The heart is constantly challenged by stresses during the ageing process. Current research suggests that ageing is accompanied by an increase in oxidative and Endoplasmic (ER) stress. Oxidative damage and ER stress is prevented by protective intracellular signaling pathways including the Unfolded Protein Response (UPR) from ER. UPR plays an important role in ageing-related human diseases and its contribution to heart disease has just begun to be recognized. A surge of new discoveries has led to the identification and characterization of IRE11, PERK, and ATF6, the highly conserved UPR machinery that maintains ER homeostasis. Little information is available about the specific roles of IRE11 in heart diseases. In preliminary studies, ectopic expression of IRE11 in cultured cardiomyocytes was sufficient to induce transient downstream activity but not sustained downstream ER stress signaling. The transient nature of IRE11 downstream activity indicates the existence of a potent negative regulator for IRE11 activity in cardiomyocytes. Protein Phosphatase 2C (PP2Ce) was identified as a component of an IRE11 signaling complex. PP2Ce is highly enriched in heart and is exclusively targeted to ER membrane. It possesses specific phosphatase activity against IRE11 and functions as a potent negative regulator of IRE11 signaling activity. These interesting new findings lead to the hypothesis that PP2Ce functions as an endogenous negative regulator of IRE11 in heart and modulates ER stress response in response to ageing and other pathological stresses. This hypothesis will be tested with biochemical, cellular and whole-heart studies Specific aim 1: Characterize the functional impact of IRE11 PP2Ce mediated signaling in cardiomyocytes. Neonatal Rat Ventricular Myocytes (NRVM) will be used as an in vitro model system for heart. Genetic manipulation b adenovirus-mediated gene transfer of NRVM will be used to characterize the direct impact of IRE11 activation, or IRE11 inactivation by PP2Ce, in UPR signaling under basal and oxidative stress. The impact of IRE11 PP2Ce pathway on cardiomyocytes signaling, hypertrophy, morphology, gene expression and viability will be measured.
Specific Aim 2 : Investigate the molecular mechanims of PP2Ce mediated IRE11 regulation. The molecular basis of PP2Ce mediated feedback mechanism will be determined at transcription/protein expression level, protein/protein interaction level or PP2Ce phosphatase activity level.
Specific Aim 3 : Determine the functional role of cardiac IRE11/PP2Ce signaling in vivo. Several genetic models have been developed in order to investigate the functional role if IRE11 and PP2Ce activity on cardiac function and the pathological process in response to ageing and pressure-overload. This study has great potential to uncover novel insights of disease mechanisms for heart diseases and failure and offers a comprehensive and in-depth training in an integrated and multi-disciplinary study.
Project Narrative Heart disease affects more than 79 million individuals in the U.S. and increases in prevalence with age. Treatments for heart disease remain elusive due to our limited understanding of fundamental mechanisms of both ageing and heart disease. This investigation seeks to uncover mechanisms of protection from oxidative stress, which accompanies ageing. Importantly, this study also aims to identify novel therapeutic strategies.