Abnormal cardiac development in the absence of heart glycogen

Abstract

Glycogen serves as a repository of glucose in many mammalian tissues. Mice lacking this glucose reserve in muscle, heart, and several other tissues were generated by disruption of the GYS1 gene, which encodes an isoform of glycogen synthase. Crossing mice heterozygous for the GYS1 disruption resulted in a significant underrepresentation of GYS1-null mice in the offspring. Timed matings established that Mendelian inheritance was followed for up to 18.5 days postcoitum (dpc) and that approximately 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.

FIG. 1.

Disruption of the GYS1 gene. LTR, long terminal repeat; β-Geo, β-galactosidase-neomycin phosphotransferase fusion gene (45); pA, polyadenylation sequence; SA, splice acceptor sequence; SD, splice donor sequence; PGK, phosphoglycerate kinase 1 promoter; BTK, Bruton's tyrosine kinase; OST, Omnibank sequence tag.

FIG. 2.

Morphological and histological analyses of GYS1-deficient mice. (a) At E14.5, GYS1-deficient embryos have prominent vessels congested with blood and hemorrhagic livers. Red arrows indicate congested blood vessels; yellow arrows indicate livers. (b) Dark-field images of sagittal sections of E14.5 embryos. White arrows indicate the autofluorescence of red blood cells pooled in the hearts and blood vessels throughout GYS1-deficient embryos. ht, heart; lv, liver; lu, lung. (c) The majority of GYS1-deficient mice fail to inflate the lungs at the time of birth and die within 5 to 10 min after birth. (d) At E18.5, GYS1-deficient mice have significantly smaller ventricles but dilated atria. Ra, right atrium; La, left atrium; Rv, right ventricle; Lv, left ventricle. (e and f) Transverse sections of wild-type and GYS1-deficient embryos at E14.5. Mutant hearts have thin ventricular walls and dilated atria and show abnormal formation of the ventricular septum. Note the huge amount of pooled blood in mutant atria and blood vessels, suggesting very poor cardiac function. Sep, septum. (g and h) Histological sections of wild-type and GYS1-deficient lungs (left lobes). Note severe pulmonary congestion in the mutant lungs. White arrows indicate pooled blood in pulmonary vessels. (i and j) Periodic acid-Schiff (PAS) staining of heart sections from wild-type and GYS1-deficient embryos at 18.5 dpc. Staining was done to visualize the level of glycogen content in cardiomyocytes. Mutants do not appear to have glycogen in cardiomyocytes. (k and l) Trichrome (Masson) staining. Staining was done to visualize abnormal fibrosis (arrows) in aged GYS1-deficient adult ventricles compared to those in age-matched littermate controls.

FIG. 3.

Glycogen biosynthesis in skeletal muscle. Glycogen content (a), glycogen synthase activity (b), glycogen synthase activity ratio (c), and glycogen synthase protein expression (d) were determined for skeletal muscle from GYS1 wild-type, heterozygous, and homozygous null 7- to 9-month-old male mice as described in Materials and Methods. Values are reported as means and standard errors of the mean (n = 5 to 9). An asterisk indicates a P value of <0.0005 for comparisons with wild-type measurements.

FIG. 4.

Glycogen biosynthesis in cardiac muscle. Hearts from GYS1 wild-type, heterozygous, and homozygous null 5- to 9-month-old male mice were analyzed for glycogen content (a), glycogen synthase activity (b), glycogen synthase activity ratio (c), and glycogen synthase protein expression (d) as described in Materials and Methods. Values are reported as means and standard errors of the mean (n = 3 to 6). An asterisk indicates a P value of <0.05 for comparisons with wild-type measurements. nd, not detectable.

FIG. 5.

Glycogen phosphorylase activity in skeletal muscle and cardiac muscle. Glycogen phosphorylase activity ratio (a and c) and total activity (b and d) were determined for cardiac muscle (a and b) and skeletal muscle (c and d) from 5- to 9-month-old male mice as described in Materials and Methods. Values are reported as means and standard errors of the mean (n = 3 to 6). An asterisk indicates a P value of <0.0005 for comparisons with wild-type measurements; a number sign indicates a P value of 0.02 for comparisons with −/− measurements.