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Title: Oxidative stress in freshly isolated cardiomyocytes and hearts during different stages of postnatal development
Author: Martin, Sarah
ISNI:       0000 0004 2722 5277
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2012
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The growing myocardium undergoes different stages of postnatal development during which the heart may respond differently to cardiac insults such as ischaemia and reperfusion (I/R). The vulnerability of the rat myocardium to I/R changes during different stages of postnatal development with 14 days old being the most resistant to I/R related injury. Mechanisms underlying I/R injury involve calcium (Ca2+) loading and the production of reactive oxygen species (ROS). The extent to which the heart is affected by the generated ROS is determined by the ability of the heart to protect against ROS and the amount of ROS that is actually produced under such conditions. Protection against ROS comes mainly in the form of endogenous antioxidants. Little is known about the role of myocardial ROS turnover or the extent of Ca2+ loading that occur during cardiac insults at different ages post-partum. This is largely due to difficulties associated with isolating and working with cardiomyocytes from different age groups during postnatal development. Therefore, the aim of this study is to investigate postnatal developmental changes in the expression and activity of myocardial antioxidant enzymes and the effect that stress, caused by metabolic inhibition, has on the viability, contractility and cytosolic Ca2+ in isolated cardiomyocytes from selected age groups throughout postnatal development compared to adult. Age related differences In antioxidant enzymes were determined by the use of Western" blotting, RT-PCR and enzyme activity assays using ventricular tissue homogenates. Additionally, conditions for cardiomyocytes isolation for different age groups were optimised and isolated cardiomyocytes were characterised according to morphology, function and Ca2+ mobilisation, to determine whether these can also be used to explain the changes in vulnerability to I/R during postnatal development. Intracellular changes in Ca2+ and other metabolites (e.g. ROS) in superfused isolated cardiomyocytes from different ages were monitored under normal conditions and when cardiomyocytes were exposed to simulated hypoxic stress (metabolic inhibition). This work shows significant age related differences in the myocardial expression and activity of antioxidant enzymes. In contrast to other antioxidant enzymes, catalase activity, protein and gene expression Increase during development to reach 2 maximum at 14-21 days before falling back to lower levels in adult. These observations suggest that catalase may be responsible for the observed changes in resistance to I/R injury in intact heart during postnatal development. Experiments on superfused and electrically stimulated cardiomyocytes exhibited age related increases in percentage twitch contraction and in the amplitude and rate to peak of Ca2+ transients under normal conditions. There were also changes in ROS and glutathione levels during development. These developmental characteristics of isolated cardiomyocytes under normal conditions do not correlate with I/R vulnerability profile during development and therefore do not offer an explanation for changes in vulnerability to I/R injury in intact heart. In contrast, the age related response of cardiomyocytes to metabolic inhibition (addition of sodium cyanide) was found to follow a pattern that is opposite to the one seen in intact perfused heart with 14 days old being most vulnerable whilst adult is least vulnerable to this insult. This "apparent" contradiction can be explained by the fact that cyanide is also an inhibitor of catalase. This is consistent with our finding that catalase expression and activity is highest in 14 days old age group, thus supporting the view that catalase is critical in determining developmental changes in vulnerability to I/R. Overall, this study has attempted to tackle an important (and difficult) area of developmental cellular cardiology and shows that the interpretation of novel data from work using isolated cardiomyocytes from different stages of development must be approached with caution. It also supports the view that antioxidants and oxidative stress are key in determining the developmental changes in vulnerability to I/R injury with endogenous catalase playing a central role.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available