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Department o Biochemistry and Molecular Biology,1 Herman B. Wells Center for Pediatric Research, Department of Pediatrics, School of Medicine,3 Center for Diabetes Research, Indiana University, Indianapolis, Indiana 46202-51222
Received 6 February 2004/ Accepted 15 May 2004
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ABSTRACT |
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90% of GYS1-null animals died soon after birth due to impaired cardiac function. Defects in cardiac development began between 11.5 and 14.5 dpc. At 18.5 dpc, the hearts were significantly smaller, with reduced ventricular chamber size and enlarged atria. Consistent with impaired cardiac function, edema, pooling of blood, and hemorrhagic liver were seen. Glycogen synthase and glycogen were undetectable in cardiac muscle and skeletal muscle from the surviving null mice, and the hearts showed normal morphology and function. Congenital heart disease is one of the most common birth defects in humans, at up to 1 in 50 live births. The results provide the first direct evidence that the ability to synthesize glycogen in cardiac muscle is critical for normal heart development and hence that its impairment could be a significant contributor to congenital heart defects. |
INTRODUCTION |
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-1,4-glycosidic linkages, the basic polymerizing linkages of the polysaccharide. The branch points, introduced by the branching enzyme (EC 2.4.1.18), are formed by -1,6-glycosidic linkages. Mammals express two isoforms of glycogen synthase, encoded by the GYS1 and GYS2 genes. GYS1, encoding the muscle isoform of glycogen synthase, is expressed in skeletal muscle, cardiac muscle, adipose tissue, kidneys, and brain (29). GYS2 expression has been found only in liver (30). Many cells can synthesize glycogen but, in absolute amounts, the major stores are in the liver and skeletal muscle. Although its basic role as an energy reserve is common to all cells, there are differences in glycogen function between tissues. Liver glycogen contributes primarily to blood glucose homeostasis, being synthesized during periods of nutritional sufficiency and subsequently converted to glucose, which is released into the bloodstream to combat hypoglycemia (34). In the fed state, glycogen is also synthesized in muscle, where it functions as an energy reserve to fuel contraction (34). Glycogen provides a portion of the glucose utilized by the aerobic working adult heart (16) and is preferentially oxidized compared to exogenous glucose (24, 26). Although long-chain fatty acids are normally the major substrates for the adult heart, when blood glucose and insulin concentrations are high, such as after a meal, glucose becomes a more prominent energy source (41). Various stresses also provoke greater reliance on glycogen. When the workload of the heart is increased, such as after stimulation by epinephrine (12, 23) and/or increased exercise (24, 26), glycogen provides a readily accessible source of additional energy. In ischemia, glucose assumes a more important role as a fuel for the heart, since fatty acid oxidation is restricted and glycogen supplies glucose for anaerobic glycolysis (16). An analogous protective role may occur in hibernating animals, which exhibit dramatically increased cardiac glycogen levels (19) similar to those seen in the fetal heart (16).
The ontogeny of glycogen accumulation and glycogen synthase activity have been examined in a number of mammals. Relatively low levels of glycogen are present in fertilized mouse eggs (38); however, there is evidence of high glycogen synthase activity in one-cell mouse embryos (8), and by the two-cell stage, significant glycogen accumulation can be measured (39). Several tissues from a variety of mammals have been analyzed for the appearance of glycogen and glycogen synthase activity in later stages of embryonic development (7, 15, 25). In the developing lung, the glycogen level is low early in gestation and high from midgestation until term, when it declines (7, 14, 15). Liver glycogen is present at a low level until late gestation (7, 14, 15, 25), consistent with a role in providing glucose for the newborn prior to the development by the liver of the capacity to produce glucose by gluconeogenesis (22). The concentration of fetal skeletal muscle glycogen has already reached the adult level when glycogen accumulation is just beginning in the liver. By late gestation, the level of muscle glycogen is higher than that in adult muscle (15). Cardiac glycogen is present at high levels during early to midgestation before falling to low levels at the time of birth (7, 14, 15, 25). The facts that glycogen is present and that its level changes during the embryonic development of several tissues suggest that it may have a specific role, but few studies have provided definitive evidence regarding this point.
