Redox regulation and oxidative stress… (F31HL189410) – Project Grant to President And Fellows Of Harvard…

Abstract: Mammalian birth is accompanied by a significant shift in oxygen tension, triggering cascades of adaptive mechanisms in neonatal cells and tissues. For example, cardiac muscle cells, known as cardiomyocytes, rewire their cellular energy metabolism, undergo hypertrophic growth, and ultimately transition to a quiescent state following parturition. This loss of proliferative capacity, a signature of cardiomyocyte maturity, instates a barrier to adult heart regeneration, and remains a significant hurdle in the development of cardiac regenerative therapies. Evidence suggests that increased relative oxygen exposure augments levels of reactive oxygen species (ROS) in cardiomyocytes following birth, perhaps driving some of these maturational processes. However, the detailed mechanisms of redox regulation in this context remain ILL-defined. Hydrogen peroxide (H2O2), a nonradical ROS, is now recognized as a pleiotropic signaling molecule, wherein steady-state, low levels are essential for basic cellular processes, and moderate levels invoke hormetic responses. Moreover, H2O2 can tune protein function via reversible covalent modifications of protein cysteine residues, such that cysteine thiol oxidation is now considered a major mode of post-translational regulation. Broadly deciphering the direct protein targets of cysteine thiol oxidation in vivo with absolute stoichiometric quantitation has been a major technical challenge in redox biology. To address this unmet need, our lab developed a method named cysteine reactive phosphate tag (CPT) proteomics, which enables broad, quantitative annotations of the cysteine redox proteome. I hypothesize that following birth and ambient oxygen exposure, cysteine thiol oxidation on select proteins drives cardiomyocyte maturation and cell cycle exit. In aim 1, i will leverage CPT proteomics to map protein targets of extensive cysteine thiol oxidation in pre- and postnatal murine hearts. I will perform protein and site-level analyses to define and stratify redox-regulated proteins that change following birth. In aim 2, i will test if H2O2 is sufficient for driving cardiomyocyte maturation in vivo using a transgenic mouse line generated by our lab, in which we can selectively generate cardiomyocyte-specific H2O2 in utero. Further, because supraphysiologic concentrations of H2O2 can disrupt oxidant balance and lead to dysregulation of redox signaling, i predict premature, prolonged exposure to H2O2 at critical stages in cardiac development promotes dysregulation of existing redox networks, underlying several features of neonatal cardiac pathology. Thus, i will leverage this model to investigate how extended exposure to H2O2 during cardiac development remodels cysteine oxidation networks, and investigate how these findings map onto established disease-relevant protein families. Together, this knowledge will inform our understanding of redox regulation in postnatal cardiac development, which may be harnessed to improve neonatal cardiac care, and optimize adult cardiac regenerative strategies.

Redox regulation and oxidative stress in cardiac development and regeneration

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