Hypothesis: Overexpression of the mitochondrial NAD+ transporter Slc25a45 specifically raises matrix NAD+ concentration, which activates SIRT3 to deacetylate key complex I subunits (e.g., NDUFA9) and promotes respirasome (supercomplex I+III+IV) assembly. This structural tightening reduces electron slip and proton leak, thereby increasing the P/O ratio (ATP produced per oxygen consumed) without requiring a rise in total cellular NAD+ or mitochondrial biogenesis. Conversely, Slc25a45 knockdown will lower matrix NAD+, diminish SIRT3 activity, increase complex I acetylation, destabilize supercomplexes, elevate ROS, and decrease coupling efficiency even if cytosolic NAD+ pools remain unchanged.

Testable predictions: 1) In C2C12 myotubes, adenoviral Slc25a45 overexpression will increase matrix NAD+/NADH ratio measured by mito‑SoNar sensor, while total NAD+ (cytosol+mitochondria) stays constant. 2) SIRT3 activity, assessed by deacetylation of SOD2 and NDUFA9, will rise concomitantly. 3) Blue‑native PAGE will show higher abundance of I+III+IV supercomplexes in Slc25a45‑overexpressing cells. 4) Seahorse XF analysis will reveal elevated ATP‑linked OCR and reduced proton leak, yielding a higher P/O ratio. 5) ROS production (MitoSOX) will decline. 6) The same manipulations in Slc25a45‑knockdown cells will produce the opposite matrix NAD+ drop, SIRT3 inhibition, supercomplex disassembly, increased leak, and lower P/O ratio despite unchanged total NAD+. 7) Rescue experiments with a mitochondria‑targeted NAD+ biosynthetic enzyme (NMNAT3) should mimic Slc25a45 overexpression, confirming that the effect hinges on matrix NAD+ rather than transporter scaffolding.

Falsification: If Slc25a45 alteration fails to change matrix NAD+/NADH ratio, SIRT3 activity, supercomplex abundance, or P/O ratio while total NAD+ varies, the hypothesis is refuted. Similarly, if matrix NAD+ rises but coupling efficiency does not improve, or if improvements occur without changes in supercomplex assembly, the proposed mechanistic link is invalid. This framework shifts focus from bulk NAD+ supplementation to spatial NAD+ flux and its direct impact on ETC architecture, offering a precise, falsifiable route to genuine mitochondrial optimization.