In the present work, we analyzed a mouse model (muscle glycogen synthase knockout [MGSKO] mice) in which the GYS1 gene has been disrupted, thereby eliminating the ability to store glycogen in skeletal muscle and cardiac muscle. About 90% of the homozygous null offspring from crosses of heterozygotes died perinatally due to abnormal cardiac development at between 11.5 days postcoitum (dpc) and 18.5 dpc. Congenital heart disease is one of the most common birth defects in newborns. Reports of the incidence of moderate to severe defects range from 6 to 19 per 1,000 live births (11, 28). The present work suggests that glycogen plays a critical role as an energy source during cardiogenesis and that impaired glycogen synthesis may contribute to congenital heart disease.
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MATERIALS AND METHODS |
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30 mg) were homogenized in 10 or 30 volumes, respectively, of buffer containing 50 mM Tris-HCl (pH 7.8), 10 mM EDTA, 2 mM EGTA, 0.1 mM N-p-tosyl-L-lysine chloromethyl ketone, 2 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 50 mM mercaptoethanol, and 10 µg of leupeptin/ml with a Tissue Tearor model 285-370 (Biospec Products, Inc.) at the maximum setting (35,000 rpm) for 20 s. Tissue homogenates were used for Western analysis and assays of glycogen synthase and phosphorylase activities as described below. Protein content was determined by the method of Bradford (9). Western analysis. For Western analysis, a homogenate (25 µg of protein) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes and incubated with antibodies raised against rabbit muscle glycogen synthase (4). Detection was done with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Levels of protein expression were quantitated by densitometric scanning of autoradiograms.
Enzyme activities. Glycogen synthase activity in tissue homogenates was determined by measuring the incorporation of glucose from UDP-[U-14C]glucose into glycogen in the presence or absence of glucose-6-phosphate by the method of Thomas et al. (43) as described by Suzuki et al. (40). Reactions were carried out at 30°C typically for 10 to 15 min. Phosphorylase activity in tissue homogenates diluted 1:4 (heart) or 1:6 (muscle) was determined by monitoring the reverse reaction, in which the incorporation of [14C]glucose from [14C]glucose-1-phosphate (21) into glycogen in the absence or presence of AMP is measured as described by Suzuki et al. (40). The activity ratio is defined as the enzyme activity measured in the absence of an allosteric effector divided by that determined in the presence of 7.2 mM glucose-6-phosphate for glycogen synthase or 3 mM AMP for phosphorylase under standard conditions. Rabbit liver glycogen (Sigma-Aldrich) was deionized by passage through an MBD-22 (Resintech, Inc.) mixed-bed exchanger prior to use.
Determination of glycogen content.
Glycogen content was estimated by measuring glucose released by amyloglucosidase digestion of ethanol-precipitable material from muscle or liver tissue (40). Samples of frozen muscle and heart tissue (30 to 60 mg) were boiled in 200 µl of 30% (wt/vol) KOH for 30 min with occasional shaking. After cooling, 67 µl of 1 M Na2SO4 and 535 µl of ethanol were added. Next, samples were heated at 100°C for 2 min and centrifuged at 17,500 x g for 20 min at 4°C to collect glycogen. The glycogen pellet was suspended in water (100 µl), 200 µl of ethanol was added, and centrifugation as described above was used to harvest glycogen. This ethanol precipitation step was repeated, and the glycogen pellet was dried in a Speed-Vac. Dried glycogen pellets were suspended in 100 µl of amyloglucosidase (0.3 mg/ml in 0.2 M sodium acetate [pH 4.8]) and incubated at 37°C for 3 h to digest glycogen. To determine the glucose concentration in the samples, an aliquot (50 µl) of digested glycogen was added to 950 µl of a solution containing 0.3 M triethanolamine (pH 7.6), 0.4 mM MgCl2, 0.9 mM NADP, 1 mM ATP, and 1 µg of glucose-6-phosphate dehydrogenase/ml. The absorbance at 340 nm was read before and after the addition of 1 µg of hexokinase (5).
Timed matings. For timed matings, breeding pairs or trios were established by introducing female mice into cages with male mice in the late afternoon. On the following morning, females were checked for vaginal plugs indicative of mating. The date when a plug was found was designated embryonic day 0.5.
