Mitochondria are ancient endomembrane organelles found in eukaryotic cells that have the ability to be a driving force in evolution, producing ATP through respiration. Inducing mutations in either the nuclear or mitochondrial DNA genes encoding proteins required for aerobic ATP production leads to a variety of human mitochondrial diseases irrespective of the organ. Complications that develop in diabetes mellitus affecting the vascular and multiorgan systems are causally and highly associated with overproduction of reactive oxygen species (ROS) induced by hyperglycemia [
1,
2,
3]. Additionally, the critically ill patients often depict levels of glucose in the blood and this aggravates the multiorgan injuries [
4]. High glucose conditions induce the metabolic substrate entry into the mitochondria and this weakens the electron transport system culminating in the over production of ROS [
1]. A deficiency of mitochondrial protein synthesis was observed in the drosophila mutant tko25t characterized by respiratory and oxidative phosphorylation defects leading to developmental delay as well as being sensitive to seizures by mechanical stress. It was uncovered that the mutant effects are aggravated by high dietary sugar intake levels in dose dependent fashion. Series of metabolic abnormalities have also been found out as a result of high sugar intake diets and some of the lethal effects include decreased NADPH and ATP productions and an increase in the levels of lactate and pyruvate [
5]. Glycation of extracellular proteins ensue when there is an abnormally high concentration of glucose such as in the case of diabetic hyperglycemia [
12]. Under high glucose conditions, there is an abnormally high concentration of glucose in the neurons because absorption of glucose by neurons is insulin independent. Under such instances, glucose is oxidized to form reactive carbonyls and ROS, ultimately activating MAP kinases and this has effects on the phenotype of the cells [
13]. Calcium (Ca
2+) plays a critical role in mediating many important biological functions and has been implicated as an intracellular regulatory factor in many physiological and pathological processes in the cell. A disruption of intracellular Ca
2+ homeostasis is frequently associated with the early development of cell injury. Research has established pathological mechanisms by which intracellular Ca
2+ overload triggers either necrotic or apoptotic cell death. From studies on different tissues in a variety of pathological conditions a general consensus emerges on the role of mitochondrial Ca
2+ overload as a pivotal link between cellular alterations and mitochondrial dysfunction [
19]. Mitochondrial Ca
2+ uptake is driven by mitochondrial membrane potential (∆Ψm) [
20]. Under conditions of oxidative stress, mitochondrial Ca
2+ cycling can reach critical levels, leading to increased energy expenditure and a dramatic fall in ∆Ψm. Recent work has shown that a fall in mitochondrial ∆Ψm is an early event in apoptosis [
21]. Researchers presume the dependence of mitochondrial Ca
2+ uptake on the membrane potential and the intracellular distribution of the organelle, both of which may be altered in mitochondrial diseases [
22]. Mitochondrial Permeability Transition (MPT) is the key switch to turn on cell apoptotic pathway. The MPT pore is a mega channel complex that contains an adenine nucleotide translocase (ANT), cyclophilin-D (CyP-D) and a voltage-dependent anion channel (VDAC). The complexity of the pore structure demonstrates how MPT can act as multiple sensors for various cellular messages originated from both intra as well as extra mitochondrial environment including Ca
2+ overload, membrane potential depolarization, oxidative pressure, as well as receptors connected cellular signals [
23]. The channel complex opening occurs as a result of the binding of CyP-D to ANT in the inner mitochondrial membrane. CyP-D binding enhances the ability of the ANT to undergo a conformational change triggered by Ca
2+. Binding of ADP or ATP to a matrix site of the ANT antagonizes this effect of Ca
2+. Modification of other ANT thiol groups inhibits ADP binding and sensitizes the MPT to Ca
2+ [
24]. Increased membrane potential changes the ANT conformation to enhance ATP binding and hence inhibit the MPT [
25]. In isolated mitochondria, opening of MPT pore leads to collapse of the electrochemical gradient of H
