gestation, as determined by ultrasound (8). Placentas were also collected with informed consent after spontaneous preterm labor with or without intrauterine infection from pregnancies between 24 and 34 wk gestation. Intrauterine infection was assigned if any of the following clinical signs were present: maternal fever, maternal tachycardia, fetal tachycardia, uterine tenderness and foul-smelling liquor, and neonatal sepsis (9). Placentas were collected at term after elective cesarean section before the onset of labor (indications: previous cesarean or malpresentation) and after spontaneous labor and uncomplicated vaginal delivery from pregnancies between 37 and 42 wk (9). Placental pieces were rinsed briefly in PBS and immediately frozen in liquid nitrogen for either total RNA or protein extraction.

Placental cultures

Placental explants were established by finely mincing the placenta and distributing the pieces in a 12-well plate in DMEM-medium 199 (Irvine Scientific, Santa Ana, CA) containing 10% heat-inactivated fetal calf serum (Life Technologies Ltd., Auckland, New Zealand). These methods were fully described previously (10).

Deoxyglucose uptake

After overnight incubation, explants were washed three times with PBS to remove any residual fetal calf serum. PBS containing 1 µCi [¹⁴C]deoxyglucose in the presence or absence of 1 µg/ml myostatin was added and incubated for 20 min. One placental explant was removed and serially rinsed in cold PBS. The explants were dissolved in 1 M NaOH overnight, and protein levels were determined by bicinchoninic acid (BCA) assay (Sigma-Aldrich Corp., St. Louis, MO). Radioactivity was determined by adding 0.75 ml Starscint scintillation fluid (PerkinElmer Life and Analytical Sciences, Boston, MA) to the lysed samples and counted in a 1214 Rackbeta liquid scintillation counter (LKB Wallac, Gaithersburg, MD). Uptake was calculated as counts per minute per milligram of protein and is expressed as a percentage of the control value.

Western blot analysis

Lysis buffer with an enzyme inhibitor (Complete, Roche Diagnostics NZ, Auckland, New Zealand) was added to 100 mg placental tissue. Samples were homogenized on ice and then centrifuged at 11,000 x g for 10 min. Supernatant was recovered, mixed with Laemmli loading buffer (11), boiled for 5 min, and then stored at –20 C until analysis. The protein concentration of the supernatant was determined using the BCA assay (Sigma-Aldrich Corp.). Ovine skeletal muscle was processed as described above and used as a positive control for myostatin. Protein from each placental sample and the control muscle sample was loaded and separated in a 12% sodium dodecyl sulfate-polyacrylamide gel, then transferred to a nitrocellulose membrane. Membranes were stained with Ponceau S to verify transfer of protein, then incubated overnight with goat antimyostatin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were washed in PBS and Tween, incubated with horseradish peroxidase-conjugated rabbit antigoat antibody (Sigma-Aldrich Corp.) for 2 h, and washed again in PBS and Tween. Bound horseradish peroxidase activity was detected with enhanced chemiluminescence (Super Signal, Pierce Chemical Co., Rockford, IL), then blots were exposed to film, after which the relative ODs were determined using a densitometer (GS 800, Bio-Rad Laboratories, Inc., Auckland, New Zealand) and Quantity One software (Bio-Rad Laboratories).

RNA extraction and real-time PCR

Frozen placental samples were homogenized on ice in TRIzol reagent (Invitrogen Life Technologies, Inc., Gaithersburg, MD) for 30 sec at 13,500 rpm using an Ultra Turrax homogenizer (Janke and Kunkel, Staufen, Germany). Debris was removed by centrifugation for 10 min at 10,000 x g at 4 C, and total RNA was isolated using the TRIzol protocol (Invitrogen Life Technologies, Inc.). RNA was resuspended in diethyl pyrocarbonate-treated water, and the final concentration was determined by measuring absorbance at 260 nm. First strand cDNA synthesis was performed using a Superscript II Pre-Amplification kit (Invitrogen Life Technologies, Inc.) and 1.25 µg total RNA from placental samples, according to the manufacturer’s protocol. Oligonucleotide primers for human myostatin mRNA (Mstn FP, 5'-TGGTCATGATCT TGCTGTAACCTT-3'; Mstn RP, 5'-TGTCTGTTACC TTGACCTCTAAAA-3') and RNA polymerase II (RPII FP 5'-GGTGGAGCTGGATCGGAAGCACAT-3' and RPII RP 5'-CGATGCAGCGCAGGAAGACAT-3') as a reference. PCR was conducted on a LightCycler 2.0 PCR Amplification System and Detection System (Roche Diagnostics Ltd.), and all reactions were performed in duplicate. Each 10-µl reaction contained 2.5 µl cDNA, 2 µl LightCycler Faststart DNA MasterPlus SYBR Green I mix, and 500 nM of each forward and reverse primer. The reactions were conducted using the following parameters: 95 C for 10 min, followed by 55 cycles of 1 min at 95 C, 10 sec at 60 C, and 7 sec at 72 C for Mstn and 42 cycles of 5 sec at 95 C, 10 sec at 60 C, and 15 sec at 72 C for RPII. At the end of the reaction, melting curve analysis and agarose gel electrophoresis (data not shown) was performed to verify the identity and specificity of amplification products. Standard curves were created using cDNA from a pooled placental sample serially diluted (1:10) by plotting values for log cDNA quantity (in arbitrary units) vs. cycle threshold (the cycle number at which fluorescence exceeds background). The cycle threshold for each of the unknown samples was used to calculate the amount of candidate and reference mRNA relative to the internal standard. For each sample, results were normalized by dividing the amount of candidate mRNA by the amount of reference RPII mRNA. Each sample was assayed in duplicate, and a no template control was included in every reaction.