Histological analysis. Pregnant mice were sacrificed at various stages of gestation by cervical dislocation. Embryos were harvested, and either whole embryos or isolated hearts were fixed in 10% neutral buffered formalin or formalin-ethanol-acetic acid solution by standard methods. Adult hearts were isolated from mice sacrificed by cervical dislocation. Fixed samples were embedded in paraffin, sectioned (5 µm), and stained with hematoxylin-eosin to examine organ morphology.
Thymidine labeling of cardiomyocytes. To analyze the proliferative activity of the developing heart, timed-mated females were injected intraperitoneally with 200 µCi of [3H]thymidine (28 Ci/mmol; 1 mCi/ml; Amersham Biosciences Corp.). Embryos were harvested after a 3-h labeling period, followed by fixation (10% neutral buffered formalin) and paraffin sectioning. Deparaffined sections were stained with Hoechst stain in phosphate-buffered saline to identify the cell nucleus. The Hoechst-stained slides were coated with photographic emulsion (Polysciences, Inc., Warrington, Pa.) and further processed for autoradiography. The [3H]thymidine labeling index was defined as the percentage of labeled nuclei out of the total number of nuclei.
Echocardiography and blood pressure. To assess left ventricle function and dimensions, mice were subjected to echocardiography (35) at the Vanderbilt University School of Medicine Mouse Metabolic Phenotyping Center. Conscious animals were imaged by use of a 15-Mhz linear transducer; optimal parasternal long- and short-axis views were achieved by adjusting gain settings for visualizing endocardial and epicardial walls. Two-dimensional targeted M-Mode echocardiographic images were taken at the level of the papillary muscles. Measurements represent the average of three to five beats with the leading-edge technique. A trained echocardiographer reviewed and interpreted data from each mouse.
Blood pressure was also measured at the Vanderbilt University School of Medicine Mouse Metabolic Phenotyping Center; the tail cuff method was used (31). Mice were acclimated to the cuff apparatus on three successive days. Systolic pressure was measured twice for each mouse.
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RESULTS |
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Heart morphology in surviving adult MGSKO mice. Approximately 10% of the MGSKO mice survived, without an overt phenotype. Histological examination of hearts from 8- to 9-month-old mice revealed no large differences in heart morphology between wild-type and GYS1–/– adult mice (data not shown). Mice were subjected to both electrocardiography and echocardiography. Electrocardiography of 4- to 7-month-old male mice indicated no gross abnormalities in the hearts of null mice (data not shown). Mice at 5 months of age were examined by echocardiography, and no major differences in left ventricular dimensions or fractional shortening velocity were detected (Table 2). A trend toward a larger left ventricle mass was observed for GYS1–/– mice (Table 2), consistent with the significantly higher weights of GYS1–/– hearts (5.49 ± 0.18 [mean and standard error of the mean] and 4.42 ± 0.08 mg of heart/g of body weight for –/– mice and +/+ mice, respectively) (P = 0.0003). Tail cuff blood pressure measurements indicated no differences in systolic blood pressures (131 ± 5 and 121 ± 4 mm Hg for –/– mice and +/+ mice, respectively; n = 8 to 9). Heart rates measured at the same time (622 ± 23 and 643 ± 25 min–1 for –/– mice and +/+ mice, respectively) were also not significantly different. However, histological examination of the hearts of older male mice (12 to 16 months of age) revealed the presence of significant fibrosis in the GYS1–/– animals compared to the wild-type animals (Fig. 2k and l).
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50% in skeletal muscle or heart samples from heterozygous mice and was at the limit of detection of the assay in samples from GYS1–/– mice (Fig. 3b and 4b). The glycogen synthase activity ratio in skeletal muscle from heterozygous mice was not different from that in wild-type mice (Fig. 3c), but there was a small but significant increase in hearts from heterozygous mice relative to those from wild-type mice (Fig. 4c). The level of glycogen synthase protein, as judged by Western analysis, correlated with glycogen synthase activity (Fig. 3d and 4d).
