Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy

Abstract

Mutations in PRKAG2, the gene for the gamma 2 regulatory subunit of AMP-activated protein kinase, cause cardiac hypertrophy and electrophysiologic abnormalities, particularly preexcitation (Wolff-Parkinson-White syndrome) and atrioventricular conduction block. To understand the mechanisms by which PRKAG2 defects cause disease, we defined novel mutations, characterized the associated cardiac histopathology, and studied the consequences of introducing these mutations into the yeast homologue of PRKAG2, Snf4. Although the cardiac pathology caused by PRKAG2 mutations Arg302Gln, Thr400Asn, and Asn488Ile include myocyte enlargement and minimal interstitial fibrosis, these mutations were not associated with myocyte and myofibrillar disarray, the pathognomonic features of hypertrophic cardiomyopathy caused by sarcomere protein mutations. Instead PRKAG2 mutations caused pronounced vacuole formation within myocytes. Several lines of evidence indicated these vacuoles were filled with glycogen-associated granules. Analyses of the effects of human PRKAG2 mutations on Snf1/Snf4 kinase function demonstrated constitutive activity, which could foster glycogen accumulation. Taken together, our data indicate that PRKAG2 mutations do not cause hypertrophic cardiomyopathy but rather lead to a novel myocardial metabolic storage disease, in which hypertrophy, ventricular pre-excitation and conduction system defects coexist.

Figure 1

Identification of three PRKAG2 mutations inherited in six families and the clinical consequences of these mutations. (a) Pedigrees indicate clinical findings of cardiac hypertrophy (left half filled), WPW (right upper quadrant filled), or conduction system disease (right lower quadrant filled) in individuals with a PRKAG2 mutation (+). Open symbols denote unaffected individuals, and shading denotes uncertain clinical status. (b) Sequence traces demonstrating G995A substitution (exon 7), C1289A (exon 11), and A1553T (exon 14), encoding Arg302Gln, Thr400Asn, and Asn488Ile substitutions, respectively. (c) Comparison of PRKAG protein sequences demonstrates evolutionary conservation of residues altered by mutation. Note that human PRKAG2 Arg302Gln mutation is homologous to R200Q mutation in RN^– pigs.

Figure 2

Histopathology of left ventricular sections obtained from individuals with PRKAG2 mutations. (a) Longitudinal section of left ventricular myocardium from a 26-year-old individual with PRKAG2 mutation Asn488Ile who died suddenly. Note vacuolated myocytes, and lack of myofiber disarray or fibrosis (H&E; bar = 100 μm). (b) High-power magnification of an endomyocardial biopsy (H&E; bar = 100 μm) from a 39-year-old individual with PRKAG2 mutation Thr400Asn shows profound vacuolization (arrows). (c) Homogenous inclusions within vacuoles (arrows and inset) stained positive with PAS are mostly diastase-resistant. (d) Electron micrograph (uranyl acetate and lead citrate) of a sample described in a (bar = 1 μm). Note the large, irregular sarcoplasmic inclusion (arrows) within a large vacuole, and normal-appearing sarcomeres (arrowhead). (e) Higher magnification (bar = 1 μm) demonstrates that the inclusion is composed of a central core of homogenous, electron-dense droplets surrounded by osmiophilic granular and fibrillar material (star).

Figure 3

Snf1-Snf4 two-hybrid interactions in the presence and absence of glucose. β-Galactosidase activity was measured in cell cultures grown in SD glucose (a) or galactose/raffinose (b) media. The activity from three cultures from different colonies is reported in units (hydrolysis of 1 μmol CPRG/min/cell), mean ± SD. WT, wild-type. *P < 0.02, **P < 0.005 versus wild-type Snf4.