IF a computationally designed quadruple-domain chimeric outer membrane protein — TOMM20(1–33)-[G4S]4-TAT(YGRKKRRQRRR)-[G4S]4-C2(299–387)-[G4S]4-LIR-decoy(DDFELL) (hereafter "TC2L"), where the LIR-decoy is a hexapeptide competitive inhibitor of LC3-family autophagy receptors — is expressed on the outer mitochondrial membrane of C2C12 myoblast-derived donor mitochondria via lentiviral transduction (EF1α promoter, N-terminal HA tag) and delivered as three intramyocardial injections (1×10⁹ organelles per dose; days 0, 7, 14) to 22-month-old male C57BL/6J mice with isoproterenol-induced cardiac injury (80 mg/kg s.c. ×2 days; target: 20–30% LVEF reduction),

THEN, compared to wild-type C2C12 mitochondria and vehicle controls:

1. Donor mtDNA retention (assessed by SNP-specific qPCR at mt-CO1 locus) will be ≥3-fold higher at 4 weeks post-final injection (vs. wild-type mitochondria which are predicted to show near-complete clearance by week 2),
2. Cardiac Complex I and Complex IV activity (Seahorse XF96 at 4 weeks) will be restored to ≥70% of young (4-month) reference levels (vs. ≤45% in wild-type mitotherapy and ≤30% in vehicle),
3. Left ventricular ejection fraction (echocardiography, 4-week endpoint) will recover by ≥12 percentage points above vehicle,

BECAUSE of the following step-by-step causal chain:

1. The TOMM20(1–33) N-terminal transmembrane anchor spontaneously inserts into the outer mitochondrial membrane (OMM) during organelle biogenesis in C2C12 myoblasts, projecting all downstream domains into the cytosol-equivalent external space without disrupting membrane potential or respiratory complex assembly — the transmembrane anchor has been validated as a scaffold for surface display that does not compromise mitochondrial respiration or ΔΨm when correctly oriented (McCully et al., 2022, as cited in Evidence Set Section 4).

2. RFdiffusion-generated rigid backbone scaffolding between the TOMM20 anchor and the downstream TAT, C2, and LIR-decoy domains — replacing purely unstructured (Gly-Ser)n tethers with de novo structured connectors — ensures each functional domain is stereochemically projected beyond the OMM glycocalyx and glycoprotein density without steric occlusion, a capability demonstrated by RFdiffusion "motif scaffolding" that fixes transmembrane anchor and effector domain coordinates during backbone generation (Watson et al., Nature, 2023, as cited in Evidence Set Section 5).

3. Upon intramyocardial injection into the aged, isoproterenol-injured myocardium, the C2 domain (MFGE8 residues 299–387) binds phosphatidylserine (PS) externalized on the sarcolemmal surface of apoptotic and ischemic cardiomyocytes, concentrating donor mitochondria at the site of maximal bioenergetic need; PS externalization is a hallmark of both ischemic and aged cardiomyocytes (Evidence Set Section 4, C2 domain "homing" effect described).

4. **The TAT-derived CPP (YGRKKRRQRRR) displayed on the OMM surface facilitates rapid in...

SENS category: MitoSENS