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GSL25 and GSL30 mice were crossed with MGSKO mice to generate progeny heterozygous for endogenous GYS1 but expressing the transgene (MGSKO+/–/L25 and MGSKO+/–/L30, respectively). These mice then were mated with MGSKO+/– and MGSKO–/– animals to generate mice null for endogenous muscle glycogen synthase but expressing the transgene (MGSKO–/–/L25 and MGSKO–/–/L30, respectively). Crossing of GSL25 mice with MGSKO mice resulted in a threefold increase in the survival of pups null for endogenous GYS1 but carrying the transgene, compared to null mice not carrying the transgene (Table 3). Crossing MGSKO mice with GSL30 mice, which have much higher glycogen synthase expression levels, completely rescued the lethality associated with the null disruption of GYS1, so that the number of observed MGSKO–/–/L30 mice was that expected for Mendelian inheritance (Table 3).
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DISCUSSION |
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10% surviving birth. The viable mice, however, were for the most part normal, indicating that life is possible in the absence of glycogen in a variety of important organs. The presence of glycogen in various tissues during embryonic development was recognized almost 150 years ago (6). Studies of rat and rabbit (7) indicated a large accumulation of glycogen in developing heart. Approximately 2% of the cell volume of an adult cardiomyocyte is occupied by glycogen, while in fetal heart cells, glycogen can comprise up to 30% of the cell volume (36). In the rabbit (7), glycogen begins to accumulate in the heart on day 18 of embryonic development, corresponding to day 16 dpc in the mouse (10, 17a, 42). The maximum accumulation occurs between days 22 and 24, at which time the heart contains approximately 40 times the amount of glycogen found in the adult heart (7). In rat heart, massive amounts of glycogen are found in both 14.5 and 18.5 dpc embryos, corresponding to 13 and 17 dpc mouse embryos (1, 10, 17a, 42). Glycogen accumulation in the mouse embryo seems to have been analyzed only in preimplantation embryos. While there may be species differences in the profile of glycogen accumulation, rat and rabbit embryos have a high heart glycogen content at a stage of embryonic development approximately equivalent to when heart abnormalities are first observed in GYS1-null mice. Dawes et al. (14, 15) hypothesized that cardiac glycogen may function to allow embryos and newborn pups to survive anoxia. Our results indicate that the 10% of MGSKO pups that survive do so in the absence of cardiac glycogen. The more important role of cardiac glycogen appears to be during embryonic development.
At the stage of embryonic development where we observed a transition from normal to abnormal morphology in the hearts of GYS1-null embryos (i.e., 11.5 to 14.5 dpc), there appear to be no reports in the literature characterizing cardiac glycogen metabolism. The only study in this time frame was in rabbits at the stage corresponding to 13 dpc in mouse. At this stage heart had accumulated high levels of glycogen and exhibited glycogen synthase activity. Interestingly, during this period cardiac growth is an important event in response to the rapid increase of hemodynamic load and maintaining normal proliferative activity in cardiomyocytes is critical to producing a normal heart. The demonstration that the absence of glycogen led to abnormalities suggests that glycogen is required to maintain normal cardiomyocyte growth and chamber maturation at midgestation. Smaller hearts, less proliferative activity, and poor cardiac function in the GYS1-null embryos suggest that glycogen is an important energy source for developing cardiomyocytes to proliferate and to support adequate circulation for the developing embryos.
Why can 10% of embryos pass through this stage without cardiac glycogen? There are several possible explanations for this strong but not completely obligate requirement for glycogen. First, there is the issue of the genetic background. The mice analyzed have mixed 129/SvJ and C57BL/6J (75% C57BL/6J and 25% 129/SvJ) backgrounds, and there could be some other genetic factors, even in littermates, that combine with the absence of GSY1 to cause the observed pathology. We consider this possibility unlikely. The MGSKO mice have now been backcrossed six times into the SvJ background and eight times into the C57 background. Breeding heterozygotes of these F6 and F8 mice is not resulting in any increase in the proportion of GYS1–/– survivors (unpublished data). A second possibility is if the embryos that survive the critical stage in development have undergone an adaptive response. This reasoning is proposed by Meeson et al. (33) to explain their observation of partial embryonic lethality in embryos null for myoglobin. The majority of mice lacking myoglobin die at midgestation, but a small number survive by mounting an adaptive response via reprogramming gene expression (33). A third possibility is that glycogen is an energy source for completion of a certain developmental phase, as discussed above, but there is enough variability that statistically some embryos are successful even in its absence.
