Ghouri, Iffath Ayesha
A study of mitochondrial redox state in cardiac muscle.
PhD thesis, University of Glasgow.
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This thesis describes the use of intrinsic fluorescence measurements as a means for examining mitochondrial function in different cardiac preparations and phenotypes.
Cardiac myocytes are intrinsically fluorescent and spectroscopic analysis of rabbit ventricular myocytes indicated that the majority of this fluorescence
arises from the metabolic coenzymes nicotinamide adenine dinucleotide in the reduced state (NADH) and flavin adenine dinucleotide (FAD) in the oxidised state. Calibration of the NADH and FAD fluorescence signal with the mitochondrial inhibitors sodium cyanide (NaCN) and carbonyl cyanide p-
(trifluoromethoxy) phenylhydrazone (FCCP) enabled calculation of mitochondrial redox states. Redox measurements reflect the balance between reduced and
oxidised forms of the NAD and FAD pools and provide an index for assessing mitochondrial function in cells and tissue. The major advantage of this technique is that the intrinsically fluorescent nature of these metabolites
obviates the need for exogenous indicators of mitochondrial function, which can themselves influence mitochondrial behaviour.
Mitochondrial redox state was established using a variety of fluorescence techniques. Values for NADstate represent the proportion of the NADH/NAD+ redox couple in the reduced state. Calculation of NADstate using single photon, two
photon and wide-field epifluorescence microscopy revealed very similar values ranging from 0.57±0.18 to 0.59±0.17 (mean±SD). FAD fluorescence measurements were used to establish FADstate (the proportion of the FADH2/FAD
redox couple in the oxidised state). However, FAD fluorescence could only be detected by epifluorescence and single photon excitation fluorescence microscopy. Once again, comparable values of 0.17±0.10 and 0.18±0.07
respectively were obtained, thus demonstrating the reproducibility of the technique. Attempts were made to perform these measurements in intact cardiac tissue preparations. However, difficulties encountered with the delivery of mitochondrial inhibitor to specific areas of tissue and problems with inner filter effects complicating the interpretation of fluorescence recordings meant that this was not possible.
Measurements of intrinsic fluorescence were utilised in order to assess the mitochondrial redox response of cardiac cells to increased energy demand. Isolated rabbit ventricular myocytes were field stimulated and fractional
shortening was simultaneously recorded with epifluorescence measurements of NADH and FAD. Cells were paced at 0.5Hz and the stimulation frequency step increased to 1Hz, 2Hz and 3Hz in order to increase work intensity and energy demand. Step increasing stimulation frequency resulted in a decrease in NADH fluorescence and an increase in FAD fluorescence before reaching an essentially steady state. This indicated oxidation of the cell environment, suggesting a transient mismatch between metabolite supply and demand. The magnitude of this response was related to stimulation frequency, with the biggest responses taking place at the highest work intensity. Reducing work intensity back to 0.5Hz pacing resulted in immediate recovery of metabolite fluorescence.
Investigation into the redox response to increased work intensity in the stroke prone spontaneously hypertensive rat (SHRSP) model of cardiac hypertrophy found that energy supply and demand matching was in fact improved in these
cardiomyocytes compared to Wistar Kyoto (WKY) control myocytes. Work intensity was increased from 1Hz to 2, 4 and 6Hz pacing and the oxidative response to increased workload was found to be significantly less in SHRSP cardiomyocytes compared to WKY myocytes (p<0.01). This was despite similar
levels of contractile work being performed by the two groups and may be related to the young age of the animals (16 weeks). At this age, hypertrophy of the SHRSP hearts is likely to still be in the compensated state and mitochondrial function may indeed be improved rather than detrimentally affected at this stage.
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