Genetic deletion of the mitochondrial phosphate carrier desensitizes the mitochondrial permeability transition pore and causes cardiomyopathy

The mitochondrial phosphate carrier (PiC) is critical for ATP synthesis by serving as the primary means for mitochondrial phosphate import across the inner membrane. In addition to its role in energy production, PiC is hypothesized to have a role in cell death as either a component or a regulator of the mitochondrial permeability transition pore (MPTP) complex. Here, we have generated a mouse model with inducible and cardiac-specific deletion of the Slc25a3 gene (PiC protein). Loss of PiC protein did not prevent MPTP opening, suggesting it is not a direct pore-forming component of this complex. However, Slc25a3 deletion in the heart blunted MPTP opening in response to Ca²⁺ challenge and led to a greater Ca²⁺ uptake capacity. This desensitization of MPTP opening due to loss or reduction in PiC protein attenuated cardiac ischemic-reperfusion injury, as well as partially protected cells in culture from Ca²⁺ overload induced death. Intriguingly, deletion of the Slc25a3 gene from the heart long-term resulted in profound hypertrophy with ventricular dilation and depressed cardiac function, all features that reflect the cardiomyopathy observed in humans with mutations in SLC25A3. Together, these results demonstrate that although the PiC is not a direct component of the MPTP, it can regulate its activity, suggesting a novel therapeutic target for reducing necrotic cell death. In addition, mice lacking Slc25a3 in the heart serve as a novel model of metabolic, mitochondrial-driven cardiomyopathy.The mitochondrial oxidative phosphorylation (OXPHOS) system is the primary source of cellular energy production. Defects in OXPHOS occur with a frequency of 1 in 5000 live births¹ and underlie a wide range of mitochondrial disorders that often affect multiple organ systems and tissues with high oxidative energy demands, such as brain, skeletal muscle, and heart.² Cardiac phenotypes associated with mitochondrial disease are diverse, and can range from cardiomyopathies to cardiac conduction defects.^{3, 4, 5}The mitochondrial phosphate carrier (PiC) is a member of the solute carrier 25A family that has a critical role in OXPHOS, serving as the primary route for inorganic phosphate (Pi) import into the mitochondrial matrix.^{6, 7} PiC, together with the adenine nucleotide translocator (ANT) and the ATP synthase, forms the ATP synthasome whereby all of the metabolites needed to generate ATP are within one immediate microdomain.^{8, 9} The importance of PiC in facilitating energy production is highlighted by the profound disease phenotype observed in patients presenting with mutations in the skeletal muscle-specific isoform of this gene.^{10, 11} Such patients present with a multisystemic disorder characterized by muscle hypotonia, lactic acidosis, severe hypertrophic cardiomyopathy, and shortened lifespan.^{10, 11} Similarly, patients with SLC25A4 (ANT1 protein) deficiency present with cardiomyopathy,¹² as do mice lacking the Slc25a4 gene,¹³ likely due to a similar molecular defect in the efficiency of ATP production within the mitochondria.In addition to its role in mitochondrial energy metabolism, PiC has been implicated in regulating cell death by serving either as a modulator or a direct component of the mitochondrial permeability transition pore (MPTP).^{14, 15, 16} The MPTP is a non-selective channel that forms in response to Ca²⁺ overload and oxidant stress that allows inner-membrane permeability to solutes up to 1500 Da in size, leading to loss of mitochondrial membrane potential, mitochondrial swelling and rupture, and eventually cell death through necrosis.¹⁵ Structurally, the MPTP complex has been proposed to be comprised of the ATP synthase^{17, 18} and to be regulated by cyclophilin D (CypD),^{19, 20} ANT,²¹ and the pro-apoptotic proteins Bax and Bak in the outer mitochondrial membrane.^{22, 23} PiC has also been suggested to be an inner-membrane component of the MPTP because it can form nonspecific channels in lipid membranes and because the MPTP is known to be activated by Pi.^{24, 25, 26, 27} Finally, PiC directly interacts with CypD in the mitochondrial matrix, which is a verified regulator and component of the MPTP.¹⁶Saccharomyces cerevisiae lacking PiC have altered MPTP characteristics with a smaller pore size, suggesting it might directly participate in the mitochondrial permeability pore.²⁸ However, partial reduction of PiC by siRNAs in cultured cells had no effect on mitochondrial permeability activity, suggesting that PiC is not required for MPTP function.²⁷ Definitive genetic proof of PiC''s involvement in MPTP formation/function is currently lacking.In the present study, we tested the role of PiC in MPTP regulation and cell death in vivo using a mouse model with inducible cardiomyocyte-specific deletion of the Slc25a3 gene (encodes PiC). We found that cardiac mitochondria depleted of PiC were able to undergo permeability transition, suggesting that PiC is not a requisite component of the MPTP. However, the extent of Ca²⁺-induced MPTP opening was blunted, suggesting that PiC serves to regulate this activity. Furthermore, Slc25a3 deletion produced a unique mouse model of mitochondrial-driven hypertrophic cardiomyopathy that recapitulates features observed in human patients with phosphate carrier deficiency and metabolic cardiomyopathy.