The mitochondrial phosphate carrier (PiC), encoded by the nuclear gene SLC25A3, was purified more than 30 years ago. It has been long believed that PiC serves as the primary means of phosphate (Pi) uptake into mitochondria for oxidative phosphorylation (oxphos) and as the main buffering species for the vast amount of calcium that mitochondria can take up. However, only very recently have mutations in human PiC been discovered, and a PiC floxed mouse has been generated, allowing PiC function to be directly studied in vivo for the first time. Human PiC mutations result in a severe clinical phenotype, with onset soon after birth and striated muscle as a key affected tissue. Similarly, mice with cardiac-specific deletion of the PiC had abnormal cardiac function; however, the extent to which this was caused by a defect in oxphos, dysregulated Ca2+ signaling related to poor mitochondrial Ca2+ buffering, and/or to matrix swelling caused by mitochondrial Ca2+ uptake in the absence of Pi uptake is unknown. Yet, these are fundamental questions about a basic process, namely mitochondrial Pi uptake that supports energy production and Ca2+ homeostasis. Here we hypothesize that perturbation of PiC in skeletal muscle causes a bioenergetic and Ca2+ handling deficit that worsens when energy demand rises. Because fusion of the inner mitochondrial membrane requires mitochondrial ATP, we further hypothesize that the bioenergetics deficit will lead to impaired mitochondrial fusion-fission dynamics. These hypotheses will be tested using new genetic models: mice with skeletal muscle-specific PiC depletion, and myotubes and fibroblasts from PiC deficient individuals with natural SLC25A3 mutations.
Aim 1 will test the dependence on PiC of mitochondrial Pi uptake and oxidative phosphorylation, and will also consider whether alternate transport mechanisms counter PiC deficiency. We will also investigate how PiC deficient skeletal muscle responds to PiC deficiency in terms altered glycolytic flux and nutrient signaling, exercise tolerance and mechanical function.
Aim 2 will test if PiC deficiency causes dysregulation of cytoplasmic and mitochondrial Ca2+, and Ca2+ regulated functions. Pi uptake is required for effective mitochondrial Ca2+ handling and a Ca2+ rise triggers each muscle contraction. Dysregulation of mitochondrial Ca2+ is also a main trigger of mitochondrial restructuring and cell death. Thus it is imperative to determine the consequence of PiC deficiency in terms of Ca2+ homeostasis. From preliminary results we hypothesize that PiC deficiency impairs mitochondrial Ca2+ handling, leading to cytoplasmic Ca2+ dysregulation, mitochondrial fragmentation, membrane permeabilization and cell death. To test this hypothesis, multiparameter Ca2+ and functional assays will be carried out in the genetic models presented by the mice and the patient cells. Studies from both Aims will help to delineate 1) the extent of PiC's biological functions in skeletal muscle, 2) the (mal)adaptive mechanisms due to severe PiC deficiency and mitochondrial dysfunction, which are poorly understood for mutations in nuclear DNA-encoded mitochondrial proteins.
Primary mitochondrial diseases can readily identified through genetic screening; yet, how they cause disease remains poorly understood. We will investigate the pathogenesis of newly described mutations in the nuclear-encoded mitochondrial phosphate carrier (PIC) using patient muscle cells and a new mouse model of skeletal muscle-specific PiC depletion. These experiments are expected to reveal 1) the PiC's biological functions in skeletal muscle; 2) (mal)adaptive mechanisms due to severe PiC depletion and mitochondrial dysfunction, which is poorly understood for mutations in nuclear DNA-encoded mitochondrial proteins.
