Although mutations in the subunit of AMP-activated protein kinase (AMPK) result in excessive glycogen accumulation and cardiac hypertrophy, the mechanisms by which this occurs have not been well defined. As greater than 65% of cardiac AMPK activity is associated with the 1 subunit of AMPK, we investigated the effects of expression of an AMPK-activating 1 subunit mutant ( 1 R70Q) on regulatory pathways controlling glycogen accumulation and cardiac hypertrophy in neonatal rat cardiac myocytes. While expression of 1 R70Q displayed the expected increase in palmitate oxidation rates, rates of glycolysis were significantly depressed. Additionally, glycogen synthase activity was increased in cardiac myocytes expressing 1 R70Q, as a result of both increased expression and decreased phosphorylation of glycogen synthase. The inhibition of glycolysis and increased glycogen synthase activity were correlated with elevated glycogen levels in 1 R70Q-expressing myocytes. In association with the reduced phosphorylation of glycogen synthase, glycogen synthase kinase (GSK)-3 protein and mRNA levels were profoundly decreased in the 1 R70Q-expressing myocytes. Consistent with GSK-3 as a negative regulator of hypertrophy via inhibition of nuclear factor of activated T cells (NFAT), the dramatic downregulation of GSK-3was associated with increased nuclear activity of NFAT. Together, these data provide important new information as to the mechanisms by which a mutation in the subunit of AMPK causes altered AMPK signaling and have identified multiple pathways involved in regulating both cardiac myocyte metabolism and growth that may contribute to the development of the mutant-associated cardiomyopathy.