Optimizing Optical Spectroscopy to Advance the Study of Cardiac Metabolism in the Isolated Perfused Heart Open Access
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Cardiovascular diseases account for 30% of all deaths in the United States yearly and cause severe financial and manpower setbacks for the society. Cardiac mitochondrial DNA mutations are also linked to the development of degenerative cardiac disorders. To be able to translate metabolic results to in vivo, metabolic research is moving from isolated organelles or cells to isolated perfused hearts (established by Oskar Langendorff in 1895). The isolated perfused heart is a valuable tool used to study cardiac function and disease. Data from this model coupled with isolated organelles and cells models led to a greater understanding of cardiac metabolism. This model however still has its limitations. The purpose of the studies presented in this dissertation is to implement and validate new techniques to further the study of cardiac metabolism in the isolated perfused heart. The work in this dissertation starts with the development of a LabVIEW-based program for multiwavelength data acquisition and linear least-square analysis to obtain differential optical absorbance density for each cardiomyocyte chromophore. Following the program, side-firing light catheters were developed and fabricated or adapted to perform true transmural optical absorbance spectroscopy in isolated perfused hearts for the first time. The program and light catheters were then used to evaluate oxygenation and cardiac function in isolated saline perfused rabbit hearts. The data showed that saline perfused hearts are 15 – 20% hypoxic, unlike the heart in vivo which is close to 100% oxygenated. The data also showed a near 4-fold increase in signal to noise ratio compared to the previously used reflective absorbance spectroscopy, along with resolving the issues of scattering and path-length in the problematic reflective imaging mode. This new technique was then used to further metabolic studies along the electron transport chain in the isolated perfused heart. In complex III, the absorbance mole fraction of heme bL to cytochrome b was measured along with varied membrane potential in isolated mitochondria experiments and a linear model of the relationship between these two measurands was developed. This model was then used to calculate for the first time absolute mitochondrial membrane potential in perfused hearts without the use of exogenous probes, just by measuring cytochrome’s absorbance. Finally, the characteristics of the catalytic intermediates of complex IV were investigated during its reduction of oxygen to water. The presence of the P and F species of heme a3 was confirmed in in vivo-like conditions during turnover in the presence of oxygen. The well-known R species was confirmed to only be present when oxygen is limited, and to have a 3 times higher extinction coefficient compared to the P and F species. Finally, using oxygen consumption and membrane potential, the rate limiting steps during turnover were modelled, along with their effects on thereactions driving force, ∆G. This dissertation present new tools to investigate as never before done parameters important to cardiovascular metabolism in the isolated perfused heart. It also presents the limitations of this well-established model, and the potential need to move the research to in vivo.