Mitochondrial pp60Src and cellular metabolism in pulmonary vascular disease associated with CHD

Florida International University, Miami FL

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Abstract

ABSTRACT Children born with congenital heart defects that cause pulmonary over-circulation develop abnormal pulmonary vascular reactivity. Although survival has improved, they are at significant risk for developing pulmonary vascular disease and continue to suffer morbidity and late mortality. Even early pulmonary endothelial dysfunction, with abnormal vascular reactivity, causes substantial morbidity and mortality. There is an intimate and complex relationship between cellular metabolism and endothelial dysfunction. However, how these processes are linked is unresolved, and unraveling novel connections will be the central goal of this Project. Our group is at the forefront of investigating the role played by post-translational modifications (PTMs) in the metabolic reprogramming associated with pulmonary vascular disease. In the current funding period, we demonstrated that modulating pp60Src activity alters phospho-tyrosine (pY) levels within the mitochondria and changes the assembly and activity of electron transport chain (ETC) Complex I. We identified the Complex I substrates as NDUFAF5, NDUFS1, NDUFV2, and the specific pY sites. We demonstrated that mitochondrial pp60Src activity is reduced in pulmonary arterial endothelial cells (ECs) isolated from our lamb model of pulmonary over- circulation (Shunt). This correlates with decreased Complex I assembly and activity. Exciting pilot data show that decreases in mitochondrial pp60Src are due to a reduction in the expression of downstream tyrosine kinase/docking protein-4 (dok-4). Restoring dok-4 levels in Shunt EC rejuvenates mitochondrial bioenergetics and mitigates the hyperproliferative angiogenic phenotype. c-Jun N-terminal kinase (JNK) activity is increased in Shunt EC and exhibits increased mitochondrial localization. JNK can phosphorylate SH3 homology- associated BTK binding protein (SAB) on the outer mitochondrial membrane. Blocking JNK-mediated SAB phosphorylation increases mitochondrial pp60Src levels in Shunt EC. Based on these extensive data, Project #2 will test the overall hypothesis that pulmonary over-circulation-dependent disruption of endothelial cell metabolism involves a loss of dok-4 expression, increased JNK-mediated SAB phosphorylation, and attenuated mitochondrial import of pp60Src. Three Specific Aims (SA) are proposed to test this highly novel hypothesis. SA #1 will Investigate how the dysregulation of dok-4 disrupts mitochondrial pp60Src import and affects mitochondrial bioenergetics and NO signaling. SA #2 will investigate how the loss of mitochondrially localized pp60Src attenuates Complex I activity and stimulates metabolic rewiring to develop a hyperproliferative antiapoptotic angiogenic EC phenotype. SA#3 will evaluate the therapeutic potential of stimulating mitochondrial pp60Src activity to prevent the development of the pulmonary vascular disease associated with pulmonary over- circulation. The successful completion of our proposed studies should yield new mechanistic insights and identify new targets amenable to therapeutic intervention in children born with congenital heart defects that result in pulmonary over-circulation.