# Modeling the Effects of Calcium Overload on Mitochondrial Ultrastructual Remodeling
## 3 Results and Discussion  
Oxidative phosphorylation was inhibited in the calcium overload state [5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21]. As the calcium load increased, the maximal ADP-stimulated respiration was significantly inhibited, as shown in Figure 1. This phenomenon is not associated with the well-known mitochondrial permeability transition phenomenon [8]. At these calcium loads, the mitochondrial calcium, membrane potential, and respiration were stable [8; 14]. The inhibitory effect began at calcium loads of around 50 nmol/mg of mitochondrial protein. This value was calculated based on the quantified calcium uptake dynamics of the residual calcium in one of our prior studies [22]. This inhibition got progressively worse as the calcium load increased up to 500 nmol/mg. Therefore, calcium loads in the range of 50-500 nmol/mg maximally stimulated the ADP-respiration rates to decrease in a titratable manner. The cause of this inhibition was suspected to originate from calcium phosphate granule-induced cristae remodeling in an earlier study [14]. Below this range, calcium is beneficial for mitochondrial function as it stimulates matrix dehydrogenases [4]. Above this range, mitochondria undergo mitochondrial permeability transition, deenergize, and become net ATP consumers. Specifically, the mitochondrial inner membrane becomes permeable to solutes in the low kDa range, effectively preventing their ability to store chemical energy in the form of a proton electrochemical gradient. Decades of research has been done on this phenomenon [72; 73; 74; 75], but little is known about how mitochondria respond to calcium loads below the level which induces this phenomenon and above those used for energy homeostasis. Therefore, we explored how calcium loads that do not cause permeability transition but inhibit oxidative phosphorylation alter the mitochondrial ultrastructure. In the absence of calcium, mitochondria possess an intact and dense cristae network, as shown in Figure 2. The cryo-electron micrograph and a \(\sim\)10 nm thick portion of the corresponding 3D reconstruction show that in the normal state, the internal structure of the cristae network is highly interconnected. In the cryo-EM image, rows of ATP synthase molecules can be seen spiraling down the cristae, with their F1 portion sticking out into the matrix space. The intermembrane and cristae spaces are nearly equal to that of the matrix space. This ensures near-optimal metabolic efficiency by maximizing both the inner membrane surface area and the matrix water space. The inner membrane houses all the proton pumps, transporters, and enzymes required to establish, maintain, and utilize the proton electrochemical gradient for ATP production. The matrix space contains nearly all the enzymes that produce the reducing equivalents used to run the proton pumps. In addition, the cristae junction widths are tight and around 10 nm in diameter (see Figure 3D). The cristae junction serves as a gateway to allow some metabolites, such as ATP, ADP, and Pi into the cristae lumen to be rapidly transported outside the cristae and mitochondria. These junctions also serve to lock key metabolites such as cytochrome c in the cristae lumen. Cytochrome c is a small (~15 kDa) mobile electron carrier that is analogous in function to nicotinamide adenine dinucleotide in the matrix and ubiquinone in the membrane lipid space. By maintaining the cristae network and matrix spaces in the form discussed above, mitochondrial ATP production rates are maximized. In the calcium-overloaded state, the mitochondrial ultrastructure was significantly altered, as is shown in Figure 3. The presence of calcium phosphate granules in the matrix space had a devastating effect on the intermembrane and cristae spaces. The matrix space was significantly expanded, with the intermembrane and cristae spaces being dramatically reduced. The matrix space expanded due to the increased osmotic pressure in the matrix that occurred when large amounts of calcium were taken up by the mitochondria.