Biology subjects

Bazil, J. N.

Publications and source records attributed to Bazil, J. N..

4 recordsLinked to original sources

bioRxiv2026

Systems Analysis of Carboxylate Transport and Oxidation Pathways in Cardiac Mitochondria

Experimental assessment and computational modeling were used to analyze substrate transport, tricarboxylic acid cycle kinetics, and oxidative phosphorylation in suspensions of purified cardiac mitochondria. The kinetics of ATP synthesis and carbohydrate oxidation, including during hypoxia and reoxygenation, were investigated using various substrate combinations and conditions. Model simulations fit to transient respiration and NAD(P)H measurements reveal novel insights into pyruvate dehydrogenase regulation, regulation of mitochondrial leak, and the clearance of oxaloacetate during respiration on succinate. High concentrations of succinate induced increased mitochondrial leak respiration driven in part by ROS-activated uncoupling. Oxidative phosphorylation under succinate-fueled respiration was inhibited by rapid buildup of oxaloacetate, inhibiting succinate dehydrogenase. Malic enzyme and oxaloacetate decarboxylase activities represent potenital pathways for removal of oxaloacetate, with glutamate further enhancing clearance. The developed model captures the observed transient behaviors as well as steady-state relationships between ATP synthesis rate and phosphate metabolite levels, lending a new systems-level understanding of mitochondrial energy metabolism. In sum, these findings offer a framework for simulating and interpreting mitochondrial function in vitro and in vivo. Key PointsThis study uses experiments and computer simulations to probe the interactions between substrate transport processes, TCA cycle kinetics, redox state, and oxidative ATP synthesis in cardiac mitochondria. The developed kinetic model simulates mitochondrial metabolism in vitro and represents a framework for integrative modeling of cardiac energy metabolism. Model-based analysis identifies a kinetic model of pyruvate dehydrogenase (PDH) deactivation during leak-state respiration and activation during oxidative phosphorylation. High levels of cation leak during respiration on succinate are explained by a ROS-dependent activation of uncoupling.

biochemistry↗

bioRxiv2025

Various Optimization Strategies for the Isolation of Mitochondria from Sprague-Dawley Rat Liver Tissue

Mitochondria are key organelles that establish a very large free energy drop for the ATP hydrolysis reaction in the cytoplasm of cells, which provides energy required for the cell maintenance of homeostasis. They are commonly isolated from living tissue as it makes them easier to interrogate at the biochemical level. Thus, isolated mitochondria of sufficient quality are desired to improve translational impact. To gain a greater insight into the isolation process and optimize our current isolation protocol for quality, we implemented various fine-tuning modifications to our standard homogenization, centrifugation, and purification steps with isolated rat hepatocyte mitochondria. The following modifications we tested were: i) different homogenization speeds (10,000, 14,000, and 18,000 rpm) with varying time intervals (10, 20, and 30 sec), ii) addition of an additional purification spin, and iii) use of density gradients to further purify the isolated mitochondria from non-mitochondrial contaminants. Mitochondrial quality was approximated using the well-established respiratory control ratio (RCR). The data reveal that our original protocol yields isolated mitochondria with acceptable quality and the optimization attempts produced similar or worse mitochondrial isolates. In addition, an extra purification spin decreased RCR values and therefore is not recommended. We note that the use of density gradients did not improve the RCR, but it did remove presumed peroxisomal contamination. While this protocol can be further enhanced using additional metrics, the data indicate that our current isolation protocol is sufficiently effective since additional modifications did not yield major improvements.

biochemistry↗

bioRxiv2021

Identifying Site-specific Superoxide and Hydrogen Peroxide Production Rates from the Mitochondrial Electron Transport System Using a Computational Strategy

Mitochondrial reactive oxygen species (ROS) play important roles in cellular signaling; however, certain pathological conditions such as ischemia/reperfusion (I/R) injury disrupt ROS homeostasis and contribute to cell death. A major impediment to developing therapeutic measures against oxidative stress induced cellular damage is the lack of a quantitative framework to identify the specific sources and regulatory mechanisms of mitochondrial ROS production. We developed a thermodynamically consistent, mass-and-charge balanced, kinetic model of mitochondrial ROS homeostasis focused on redox sites of electron transport chain complexes I, II, and III. The model was calibrated and validated using comprehensive data sets relevant to ROS homeostasis. The model predicts that complex I ROS production dominates other sources under conditions favoring a high membrane potential with elevated NADH and QH2 levels. In general, complex I contributes to significant levels of ROS production under pathological conditions, while complexes II and III are responsible for basal levels of ROS production, especially when QH2 levels are elevated. The model also reveals that hydrogen peroxide production by complex I underlies the non-linear relationship between ROS emission and O2 at low O2 concentrations. Lastly, the model highlights the need to quantify scavenging system activity under different conditions to establish a complete picture of mitochondrial ROS homeostasis. In summary, we describe the individual contributions of the ETS complex redox sites to total ROS emission in mitochondria respiring under various combinations of NADH- and Q-linked respiratory fuels under varying work rates.

systems biology↗

bioRxiv2020

The Mitochondrial Permeability Transition Phenomenon Elucidated by Cryo-EM Reveals the Genuine Impact of Calcium Overload on Mitochondrial Structure and Function

Mitochondria have a remarkable ability to uptake and store massive amounts of calcium. However, the consequences of massive calcium accumulation remain enigmatic. In the present study, we analyzed a series of time-course experiments to identify the sequence of events that occur in a population of guinea pig cardiac mitochondria exposed to excessive calcium overload that cause mitochondrial permeability transition (MPT). By analyzing coincident structural and functional data, we determined that excessive calcium overload is associated with large calcium phosphate granules and inner membrane fragmentation, which explains the extent of mitochondrial dysfunction. This data also reveals a novel mechanism for cyclosporin A, an inhibitor of MPT, in which it preserves inner membrane architecture despite the presence of massive calcium phosphate granules in the matrix. Overall, these findings establish a mechanism of calcium-induced mitochondrial dysfunction and the impact of calcium regulation on mitochondrial structure and function.

cell biology↗