Cardiovascular instability is a common feature of NICU infants that often leads to compromised cerebral autoregulation, hypoxic-ischemic brain injury, and intraventricular hemorrhage. Our recent work suggests that postnatal cardiovascular instability involves depressed function of Myosin Light Chain Kinase (MLCK), the rate- limiting enzyme responsible for initiation and regulation of vascular contraction. Because rates of mRNA transcription for MLCK vary little with age and hypoxia, our results implicate changes in mRNA translation, MLCK degradation, and MLCK activity as the main mechanisms that govern neonatal MLCK function. First, we will examine effects of micro-RNAs on MLCK translation. Numerous micro-RNAs are induced by hypoxia and influence contractile protein expression directly through binding to transcripts, and indirectly by influencing smooth muscle differentiation. To explore these mechanisms we have developed surgical methods that enable the in vivo adenoviral transfection of pre-term fetal lambs, in utero. This approach offers unprecedented opportunities to explore the molecular roles of micro-RNAs in fetal responses to hypoxic stress, particularly as related to regulation of MLCK function. Second, we will examine the roles of ubiquitination and protein degradation in fetal vascular responses to hypoxia. Despite the recognized importance of ubiquitination, it has not been studied in fetal lambs, their cerebral arteries or their responses to hypoxia. Our findings demonstrate that expression of some ubiquitin ligases is age-dependent and for others is potently upregulated by chronic hypoxia. These results advance the novel idea that changes in protein degradation are intimately involved in fetal vascular adaptation to chronic hypoxia. Third, we will examine effects of hypoxia on MLCK activity, in situ. Using novel methods to measure high-speed transients in cytosolic calcium and myosin light chain phosphorylation in whole arteries, we have found that MLCK velocity is enhanced by chronic hypoxia in fetal but not adult arteries. Our confocal methods further suggest that colocalization of MLCK with its substrate is stronger in fetal than adult arteries, and is significantly altered by chronic hypoxia, suggesting a new role for MLCK compartmentalization in regulation of fetal cerebrovascular contractility. Overall, further study of the mechanisms identified by our recent work promises to reveal multiple important new features of the molecular, cellular, and tissue level regulation of MLCK function, and offers new understanding of how these mechanisms might be leveraged to improve clinical management of postnatal cardiovascular instability.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Program Projects (P01)
Project #
5P01HD083132-03
Application #
9438918
Study Section
Special Emphasis Panel (ZHD1)
Project Start
Project End
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Loma Linda University
Department
Type
DUNS #
009656273
City
Loma Linda
State
CA
Country
United States
Zip Code
92350
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