Statistical analyses and presentation of data

Deoxyglucose uptake rates in human placental explants were expressed as a percentage of the control value to allow the results of multiple experiments to be pooled and analyzed collectively. Differences in myostatin protein in human preterm and term placentas and in deoxyglucose uptake of human term placental explants in the presence and absence of myostatin, were assessed by Mann-Whitney U test for nonparametric data. Myostatin protein data in human preterm and term placentas was assessed for correlation by Spearman’s correlation test. P 0.05 was considered significant for the Mann-Whitney and Spearman tests.

Results

Myostatin precursor and dimer proteins were found consistently in human preterm and term placentas. A representative Western blot (Fig. 1) shows two preterm and two term human placental samples with expression of myostatin precursor protein (52 kDa) and myostatin dimer protein (28 kDa). The complete dataset was calculated and expressed as a ratio of myostatin dimer to precursor in Fig. 2. There was no difference in myostatin levels between preterm infection and no infection groups, and therefore, they were grouped together. Similarly, there was no difference in myostatin proteins between labor and no labor (cesarean section) groups; they were pooled into one term placental group. The expression of myostatin protein in human placentas was significantly less at term compared with expression in preterm placentas (P < 0.001). A statistically significant negative correlation was found between myostatin levels and gestational age (Spearman correlation coefficient = –0.823; P < 0.001), indicating reduced myostatin actions near term.

View larger version (45K): [in this window] [in a new window]   FIG. 1. Myostatin (Mstn) precursor and dimer protein in human samples of preterm and term placentas by Western blotting. Skeletal muscle served as a positive control. Placentas were collected at preterm deliveries and at term by elective cesarean section or spontaneous vaginal delivery (n = 8/group).

View larger version (8K): [in this window] [in a new window]   FIG. 2. Myostatin protein in human preterm and term placentas, expressed as a ratio of myostatin to precursor (mean ± SEM; n = 8). Placentas were collected at preterm deliveries and at term by elective cesarean section or spontaneous vaginal delivery. The significant difference between the means of preterm compared with term placentas was established by Mann-Whitney; P 0.05 (*) was considered significant (***, P 0.001). Myostatin in term placentas tends to decrease, whereas myostatin in preterm placentas increases (Spearman correlation coefficient = –0.823; P < 0.001).

View larger version (13K): [in this window] [in a new window]   FIG. 3. Myostatin mRNA in human early, preterm, and term placentas determined by real-time PCR (mean ± SEM; n = 6). Placentas were collected at terminations, at preterm deliveries, and at term by elective cesarean section or spontaneous vaginal delivery (n = 6/group). Statistical analysis showed no significant difference.

View larger version (12K): [in this window] [in a new window]   FIG. 4. Effects of myostatin on deoxyglucose uptake by human term placental explants (mean ± SEM; n = 5). Explants were isolated and the following day, after rinsing in PBS, were treated in the presence and absence of myostatin (1 mg/ml) in combination with [14C]deoxyglucose (1 µCi/ml). Explants were removed, serially rinsed in PBS, and lysed in 1 M NaOH overnight. The protein content was then determined by BCA, and the amount of [14C]deoxyglucose incorporated was measured (placentas collected at elective cesarean section; significance between the means of untreated control explants compared with the treated ones was established by Mann-Whitney). P 0.05 (*) was considered significant. **, P 0.01.

Although myostatin is considered predominantly a negative controller of muscle fiber hyperplasia and hypertrophy (1), it has recently been shown to be expressed in other tissues and other species (12). This indicates that myostatin may have physiological roles other than muscle growth. Our results show that human placental tissues express myostatin throughout gestation and that myostatin decreases with gestational age. This is consistent with our unpublished observations which have localized myostatin to the human chorionic villi and found that myostatin in murine placenta is highest at midgestation and low at term.

Myostatin has also been associated with hyperglycemia in mice (6). Our results show that acute myostatin treatment enhances glucose uptake of human placental tissues, indicating that myostatin may be involved in the control of maternal-fetal/nutrient balance. However, fetuses from myostatin-null mice are larger than controls (1), indicating an increase in fetal placenta nutritional supply, which is not in agreement with the results from our short-term glucose uptake incubations. Possible explanations include changes in placental glucose transporters near term or insufficient time of myostatin incubations to alter placental glucose transporters (13, 14), because glucose transport response to a stimulus is biphasic (15). Alternatively, endogenous myostatin could be down-regulated by myostatin in the medium, or it could affect the posttranslational modification (11). The novel findings from these studies clearly indicate that myostatin is involved in the maternal/fetal nutrient partitioning of the human placenta.

Acknowledgments

We are grateful to the theater staff at Auckland Hospital for the collection of placentas.

Footnotes

This work was supported by grants from the National Research Center for Growth and Development and Auckland University Research Committee.

M.D.M., C.C.O., K.-C.L., C.D.M., and J.J.B. have nothing to declare.

First Published Online February 7, 2006

Abbreviation: BCA, Bicinchoninic acid.

Received October 27, 2005.

Accepted January 27, 2006.

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