+ i.e. causing mitochondrial membrane potential depolarization, thereby, annihilates the driving force needed for ATP production and triggers the production of ROS. Moreover, the pore opening also releases several apoptotic proteins, such as cytochrome c, apoptosis inducing factors (AIF), and procaspase-9, and turns on cellular apoptotic cascades that eventually lead the cell to the 'point of no return' journey of apoptotic pathway. The MPT is regulated by a variety of cellular physiological and pathological effectors such as mitochondrial Ca
2+ overload transients, oxidative stress, and depolarization of mitochondrial membrane potential in the concentration of polyamines [
26]. The voltage and Ca
2+ threshold at which MTP pore opening occurs are modulated by a variety of agents and conditions [
27]. Cyclosporin A and its non-immunosuppressive analogue N methyl-Val-4 cyclosporin A (PKF220-384) both inhibit opening and prevent the translocation of cyclophilin D from the matrix to the membranes of cortical mitochondria [
28]. Although existence of transient or flicker opening of MPT has long been debated from different aspects, using isolated mitochondria, MPT pore flicker has recently been elegantly demonstrated. Mitochondria immobilized on coverslips were imaged using tetramethylrhodamine methyl ester as membrane-potential indicator; pore opening was triggered by the generation of mROS on photodecomposition of the indicator. Pore flicker was apparent as a transient depolarization inhibited by Cyclosporin A. This phenomenon has been suggested as a protective mechanism against mitochondrial Ca
2+ overload and mitochondrial membrane potential depolarization [
29]. It is not uncommon to find a decrease of mitochondrial functions in neurodegenerative diseases, this is critical as far as mitochondrial biology is concerned, but the molecular basis to confirm this is elusive. The human mtDNA is a double membrane circular molecule of 16,569 bp and it critically encodes 13 polypeptides of the mitochondrial respiratory chain for oxidative phosphorylation, 22 tRNAs and two small and large subunits of rRNA for protein synthesis [
24]. The F1F0-ATPase (ATP synthase or complex V) is the enzyme that is responsible for catalyzing the final step of oxidative phosphorylation. It does so by coupling the translocation of the protons that are in the intermembrane space and shuttling them to the mitochondrial matrix for ATP synthesis to take place. In the mammals, the ATP synthase structure consists of a soluble portion denoted F1 in which catalyzing ATP synthesis takes place. It also has the F0 portion that is found embedded in the inner membrane of mitochondria and serves as a proton channel. Proton shuttling in the F0 takes place at the interface between ATP6 subunit and the C-ring, enhances conformational changes or modifications that are transmitted to the F1 portion, hence providing energy for the synthesis of ATP via rotation of the stalk [
30]. Mitochondrial DNA (mtDNA) encoded ATP6 subunit mutations cause complex disorders with varying and heterogeneous expressions as well as severity, this ranges from adult onset Neurogenic Muscle Weakness, Ataxia and Retinitis Pigmentosa (NARP) syndrome to a fatal infantile subacute necrotizing encephalomyelopathy, a maternally inherited form of Leigh Syndrome (MILS). The most frequent and first mutation that is associated with NARP/MILS is the ATP6 mutation (T8993G) which results in the substitution of a highly conserved amino acid leucine to arginine (L156R) [
31]. NARP cybrids can be established when human osteosarcoma 143B cells are depleted and gotten rid of the mtDNA by exposing the cells to ethidium bromide and obtain a cell line that is devoid of mtDNA (ρº rho) cells. Human skin fibroblasts that are obtained from a patient that is clinically confirmed to have NARP syndrome are enucleated and fused with the ρº cells to create a 98% mutant type (NARP) cybrids [
32]. The effects of high glucose toxicity on NARP cells has not been explored. Our research group hereby hypothesized that mitochondrial calcium [Ca
2+]
m overload modulates high glucose toxicity on respiratory chain defects in NARP cells. The ultimate aim of this research is to investigate how mitochondrial calcium [Ca
2+]
m overload in tandem with oxidative stress modulate high glucose toxicity on respiratory chain defect-augmented mitochondrial reactive oxygen species (ROS) production, membrane potential (ΔΨ) depolarization, cardiolipin (CL) remodeling, calcium (mCa
2+) homeostasis and mitochondrial permeability transition pore (MPTP) including transient (t-MPT) and permanent-MPT (p-MPT).