IF aged C57BL/6J mice (22–24 months, both sexes), pre-stratified by elevated skeletal muscle and cardiac MLCL/CL ratio (≥3-fold above young 3-month reference, quantified via LC-MS/MS) as a proxy for accumulated inner mitochondrial membrane (IMM) oxidized cardiolipin burden, receive a combinatorial regimen of (a) elamipretide (SS-31, 3 mg/kg/day subcutaneous injection, continuous osmotic minipump) to arrest ongoing cytochrome c-mediated CL peroxidase activity, plus (b) mitochondria-targeted overexpression of calcium-independent phospholipase A2 gamma (iPLA2γ) via intravenous AAV9-iPLA2γ (driven by the MCK or αMHC promoter for tissue specificity), administered simultaneously over 8 weeks to 24-month-old mice,

THEN the following outcomes will be observed relative to elamipretide monotherapy controls (also aged, also biomarker-stratified) and to young-reference animals:

• MLCL/CL ratio will decrease by ≥50% in cardiac and skeletal muscle mitochondria (LC-MS/MS lipidomics)
• Tetralinoleoyl-CL (TLCL) content will increase by ≥30% of total CL pool (LC-MS/MS)
• ETC supercomplex (Complex I+III₂+IV, "respirasome") abundance will increase ≥35% by Blue Native-PAGE densitometry
• Cristae junction width and frequency will be restored toward young-reference values (≥25% increase, transmission electron microscopy morphometry)
• Maximal mitochondrial oxygen consumption rate (OCR) and Complex I-linked State 3 respiration will improve ≥25% above elamipretide monotherapy (Seahorse XF96 on isolated mitochondria)
• Grip strength and exercise capacity (treadmill exhaustion) will improve ≥20% vs. aged vehicle controls,

BECAUSE the following mechanistic chain connects the intervention to the outcomes:

1. Accumulated oxidized CL species are the primary unrepaired damage substrate. In aged mitochondria, ALCAT1 upregulation and declining tafazzin remodeling activity result in progressive CL peroxidation and MLCL accumulation. This is a structural damage legacy that SS-31 alone, acting purely as a CL-binding shield, cannot retroactively remove — it can only halt further peroxidation (SS-31 prevents further CL peroxidation by inhibiting cytochrome c peroxidase activity)[https://pubmed.ncbi.nlm.nih.gov/33586053/]. Existing oxidized CL species remain embedded in the IMM and continue to disrupt supercomplex geometry even after SS-31 treatment begins.

2. SS-31 halts the upstream peroxidase reaction, creating a protected window for lipid repair. Elamipretide penetrates the heme crevice of cytochrome c bound to CL, preventing the conformational change that enables peroxidase activity. Without this arrest, any newly synthesized CL would immediately be re-oxidized (elamipretide stabilizes mitochondrial cristae and improves mitochondrial function in heart failure)[https://pubmed.ncbi.nlm.nih.gov/33021362/]. SS-31 thus creates the biochemical permissive environment in which downstream lipid repair can occur without futile cycling.

3. **iPLA2γ selectively ex...

SENS category: LysoSENS

Key references: • doi.org/10.1038/s41467-018-07253-3]. • doi.org/10.1101/2024.07.11.603085]. • doi.org/10.2217/nnm.14.161].