Activated glycogen synthase-3 beta suppresses cardiac hypertrophy in vivo

Abstract

The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium/calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) phosphorylates NFAT proteins and antagonizes the actions of calcineurin by stimulating NFAT nuclear export. To determine whether activated GSK-3 can act as an antagonist of hypertrophic signaling in the adult heart in vivo, we generated transgenic mice that express a constitutively active form of GSK-3 beta under control of a cardiac-specific promoter. These mice were physiologically normal under nonstressed conditions, but their ability to mount a hypertrophic response to calcineurin activation was severely impaired. Similarly, cardiac-specific expression of activated GSK-3 beta diminished hypertrophy in response to chronic beta-adrenergic stimulation and pressure overload. These findings reveal a role for GSK-3 beta as an inhibitor of hypertrophic signaling in the intact myocardium and suggest that elevation of cardiac GSK-3 beta activity may provide clinical benefit in the treatment of pathologic hypertrophy and heart failure.

Figure 1

α-MHC-GSK-3βS9A transgene expression. Heart extracts were prepared from transgenic and nontransgenic littermates and analyzed for expression of GSK-3 by Western blot analysis as described in Materials and Methods. (Upper) Anti-hemagglutinin antibody detects the epitope-tagged GSK-3βS9A protein in extracts from transgenic lines 10 and 12. (Lower) Anti-GSK-3 antibody detects endogenous GSK-3 isoforms, as well as exogenous GSK-3S9A. The GSK-3βS9A protein is expressed at a level ≈8-fold higher than endogenous α and β isoforms.

Figure 2

Effects of GSK-3βS9A on cardiac hypertrophy induced by activated calcineurin. (A) Hearts were removed from 4-week-old mice of the indicated genotypes. (Scale bar, 2 mm.) Histological sections stained with hematoxylin and eosin (H&E) are shown at ×40 magnification. (Scale bar, 0.02 mm.) (B) Heart-to-body-weight ratios of 4- and 9-week-old mice of the indicated genotypes were determined. Heart sizes of at least six mice of each genotype were determined. Values represent mean ± SD. (C) Cardiomyocyte area was determined in histological sections from 4-week-old mice shown in A, as described in Materials and Methods. Values represent mean ± SD. (D) Expression of transcripts for β-MHC, ANF, and BNP in RNA samples from 4-week-old mice of the indicated genotypes were determined as described in Materials and Methods.

Figure 3

Effects of GSK-3βS9A on cardiac hypertrophy induced by isoproterenol stimulation and pressure overload. (A) Masson-trichrome staining of hematoxylin and eosin (H&E)-stained histological sections of hearts from animals implanted with miniosmotic pumps that delivered saline or isoproterenol. (Scale bar, 2 mm.) Note that the heart from the GSK-3βS9A transgenic treated with isoproterenol developed an atrial thrombosis. (B) Heart-to-body-weight ratios of mice of the indicated genotypes were determined. Heart sizes of at least six mice of each genotype were determined. (C) The ratio of LV mass to tibia length of mice of the indicated genotypes were determined 21 days after thoracic aortic banding (TAB). Sham-operated littermates served as controls. At least five mice of each genotype were analyzed. Values represent mean ± SD.

Figure 4

Calcineurin-induced nuclear localization of NFAT is antagonized by activated GSK-3S9A. (A) Representative view of a cardiomyocyte nucleus from an α-MHC-calcineurin transgenic mouse immunostained for NFATc1 expression, as described in Materials and Methods. NFAT staining is observed as punctate dots in the nucleus. Propidium iodide (red) was used as a nuclear counter stain. (B) The graph shows the relative number of NFAT-positive nuclei in sections of cardiac ventricles of mice of the indicated genotypes. The number of NFAT-positive nuclei in sections from α-MHC-calcineurin transgenic mice was set at 100%.

Figure 5

A model depicting the antagonistic effects of calcineurin and GSK-3 on NFAT activity and cardiac hypertrophy. Numerous hypertrophic stimuli activate calcineurin and Akt. Calcineurin dephosphorylates NFAT proteins, which results in their translocation into the nucleus and activation of the hypertrophic gene program. GSK-3β antagonizes calcineurin signaling by phosphorylation NFAT proteins. GSK-3β is inactivated by phosphorylation of serine-9 by Akt and other kinases. GSK-3β also has other substrates that could influence hypertrophic signaling. Calcineurin activity is also inhibited by myocyte-enriched calcineurin-interacting protein (MCIP).