Glycogen syntheses kinase-3b (GSK-3b) is a ubiquitously expressed serine/heroine protein kinase that has multiple functions in cells, including growth, death and metabolism. GSK-3b is active under resting conditions and inhibited via phosphorylation at Serine9 (S9) by upstream protein kinase B/Akt. It has been shown that S9 phosphorylation of GSK-3b is highly elevated in the heart of old animals and in stress-induced cellular senescence. Previous work done by the applicant revealed that inhibition of GSK-3b inhibits autophagy in the heart under stress conditions. Accumulating lines of evidence suggest that a decrease in autophagy activity may play a major role in accumulation of altered macromolecules and organelles, causing aging-related abnormalities. Our hypotheses are: 1) GSK-3b regulates autophagy by phosphorylating Unc-51 like kinase 1 (Ulk1), and 2) GSK-3b modulates cardiac aging through Ulk1-dependent regulation of autophagy. There are four specific aims in this proposal to test our hypotheses.
In Aim 1, it will be determined if GSK-3b phosphorylates Ulk1 and if so, which residue of Ulk1 is phosphorylated by GSK-3b. An in vitro kinase assay and mass spectrometry will be used in this aim.
In Aim 2, it will be determined if phosphorylation of Ulk1 by GSK-3b participates in modulation of autophagy. Glucose deprivation of neonatal rat cardiac myocytes will be used as a model to stimulate autophagy. We have generated adenovirus harboring mRFP-GFP-LC3 to study autophagic flux.
In Aim 3, it will be determined, in constitutively active GSK-3bS9A knock-in mice and GSK- 3bS9A/mRFP-GFP-LC3 bigenic mice, if aging-associated changes, including autophagy, in the heart are attenuated. Postmortem measurements of organ weight, left ventricular catheterization, pressure-volume loop analysis, echocardiography, measurements of cardiac fibrosis, cardiac myocyte size, apoptosis and necrosis will be applied to characterize the aging-related changes in the heart. Immunoblotting of aging marker will also be carried out. Immunoblotting of p62 and LC3, and measurements of red and yellow puncta in images taken from cardiac sections of mRFP-GFP-LC3 transgenic mice and the bigenic mice will be employed to evaluate autophagy and autophagic flux in vivo.
In Aim 4, it will be determined, in GSK-3bS9A/Ulk1+/- bigenic mice, if knock-out of Ulk1 attenuates the effects of GSK-3b on aging-associated changes in the heart. Information obtained from the proposed studies will provide insights into the role of GSK-3ss and autophagy in aging and help to develop strategies specifically targeting GSK-3b and autophagy to modulate the adverse effects of aging.
The prevalence of heart diseases increases dramatically in aging populations. The research project proposed in this application will obtain information to help developing strategies to modulate the aging-related abnormalities in the heart.