Our immediate thought, given the defects in cardiac development and signs of impaired cardiac function in the affected embryos, is that the death of the MGSKO mice is attributable to defects in the heart. After switching at birth to a circulation completely independent of the mother, the defective heart would fail in the face of the increased load and perhaps contribute to the failure of the lungs to inflate (13, 27). Some support for lack of cardiac glycogen causing perinatal mortality came from expressing the glycogen synthase transgene of the GSL mice in the MGSKO background. In hearts from GSL25 mice, the glycogen synthase activity and glycogen levels are indistinguishable from wild-type but in the GSL30 line there is a sevenfold increase in cardiac glycogen. The observation that the glycogen synthase transgene from the GSL30 line completely restored viability to mice lacking a functional GYS1 gene while that from GSL25 does not, supports the hypothesis that the need for glycogen in cardiac development is the cause of death in pups unable to synthesize this polymer. A more definitive demonstration should come from expression of glycogen synthase driven by a heart specific promoter in the MGSKO background to see if this restores viability to the offspring. We are currently crossing transgenic mice expressing glycogen synthase under the control of the atrial natriuretic factor promoter with MGSKO mice.
Adult hearts from MGSKO mice less than 1 year old were normal by our analyses, except for their lack of glycogen and glycogen synthase activity and a larger cardiac mass. Other than by size, the hearts were normal morphologically. Analysis by echocardiography revealed no major alterations in cardiac dimensions in male MGSKO mice, except for a trend toward increased left ventricular mass, consistent with overall heart weight. Functionally, no abnormalities were observed. Likewise, electrocardiograms were not anomalous. However, we cannot exclude the possibility that the hearts might have behaved abnormally in response to specific stresses, such as ischemia or ß-adrenergic stimulation. The only cardiac anomaly detected was in older MGSKO mice, more than 1 year old, in which fibrosis became apparent. How this observation is related to cardiac glycogen is not readily apparent but could be linked to reduced ability of the myocytes to cope with stresses as the animals grow older. Most glycogen-based aberrations in hearts have been linked to the hyperaccumulation of glycogen, such as in patients with various glycogen storage diseases (reviewed in references 17 and 18) or Wolff-Parkinson-White syndrome (2). A mouse model involving overexpression of a mutant form of the 
2 subunit of AMP-activated protein kinase causes glycogen hyperaccumulation (30-fold above that in the wild type) in the heart (3). These mice suffer from ventricular hypertrophy and electrophysical abnormalities. It will be worthwhile to study more extensively hearts from MGSKO mice in terms of stress and cardiac function upon aging.
In summary, from the analysis of genetically engineered mice, we have established that animals can exist, ostensibly normally, in the absence of the storage polymer glycogen in several important tissues, including skeletal muscle and heart. However, we found that lack of glycogen had a severe impact on cardiac development, resulting in perinatal mortality. It is interesting that congenital heart disease in humans is extremely common, resulting in a variety of abnormalities of differing severity (11, 28). As many as 1 in 50 live births may involve a moderate to serious cardiac deformity, the cause of which is often not known. Many of these defects likely result from genetic or environmental challenges to the programs governing gene expression patterns, cell fate determination, and tissue formation. Analysis of MGSKO mice, however, has demonstrated that a simple metabolic defect can also have grave consequences for cardiac development and it is not impossible that impaired glycogen metabolism contributes to congenital heart disease in humans. Mice lacking cardiac glycogen should provide valuable insights into the role of glycogen in heart development and function.
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ACKNOWLEDGMENTS |
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We thank David Wasserman and Owen McGuiness of Vanderbilt University and Beth Thurberg of Genzyme Corporation, Framingham, Mass., for helpful advice and discussions. We are indebted to Cheryl Bock of the Comprehensive Cancer Center, Duke University Medical Center, for karyotyping ES cells. Carlie R. Cope provided invaluable technical assistance.
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FOOTNOTES |
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