Abstract
Hypoxic exposure during development can have a profound influence on offspring physiology, including cardiac dysfunction, yet many reptile embryos naturally experience periods of hypoxia in buried nests. American alligators experimentally exposed to developmental hypoxia demonstrate morphological and functional changes to the heart that persist into later life stages; however, the molecular bases of these changes remain unknown. We tested if targeted and persistent changes in steady-state protein expression underlie this hypoxic heart phenotype, using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics. Alligator eggs were reared under normoxia or 10% hypoxia, then either sampled (embryo) or returned to normoxia for 2 years (juvenile). Three salient findings emerge from the integrated analysis of the 145 differentially expressed proteins in hypoxia-reared animals: (1) significant protein-protein interaction networks were identified only in up-regulated proteins, indicating that the effects of developmental hypoxia are stimulatory and directed; (2) the up-regulated proteins substantially enriched processes related to protein turnover, cellular organization, and metabolic pathways, supporting increased resource allocation towards building and maintaining a higher functioning heart; and (3) the juvenile cardiac proteome retained many of the signature changes observed in embryonic hearts, supporting long-term reprogramming of cardiac myocytes induced by hypoxia during critical periods of development.
Highlights
Developmental plasticity is the process by which phenotypic variation in offspring is induced by environmental variables, and its contribution to everything from evolutionary processes[1] to human disease pathology[2,3,4,5] is widely recognized
Recent longitudinal studies of reptiles that were exposed to hypoxia during embryonic development and returned to normoxia after hatching, suggest that some of these phenotypic responses are maintained into later life stages
In keeping with our main objective of determining the impacts of developmental hypoxia exposure on the cardiac proteome, only protein expression changes related to oxygen (Fig. 1a) and interaction (Fig. 1b) are considered
Summary
Developmental plasticity is the process by which phenotypic variation in offspring is induced by environmental variables, and its contribution to everything from evolutionary processes[1] to human disease pathology[2,3,4,5] is widely recognized. Joyce and colleagues[19] reported that the increase in relative heart mass of alligators exposed to developmental hypoxia is maintained in 4 y old juveniles, and demonstrated increased β-adrenergic sensitivity during exercise in these animals[19] Combined, these studies provide compelling evidence that developmental hypoxia exposure reprograms heart morphology and function to augment performance under conditions where oxygen is limiting We used quantitative shotgun proteomics to simultaneously identify and quantify the relative abundance of hundreds of proteins in the hearts of embryonic and juvenile alligators that were either reared continuously in normoxia, or were exposed to hypoxia during early development This powerful, comprehensive approach revealed a suite of targeted changes in protein abundance induced by hypoxia that occur during embryonic development and are carried forward into later life stages. These protein expression changes contribute to a significant enrichment of functional pathways related to protein turnover, cellular organization, and cellular metabolism, and these findings are discussed in relation to increased performance of the heart in hypoxia
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