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Biological/Clinical Application 1

PI: David Kass            Collaborators: David Graham, Jennifer Van Eyk, Akhilesh Pandey


G-coupled receptor signaling PTM in HF

The Kass laboratory has pioneered research into the role of cGMP/PKG modulation of maladaptive hypertrophic remodeling to pressure-overload, revealing a broad transcriptional and PTM program that allows the heart to counter sustained stress with better function, less hypertrophy, and without developing HF.  Improvement occurs in metabolic proteins with reduction of oxidant stress stimuli, and in PTM changes that enhance myocyte contraction. Another mechanical stress model involves cardiac dysynchrony in hearts with a conduction block so that one part of the ventricle contracts later than the other.  The heart becomes markedly heterogeneous, and with O’Rourke, Van Eyk and other collaborators, we have shown this results in a marked dysfunction in G-coupled receptor signaling.  A therapy known as cardiac resynchronization (CRT), first clinically pioneered in the U.S. by Dr. Kass and colleagues, has recently been shown to reverse many of these molecular changes.

Overall Research Plan: We will advance and utilize proteomic methods to define the plasma membrane micro-domain and b-adrenergic signaling pathways in models of mechanical stress-induced cardiomyopathy, focusing on changes that are reversed by successful treatments for these disorders in mice and then, target these in humans. 

Goal 1: Using quantitative mass spectrometry and new methods of membrane/targeted MRM, we will assess changes in beta-receptor PTMs in models of sustained cardiac mechanical stress.

Rationale:  Studies in mice subjected to trans-aortic constriction and then treated with the nitric oxide synthase co-factor BH4 revealed the capacity of the latter to suppress maladaptive remodeling and reverse fibrosis and hypertrophy. CRT improves LV function and survival in patients with HF and contractile dyssynchrony, and it is the only treatment known that both acutely and chronically improves heart systolic function, but also reduces long-term mortality.

Goal 2:  Detailed targeted quantitative analysis of the beta-adrenergic signaling pathways in mouse models of HF.

Rationale:  While the importance of b1-AR and b2-AR and associated signaling in HF is unquestioned, the low abundance of these proteins and inadequate affinity of existing Abs for pull-down studies has made this difficult to assess. Therefore, we will target the known proteins within this pathway using enrichment strategies and MRM to obtain data about the quantities of the proteins and stoichiometry of each known phosphorylation site.

Goal 3:  Develop MRM methods to probe the membrane domains and beta-adrenergic signaling pathways in human heart biopsies.

Rationale: We expect to find many membrane domain protein changes in one or more of the animal models, each potentially representing an important modifier of outside-in (and inside-out) signaling in the development of HF. Those that are particularly reversed by treatment will provide equally important insight into potential therapeutic pathways.