Direct proteasomal enhancement contributes to PKG-triggered cardioprotection

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DESCRIPTION (provided by applicant): Direct proteasomal enhancement contributes to PKG-triggered cardioprotection Ischemic heart disease (IHD) is the most common heart disease in the US, a leading cause of morbidity and mortality in humans. Ischemia/reperfusion (I/R) injury is an important pathological process during not only the intervention but also the natural disease progress of IHD. Besides genetic mutations, cardiac hypertrophy and ischemia/reperfusion (I/R) can both rapidly increase production of misfolded proteins as well. Under normal conditions, cardiomyocytes remove misfolded proteins via the ubiquitin proteasome system. The highly regulated process requires ubiquitination of the targeted protein followed by its transfer to the proteasome for degradation. Cardiac proteasome functional insufficiency has been implicated in the accumulation of ubiquitinated proteins that characterize most heart diseases. Improving proteasome function would be a strategic approach to enhance removal of ubiquitinated proteins; however a known method to do so remains elusive. Posttranslational modifications of proteasome subunits elicit instant adaptation of proteasome activities to receptor stimulation/stress, and targeted modifications could prove of significant therapeutic benefit. The aim of the research project of this postdoctoral fellowship is to elucidate a novel mechanism of PKG manipulation on the proteasome that results in increased proteasome activity and determine if the proteasome enhancement contributes to PKG-triggered cardioprotection. Results of the research would enable development of a more tactful approach to treat heart disease by way of improving proteasome function. PKG activity can be increased by phosphodiesterase 5 inhibitors (e.g. sildenafil) that have known positive effects on cardioprotection, though their mechanism of protection is unknown. The specific aims of this proposal couple mechanistic analysis of the PKG pathway with physiological effect of PKG treatment. The first aim of the project will employ a comprehensive proteomics approach to determine the site(s) of posttranslational modification by PKG on the proteasome and assess PKG-induced changes in proteasome activity. This will enable, and is essential to, determination of the cause-effect relationship between the specific PKG phosphorylation and proteasome functional change. The second aim will test the hypothesis that proteasomal enhancement by PKG manipulation contributes to cardioprotection. A unique mouse model of cardiomyocyte-restricted moderate expression of a peptidase-disabled proteasome subunit will undergo I/R surgery with comparative sildenafil and placebo treatments. The work will delineate a novel mechanism by which proteasome function is regulated and yield significant new insight into how the PKG pathway protects the heart. This will ultimately facilitate the search for new measures to prevent and/or more effectively treat heart disease.