Heart disease is a leading global cause of death and results in significant economic and healthcare burdens. A critical factor involved is mitochondrial dysfunction induced by calcium overload. This form of dysfunction plays a major role in the injuries that develop from acute myocardial infarctions. Thus, it essential to develop strategies to target these organelles to enhance overall functionality. This thesis examines the critical role of mitochondria in cellular energetics, with a focus on the effects of calcium on mitochondrial structure and function and proposes strategies to advance our understanding.It is proposed that i) mitochondrial structure is a critical regulator of cellular energetics, and ii) inhibition of oxidative phosphorylation is a calcium-related phenomenon that involves ultrastructural changes. To investigate the mechanisms behind the inhibitory effect of mitochondrial calcium overload on ADP-stimulated respiration, high-resolution respirometry and cryo-electron microscopy were used. The findings show that calcium accumulation leads to the formation of calcium phosphate deposits, outer membrane rupture, inner membrane fragmentation, and evisceration. This results in loss of respiratory control.High-resolution, cryo-EM images of isolated mitochondria exposed to various conditions designed to elucidate how calcium overload impacts structure and function were collected. From these data, 3D reconstructions were generated for morphometric analysis. The results show that higher levels of calcium can lead to a significant reduction in the density of the cristae network, which ultimately impacts the integrity of the cristae. The impact of calcium phosphate deposits on the mitochondrial matrix is also apparent, resulting in looser and thinner matrices in mitochondria loaded with calcium compared to control or treated-mitochondria with the permeability transition pore inhibitor Cyclosporin A. The study raises questions about the potential link between cristae destabilization and the permeability transition pore, which can lead to mitochondrial rupture and cytochrome c release.In addition, a phase separation-based approach was used to study the dynamics of mitochondrial ultrastructure pattern formation, which defined the mitochondrial ultrastructure as a two-component system consisting of the inner membrane space and the matrix space, with the outer membrane serving as the domain boundary. The study mimicked the observed effects of calcium phosphate deposits, where calcium phosphate induces devastating remodeling effects on the inner membrane, resulting in matrix expansion, intermembrane space contraction, and cristae remodeling. Finally, the thesis discusses a few compound libraries that were screened to find drugs capable of protecting isolated mitochondria from calcium overload and oxidative stress. Eleven compounds passed the initial screening for protection against calcium overload. Future directions for this line of research should expand the drug screening approach to validate the compound hits and investigate the potential for combination therapies using multiple drug candidates. Overall, these findings provide a promising starting point for further research into the development of effective treatments and preventative measures for mitochondrial dysfunction and related diseases.
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